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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 5  |  Issue : 2  |  Page : 88-92

Evaluation of two diagnostic methods for dengue virus infection and its correlation with thrombocytopenia


Department of Microbiology, Government Medical College, Surat, Gujarat, India

Date of Web Publication14-Apr-2016

Correspondence Address:
Tanvi H Panwala
Department of Microbiology, Government Medical College, Surat - 395 001, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.180426

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  Abstract 

Context: Early definitive diagnosis of dengue virus (DENV) infections is essential for the timely management of dengue infections. Aims: The aim of the present study is to compare results of both tests (IgM enzyme-linked immunosorbent assay [ELISA] and nonstructural protein 1 [NS1]) and to prove that use of NS1 with IgM ELISA tests improves the dengue laboratory diagnosis. Settings and Design: Tertiary Care Hospital, Gujarat and retrospective cross-sectional study. Subjects and Methods: One thousand three hundred forty-three blood samples were screened from clinically suspected cases of dengue fever reporting at Tertiary Care Hospital, Gujarat from August to November 2014. The samples were divided into three study groups according to the tests performed. In Group 1, 840 samples were subjected for dengue IgM ELISA only. Group 2 comprised 379 samples tested for NS1 antigen (Ag) only. Group 3 comprised 124 samples tested by both methods NS1 Ag detection test and IgM ELISA. Statistical Analysis Used: As the study has compared three different groups, Statistical proportion method used and data entry was done in Microsoft office excel version 2010. Results: In Group 1, 145 patients (76.3%) found positive between 6 and 10 days of fever. More number of cases (89.4%) gave positive reaction in NS1 Ag detection test at <5 days of fever in Group 2. In Group 3, of the forty positive samples, 19 (47%) samples gave positive reaction in NS1 Ag detection test and negative in IgM ELISA with <5 days of fever history. Ten patients (25%) showed positive IgM ELISA and negative for NS1 at more than 10 days of fever. Eleven patients (27.5%) showed both the tests positive between 6 and 10 days of fever. Conclusions: If NS1 Ag detection test used in combination with IgM ELISA on a single serum sample of suspected patients will increase the sensitivity of detection, especially in areas with higher prevalence of secondary DENV infections.

Keywords: Antigen, dengue, IgM enzyme-linked immunosorbent assay, thrombocytopenia


How to cite this article:
Panwala TH, Mulla SA. Evaluation of two diagnostic methods for dengue virus infection and its correlation with thrombocytopenia. Int J Health Allied Sci 2016;5:88-92

How to cite this URL:
Panwala TH, Mulla SA. Evaluation of two diagnostic methods for dengue virus infection and its correlation with thrombocytopenia. Int J Health Allied Sci [serial online] 2016 [cited 2019 Sep 20];5:88-92. Available from: http://www.ijhas.in/text.asp?2016/5/2/88/180426


  Introduction Top


Dengue has become a major global public health problem in tropical and subtropical region, especially in developing countries. Approximately 2.5 billion people, living mainly in urban areas, are estimated to be at risk of acquiring dengue infection (DI).[1] It affects up to 100 million people each year, with 500,000 cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) and 30,000 deaths, mostly among children.[2] It is challenging for us to control the DI as it requires not only effective control of vectors but also rapid, accurate, and early diagnosis of DI, so it can assist in patient management by directing clinical attention to the appearance of major warning signs of severe or life-threatening complication, e.g., rapid rise of hematocrit, poor peripheral perfusion. Other advantages for early diagnosis of dengue case are, it prevents unnecessary, expensive usage of antibiotics and provides important data on the epidemiology and health burden of dengue. Several laboratory methods such as viral isolation, genomic RNA, antigen (Ag), and antibody detection methods are available to diagnose DIs. Virus isolation and genomic RNA detection need a specialized laboratory with trained personnel which are not available in hospital setting. Serologic test which detect dengue specific IgM immunoglobulin and IgG immunoglobulin by enzyme-linked immunosorbent assay (ELISA) are the most commonly used methods.[3] Recently, a new biomarker dengue virus (DENV) nonstructural protein 1 (NS1) Ag detection is also important serologic method for early diagnosis of DI. NS1 Ag, highly conserved glycoprotein produced in both membrane-associated and secretion forms, is abundant in the patient's serum during early stage of infection.[4] During the acute stage, the presence of IgM antibodies alone suggests that primary infection and detection of newly formed IgM antibodies are possible only after the viremia phase ends or fever subsides.[1] IgM antibodies level increases rapidly and appears to peak about 2 weeks after the onset of symptoms, then decreases to undetectable levels over 2–3 months. In contrast to IgM, NS1 Ag can be detected before the formation of antibodies.[5],[6] It is detectable in blood from the 1st day after the onset of fever up to day 9, it is still detectable even when viral RNA is negative by reverse transcription polymerase chain reaction (RT-PCR) (Alcon et al., 2002). Several studies revealed the detection rate of NS1 Ag is higher in acute primary dengue than in acute secondary DI.[7],[8],[9] Detection of dengue NS1 Ag represents the newer approach for the diagnosis of acute dengue cases. Hence, the aim of the present study is to compare results of both tests (IgM ELISA and NS1) and to prove that use of NS1 with IgM ELISA tests improve the dengue laboratory diagnosis.


