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Typhoid Fever
I. Organism Information
A. Taxonomy Information
1. Species: a. Salmonella enterica : i. Ontology: UMLS:C0445750 ii. GenBank Taxonomy No.: 28901 iii. Description: The genus Salmonella was named for the pathologist Salmon, who first isolated the organism from animal intestines (Crum, 2003). The bacterial genus Salmonella is divided into two species, Salmonella bongori and S. enterica. S. enterica itself is comprised of six subspecies: they are S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. indica, and S. enterica subsp. houtenae, or I, II, IIIa, IIIb, IV, and VI, respectively. Of these six subspecies, only subspecies I is associated with disease in warm-blooded animals. Subspecies I of Salmonella enterica is responsible for almost all Salmonella infections of warm-blooded animals. Within subspecies 1 there are over 2,300 known serovars that differ in their prevalence and the diseases that they cause in different hosts. Only a few of these serovars are responsible for most Salmonella infections in humans and domestic animals (Porwollik et al., 2002). Subgroup I accounts for most human disease and contains the serotype typhi (Crum, 2003). Salmonella are divided into distinct serologic groups (A through E) on the basis of their somatic O antigens. While all group D organisms, such as S typhi possess O antigen 9, about 60 of the 78 group D serotypes including S typhi also have O antigen 12. Thus, infection by any of the group D serotypes can produce antibodies that can react with the O antigen used in the Widal reaction. Also, since all groups A and B organisms possess O antigen 12, cross-reactions with O antibody of group D serotype can occur with any of the group A and B serotype O antigens (Olopoenia et al., 2000). S. enterica serovars are defined by antigenic variation at lipopolysaccharide moieties (O antigen), flagellar antigens (H antigen), and capsular polysaccharides (Vi antigen) (Porwollik et al., 2002). Salmonella enterica serovar Typhi is a member of the genus Salmonella in the family Enterobacteriaceae. The Salmonella genus contains two species, enterica and bongori. S. enterica is further subdivided into six subspecies (enterica, salamae, arizonae, diarizonae, houtenae and indica) containing 2443 serovars. Most of the salmonellae that cause disease with some exceptions, are in the subspecies Salmonella enterica subspecies enterica. The agents that cause enteric fever are therefore Salmonella enterica subspecies enterica serovar Typhi (commonly referred to as S. enterica serovar Typhi) and serovars Paratyphi A, B, and C) (Parry, 2006). Synonym: "Salmonella enterica" (ex Kauffmann and Edwards 1952) Le Minor and Popoff 1987 (NCBI Taxonomy). Using the Kauffman-White scheme, over 2300 Salmonella serotypes have been defined on the basis of the somatic O antigens, surface Vi (virulence antigen), and flagellar H antigens (Crum, 2003). iv. Variant(s): Salmonella enterica subsp. enterica serovar Typhi : GenBank Taxonomy No.: 90370 Parent: Salmonella enterica Description: Typhoid fever is an acute systemic infection caused by the bacterium Salmonella enterica Typhi. Salmonella enterica serovars Paratyphi A, B and C cause the clinically similar condition, paratyphoid fever. Typhoid and paratyphoid fevers are collectively referred to as enteric fevers. In most endemic areas, approximately 90% of enteric fever is typhoid (Parry, 2006). Although the organism of typhoid fever is commonly referred to as S. typhi, it should be recognized that typhi represents the serotype, not the species (Crum, 2003). Synonym: Salmonella enterica ser. typhi, Salmonella enterica serotype Typhi, Salmonella choleraesuis typhi, Salmonella choleraesuis typhi, Salmonella choleraesuis serovar Typhi, Salmonella enterica serovar Typhi (NCBI Taxonomy). Synonyms: Typhusbacillen, Bacterium typhosum, Bacillus typhosus, Bacillus typhus, Bacillus typhi abdominalis, bacillus typhicus, bacterium typhi, Acystia typhi, Bacterium (Eberthella) typhi, Eberthus typhosus, Eberthella typhi, Eberthella typhosa, Salmonella typhi, Salmonella typhosus, Typhus Typhus, Salmonella typhosa (Kelterborn et al., 1967). Salmonella enterica subsp. enterica serovar Typhi str. CT18 : GenBank Taxonomy No.: 220341 Parent: Salmonella enterica subsp. enterica serovar Typhi Description: Salmonella typhi CT18 was isolated in December 1993, at the Mekong Delta region of Vietnam, from a 9-year-old girl who was suffering from typhoid. The strain was isolated from blood using routine culture methods (Parkhill et al., 2001). Synonym: Salmonella enterica subsp. enterica serovar Typhi CT18, Salmonella enterica subsp. enterica serovar Typhi strain CT18, Salmonella typhi CT18 (NCBI Taxonomy). Agglutination reactions can divide organisms into serogroups A, B, C1, C2, D and E based on the O antigen (Crum, 2003). Using this schema, S. typhi is classified as group D, but other Salmonella are also contained within this serogroup (Crum, 2003). Salmonella enterica subsp. enterica serovar Typhi Ty2 : GenBank Taxonomy No.: 209261 Parent: Salmonella enterica subsp. enterica serovar Typhi Description: Isolated in the USSR in 1918 (Kothapalli et al., 2005). This strain was the foundation for vaccine development and was the parent of mutant strains Ty21a and CVD908 and their derivatives, used in trials of live attenuated vaccines (Deng et al., 2003). Synonym: Salmonella enterica subsp. enterica serovar Typhi str. Ty2 (NCBI Taxonomy) Salmonella paratyphi. : GenBank Taxonomy No.: 54388 Parent: Salmonella enterica Description: Synonym: Salmonella enterica subsp. enterica serovar Paratyphi A, Salmonella choleraesuis subsp. choleraesuis serovar Paratyphi A, Salmonella paratyphi-a, Salmonella choleraesuis choleraesuis (serotype paratyphi A), Salmonella paratyphi A, Bacterium paratyphi typhus A, Bacterium paratyphi "Salmonella paratyphi-A" (Brion and Kaiser 1902) Castellani and Chalmers 1919 (NCBI Taxonomy). Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 : GenBank Taxonomy No.: 295319 Parent: Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 Description: Serovar Paratyphi A is the second most prevalent cause of typhoid, responsible for one third of cases or more in southern and eastern Asia (McClelland et al., 2004). Paratyphi A and Typhi cause a similar illness, with relapsing fever. Paratyphi A generally causes a milder disease but has been particularly virulent in some outbreaks (McClelland et al., 2004). There are moderately effective vaccines for Typhi, with weak cross-protection against Paratyphi A. A vaccine for Paratyphi A is under development (McClelland et al., 2004). Paratyphi A is increasingly isolated in India, and in some regions of China it is emerging as the most frequent cause of enteric fever (Cooke et al., 2006). Other epidemiological studies also highlighted several recent outbreaks (e.g. in India and Thailand) caused by Salmonella paratyphi-A and emphasized that this microorganism can also be associated with severe, even fatal, disease (Pang et al., 1998) Synonym: Salmonella enterica ser. typhi, Salmonella enterica serotype Typhi, Salmonella choleraesuis typhi,
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Salmonella choleraesuis serovar Typhi, Salmonella enterica serovar Typhi (NCBI Taxonomy). Synonyms: Paracolon bacillus, typhusahnliche Bazillen, Paratyphusbazillus, Bacterium paratyphi Typhus A, Bacillus paratyphosus A, Salmonella paratyphi A, Salmonella paratyphosus A, Salmonella paratyphi, Typus A (Kelterborn et al., 1967). Salmonella enterica subsp. enterica serovar Paratyphi B. Synonym: Salmonella paratyphi B. : GenBank Taxonomy No.: 57045 Parent: Salmonella enterica Description: Salmonella enterica serotype paratyphi B causes sporadic gastroenteritis and, less frequently, paratyphoid fever (Desenclos et al., 1996). Synonyms: Bacille paratyphique, Paratyphusbazillus, Bacillus bremensis febris gastricae, Bacterium paratyphi Typus B, Bacillus paratyphosus B, Salmonella paratyphi B, Bacillus paratyphosus B Schottmuller type, Group IV, Salmonella paratyphosus B, Salmonella schottmuelleri, Typus Schottmuller (Kelterborn et al., 1967). Salmonella enterica subsp. enterica serovar Paratyphi B str. SPB7. Synonym: Salmonella enterica subsp. enterica serovar Paratyphi B SPB7, Salmonella enterica subsp. enterica serovar Paratyphi B strain SPB7. : GenBank Taxonomy No.: 272994 Parent: Salmonella enterica subsp. enterica serovar Paratyphi B. Synonym: Salmonella paratyphi B. Description: Salmonella enterica subsp. enterica serovar paratyphi B strain SPB7. This strain causes paratyphoid fever and was isolated from a female patient in Malaysia, 2002 (NCBI Taxonomy). Salmonella enterica subsp. enterica serovar Paratyphi C. : GenBank Taxonomy No.: 57046 Parent: Salmonella enterica Description: Unlike S. typhi, S. paratyphi C is pathogenic for animals as well as for humans. This pathogen is only a minor cause of enteric fevers in South America, Asia, and the United States. However, in certain parts of Africa, occasional outbreaks of enteric fever caused by S. paratyphi C have been reported (Daniels et al., 1989). Bacillus paratyphosus Beta5, Bacillus Erzindjan, Gaertner-paratyphoid group, Para-C, Paratyphoid C bacillus, Salmonella paratyphi C, Bacillus paratyphosis B, Bacillus paratyphosis C, Paratyphus CII, Paratyphus N 1, Salmonella hirschfeldii, Typus Orient, Salmonella orientalis (Kelterborn et al., 1967).

B. Lifecycle Information :
1. Typhoid bacillus : a. Size: These organisms are gram-negative, facultative anaerobic, nonencapsulated, flagellated bacilli in the family of Enterobacteriaceae (Crum, 2003). These are motile bacilli 1-3um, in length and 0.5-0.7um in thickness (Huckstep et al., 2002). b. Shape: Rods: Singles, Pairs (NCBI Taxonomy). c. Picture(s): i. SEM Image of Salmonella typhi (Dennis Kunkel Microscopy, Inc.):

Description: Scanning Electron Micrograph of Salmonella typhi - Gram-negative, enteric, rod prokaryote (dividing); causes typhoid fever. Magnification: x3,220 (Dennis Kunkel Microscopy, Inc.). ii. Spread of Typhoid (Huckstep):

Description: The spread of typhoid (Huckstep) d. Description: The Salmonella, members of the family Enterobacteriaceae, are gram-negative, noncapsulated bacilli that do not form spores (Edwards, 1999). Oxygen requirement: Facultative; Optimum temperature: 37 degrees C (NCBI Taxonomy). The chemical structure of Salmonella LPS is, in fact, well-known. It is composed of repeating units of an oligosaccharide (O) chain joined to a polysaccharide-lipid A backbone. The O chain of S. typhi, which bears the O9 and O12 antigens, distinguishes this organism from > 99% of other subtypes (serotypes) of Salmonella, and the organisms (serogroup D) that share this O chain can be further distinguished from S. typhi based on their flagellar and capsular antigens. The O9 antigen is highly specific to Salmonella serogroup D, as it contains a sugar that is extremely rare in nature, alpha-d-tyvelose (Lim et al., 1998).

C. Genome Summary:
1. Genome of Salmonella enterica subsp. enterica serovar Typhi (Parkhill et al., 2001): a. Description: Here we have sequenced the 4,809,037- base pair (bp) genome of a S. typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands (Parkhill et al., 2001). b. Salmonella enterica serovar Typhi CT18 (NCBI Taxonomy): i. GenBank Accession Number: AL513382 AL513383 AL513384 ii. Size: Chromosome Total size: 4,809,037 bp; pHCM1 Total size 218,150 bp; pHCM2 Total size 106,516 bp (Parkhill et al., 2001). iii. Gene Count: Chromosome: 4,599, pseudogenes 204; pHCM1: 249, pseudogenes 8; pHCM2: 131 (Parkhill et al., 2001) CT18 harbours a 218,150-bp multiple-drug-resistance in H1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis (Parkhill et al., 2001). iv. Description: Salmonella typhi CT18 harbours two plasmids, the larger of which (pHCM1) is conjugative and encodes resistances to all of the first-line drugs used for the treatment of typhoid fever (Parkhill et al., 2001). In the genome of S. typhi, >200 pseudogenes could be identified, representing 4.4% of the coding capacity (Parkhill, 2002). c. Salmonella enterica serovar Typhi strain Ty2 (Deng et al., 2003): i. GenBank Accession Number: AE014613 ii. Size: The genome of Ty2 consists of a single, circular chromosome of 4,791,961 bp with an average G+C content of 52.05% (Deng et al., 2003). iii. Gene Count: For Ty2, we predicted 4,339 ORFs, 206 pseudogenes, and 101 RNA genes by comparison with the CT18 annotations (Deng et al., 2003). A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes (Deng et al., 2003). iv. Description: A comparison of Ty2 with CT18 reveals that more than 98% of the genome sequence is shared in these two S. enterica serovar Typhi strains (Deng et al., 2003). d. Salmonella enterica serovar Paratyphi A ATCC 9150 (McClelland et al., 2004):
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i. ii. iii. iv.

GenBank Accession Number: CP000026 Size: 4,585,229 bp (McClelland et al., 2004) Gene Count: 4,263 protein coding sequences (McClelland et al., 2004). Description: Typhi and Paratyphi A are members of different O antigen serogroups, D1 and A, based on a minor difference in lipopolysaccharide sugars. Despite this difference, microarray data on shared gene content indicates that Paratyphi A and Typhi are closely related, with Paratyphi A more distant from other members of serogroup A12. Lateral transfer of serogroup genes has been observed, and so it is possible that Paratyphi A arose by transfer of serogroup A genes into a strain very similar to Typhi (McClelland et al., 2004).

II. Epidemiology Information

Typhoid (enteric) fever is a systemic infection in man that is caused by Salmonella serotypes which are strictly adapted to humans or higher primates, including Salmonella enterica serotypes Typhi, Paratyphi A, Paratyphi B, and Paratyphi C. The disease is currently rare in the United States and Europe but endemic in Asia, Africa and South America from where it can be imported by foreign travel (Santos et al., 2001). Investigators from the US Centers for Disease Control and Prevention estimate that there are 21.6 million typhoid cases annually, with the annual incidence varying from 100 to 1000 cases per 100 000 population. The global mortality estimates from typhoid have also been revised downwards from 600 000 to 200 000, largely on the basis of regional extrapolations. Recent population based studies from South Asia suggest that the incidence is highest in children aged less than 5 years, with higher rates of complications and hospitalisation, and may indicate risk of early exposure to relatively large infecting doses of the organisms in these populations (Bhutta, 2006). This threat is especially pronounced in the Southeast Asian region. As a consequence of economic growth, extensive, reciprocal movements of migrant workers are occurring between the neighboring countries of Malaysia, Thailand, and Indonesia, which has one of the highest incidences of typhoid fever in the world at more than 1,000 cases per 100,000 inhabitants (Thong et al., 1995). During the surveillance period, 285 S. Typhi episodes and 84 S. Paratyphi A episodes were detected at the 4 sites. In Indonesia, 14% of enteric fever episodes were caused by S. Paratyphi A, in Pakistan 15%, in India 24%, and in China 64%. The highest S. Typhi incidence was observed in Pakistan (394/100,000/year), and the lowest S. Typhi incidence was found in China (15.2/100,000/year). The highest S. Paratyphi A incidence was also seen in Pakistan. (72/100,000/year), and the lowest S. Paratyphi A incidence was seen in Indonesia (13.7/100,000/year) (Ochiai et al., 2005). In the USA the annual incidence dropped from 7.5 per 100000 in 1940 to 0.2 per 100000 in the 1990s, and the proportion of cases related to foreign travel increased from 33% in 1967-72 to 81% in 1996-97 (Connor et al., 2005). The annual report of the Centers for Disease Control and Prevention (CDC) for the year 2001 registered 360 different serovars in human infections in the U.S. Approximately 50% of these infections were caused by only three Salmonella serovars, specifically Typhimurium, Enteritidis, and Newport. The 12 most prevalent Salmonella serovars were responsible for >70% of all human Salmonella infections. Similarly, 41.8% of all veterinary infections were attributed to only two Salmonella serovars, namely, Typhimurium and Newport. The 10 most prevalent veterinary serovars caused 70% of all infections (Porwollik et al., 2002). Paratyphoid fever is estimated to have caused an additional 5.4 million illnesses in 2000. This number is based on an estimated one case of paratyphoid fever for every four cases of typhoid fever. Studies from India and Nepal suggest that in some settings and times, paratyphoid fever caused by S paratyphi A can contribute up to half of all cases of typhoid fever (Bhan et al., 2005).

A. Outbreak Locations:
1. Vietnam: In South Viet-nam, typhoid fever remains a considerable intestinal infection. Between 1990 and 1993, among 15 districts in the South of the country, a total per year of 3,853 to 9,179 cases was registered: from 8 to 31 led to death. Recently a large epidemic of typhoid fever broke out in the An Minh district (territory of Kien Giang, South Viet-nam), affecting 3,049 people and bringing on two cases of death. Among the 574 blood samples, 266 strains of S. Typhi, 22 S. Paratyphi A and 2 S. Choleraesuis have been isolated. Our investigations on the spot led to some epidemiologic and clinical reflexions and enabled us to estimate the effectiveness of quinolones in the treatment for typhoid fever. The epidemic may be ascribed to different causes: lack of pure water supply in rural area; fecal pollution caused by inhabitants of this endemic area defecating directly in the waterways; ingestion of contaminated food, especially vegetables sprayed with polluted water; quite low level of public sanitation and individual hygiene (Nguyen et al., 1993). 2. Bangalore, India: Six hundred and eighty five blood cultures from children clinically diagnosed as enteric fever yielded 176 salmonella strains showing isolation success rate of 25.7%, S. typhi were 164 (93.2%), S. paratyphi A 5 (2.8%), S. choleraesuis 4 (2.3%) and S. typhimurium 3 (1.7%). Antibiogram of 164 isolates of S. typhi showed triple drug resistance (TDR) in 156 strains (95.1%) to chloramphenicol, ampicillin and cotrimoxazole, and sensitivity of 90.2% and 95.1% to norfloxacin and ciprofloxacin respectively. Minimum inhibitory concentrations (MIC) of chloramphenicol were between 360 mcg and 640 mcg per ml. Phage types of 38 strains of TDR S. typhi were predominantly E1 and 0 with prevalences of 47.4% and 36.8% respectively in this region. All children with S. typhi isolates sensitive to quinolones in Vitro responded well to these drugs with almost no relapse and hence, the newer generation of quinolones could be considered as the first choice in the primary treatment of enteric fever (Rathish et al., 1995). 3. Myamar: In September 2000, an outbreak of typhoid fever was reported in a rural village of Central Myanmar. The authors investigated the outbreak in the affected village. A suspected case was a person suffering from fever with either constipation, abdominal pain, diarrhoea/bloody diarrhoea. A probable case was a suspected case who had positive result on the diazo urine test or widal test. Based on probable cases, the authors conducted a case-control study comparing history of contact with the cases, water source, and personal hygiene. Control was a person living in the village was not ill and having a negative result for diazo urine test. Among 49 suspected cases, 33 were probable. Attack rate was 1.2%. Three cases had a positive culture for Salmonella typhi and were not drug resistant. The following risk factors were identified: drinking unboiled river water (adjusted OR 12.5, 95% CI 2.8-75.3), history of contact with other patients before the illness (adjusted OR 22, 95%CI 3.5-76.2), no hand washing with soap after defecation (adjusted OR 0.15, 95% CI 0.03-0.81). Environmental investigation result showed that most of the households had unsanitary latrine and some latrines were constructed near the edge of a river. The outbreak subsided quickly after intervention (Aye et al., 2004). 4. Turkey: Salmonella typhi leads to typhoid fever outbreaks due to the contamination of drinking water. In this study, a typhoid fever outbreak due to drinking water contamination in Ahmetli village, Ergani-Diyarbakir, in the period of December 25, 2001-January 4, 2002 was evaluated. A total of 181 suspicious cases were admitted to different health care centers during the outbreak and 71 (39.2%) of them were hospitalized. Gruber-Widal test could be performed for only 8 hospitalized patients, and were found positive in 6 of them. S. typhi was isolated from 3 of the 26 blood cultures and two of the 73 stool cultures of the patients. All village inhabitants were visited and suspicious cases were screened for salmonellosis. It was shown that the village-drinking water was contaminated with sewage. No S. typhi was isolated from the drinking water since it was previously disinfected. Maintenance of drinking water supply system, individual chloride usage and education were recommended, as safety cushions (Ceylan et al., 2003). 5. Bharatpur, Nepal: In the summer of 2002, a total of 5963 cases of typhoid fever were recorded in Bharatpur, Nepal (population, 92,214) during a 7-week period. The majority of the isolates, including 1 from the municipal water supply, were multidrug resistant. To the best of our knowledge, this outbreak is the largest singlepoint source outbreak of multidrug-resistant typhoid fever yet reported, and it was molecularly traced to the city's single municipal water supply. Isolates were uniformly resistant to nalidixic acid, there was a decrease in their susceptibility as measured by minimum inhibitory concentrations (MIC) of fluoroquinolones, and 90% of isolates obtained were resistant to more than 1 antibiotic (Lewis et al., 2005). 6. New York, USA: In the summer of 2000, an outbreak of endemic typhoid fever involved 6 people in Queens, NY, and a seventh person in Manhattan, NY. An investigation by the New York City Department of Health traced the likely source to an immigrant employee working at a local restaurant (Yoon et al., 2004). 7. Ohio, USA: In August 2000, the Ohio Department of Health reported a cluster of men with typhoid fever who denied having traveled abroad. To determine the cause and the extent of the outbreak, an epidemiological investigation was initiated in which 7 persons in Ohio, Kentucky, and Indiana with culture-confirmed Salmonella enterica serotype Typhi infection and 2 persons with probable typhoid fever were evaluated; all were men, and all but one reported having had sex with 1
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asymptomatic male S. Typhi carrier. We document sexual transmission of typhoid fever, which may be acquired by means of oral and anal sex, as well as via food and drink (Reller et al., 2003). 8. Ivory Coast (Africa): In 2001, an outbreak of typhoid fever occurred among the members of the French Armed Forces. All had received a typhoid vaccination as per the immunization schedule practiced in the Armed Forces (every 5 years). A retrospective cohort study was conducted in 94 personnel. Compliance with food hygiene rules could have prevented 24 cases of typhoid fever. Nevertheless, repeat vaccination against typhoid fever is now conducted every 3 years in the French Forces, in compliance with the manufacturers' recommendations (Michel et al., 2005). 9. Chechan Republic (Russia): During the period from December 1999 to April 2000 the disease was diagnosed in 43 persons. The cases of the disease were registered in 3 settlements: Lermontov-Yurt, Kulary and Sernovodsk. The pronounced focal character of the disease was noted with the prevalence of family foci with two or more cases of the disease. This conclusion was based on the fact that at the initial stages of the formation of the foci the leading role belonged to the water factor of the transmission of the disease: water from irrigation ditches used by the population for both drinking and household needs. The unusual season when this outbreak took place (the cold time of the year) was due to highly unfavorable sanitary and hygienic conditions formed as the result of the antiterrorist operation (Grizhebovskii et al., 2001). 10. France: A case finding study of S paratyphi B infection between 15 August and 30 November 1993; a pair matched case-control study; an environmental investigation at a processing plant that produced a raw goats' milk cheese incriminated in the outbreak; phage typing and genotyping of food and human S paratyphi B isolates (Desenclos et al., 1996). The outbreak began during the second week of August 1993 and continued till the second week of November. Most of the infections were due to phage type 1 var 3. Cases were distributed nationally; however, the incidence was greatest in Poitou-Charentes (a traditional area of goats' milk cheese production) and the surrounding regions. The isolation rate was highest among infants and children aged 1 to 5 years. This large nationwide outbreak of salmonellosis was caused by unpasteurised goats' milk cheese made in a single plant. Evidence comes from the results of the case-control study and the isolation from cheese A and goats' milk of an S paratyphi B strain of the same phage type and IS 200 pattern as the epidemic strain (Desenclos et al., 1996).

