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CTQ #9 (1.

2 pts)
Instructions
Read the journal article, Nosocomial Acquisition of Dengue, by Dirk Wagner et al. and revie
w the othersupplemental resources added to module, to answer the following questions. Not
e that
additional questions may be asked of you as a group, so it is in your best interest to review a
ll of these resources and/or have them accessible on 12.1.14.
1. What is the significance of leukocyte data shown in Table 1, relative to
understanding the course of dengue infection?
Leukopenia occurs during the acute phase of when the leukocyte value is less
than 4000, and is commonly seen near the end of the febrile phase (acute)
following dengue infection as a result of viral destruction of bone marrow
precursor cells, and resulting in the associated bone pane. The decrease in
leukocytes caused by dengue infection allows for relatively easy diagnosis
because most viral infections result in an increased leukocyte count.
1. What is the significance of the RTPCR results? Can you explain why the virus is not
detectable with PCR for Day 4i?
The virus was not detectable with PCR for Day 4i because the level of the virus
was too low to detect. Dengue viremia is highest in the first 3-4 days following
fever onset, and quickly falls to undetectable levels over the next few days as
fever abates and anti-dengue IgM antibodies increase.
2. Estimate the length of the prodrome period for the nurse, and justify your response.
The nurse experienced cephalgia and myalgias after an incubation period of 4
days. These symptoms signified the start of the prodrome period, which lasted
roughly 7 days, before the febrile phase began. The positive RT-PCR results on
day 2n indicate the test was performed during the acute phase (0-5 days) of the
disease.
3. Currently, Dengue Fever is endemic to Northern Mexico, but is not endemic in the
southern U.S. List 2 factors do you believe could contribute to an emergence of
dengue fever cases in the U.S.? Explain your rationale clearly.
The emergence of dengue fever cases in the U.S. is due largely to travel out of
the U.S., especially to the tropics, because travelers often return home without
knowing they are infected due to the 3-14 day incubation period of dengue. The
traveler may then be bit by a mosquito in the U.S, which can subsequently bite
and infect others in close proximity, introducing new serotypes/genotypes to
people with differing levels of immunity. Another factor which would contribute
to an emergence of dengue fever cases in the U.S. would be an increase in
unplanned urbanization resulting in over-crowded, run-down neighborhoods with
no air-conditioning in areas lacking proper public health systems.
4. In the second set of results, EIA was used to quantify antidengue antibodies in
Nancys blood. What do the results tell you? What new, complementary information is
gained from the EIA report?
During the acute phase of Dengue infection (disease day 5-6), IgM is the first
class of antibody secreted in response to a primary (recent) Dengue infection,

and is followed by IgG secretion, which is observed in Nancys results. A


secondary Dengue infection is indicated in the results of the index patient due
to the positive IgG results and negative IgM results on day 0 i. This is an
indication of secondary infection because following exposure to the Dengue
virus, memory B cells express IgG on their cell membranes, which is then
secreted following a secondary infection.

the predominant antibody class present switches to IgG. In people who


have been previously exposed to a pathogen, memory B cells exist, and
these express IgG bound to their cell membranes. Upon stimulation by
re-infection, they diff erentiate into plasma cells that secrete IgG that
is directed against the pathogen. In the second set of results, EIA was
used to quantify anti- dengue antibodies in Nancys blood.

An EIA (enzyme-linked-immunosorbent assay, sometimes also called


an ELISA) is a frequently used technique that allows medical
personnel and researchers to identify and quantitate proteins such
as antibodies, antigens, or other immune system chemicals that are
present in biological uids. Antibodies are proteins produced by
white blood cells called B cells. Th ey bind in a highly specifi c lock
and key fashion to viruses, bacteria, fungi, and to pieces of viruses,
bacteria, and fungi. Th e immune system uses these antibodies
almost like handles that aid in clearance of the infecting organisms
as well as to tag pathogens and identify them as foreign, which also
facilitates clearance and healing. Th ere are several diff erent
classes of antibody (or immunoglobulin, as the antibody protein is
properly named), including IgM, IgG, IgD, IgA and IgE. What do the
results tell you? What new, complementary information is gained
from the EIA report?
5. If Nancy is the nurse: Nancys EIA report shows a negative IgM
ELISA during viral onset, but a positive IgM ELISA on day 8 n, which
indicates a recent infection
If Nancy is the index patient: The positive IgG ELISA test on day 1 i
indicates the index patient had had a past dengue viral infection.

