Bagian Patologi Klinik FK UNHAS

Pengamatan Pada : - Eritrosit, Lekosit, Trombosit - Manifestasi : Anemia, lekositosis /lekopeni dan DIC Lekositosis - Umumnya Netrofil , bentuk muda - Netrofilia lanjut infeksi kronik - Netrofilia menghebat + sel muda reaksi leukemoid * Non-Ganas > 25-30 x 106/l * Inflamasi, Stress, Trauma.

Lekopeni * Netropenia, mis Demam Tifoid, Brucellosis * Infeksi hebat netropenia hebat prognosis buruk Perubahan morfologik pada sepsis * Dohle Bodies * Granulasi Toksik * Vakuolisasi Eosinofilia : * Non-bakterial, biasanya alergi/infeksi parasit

 Anemia

* Bisa timbul sekalipun cadangan besi cukup * Anemia akut : perdarahan / destruksi eritrosit (misalnya : cold aglutinin sehubungan dengan Mycoplasma pneumoniae) * Anemia kronik, dengan : - Cadangan besi yang normal atau meninggi di sistem retikuloendotelial - Penurunan besi dalam plasma - Penurunan TIBC

Infeksi serius + bakteremia * Gram negatif DIC (gram positif kurang) - Trombus - PT memanjang - FDP - Fibrinogen Trombositopenia bisa juga menjadi tanda sepsis bakterial dan bisa bermanfaat dalam mengobservasi respon pasien terhadap terapi.

(A) Direct Detection

1. Electron Microscopy
- direct demonstration of viruses - standard morphology of virus particles - catch-all method - helpful for virus that fails to grow in cell culture - virus may be recovered direct from clinical sample and needs to be purified or concentrated

Direct Detection: Electron Microscopy

106 virus particles per ml required for visualization X 50,000-60,000 magnification normally used Viruses may specimens: Faeces be detected in the following

Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus HSV,VZV papillomavirus, orf molluscum contagiosum

Vesicle Fluid Skin scrapings

Direct Detection: Electron Microscopy

Adenovirus

Enterovirus

Rotavirus

(courtesy of Linda Stannard, University of Cape Town, S.A.)

2. Virus Isolation
Methods of isolation: (A) (B) (C) Cell Culture Chick Embryo/ Eggs Animals (eg: Suckling mouse brain inoculation)

Specimens for viral isolation: Collect during acute phase Intact cells important Prompt delivery Refrigerate if stored less 24 h

Seal well if stored on dry ice (pH change)

(A) Cell Cultures

(B) Catch-all cell culture-based Labour and resource intensive CPE and haemadsorption based Roller tubes, flasks or multi-well plates

Chick Embryo/ Eggs

now rarely used

demonstration of poxviruses and propagation of orthomyxoviruses and paramyxoviruses

Virus Isolation: Cell culture

Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells. (Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)

Viruses readily isolated by cell culture Herpes Simplex Cytomegalovirus Adenovirus Poliovirus Coxsackie B viruses Echoviruses Influenza viruses Parainfluenza viruses Mumps Respiratory Syncytial virus

Less frequently isolated viruses Varicella-Zoster Measles Rubella Rhinoviruses Coxsackie A viruses

Virus Isolation: Cell culture

Problems with cell culture:

- Long period (up to 4 weeks) required for result - Often very poor sensitivity (depends on a large extent on the condition of the specimen) - Susceptible to bacterial contamination - Susceptible to toxic substances which may be present in the specimen. - Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus

Direct Detection

3. Detection of Viral Antigens

Tests include: Immunofluorescence (IFA) Enzyme Immuno Assays (EIA) Agglutination

2 methods: (A) Ab-staining technique (i) Immunofluorescence technique - label used is fluorescein/rodamine dye immunofluorescence microscope (ii) Immunoperoxidase technique - label used is horseradish peroxidase light microscope (B) Other assays: Enzyme immunoassay (EIA/ELISA) Radio Immunoassay (RIA) Latex agglutination Reverse passive haemagglutination (RPHA) Eg: HbsAg (serum) p24 HIV Ag (serum)

Direct Detection: Antigen detection (IF)

Positive immunofluorescence test for rabies virus antigen. (Source: CDC)

(Virology Laboratory, Haven Hospital)

Yale-New

Direct Detection: Antigen detection

Direct Detection

4. Nucleic Acid (NA) Detection - Methods based on the detection of viral genome molecular methods. - PCR and related techniques - Molecular techniques: 2 types: (a) No amplification involved (e.g hybridisation with nucleic acid probes) (b) Amplification (PCR, LCR, NASBA etc.)