B. Transmission Information:
1. Ontology: UMLS:C1444005 From: Human To: Human Mechanism: Food and Water. The common mode of infection is by ingestion of an infecting dose of the organism, usually through contaminated water or food. Although the source of infection may vary, person to person transmission through poor hygiene and sewage contamination of water supply are the most important (Bhutta, 2006). The infection is transmitted by ingestion of food or water contaminated with faeces. Epidemiological data suggest that water borne transmission of S. Typhi usually involves small inocula, whereas food borne transmission is associated with large inoculum (Ismail, 2006). Most cases occur among persons who have traveled to or among visitors from areas where disease is endemic. Food or waterborne outbreaks occur as a result of fecal contamination with Salmonella enterica serotype Typhi by ill or asymptomatically infected persons (Reller et al., 2003). 2. Ontology: UMLS:C0015733 From: Human To: Human Mechanism: Feces. Both ill people and carriers shed S. typhi in their faeces. S. typhi can be passed in the faeces of patients who are acutely ill with typhoid fever or are chronic carriers. Direct faecal-oral transmission may occur if vegetables fertilised with human waste are eaten raw, while shellfish that have been harvested from sewage-contaminated beds, and milk products contaminated by worker's hands all may result in typhoid infection (Xavier, 2006). 3. Ontology: UMLS:C1444005, SNOMED:A6941861 From: Human To: Human Mechanism: Sexual Transmission. In this outbreak, onset of typhoid fever after oral-anal or oral-penile sex documents ingestion as a recognized mode of sexual transmission of enteric pathogens. That illness also occurred after receptive anal sex only suggests that another mode of sexual transmission-direct intestinal inoculation-can also occur. The plausibility of anal intercourse as a risk factor for typhoid fever is corroborated by reports of transmission of S. Typhi infection via an unsterilized fiber-optic endoscope, polyvinyl duodenal tube, or rectal tube (Reller et al., 2003). 4. Ontology: UMLS:C1444005 From: Human To: Human Mechanism: Mechanical Transmission. Typhoid fever was the scourge of 19th- and early 20th-century armies. During the Spanish-American War (1898) and the Anglo-Boer War (1899 - 1902), typhoid killed more soldiers than enemy bullets. Walter Reed and his coworkers investigated the cause of the typhoid epidemics in the U.S. Army camps and concluded that, next to human contact, the housefly (Musca domestica) was the most active agent in the spread of the disease. British medical officers in South Africa, facing even worse typhoid epidemics, reached the same conclusion. The experiences of the American and British armies finally convinced the medical profession and public health authorities that these insects conveyed typhoid. The housefly was now seen as a health menace. Military and civilian sanitarians waged fly-eradication campaigns that prevented the housefly's access to breeding places (especially human excrement), and that protected food and drink from contamination. Currently, M. domestica is recognized as the mechanical vector of a wide variety of viral, bacterial, and protozoal pathogens. Fly control is still an important public health measure in the 21st century, especially in developing countries (Cirillo, 2006).

C. Environmental Reservoir:
1. Human : a. Description: The main reservoir for S. typhi is the human intestinal tract, although people with typhoid fever also carry the bacteria in their bloodstream. In addition, a small number of carriers recover from typhoid fever but continue to carry the bacteria. Both ill people and carriers shed S. typhi in their faeces (Xavier, 2006). Human beings are the only reservoir for Sal typhi. Infected persons shed Sal typhi in their feces from the first week of illness through convalescence. About 30% of those infected may become transient carriers who excrete Sal typhi in their feces or urine for weeks or months; 5% become long-term carriers . Although the infective dose is thought to be small, chronic carriers shed from 10(6) to 10(11) microorganisms per gram of feces. The typical carrier is a woman older than 40 years of age. The longest duration of the carrier state reported in the literature was 52 years. Typhoid fever and typhoid carriers are more common in economically disadvantaged countries than in the United States (Snyder et al., 1996) The transmission of S. typhi most often occurs by food or water which has been contaminated by feces or urine from infected humans. The organism appears to be a parasite only of the human, and if multiplication occurs outside the human body any increase in numbers is negligible. The epidemiology of typhoid fever literally depends upon the transmission of fecal or urine contamination from an infected individual to something to be ingested by susceptible individuals (Gutherie et al., 1991). Typhoid is usually contracted by ingestion of food or water contaminated by fecal or urinary carriers excreting S. enterica serovar Typhi. In addition, these bacteria can survive for prolonged periods in water, ice, dust and dried sewage and these may become sources of infection. In endemic areas, peaks of transmission occur in dry weather or at the onset of rains. Risk factors for disease include eating food prepared outside of the home, such as ice creams or flavoured ice drinks from street vendors, drinking contaminated water and eating vegetables and salads hat have been grown with human waste as fertilizer. A close contact or relative with recent typhoid fever, poor housing with inadequate food and personal hygiene and recent consumptions of antimicrobials are further risk factors. Transmission of typhoid has also been attributed to flies, laboratory mishaps, unsterile instruments and anal intercourse (Parry, 2006). b. Survival Information: The salmonellae grow best at 37 C and can survive freezing sterile water and drying for several weeks and sometimes longer. They are killed by a temperature of 60 C for 15 minutes, and rapidly by boiling (Huckstep et al., 2002). The pathogen can survive for days in groundwater, pondwater, or
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seawater, and for months in contaminated eggs and frozen oysters (Bhan et al., 2005). Chronic carrier is determined as excretion of S. Typhi in stools or urine for longer than one year after the onset of acute typhoid fever. Short-term carriers also exist but their epidemiological role is not as important as that of chronic carriers. Some patients excreting S. Typhi have no history of typhoid fever (Ismail, 2006).

D. Intentional Releases:
1. Intentional Release information : a. Description: Contaminating water and food supply. In the field of low level clandestine warfare Ishii found severe intestinal pathogens, such as the causative agents of typhoid fever, dysentery and cholera, to be highly effective (Williams et al., 1989). Pingfan struggled to supply part of the massive quantity of germs required for the operation, producing 130 kg of anthrax and paratyphoid which were placed in special glass bottles, sealed with paraffin wax and cellophane, put fifteen or sixteen to a box marked water supply and ferried by truck to the Unit's aerodrome. Together with Unit 731's expeditionary party, the germs were flown to China by order of Kwantung Army Commander Umezu. The rest of the Chekiang Campaign germ payload was supplied by another large Ishii BW unit at Nanking, and its satellite sub-units (Williams et al., 1989). In 1939, the Japanese military poisoned Soviet water sources with intestinal typhoid bacteria at the former Mongolian border (Public Health Services). b. Emergency contact: c. Delivery mechanism: Food and water. This method of warfare could involve contaminating water supplies or releasing plague-injected rats soaked with fleas into areas of dense population. Kajitsuka recalled some of the techniques: Speaking about the infection of foodstuffs, Ishii told me that in the researches in this field, the germs of cholera, dysentary, typhoid and paratyphoid were being used, and that vegetables, fruit, fish and meat were so infected. Vegetables were found to be the most suitable for warfare: especially such as had numerous leaves, cabbage, for example; root crops, having smooth surfaces, proved to be less suitable. The injection of bacteria into food products, for example, was found to be more effective than infecting their surfaces. The most suitable medium for spreading infectious diseases, according to what Ishii said, were vegetables; next in order came fruit, fish and last meat (Williams et al., 1989). Flasks. During the operation that followed, the germs of cholera, dysentary, typhoid, plague, anthrax and paratyphoid were all used. Flasks containing pure germs were poured by BW cadres into reservoirs, wells and rivers. Containers were also tossed into houses. Three planes from Nanking Unit fighter escort dropped germs from the air (Williams et al., 1989). Floating bottles. Floating bottles were also invented. The floating bottle is a long necked flask of about 5 liters capacity. the bacterial suspension is put into the flask, the mouth of which is provided with explosive as well as a clock-like timer. The bottle is so managed that when put in water only the mouth of the bottle is exposed above the water surface. This bacterial apparatus is used to attack streams, swimming grounds at the sea sides, and dockyards in which vessels are gathering. It is also used in special conditions of battle. For instance when the combating forces of both sides are located on the banks of a river, and one side is on the upper stream while the other is on the lower stream, the former having ascertained that the enemy is using the river for drinking and bathing purposes, the floating bottle can be used. The velocity of the stream is determined, the clock-like timer is properly set so that when the bottle reaches the enemy front by floating, it explodes. the contents of the bottle are set free and the stream water is contaminated. In the case of sea water the speed and the direction of the tide should be determined before this method of attack is applied. Dysentary bacilli, typhoid bacilli and cholera vibrios are used in bacterial attacks for river water, while the cholera vibrios are most suitable for sea water (Williams et al., 1989). d. Containment: Typhoid fever is a demonstrated hazard to laboratory personnel. Laboratory hazards: The agent may be present in feces, blood, gallbladder (bile) and urine. Humans are the only known reservoir of infection. Ingestion or parenteral inoculation of the organism represent the primary laboratory hazards. The importance of aerosol exposure is not known (CDC). Recommended precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities utilizing known or potentially infectious clinical materials and cultures (CDC). Biosafety Level 3 practices and procedures are recommended for activities likely to generate aerosols or for activities involving production quantities of organisms. Licensed vaccines, which have been shown to protect 70-90% of recipients, may be a valuable adjunct to good safety practices in personnel regularly working with cultures or clinical materials which may contain S. typhi (CDC).

III. Infected Hosts

A.

Human:
1. Taxonomy Information: a. Species: i. Human : Ontology: UMLS:C0086418 GenBank Taxonomy No.: 9606 Scientific Name: Homo sapiens (NCBI Taxonomy) Description: Actually, any Salmonella strain or serovar may be cause such a systemic infection, but typhoid is considered by many to be caused only by S. typhi, while other enteric fever infections are considered typhoidal in nature, or a continued fever type of disease (Gutherie et al., 1991). Typhoid fever the infection caused by S. typhi or, in some cases, by the S. paratyphi strains, A, B, or C is a systemic infection characterized by fever, headache, enlargement of the spleen, rose spots on the abdominal surface, and constipation more often than diarrhea. Frequently, the infection is characterized by prostration and septicemia. The incubation period in such an infection may be as long as 3 weeks following ingestion of the infectious dose of organisms (Gutherie et al., 1991). Signs and symptoms of typhoid fever include a persistently high fever, headache, malaise, lethargy, skin rash, loss of appetite, hepatosplenomegaly, and bradycardia. Older children and adults usually become constipated; younger children may have diarrhea. Not all patients experience classic symptoms, however, so stool and blood cultures should be performed on patients with persistent high fevers who've recently traveled to a developing country (Snow et al., 2006). 2. Infection Process: a. Infectious Dose: Infectious dose is between 1000 and 1 million organisms given orally (Bhan et al., 2005). The causative organism is the bacterium Salmonella enterica serovar Typhi; S. enterica Paratyphi types A and B cause paratyphoid fever, a less common but clinically similar enteric fever. Ingestion of these highly infectious organisms resulted in typhoid fever in up to 55% of study volunteers who ingested 10(5) organisms (Guzman et al., 2006). It was stated that Hornick and co-workers had determined that when 14 persons were tested with doses of S. typhi cells, none became infected. But when 116 persons were tested with 10,000 cell doses, 32 became infected, and with 10 million cells, 16 of 32 were infected (Gutherie et al., 1991). b. Description: The infection is transmitted by ingestion of food or water contaminated with faeces. Established risk factors are contaminated water supply, eating ice cream, flavoured iced drinks or food from street vendors, and raw fruit and vegetables grown in fields fertilised with sewage. Other reported risk factors include a history of contact with other patients before illness, not using soap for washing hands, poor housing, and past evidence of infection with Helicobacter pylori. The mechanism of increased risk of typhoid in individuals with chronic H. pylori infection is postulated to be reduced gastric acidity. Factors within the household (e.g., poor personal hygiene and housing) might be more important risk factors for typhoid, whereas factors outside the household (e.g., food from street vendors, flooding) are more important for paratyphoid fever. A possible reason proposed for this difference was the higher infective dose necessary for paratyphoid, which is more likely to be present in food from street vendors. Although chronic typhoid carriers are important for survival of the pathogen, they are less important as a direct source of infection in endemic

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areas than contaminated water or food. In the USA, up to 30% of infections are due to exposure from previously or newly diagnosed chronic carriers (Bhan et al., 2005). 3. Disease Information: a. Typhoid (i.e., Typhoid_fever) : i. Pathogenesis Mechanism: When Salmonella are ingested they must survive the acid pH of the stomach to set up infection. If surviving in adequate numbers, the bacteria reaching the small intestine may penetrate the mucosa of the intestine to the midlayer of this membrane where they are engulfed into the epithelial cells. Salmonella penetrate epithelial cells, causing an inflammatory response in the small bowel and the colon. The presence of the bacteria in this location results in an inflammatory reaction, and depending on the serovar involved, the infection may progress past this tissue into the deeper layers of the mucosa of the intestinal wall. The salmonellosis, the diarrheal symptoms result from the inflammatory reaction which has been elicited in the small intestine. In typhoid fever, when the organisms invade the tissues, the response elicited is monocytic in nature. The monocytes engulf the bacteria, which are not killed, but which may continue to grow within the cells. The migration of the monocytes following this growth is important in the spread of bacteria throughout the body tissues. In the first week, Peyer's patches may form with necrosis which may cause intestinal bleeding or even bowel perforation. Typhoid fever the infection is caused by S. typhi or, in some cases, by the S. paratyphi strains A, B, or C, is a systemic infection characterized by fever, headache, enlargement of the spleen, rose spots on the abdominal surface, and constipation more often than diarrhea. Frequently, the infection is characterized by prostration and septicemia (Gutherie et al., 1991). S. typhi is ingested in contaminated food or water. It passes through the stomach and invades the gut epithelium, possibly in the distal ileum. Active attachment promoted by the bacterium may be necessary before invasion can occur and this most likely involves unidentified adhesion molecules on the bacterium interacting with receptors on the host cell (House et al., 2001). Salmonella spp. invade epithelial cells in vitro by a process of bacterial-mediated endocytosis, involving cytoskeletal re-arrangement, disruption of the epithelial cell brush border and the subsequent formation of membrane ruffles. An adherent and invasive phenotype of S. enterica is activated under conditions similar to those found in the human small intestine (high osmolarity, low oxygen). The invasive phenotype is mediated, in part, by salmonella pathogenicity island (SPI)-1, a 40 kb region of the chromosome which encodes regulator proteins (e.g. HilA), a type III secretion system (TTSS) that delivers bacterial proteins from the salmonella cytosol into the host cell, and several effector proteins which induce changes within the host cell and promote bacterial uptake (House et al., 2001). The small intestine and particularly the Peyer's patches of the terminal ileum provide the portal of entry facilitating dessemination to systemic sites. S. typhi both survives and replicates in the underlying lymphoid tissue of the Peyer's patches since experimentally infected chimpanzees were found to contain as many as 6.2 x 10(7) bacilli at this site just four days post inoculation. By way of this route bacteria may proceed to disseminate via the lymphatics to the regional lymph nodes and pass into the blood via the thoracic duct resulting in transient primary bacteremia (Tsolis et al., 1999). Shortly following invasion of the gut epithelium, invasive Salmonella spp. encounter macrophages within the gut-associated lymphoid tissue. The interaction between Salmonella and macrophage results in an alteration in the expression of a number of host genes including those encoding pro-inflammatory mediators (House et al., 2001) The ability of S. typhi survive in human macrophages in vitro suggests that this pathogen resides within an intracellular compartment during growth in liver and spleen. Bacterial growth in liver and spleen leads to splenomegaly and hepatomegaly (Tsolis et al., 1999) At the cellular level, the Salmonella species that cause typhoid and non-typhoid disease cannot be distinguished; a key question is what determines host specificity. Helmut Tschape (Robert Koch Institute, Wernigerode, Germany) discussed the involvement of enterotoxins, which induce the efflux of water and electrolytes, in the pathogenesis of gastroenteritis caused by salmonellae. Seroepidemiological and immunological studies suggesting the possible involvement of the Vi antigen (Shou-Sun Szu, National Institutes of Health, Bethesda, MD, USA), flagellin (Suttipant Sarasombath, Mahidol University, Bangkok, Thailand) and outer-membrane proteins (Edmund Calva, Institute of Biotechnology, Cuernavaca, Mexico) emphasize the complexity of Salmonella pathogenesis (Pang et al., 1995).

ii. Incubation Period: The incubation period in such an infection may be as long as 3 weeks following ingestion of the infectious dose of organisms (Gutherie et al., 1991). S. enterica serovar Typhi infections result in a clinical syndrome that varies in severity. After ingestion of the bacteria, an incubation period follows usually lasting 8 to 14 days (range 3- 60 days) (Parry, 2006).

iii. Prognosis: Clinical disease associated with S paratyphi A is indistinguishable from that of S typhi (Connor et al., 2005). The median (IQR) age of the typhoid patients was 7 (5 to 14 years) (House et al., 2001). The median (IQR) duration of illness for the typhoid patients was 8 (5 to 14 days) (House et al., 2001). Patients were hospitalized for an average of 9.8 days (Hoffner et al., 2000). Untreated, the fever persists for two weeks or more and defervescence occurs slowly over the following 2-3 weeks. Convalescence may last for 3-4 months. If an appropriate antibiotic is given the fever gradually falls over 3-4 days. The duration of untreated illness prior to the initiation of therapy influences the severity of the disease (Parry, 2006). A chronic carrier state, defined as a persistence of S. typhi in the stool for more than 1 year, may occur in up to 3% of treated patients (Hoffner et al., 2000). All Salmonella can cause disease but severe systemic infections are primarily caused by a few lineages. Paratyphi A and Typhi are the deadliest human restricted serovars, responsible for 600,000 deaths per annum (Didelot et al., 2007). About 10% of people recovering from untreated typhoid fever may excrete S typhi in the stools for at least 3 months. Between 1% and 5% of typhoid patients become chronic carriers (defined as excretion of S typhi in urine or stools for more than one year), and the rate is higher for women, those older than 50 years, and patients with schistosomiasis, cholelithiasis, carcinoma of the gall bladder, and other gastrointestinal malignancies. Most chronic carriers are asymptomatic and almost a quarter may have had no history of typhoid fever (Bhan et al., 2005). Chronic biliary carriage may occur in 2-5% of cases, even after treatment. Biliary carriage is defined as continued shedding of the organism for more than a year, and is a public-health risk, especially for infected individuals who work in the food industry (Connor et al., 2005).

iv. Diagnosis Overview: The definitive diagnosis of typhoid fever depends on the isolation of S. Typhi from blood, bone marrow or a specific anatomical lesion. Blood and bone marrow aspirate cultures are the gold standard for the diagnosis of typhoid fever. Duodenal aspirate culture has also proved highly satisfactory as a diagnostic test by author but has not found widespread acceptance because of poor tolerance of duodenal aspiration, particularly in children (Ismail, 2006). Although clinical diagnosis of typhoid may be difficult, there are indications that simple algorithms can be developed for diagnosis and patient triage in endemic areas. Such algorithms would have implications for diagnostic and treatment protocols in endemic areas: in particular, diagnosis and triage of typhoid among febrile children must be included among the protocols for integrated management of childhood illnesses in South Asia, which currently largely focus on malaria as a cause of fever without localising signs (Bhutta, 2006). Although the mainstay of diagnosing typhoid fever is a positive blood culture, the test is positive in only 40-60% of cases, usually early in the course of the disease. Stool and urine cultures become positive after the first week of infection, but their sensitivity is much lower. In much of the developing world, widespread antibiotic availability and prescribing is another reason for the low sensitivity of blood cultures. Although bone marrow cultures are more sensitive, they are difficult to obtain, relatively invasive, and of little use in public health settings. Other haematological investigations are non-specific. Blood leucocyte counts are often low in relation to the fever and toxicity, but the range is wide; in younger children leucocytosis is a common association and may reach 20 000-25 000/mm. Thrombocytopenia may be a marker of severe illness and accompany disseminated intravascular coagulation. Liver function test results may be deranged, but significant hepatic dysfunction is rare. The classic Widal test measures antibodies against O and H antigens of S typhi and is more than 100 years old. Although robust and simple to perform, this test lacks sensitivity and specificity, and reliance on it alone in areas where typhoid is endemic may lead to overdiagnosis. Newer diagnostic tests have been developedsuch as the Typhidot or Tubex, which directly detect IgM antibodies against a host of specific S typhi antigens-but these have not proved to be sufficiently robust in large scale evaluations in community settings. A nested polymerase chain reaction using H1-d primers has been used to amplify specific genes of S typhi in the blood of patients and is a promising means of making a rapid diagnosis (Bhutta, 2006). Despite these new developments, the diagnosis of typhoid in much of the developing world is made on clinical criteria. This poses problems, since typhoid fever may mimic many common febrile illnesses without localising signs. In children with
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multisystem features, the early stages of enteric fever may be confused with conditions such as acute gastroenteritis, bronchitis, and bronchopneumonia. Subsequently, the differential diagnosis includes malaria; sepsis with other bacterial pathogens; infections caused by intra-cellular organisms such as tuberculosis, brucellosis, tularaemia, leptospirosis, and rickettsial diseases; and viral infections such as dengue fever, acute hepatitis, and infectious mononucleosis. There is thus an urgent need to develop a multipurpose "fever stick" that may allow the rapid and specific diagnosis of common febrile illnesses, especially malaria, dengue fever, and typhoid (Bhutta, 2006).

