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Importance of concurrent testing of dengue specific serological markers and platelet enumeration.

INTRODUCTION: Dengue is an acute febrile arboviral disease found largely in the tropics and subtropics. There are four distinct but antigenically related serotypes of dengue virus and its transmission is by mosquito principally Aedes aegypti. (1) Infection with one serotype induces lifelong immunity against reinfection by the same serotype but only partial protection against other serotypes. (2,3) Dengue infections vary in severity ranging from asymptomatic infection to dengue fever and the severe disease such as dengue hemorrhagic fever/dengue shock syndrome. (4) It carries a significant mortality if the diagnosis and treatment are delayed. (5) So it is imperative to have a rapid, sensitive and easy to use diagnostic assay for early detection of the disease. (6)

Laboratory confirmation relies on isolation of virus in cell culture, identification of viral RNA but these methods have restricted scope as routine procedure. Serological tests based on determination of dengue specific IgG/IgM antibodies have been the mainstay of diagnosis because of their ease of use. But the sensitivity and specificity of these assays is strongly influenced by the quality of antigen used. (7) Also these assays have poor sensitivity and specificity in first four days of illness. (8) Recently detection of secreted NSI antigen in the bloodstream has emerged as sensitive and specific diagnostic tool during acute phase of illness. (9)

In peripheral health clinics of resource poor countries, platelet count is also considered to be the reliable parameter to support the diagnosis of dengue infection as this can be determined by microscopy. (10) Considering this fact the present study was conducted to determine the association between dengue specific serological markers and platelet counts.

MATERIAL AND METHODS: The present study was conducted at a tertiary teaching care center from September 2011 to December 2011 after receiving approval from institutional ethical committee. A total of 233 serum samples were screened from clinically suspected cases of dengue fever. The dengue serology was performed according to manufacturer instructions using SD Dengue Duo by standard diagnostics (Korea). This is an invitro immunochromatographic one step assay to detect NSI antigen and differential IgG/ IgM antibodies to dengue virus. The individual reports were available in 1-2 hours. Platelet counts of all positive cases were recorded. Platelet counts of all 100 dengue negative cases were recorded as well. Apart from inbuilt controls in the test; no independent controls were used for IgG/ IgM and NSI antigen.

RESULTS: Of the total 233 serum samples tested, 78 samples turned positive for one or more of three serological markers. Of 78 samples NS1 antigen was detected alone in 26 cases (33.33%), IgG+IgM was detected in 7 cases (8.97%), NS1+IgG in 7 cases (8.97%), NS1+IgG+IgM in 27cases (34.6%). Platelet count less than 100000/ul was detected in 48 cases (61.53%). Out of the 100 cases that were negative for dengue infection thrombocytopenia was detected in 28 % cases.

DISCUSSION: In this study two parameters were studied. First the distribution of dengue specific serological markers was analyzed. In this study, out of total 78 positive cases, 26 cases were exclusively positive for NSI antigen. These were the cases with primary infection and could transmit the infection if bitten by mosquito. Srivastava et al also reported the NSI antigen positivity to be around 23.3 %. (11) Similarly there were 7 cases that were positive for NS1+IgG. Without NSI screening these would have been overlooked and labeled as negative. This is because IgG alone is considered to be less reliable marker for dengue infection and its positivity is due to subclinical infection and its levels are higher in endemic areas (12). So inclusion of NS1 antigen testing in test panel helped in detecting these 33 cases (26 NSI antigens, 7 IgG+NSI antigen) which would have been missed without NSI antigen screening. NSI testing does not require repeat testing due to its high specificity and can reliably diagnose the disease from day 1 of illness.

There were 4 cases who were exclusively IgM positive, these patients had a primary infection presenting during later phase of illness. Similarly there was 1 case that was positive for IgG only, probably the patient presented during secondary infection. 7 Cases were positive for IgG+IgM; these were cases with primary and secondary infection who presented in later stage of illness. Furthermore there were 155 cases that were negative for serological markers. In such patients dengue infection should be ruled out by testing for viral replication by cell culture or RNA detection methods, immunofluorescence, immunohistochemistry. (13)

Secondly in this study correlation between platelet count and dengue serological marker was studied. Thrombocytopenia occurred in 61.5% cases with acute dengue infection. Platelet counts were low in NS1 positive cases. The counts were lowest in patients with NSI+IgM+IgG. Statistical analysis of NSI positive cases only (33.33%) with NSI+IgG+IgM positivity (34.6%) showed that thrombocytopenia was significantly associated with NSI+IgG+IgM positive cases than with NS1 only ([chi square] = 3.84, p value=0.049).The role of antibodies is well defined in causing disease pathogenicity and thrombocytopenia.

The platelet counts were low in 28% with negative triple serological marker. But association of thrombocytopenia with dengue positivity was more significant than in dengue negative cases ([chi square] = 20.15, p value=0.0001). Thrombocytopenia is observable in other viral diseases, collagen vascular disorders, drug induced thrombocytopenia.14

The various limitations in the study were that no gold standard test was used for authentication as facilities for these methods were not available in our study. Also precise details about the duration of fever were not available which would have helped us to pick additional NSI positive cases.