WNV+Dengue Review RASH VIDEO


Flavici

Positive strand RNA viruses


Icosohedral capsids and envelope
Dengue Fever
They have the abil to attach to variety of diff kinds of cells tropism = viruses can
only attack tis they have struc for, but flaviviruses are diverse and can attach to
many diff wbcs particularly those with the antibody the antibodies actually
enhance the flavivirus
Many ppl are infected with the virus but remain subclinical or remain asymptomatic
for their entire lives.
Intro of virus by way of arthropod (mosquito) asymptomatic or mild symptoms
during prodomic period
3-7 days virus is now infrecting macrophages, liver, spleen, lymphnodes produc
of Abs which allow virus to spread more.
Dengue- rare in US prevalent in Mexico ades mosquitos geographic distribution
geog range extends only to rio grande that borders mexico and texas provides us
a saftety net for dengue cases, but not west nile cases

NOTES FOR CTQ 9:


-

correlation of Dengue viremia with the presence of fever


Dengue viremia is typically highest in the first three to four days after onset
of fever but then falls quickly to undetectable levels over the next few days.

The level of viremia and fever usually follow each other closely, and antidengue IgM anti-bodies increase as fever abates.

In primary infections, immunoglobulin M (IgM) is detected 5 or more days after the onset of illness
in the majority of infected individuals and immunoglobulin G (IgG) is detected from 1015 days

Fever typically abates with the cessation of viremia


Molecular testinga PCR test that detects the presence of the virus itself is generally considered the most
reliable means of diagnosis, but the test is not widely available. A positive result from a PCR is considered
conclusive. A negative result on a PCR test may indicate that no infection is present or that the level of virus
is too low to detect, as may happen if the test was performed after the 5-day window during which the virus
is present in the sample collected for this test. If very recent exposure is suspected, repeating the test at a
later time may be warranted

The global incidence of epidemic and endemic dengue fever has increased
substantially and is estimated at 50100 million cases per year. International travel
leads to imported cases in countries of the Northern Hemisphere (1), where dengue
fever is an important differential diagnosis of fever in travelers returning from the
tropics. Occupational needlestick injuries continue to pose a substantial risk for
healthcare workers and occur at rates of 1.0 to 6.2 per 100 person-years
6.

The dengue virus is most commonly introduced into the U.S. population by returning travelers
who contract the disease abroad, according to Tomashek. In fact, dengue has become the
leading cause of illness among travelers returning with acute fevers.

7.

And as people increasingly chose to travel to remote locations, cases of travelers bringing the
disease back to the United States have increased.

8.

If you were to travel to Puerto Rico and get bitten by a mosquito thereand return home and
you live in Southern Florida, you could then transmit that virus in your blood to a mosquito if
you had one in your house and it bit you, Tomashek said. Then, within another week (that
mosquito) could transmit the virus to all the people in your household.

9.

Other factors contributing to climbing rates of dengue fever throughout the world include
increasing rates of pesticide resistance among the two types of mosquitos responsible for
transmitting dengue the Aedes aegypti and Asian tiger mosquitos and a lack of good
methods by which to control the disease.

10. We havent been effective at reducing the population or spread of Aedes aegypti, Tomashek
said. We are all in search of a more effective, more sustainable vector control method. We
dont have one now, and no country does.

Data from other countries supports the finding that the severity of secondary dengue infections
appears to intensify with longer intervals between infections.[7, 8] Since then, dengue fever and
dengue hemorrhagic fever cases have progressively increased.
United States
Factors believed to be responsible for the spread of dengue include the following:

Explosive population growth

Unplanned urban overpopulation with inadequate public health systems

Poor control of standing water and vectors

Viral evolution

Increased international recreational, business, and military travel to endemic areas