v. Symptom Information : Syndrome -- Typhoid fever: Description: Salmonellosis in the human occurs in a wide variety of forms presenting a broad clinical spectrum. The disease may occur solely as an intestinal infection, termed salmonellosis or salmonella gastroenteritis, as a focal infection in any organ of the body, or as a systemic febrile infection. The clinical symptoms of the intestinal infections vary from asymptomatic, or no symptoms, to a most severe diarrhea with fever and nausea. Prolonged fever does not occur with infections other than those caused by Salmonella typhi, which is classified as enteric fever (Gutherie et al., 1991). Signs and symptoms of typhoid fever include a persistently high fever, headache, malaise, lethargy, skin rash, loss of appetite, hepatosplenomegaly, and bradycardia. Older children and adults usually become constipated; younger children may have diarrhea. Not all patients experience classic symptoms, however, so stool and blood cultures should be performed on patients with persistent high fevers who've recently traveled to a developing country (Snow et al., 2006). After ingestion of S typhi, an asymptomatic period follows that usually lasts 7-14 days (range 3-60 days). The onset of bacteraemia is marked by fever and malaise. Patients typically present after the onset of fever with influenza-like symptoms with chills (although rigors are rare), a dull frontal headache, malaise, anorexia, and nausea, but with few physical signs (Connor et al., 2005). Acute typhoid fever is characterized by prolonged fever, disturbances of bowel function (constipation in adults, diarrhoea in children), headache, malaise and anorexia. Cough is common in the early stage of the illness (Ismail, 2006). Fever often occurs in a stepwise fashion with 5-7 days of daily increments in maximal temperature of 0.5-1 C, with the height of fever usually occurring in the afternoon. There follows a period of 10-14 days of sustained fever of 39-41 C (Connor et al., 2005). Confirmed case of typhoid fever is defined, according to the World Health Organization (WHO), as a patient with fever (>38 C) that has lasted for at least three days, with a laboratory confirmed positive culture of S. Typhi. Probable case of typhoid fever is a patient with fever (>38 C) that has lasted for >3 days, with a positive serodiagnosis or antigen detection test but without S. Typhi isolation. Chronic carrier is determined as excretion of S. Typhi in stools or urine for longer than one year after the onset of acute typhoid fever (Ismail, 2006). Some patients excreting S. Typhi have no history of typhoid fever (Ismail, 2006). Hepatomegaly and splenomegaly may exist. Relative bradycardia is considered common in typhoid fever, although it is not specific for it. Rose spots-blanching erythematous maculopapular lesions usually 2 - 4 mm in diameter-are reported in 5 - 30% of cases, and usually occur on the abdomen and chest. Rose spots are easily missed in dark-skinned patients. In one study that compared S typhi with S paratyphi in travellers, rose spots were not found in S paratyphi infection (Connor et al., 2005). More serious complications-e.g., gastrointestinal bleeding, intestinal perforation, and typhoid encephalopathy-that may occur in 10-15% of typhoid patients in endemic countries (Connor et al., 2005). S. typhi is not typically associated with acute diarrhoea, suggesting that the initial interaction between this serovar and the human gut is less inflammatory than that seen with enteritis-causing Salmonella spp (House et al., 2001). A high proportion of S. typhi in the bone marrow of the human host is intracellular and in the blood this percentage increases as the disease progresses (House et al., 2001) Clinical evidence of enteric fever consisted of the presence of at least six of the following features- history of fever (essential), fever of at least 38.0 centigrade documented in a hospital (essential), residence in a known typhoid-endemic area, family history of enteric fever, hepatomegaly, doughy abdomen on palpation, ileus, elevated serum C-reactive protein concentration, leucocytosis or leucopenia, failure of fever to resolve after exclusion of other causes, response to chloramphenicol therapy, development of complications including intestinal haemorrhage and perforation (Choo et al., 1999). Clinical features of paratyphoid fever are similar to those of typhoid fever but are usually milder with a shorter incubation period. S paratyphi A or paratyphi B can manifest with jaundice, thrombosis, and systemic infections. S paratyphi B might occasionally have an onset similar to non-specific salmonella gastroenteritis. Gastrointestinal symptoms are usually not present with S paratyphi C but there have been cases with systemic complications such as septicaemia and arthritis. A relapse rate of 8% has been reported with S paratyphi A (Bhan et al., 2005). Observed: Typhoid is estimated to have caused 21.6 million illnesses and 216,500 deaths globally in 2000. The incidence of typhoid was high (>100 cases per 100000 population per year) in south-central Asia, southeast Asia, and possibly southern Africa, medium (10-100 cases per 100,000) in the rest of Asia, Africa, Latin America, and Oceania, except for Australia and New Zealand, and low in the other parts of the world (<10 cases per 100,000). These estimates are based on blood culture-positive cases in 22 population-based studies, many of which were done after publication of the previous global estimates (Bhan et al., 2005). Where typhoid is endemic, most cases in health facilities are children aged 5-19 years and young adults. Recent population based studies from India, Indonesia, and Vietnam suggest that in some settings typhoid fever is also common in 1-5 year-old children. Data from hospital-based studies in Bangladesh and Thailand and support these findings. Surveillance data from the USA indicate that the proportion of cases of typhoid is constant over the first 25 years of life. Typhoid fever is more common in urban than in rural areas, but studies from two rural settings in Nepal and Vietnam also found a high disease burden. Most cases in developed countries arise in travellers, but domestically acquired disease is still reported. A total of 1393 typhoid cases were reported between 1994 and 1999 in the USA, 74% of which were related to travel and the rest of which were acquired domestically; 7% of total cases were part of recognised outbreaks (Bhan et al., 2005). Case fatality rates in endemic countries have been reported as high as 30% (Connor et al., 2005)

Symptoms Shown in the Syndrome: Fever: Ontology: UMLS:C0015967 Description: The most common clinical manifestations seen in patients in this group were prolonged fevers and headaches, followed by abdominal pain and diarrhea (Hoffner et al., 2000). Typically, there is a 7- to 14-day asymptomatic period after ingestion of the bacterium, followed by onset of bacteremia marked by fever and malaise (Bal et al., 2004). A fever exhibiting a stepladder rise over the first week, with a plateau in the second week and a gradual fall in the third or fourth week is typical (Bal et al., 2004). Confirmed case of typhoid fever is defined, according to the World Health Organization (WHO), as a patient with fever (>38 C) that has lasted for at least three days, with a laboratory confirmed positive culture of S. Typhi. Probable case of typhoid fever is a patient with fever (>38 C) that has lasted for > 3 days, with a positive serodiagnosis or antigen detection test but without S. Typhi isolation (Ismail, 2006). Observed: Fever was present in all patients (100%) (Chowta et al., 2005). Gastroenteritis : Description: The disease may occur solely as an intestinal infection, termed salmonellosis or salmonella gastroenteritis, as a focal infection in any organ of the body, or as a systemic febrile infection. The clinical symptoms of the intestinal infections vary from asymptomatic, or no symptoms, to a most severe diarrhea with fever and nausea (Gutherie et al., 1991). Observed: Acute gastroenteritis (7 or 9.7%) (Choo et al., 1999). Gastroenteritis 16 (2.6%) (Ellis et al., 2006). Diarrhoea: Description: Coated tongue, alteration of bowel habits varying from constipation in adults to diarrhoea in children, tender abdomen, hepatomegaly, and splenomegaly are often present (Bhan et al., 2005). A short episode of diarrhea and vomiting sometimes occurs in the first day or two of an attack of typhoid fever though constipation rather than diarrhea characterizes the later stages of the disease (Vaishnavi et al., 2006).
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Observed: Diarrhoea was seen in 20.4% patients (Chowta et al., 2005). Headache : Description: Acute non-complicated typhoid fever has an incubation period of 10-14 days and is usually associated with prolonged low-grade fever, dull frontal headache, malaise, myalgia, a dry bronchitic cough, anorexia, and nausea (Bhan et al., 2005). Observed: 18.1% (Chowta et al., 2005). Abdominal pain: Description: Coated tongue, alteration of bowel habits varying from constipation in adults to diarrhoea in children, tender abdomen, hepatomegaly, and splenomegaly are often present (Bhan et al., 2005). Observed: 11.3% (Chowta et al., 2005). Body ache : Description: Observed: 2.3% (Chowta et al., 2005). Dry cough: Description: A nonproductive cough is common, and epistaxis may occur but is rare (Bal et al., 2004). Observed: 6.8% (Chowta et al., 2005). Constipation: Description: Constipation is almost as likely as diarrhea to be a symptom of this enteric illness (Bal et al., 2004). Constipation is generally more common in adults, but in young children and adults with HIV infection diarrhoea predominates. S. enterica serovar Paratyphi causes a similar, although less sever syndrome (Parry, 2006). Observed: Constipation was present in 9.09% patients (Chowta et al., 2005). Anorexia: Description: Acute typhoid fever is characterized by prolonged fever, disturbances of bowel function (constipation in adults, diarrhoea in children), headache, malaise and anorexia (Ismail, 2006). Observed: Vomiting: Description: Nausea and vomiting are infrequent in uncomplicated typhoid but are seen with abdominal distension in severe cases (Parry, 2006). Observed: Vomiting was present in 20.4% patients (Chowta et al., 2005). Hepatomegaly: Description: Other atypical gastrointestinal symptom observed was isolated hepatomegaly (Dutta et al., 2001). Isolated hepatomegaly is uncommon in typhoid fever except perhaps in multidrug resistant typhoid fever (Dutta et al., 2001). Observed: Hepatomegaly was observed in two (6.2%) patients; however, none of the patients had jaundice (Dutta et al., 2001). Hepatomegaly was detected in 18% patients (Chowta et al., 2005). Toxemia: Description: High fever, toxaemia, constipation during first week of fever, diarrhoea during second week of fever, mild splenomegaly and leucopenia/neutropenia, complicated by encephalopathy, intestinal haemorrhage and perforation during third week of fever are the typical manifestations of typhoid fever (Dutta et al., 2001). Observed: Splenomegaly: Description: All 26 cases showed diffuse enlargement of the spleen. In 22 cases the spleen showed a normal echotexture. 4 cases had abscess formation in the spleen (Mateen et al., 2006). Splenomegaly persisted in all 26 patients even at the follow up scan done on the 15th day. However, there was a significant decrease in the size of the spleen (Mateen et al., 2006). Observed: Splenomegaly was present in 34.09% patients (Chowta et al., 2005). Coated tongue : Description: Patients can have a furry, or coated, tongue (Bal et al., 2004). Thus, the evaluation of any patient presenting with symptoms suggestive of typhoid fever warrants a thorough oropharyngeal examination, since a coated tongue, if present, may provide an important diagnostic clue in an otherwise nonspecific presentation (Bal et al., 2004). Observed: In one study, coated tongue was found to be 1 of 4 specific clinical markers for typhoid fever (in addition to high fever, loose bowel movements and bradycardia), with a reported specificity of 94% (Bal et al., 2004). Ileus : Description: One third of patients developed complications, such as hepatitis, bone marrow suppression, paralytic ileus, myocarditis, psychosis and osteomyelitis (Malik, 2002). Salmonella infection in the gut involves local inflammation, lymphatic absorption of toxins and direct portal spread of infection, so there is intestinal infection of variable extent. Intestinal perforation and paralytic ileus are commonly reported complications (Malik, 2002). Intestinal perforation associated with typhoid in children has a high mortality and its surgical treatment leads to other complications whereas ileus is a relatively mild complication with a good prognosis (Malik, 2002). Dilated loops of bowel may be palpated indicating an ileus (Parry, 2006).

Salmonellosis, ulcer of ileum in typhoid fever (Pathology):

Description: Salmonellosis, ulcer of ileum in typhoid fever (Pathology) Observed: Hospital based patients 12(1); Community based cohort 1 (0.3) (Bhutta, 2006). Intestinal perforation: Description: Intestinal (usually ileal) perforation is the most serious complication occurring in 1-3% of hospitalized patients. Perforation may be present with acute abdomen or more covertly with simple worsening of abdominal pain (Parry, 2006). Observed: Miscarriage : Description: Pregnancy: Typhoid fever in pregnancy may be complicated by miscarriage, although antimicrobial treatment has made this less common (Parry, 2006). Vertical intra-uterine transmission from a typhoid-infected mother may lead to neonatal typhoid, a rare but severe and life-threatening complication (Parry, 2006). Observed: Gastrointestinal bleeding: Description: Although many complications have been described, gastrointestinal bleeding, intestinal perforation and typhoid encephalopathy are the most
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important (Parry, 2006). Observed: Gastrointestinal bleeding occurs in up to 10% of patients and results from erosion of a necrotic Peyer's patch through the wall of an enteric vessel. Usually the bleeding is slight and resolves without the need for blood transfusion. In 1-2% of cases, bleeding is significant, and can be rapidly fatal if a large vessel is involved (Parry, 2006). Two devastating complications of typhoid fever are gastrointestinal hemorrhage and perforation (Hoffner et al., 2000). Typhoid encephalopathy: Description: Typhoid encephalopathy, often accompanied by shock, is associated with high mortality. Patients may display the 'typhoid" faces, a thin, flushed face with a staring, apathetic expression. Mental apathy may progress to an agitated delirium, frequently accompanied by tremor of the hands, tremulous speech and gait ataxia, and then muttering delirium, twitchings of the fingers and wrists, agitated plucking at the bedclothes, and a staring, unrousable stupor (Parry, 2006). Encephalopathy or typhoid state is a well-recognised entity in typhoid fever. This occurs typically in third week of illness. Early encephalopathy (i.e., during first week), as observed in one of our patients is uncommon. It must be considered as a grave sign; nevertheless, our patient improved (Dutta et al., 2001). Observed: Early encephalopathy i.e., encephalopathy during the first week of illness, was one of the grave complications observed in one (3.1%) of our patients (Dutta et al., 2001). Intestinal perforation: Description: Perforation may present with an acute abdomen or more covertly with simple worsening of abdominal pain, rising pulse and falling blood pressure in an already sick patient (Parry, 2006). Patients with typhoid perforation require resuscitation with fluids, gastric suction, blood transfusions and administration of oxygen followed by surgery. Perforations are found usually in the ileum but may also occur in the caecum and proximal large bowel (Parry, 2006). Early intervention is crucial, as mortality rates increase if there is a long delay between perforation and surgery. Mortality rates between 10 and 32% (Parry, 2006). Observed: Intestinal (usually ileal) perforation is the most serious complication occurring in 1-3% of hospitalized patients (Parry, 2006). Two devastating complications of typhoid fever are gastrointestinal hemorrhage and perforation. Perforation classically occurs at the Peyer's patches of the terminal ileum. Only one patient in our study suffered from a perforated bowel, and none experienced gastrointestinal hemorrhage (Hoffner et al., 2000). Leukocytosis : Description: Blood leucocyte counts are often low in relation to the fever and toxicity, but the range is wide; in younger children leucocytosis is a common association and may reach 20 000-25 000/mm(3) (Bhutta, 2006). Leukocytosis: Most clinical and laboratory findings were similar in those infected with S. Typhi or S. Paratyphi A (Parry, 2006). Salmonella Paratyphi B appeared to have a different clinical spectrum. It was associated with disease in children; on three occasions it caused a self-limited disease that subsided within a few days without antibiotic treatment. Laboratory findings were also different from classical enteric fever in that patients usually had leukocytosis, a higher level of platelets, and normal liver function test results (Parry, 2006). Observed: Rose spots : Description: Other signs and symptoms of typhoid fever include prolonged fever, relative bradycardia and a rash consisting of rose spots (pinkish lesions 1-4 mm in diameter) usually on the abdomen and chest (Bal et al., 2004, Cunha, 2006). These rose spots are small blanching erythematous maculopapular lesions typically on the abdomen and chest. Melanesian typhoid patients may develop purpuric macules that do not blanch (Parry, 2006).

Rose spots (image):