Direct Detection: NA detection

Advantages of PCR:

Extremely high sensitivity, may detect down to one viral genome per sample volume Fast turnaround time (5 to 10 hours) Clinical samples small in size/volume Detect viruses that are unculturable Does not rely on viability of virus or intact genome Only target NA amplified non infectious

Direct Detection: NA detection

Disadvantages of PCR
Reagents and equipment expensive High degree of operator skill required Extremely liable to contamination (surfaces, reagents, equipment) A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.

Direct Detection: NA detection

Polymerase chain reaction

Direct Detection: NA detection

Conventional PCR

RT PCR

Direct Detection: NA detection

Post PCR Analysis:

1. 2. 3. 4.

Agarose gel with ethidium bromide staining Southern/dot blot Restriction fragment length polymorphism (RFLP) NA sequencing

Direct Detection: NA detection

Gel Electrophoresis

Direct Detection: NA detection

Southern Blot

(B) Indirect Detection: Serology

-

Serology forms the mainstay of viral diagnosis.

Viruses are good antigens and infection elicits a prompt antibody response and can remain at a high level for many years after infection. Following exposure, the first antibody to appear is IgM (7 to 10 days), which is followed by a much higher titre of IgG.

Indirect Detection: Serology

Note that during reinfection, IgM may be absent or present at a low level transiently

Indirect Detection: Serology

Criteria for Diagnosing Primary Infection

(a) Presence of IgM - the earliest Ab to appear - problems with IgM assays: interference by rheumatoid factor (ab IgM class) (false positive) re-infection (absent or low level) unexplained persistence of IgM years after primary infection

(b) Rise in titre - 4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera - 2 serum samples [acute (before 7days of illness) and convalescence (1-2 weeks later)

Indirect Detection: Serology

(c)

Seroconversion - changing from a previously antibody negative state to a positive state e.g. seroconversion against HIV following a needle-stick injury, or against rubella following contact with a known case. A single high titre of IgG/total ab (High stationary titre) - considerably higher than the general population this is a very unreliable means of serological diagnosis since the cut-off is very difficult to define.

(d)

Indirect Detection: Serology

Criteria for Diagnosing Re-infection/Re-activation

Difficult to differentiate re-infection/re-activation from a primary infection.

Under most circumstances, it is not important to differentiate between a primary infection and re-infection.
Important under certain situations, such as rubella infection in the first trimester of pregnancy: primary infection is associated with a high risk of fetal damage whereas re-infection is not IgM is usually low or absent in cases of re-infection/ reactivation A sharp large rise in antibody titres is found in re-infection

Indirect Detection: Serology

Serological Tests
(i) Neutralization test
- measures Abs that neutralize virus infectivity - Ab prevents virus infection of cells - Ab can be detected by neutralization of virus CPE in tissue culture - variation, plaque reduction test - highly specific, can type virus strains - time-consuming and laborious

Serology: Serology Tests

(ii) Complement fixation test (CFT)

- not all viruses will agglutinate RBCs. - CFT can get around this problem by using complement to detect the presence of antibody. - not to react with an antigen or an antibody alone but to enter into combination with antigen-antibody complexes (mixture of ab, ag and complement) - indicator system used in CFT is sheep red blood cells (RBCs) use for herpesviruses and respiratory viruses

(can be used to identify either viral antigens or antibodies)

Serology Test: CFT

Complement fixation test (CFT)

Positive or reactive test

The complement is bound to an antigen-antibody complex, and is not free to interact with sensitized RBCs so they remain unlysed and settle to the bottom of the well to form a button.

Negative or nonreactive test The complement remains free since there is no antigen-antibody complex for it to bind to, and it interacts with the sensitized RBCs causing them to lyse

 Ag

+ Serum (Ab pos)+ complemen Complemen fixed + RBC (sheep) RBC unlysed POSITIF TEST
+ Serum (Ab neg)+ complemen Complemen unfixed + RBC (sheep) RBC lysed NEGATIF TEST

Ag

Serology Test: CFT

Complement fixation test (CFT)

Reliable only when test carefully standardized. All reagents involved must be used at optimal reactivity. All reagents to be carefully prepared and standardized to insure a completely balanced system titrations of sheep RBCs, hemolysin, complement and antigens. Rather difficult and time consuming (2 days to complete) Many of the test antigens and antisera are not available commercially.