  Subjects and Methods Top


Patients and study design

This was a retrospective cross-sectional study. One thousand three hundred forty-three blood samples were screened from clinically suspected cases of dengue fever (DF) (presented with the sign and symptoms of fever, headache, and joint pain which were suggestive of the dengue or DHF or DSS) reporting at Tertiary Care Hospital, Surat, Gujarat, from August to November 2014. WHO criteria were followed for inclusion or exclusion of a case of DI and their categorization as DF/DHF.[9] A specially designed, semi-structured questionnaire form was used to collect the data on the demographic factors such as age, sex, and residence, in addition to the data on the history of the illness, the possible risk factors and the results of the investigations. The samples were divided into three study groups according to the tests done.

Group 1 comprised 840 samples obtained from patients of suspected DF cases and samples were subjected for dengue IgM ELISA only. Group 2 comprised 379 samples obtained from suspected dengue patients and tested for NS1 Ag only. Group 3 comprised 124 patients and samples were tested by both methods NS1 and IgM ELISA. The collected data were part of the diagnostic services, so used in the present study.

Dengue IgM enzyme-linked immunosorbent assay

All the samples were tested for the presence of dengue specific IgM antibodies using MAC-ELISA, developed and commercialized by the National Institute of Virology, Pune, and recommended by the National Vector Borne Disease Control Programme. Tests were done and results were read as per the literature provided.

Dengue nonstructural protein 1

The test was performed using new Platelia™ Dengue NS1 Ag assay (Bio-Rad, Marnes-la-Coquette, France). This is a single-step sandwich-format microplate enzyme immunoassay to detect DENV NS1 Ag in human serum or plasma. Murine monoclonal antibodies (MAbs) are used to capture and detect NS1 Ag. Samples and controls were directly and simultaneously incubated with the conjugate for 90 min at 37°C within the microplate wells sensitized with MAb. If NS1 Ag presents in the sample, an immune-complex MAb-NS1 – MAb/peroxidase will be formed. After a washing step, the presence of immune-complex was demonstrated by distribution in each well of a chromogenic solution initiating a color development reaction. After 30 min of incubation at room temperature, the enzymatic reaction was stopped by adding of an acid solution. The optical density reading obtained with a spectrophotometer set at 450/620 nm is proportional to the amount of NS1 Ag present in the sample. The presence of NS1 Ag in an individual sample was determined by comparing the optical density reading of the sample to the optical of the calibrator. Sample results were expressed in terms of index value. According to the manufacturer's recommendations, samples were considered (i) nonreactive for DENV NS1 Ag if index value <0.5, (ii) equivocal for DENV NS1 Ag if between 0.5 and 1.0, and (iii) reactive for DENV NS1 Ag if index value 1.0 or more was obtained.

Platelet count

From ethylenediaminetetraacetic acid blood samples, platelet count was done and interpreted as normal, when the count was between 150,000 and 450,000 and DHF, when the count was <100,000/cmm (WHO cut off for platelet count for DHF).


  Results Top


In Group 1, of the 840 samples, 190 (22.6%) were positive for IgM only [Table 1]. In Group 2, of the 379 samples, 114 (30%) were positive for NS1 Ag only. In Group 3, of 124 samples, 29 (23.3%) samples were positive either by NS1 or IgM ELISA and 11 (8%) samples were positive by both testing methods.
Table 1: Seropositive cases by testing with different parameter for dengue infection

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[Table 2] shows the distribution of seropositive cases according to their days of fever. In Group 1 where IgM ELISA test was done, 145 patients (76.3%) found positive between 6 and 10 days of fever. In contrast to IgM ELISA, more number of cases (89.4%) gave positive reaction in NS1 Ag detection test at <5 days of fever. In Group 3, of the forty positive samples, 19 (47%) samples gave positive reaction in NS1 Ag detection test and negative in IgM ELISA with <5 days of fever history. Ten patients (25%) showed positive for IgM ELISA and negative for NS1 at more than 10 days of fever. Eleven patients (27.5%) showed both the test positive between 6 and 10 days of fever.
Table 2: Distribution of seropositive cases according to their days of fever

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[Table 3] shows age- and sex-wise distribution of positive cases and more number of cases was found between 16 and 30 years. In 114 NS1 positive cases, thrombocytopenia was evident in 72 (63%) cases. In contrast, when IgM antibodies were considered for the diagnosis of DI, thrombocytopenia was noted in 147 of 190 (77.3%) cases and of 11 cases positive for both NS1 and IgM, thrombocytopenia was detected in 8 (72.7%) cases.
Table 3: Distribution of seropositive cases according to their age and sex

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[Chart 1] shows the correlation of platelets count in seropositive cases which were tested by different serological methods.