Description: Observed: Rose spots are reported in 5-30% of cases but are easily missed in dark-skinned patients (Parry, 2006). Rose spots, described as blanching 2 to 4 mm diameter papules classically found on the upper abdomen and lower chest, have been reported to occur in 30 to 50% of patients (Hoffner et al., 2000). Myocarditis : Description: Electrocardiographic changes suggestive of myocarditis are rather common in typhoid fever but the incidence of true myocarditis is low both in adult patients and children (Malik, 2002). Observed: A total number of 44 cases of typhoid fever were studied. Out of these 21 (47.7%) were males and 23 (52.3%) were females (Chowta et al., 2005). Myocarditis was observed in two patients (Chowta et al., 2005). Burning micturition: Description: Thirty-two (age >13 years, 19 males and 13 females) culture-positive typhoid fever patients were admitted during the period. The mean age of the patients was 23.9 years (range 13-55 years. 15 (46.9%) of 32 patients had atypical manifestations. Burning micturition was observed in 5 (15.6%) patients (Dutta et al., 2001). Urinary examination, however, revealed no abnormality and the symptom resolved with treatment of typhoid fever (Dutta et al., 2001). Observed: Burning micturition was observed (15.6%) (Dutta et al., 2001). vi. Treatment Information: Antibiotics : Historically, typhoid fever was treated with chloramphenicol, ampicillin, or trimethoprim-sulfamethoxazole (co-trimoxazole) (Connor et al., 2005). The introduction of fluoroquinolones was a major advance. The drugs were found to be highly effective, well-tolerated, and could be administered orally. Antibiotics such as ciprofloxacin quickly became first-line agents (Connor et al., 2005). Currently, the recommendation for first-line therapy is ceftriaxone 2 g daily. Where isolates have been found to be fluoroquinolone sensitive, standard doses of a fluoroquinolone e.g., ciprofloxacin 500 mg twice daily may be used (Connor et al., 2005). Another promising option that has been tested in the past few years is azithromycin. This agent shows similar results to ciprofloxacin and ofloxacin. Furthermore, oral azithromycin was comparable to intravenous ceftriaxone in uncomplicated typhoid fever in children and adolescents (Connor et al., 2005). Applicable: Typhoid fever should be a serious consideration in an endemic area when a fever lasts longer than a week, even by the fifth day if there is severe toxaemia. The decision to send laboratory investigations and initiate empirical antimicrobial therapy depends largely on clinical judgment. Initial choice of antibiotic depends on the sensitivity patterns of S typhi and paratyphi isolates in the area. The isolates can be broadly classified as sensitive to first-line antimicrobials, multidrug resistant but nalidixic-acid sensitive, and nalidixic-acid resistant (often also multidrug resistant) (Bhan et al., 2005). Contraindicator: In late 1987, there was an outbreak of typhoid fever in China caused by strains resistant to all the first line antimicrobials (ampicillin, cotrimoxazole, and chloramphenicol). During 1989-1990, there were reports of similar S typhi strains from India, Pakistan, and the Arabian Gulf. Such multidrug resistant typhoid is now reported from many parts of the world. Multiple antimicrobial resistance is mediated through pHCM1 plasmid (Bhan et al., 2005). Complication: Most carriers (defined as individuals who excrete S typhi in their stools or urine for more than a year) without gallstones can be cured by a long course of antimicrobials. Almost 80% of carriers were cured by 750 mg of ciprofloxacin twice daily for 28 days, and 11 out of 12 carriers treated with 400 mg norfloxacin twice daily had negative stool and bile cultures for S typhi after 28 days of treatment. In patients with gallstones, cholecystectomy along with antibiotic therapy might be required (Bhan et al., 2005). Success Rate: Fluoroquinolones (ciprofloxacin, ofloxacin, and pefloxacin) are the most effective drugs for treatment of typhoid fever caused by isolates that are not quinolone resistant, 159-163 with a clinical cure rate of about 98%, fever clearance time of about 4 days, and relapse and faecal carriage rates of less than 2%. Chloramphenicol, the traditional first-line drug of choice, is less effective than fluoroquinolones in all these respects and in terms of persistence of the
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organism in bone marrow, even for treatment of patients with fully sensitive isolates (Bhan et al., 2005). Drug Resistance: By 1972, chloramphenicol resistance became widespread and, since 1989, resistance to all three has been noted in strains in India, Pakistan, China, and the Persian gulf. Resistance appears to be plasmid mediated, allowing the simultaneous acquisition of resistance to multiple drugs and the emergence of multidrug-resistant strains. 50-80% of isolates from China and the Indian subcontinent are now multidrug resistant, and 93% of isolates in an outbreak in Tajikistan were multidrug resistant (Connor et al., 2005). A disturbing trend, however, has been an increase in fluoroquinolone resistance, which is not plasmid mediated but rather the result of chromosomal mutation. In an outbreak in Tajikistan, 82% of isolates were ciprofloxacin resistant. This resistance has necessitated higher, more prolonged doses of quinolones (10-14 days or longer). In a study done in Vietnam, quinolone resistance increased from 4% in 1993 to 76% in 1998. These quinolone-resistant strains were noted to be sensitive to ceftriaxone, cefixime, and azithromycin but the clinical response was slower, with fever clearance taking 7 days or more and failure rates of >20% (Connor et al., 2005). Our clinical experience with travellers shows a slower response to treatment in recent years, even when the pathogen is sensitive in vitro to quinolones or ceftriaxone. The same phenomena occurs with S paratyphi A (Connor et al., 2005). Amoxicillin: The aminopenicillins have activity against H. influenzae, E. coli, P.mirabilis and Salmonella and some Shigella species, while also retaining activity against penicillin-sensitive gram-positive bacteria. However, many Enterobacteriaceae, H. influenzae, Salmonella and Shigella species are resistant to these penicillins because of beta-lactamase production of these organisms. Bacampicillin and pivampicillin are esters of ampicillin that are hydrolyzed during absorption to liberate ampicillin; this results in increased bioavailability and serum concentrations of ampicillin (MICROMEDEX 2001). Amoxicillin has the same in vitro activity as ampicillin, although amoxicillin has slightly better activity against Enterococcus faecalis, E. coli and Salmonella sp. (Typhoid fever treatment)- Amoxicillin and ampicillin are used in the treatment of typhoid fever caused by Salmonella typhi (MICROMEDEX 2001). Applicable: Uncomplicated typhoid at home: Fully susceptible: Second-line oral drug: Antibiotic: Amoxycillin; daily dose 75-100 mg/kg; Days: 14 (Bhan et al., 2005). Severe typhoid in hospital: Fully susceptible: Second-line oral drug: Antibiotic: Ampicillin; daily dose 100 mg/kg; Days: 10-14 (Bhan et al., 2005). Typhoid fever: Intramuscular or intraveneous, 25 mg per kg of body weight every six hours. Adult prescribing limits: 4.5 grams per day (MICROMEDEX 2001). Contraindicator: Allergy: Patients allergic to one penicillin may be allergic to other penicillins (MICROMEDEX 2001). Fertility: Studies in mice and rats at doses up to 10 times the human dose of amoxicillin revealed no evidence of impaired fertility (MICROMEDEX 2001) Pregnancy: Penicillins cross the placenta. Adequate and well-controlled studies in humans have not been done to determine whether penicillins are teratogenic; however, penicillins are widely used to in pregnant women and problems have not been documented (MICROMEDEX 2001). Studies in mice and rats at doses up to 10 times the human dose of amoxicillin revealed no evidence of harm the fetus (MICROMEDEX 2001). Breast-feeding: Penicillins are distributed into breast milk, some in low concentrations. Although significant problems in humans have not been documented, the use of penicillins by nursing mothers may lead to sensitization, diarrhea, candidiasis, and skin rash in the infant (MICROMEDEX 2001). Pediatrics: Many penicillins have been used in pediatric patients and no pediatrics specific problems have been documented to date. However, the incompletely developed renal function of neonates and young infants may delay the excretion of renaly eliminated penicillins (MICROMEDEX 2001). Phenylketonuria: The 200-mg and the 400-mg chewable tablets of amoxicillin contain 1.8 mg and 3.6 mg phenylalanine, respectively, produced through the metabolism of aspartame. These dosage forms should be used with caution, if at all, in patients with phenylketonuria. The other strengths and dosage forms of amoxicillin do not contain phenylalanine (MICROMEDEX 2001). Geriatrics: Penicillins have been used in geriatric patients and no geriatrics-specific problems have been documented to date. However, elderly patients are more likely to have age-related renal function impairment, which may require an adjustment in dosage in patients receiving penicillins (MICROMEDEX 2001). Dental: Prolonged use of penicillins may lead to the development of oral candiasis (MICROMEDEX 2001). Relapse: Standard treatment with chloramphenicol or amoxicillin is associated with a relapse rate of 5-15% or 4-8% respectively, whereas the newer quinolones and third generation cephalosporins are associated with higher cure rates (Bhutta, 2006). Complication: Oral Contraceptives: May also interact with oral contraceptives (MICROMEDEX 2001). The amoxicillin products containing aspartame (200mg and 400-mg chewable tablets) are hazardous to patients with phenylketonuria (MICROMEDEX 2001). Success Rate: 27.3% (Chowta et al., 2005). Drug Resistance: 72.7% (Chowta et al., 2005). Chloramphenicol and amoxicillin may need to be reconsidered as the drugs of choice in cases of enteric fever because of the increased susceptibilities of such cases to these drugs > 90% for reemerging isolates of S. Typhi (Chandel et al., 2000). Greater than 32 MIC (ug/ml), broth microdilution 256 MIC (ug/ml) for the isolate of Salmonella enterica serovar Paratyphi A (Adachi et al., 2005). Fluoroquinolones : Fluoroquinolones are broad-spectrum anti-infectives, active against a wide range of aerobic gram-positive and gram-negative organisms. They are active in vitro against most Enterobacteriaceace, including Salmonella enteritidis and Salmonella typhi. Oral ciprofloxacin is indicated in the treatment of typhoid fever caused by susceptible strains of S. typhi (MICROMEDEX 2001). The defervescence period for ciprofloxacin is about 3 - 5 days according to the literature and for cephalosporin is about three days. But in the present study we have observed that the defervescence period was comparatively longer; about eight days for ciprofloxacin and about six days for cephalosporins (Chowta et al., 2005). Applicable: Uncomplicated typhoid at home: Fully susceptible: Antibiotic: Fluoroquinolone; daily dose 15 mg/kg; Days: 5-7 (Bhan et al., 2005). Uncomplicated typhoid at home: Multidrug-resistant: Antibiotic: Fluoroquinolone; daily dose 15 mg/kg; Days: 5-7 (Bhan et al., 2005). Severe typhoid in hospital: Fully susceptible: Antibiotic: Fluoroquinolone; daily dose 15 mg/kg; Days: 10-14 (Bhan et al., 2005). Severe typhoid in hospital: Multidrug-resistant: Antibiotic: Fluoroquinolone; daily dose 15 mg/kg; Days: 10-14 (Bhan et al., 2005). Fluorquinolones have been the drug of choice for treatment of typhoid and paratyphoid fever since the beginning of the 1990s. In recent years, however, strains with decreased susceptibility to quinolones have emerged, and clinical treatment failure is a serious concern. S. typhi and Paratyphi A with decreased susceptibility to fluoroquinolones emerged on the Indian subcontinent, Southeast Asia and Central Asia in the mid-1990s. On disk diffusion testing, these strains were NA (Nalidixic acid)-resistant, and susceptible to ofloxacin or ciprofloxacin; however the MICs of ciprofloxacin increased to 0.25-4 ug/ml, 10- to 100-fold higher than the usual NA-susceptible strains. Several epidemics of typhoid and paratyphoid fever caused by NA-resistant strains with clinical failure of quinolone treatment have been reported. Our findings strongly suggest that high-level of quinolone resistance was induced through the long-term carrier state of S. Paratyphi A under selective pressure of frequent quinolone administration (Adachi et al., 2005). We investigated the use, treatment results and safety of ciprofloxacin in the treatment of childhood typhoid and paratyphoid infections in an industrialized country. The study was retrospective, and the material consisted of children admitted to Hvidovre or Glostrup University Hospitals from 1991 to 1995, and treated with ciprofloxacin for a culture proven diagnosis of typhoid fever. 21 children were included, 18 had positive cultures for Salmonella typhi and 3 had positive cultures for S. paratyphi A. All isolates were fully susceptible to ciprofloxacin. The median duration of treatment was 10 days, median oral dose was 24 mg/kg/d and the median intravenous dose was 15 mg/kg/d. Within 4 days after start of treatment all subjects had a normal body temperature. No subjects had clinical or microbiological relapse and all stool cultures after end of treatment were negative (Thomsen et al., 1998). Contraindicator: Drug resistance (MDR) is a serious emerging threat to the treatment of infectious diseases. MDR S. typhi are resistant to commonly available antibiotics, and clinical resistance to Fluoroquinolones, the most effective antimicrobials for the treatment of typhoid fever, has been reported (Parkhill et al., 2001). Salmonella spp. with reduced susceptibility to fluoroquinolones have higher than usual MICs to these agents but are still considered "susceptible" by NCCLS criteria. Delayed treatment response to fluoroquinolones has been noted, especially in cases of enteric fever due to such strains. We reviewed the ciprofloxacin susceptibility and clinical outcome of our recent enteric fever cases. Salmonella enterica Serotype Typhi (S. Typhi) and Serotype Paratyphi (S. Paratyphi) blood culture isolates (1998-2002) were tested against nalidixic acid by disk diffusion (DD) and agar dilution (AD) and to ciprofloxacin by AD using NCCLS methods and interpretive criteria. Reduced fluoroquinolone susceptibility was defined as a ciprofloxacin MIC of 0.125-1.0 mg/L (Slinger et al., 2004). Seven of 21 (33%) S. Typhi and S. Paratyphi isolates had reduced susceptibility to fluoroquinolones (MIC range 0.125-0.5 mg/L) (Slinger et al., 2004). Our cases provide further evidence that reduced fluoroquinolone susceptibility of S. Typhi and S. Paratyphi is clinically significant. Laboratories should test extraci.vbi.vt.edu/pathinfo/pathogens/enterica.html 10/25