So we conclude that concurrent detection of NSI antigen with IgG/IgM antibodies will help in detection of additional dengue positive cases which escape detection and its testing must be included in test panel. The use of one step ICT based test would enable rapid and easy detection of dengue infection in resource poor settings. Also simultaneous estimation of thrombocytopenia will help clinician to offer appropriate therapy.

DOI: 10.14260/jemds/2015/1559

BIBLIOGRAPHY:

(1) Guzman, M. G. & Kouri, G. Dengue: an update. Lancet Infect. Dis. 2002; 2:33-42 (2002).

(2) Halstead, S. B. Pathogenesis of dengue: challenges to molecular biology. Science 1988; 239:476481.

(3) Kurane, I. Dengue hemorrhagic fever with special emphasis on immunopathogenesis. Comp. Immunol. Microbiol. Infect. Dis. 2007; 30:329-334.

(4) Tricou V, Vu HT, Quynh NV, Nguyen CV, Tran HT, Farrar J, Wills B, Simmons CP. Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses. BMC Infect Dis. 2010; 10:142.

(5) Gibbons RV, Vaughn DW. Dengue: an escalating problem. BMJ 2002; 324: 1563-6.

(6) Datta S, Wattal C. Dengue NS1 antigen detection: A useful tool in early diagnosis of dengue virus infection. Indian J Med Microbiol 2010; 28:107-10.

(7) Rosanna W. Peeling et al. Evaluation of diagnostic tests: dengue. Nature Reviews Microbiology December 2010:S30-S37.

(8) Peeling RW, Artsob H, Pelegrino JL, Buchy P, Cardosa MJ, Devi S, et al. Evaluation of diagnostic tests: Dengue. Nat Rev Microbiol 2010; 8: S30-7.

(9) Alcon S, Talarmin A, Debruyne M, Falconar A, Duebel V, Flamand M. Enzyme-Linked Immunosorbent Assay Specific to Dengue Virus Type 1 Nonstructural Protein NS1 Reveals Circulation of the Antigen in the Blood during the Acute Phase of Disease in Patients Experiencing Primary or Secondary Infections. J Clin Microbiol 2002; 40:376-81.

(10) Young PR, Hilditch PA, Bletchly C, Halloran W. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. J Clin Microbiol 2000; 38:1053-7.

(11) Shrivastava A, Dash PK, Tripathi NK, Sahni AK, Gopalan N, Lakshmana Rao PV. Evaluation of a commercial dengue NS1 enzyme-linked immunosorbent assay for early diagnosis of dengue infection. Indian J Med Microbiol 2011; 29:51-5.

(12) Pei-Yun S, Jyh-Hsiung H. Current Advances in Dengue Diagnosis. Clin Diagn Lab Immunol 2004; 11:642-50.

(13) Centers for Disease Prevention and Control. Dengue: Laboratory criteria for diagnosis for case definitions. From http://www.cdc.gov/dengue/ clinicalLab/case Def.

(14) Peters CJ. Infections caused by arthropod- and Rodent -borne viruses. In: Fauci AS, editor. Harrison's principles of Internal Medicine. 17 Th Ed. New York: McGraw-Hill Medical Publishing Division; 2008. p. 1226-39.

Manju Bala [1], Prerna Aggarwal [2], Maninder Kaur [3], Ashwini Manhas [4]

AUTHORS:

[1] Manju Bala

[2] Prerna Aggarwal

[3] Maninder Kaur

[4] Ashwini Manhas

PARTICULARS OF CONTRIBUTORS:

[1] Assistant Professor, Department of Microbiology, Gian Sagar Medical College & Hospital, Banur.

[2] Associate Professor, Department of Microbiology, Gian Sagar Medical College & Hospital, Banur.

[3 ]Assistant Professor, Department of Microbiology, Gian Sagar Medical College & Hospital, Banur.

[4]. Assistant Professor, Department of Microbiology, Gian Sagar Medical College & Hospital, Banur.

FINANCIAL OR OTHER COMPETING INTERESTS: None

NAME ADDRESS EMAIL ID OF THE CORRESPONDING AUTHOR:

Dr. Manju Bala, Assistant Professor, Department of Microbiology, H. No. 4566-B, SEC-70, Mohali.

E-mail: suvanarora15@gmail.com

Date of Submission: 03/12/2013.

Date of Peer Review: 04/12/2013.

Date of Acceptance: 12/12/2013.

Date of Publishing: 01/08/2015

Table 1: Association of dengue specific
serological marker with platelet count


Dengue Specific   Total        Platelet Count Less
Serological       Cases (%)    Than 100000/ul (%)
Marker

NS1 antigen       26(33.33%)        16(61.5%)
IgM                4(5.12%)          1(25%)
IgG                1(1.28%)             0
NS1+IgG+IgM       27(34.6%)         23(85.1%)
NS1+IgG            7(8.9%)          4(57.14%)
NS1+IgM            6(7.69%)         2(33.3%)
IgG+IgM            7(8.9%)          2(28.5%)
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Article Details
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Title Annotation:ORIGINAL ARTICLE
Author:Bala, Manju; Aggarwal, Prerna; Kaur, Maninder; Manhas, Ashwini
Publication:Journal of Evolution of Medical and Dental Sciences
Article Type:Report
Date:Aug 3, 2015
Words:1598
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