Serology Test: CFT

(iii) Haemagglutination-inhibition (HAI) test - many viruses have the ability to agglutinate erythrocyte of various species (e.g. influenza, parainfluenza, adenoviruses, rubella, alphaviruses, flaviviruses) - this agglutination can be inhibited by specific Ab - if virus Ab present can see button - mostly type specific except in flaviviruses (crossreact) - IgG, IgM and IgA - main use today, influenza and parainfluenza

Indirect Detection: Serology

(iv)
-

Enzyme-linked Immunoassay

The most commonly used sensitivity (ability to detect small amounts of Ag or Ab), specificity (ability to discriminate between closely related but antigenically different molecules) Ease of automation (large numbers of tests). Generally considered safer than radioisotopes used in RIA (radioimmunoassay).

False positives can result from impure reagents

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Indirect Detection: Serology

To detect antibody (indirect ELISA)

No Abs

Abs present

LABORATORY DIAGNOSIS (ANTIBODY TEST: ELISA)

ELISA Test Kit

ELISA Machine

Indirect Detection: Serology

Rapid Test

Tests that can be performed in less than 30 minutes, easy to perform.

Disadvantages: subjective interpretation more expensive sensitivity may not match the ELISA

Indirect Detection: Serology

long length of time required for diagnosis for paired acute and convalescent sera

mild local infections such as HSV genitalis may not produce a detectable humoral immune response
Extensive antigenic cross-reactivity between related viruses may lead to false positive results immunocompromised patients often give a reduced or absent humoral immune response Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react nonspecifically giving a false positive result Patients given blood or blood products may give a false positive result due to the transfer of antibody.

LABORATORY DIAGNOSIS OF DENGUE

Methods

Virus Isolation Serological Test Genome Detection

Method (1) Cell culture: Mosquito cell lines Primitive cell lines

(2) Mosquitoes: (3) Animals:

Larvae Adult Suckling mice

Virus Isolation- C6/36 Cells

Cell culture

Cytopathic effect

Larvae Inoculation: Toxorhynchites splenden

Suckling mice

Serological Tests
1. Rising Ab titre Detection of specific IgG Ab Four-fold or greater rise in Ab titre against a virus 2. Detection of specific IgM antibody 3. High stationary titre

IgG

Onset of Sx

Onset of Sx

IgG Cut Off

Virus

IgM

Virus

IgM

IgM Cut Off

1o Infection

2o Infection

Serological Tests
1. Traditional Techniques Haemagglutination Inhibition Test (HI) Neutralization Test (NT) Complement Fixation Test (CFT) 2. Newer Techniques ELISA-IgM Dengue Blot assay Pan Bio Dengue Rapid Test

Serological Profile Of Dengue Virus Infection

Titre Neutralising Ab Viraemia Complement Fixing Ab Haemagglutination Inhibition Ab

IgM Ab

20 Infection Days

60 Weeks

3 Months Years

TRADITIONAL TECHNIQUES Detection of specific IgG antibody -2 serum sample -acute and convalescence

(1)

Haemagglutination Inhibition (HI) -Test very pH dependent -Takes 3-5 days to complete -Able to differentiate between primary and secondary -Problem of original antigenic sin

(2)

Neutralization Test (NT) -The most specific and sensitive -Useful for research purposes -Laborious -At least 5 days to complete Complement Fixation Test (CFT) -Less sensitive than HI or NT -CFT can be used to show rises in Ab titer later in infection than other serological tests

(3)

NEWER TECHNIQUES
ELISA 1. Anti-dengue IgM - Infeksi Primer, akut 7-10 hari 2. IgG (post/kronik) - Infeksi sekunder, sesudahnya
BLOT ASSAY (i) IgG (ii) IgM

Dengue Blot Assay

Positive Control

Negative Control

Invalid

Panbio Dengue Rapid Test

Primary Dengue Secondary Dengue

M G C

M G C

Secondary Dengue Suspected

Negative

M G C

M G C

HASIL IgG + + IgM + + -

INTERPRETASI

D Sekunder D Primer Duga D sekunder Non-D Primer sangat dini

Genome Detection
(1) Polymerase Chain Reaction
Universal primers for Dengue virus followed by Specific primers for 4 serotypes Multiplex PCR 1 set of primers which amplify different sites and sizes of each serotypes Serotyping/Surveillance -Specimen within 5 days of symptoms -Serum/Plasma/PBMC/Biopsy samples -Expensive

(2) DNA Sequencing

- Genotyping/Molecular epidemiology study

RT-PCR using consensus primers

RT-PCR for detection of dengue serotypes

Laboratory findings * Hematology - Leukopenia - Trombocytopenia - serum aminotransferase (AST, ALT) elevations * The diagnosis is made by lab test seroloimmunology 1. Hemaglutination tests 2. Complement Fixation test 3. Neutralization Test - IgM ELISA or - paired serology during recovery or - by antigen-detection ELISA or - RT-PCR during the acute phase * Virus is readily isolated from blood in the acute phase if mosquito inoculation or mosquito cell culture