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intestinal Salmonella spp. for reduced fluoroquinolone susceptibility (Slinger et al., 2004). Complication: Fluoroquinolones are considered the most effective drugs for the treatment of typhoid fever and hence are widely used in the empiric treatment of acute undifferentiated febrile illnesses in India. Recent reports of Salmonella enterica serotype Typhi (S. Typhi) strains with increasing minimum inhibitory concentration (MIC) of ciprofloxacin have raised the fear of potential treatment failures. In this case series of 109 consecutive patients hospitalized with typhoid fever (S. Typhi grown from blood), we documented clinical failure (fever persisting > 6 days) in 25 of 46 (54.3%) adults who could be evaluated. Among these, eight (17.4%) had microbiological failure (S. Typhi recovered from blood after 6 days of ciprofloxacin therapy) despite adequate serum ciprofloxacin levels, and all required alternative drugs for treatment. These 8 S. Typhi strains, although susceptible to ciprofloxacin (MIC less than 1 microg/mL) (NCCLS 2000), had MICs (median MIC 0.5 microg/mL) that were increased 15-fold compared to S. Typhi strains from patients with typhoid fever seen at our center in 1995 (median MIC 0.032 microg/mL), and were nalidixic acid resistant S. Typhi (NARST) (MIC > or equal to 32 microg/mL). The poor treatment outcomes with ciprofloxacin therapy in patients infected with NARST strains that exhibit an increased ciprofloxacin MIC call for a need to revise the ciprofloxacin breakpoints for S. Typhi (Rupali et al., 2004). Relapse or complications were observed in 11 other patients (8.1%). Three had relapses, two had endocarditis, two had splenic abscesses, one had jaundice (due to quinolones or enteric fever), one had an intestinal perforation, one had hemolytic anemia, and one had reactive arthritis (Meltzer et al., 2006). Success Rate: 81.9% (Chowta et al., 2005). Drug Resistance: Quinolone resistant isolates of Salmonella enterica serovar Typhi have reduced susceptibility to ciprofloxacin, with minimum inhibitory concentrations between 0.1 mg/l and 1 mg/l compared with wild type strains (<0.1 mg/l). The currently agreed definition for resistance is a minimum inhibitory concentration >1 mg/l, and such isolates are therefore reported as being susceptible to ciprofloxacin by disc sensitivity testing,2 although systemic infections with these isolates respond poorly to ciprofloxacin and ofloxacin (Parry et al., 2006). This problem is illustrated by two recent cases of typhoid fever. Each isolate of S typhi was reported as susceptible to ciprofloxacin (Parry et al., 2006). However, both patients remained feverish and unwell after seven days' treatment with oral ciprofloxacin. After changing to intravenous ceftriaxone, symptoms quickly resolved, and both patients recovered (Parry et al., 2006). The incidence of enteric fever caused by Salmonella enterica serotype Paratyphi A has been increasing in India since 1996. In 1998, the incidence of enteric fever caused by drug-resistant S. Paratyphi A abruptly increased in the New Delhi region. In the first 6 months of 1999, 32% of isolates were resistant to both chloramphenicol and cotrimoxazole and another 13% were resistant to more than two antibiotics (Chandel et al., 2000). S. Paratyphi A, which causes 1%-15% of enteric fever cases in India, has been increasing since 1996. Our study found that 32% of isolates from the New Delhi region had decreased susceptibility to ciprofloxacin (MIC > 2.0 mg/L), the drug of choice for enteric fever in India. One sequella of this increased resistance was delay in the resolution of symptoms. Although strains may appear sensitive at this level, when subjected to ciprofloxacin-susceptibility testing by disc diffusion, treatment failure may still occur. The mechanisms proposed for quinolone resistance involve alteration in the permeability of the drug (outer membrane protein gene mutation) or alteration of the target enzyme DNA gyrase within the treated bacterium as its adaptive reflex. Since resistance to quinolones is independent of resistance to other drugs that are mainly plasmid mediated, it may occur in otherwise sensitive strains (Chandel et al., 2000). Enteric fever (typhoid) is classically caused by Salmonella enterica serotype Typhi, but a similar syndrome may be observed with S. Paratyphi A and other serotypes. Outbreaks of enteric fever associated with S. Paratyphi A have rarely been reported in India. Although multidrug-resistant outbreaks of S. Typhi with an increase in numbers of strains with decreased susceptibility to ciprofloxacin have occurred, cases of drug-resistant S. Paratyphi A have been relatively uncommon. We report a sudden increase in enteric cases caused by drug-resistant S. Paratyphi A unresponsive to ciprofloxacin therapy (Chandel et al., 2000). Etest: > 32 MIC(ug/ml), broth microdilution 258 MIC (ug/ml) for the isolate of Salmonella enterica serovar Paratyphi A (Adachi et al., 2005). Our findings strongly suggest that high-level quinolone resistance was induced through the longterm carrier state of S. Paratyphi A under selective pressure of frequent quinolone administration (Adachi et al., 2005). 18.1% (Chowta et al., 2005). The emergence of bacterial resistance to fluoroquinolones, and to cross-resistance within this class of antimicrobial agents, has become a significant concern. Decreased susceptibility among Enterobacteriaceae, including E. coli, K. pneumoniae and Salmonella, has been reported worldwide (MICROMEDEX 2001) The last two decades have seen a change in the pattern of enteric fever with the emergence of multidrug-resistant strains (MDRS), particularly strains resistant to nalidixic acid (Walia et al., 2005). Of 377 blood culture-positive cases, 80.6% were Salmonella typhi and 19.4% Salmonella paratyphi A; 21.7% were children aged under 5 years and 6.1% were under 2 years (Walia et al., 2005). Complete resistance to ciprofloxacin (MIC >4 microg/ml) was detected in only two isolates, both Salmonella paratyphi A. Minimal inhibitory concentrations (MICs) of ciprofloxacin for NARS were increased (0.125-0.5 microg/ml) but were within National Committee for Clinical Laboratory Standards susceptibility ranges (Walia et al., 2005). Chloramphenicol: Outbreaks of chloramphenicol-resistant typhoid were reported in 1972. At this time, the isolates were still sensitive to co-trimoxazole and ampicillin or amoxycillin. In the late 1980s and 1990s, outbreaks of typhoid caused by organisms resistant to chloramphenicol, co-trimoxazole, ampicillin, and amoxicillin were reported (Bhan et al., 2005). Applicable: Uncomplicated typhoid at home: Fully susceptible: Second-line oral drug: Antibiotic: Chloramphenicol; daily dose 50-75 mg/kg; Days: 14 - 21 (Bhan et al., 2005). Severe typhoid at home: Fully susceptible: Second-line oral drug: Antibiotic: Chloramphenicol; daily dose 100 mg/kg; Days: 14 - 21 (Bhan et al., 2005). Contraindicator: Standard treatment with chloramphenicol or amoxicillin is associated with a relapse rate of 5-15% or 4-8% respectively, whereas the newer quinolones and third generation cephalosporins are associated with higher cure rates (Bhutta, 2006). Complication: Chloramphenicol, the traditional first-line drug of choice, is less effective than fluoroquinolones in all these respects and in terms of persistence of the organism in bone marrow, even for treatment of patients with fully sensitive isolates (Bhan et al., 2005). Success Rate: 36.4% (Chowta et al., 2005). Drug Resistance: Out of 59 serotypes; 33 were also resistant to chloramphenicol and tetracycycline (). Chloramphenicol and amoxicillin may need to be reconsidered as the drugs of choice in cases of enteric fever because of the increased susceptibilities of such cases to these drugs >90% for reemerging isolates of S. Typhi (Chandel et al., 2000). 7 (32%) of 22 isolates were resistant to both chloramphenicol and cotrimoxazole (Chandel et al., 2000). 63.6% (Chowta et al., 2005). Today due to its changing modes of presentation, as well as the development of multidrug resistance, typhoid fever is becoming increasingly difficult to diagnose and treat. Improved standards of public health have resulted in a marked decline in the incidence of typhoid fever in developed countries. The emergence of strains of Salmonella typhi resistant to multiple antibiotics poses a serious problem. Chloramphenicol was considered the antimicrobial gold standard for the treatment of typhoid fever till 1948. But in the last two decades there has been increase in the resistance of strains of S. typhi to chloramphenicol. It was first reported in Britain, in 1950 and in India in 1972. Gradually, resistance to multiple antibiotics developed. The first major epidemic of multidrug resistant S. typhi was reported in 1972 in Mexico. Since then, an increasing frequency of antibiotic resistance has been reported from all parts of the world, but more so from the developing countries. The uses of chloramphenicol, ampicillin and co-trimoxazole have become infrequent and quinolones have become the first line of treatment of typhoid fever. However, over the last few years there has been increase in the defervescence period in patients treated with quinolones (Chowta et al., 2005). Sulfamethoxazole and Trimethoprime : Sulfamethoxazole and trimethoprime is used as an alternative agent in the treatment of paratyphoid and typhoid fevers caused by susceptible strains (MICROMEDEX 2001). Sulfonamides, such as sulfadiazine and sulfamethoxazole, used together with trimethoprime, produce synergistic antibacterial activity. These sulfonamides, in combination with Trimethoprime, are active in vitro against many gram-positive and gram-negative aerobic organisms (MICROMEDEX 2001). Applicable: Uncomplicated typhoid at home: Fully susceptible: Second-line oral drug: Antibiotic: Trimethoprim-sulpfamethoxzole; daily dose 8 mg/kg (trimethoprim) and sulfamethoxazole 40 mg/kg daily dose; Days: 14 (Bhan et al., 2005). Sulfamethoxazole and oral Trimethoprime: Usual adult and adolescent dose: Antibacterial- Oral, 800 mg of sulfadiazine and 160 mg of trimethoprime every 12 hours (MICROMEDEX 2001). Infants 2 months of age and over and children upto 40 kg of body weight- Oral, 20 to 30 mg of sulfamehtoxazole and 4 to 6 mg of trimethoprime per kg of body weight every twelve hours. Children
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3 months to 12 years of age: Oral, 7 mg of sulfadiazine and 1.5 mg of trimethoprim per kg of body weight every twelve hours (MICROMEDEX 2001). Sulfamethoxazole and Trimethoprime for injection concentrate USP: Usual adult and adolescent dose: Antibacterial- Intravenous infusion, 10 to 12.5 mg of sulfamethoxazole and 2 to 2.5 mg of trimethoprime per kg of body weight every six hours, 13.3 to 16.7 mg of sulfamethoxazole and 2.7 to 3.3 mg of trimethoprime per kg of body weight every eight hours; or 20 to 25 mg of sulfamethoxazole and 4 to 5 mg of trimethoprime per kg of body weight every twelve hours (MICROMEDEX 2001). Contraindicator: Use in children: Sulfadiazine and trimethoprime combination is contraindicated in infants up to 3 months of age and sulfamethoxazole and trimethoprime combination is contraindicated in infants up to 2 months of age for most indications since sulfonamides may cause kernicterus in neonates; however, sulfamethoxazole and trimethoprime combination is indicated in all infants born to human immunodeficiency virus (HIV)-infected mothers, starting at 4 to 6 weeks (MICROMEDEX 2001). Complication: Pregnancy: Sulfonamides and trimethoprime cross the placenta; trimethoprime may interfere with folic acid metabolism, use is not recommended at term since sulfonamides may cause jaundice, hemolytic anemia, and kernicterus in neonates. Breast-feeding: Sulfonamides and trimethoprime are distributed into breast milk; sulfonamides my cause kernicterus in nursing infants; trimethoprime may interfere with folic acid metabolism (MICROMEDEX 2001). Use in the elderly: Elderly patients, especially those also taking diuretics, may be at increased risk of severe side effects (MICROMEDEX 2001) Success Rate: Cotrimoxazole: 4 (36.4%) (Chowta et al., 2005) Drug Resistance: Cotrimoxazole: 63.6% (Chowta et al., 2005). Cotrimoxazole: 7 (32%) of 22 isolates were resistant to both chloramphenicol and cotrimoxazole (Chandel et al., 2000) Third generation cephalosporins : Treated early, typhoid fever usually responds well to appropriate antibiotics. At one time, ampicillin and sulfamethoxazole/trimethoprim were drugs of choice, but increasing antibiotic resistance has prompted wider use of third-generation cephalosporins and quinolone derivatives, according to the World Health Organization. The CDC says that antimicrobial therapy "should be guided by local data on antimicrobial sensitivity" (Snow et al., 2006). The defervescence period for ciprofloxacin is about 3 - 5 days according to the literature and for cephalosporin is about three days. But in the present study we have observed that the defervescence period was comparatively longer; about eight days for ciprofloxacin and about six days for cephalosporins (Chowta et al., 2005). Applicable: Uncomplicated typhoid at home: Nalidixic-acid resistant Antibiotic: Azithromyin or cefixime; daily dose Azithromycin 8-10 mg/kg; Days: 7; Cefixime 20 mg/kg; Days: 7-14 (Bhan et al., 2005). Severe typhoid in hospital: Nalidixic-acid resistant Antibiotic: Ceftriaxone or cefotaxime; daily dose Ceftriaxone 60 mg/kg; Days:10-14; cefotaxime 80 mg/kg; Days: 10-14 (Bhan et al., 2005). Success Rate: Azithromycin, and cefixime, an oral third-generation cephalosporin, have a clinical cure rate of over 90% with a fever clearance time of 5-7 days and relapse and faecal carriage rates of less than 4% in typhoid fever (Bhan et al., 2005). Third generation cephalosporins and azithromycin have proved effective in endemic areas (Parry et al., 2006). 100% (Chowta et al., 2005). 4. Prevention: a. Whole cell vaccine : Ontology: UMLS:C0042196 Description: The live oral vaccine is an attenuated S typhi strain, Ty21a, which is a mutant of Ty2 with a uridine diphosphogalactose 4-epimerase defect. The strain lacks the virulence (Vi) antigen and is thus avirulent but contains immunogenic cell wall polysaccharides (Connor et al., 2005). Whole cell vaccines against typhoid fever: Route of administration- Parenteral; Adverse systemic reactions- 10-20%; Adverse local reactions- 10-50%; Protection rate- 60-80%; duration of protection7 years; Booster doses/revaccination- 3 years; Medical professional needed for vaccination- yes. Parenteral whole cell typhoid vaccines were obtained by inactivating virulent microorganisms with heat or chemicals. The destruction of some heat-labile antigens during preparation was believed to compromise the overall efficacy of heat-inactivated, phenol-preserved vaccine. This problem was in part solved by the use of acetone for inactivation, followed by drying. The establishment of appropriate quality control tests (e.g., potency and killing) was very difficult for the inactivated whole cell vaccines (Guzman et al., 2006). Efficacy: Rate: These studies demonstrated the efficacy of vaccines consisting of S. Typhi inactivated by heat and phenol or by acetone ranged from 51% to 88% in children and young adults (Guzman et al., 2006). A meta-analysis of published studies of the live oral vaccine Ty21a showed that the overall protective efficacy was 71% for 5-9 year-olds, and 63% for 10-14 year-olds (Connor et al., 2005). In a case control study of travellers to India, protective efficacy was only 23% with the Ty21a oral vaccine (Connor et al., 2005). Duration: Primary vaccination consists of one enteric coated capsule or lyophilised sachet on alternate days for three to four doses (Connor et al., 2005). Protection persisted for up to 7 years (Guzman et al., 2006). Contraindicator: Complication: Although these early parenteral vaccines were clearly able to confer protection against typhoid fever, their global use as public health tools for routine vaccination was undermined by their high reactogenicity: the parenteral whole cell vaccines caused fever (6-30% of the recipients), headache (10%) and severe local pain (up to 35%). Furthermore, up to 10% of the vaccinees missed work or school owing to the vaccination (Guzman et al., 2006). This vaccine needs to be refrigerated, cannot be given to children under 6 years of age, and relies on the traveller's compliance and memory to complete the three to four required doses (Connor et al., 2005). The live attenuated vaccine is theoretically contraindicated in pregnancy and in those with cell-mediated immunosuppression (Connor et al., 2005). b. Vi-based parenteral subunit vaccine : Ontology: UMLS:C0042196 Description: The whole cell vaccines have been replaced by the well tolerated Vi-based parenteral subunit vaccine and the oral attenuated live bacterial vaccine Ty21a (Guzman et al., 2006). The CDC recommends typhoid vaccination for people traveling to developing countries in Africa, Asia, the Indian subcontinent, Central and South America, and the Caribbean. Two vaccines are available in the United States: a live attenuated vaccine given orally (Vivotif Berna vaccine manufactured from the Ty21a strain of S. typhi) and an I.M. vaccine (Typhi Vi or ViCPS). The oral vaccine is taken in multiple doses. Adults and children age 6 and older should take one capsule every other day for a total of four doses. The regimen should be completed 1 week before travel. Tell patients to take each dose 1 hour before meals with cool water and to keep the capsules refrigerated. The one-dose parenteral vaccine is an option for children ages 2 to 6, immunocompromised patients, and those who may not adhere to the oral dosage regimen (Snow et al., 2006). The Vi antigen is a capsular polysaccharide antigen that allows S typhi to survive in blood leading to septicaemia. The parenteral Vi vaccine contains only the purified antigen and produces rapid seroconversion following one dose (Connor et al., 2005). Subunit vaccines that are generated using purified Vi capsular polysaccharide of S. Typhi. A subunit vaccine was developed from wild type S. Typhi strain Ty2 on the basis of non-denatured purification of the Vi polysaccharide. Vi consists of ([alpha]1-4),2-deoxy-2-N-acetyl galacturonic acid, which is partially O-acetylated at carbon 3 and forms a capsule that protects the bacteria against complement-mediated lysis and phagocytosis. For vaccine production, the Ty2 cells are cultured in large-scale bioreactors. The capsular polysaccharide is precipitated from the culture supernatant, purified and vacuum dried before the antigen is resuspended in buffer. The vaccine also contains phenol as a preservative. Initial Vi purification attempts failed as the polysaccharide required for its immunogenicity was denatured. Only the establishment of more gentle antigen purification processes, that retain the original structure of the polysaccharide, resulted in the successful development of potent Vibased vaccine. The vaccine is administered as a single intramuscular or subcutaneous dose containing 25 ug of non-denatured Vi-antigen (Guzman et al., 2006). Efficacy: Rate: The vaccine is safe to be coadministered with other travel vaccines as well as antimalarials with no diminution in antibody response. However, because it is a polysaccharide vaccine there is no boosting effect from revaccination and the duration of protection appears to be 2-3 years. A new S typhi Vi conjugate vaccine
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has been developed, conjugating Vi to a carrier protein to enhance immunogenicity (Connor et al., 2005). The efficacy of the Vi polysaccharide was assessed in the late 1980s in field trials in typhoid-endemic areas in Nepal and South Africa. In South Africa, the vaccine was given to more than 11,000 children 6-14 years of age and exhibited a protective efficacy of 64% during the first 21 months after vaccination and an efficacy of 55% over 3 years. In Nepal, the vaccine was tested in 6900 individuals 5-44 years of age and resulted in 72% protective efficacy over 17 months. Two weeks after immunisation, about 80% of vaccines exhibit a fourfold rise in antibody titres. Hence, it is recommended that an interval of at least 2 weeks exist between immunisation and expected exposure. Vi capsular polysaccharide is well tolerated and safe (Guzman et al., 2006). Currently, a monovalent Vi-based vaccine is available, as well as two bivalent vaccines combining Vi-antigen with a hepatitis A vaccine (Guzman et al., 2006). Duration: Revaccination is recommended every 2 years in the US (Guzman et al., 2006). Contraindicator: An important drawback of the parenteral administration of Vi polysaccharide vaccines lies in their inability to stimulate mucosal immunity and the fact that revaccination does not elicit any booster effect, as shown in clinical trials. This absence of a booster effect is due to the fact that immune responses against polysaccharides do not involve T cells, therefore immunological memory cannot be established. Furthermore, these vaccines are not very effective in infants or toddlers (Guzman et al., 2006). Complication: The most common side-effects are pain, redness and induration at the injection site, and fever. In very rare cases, allergic reactions and rashes have been observed (Guzman et al., 2006). c. Live attenuated oral vaccine S. Typhi Ty21a: Ontology: UMLS:C0042196 Description: The CDC recommends typhoid vaccination for people traveling to developing countries in Africa, Asia, the Indian subcontinent, Central and South America, and the Caribbean. Two vaccines are available in the United States: a live attenuated vaccine given orally (Vivotif Berna vaccine manufactured from the Ty21a strain of S. typhi) and an I.M. vaccine (Typhi Vi or ViCPS).The oral vaccine is taken in multiple doses. Adults and children age 6 and older should take one capsule every other day for a total of four doses. The regimen should be completed 1 week before travel. Tell patients to take each dose 1 hour before meals with cool water and to keep the capsules refrigerated. The one-dose parenteral vaccine is an option for children ages 2 to 6, immunocompromised patients, and those who may not adhere to the oral dosage regimen (Snow et al., 2006). Attenuated strains administered orally mimic the mucosal and systemic immune responses elicited by natural infection (Guzman et al., 2006). Ty21a is an attenuated mutant strain of S. Typhi Ty2 that is safe and protective as a live oral vaccine. This mutant was isolated in the early 1970s by chemical mutagenesis and has a GalE- and Vi-negative phenotype. The mutation of the galE gene results in a complete deficiency of the enzyme uridine diphosphate (UDP)-galactose-4-epimerase, which is responsible for the conversion of UDP-glucose to UDP-galactose and vice versa. Because of this enzyme deficiency, UDP-galactose cannot be metabolised and accumulates in the cytoplasm to cause cell lysis and attenuation, when galactose is present in the growth medium. However, galE deficiency alone was found to result in premature lysis when administered to mice, thereby preventing the elicitation of an adequate immune response. Therefore, strain Ty21a resulted from a further selection for reduced levels of enzymes involved in the synthesis of UDP-galactose from exogenous galactose, namely galactose permease, galactokinase, and galactose-1-phosphate-urydiltransferase. Since galactose is incorporated into the lipopolysaccharide (LPS) core moiety via UDP-galactose, the absence of galE leads to the formation of rough LPS, i.e., LPS devoid of part of the core and the O-antigen. Since the O-antigen is the main antigenic determinant on the cell surface, Ty21a is supplied with a source of exogenous galactose during production of the vaccine. This enables bacteria to generate UDP-galactose by an alternative route, thereby expressing complete immunogenic LPS (Guzman et al., 2006). Although the immunogenic properties of wild type S. Typhi are maintained when Ty21a is grown under appropriate conditions, the galE phenotype contributes to strain attenuation in vivo. As a result of the mutagenesis method used during the generation of the vaccine strain, further spontaneous mutations were generated. These mutations included the via and ilvD genes, leading to the loss of the Vi capsular polysaccharide and an auxotrophic phenotype for isoleucine and valine, respectively, and a mutation precluding H2S utilisation. An additional mutation in the rpoS gene, which also contributes to the avirulence of the Ty21a strain, was inherited from wild type parental strain Ty2. It is likely that the poor capacity of Ty21a to survive starvation conditions and resist various environmental stresses results, at least in part, from the rpoS mutation . This, combined with the low shedding rate, reduces the environmental risks posed by use of Ty21a. The Ty21a strain is the active constituent of Vivotif (Berna Biotech Ltd., Switzerland), currently the only licensed live oral vaccine against typhoid fever (Guzman et al., 2006). The additional mutations present in Ty21a are therefore instrumental in the added safety level of Ty21a. Clinical trials have also shown either a limited and transient level or a complete lack of shedding in the stools of volunteers depending on the administered dose of Ty21a (Guzman et al., 2006). Further studies showed a lack of faecal excretion of Ty21a upon administration of the commercial formulation. The inability to culture Ty21a from the small intestine suggests that the strain has a limited ability to proliferate in vivo. Neither person-to-person transmission nor invasion of the bloodstream has been observed in vaccinees. The very low excretion rate of Ty21a combined with its genetic attenuation significantly reduces its ability to survive in humans and the environment (Guzman et al., 2006). Efficacy: Rate: Oral vaccination is generally associated with lower rates of side-effects and higher acceptance by vaccines and can be logistically simpler (Guzman et al., 2006) The efficacy of Ty21a was assessed in a large number of clinical trials, with over 500,000 vaccinated adults and children. Excellent tolerability and an overall protective efficacy of 67-80% (applying three doses of enteric-coated capsules or a liquid formulation) were demonstrated for up to 7 years. The field studies conducted in Santiago (Chile) confirmed the efficacy and tolerability of Ty21a, and provided evidence of indirect protection (herd immunity). The incidence of typhoid fever fell in the placebo control group in the first field trial during the years in which Ty21a field trials were performed in other parts of Santiago and started to rise again when vaccination with Ty21a was not carried out (Guzman et al., 2006). Two possible mechanisms have been suggested for the herd immunity effect of Ty21a. Firstly, individuals vaccinated with Ty21a have significantly reduced excretion of virulent Salmonella in comparison with the non-vaccinated population, thereby resulting in reduced contamination of water supplies. Secondly, fewer temporary carriers (i.e., children with sub-clinical or incubating acute infections) may reduce the transmission of the disease. The optimum booster schedule for Vivotif vaccine has not been determined. Efficacy has been shown to persist for at least 7 years. Further, there is no experience with Vivotif vaccine as a booster in persons previously immunised with parenteral typhoid vaccine (Guzman et al., 2006). The excellent safety and tolerability profile of Ty21a was further confirmed in more than 200 million vaccinees during its over 20 years use worldwide (Guzman et al., 2006). Duration: The capsules are administered orally, one hour before meals, and in three doses (four doses in the US and Canada) within a time frame of 1 week. The full course is recommended to be completed 1 week prior to potential exposure (Guzman et al., 2006). In the US, it is recommended that a re-immunisation dose consisting of four vaccine capsules taken on alternate days be given every 5 years under conditions of repeated or continued exposure to typhoid fever (Guzman et al., 2006). Complication: Recent post-marketing surveillance has identified only mild and infrequent adverse events associated with Ty21a. In the 10 years from 1990 to 2000, more than 38 million people were vaccinated with Ty21a with only 743 spontaneous reports of adverse events, an incidence of 0.002%. The most common adverse events reported with Ty21a were mild and transient gastrointestinal disturbances, followed by general symptoms such as pyrexia. The multiple mutations of Ty21a collectively render it genetically stable. Reversion to virulence has not been observed in vitro or in vivo (Guzman et al., 2006). d. Destroying contaminated food: Description: The foods most likely to be contaminated are meats. Since the contamination for transmission must come after the food has been processed or prepared for consumption, those foods which are allowed to stand for some considerable period of time after being cooked before they are served or the foods which are not heated before service are the ones most likely to be involved in this transmission. Food may be contaminated by persons who are carriers and who use less than desirable hygienic methods in preparation or who use contaminated ingredients, including contaminated water used as an ingredient or used for cleaning utensils. When these contaminations are followed by inadequate or no cooking, and the food is then allowed to stand, multiplication of bacteria in the food will be sufficient to provide an infectious dose to the consumer (Gutherie et al., 1991). e. Water treatment:
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Ontology: UMLS:C0597684 Description: The control of typhoid fever was greatly enhanced by the treatment of water supplies for general consumption; however, this was not an absolute control since the organism does readily establish a carrier condition in recovering patients, and in some of these individuals the condition may be permanent (Gutherie et al., 1991). f. Personal hygiene : Ontology: UMLS:C0237122 Description: S. typhi lives in the gastrointestinal tract and bloodstream of people who are acutely ill and those who are asymptomatic chronic carriers. It's transmitted via the fecal-oral route, usually through contact with contaminated food or water. Although uncommon in industrialized nations, typhoid fever is endemic in areas where sanitation is poor. Vaccination helps protect against the disease, but the most important safeguards are good food handling practices and water sanitation (Snow et al., 2006). g. Travel related precautions : Ontology: UMLS:C0040802 Description: Travel to rural areas with poor sanitation was associated with a higher risk. Not following food and water precautions and not receiving pretravel consultation increased the risk ten times (Connor et al., 2005). Vaccination against typhoid fever is not the main preventative measure. Indeed, avoidance of potentially contaminated food and drink should be the basic advice given to travellers, because the ingestion of a large inoculum of S. enterica Typhi may result in infection despite immunisation (Papadimitropoulos et al., 2004). Vaccination protects only 50% to 80% of travelers, so tell your patient to take precautions throughout her travels, even if she's been vaccinated, to protect herself from all enteric illnesses, including hepatitis A, cholera, and traveler's diarrhea. Teach her the adage "Boil it, cook it, peel it, or forget it" and advise her to: buy bottled water. If bottled water isn't available, she should boil tap water for at least 5 minutes before drinking, cooking, or brushing her teeth with it. Avoid ice in beverages and in desserts and treats that contain ice. Eat well-cooked food that's still steaming hot. Avoid raw foods, including garden or fruit salads . Peel fresh fruits before eating them. Wash (her) hands with soap and water, vigorously wash the outside of the fruit, then peel it. Avoid foods sold by street vendors (Snow et al., 2006). Current vaccines offer only moderate protection against S typhi and no protection against S paratyphi, which has become the dominant pathogen among travellers. Thus, there is a great need for a combined vaccine, particularly with increasing antibiotic resistance in both S typhi and S paratyphi (Connor et al., 2005). 5. Model System: a. Mouse : i. Ontology: UMLS:C0025914 ii. Model Host: Mus musculus iii. Description: From the standpoint of human disease, Salmonella serotypes can be divided into three groups that cause distinctive clinical syndromes, typhoid fever, bacteremia and enteritis. Animal models are frequently used to study the virulence mechanisms of Salmonella serotypes that are important for two of these human disease syndromes, typhoid fever and enteritis (Santos et al., 2001). Mouse: Initial studies revealed that the distribution of bacteria in tissue of mice infected with serotype Typhimurium is similar to that in typhoid fever patients. These early reports therefore established murine typhoid as an animal model for the study of Typhoid fever. Serotype Typhimurium is a natural pathogen for rodents as shown by its frequent association with disease in this animal reservoir. The interaction of serotype Typhimurium with one of its natural hosts is considered by many to be a better model for studying typhoid fever than more recently developed artificial systems (e.g., serotype Typhi infection of mice treated with iron). susceptible mouse lineages (e.g., Balb/c) are widely used to study the pathogenesis of serotype Typhimurium infections. These animals show signs of disease (i.e. elevated temperature as indicated by ruffled fur) between 4 - 8 days post oral infection, however, diarrhea does not develop. Gross pathology of the intestine commonly reveals enlarged Peyer's patches and a thickening of the ileal mucosa. A diffuse enteritis is present in the small intestine characterized by a predominantly mononuclear leukocyte infiltrate as well as edematous villi which become shortened in height. Follicular hyperplasia of the lymphoid tissue, capillary thrombosis, hemorrhage, and ulcerations may be present in the terminal ileum at areas of Peyer's patches. The intestinal epithelium in other areas of the intestine remains largely intact. The intestinal pathology and inflammatory reaction in mice is hence more similar to that of typhoid fever patients than it is to that caused by serotype Typhimurium in human intestines (Santos et al., 2001). One obvious limitation of the mouse model of typhoid fever is that serotype Typhimurium causes enteritis rather than typhoid fever in humans. Thus, humans and mice exhibit strikingly different host responses to serotype Typhimurium infections (Santos et al., 2001). b. Human: i. Ontology: UMLS:C0086418 ii. Model Host: Homo sapiens iii. Description: Human: In human volunteers, fever is the first symptom and manifests after a median incubation period of 5 - 9 days, depending on the challenge dose. Typhoid fever patients are often constipated during the early stages of infection but about one third develop diarrhea subsequent to the onset of fever. Biopsies taken from the upper small intestine as early as three days post experimental infection of volunteers with serotype Typhi reveal diffuse enteritis caused predominantly by a mononuclear leukocyte infiltrate. Similarly, mucosal thickening of the ileum due to a polymorphonuclear-poor, mononuclear-rich infiltrate is characteristic in typhoid fever patients. Tissue colonization of serotype Typhi may produce capillary thrombosis in Peyer's patches of the terminal ileum which can result in hemorrhage, necrosis (usually observed in the second week of infection), ulceration and intestinal perforation (usually observed in the third week). Enlargement of mesenteric lymph nodes, liver and spleen is accompanied by granulomatous lesions. The low-level bacteremia detected in most patients is important for the systemic spread of bacteria but endotoxin appears not to play a major role in the pathogenesis of typhoid fever (Santos et al., 2001). c. Rhesus Monkey: i. Ontology: UMLS:C0024400 ii. Model Host: Macaca mulatta iii. Description: Rhesus Monkey: While rhesus monkeys are resistant to infection, typhoid fever can be induced by oral infection of chimpanzees with serotype Typhi. In this animal model, mucosal thickening of the ileum and enlargement of Peyer's patches is due to a diffuse enteritis characterized by infiltration of predominantly mononuclear leukocytes. Furthermore, a reactive follicular hyperplasia of lymphoid follicles of Peyer's patches is noted (Santos et al., 2001).

IV. Labwork Information

A. Biosafety Information:
1. Biosafety information for : Salmonella enterica subsp. enterica serovar Typhi (CDC): Biosafety Level: Recommended precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities utilizing known or potentially infectious clinical materials and cultures. Biosafety Level 3 practices and procedures are recommended for activities likely to generate aerosols or for activities involving production quantities of organisms. Licensed vaccines, which have been shown to protect 70-90% of recipients, may be a valuable adjunct to good safety practices in personnel regularly working with cultures or clinical materials which may contain S. typhi (CDC). Applicable: Laboratory Hazards: The agent may be present in feces, blood, gallbladder (bile), and urine. Humans are the only known reservoir of infection. Ingestion and parenteral inoculation of the organism represent the primary laboratory hazards. The importance of aerosol exposure is not known (CDC: Bacterial
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Agents). Precautions: Recommended Precautions: Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious clinical materials and cultures. Biosafety Level 3 practices and procedures are recommended for activities likely to generate aerosols or for activities involving production quantities of organisms (CDC: Bacterial Agents). Disposal: The salmonellae grow best at 37 degrees C and can survive freezing sterile water and drying for several weeks and sometimes longer. They are killed by a temperature of 60 degrees C for 15 minutes, and rapidly by boiling (Huckstep et al., 2002).

B. Culturing Information:
1. Plate culture : a. Description: When isolated from clinical specimens, colonies of S. enterica serovar Typhi are non-lactose fermenting and produce a characteristic biochemical pattern in Kligler iron agar (acid but without gas, an alkaline slant and a moderate amount of hydrogen sulfide production) (Parry, 2006). With the standard broth culture, S. Typhi is found in the blood of 30-90% of patients with clinically suspected cases of typhoid fever (Massi et al., 2005). . b. Medium: i. Plating media: Plating media for the isolation of salmonella can be subdivided into three groups according to the selective agents used. These are the bile salt agars, the brilliant green agars and bismuth sulphite agar (Busse, 1995). Bile salt media: Bile salt agars show the widest variation. They comprise the deoxycholate citrate agars originally proposed by Leifson, Salmonella Shigella (SS) agar, Hektoen Enteric agar and XLD agar. Plain deoxycholate agar and XLD agar contain bile salts and citrate in amounts of l-2 g/l. With the more selective deoxycholate citrate agars the content of bile salt is increased to 5-10 g/l. The content of citrate goes up to 10-20 g/l. Some formulations like Leifson agar are citrate-dominated whereas others like Hektoen agar contain bile salts as the main selective substance. SS agar contains both substances in considerable amounts. Thiosulphate as a source of H(2)S is included in most of these agars to detect hydrogen sulfide positive colonies (Busse, 1995). Brilliant green media: On Brilliant green agar salmonella is detected by its inability to ferment lactose or lactose and sucrose, giving pink-coloured colonies (Busse, 1995). Using pure strains the authors showed that MLCB is more selective than brilliant green agar. Salmonella strain grew well, whereas certain competitive enterobacteria which still grow on brilliant green agar, did not develop on LCB (Busse, 1995). Bismuth Sulphite Agar: Bismuth sulphite agar is usually said to be particularly useful for detecting S. typhi (Busse, 1995). ii. Selective enrichment media (Rappaport- Vassiliadis medium (RV): The original Rappaport medium contains rather high amounts of malachite green and magnesium chloride. In addition the medium has a very low pH of 5.2. The medium was developed for the enrichment of S. paratyphi and other serotypes known to be relatively resistant to brilliant green. Magnesium chloride, was shown to counteract the toxic effect of the dye for salmonella (Busse, 1995). c. Optimal Temperature: Selenite cystine: 37 degrees C, Tetrathionate bile 37 degrees C (Busse, 1995). Rappaport-Vassiliadis medium (RV): Vassiliadis et al. recommended incubation of RV at 43 degrees C. This achieved maximum selectivity, but any deviation above 43 degrees C may be lethal for salmonella. For this reason most workers follow the ISQ recommendation to incubate at 42 degrees C (Busse, 1995) d. Picture(s): i. Culture of typhoid bacillus (Huckstep):