All of these factors must be addressed to control the spread of dengue and other mosquito-borne
infections. Unplanned urbanization is believed to have had the largest impact on disease
amplification in individual countries, whereas travel is believed to have had the largest impact on
global spread
Compared with patients with mild infection, those with severe infection (dengue hemorrhagic
fever grade II or worse) had a higher leukocyte count (3580 vs 3050 cells per L; P = .04), and
fewer had leukopenia on admission (70% vs 89%; P = .03). They also had a lower percentage of
typical lymphocytes (24% vs 40%; P = .02). Two predictors were identified; either one classified
19% of all admitted patients as being at low risk. Typical lymphocyte counts of <40% excluded
patients with mild disease with 89% sensitivity and 24% specificity (negative predictive value:
77%; positive predictive value: 45%). A combination of parameters [(white blood cells per L) +
470 (% typical lymphocytes) + 5 (atypical lymphocytes per L) 14 950] improved the
sensitivity and specificity to 92% and 26% (negative predictive value: 82%; positive predictive
value: 46%).
CONCLUSIONS: The absence of leukopenia and a low percentage of typical lymphocytes predict
severe dengue illness. Simple hematologic parameters may be used to reduce unnecessary
admissions of patients with suspected dengue infection in the absence of more sophisticated
predictors.

A range of findings in peripheral blood is seen mostly during dengue viral infections.
Heamo-concentration and raised haematocrit are well known findings in patient of
Dengue haemorrhagic fever.7 A total decrease in the leukocyte count during the
illness is mainly due to a decrease in granulocytes, i.e., neutrophils. Presence of
atypical lymphocytes with activated lymphocytes and even plasma cells along with
thrombocytopenia is reported consistently along with other laboratory findings.8 A
number of studies have shown that early in the course of illness, patients with
either primary or secondary dengue infections exhibit a fall in the leukocyte count
associated with a rise in the percentage of lymphocytes and this finding is in
parallel to marrow suppression during acute phase.9,10 Most prominently increase
in the percentage and total number of lymphocytes and an increase in the
percentage and number of atypical lymphocytes.11 Atypical lymphocytes are
frequently seen in a variety of viral illnesses, including infectious mononucleosis,
herpes, rubella, influenza and viral hepatitis. The exact function of atypical
lymphocytes is unclear, they incorporate increased amounts of [3H] thymidine into
deoxyribonucleic acid and are similar in appearance to lymphocytes which undergo
blast transformation after stimulation with mitogens (such as
phytohemagglutinin).12 Thus, it is possible that atypical lymphocytes represent a
response to non-specific viral stimulation or to specific viral antigens due to
recognition followed by transformation.11 The potential importance of this in DHF
stems rom studies which have demonstrated that the dengue virus in vitro invades
and replicates poorly in resting lymphocytes but well in stimulated transformed B
lymphoblast cells.13 We observed a marked increase in concentration of

Plamacytoid Lymphocytes and even Plasma cell in five of our patients. Similar
findings have been reported by John Gawoski and Winnie Ooi14 and it mainly
represents that serum immunoglobulin production is enhanced during dengue viral
infection, these are mostly against the specific serotype and obviously not
protective to the infections caused by other serotypes.15 It has been suggested that
the atypical lymphocytes in secondary dengue infection can be representative of
augmented immune response in an attempt to control the spread of dengue virusinfected cells.16 Differences between the samples at the time of
admission and collected later during convalescence were also found in the
concentrations of eosinophils. Similar findings have been reported for other viral
infections, where, in response to inflammation during the acute phase, eosinophil
concentrations fell. During convalescence, the eosinophil concentrations rise to
normal or even more.17 Eosinophilia during the convalescent phase of dengue fever
has been reported consistently in a number of studies.18,19 An increase in the total
number of basophils is also seen in the convalescent period. The increase in
basophils may be due to element of recovery from the bone marrow suppression
during convalescence.9 The most significant laboratory abnormality seen in our
patients was thrombocytopenia, as observed in other studies.20 This is thought to
be due to depression of bone marrow observed in acute stage of dengue virus
infection.9 Other explanations are direct infection of the megakaryocytes by virus
leading to increased destruction of the platelets or the presence of antibodies
directed against the platelets.21 Thrombocytopenia may result from by destruction
of peripheral platelet or bone marrow megakaryocytes by viruses which
consequently reduce the platelet production.22 Haemorrhagic manifestations are
very common with severe thrombocytopenia and severity of haemorrhagic
tendency correlates with the platelet counts.11 Coagulopathy is also frequent in
most patients with dengue fever. In our study, prolongation of aPTT was quite
common, whereas PT was normal in majority of our patients (76%) where as the
samples labelled as having prolonged timing were (24%), similar results have been
reported in other studies.23,24 CONCLUSION Peripheral blood parameters are very
helpful for disease monitoring and can be useful in prediction of prognosis. These
indicators, if rightly and timely assessed can be of value for better care of
complicated cases. REFERENCES
Dengue infection is a common tropical disease with wide range of clinical
illness ranging from a nonspecific viral syndrome to severe and fatal
hemorrhagic disease. However, the fever is the most common presentation
of this illness. Our finding support that all of our subjects present high grade fever (average > 38 degree Celsius) 3 - 5. Hence, the history of high
fever, which is not relieved by ingestion of self prescribed
acetaminophen could be a useful point for the general practitioner in the
tropical countries that faced with those patients in the endemic season.
Here, we detected that the total white count is not useful for diagnosis.
The average level of the total white count is not high and the range is
wide. Indeed, the leukocyte disorder in the patients with dengue infection
is described as lymphocytosis3 - 5. Our finding also support this fact, high
average lymphocyte count can be observed. However, the range of