Diagnosis Demam Dengue/Demam Berdarah Dengue

Suspicion of dengue infection

Delay after onset of fever Unknown or 5 days NS1 Antigen + >5 hari

Antisipated Diagnosis IgM/IgG Serologis Improve patient Management + Late acute or past-infection

Dengue is unlikely

Confirmed early IgM serology infection

Acute infection
+ -

Presumably early early acute infection Acute infection is unlikely A second sample isrequested for confirmation EARLY DIAGNOSIS LATE DIAGNOSIS

 In

dengue

Present by the 2nd day of fever By the 4th or 5th day, the wbc count 2000 to 4000/ml, 20 to 40 % granulocytes Moderate albuminuria and a few casts may be found - Dengue may be confused with Colorado tick fever, typhus, yellow fever, or other hemorhagic fevers. Serologic diagnosis may be made by - Hemagglutination inhibiting and complement fixation test using paired sera - but is complicated by cross-reactions with other flavivirus antibodies

In dengue hemorrhagic fever

Hct >50 %: ipresent during shock WBC count in 1/3 of patients Coagulaive abnormalities - Trombocytopenia (<100.000/ml) - positive tourniquet test - prolonged PT - Minimal proteinuria may be present. - AST levels may be moderately - Serologic test usually show high complement fixation antibodies titers againts flavivirus, suggestive of a secondary immune response

WHO clinical criteria for diagnosis of dengue hemorrhagic fever :

Acute onset of high, continous fever lasts for 2 to 7 days Hemorrhagic manifestations, including at least a positive tourniquet tes and petechiae, purpura, ecchymoses, bleeding gums, hematemesis or melena Hepatomegaly Trombocytopenia (<100.000/ml);or hemoconcentration (Hct increased by >20%) Those with dengue shock syndrome also have a rapid weak pulse with narrowing of the pulse pressure (<20 mmHg) or hypotension with cold, clammy skin and restlessness.

Laboratory tests are generally not necessary (viral cultures and Tzanck smear will confirm diagnosis in patient with atypical presentation) Antibody to appropriate serotype : - Seroconversion - Increase - Direct immunofluorescent antibodies slide test (rapid diagnosis) Tzanck preparation : - Base of lesion - Multinucleate giant cells

Laboratory

test are generally not necessary (viral cultures and Tzanck smear will confirm diagnosis in patient with atypical presentation) The Tzanck preparation shows multinucleate giant cells for both varicella-zoster virus and HSV

 Darah

- lekopeni - serum amilase dalam 10 hari - Serologi : * cold agglutinin * IgM , max 2 minggu, menetap 6-9 bulan; kadar serum konvalesens 4x dr pd serum akut * tes fixasi komplement thd atb positif minggu pertama Biakan - Virus dari ludah 1-5 hari

 Komplikasi

: - Inflamasi testis / ovarium : lekositosis, LED - Pankreatitis : lekositosis, amilase , hiperglikemia - Meningitis : sel LCS < 500/l, mononuklear; glukosa normal, protein agak (20-125 mg/dl)

 Temuan

Laboratorium - Darah : * Lekosit terutama limfosit dan segmen * Serologi : EIA - IgM : Fase akut ( 1-2 hari) - IgG : >10 hari - Sekret : * Apusan + pulasan imunofluorosen * Pulasan Tzanck : Multinucleated Giant Cells - Biakan : * Bahan : sekret resp dan urin * Identifikasi : jaringan

 Tes

Lab yang bisa dilakukan: - Sediaan Apus : * Bahan : Kerokan dasar vesikel * Pulasan : Tzanck Multinucleated Giant Cells * Sensitivitas 60 % - Darah : * Serologi : - Titer atb serum konvalesent 4x dpd serum akut. - Hemaglutinasi - ELISA - FAMA * PCR : deteksi DNA Virus

 Giemsa

stained

* Show inclusion bodies within many large cells or extracellularly

 DNA

typing : Circular doubel stranded , 8000 bp * Crosshybridization : - > 50% : type seperation - < 50% : subtype seperation

 Generally

not necessary Gram stain and C&S to confirm the diagnosis when the clinical presentation is unclear Sedimentation rate parallel to activity of the disease Anti-DNAse B and anty hyaluronidase Urinalysis : hematuria with erytrocyte casts and proteinuria in patients with acute nephritis