Description: Culture of typhoid bacillus (Huckstep). 2. Blood culture : a. Description: Widely regarded as the gold standard, but sensitivity may be low in endemic areas with high rates of antibiotic use--hence true specificity is difficult to estimate (Bhutta, 2006). Although the mainstay of diagnosing typhoid fever is a positive blood culture, the test is positive in only 40-60% of cases, usually early in the course of the disease (Bhutta, 2006). Blood culture is not always feasible particularly when smaller children are involved (Vaishnavi et al., 2006). b. Medium: i. This should include both whole blood in citrate solution, and clotted blood in a plain sterile bottle (Huckstep et al., 2002). c. Note: Sensitivity range 40-80% (Bhutta, 2006). S. paratyphi-A: No. (%) of specimens positive in Blood-TSB: 32(52); Blood-bile: 40(67); Clot-bile: 40(65); clotstreptokinase 42(68) (Escamilla et al., 1986). S. typhi: No. (%) of specimens positive in Blood-TSB: 32(69); Blood-bile: 39(84); Clot-bile: 39(84); clotstreptokinase 36(78) (Escamilla et al., 1986). The results of blood cultures in patients with typhoid fever depend on many factors, including the volume of blood samples, the bacteremic level of S. Typhi, the type of culture medium used, and the length of the incubation period (Massi et al., 2005). 3. Direct plating of the buffy coat : a. Description: The concentration of typhoid bacilli in the buffy coat (presumably in monocytes and polymorphonuclear leukocytes) means that direct plating of this cell layer provides an alternative and more rapid method of diagnosis than whole-blood broth culture. Colonies were usually evident after an overnight incubation (Wain et al., 1998). To determine the number of intraleukocytic CFU in blood samples, quantitative cultures were performed on peripheral blood buffy coats. This study was performed on a randomly chosen subgroup of patients. Heparinized whole blood (2.5 or 5 ml) taken at the same time as QBC was centrifuged at 2,700 x g for 10 min. The plasma was removed carefully with a sterile plastic pipette, and 0.1 ml including the buffy coat layer was aspirated with a sterile 1 ml syringe. Quantitative cultures were performed on this 0.1ml sample by mixing it with 19 ml of molten Columbia agar, pouring the mixture as an agar plate, and incubating it for up to 4 days. Peripheral blood phagocytes containing more than one bacterium will produce a single colony in solid culture medium. Thus, for whole blood and buffy coat samples, results were expressed as CFU per milliliter. To quantitate precisely the number of intracellular bacteria per infected phagocyte, a second 0.1 ml buffy coat sample was taken and the leukocytes were lysed by incubation with 0.1 ml of 0.1 % digitonin for 10 min at 37 degrees C. This should have released the intracellular bacteria. Quantitative cultures were then performed on this sample in the same way as for the first sample, i.e., by mixing with 19 ml of molten Columbia agar, pouring the mixture as an agar plate, and incubating it at 37 degrees C for up to 4 days (Wain et al., 1998). In order to exclude the possibility that the localization of bacteria in the buffy coat was an artifact of the centrifugation step, 10 ml of blood from a healthy volunteer was seeded with 10(4) CFU of S. typhi and centrifuged immediately at 2,700 x g for 10 min. A 0.1 ml volume of buffy coat and 0.1 ml of the erythrocyte layer were collected with sterile 1 ml syringes, mixed with 19 ml of molten Columbia agar, and allowed to set as described above. Bacterial colonies were counted after 4 days of incubation at 37 degrees C (Wain et al., 1998). The number of S. typhi bacteria per milliliter of blood (QBC) was estimated from the number of CFU on each pour plate (Wain et al., 1998). In patients who were broth culture positive but QBC negative, the quantitative value was estimated from the total volume of blood cultured: 0.7 to 1.0 CFU/ml from 1.5 ml of blood and 0.3 to 1.0 CFU/ml from 3 ml of blood (Wain et al., 1998). b. Medium: i. Brain heart infusion broth, sheep blood agar (Wain et al., 1998). c. Optimal Temperature: 37 degrees C (Wain et al., 1998). d. Note: Antibiotic disc sensitivities were performed (Wain et al., 1998). Organisms resistant to chloramphenicol, ampicillin, trimethoprim, and sulfamethoxazole but
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sensitive to ofloxacin and ceftriaxone were described as multidrug resistant (Wain et al., 1998). Venous blood for quantitative culture and for broth culture was taken before administration of antimicrobials for typhoid fever. The blood (3 to 9 ml from adults and 1.5 to 6 ml from children) was collected in a sterile heparinized tube and transported immediately to the laboratory. Quantitative whole-blood cultures (QBC) were carried out by a pour plate method. In brief, three measured aliquots of blood (usually 1 ml; 0.5 ml for small children) were mixed with 19 ml of molten (50 C) Columbia agar in a sterile petri dish, allowed to set, and then incubated at 37 C. After 2 to 4 days colonies were counted and recorded as CFU per milliliter. Up to five colonies were picked from the surface of the agar for identification. After reincubation for 24 h standard biochemical tests and agglutination with the specific antisera were performed. Plates were discarded as negative if no colonies were visible after 4 days of incubation (Wain et al., 1998). 4. Stool culture : a. Description: This will require a selective culture medium. It is not positive as often as blood culture. It is, however, a valuable diagnostic test, and several stool specimens should always be cultured. Contrary to the usual teaching, it is often positive before the third week of illness, and may be positive at any stage of the disease (Huckstep et al., 2002). Stool samples were processed using Salmonellas isolation method. Each sample was first enriched in selenite - F-broth incubated at 37 degrees C for 24 - 48 hours. Growth from the broth subcultured onto brilliant green Mac Conkey agar incubated at 37 degrees C for 18 - 24 hours. Nonlactose fermenting colonies on this medium were subcultured onto Wilson and Blair's bismuth sulphite agar incubated at 37 degrees C for 18-24 hours. Growth showing central sheen, which indicated Salmonella organism was subcultured into nutrient agar for further characterization (Ngwu et al., 2003). Stool cultures were plated in MacConkey and deoxy-cholate-citrate agar both directly and after initial incubation in selenite broth. Specific identification in positive cultures was by a panel of biochemical and agglutination tests (Choo et al., 1999). 30 % (Bhutta, 2006). b. Medium: i. Selenite - F broth, brilliant green Mac conkey agar, Wilson and Blairs's bismuth sulphite agar (Ngwu et al., 2003). c. Optimal Temperature: 37 degrees C (Ngwu et al., 2003). d. Note: Sensitivity lower in developing countries and not used routinely for follow-up (Bhutta, 2006). Stool and urine cultures become positive after the first week of infection, but their sensitivity is much lower (Bhutta, 2006).