lymphocyte as well as neutrophil count is wide, therefore, the implication


directly to the dengue infection is limited. Concerning the correlation
between body temperature and total white blood cell count and
differential white blood cell count, non - significant correlation could be
detected. Therefore, the fever and the lymphocytosis in the patients with
dengue fever is only a co - presentation. Although the fever is believed to
due to the cytokine excreted from the lymphocyte the lymphocyte count is
not relating to the body temperature level. The explanation might be due
to the fact that the patients usually got the antipyretic drug before
visiting the physician, which can be the modifying factor for the body
temperature. Another fact is that the immunological response of the host
is an important factor in the course of disease 8. The main response to
this infection is the lymphocytosis, as detected in this study. However,
mainly the qualitative not quantitative response might be more
responsible for the host versus pathogen interaction mechanism, fever.
Indeed, sometimes those qualitative responses resulted in the severe
hypovolemic shock known as dengue shock syndrome 9 - 10. However it
cannot conclude that fever and leukocyte disorder are without any
relation, justified by the data showing absence of correlation since both
fever and WBC changes still could relate to a single pathophysiologic
process (e.g. cytokine production). It can be said in another way that the
pathobiology change of white blood cell in dengue infection is very
complex and required further comprehensive study to assessment.
Similarly, the conclusions that fever is useful in diagnosis of dengue or
that total white
Fever and leukocyte in dengue
Advance Laboratory Medicine International 2012; 3(1): 9 - 13.
12
12
count is not useful for diagnosis cannot be reached based on the data
presented, as there are no comparison groups without dengue infection.
Furthermore, since inclusion in the study was based on presentation to a
referral center, it is impossible to draw conclusions about the overall
frequency of fever in dengue infections. To catch up with those mentioned
limitations, the authors plan for future collaborative study to answer the
research question in this report.
CONCLUSION
Conclusively, the fever is common in the patient with dengue infection.
The lymphocytosis, not leukocytosis is the common leukocyte disorder in
these patients. The correlation between body temperature and total white
blood cell count and differential white blood cell count is poor and not
significant. Therefore, the fever and the leukocyte disorder in the patients
with dengue fever is only a co - presentation without any relation.

ACKNOWLEDGEMENT Total White Blood Cells Count: In case of dengue, this


test will reveal leukopenia. The presence of leukocytosis and neutrophilia excludes
the possibility of dengue and bacterial infections (leptospirosis,
meningoencephalitis, septicemy, pielonephritis etc.) must be considered.