(A) Screening Test

Enzyme-linked Immunosorbent Assay =ELISA

Simultaneously detection of antibodies to HIV-1 and HIV-2 in human serum or plasma. Detect antibodies directed against major group of HIV-1 and HIV-2 proteins: HIV-1: envelope protein of gp41 core structural protein of p24 HIV-2: envelope protein of gp36 Methods: indirect, competitive, antigen sandwich

ELISA for HIV antibody

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Agglutination Test
Requires greater than 30 minutes but has procedures that can be performed easily without instrumentation. Within this class of tests are agglutination assays in which antigen-coated particles (red blood cells [RBC], latex particles or gelatin particles) are allow to react with serum antibodies to form visible clumping (agglutination). If RBC are used, the technique is termed passive haemagglutination (PHA); latex particles is known as latex agglutination (LA); gelatin particle is known as particle agglutination (PA).

Agglutination Test
Good sensitivity, low cost and ease of performance. It incorporates a quality system to detect nonspecific antibodies directed toward the gelatin particles themselves; sample need to be treated by adsorption and retested. False-negative reaction due to prozone (inhibition agglutination when excess of antibody present, events optimal combination) - perform dilution. Results can be obtained within 2 hours. of

Particle Agglutination Test

(B) Supplementary/Confirmation Tests

1. Western Blot (WB)
- gold standard for validation of HIV results. - Based on electrophoretic technique to separate HIV antigens derived from viral lysate grown in culture and the separate antigens are transferred (blotted) onto nitrocellulose membrane. - Commercial WB are supplied with individual strips of nitrocellulose containing the blotted, separated HIV antigens. Application of ELISA technique in detection of specific antibodies to each viral antigens. - Modified WB has the ability to identify and differentiate infections by HIV-1 and HIV-2.

Western Blot

Line Immunoassay (LIA)

Another alternative to the classic Western blot. Recombinant or synthetic peptide antigens are applied onto nylon strip rather than electrophoresed as in the Western blot.

Use of artificial antigens decreases the presence of contaminating substances derived from cell culture that can cause interference and sometimes false reactions.
A number of reports have verified that the accuracy is equivalent to the Western blot.

LABORATORY DIAGNOSIS (ANTIBODY TEST: SUPPLEMENTARY/CONFIRMATORY)

Line Immunoassay

Interpretation criteria for Immunoblot tests

Organization American Red Cross Minimum band requirements for reactive pattern At least one band from each gene product group: gag AND pol AND env

Centres for Disease Control (CDC)

World Health Organization (WHO) Du Pont

Any two of p24, gp41 or gp120/160

Two env bands (gp160, gp120 or gp41) +/- pol bands, +/- gag bands p24 AND p31 AND gp41 or gp120/160

(C) Other Antibody Test: Rapid Test

less than 30 minutes, easy to perform.

Qualitative, intended for use as point-of-care

Technical errors are common, must be performed carefully by experienced personnel. Application includes: emergency rooms, physician's offices, autopsy rooms. Not recommended for use in a blood transfusion centre or home-use. Example: (i) "dot blot" or "immunoblot (incorporate a built-in control that indicates that the test was performed correctly) (ii) dipsticks (antigen is attached on the "teeth" of comblike devices.

Several of these rapid tests have the ability to differentiate HIV-1 and HIV-2. Disadvantages: subjective interpretation more expensive sensitivity may not match the ELISA Result obtained, must always be confirmed Types of samples: blood, serum, plasma, saliva and urine

HIV RAPID TEST KIT

HIV Testing Strategies

WHO recommends three testing strategies to:
-Maximize accuracy -Minimize cost Choice of strategy depends upon: -objective of test -prevalence of HIV in population

WHO Testing Strategies

Objective of testing Transfusion/donation safety Surveillance Clinical signs/ symptoms of HIV infection/AIDS Asymptomatic

Prevalence of Infection All >10% <10%

Testing Strategy I I II

all >10% <10%

II II III

Diagnosis

STRATEGY I: All samples are tested with one ELISA or rapid/simple assay. Samples that is reactive is considered HIV Ab positive.

STRATEGY II:
All samples are first tested with one test. Any reactive samples are subjected to second test based on different principle and/or a different antigen preparation. STRATEGY III: All samples are first tested with one test. Any reactive samples are subjected to second test based on different principle and/or a different antigen preparation. Requires a third test if samples are found reactive on the second test.