C. Diagnostic Tests :
1. Organism Detection Tests: No organism detection tests available here. 2. Immunoassay Tests: a. Widal tube agglutination test (tube dilution and slide agglutination): i. Ontology: UMLS:C0430432, SNOMED:143236008 ii. Time to Perform: 2-to-7-days iii. Description: The Widal agglutination test, developed by F Widal in 1896 to aid in the diagnosis of typhoid fever, utilises a suspension of killed Salmonella typhi as antigen, to detect typhoid fever in serum from suspected S typhi-infected patients who present with febrile illness (Olopoenia et al., 2000). The test was based on demonstrating the presence of agglutinin (antibody) in the serum of an infected patient, against the H (flagellar) and O (somatic) antigens of Salmonella typhi. While the definitive diagnosis of typhoid fever depends on the isolation of S typhi from blood, stools, urine or other body fluids, the role of the Widal test had been to increase the index of suspicion for the presence of typhoid fever by demonstrating a positive agglutination during the acute and convalescent period of infection with evidence of a four-fold rise of antibody titre (Olopoenia et al., 2000). The Widal test reaction involves the use of bacterial suspensions of S typhi and S paratyphi `A' and `B', treated to retain only the 'O' and 'H' antigens. These antigens are employed to detect corresponding antibodies in the serum of a patient suspected of having typhoid fever. The IgM somatic O antibody appears first and represents the initial serologic response in acute typhoid fever, while the IgG flagella H antibody usually develops more slowly but persists for longer (Olopoenia et al., 2000). Two types of agglutination techniques are available: the slide test and the tube test. The slide test is rapid and is used as a screening procedure. Using commercially available antigens of S typhi, a drop of the suspended antigen is added to an equal amount of previously prepared serum. An initial positive screening test requires the determination of the strength of the antibody. This is done by adding together equal amounts of antigen suspension and serially diluted serum from the suspected patient (Olopoenia et al., 2000). A mixture of suspended antigen and antibody is incubated for up to 20 h at 37 C in a water bath. Agglutinations are visualised in the form of pellets, clumped together at the bottom of the test tube. Results are scored from 0 to 4(+) positive agglutination (Olopoenia et al., 2000). Serum was considered positive for Widal test when anti-O or anti-H titer was (> or equal to) 160 (Mekara et al., 1990). The levels are measured by using serial dilutions of sera. Usually, O antibodies appear on days 6-8 and H antibodies on days 10-12 after the onset of the disease. The test is usually performed on acute and convalescent sera to detect the rising titers. The test has only moderate sensitivity and specificity (Ismail, 2006). Classic and inexpensive. Despite mixed results in endemic areas, still performs well for screening large volumes. May need standardisation and quality assurance of reagents (Bhutta, 2006). Of the indirect tests available for the diagnosis of enteric fever, the most established is the Widal which has enjoyed widespread use as an adjunct to clinical assessment and bacterial isolation (Choo et al., 1999). The Widal test was carried out using antigens from Wellcome Diagnostics and standard serial dilutions of serum in normal saline starting at 1:40. A positive Widal test was taken as a titre of at least 1 in 80 (Choo et al., 1999). The Widal test does not provide results for some 2 days after blood sampling (Choo et al., 1999). The usefulness of the Widal test in diagnosing childhood typhoid fever in endemic areas was investigated. The test was done on 150 children with other febrile illnesses and 98 bacteriologically proved cases of typhoid fever. Of the 150 children with nontyphoidal fever, only one had an H agglutinin titer of 1:50. Using an H or O agglutinin titer of 1:50 or more as a criterion for diagnosis, a positive Widal test was found in 88% of typhoid fever cases on the first occasion on which the test was done. If the test was repeated at least 94% of the typhoid cases had a significant result. The Widal test is a useful diagnostic test in children in endemic areas, provided interpretation of the test is made against background information relating to agglutinin levels in normal children in the region (Chow et al., 1987). Approximate cost (U.S. dollars): 0.50, No. of tests/kit: 55; Reaction time: 5 minutes, Temperature for storage: 2-8 degrees C; Amount of serum needed: 300 ul +- (two dilutions) (Olsen et al., 2004) iv. False Positive: Widal 1:80, positive predictive value 19.6% (Nizami et al., 2006). Sensitivity 47-77 %; Specificity 50-92 % (Bhutta, 2006). v. False Negative: Widal 1:80, negative predictive value 90.2% (Nizami et al., 2006). b. Typhidot test: i. Ontology: UMLS:C0014441 ii. Time to Perform: 1-hour-to-1-day iii. Description: A recently developed dot enzyme immunosorbent assay (Typhidot) using a 50 kD outer membrane protein (OMP) from Salmonella typhi is, however, a cost-effective and more rapid alternative to the Widal test with at least comparable sensitivity and specificity. The Typhidot allows separate evaluation of the presence of specific serum immunoglobulin (Ig) g and m antibodies to the OMP (Choo et al., 1999). In areas of high endemicity, Typhidot identification of specific IgG but not IgM in the serum of a febrile child is taken as a positive result for enteric fever but has two explanations. Firstly, there has been a boosting of specific IgG antibody levels in response to re-infection with S. typhi. In this situation, a marked IgG response may interfere with IgM antigen binding in the Typhidot IgM test, giving a false negative IgM result. Secondly, the patient has residual detectable IgG antibody from recent enteric fever and a different new infection. Because specific IgG may persist for 6 months or more after successful treatment of enteric fever while a positive IgM test typically lasts
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3 months, the child may be in the 'window' period when only Typhidot IgG is positive. To increase diagnostic accuracy in these situations, a modification of the original Typhidot test has been developed in which initial inactivation of total serum IgG aims to unmask the presence of specific IgM (the Typhidot-M test) (Choo et al., 1999). Typhidot takes three hours to perform. It was developed in Malaysia for the detection of specific IgM and IgG antibodies against a 50 kD antigen of S. Typhi (Ismail, 2006). In the case of Typhi-dot test, 1:100 dilutions of patient serum were applied to standard aliquots of 0.3 ug purified OMP dotted on to nitrocellulose strips. After 1 hour, horseradish peroxidase-conjugated antiserum to human IgG or IgM was added. The presence of antigenantibody complexes was assessed visually from the resultant colour change in comparison to that from positive control sera. Because only one dilution was used, a positive Typhidot test was taken as an IgG and /or IgM titre of at least 1:100. In the case of the Typhidot-M test, diluted sera were first added to a buffered inactivation solution containing anti-human IgG. Inactivated control sera were used for visual comparsion (Choo et al., 1999). Typhidot test makes use of the 50 kD antigen to detect specific IgM and IgG antibodies to S. Typhi. It has undergone full-scale multinational clinical evaluation of its diagnostic value. This dot enzyme immuno assay (EIA) test offers simplicity, speed, specificity (75%), economy, early diagnosis, sensitivity (95%) and high negative and positive predictive values. The detection of IgM reveals acute typhoid in the early phase of infection, while the detection of both IgG and IgM suggests acute typhoid in the middle phase of infection. In areas of high endemicity, where the rate of typhoid transmission is high, the detection of specific IgG increases. Since IgG can persist for more than two years after typhoid infection, the detection of specific IgG cannot differentiate between acute and convalescent cases. Evaluations of Typhidot and Typhidot-M in clinical settings showed that they performed better than the Widal test and the culture method (Ismail, 2006). The Typhidot tests can both be completed within 3 h of venesection without the need for special training or equipment (Choo et al., 1999). Approximate cost (U.S. dollars): 2.14; No. of tests/kit: 56; Amount of serum need: 2.5 ul; reaction time: 60 minutes, temperature for storage: 2-8 degrees C (Olsen et al., 2004). iv. False Positive: Typhidot identification of specific IgG but not IgM in the serum of a febrile child is taken as a positive result for enteric fever but has two explanations. Firstly, there has a been a boosting of specific IgG antibody levels in response to re-infection with S. typhi. In this situation, a marked IgG response may interfere with IgM-antigen binding in the Typhidot-IgM test, giving false negative IgM result. Secondly, the patient has residual detectable IgG antibody from recent enteric fever and a different new infection. Because specific IgG may persist for 6 months or more after successful treatment of enteric fever while a positive IgM test typically lasts 3 months, the child may be in the 'window' period when only Typhidot IgG is positive. To increase diagnostic accuracy in these situations, a modification of the original Typhidot test has been developed in which initial inactivation of total serum IgG aims to unmask the presence of specific IgM (the Typhidot-M test). The results suggest that the tests have comparably high diagnostic accuracy (Choo et al., 1999). Typhi-dot IgG, positive predictive value 15.7% (Nizami et al., 2006). Typhi-dot IgG, no. of samples tested 210, sensitivity 80.7%, specificity 39.1% (Nizami et al., 2006). Sensitivity, specificity and efficiency of test for Typhidot and Typhidot M kits were 90%, 76.6% and 84% (Gopalakrishnan et al., 2002). v. False Negative: Sensitivity 66-88 %; Specificity 75 -91% (Bhutta, 2006). Typhi-dot IgG, negative predictive value 93.5% (Nizami et al., 2006). We have performed a comparison of the Typhidot and Typhidot-M tests in an area of peninsular Malaysia endemic for enteric fever. The results suggest that the tests have comparably high diagnostic accuracy. Their use in combination increases negative predictive value but at the cost of positive predictive value (Choo et al., 1999). Typhidot has lower sensitivity than Typhidot-M (Bhutta, 2006). c. Typhidot-M test: i. Ontology: UMLS:C0014441 ii. Time to Perform: 1-hour-to-1-day iii. Description: Typhidot M is a dot enzyme immunoassay for the detection of specific IgM to Salmonella typhi. In this test IgG is inactivated before carrying out the assay as for the Typhi dot (Gopalakrishnan et al., 2002). This theoretically allows improved detection of IgM, and thus would differentiate new from recent infections (Gopalakrishnan et al., 2002). A newer version of the test, Typhidot-M, was recently developed to detect specific IgM antibodies only. The dipstick test, developed in the Netherlands, is based on the binding of S. Typhi specific IgM antibodies in samples to S. Typhi lipopolysaccharide (LPS) antigen and the staining of bound antibodies, by an anti-human IgM antibody, conjugated to colloidal dye particles (Ismail, 2006). The test uses a nitrocellulose membrane strip dotted with the 50 kDa specific protein and a control antigen 10 ul of patient serum and controls are pre-absorbed for at least one minute with 90 ul of IgG inactivation reagent. 900 ul of sample diluent is then added into the reaction wells and the mixture incubated at room temperature on a rocker platform for one hour. The strips were washed thrice for a total of five minutes, and 1 ml of anti-human IgM conjugate was added and incubated for one hour. The strips were washed as before, and 1 ml of colour development solutions was added and incubated for 15 minutes. The reaction was stopped, by washing the strips in distilled water and the results were read. When both the dots on the test strip were as dark or darker than their corresponding dots on the positive control strip, they were reported as positive (Gopalakrishnan et al., 2002). Commercial kits evaluated were found to be less time consuming and easier to perform than Widal. The Typhidot M seems to be a practical alternative in the field and in small hospitals with lesser facilities (Gopalakrishnan et al., 2002). Higher sensitivity and specificity than classic Typhidot in some series, but other evaluations suggest that the performance may not be as robust in community settings as in hospital (Bhutta, 2006). iv. False Positive: Sensitivity 73-95 %; Specificity 75-91 % (Bhutta, 2006). Typhi-dot IgM, positive predictive value 19% (Nizami et al., 2006). v. False Negative: Typhi-dot IgM negative predictive value 93.6% (Nizami et al., 2006). d. IDL Tubex test: i. Ontology: UMLS:C0014441 ii. Time to Perform: 1-hour-to-1-day iii. Description: The new test (TUBEX) detects anti- Salmonella O9 (both immunoglobulin M [IgM] and IgG) antibodies in patients by inhibiting the binding between an anti-O9 IgM monoclonal antibody (MAb) conjugated to colored latex particles and S. typhi lipopolysaccharide (LPS) conjugated to magnetic latex particles (Lim et al., 1998). Like the Widal test, TUBEX consists of one step, and the result is read visually based on the appearance of the liquid contents in the tube. On the other hand, a specially designed set of tubes is used in TUBEX, and, instead of whole bacterial cells, S. typhi LPS adsorbed to magnetic particles is used as the detecting reagent. To make the test more specific, a monoclonal antibody (MAb) which recognizes the immunodominant O9 determinant in S. typhi LPS is also used; the antibody is conjugated to colored latex particles (Lim et al., 1998). White latex particles (diameter, 0.8 um) were sensitized with purified anti-O9 antibody or S. typhi LPS by passive adsorption (Lim et al., 1998). When the antibody-conjugated particles bind to the antigen-sensitized magnetic particles, and the latter are sedimented by use of a magnet, the color of the liquid (supernatant) in the tube changes (Lim et al., 1998). When magnetic particles coated with antigen (S. typhi LPS) are mixed with blue latex particles coated with anti-S. typhi LPS (O9) antibody, the two types of particles will bind to each other. When the magnetic particles are sedimented to the bottom of the tube by use of a magnet at the end of the experiment, the blue latex particles are also brought down. This would leave behind a clear supernatant if not for the fact that control (bovine serum albumin (BSA) coated) red latex particles are also added to the reaction mixture and remain suspended in the solution throughout the experiment. This makes the supernatant red when the blue particles are sedimented, which is easier to see than a colorless supernatant. When a patient's anti-O9 antibodies are present in the reaction mixture, they will inhibit the binding of the blue particles to the magnetic particles. Consequently, the supernatant remains purplish blue (unchanged from the beginning) due to the presence of blue particles and also, in a lower concentration, of red particles (Lim et al., 1998). The infection-specific anti-O9 antibodies in typhoid patients are detected by their ability to block the binding between the two types of particles, hence, no change in color. Transformation of the ELISA to the TUBEX test was made possible by the demonstration that magnetic particles could be conveniently used to separate reacted from unreacted indicator latex particles and, more importantly and more recently, by the development of a special tube which allows efficient reaction to take place (Lim et al., 1998). The test kit kept well at 4 degrees C, as shown by the fact that only a slight loss of activity was detected after 9 months of storage (Lim et al., 1998). Sensitivity 65-88 %, Specificity 6389 % (Bhutta, 2006). In the examination of 16 stored sera obtained from 14 patients with proven cases of typhoid fever and 78 serum samples from 75 subjects without typhoid fever, TUBEX was found to be 100% sensitive and 100% specific (Lim et al., 1998). No of samples tested: 210, sensitivity 26.9 %, specificity 88 % (Nizami et al., 2006).
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iv. False Positive: Positive predictive value 24.1 % (Nizami et al., 2006). v. False Negative: Negative predictive value 89.5 % (Nizami et al., 2006). e. Serotype Typhi IgM dipstick assay: i. Ontology: UMLS:C0014441 ii. Time to Perform: 1-hour-to-1-day iii. Description: The dipstick test, developed in the Netherlands, is based on the binding of S. Typhi specific IgM antibodies in samples to S. Typhi lipopolysaccharide (LPS) antigen and the staining of bound antibodies, by an anti-human IgM antibody, conjugated to colloidal dye particles (Ismail, 2006). Serum samples were diluted (1/50) in the detecting reagent (containing dye-labeled, anti-human IgM antibodies). Nitrocellulose dipsticks coated with heatinactivated serotype Typhi were immersed in the diluted serum and incubated at room temperature for 4 h. The strips were then washed and dried at room temperature. The sera were graded (0 to 4) according to the staining intensity of the colored band corresponding to the antigen (House et al., 2001). Sensitivity 0.77 and specificity 0.95 (House et al., 2001). Multi-Test Dip-S-Ticks: Approximate cost (U.S. dollars)/specimen: 10, No. of tests/kit: 50; Amount of serum needed 10 ul; Reaction time 90 minutes; Temperature for storage 2-8 degrees C (Olsen et al., 2004). iv. False Positive: PPV 0.94 (House et al., 2001). v. False Negative: NPV 0.80 (House et al., 2001). f. PanBio ELISA: i. Ontology: UMLS:C0014441 ii. Time to Perform: 1-hour-to-1-day iii. Description: The PanBio utilises a direct ELISA format. Briefly, the Salmonella typhi antigen coated microwell strips, diluted absorbed sample sera, control sera and cutoff calibrators were added and incubated at 37 degrees C for 20 minutes. Residual serum was washed, and HRP conjugates anti human IgM or anti human IgG was added and incubated for another 20 minutes at 37 degrees C. The microwells were washed and the colourless substrate system, tetra-methyl benzidine (TMB) and hydrogen peroxide, was added and incubated at room temperature for 10 minutes. The reaction was stopped and the wells were read at a wavelength of 450 mm (Gopalakrishnan et al., 2002). The PanBio ELISA kits reflected a 78% sensitivity, 80% specificity (Gopalakrishnan et al., 2002). Comparsion of the PanBio ELISA with results of combined Typhidot and Typhidot M test showed PanBio ELISA to have a lower sensitivity , PPV, NPV and efficacy but a higher specificity thus reflecting on the antigen used in the assay which seemed to be more specific for S. typhi (Gopalakrishnan et al., 2002). iv. False Positive: PPV of 68.4% (Gopalakrishnan et al., 2002). v. False Negative: NPV of 87.4% (Gopalakrishnan et al., 2002). g. Indirect Sandwich ELISA: i. Ontology: UMLS:C0014441 ii. Time to Perform: 1-hour-to-1-day iii. Description: In this study, the serum antibody responses to the LPS and flagellum antigens of serotype Typhi were investigated (House et al., 2001). In-house indirect sandwich ELISAs were established to detect anti-LPS IgA, IgM, and IgG and antiflagellum IgG (House et al., 2001). The sera from typhoid patients were 100% positive for IgG anti-protein, 94.44% positive for IgG anti-LPS and 88.89% positive for IgM anti-protein as well as anti-LPS. In contrast, only 61.11% and 83.33% of these sera gave positive Widal tests for anti-O and anti-H antibodies (Mekara et al., 1990). Immulon 1b flat-bottom 96-well microtiter plates were coated overnight at 4 C with 100 ul of either 1 ug of antigen/ml in coating buffer (0.1 M carbonate buffer (pH 9.4), antigen positive) or coating buffer alone (antigen negative). The plates were blocked for 1 h at 37 C with 200 ul of phosphate-buffered saline containing 1% bovine serum albumin (BSA). Sera were either assayed at a single dilution (1/1,000 for anti-LPS IgG, 1/500 for anti-LPS IgA, or 1/250 for anti-LPS IgM and antiflagellum IgG) or serially diluted (starting at a dilution of 1/50). Sera were diluted in phosphate-buffered saline containing 0.1% BSA and 0.05% Tween 20, 100 ul was applied to the appropriate wells, and the plates were incubated for 4 h at room temperature. Bound antibodies (IgA, IgG, or IgM) were detected using heavy-chain-specific goat antibodies directly conjugated to alkaline phosphatase. The latter were diluted (anti-IgG, 1/5,000; anti-IgA, 1/500; and anti-IgM, 1/2,500) in Tris-buffered saline containing 0.1% BSA and 0.05% Tween 20. One hundred microliters was added to each well, and the plates were incubated overnight at 4 C. One hundred microliters of p-nitrophenyl phosphate (1 mg/ml) was added to each well, and the plates were incubated at ambient temperature in the dark for 30 to 40 min. The absorbance at 405 nm (reference filter, 450 nm) was determined using an automated ELISA reader (Bio-Rad). For sera assayed at a single dilution, antibody levels were expressed in optical density (OD) units. These were taken as the mean OD of three wells with antigen minus the OD of a single well without antigen. For the titration assays, sera were assayed in triplicate (two wells antigen positive and one well antigen negative), and the titer was taken as the highest dilution giving a net OD (mean OD of antigen-positive wells minus OD of antigen-negative well) of > or equal to 0.3 (anti-LPS IgG) or > or equal 0.2 (all other antibodies). Six standards were included on each plate, and the OD or titer of the samples was adjusted accordingly. Blank wells with no sera were included to monitor background (House et al., 2001). Samples were graded as 0 to 10 according to the color of the reaction mixture at the end of the procedure. Those with a grade of >2 were considered positive (House et al., 2001). iv. False Positive: At a specificity of [greater or equal to] 0.93, the sensitivities of the different tests were 0.75, 0.55, and 0.52 for the anti-LPS IgM, IgG, and IgA ELISAs, respectively; 0.28 for the antiflagellum IgG ELISA (House et al., 2001). Among individuals with typhoid fever who had positive blood cultures ELISA tests were positive in 89-100%, whereas the Widal test is positive in only 61-83% (Mekara et al., 1990). v. False Negative: Among healthy controls, 7.5-17.5% had positive Widal tests but only 0-5% were reactive in the ELISA assays (Mekara et al., 1990). The Widal test may give false negative results in 16-39% of cases. In comparison, there were no false negative ELISA tests for IgG anti-protein and only 6% for IgG anti-LPS (Mekara et al., 1990). h. Diagnostic method for detection of Salmonella enterica serovar Typhi in fecal specimens : i. Ontology: UMLS:C0001801 ii. Time to Perform: 1-hour-to-1-day iii. Description: Laboratory diagnosis requires isolation and identification of the organism from the patient's blood or feces (Vaishnavi et al., 2006). Isolation of S. typhi from the feces, blood and other clinical specimens is the most reliable means of confirming an infection. During the incubation phase of the disease, the bacilli may be occasionally cultivated from the faeces and rarely from the blood of the so called 'precocious carrier' and subclinical cases (Vaishnavi et al., 2006). Widal test based on bacterial agglutination has remained the most widely used test even though it is neither specific nor sensitive and the result can be obtained only after the second week when antibodies are formed (Vaishnavi et al., 2006). Faecal specimens were inoculated into selenite F broth and incubated at 37 degrees C for 6 hours or overnight (Vaishnavi et al., 2006). After incubation, the broth culture was centrifuged at 1000 rpm for 10 minutes for the debris to settle down. The supernatant obtained was checked for the presence of Vi antigen with the respective test reagent. The supernatants testing positive with Vi antigen were heated for 1 hour and then checked for O9 antigen and H-d antigen. Those samples not giving a positive reaction with Vi antigen were checked without heating for O9 antigen and H-d antigens. Strong agglutination occurring within 1 minute was taken as positive for the respective test reagent. Latex beads coated with normal rabbit antiserum constituted a negative control. All samples that gave strongly positive reactions with at least two test reagents were considered to have S. typhi (Vaishnavi et al., 2006). Diagnostic tests for typhoid fever employing antibody responses may not correctly detect the present disease as antibody formation may be delayed or antibodies may be present due to vaccination or may be there due to subclinical infection (Vaishnavi et al., 2006). 3. Nucleic Acid Detection Tests: : a. Polymerase chain reaction:
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i. Ontology: UMLS:C0032520 ii. Time to Perform: 1-hour-to-1-day iii. Description: Yield of blood culture in clinically suspected cases of typhoid fever is low, whereas indirect serological diagnostic tests are unreliable. Hence, polymerase chain reaction (PCR) based detection of Salmonella enterica Serovar typhi was used as an aid for diagnosis of typhoid fever in addition to other diagnostic tests (Nizami et al., 2006). Of several indirect methods used for the detection of infective organisms, the polymerase chain reaction (PCR) has emerged as a reliable method for detection of their genome. It is thought that as few as 10 organisms could be detected using specific PCR. Thus PCR may be helpful in the early diagnosis of infectious diseases, especially typhoid fever, the morbidity and mortality of which are likely to be reduced significantly if they are treated early and promptly (Nizami et al., 2006). Most investigators have used either flagellar or ViaB gene for PCR for detection of S. enterica S. typhi (Nizami et al., 2006). This PCR is nested, the PCR targeting the nucleotide sequence encoding the Vi antigen (called the ViaB region) (Nizami et al., 2006). iv. Primers: External PCR Forward: R1 forward: 5 prime end-GTT ATT TCA GCA TAA GGA AG-3 prime end (Nizami et al., 2006) Reverse : R1 reverse: 5 prime end-ACT TGT CCG TGT TTT ACT C-3 prime end (Nizami et al., 2006) Product Name: ViaB gene sequence Size: 599 bp Product source: Salmonella enterica subsp. enterica serovar Typhi Internal PCR Forward: R2 forward: 5 prime end-GTG AAC CTA AAT CGC TAC AG-3 prime end (Nizami et al., 2006) Reverse : R2 reverse: 5 prime end-CTT CCA TAC CAC TTT CCG-3 prime end (Nizami et al., 2006) Product Name: 307 bp Product source: Salmonella enterica subsp. enterica serovar Typhi v. False Positive: Positive predictive value 45.4 % (Nizami et al., 2006). vi. False Negative: Negative predictive value 91.7% (Nizami et al., 2006). b. Multiplex PCR: i. Ontology: UMLS:C0032520 ii. Time to Perform: 1-hour-to-1-day iii. Description: The PCR primers for O, H, and Vi antigen genes, tyv (rfbE), prt (rfbS), fliC-d, fliC-a, and viaB, were designed and used for the rapid identification of Salmonella enterica serovars Typhi and Paratyphi A with multiplex PCR (Hirose et al., 2002). The classic diagnosis method for typhoid fever or paratyphoid fever requires at least 4 or 5 days for positive results. A rapid, alternative method is needed for the diagnosis of typhoid fever or paratyphoid fever (Hirose et al., 2002). In this study, we developed a more specific diagnosis method for both typhoid fever and paratyphoid fever based on a multiplex PCR technique that detected the Vi antigen gene (viaB), H antigen genes (fliC-d and fliC-a), and O antigen synthesis genes (tyv and prt). This system enabled us to identify and differentiate serovars Typhi and Paratyphi A, which are clinically important human pathogens, by only a single PCR, when we isolated the bacteria from blood or stool cultures from clinical patients (Hirose et al., 2002). We designed the primers tyv-s and tyv-as for detection of the tyvelose epimerase gene (tyv, previously called rfbE) and the primers fliCcom-s and fliCd-as for detection of the fliC-d gene (phase-1 flagellin gene for d antigen [H:d]) of Salmonella serovar Typhi. The primers parat-s and parat-as were designed for detection of a paratose synthase gene (prt, previously called rfbS), and the primers fliCcom-s and fliCa-as were designed for detection of a fliC-a gene (phase-1 flagellin; H:a) (Hirose et al., 2002). The gene prt encodes CDP-paratose synthase (Hirose et al., 2002). The multiplex PCR using five sets of primer pairs, which were targeted for the viaB, prt, tyv, fliC-d, and fliC-a genes, correctly identified Salmonella serovars Typhi and Paratyphi A and differentiated the two serovars by the combinations of the different-size bands produced: four positive bands, which consist of viaB, prt, tyv and fliC-d PCR products, in serovar Typhi and two positive bands, which consist of prt and fliC-a PCR products, in serovar Paratyphi A (Hirose et al., 2002). iv. Primers: tyv (rfbE) Forward: tyv-s (5'-GAG GAA GGG AAA TGA AGC TTT T-3') (Hirose et al., 2002) Reverse : tyv-as (5'-TAG CAA ACT GTC TCC CAC CAT AC-3') (Hirose et al., 2002) Product Name: tvy (rfbE) Size: 615 bp Product source: Salmonella enterica subsp. enterica serovar Typhi Product GenBank Accession Number: M29682 prt (rfbS) Forward: parat-s (5'-CTT GCT ATG GAA GAC ATA ACG AAC C-3') (Hirose et al., 2002) Reverse : parat-as, (5'-CGT CTC CAT CAA AAG CTC CAT AGA-3') (Hirose et al., 2002) Product Name: prt Size: 258 bp Product source: Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 Product GenBank Accession Number: M29682 viaB Forward: vi-s (5'-GTT ATT TCA GCA TAA GGA G-3') (Hirose et al., 2002) Reverse : vi-as (5'-CTT CCA TAC CAC TTT CCG-3') (Hirose et al., 2002) Product Name: viaB Size: 439 bp Product source: Salmonella enterica subsp. enterica serovar Typhi Product GenBank Accession Number: D14156 fliC Forward: fliCcom-s (5'-AAT CAA CAA CAA CCT GCA GCG-3') (Hirose et al., 2002) Reverse : fliCd-as (5'-GCA TAG CCA CCA TCA ATA ACC-3') (Hirose et al., 2002) fliCa-as (5'-TAG TGC TTA ATG TAG CCG AAG G-3') (Hirose et al., 2002) Product Name: fliCcom-fliCd-as
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Size: 750 bp Product source: Salmonella enterica subsp. enterica serovar Typhi Product GenBank Accession Number: L21912 c. Real-time PCR: i. Ontology: UMLS:C1709846 ii. Time to Perform: 1-hour-to-1-day iii. Description: A basic PCR technique combined with a DNA probe specific to the Vi antigen of S. Typhi could be used to reinforce the clinical diagnosis of typhoid fever with a negative blood culture. However, the detection of S. Typhi using these techniques is not sensitive enough to detect fewer than 500 bacteria in a 1-ml blood sample (Massi et al., 2005). In the present study, we report the development of a TaqMan-based real-time PCRassay (TaqMan assay) for quantifying S. Typhi directly from blood specimens of patients suspected of having typhoid fever, especially in cases with a negative blood culture test result (Massi et al., 2005). We quantified the gene copies from Salmonella enterica serovar Typhi (S. Typhi) in the blood of patients suspected of having typhoid fever by using TaqMan-based real-time PCR (TaqMan assay) to target the S. Typhi flagellin gene in genomic DNAs isolated from blood samples. Of 55 blood samples taken from suspected typhoid fever patients, eight blood samples with a positive blood culture had S. Typhi loads ranging from 1.01 x 10(3) to 4.35 x 10(4) copies/ml blood, and from 47 blood samples with negative blood culture, there were 40 (85.1%) TaqMan assay-positive samples with loads ranging from 3.9 to 9.9 x 10(2) copies/ml blood. In the present study, the TaqMan assay detected more than 10(3) copies/ml blood of S. Typhi in all of the blood culturepositive samples, whereas less than 103 copies/ml blood of S. Typhi were detected in the blood culture-negative samples (Massi et al., 2005). There was a significant difference in the S. Typhi flagellin gene loads between the samples with a positive blood culture and those with a negative blood culture (P <0.005) (Massi et al., 2005). TaqMan assay may be useful for assessing S. Typhi loads, especially in cases of suspected typhoid fever with negative results from the standard blood culture test (Massi et al., 2005). iv. Primers: ST5 and ST6 Forward: forward primer (ST5) was 5'-CAACCTGGGCAATACCGTAAATAA-3' (nucleotides 1347 - 1370 in the sequence with GenBank accession number L21912) (Massi et al., 2005). Reverse : The reverse primer (ST6) was 5'-ATTCCGACTACGCAACCGAA-3' (nucleotides 1400 - 1419) (Massi et al., 2005). Real-time-probe : Probe for the H1-d flagellin gene. The sequence of the probe (ST7) was 50-FAM-TGTCTTCTGCCCGTAGCCGTATCG-TAMRA (nucleotides 1373 -1396) (Massi et al., 2005). Product Name: Flagellin Size: 506 amino acids Product source: Salmonella enterica subsp. enterica serovar Typhi Product GenBank Accession Number: CAA34436 v. False Positive: We found that a blood culture test will be positive only when the bacterial S. Typhi loads are >10(3) copies/ml blood in patients suspected of having typhoid fever. Our results showed that 85.1% of negative blood cultures were possibly false-negatives, as demonstrated by our TaqMan assay method (Massi et al., 2005). 4. Other Types of Diagnostic Tests: a. Pulsed-field: i. Ontology: UMLS:C0918200 ii. Time to Perform: 1-to-2-days iii. Description: PFGE, in particular, has been widely used recently in molecular epidemiological investigations of infections caused by a large number of bacterial pathogens, including S. typhi. In a previous study, we used PFGE to analyze S. typhi isolates from sporadic cases and from outbreaks in Malaysia. It has been proposed recently that PFGE is able to differentiate between clonally related strains (one or two band differences) and strains which represent independent clones (differences in three or more bands. Preparation of DNA for restriction endonuclease digestion and subsequent analysis by PFGE were done as described previously . S. typhi chromosomal DNA was digested with restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'CCTAGG-3'), and DNA fragments were separated by a contour-clamped homogeneous electric field gel electrophoresis method on a CHEF DR-II or CHEF DR-III system. PFGE patterns were visually assessed, assigned arbitrary pattern types, and compared by calculating a similarity coefficient (F, proportion of shared fragments between two isolates). With this method, an F value of 1.0 indicates identical patterns and an F value of 0 suggests complete dissimilarity. In relation to the significance of differences in PFGE profiles between individual strains, it has been proposed that strains with one or two band shifts caused by a single genetic event (e.g., a point mutation resulting in loss or gain of a restriction site, insertion, deletion, or chromosomal inversion) are considered to be clonally related. If a very limited number of band changes occur which are not due to a single genetic event, the strains are considered to be very closely related but distinct. Strains that differ in three or more bands are considered to be independent (Thong et al., 1995). b. Urine antigen detection: i. Ontology: UMLS:C0430420 ii. Time to Perform: 1-hour-to-1-day iii. Description: We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies to capture somatic antigen 9 (O9), flagellar antigen d (Hd), and the Vi capsular polysaccharide antigen (Vi) from the urine of persons with and without typhoid fever. Sequential urine samples were collected from 44 patients with blood culture-confirmed typhoid fever and from two control groups (Fadeel et al., 2004). Urine samples were collected from patients admitted to Abbasia Hospital (Cairo, Egypt) from June 2000 through September 2001 who had positive blood cultures for Salmonella Typhi (Fadeel et al., 2004). Urine samples were stored within three hours of collection at -20 C (Fadeel et al., 2004). Sandwich ELISAs were developed for the detection of Salmonella serotype Typhi O9, Hd, and Vi antigens. Purified bacterial antigens including lipopolysaccharide of somatic (O9) antigen, Salmonella serotype Eschberg flagellar (Hd) antigen, and Vi capsular antigen (CDC) were used to develop and characterize the different ELISAs for antigen detection (Fadeel et al., 2004). The release of S. Typhi antigens into urine was found to be intermittent and small antigens could have been destroyed by freezing and thawing. The intermittent nature of S. Typhi antigen release explains the sharp increase of assay sensitivity from 73% to 97% (for the Vi antigen) when more than one sample from the same patient were tested. A similar trend was seen with the O9 and Hd antigens (Fadeel et al., 2004). When assessed relative to date of fever onset, sensitivity was highest during the first week for all three antigens: Vi was detected in the urine of nine (100%) patients, O9 in 4 (44%) patients, and Hd in 4 (44%) patients. Sequential testing of two urine samples from the same patient improved test sensitivity. Combined testing for Vi with O9 and Hd produced a trend towards increased sensitivity without compromising specificity. The specificity for Vi exceeded 90% when assessed among both febrile and healthy control subjects (Fadeel et al., 2004). The sensitivity of the Vi antigen assay was higher than that of the O9 or Hd assays (Fadeel et al., 2004). Sensitivity 65-95% (Bhutta, 2006). c. Surveillance study: i. Time to Perform: 1-to-2-days ii. Description: During the surveillance period, persons with fever who lived in each study area were requested to visit participating healthcare providers. We collected 5-10 mL blood from adults with fever >3 days' duration into Bactec bottles. We collected 3-8 mL from children with fever >3 days' duration into
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Pediatric Bactec bottles. The bottles were incubated at 37 C for 7-10 days and visually checked for growth every day. Bottles were subcultured on MacConkey agar on days 1, 2, 4, and 7 or when turbidity was detected. Suspected colonies were screened by using Kligler iron agar, sulfide-indole-motility medium, urea agar, and citrate. Colonies that showed biochemical reactions suggestive of salmonellae were confirmed serologically by Felix-Widal tube agglutination test with specific O and H antisera (Ochiai et al., 2005).

V. References
A. Journal References:
Adachi et al., 2005: Adachi T, Sagara H, Hirose K, Watanabe H. Fluoroquinolone-resistant Salmonella Paratyphi A. Emerg Infect Dis. 2005; 11(1): 172 174. [PubMed: 15714664]. Aye et al., 2004: Aye TT, Siriarayapon P. Typhoid fever outbreak in Madaya Township, Mandalay Division, Myanmar, September 2000. J Med Assoc Thai. 2004; 87(4): 395 - 399. [PubMed: 15217177]. Bal et al., 2004: Bal SK, Czarnowski C. A man with fever, cough, diarrhea and a coated tongue. CMAJ. 2004; 170(7): 1095 - 1095. [PubMed: 15051692 ]. Bhan et al., 2005: Bhan MK, Bahl R, Bhatnagar S. Typhoid and paratyphoid fever. Lancet . 2005; 366(9487): 749 - 762. [PubMed: 16125594 ]. Bhutta, 2006: Bhutta ZA. Current concepts in the diagnosis and treatment of typhoid fever. BMJ. 2006; 333(7558): 78 - 82. [PubMed: 16825230]. Busse, 1995: Busse M., Media for Salmonella. Int J Food Microbiol. 1995; 26(1): 117 - 131. [PubMed: 7662516]. Ceylan et al., 2003: Ceylan A, Acemoglu H, Hosoglu S, Gul K, Ilcin E, Efe M. Typhoid fever epidemic in Ahmetli village, Diyarbakir-Ergani. Mikrobiyol Bul. 2003; 37(1): 41 - 7. [PubMed: 12838677]. Chandel et al., 2000: Chandel DS, Chaudhry R, Dhawan B, Pandey A, Dey AB. Drug-resistant Salmonella enterica serotype paratyphi A in India. Emerg Infect Dis. 2000; 6(4): 420 - 421. [PubMed: 10905982]. Choo et al., 1999: Choo KE, Davis TM, Ismail A, Tuan Ibrahim TA, Ghazali WN. Rapid and reliable serological diagnosis of enteric fever: comparative sensitivity and specificity of Typhidot and Typhidot-M tests in febrile Malaysian children. Acta Trop. 1999; 72(2): 175 - 183. [PubMed: 10206117]. Chow et al., 1987: Chow CB, Wang PS, Cheung MW, Yan WW, Leung NK. Diagnostic value of the Widal test in childhood typhoid fever. Pediatr Infect Dis J. 1987; 6(10): 914 - 917. [PubMed: 3696823]. Chowta et al., 2005: Chowta MN, Chowta NK. Study of clinical profile and antibiotic response in typhoid fever. Indian J Med Microbiol. 2005; 23(2): 125 - 127. [PubMed: 15928444]. Cirillo, 2006: Cirillo VJ. Winged sponges: houseflies as carriers of typhoid fever in 19th- and early 20th-century military camps. Perspect Biol Med. 2006; 49(1): 52 - 63. [PubMed: 16489276]. Connor et al., 2005: Connor BA, Schwartz E. Typhoid and paratyphoid fever in travellers. The Lancet Infectious diseases. 2005; 5(10): 623 - 628. [PubMed: 16183516]. Cooke et al., 2006: Cooke GS, Cooke FJ, Stone M, Turner K, Al-Nahhas A, Win Z, Wain J, Rogers TR, Friedland JS, Bamford KB. Deep-seated resistance in relapsed paratyphoid fever. Clin Infect Dis. 2006; 42(11): e92 - e94. [PubMed: 16652303]. Crum, 2003: Crum NF., Current trends in typhoid Fever. Current gastroenterology reports. 2003; 5(4): 279 - 286. [PubMed: 12864957]. Cunha, 2006: Cunha BA., Typhoid fever: the temporal relations of key clinical diagnostic points. Lancet Infect Dis. 2006; 6(6): 318 - 320. [PubMed: 16728315]. Daniels et al., 1989: Daniels EM, Schneerson R, Egan WM, Szu SC, Robbins JB. Characterization of the Salmonella paratyphi C Vi polysaccharide. Infect Immun. 1989; 10: 3159 - 3164. [PubMed: 2506132]. Deng et al., 2003: Deng W, Liou SR, Plunkett G 3rd, Mayhew GF, Rose DJ, Burland V, Kodoyianni V, Schwartz DC, Blattner FR. Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18. J Bacteriol. 2003; 185(7): 2330 - 2337. [PubMed: 12644504]. Desenclos et al., 1996: Desenclos JC, Bouvet P, Benz-Lemoine E, Grimont F, Desqueyroux H, Rebiere I, Grimont PA. Large outbreak of Salmonella enterica serotype paratyphi B infection caused by a goats' milk cheese, France, 1993: a case finding and epidemiological study. BMJ. 1996; 312(7023): 91 94. [PubMed: 8555937]. Didelot et al., 2007: Didelot X, Achtman M, Parkhill J, Thomson NR, Falush D. A bimodal pattern of relatedness between the Salmonella Paratyphi A and Typhi genomes: Convergence or divergence by homologous recombination. Genome Res. 2007; 17(1): 61 - 68. [PubMed: 17090663]. Dutta et al., 2001: Dutta TK, Beeresha, Ghotekar LH. Atypical manifestations of typhoid fever. J Postgrad Med. 2001; 47(4): 248 - 251. [PubMed: 11832640]. Edwards, 1999: Edwards BH. Salmonella and Shigella species. Clin Lab Med. 1999; 19(3): 469 - 487. [PubMed: 10549421]. Ellis et al., 2006: Ellis RD, Fukuda MM, McDaniel P, Welch K, Nisalak A, Murray CK, Gray MR, Uthaimongkol N, Buathong N, Sriwichai S, Phasuk R, Yingyuen K, Mathavarat C, Miller RS. Causes of fever in adults on the Thai-Myanmar border. American Journal of Tropical medicine and hygiene. 2006; 74(1): 108 - 113. [PubMed: 16407353]. Escamilla et al., 1986: Escamilla J, Florez-Ugarte H, Kilpatrick ME. Evaluation of blood clot cultures for isolation of Salmonella typhi, Salmonella
ci.vbi.vt.edu/pathinfo/pathogens/enterica.html 21/25