  Discussion Top


Demonstration of dengue specific IgM/IgG is the mainstay for the diagnosis of DI since from long time. The antibodies begin to appear on the 5th day of fever in primary infection.[10] Sometimes, IgM/IgG antibodies cannot be detected before the 3rd day of fever in secondary DI.[11] Therefore, there is some lag period both in primary and in secondary dengue when test will give negative results in antibodies specific tests. The NS1 Ags is a new parameter nowadays available for diagnosis of DI from day 1 of fever both in primary and secondary infections. The results of various dengue specific parameters are shown in [Table 1] and [Table 2]. Of the 379 cases which were tested for NS1 Ag only, 114 were positive only for NS1 Ag and of the 114, 102 cases were positive within first 5 days of fever. Of the 124 cases which were tested by both methods (IgM, NS1), 19 cases were positive for NS1 Ag and negative in IgM ELISA, so it is stated that 47% cases would miss if NS1 Ag test had not included in the testing panel.[12],[13]

Serological IgM ELISA and IgG ELISA are currently widely used diagnostic method for DF in routine laboratory practice. Among the two antibodies, IgG is less reliable marker in the diagnosis of DI as both clinical and subclinical infection can produce IgG, which may persist for several years affecting the interpretation of testing results.[14] IgG levels could be higher in endemic areas due to continues biting from mosquitoes so, dengue specific IgM is very good marker for acute cases, and it may also give idea about secondary DI.[10] In the endemic areas, confirmation of DI mainly depends upon the rising titer in paired serology. However, repeat testing for the same infection when first sample was negative or for confirmation of DI is almost not possible in routine clinical practice. When NS1 Ag gives positive results in first 4 days of illness, there is no need of repeat testing as NS1 is highly specific marker for the diagnosis of DI.[10]

NS1 Ag is highly conserved for all dengue serotypes, circulating high levels during first few days of illness. There is no cross reaction of dengue NS1 protein with those of other related flavivirus as it noticed in IgM ELISA.[15] Plasma viremia levels would be associated with the detection of NS1, such as virions, is the product of infected cells. Viremia level is significantly higher in patients who were positive for NS1 versus those negative for NS1.[16] One study have noted that NS1 level of dengue-2 in plasma was significantly higher in DHF than in patients with DF within 72 h of illness.[17] NS1 Ag detection test can be implemented in routine diagnostic laboratories and be easily automated. It limits the disease expansion by early screening of patients. This study concluded that NS1 alone or in combination with IgM ELISA gave better positivity as out of 114 seropositive cases, 102 (89%) samples were positive in first 5 days of illness if only NS1 Ag tested and in combination with IgM ELISA, seropositivity increased from 25% to 47%. This study also tried to find the association of dengue parameter with thrombocytopenia. Of the 344 seropositive cases, 249 (72%) showed thrombocytopenia. In 114 cases that were positive for NS1, thrombocytopenia was evident in 72 (63%) cases. In contrast, when IgM antibodies were considered for the diagnosis of DI, thrombocytopenia was noted in 147 of 190 (77%) cases and out of 40 seropositive cases tested by both methodologies, thrombocytopenia was detected in 30 (75%) cases. Association of thrombocytopenia with IgM was found to be higher. In contrast to the study of Kulkarni et al. and Saroj et al., association of thrombocytopenia with NS1 was found to be higher that is 73.7% and 71.4%, respectively. Higher association of IgM and thrombocytopenia in the present study may be due to most of the cases were referred late in tertiary care hospital from various places. This may result in delayed diagnosis and thus increasing morbidity.

The limitation of the present study is that the paired sera were not used, and this lack of serological data made it impossible to distinguish between primary and secondary DI. Another limiting factor was unable to analyze the NS1 Ag in severe DHF/DSS because lack of clinical data from physician. With the expansion of geographic range of DF and increasing the number and severity of disease, strategy should be prepared for diagnosis of DI in clinical diagnostic laboratories so early identification of infection will helpful for patients management, reducing the time between detection of first cases, based on NS1 Ag detection, and the notification of public health authorities, including vector control teams.


  Conclusions Top


The present study concluded that as in comparison to MAC-ELISA, NS1 Ag is an effective tool for the diagnosis of DENV infection, especially with in first 4 days of illness. Early detection of NS1 Ag can be useful tool for confirmation and management of DHF/DSS cases because tests such as viral culture and RT-PCR may not affordable in laboratories with limited resources. NS1 Ag if used in combination with IgM ELISA on a single serum sample of suspected patients can improve the diagnosis of acute dengue cases and even treatment and control of dengue viral infection.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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  [Table 1], [Table 2], [Table 3]



 

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