POSITIVE High Risk Group (WHO 2-tests strategy) ELISA - Reactive PA - Detected Low Risk Group (WHO 3-tests strategy) ELISA - Reactive PA - Detected Immunoblot - Positive NEGATIVE Both screening tests: EIA - non-reactive and PA - not-detected

FALSE POSITIVE The ELISA test may produce false positive results on some blood from uninfected individuals. Employs at least 2 different tests. May due to nonspecific reaction, autoimmune diseases (Systemic Lupus Erythematosus), etc FALSE NEGATIVE Test concludes HIV is not present, when in fact the person is infected This occurs when the blood is taken during the window period. If there has been exposure to risk activities, it is advisable to repeat the test 3-6 months later.

EQUIVOCAL

Results that neither clearly positive nor clearly negative (one of the screening tests is positive) To test with third test (LIA or Wblot) Follow-up sample after a minimum of 2-weeks If produces an equivocal result, person is considered to be HIV Ab Negative Units of donated blood must be discarded

Once a diagnosis of HIV infection had been made, it is important to monitor the patient at regularly for signs of disease progression and response to antiviral chemotherapy. Tests use: HIV antigen tests (p24)

Detect the presence of HIV p24 antigen Not as good as serial CD4 counts (does not check presence of HIV, monitor immune function) CD4 T-cells marker of progression of HIV infection HIV viral load in serum may be measured by assays which detect HIV-RNA e.g. RT-PCR, NASBA, or bDNA. HIV viral load has now been established as having good prognostic value, and in monitoring response to antiviral chemotherapy.

HIV viral load

Sediaan darah malaria Kegunaan sediaan darah malaria :

Menentukan ada tdknya parasit malaria Menentukan spesies & stadium plasmodium Dapat melacak 10 100 parasit/L darah Menentukan kepadatan parasit

104

Sediaan darah tebal

Cara terbaik menemukan parasit malaria Mudah dibuat Diperiksa paling sedikit 100 lapangan pandang

Sediaan darah tipis

Digunakan utk identifikasi jenis Plasmodium bila dgn sediaan darah tebal sulit ditentukan Diperiksa paling sedikit 200 lapangan pandang

105

Kelebihan sediaan darah tebal

Lebih banyak sel darah yg diperiksa Parasit lebih mudah ditemukan

Kekurangan sediaan darah tebal

Tdk dpt membandingkan ukuran Plasmodium dgn ukuran eritrosit Spesies Plasmodium sukar ditentukan

106

Kelebihan sediaan darah tipis

Morfologi eritrosit jelas Spesies Plasmodium bisa ditentukan Perbandingan ukuran Plasmodium terhdp ukuran eritrosit bisa dilihat % parasitemia bisa dihitung

Kekurangan sediaan darah tipis

Jumlah parasit dlm lap. pandang sangat sedikit.

107

Bentuk Tropozoit
Cincin kecil ( eritrosit normal) Sitoplasma biru Kromatin inti merah

Bentuk Skizon
Jarang ada dlm sirkulasi darah tepi Jk ditemukan dlm darah tepi tanda malaria berat

108

Bentuk Gametosit
Sgt khas yaitu elips (crescent) Berpigmen warna hitam Sitoplasma kuning

109

Teknik Quantitative Buffy Coat (QBC)

Prinsip : Tes Fluoresensi Eritrosit yg terinfeksi Plasmodium akan terlihat berfluoresensi di bwh mikroskop fluoresensi

Cepat namun peralatannya mahal Tdk dpt membedakan spesies Plasmodium & tdk dpt digunakan utk hitung parasit.

110

Mendeteksi Ab/Ag spesifik terhadap parasit malaria atau eritrosit yg terinfeksi Plasmodium Tes imunoserologis yg melacak Ab tdk dipakai utk keperluan diagnosis Tes imunoserologis malaria :
Radioimmunoassay (RIA) Enzyme Linked Immunoassay (ELISA) Immunochromatographi (ICT) Indirect Fluorescent Antibody Test (IFAT)

111

Radioisotop sebagai label Kadar Ag atau Ab pada sampel dpt ditentukan scr kuantitatif Low detection limit 50 parasit/L darah Sensitif Kurang praktis & berbahaya

112

Lebih praktis dibanding RIA Enzyme direaksikan dgn substrat kromogen intensitas warna sebanding dgn kadar bahan Mendeteksi Ag & Ab spesifik terhadap Plasmodium

113

Rapid Diagnostic Test Melacak Ag parasit malaria melalui pengikatan Ag oleh Ab monoklonal Ada 3 jenis Ag utama yg sering dijadikan target ICT utk mendiagnosis malaria yaitu :
Histidine-rich protein (HRP) Parasite specific lactate dehydrogenase (pLDH) Plasmodium Aldolase