12/11/13

Typhoid Fever

paratyphi-A, and Brucella melitensis. J Clin Microbiol. 1986; 24(3): 388 - 3990. [PubMed: 3093527]. Ezaki et al., 2000: Ezaki T, Amano M, Kawamura Y, Yabuuchi E. Proposal of Salmonella paratyphi sp. nov., nom. rev. and request for an opinion to conserve the specific epithet paratyphi in the binary combination Salmonella paratyphi as nomen epitheton conservandum. Int J Syst Evol Microbiol. 2000; 50(2): 941 - 944. [PubMed: 10758909]. Fadeel et al., 2004: Fadeel MA, Crump JA, Mahoney FJ, Nakhla IA, Mansour AM, Reyad B, El Melegi D, Sultan Y, Mintz ED, Bibb WF. Rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of Salmonella serotype typhi antigens in urine. Am J Trop Med Hyg. 2004; 70(3): 323 328. [PubMed: 15031525 ]. Gopalakrishnan et al., 2002: Gopalakrishnan V, Sekhar WY, Soo EH, Vinsent RA, Devi S. Typhoid fever in Kuala Lumpur and a comparative evaluation of two commercial diagnostic kits for the detection of antibodies to Salmonella typhi. Singapore Med J. 2002; 43(7): 354 - 358. [PubMed: 12437043]. Grizhebovskii et al., 2001: Grizhebovskii GM, Onishchenko GG, Taran VI, Ivanov SI, Evchenko IuM, Galimshin SS, Sychugov EV, Eremenko EI, Bakaev UN, Mezentsev VM, Briukhanov AF, Protsenko SL. Outbreak of typhoid fever in the Chechen Republic in 2000: epidemiological characterization. Zh Mikrobiol Epidemiol Immunobiol. 2001; Nov-Dec(6 Suppl): 45 - 47. [PubMed: 12718173]. Gupta et al., 2003: Gupta A, Fontana J, Crowe C, Bolstorff B, Stout A, Van Duyne S, Hoekstra MP, Whichard JM, Barrett TJ, Angulo FJ. Emergence of multidrug-resistant Salmonella enterica serotype Newport infections resistant to expanded-spectrum cephalosporins in the United States. The Journal of Infectious Diseases. 2003; 188: 1707 - 1716. [PubMed: 14639542]. Guzman et al., 2006: Guzman CA, Borsutzky S, Griot-Wenk M, Metcalfe IC, Pearman J, Collioud A, Favre D, Dietrich G. Vaccines against typhoid fever. Vaccine. 2006; 24(18): 3804 - 3811. [PubMed: 16278037]. Hirose et al., 2002: Hirose K, Itoh K, Nakajima H, Kurazono T, Yamaguchi M, Moriya K, Ezaki T, Kawamura Y, Tamura K, Watanabe H. Selective amplification of tyv (rfbE), prt (rfbS), viaB, and fliC genes by multiplex PCR for identification of Salmonella enterica serovars Typhi and Paratyphi A. Journal of clinical microbiology. 2002; 40(2): 633 - 636. [PubMed: 11825983]. Hoffner et al., 2000: Hoffner RJ, Slaven E, Perez J, Magana RN, Henderson SO Emergency department presentations of typhoid fever. The Journal of emergency medicine. 2000; 19(4): 317 - 321. [PubMed: 11074322]. House et al., 2001: House D, Bishop A, Parry C, Dougan G, Wain J. Typhoid fever: pathogenesis and disease. Curr Opin Infect Dis. 2001; 14(5): 573 578. [PubMed: 11964878]. House et al., 2001: House D, Wain J, Ho VA, Diep TS, Chinh NT, Bay PV, Vinh H, Duc M, Parry CM, Dougan G, White NJ, Hien TT, Farrar JJ. Serology of typhoid fever in an area of endemicity and its relevance to diagnosis. J Clin Microbiol. 2001; 39(3): 1002 - 1007. [PubMed: 11230418]. Ismail, 2006: Ismail TF. Rapid diagnosis of typhoid fever. Indian J Med Res. 2006; 123(4): 489 - 492. [PubMed: 16783038]. Kothapalli et al., 2005: Kothapalli S, Nair S, Alokam S, Pang T, Khakhria R, Woodward D, Johnson W, Stocker BA, Sanderson KE, Liu SL. Diversity of genome structure in Salmonella enterica serovar Typhi populations. J Bacteriol. 2005; 187(8): 2638 - 2650. [PubMed: 15805510]. Lewis et al., 2005: Lewis MD, Serichantalergs O, Pitarangsi C, Chuanak N, Mason CJ, Regmi LR, Pandey P, Laskar R, Shrestha CD, Malla S. Typhoid fever: a massive, single-point source, multidrug-resistant outbreak in Nepal. Clin Infect Dis. 2005; 40(4): 554 - 561. [PubMed: 15712078 ]. Lim et al., 1998: Lim PL, Tam FC, Cheong YM, Jegathesan M. One-step 2-minute test to detect typhoid-specific antibodies based on particle separation in tubes. J Clin Microbiol. 1998; 36(8): 2271 - 2278. [PubMed: 9666004]. Malik, 2002: Malik AS. Complications of bacteriologically confirmed typhoid fever in children. Journal of Tropical Pediatrics. 2002; 48(2): 102 - 108. [PubMed: 12022423]. Massi et al., 2005: Massi MN, Shirakawa T, Gotoh A, Bishnu A, Hatta M, Kawabata M. Quantitative detection of Salmonella enterica serovar Typhi from blood of suspected typhoid fever patients by real-time PCR. International journal of medical microbiology. 2005; 295(2): 117 - 120. [PubMed: 15969472]. Mateen et al., 2006: Mateen MA, Saleem S, Rao PC, Reddy PS, Reddy DN. Ultrasound in the diagnosis of typhoid fever. Indian J Pediatr. 2006; 73(8): 681 - 685. [PubMed: 16936362]. McClelland et al., 2004: McClelland M, Sanderson KE, Clifton SW, Latreille P, Porwollik S, Sabo A, Meyer R, Bieri T, Ozersky P, McLellan M, Harkins CR, Wang C, Nguyen C, Berghoff A, Elliott G, Kohlberg S, Strong C, Du F, Carter J, Kremizki C, Layman D, Leonard S, Sun H, Fulton L, Nash W, Miner T, Minx P, Delehaunty K, Fronick C, Magrini V, Nhan M, Warren W, Florea L, Spieth J, Wilson RK. Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid. Nature genetics. 2004; 36(12): 1268 - 1274. [PubMed: 15531882]. Mekara et al., 1990: Mekara Y, Maneekarn N, Vithayasai V, Makonkawkeyoon S. Determination of antibody from typhoid patients against lipopolysaccharide and protein antigens of Salmonella typhi. Asian Pacific Journal of Allergy and Immunology. 1990; 8(2): 95 - 101. [PubMed: 2091664 ]. Meltzer et al., 2006: Meltzer E, Yossepowitch O, Sadik C, Dan M, Schwartz E. Epidemiology and clinical aspects of enteric fever in Israel. Am J Trop Med Hyg. 2006; 74(4): 540 - 545. [PubMed: 16606981]. Michel et al., 2005: Michel R, Garnotel E, Spiegel A, Morillon M, Saliou P, Boutin JP. Outbreak of typhoid fever in vaccinated members of the French Armed Forces in the Ivory Coast. Eur J Epidemiol. 2005; 20(7): 635 - 642. [PubMed: 16119438]. Nguyen et al., 1993: Nguyen TA, Ha Ba K, Nguyen TD. Typhoid fever in South Vietnam, 1990-1993. Bull Soc Pathol Exot . 1993; 86(5 pt 2): 476 - 478. [PubMed: 7819805]. Ngwu et al., 2003: Ngwu BA, Agbo JA. Typhoid fever: clinical diagnosis versus laboratory confirmation. Niger J Med. 2003; 12(4): 187 - 192. [PubMed:
ci.vbi.vt.edu/pathinfo/pathogens/enterica.html 22/25

12/11/13

Typhoid Fever

14768191]. Nizami et al., 2006: Nizami SQ, Bhutta ZA, Siddiqui AA, Lubbad L. Enhanced detection rate of typhoid fever in children in a periurban slum in Karachi, Pakistan using polymerase chain reaction technology. Scand J Clin Lab Invest . 2006; 66(5): 429 - 436. [PubMed: 16901852]. Ochiai et al., 2005: Ochiai RL, Wang X, von Seidlein L, Yang J, Bhutta ZA, Bhattacharya SK, Agtini M, Deen JL, Wain J, Kim DR, Ali M, Acosta CJ, Jodar L, Clemens JD. Salmonella paratyphi A rates, Asia. Emerging infectious diseases. 2005; 11(11): 1764 - 1766. [PubMed: 16318734]. Olopoenia et al., 2000: Olopoenia LA, King AL. Widal agglutination test - 100 years later: still plagued by controversy. Post graduate medical journal. 2004; 76(): 80 - 84. [PubMed: 10644383]. Olsen et al., 2004: Olsen SJ, Pruckler J, Bibb W, Nguyen TM, Tran MT, Nguyen TM, Sivapalasingam S, Gupta A, Phan TP, Nguyen TC, Nguyen VC, Phung DC, Mintz ED. Evaluation of rapid diagnostic tests for typhoid fever . J Clin Microbiol. 2004; 42(5): 1885 - 1889. [PubMed: 15131144]. Pang et al., 1995: Pang T, Bhutta ZA, Finlay BB, Altwegg M. Typhoid fever and other salmonellosis: a continuing challenge. Trends Microbiol. 1995; 2(7): 253 - 255. [PubMed: 7551636 ]. Pang et al., 1998: Pang T, Levine MM, Ivanoff B, Wain J, Finlay BB. Typhoid fever--important issues still remain. Trends Microbiol. 1998; 6(4): 131 - 133. [PubMed: 9587187]. Papadimitropoulos et al., 2004: Papadimitropoulos V, Vergidis PI, Bliziotis I, Falagas ME. Vaccination against typhoid fever in travellers: a costeffectiveness approach. Clin Microbiol Infect . 2004; 10(8): 681 - 683. [PubMed: 15301670]. Parkhill, 2002: Parkhill J. The importance of complete genome sequences. Trends Microbiol. 2002; 10(5): 219 - 220. [PubMed: 11973153]. Parkhill et al., 2001: Parkhill J, Dougan G, James KD, Thomson NR, Pickard D, Wain J, Churcher C, Mungall KL, Bentley SD,Holden MT, Sebaihia M, Baker S, Basham D, Brooks K, Chillingworth T, Connerton P, Cronin A, Davis P, Davies RM, Dowd L, White N, Farrar J, Feltwell T, Hamlin N, Haque A, Hien TT, Holroyd S, Jagels K, Krogh A, Larsen TS, Leather S, Moule S, O'Gaora P, Parry C, Quail M, Rutherford K, Simmonds M, Skelton J, Stevens K, Whitehead S, Barrell BG. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature. 2001; 413(6858): 848 852. [PubMed: 11677608 ]. Parry et al., 2006: Parry CM, Karunanayake L, Coulter JB, Beeching NJ. Test for quinolone resistance in typhoid fever. BMJ. 2006; 333(7561): 260 - 261. [PubMed: 16873871]. Porwollik et al., 2002: Porwollik S, Boyd EF, Choy C, Cheng P, Florea L, Proctor E, McClelland M. Characterization of Salmonella enterica subspecies I genovars by use of microarrays. J Bacteriol. 2004; 186(17): 5883 - 5898. [PubMed: 15317794]. Rathish et al., 1995: Rathish KC, Chandrashekar MR, Nagesha CN. An outbreak of multidrug resistant typhoid fever in Bangalore. Indian J Pediatr. 1995; 62(4): 445 - 448. [PubMed: 10829903]. Reller et al., 2003: Reller ME, Olsen SJ, Kressel AB, Moon TD, Kubota KA, Adcock MP, Nowicki SF, Mintz ED. Sexual transmission of typhoid fever: a multistate outbreak among men who have sex with men. Clin Infect Dis. 2003; 37(1): 141 - 144. [PubMed: 12830419]. Rupali et al., 2004: Rupali P, Abraham OC, Jesudason MV, John TJ, Zachariah A, Sivaram S, Mathai D. Treatment failure in typhoid fever with ciprofloxacin susceptible Salmonella enterica serotype Typhi. Diagn Microbiol Infect Dis. 2004; 49(1): 1 - 3. [PubMed: 15135492]. Santos et al., 2001: Santos RL, Zhang S, Tsolis RM, Kingsley RA, Adams LG, Baumler AJ. Animal models of Salmonella infections: enteritis versus typhoid fever. Microbes Infect . 2001; 3(14-15): 1335 - 1344. [PubMed: 11755423]. Slinger et al., 2004: Slinger R, Desjardins M, McCarthy AE, Ramotar K, Jessamine P, Guibord C, Toye B. Suboptimal clinical response to ciprofloxacin in patients with enteric fever due to Salmonella spp. with reduced fluoroquinolone susceptibility: a case series. BMC Infect Dis. 2004; 4(Sept 20): 36 - 36. [PubMed: 15380025]. Snow et al., 2006: Snow M. Protecting travelers from typhoid fever. Nursing. 2006; 36(3): 73 - 73. [PubMed: 16523113 ]. Snyder et al., 1996: Ollinger-Snyder P, Matthews ME. Food safety: review and implications for dietitians and dietetic technicians. J Am Diet Assoc. 1996; 96(2): 169 - 170. [PubMed: 8557943]. Thomsen et al., 1998: Thomsen LL, Paerregaard A. Treatment with ciprofloxacin in children with typhoid fever. Scand J Infect Dis. 1998; 30(4): 355 - 357. [PubMed: 9817514]. Thong et al., 1995: Thong KL, Puthucheary S, Yassin RM, Sudarmono P, Padmidewi M, Soewandojo E, Handojo I, Sarasombath S, Pang T. Analysis of Salmonella typhi isolates from Southeast Asia by pulsed-field gel electrophoresis. J Clin Microbiol. 1995; 33(7): 1938 - 1941. [PubMed: 7665677 ]. Tsolis et al., 1999: Tsolis RM, Kingsley RA, Townsend SM, Ficht TA, Adams LG, Baumler AJ. Of mice, calves, and men. Comparison of the mouse typhoid model with other Salmonella infections. Advances in experimental medicine and biology. 1999; 473(): 261 - 274. [PubMed: 10659367]. Vaishnavi et al., 2006: Vaishnavi C, Kaur S, Singh K. Evaluation of an in-house rapid diagnostic method for detection of Salmonella enterica serovar Typhi in fecal specimens. Trop Gastroenterol. 2006; 27(1): 19 - 21. [PubMed: 16910055]. Wain et al., 1998: Wain J, Diep TS, Ho VA, Walsh AM, Nguyen TT, Parry CM, White NJ. Quantitation of bacteria in blood of typhoid fever patients and relationship between counts and clinical features, transmissibility, and antibiotic resistance. J Clin Microbiol. 1998; 36(6): 1683 - 1687. [PubMed: 9620400]. Walia et al., 2005: Walia M, Gaind R, Mehta R, Paul P, Aggarwal P, Kalaivani M. Current perspectives of enteric fever: a hospital-based study from India. Ann Trop Paediatr. 2005; 25(3): 161 - 174. [PubMed: 16156980]. Watson, 1978: Watson KC. Laboratory and clinical investigation of recovery of Salmonella typhi from blood. J Clin Microbiol. 1978; 7(2): 122 - 126. [PubMed: 632343].
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Xavier, 2006: Xavier G. Management of typhoid and paratyphoid fevers. Nursing times. 2006; 102(17): 49 - 51. [PubMed: 16700236]. Yoon et al., 2004: Yoon J, Segal-Maurer S, Rahal JJ An outbreak of domestically acquired typhoid fever in Queens, NY. Arch Intern Med. 2004; 164(5): 565 - 567. [PubMed: 15006835].

B. Book References:
Gutherie et al., 1991: Guthrie Rufus. Salmonella- The infection. 41 - 61. In: Guthrie Rufus. Salmonella.1991. CRC Press, Inc., Boca Raton, Florida. Huckstep et al., 2002: Huckstep RL. Bacteriology: The Salmonellae. 25 - 34. In: Wright FJ. Typhoid Fever and other Salmonella infections.1962. S. Livingston LTD, Edinburgh and London. Kelterborn et al., 1967: Kelterborn Eckehart. First Isolations, Names and Occurrence of the Salmonella Species Listed in the Kauffman-White Schema.. 35 - 412. In: Kauffmann F. Salmonella species1967. Offizin Andersen Nexo, Leipzig, Germany. MICROMEDEX 2001: MICROMEDEX. 1 - 3068. In: Drug Information for the Health Care Professional.2001. MICROMEDEX, Englewood, Colorado, USA. Parry, 2006: Parry CM. Epidemiological and clinical aspects of human typhoid fever. 1 - 24. In: Mastroeni P., Maskell D. Salmonella Infections: Clinical, Immunological and Molecular Aspects.2006. Cambridge University Press, New York, New York, USA. Williams et al., 1989: Williams Peter. Experiments in Manchuria. 13 - 30. In: UNIT 731- Japan's Secret Biological Warfare in World War II.1989. The Free Press, 866 Third Avenue, New York, N.Y. 10022. Williams et al., 1989: Williams Peter. Waging Germ Warfare. 63 - 80. In: UNIT 731- Japan's Secret Biological Warfare in World War II.1989. The Free Press, 866 Third Avenue, New York, N.Y. 10022.

C. Website References:
Genome: Circular representation of the S. typhi genome [ http://www.nature.com/nature/journal/v413/n6858/images/413848aa.2.jpg6 ]. image: Typhoid fever: Rose spots on abdomen of a patient with typhoid fever due to the bacterium Salmonella typhi. [ http://www.wrongdiagnosis.com/phil/images/2214.jpg ]. Huckstep: The spread of typhoid [ http://www.worldortho.com/huckstep/typhoid/chapt3-5.html ]. Huckstep: Culture of the Typhoid Bacillus [ http://www.worldortho.com/huckstep/typhoid/chapt11amp;12.html ]. CDC: Salmonella typhi: Laboratory hazards and recommended precautions [ http://www.cdc.gov/od/ohs/biosfty/bmbl/sect7c.htm#Salmot ]. Pathology: Salmonellosis, ulcer of ileum in typhoid fever. [ http://images.google.com/imgres?imgurl=http://erl.pathology.iupui.edu/C603/IMAGES/949 3.JPG&imgrefurl=http://erl.pathology.iupui.edu/C603/GENE643.HTM&h=391&w=585&sz=63&hl=en&start=27&tbnid=w9z5h4ZyNwGIKM:&tbnh=90&tbnw=135&prev =/images%3Fq%3Dtyphoid%2Bfever%26start%3D18%26ndsp%3D18%26svnum%3D10%26hl%3Den%26lr%3D%26s a%3DN ]. NCBI_Protein: Salmonella typhi: flagellin [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=397811 ]. CDC: Bacterial Agents: Agent Summary Statements Section VII-A: Bacterial Agents [ http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7a.htm#N_88_ ]. Dennis Kunkel Microscopy, Inc.: Salmonella typhi [ http://www.denniskunkel.com/ ]. NCBI Taxonomy: Salmonella enterica subsp. enterica serovar Typhi Ty2 project at Univ. Wisconsin [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? db=genomeprj&cmd=Retrieve&dopt= Overview&list_uids=371 ]. NCBI Taxonomy: Salmonella enterica subsp. enterica serovar Typhi [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=90370 ]. NCBI Taxonomy: Salmonella enterica [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=28901 ]. NCBI Taxonomy: Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=295319 ]. NCBI Taxonomy: Salmonella enterica subsp. enterica serovar Paratyphi B [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi? lvl=0&id=57045 ]. NCBI Taxonomy: Salmonella enterica subsp. enterica serovar Paratyphi C [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi? lvl=0&id=57046 ]. NCBI Taxonomy: Homo sapiens [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606&lvl =3&lin=f&keep=1&srchmode=1&unlock ]. NCBI Taxonomy: Salmonella enterica subsp. enterica serovar Typhi Ty2 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi? mode=Info&id=209261&l vl=3&lin=f&keep=1&srchmode=1&unlock ]. NCBI Taxonomy: Salmonella enterica subsp. enterica serovar Typhi str. CT18 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi? mode=Info&id=220341&l vl=3&lin=f&keep=1&srchmode=1&unlock ]. NCBI Taxonomy: Salmonella paratyphi [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=54388&lv l=3&lin=f&keep=1&srchmode=1&unlock ].

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NCBI Taxonomy: Salmonella paratyphi [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=272994&l vl=3&lin=f&keep=1&srchmode=1&unlock ]. NCBI Taxonomy: Salmonella enterica subsp. enterica serovar Paratyphi B str. SPB7 project at Washington University [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprj&cmd=Retrieve&dopt= Overview&list_uids=209 ]. Public Health Services: History of Biowarfare and Bioterrorism [ http://www.azdhs.gov/phs/edc/edrp/es/bthistor2.htm ].

D. Thesis References:
No thesis or dissertation references used.

VI. Curation Information

Curators: Abramochkin George (pathinfo@vbi.vt.edu) Date: 05.15.2004 Version: 1.0 Revision: Curators: Shallom Shamira (pathinfo@vbi.vt.edu); Date: 7 Sept 2006 Version: 0.83 Contact information: Email: pathinfo@vbi.vt.edu

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