Dengue is transmitted in 100 tropical countries, with close to 100 million people
presenting with symptoms of mild or severe disease. The 2009 WHO guidelines
establish that dengue diagnostic testing should be used to differentiate dengue
from other acute febrile illnesses. For many years, IgM ELISA seroconversion and
virus isolation were the gold standards for confirmation of a dengue case. However,
most patients present with symptoms before IgM antibodies are detectable,
requiring a second sample be obtained for conclusive results, and virus isolation
takes at least 7 days for a confirmation. Because dengue patients usually present
with symptoms when they are viremic, there have been significant efforts in
developing and improving tests for detection of dengue virus during the acute
phase of illness. The CDC DENV-1-4 Real Time RT-PCR Assay was approved by the
FDA in May 2012 for diagnosis of dengue. The assay has been configured to
determine the subtype of the infecting virus and for detection of all strains currently
circulating worldwide. Our clinical study of 371 cases showed a positive percent
agreement of 98.04% between the CDC DENV-1-4 RT-PCR assay and IgM
seroconversion, and 97.92% between RT-PCR and E gene sequencing. Following the
implementation of the CDC DENV-1-4 RT-PCR assay in 110 laboratories, we have
assessed the reproducibility of the assay worldwide. We have also compared the
sensitivity of this test with that of 10 other reference and commercial molecular
assays to provide a better understanding of their sensitivity and usefulness for early
diagnosis. These developments make it now feasible to diagnose dengue on a single
sample during acute illness globally. To evaluate this new diagnosis paradigm we
have assessed the sensitivity of RT-PCR in a clinical study of 1234 dengue
specimens. Our results show that early diagnosis of dengue is feasible during the
acute phase of the disease, without having to obtain two samples.
11 COMPETITION OF DENGUE VIRUS SEROTYPE 2 CLADES FROM NICARAGUA
REVEALS A FITNESS ADVANTAGE IN AEDES AEGYPTI MOSQUITOES Claire A. Quiner1,
Alex Ciota2, Poornima Parameswaran1, The four dengue virus (DENV) serotypes are
transmitted by Aedes aegypti and Ae. albopictus mosquitoes, causing up to 96
million cases of dengue worldwide each year. We have previously reported on a
clade replacement within the Asian-American genotype of DENV2, in which the NI-1
clade was replaced with clade NI-2B over three epidemic seasons in Managua,
Nicaragua. Here, we studied the replicative ability of DENV2 NI-1 and NI- 2B viruses
in mosquito cell lines and F2-F4 Ae. aegypti mosquitoes reared from eggs collected
in Managua. Several different pairs of NI-1 and NI-2B strains were assessed in vitro
and in vivo via a competition assay to yield an indicator of replicative fitness. In
these experiments, equal genomic copy numbers of clinical isolates of NI-I and NI2B viruses were mixed and used to infect Aag2 Ae. aegypti cells or inoculated into
Ae. aegypti mosquitoes via a blood meal. At different time-points post-infection

(p.i.), cell supernatant or mosquitoes were harvested, viral RNA was extracted, and
a 1-kb region in the NS1 gene containing single nucleotide polymorphisms (SNPs)
that distinguish clade NI-1 from NI-2B was amplified by RT-PCR. Amplicons were
sequenced, and the proportion of each strain was determined using PolySNP PERL
script software, which measures the height of the peaks of the SNPs from a
sequencing chromatogram. Co- infections of the NI-1 and NI-2B viruses in Aag2 cells
consistently showed
a significant replicative fitness advantage of NI-2B over NI-1. The mean proportion
of NI-2B over NI-1 significantly increased from the input in the carcasses of infected
mosquitoes at 3, 6, 7 and 14 days p.i.. Additionally, NI-2B disseminated more rapidly
than NI-1 viruses into mosquito legs. Importantly, NI-2B viruses were also found in
greater abundance in the salivary glands at 7 days p.i., and the trend remained
through the last time-point collected on day 21 p.i.. Furthermore, the NI-2B strains
were the dominant virus in a greater percentage of mosquito carcasses (75%,
55/73), legs (71%, 27/38), and salivary glands (76%, 23/30) on day 7 p.i. and other
time-points from 3-21 days p.i.. Finally, NI-2B was dominant in the legs (80%, 12/15)
and salivary glands (56%, 18/32) at 14 days p.i.. Together, these results
demonstrate that NI-2B clinical isolates have a modest early fitness advantage over
NI-I viruses in multiple tissues of the native vector, Ae. aegypti, which could have
contributed to the clade replacement observed in Managua. 12 DENGUE VIRUS
INFECTION OF DERMAL DENDRITIC CELLS INCREASES DURING ANTIBODYDEPENDENT ENHANCEMENT AND IS MODULATED BY AEDES AEGYPTI MOSQUITO
SALIVA Michael Schmid1, Matthew T. Aliota2, Laura D. Kramer2, Eva Harris1
1Division of Infectious Diseases and Vaccinology, School of Public Health, University
of California Berkeley, Berkeley, CA, United States, 2Wadsworth Center, Arbovirus
Laboratory, New York State Department of Health, Slingerlands, NY, United States
The four dengue virus serotypes (DENV1-4) are transmitted via the bite of infected
Aedes aegypti or Ae. albopictus mosquitoes, causing dengue, the most prevalent
arboviral disease in humans. Primary DENV infection confers life-long protective
immunity to the same serotype, while secondary infection with a distinct DENV
serotype is a major risk factor for severe disease via serotype cross-reactive
enhancing antibodies (antibody-dependent enhancement; ADE) or T cells. Skin
dendritic cells (DCs) are sentinels of the immune system and, with monocytes and
macrophages, are targets of DENV in humans; yet, little is known about initial DENV
replication in the skin or subsequent viral spread. We established a novel
intradermal (i.d.) infection model of C57BL/6 mice lacking the IFN-/ receptor
(Ifnar-/-) with DENV2 strain D220 inoculated via the ear. Intracellular staining with
monoclonal antibodies directed against DENV NS3 and envelope proteins revealed
DENV replication in epidermal Langerhans cells and, for the first time, in dermal
CD11b+ DCs and CD103+ DCs by flow cytometry. In a model of ADE, inoculation of
sub-neutralizing DENV-specific monoclonal antibodies enhanced infection of dermal
DCs 48 hours after i.d. DENV infection, increased morbidity, and induced mortality
of Ifnar-/- mice. While probing for blood, mosquitoes eject saliva into the skin.
Salivary gland extracts from female Ae. aegypti co-injected i.d. with DENV2 reduced
morbidity of mice during primary, non-enhanced conditions, but exacerbated
disease during ADE infection. We are investigating the impact of ADE and mosquito