114

Keunggulan tes ICT

Praktis Tdk membutuhkan alat pembantu lain Tdk memerlukan tenaga terampil

Kelemahan tes ICT

Hanya dpt melacak parasit > 100 parasit/L darah Membutuhkan biaya pemeriksaan yg relatif sdg Tdk dpt memberi informasi derajat parasitemia

115

Mendeteksi Ab spesifik terhdp Plasmodium Keadaan dimana parasit sangat minimal Tdk utk menentukan infeksi baru Mendeteksi keempat spesies Plasmodium Manfaat utk penelitian epidemiologi

116

Mendeteksi DNA spesifik terhadap parasit Plasmodium dlm darah penderita malaria Teknik Polymerase chain reaction (PCR) Dpt melacak sampai 5 parasit/L drh Dpt mengidentifikasi spesies Plasmodium Waktu pemeriksaan cepat Sensitivitas & spesifisitasnya tinggi Mahal

117

Jenis tes laboratorium untuk tuberkulosis terdiri dari:

Tes Mikrobiologi, terdiri dari:

Tes seluler:
Tes BTA Sputum Tes Biakan dan Identifikasi M.tuberculosis Tes Kepekaan Antibiotika

Tes molekuler: PCR Semirapid: TB-Dot, ELISA, Tb-kompleks Rapid: Mycodot, ICT-TB

Tes Serologis, terdiri dari:

 Anamnesis Pemeriksaan

Fisis Pemeriksaan Radiologis Tes Laboratorium Tes Mikrobiologi

Tes seluler: Tes Apusan BTA
Tes Biakan & Identifikasi Tes Kepekaan Antibiotika

Tes molekuler: PCR

Tes Serologis
Semirapid: TB-Dot, ELISA, Tb-kompleks Rapid: Mycodot, ICT-TB seperti Mycotec TB

Mikroskopik

Ziehl Neelsen Dekontaminasi

Kultur (pembiakan)

medium Lowenstein-Jensen

PCR

Isolasi DNA: Metode Boom Proses : denaturasi, annealing, elongasi

IS6110

Tes Imunoserologi

Deteksi Ab terhadap Ag mikobakterial spesifik atau gabungan beberapa antigen

Ag60: ELISA Ag16 : 16 kDa, spesies-spesific epitop imunodominan stadium awal infeksi M.tbc & TB primer 38kDa spesies-spesific epitop imunodominan ESAT-6 kontak baru terjadi, konversi & meningkatnya risiko penyakit

Pemeriksaan lab. meliputi : 1. Pemeriksaan bakteriologik - menegakkan diagnosis dan memantau pengobatan - kerokan kulit/mukosa hidung (pewarnaan Ziehl Neelsen) - negatif : bukan berarti tdk mengandung M. leprae - indeks bakteriologi (IB) dan indeks morfologi (IM) - IB : banyaknya kuman M. leprae tiap satuan lap. tertentu - IM : prosentase basil utuh dlm semua basil yg dihitung

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Cara menghitung IB dan IM :

IB :

total kepadatan jumlah tempat pengambilan jumlah basil utuh x 100% jumlah basil yang diperiksa

IM :

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Penilaian - negatif (-) penglihatan - 1 (+) - 2 (+) - 3 (+) - 4 (+) - 5 (+) - 6 (+)

skala algoritme Ridley : : tdk ditemukan BTA pd 100 lap. (LP) : 1 10 basil/100 LP : 1 10 basil / 10 LP : 1 10 basil/1LP : 10 - 100 basil/1 LP : 101 1000 basil/1 LP : > 1000 basil/LP

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2.

Pemeriksaan histopatologik - menegakkan diagnosis (manifestasi klinik tdk jelas) - biopsi kulit & imunohistokimia 3. Pemeriksaan imunologik - tdk utk diagnosis menentukan klasifikasi & perjalanan peny. kusta

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Tes lepromin - kemampuan individu bereaksi scr seluler thd M. leprae

- lepromin : suspensi steril dr jaringan yg dihancurkan & sbg tes kulit secara intradermal a. lepromin Mitsuda : lepromin dr suspensi jaringan, mengandung kuman M. leprae yg sdh disterilkan dlm autoklaf (manusia / binatang) b. Lepromin Dharmendra : dr ekstraksi fraksi protein dgn kloroform eter (tipe lepromatous)

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Reaksi kulit terhadap lepromin : 1. reaksi dini (reaksi Fernandez) - berbentuk infiltrat eritematosa (12 72 jam) - hipersensitivitas yg telah ada thd antigen - pembacaan : 48 jam sth penyuntikan 2. reaksi lambat (reaksi Mitzuda) - btk noduler, tampak pd hr ke-21 30 (paling jelas) - respon thd imunitas seluler - pembacaan : sth hr ke-21