saliva on DENV infection in the skin, virus spread, the immune response, and
disease severity. These studies will improve understanding of DENV transmission,
systemic spread, and factors that influence disease outcome and may inform future
vaccine development.
13 PROPHYLACTIC AND THERAPEUTIC HUMAN MONOCLONAL ANTIBODIES AGAINST
DENGUE United States Dengue virus (DENV) is the leading cause of mosquito-borne
viral disease in humans worldwide. Although primary infection with one of the four
serotypes of DENV (DENV1-4) results in lifelong immunity, secondary infection with
a different DENV serotype increases the risk for severe dengue, a potentially lethal
disease. Antibody-dependent enhancement (ADE) theory posits that cross-reactive,
sub-neutralizing levels of anti- DENV antibodies facilitate increased viral entry into
Fc receptor-bearing cells, thereby increasing viral load and disease severity. The
presence of multiple DENV serotypes, as well as the ADE phenomenon, has
hindered vaccine and drug development. As a part of multi-center collaborative
project, we have begun identifying and characterizing human monoclonal antibodies
(hMAbs) to DENV that have led to identification of three classes of potentially
therapeutic hMAbs consisting of 1) highly-neutralizing, serotype-specific hMAbs that
bind a novel hinge epitope on the envelope (E) protein; 2) strongly-neutralizing,
serotype cross-reactive hMAbs that target E; and 3) serotype cross-reactive MAbs
that suppress the activity of enhancing Abs. We have previously reported that
enhancement- suppressing MAbs engineered to be unable to bind the Fc
receptor are therapeutically effective in an ADE mouse model of lethal disease
when they are highly neutralizing AND displace pre-existing enhancing Abs. Here,
we focus on the prophylactic and therapeutic efficacy of type-specific and crossreactive highly neutralizing hMAbs in our dengue AG129 mouse model (interferon/ and - receptor-deficient mice). Mice administered a type-specific hMAb to
DENV1 (hMAb 1F4) or DENV2 (hMAb 2D22) 24 hours prior to a sublethal DENV1 or
DENV2 infection, respectively, showed robust reduction of viral load in serum and
various organs compared to mice receiving the isotype control. Mice receiving the
cross-reactive hMAbs 1N5 or 1C19 showed reduction in both DENV1 and DENV2
viral load. Evaluation of prophylactic and therapeutic efficacy against lethal
challenge and against other DENV serotypes is underway. Further study of these
and other hMAbs should identify the simplest mixture of hMAbs that can be used as
a single product for prophylactically and/or therapeutically preventing disease
caused by the four DENV serotypes and may identify epitopes useful for future
structure-based rational vaccine design.