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3.2 Tes histamin - secara intradermal pd kulit normal dilatasi kapiler bintul berwarna merah (histamin flare) - ukuran bintul merah derajat kerusakan saraf 3.3 Tes serologis - ELISA mendeteksi antibodi phenolic glicolipid-1 (PGL-1) reaksi antigen antibodi dgn enzim sbg label - imunokromatografi menggunakan antigen PGL-1 neoglycoconjugate, sensitivitas 91,7%, spesivisitas 78,1%

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3.4 Polymerase Chain Reaction (PCR) - mendeteksi adanya organisme dgn cepat dan tepat - mendeteksi sejumlah kecil basil dr biopsi kulit - kolonisasi M. leprae pd mukosa/apusan hidung penderita atau orang sehat - diagnosis pasti tipe tuberkuloid - follow-up hasil pengobatan - menggantikan pemeriksaan adanya BTA

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Tes lain: 1. Tes pengeluaran keringat - mengetahui integritas saraf kulit - tergantung pd saraf parasimpatik - respon kelenjar keringat thd obat kolinergik berkurang 2. Tes pilokarpin - melihat perubahan warna pada kulit setelah ditaburi tepung amilum - warna amilum tetap (ada kerusakan saraf)

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Diagnosis : pemeriksaan Klinik (bakteriologi, histopatologi, imunologi) Tanda-tanda kardinal : 1. Anestesi 2. Penebalan saraf di daerah yang terkena 3. Adanya lesi kulit berupa hipopigmentasi, eritema, infiltrat, nodul 4. Ditemukannya kuman tahan asam (BTA positif) Diagnosis : 2 dari 3 tanda kardinal I, terlebih BTA positif

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 Generally

not necessary Gram stain and C&S to confirm the diagnosis when the clinical presentation is unclear Sedimentation rate parallel to activity of the disease Anti-DNAse B and anti hyaluronidase Urinalysis : hematuria with erytrocyte casts and proteinuria in patients with acute nephritis

 Diagnosis

definitif tergantung pada isolasi C.diphtheriae yang diambil dari bahan lesilesi lokal Pihak laboratorium harus diberitahukan bahwa bahan disangka diphteri. Gram stains of secretion :

club-shaped organism, appear as Chinese letters

 CSF

: * Aseptic meningitis * Elevated WBCs * Elevated protein * Normal glucose

 Kultur

: * Darah : positif dalam 10 hari pertama * Tinja & urin positif dalam minggu 3-5 * Sumsum tulang Serologi : * Tes Widal : Serum sembuh 4x drpada sakit Darah rutin : lekopeni

Felix-Widal test

- measures levels of agglutinating antibodies against O and H antigens - usually appear after 5-8 days of infection - limited clinical utility in acutely ill patients because positive results may represent previous infection in endemic areas - paired titration should be preformed - In the absence of recent immunization, a high titre of antibody to O antigen > 1:640 is suggestive but not specific. - false positive (cross reactivity)

An ELISA for antibodies to the capsular polysaccharide Vi antigen is useful for detection of carriers but not for the diagnosis of acute illness

Typhidot test that detects presence of IgM and IgG in one hour (sensitivity>95%, Specificity 75%) Typhidot-M, that detects IgM only (sensitivity 90% and specificity 93%) Typhidot rapid (sensitivity 85% and Specificity 99%) is a rapid 15 minute immunochromatographic test to detect IgM.

IgM dipstick test

 Isolasi

vibrio cholerae dari bahan tinja identifikasi serogroup 01 atau 139 Serologi : * tes agglutinasi menggunakan antiserum spesifik

Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 5th Edition, 2000

 Lab

studies:

Confirm diagnosis Epidemiologic investigations

Direct

microscopy:

Dark field microscopy: at least 104 org/ml to be able to see 1 spirochete per HPF. Silver staining DF using mouse monoclonal AB IP Insitu hybridisation using DNA probes Electron microscopy

Isolation:

Possible using special media containing serum. may not be of value as diagnostic test. Generation time of the bacteria is about 12-14 days. It takes 4-8 weeks to be positive Sensitivity is very low . 1st week : Blood (Blood in EDTA bottle) CSF 2nd week: urine

 Immuno

diagnosis:

Antigen detection:
RIA, ELISA (Monoclonal antibody Based), Chemiluminiscent immunoassay Immunomagnetic antigen capture combined with fluoroimmunoassay can detect as low as 102 lepto/ml of cattle urine. For biopsy or post mortem tissues: immunohistochemistry