|Year : 2017 | Volume
| Issue : 2 | Page : 256-261
Clinical and laboratory profile of dengue viral infections in and around Mangalore, India
Tina Damodar1, Meena Dias1, Reeta Mani2, KA Shilpa2, Ashwini Manoor Anand2, V Ravi2, Jutang Tiewsoh1
1 Department of Microbiology, Father Muller Medical College, Mangalore, India
2 Department of Neurovirology, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India
|Date of Web Publication||5-Jul-2017|
Department of Neurovirology, National Institute of Mental Health and Neurosciences (NIMHANS), Bengaluru - 560 070, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Dengue (DEN) is being recognised as the world's major emerging tropical disease. Clinically, DEN may resemble other infections such as malaria, leptospirosis, and typhoid, and thus, laboratory investigations are required for definitive diagnosis. Secondary DEN infection, caused most often by dengue virus (DENV) serotypes 2 and 3, is known to present with severe disease manifestations. This study was undertaken to examine the clinical and laboratory profile of DEN viral infections and to determine the circulating serotypes in and around Mangalore, India. Materials and Methods: Serum samples from 285 clinically suspected cases of DEN in and around Mangalore between September 2013 and January 2014 were processed for detection of DEN IgM and IgG antibodies and nonstructural 1 (NS1) antigen using commercial ELISA kits. Detection of DEN viral RNA and serotyping was done by multiplex real-time reverse-transcriptase polymerase chain reaction (RT-PCR). The clinical and haematological profiles of the patients were analysed. Results: Serum samples from 83 (29%) patients were positive for DEN NS1 antigen and/or IgM antibodies. 33 (45%) out of 73 serum samples processed by multiplex real-time RT-PCR were positive for DEN viral RNA. DEN-1, -2 and -3 were the serotypes identified in this study. Fever was the most common presenting symptom followed by myalgia/arthralgia. Majority of the patients had thrombocytopaenia. Conclusion: Early detection of DEN can be achieved effectively using NS1 ELISA and IgM capture ELISA. Circulating DENV serotypes should be closely monitored for prevention of fatal outcomes in secondary infections.
Keywords: Circulating serotype, dengue, polymerase chain reaction, serology
|How to cite this article:|
Damodar T, Dias M, Mani R, Shilpa K A, Anand AM, Ravi V, Tiewsoh J. Clinical and laboratory profile of dengue viral infections in and around Mangalore, India. Indian J Med Microbiol 2017;35:256-61
|How to cite this URL:|
Damodar T, Dias M, Mani R, Shilpa K A, Anand AM, Ravi V, Tiewsoh J. Clinical and laboratory profile of dengue viral infections in and around Mangalore, India. Indian J Med Microbiol [serial online] 2017 [cited 2017 Sep 26];35:256-61. Available from: http://www.ijmm.org/text.asp?2017/35/2/256/209566
| ~ Introduction|| |
Dengue (DEN) is the most rapidly spreading viral disease in the world with a 30-fold increase in incidence in the last 50 years. An estimated 50 million DEN infections occur annually and approximately 2.5 billion people live in DEN endemic countries. Among the four serotypes of DEN (DEN 1–4), 'Asian' genotypes of DEN-2 and -3 are frequently associated with secondary DEN infections. Since DEN produces a broad-spectrum of symptoms, a diagnosis based only on clinical symptoms is unreliable. Early laboratory confirmation is valuable because some patients may deteriorate rapidly resulting in death.
During primary infection, IgM appears after 5–6 days and IgG after 7–10 days. During a secondary infection, high levels of IgG are detectable even during the acute phase and rise over the next 2 weeks, whereas IgM are low or even absent in some cases of secondary infection. IgM antibodies suggest a recent infection; however, they can persist for 2–3 months., High titres of IgG are a criterion of secondary infection. However, in secondary infection, the antibody response may be greater to the primary infecting dengue virus (DENV) serotype than to the infecting DENV serotype. Moreover, extensive cross-reactivity with other flaviviruses and between various DEN serotypes and a low sensitivity in the early stages of infection can limit the utility of serological assays. Viral nonstructural 1 (NS1) antigen is abundant in the serum of patients in the early stages of DEN infection, lasting from 1 to 9 days; therefore, NS1 antigen ELISA, especially when used together with a IgM capture ELISA, is sufficiently informative in an endemic setting. Sensitivity of NS1 ELISA in secondary DEN infections is low. Molecular assays for detection of DEN viral RNA may be more sensitive in early stages of infection, with an additional advantage of identification of the serotype as well.
Mangalore, situated in coastal Karnataka, is endemic for DEN; however, there is paucity of data regarding the predominant circulating serotypes. This study was undertaken to evaluate the utility of various diagnostic tests in patients with clinically suspected dengue fever (DF).
| ~ Materials And Methods|| |
This prospective study was carried out in the microbiology laboratory between September 2013 and January 2014 and was approved by the Institutional Ethics Committee of Father Muller Medical College, Mangalore. The study included a total of 285 patients with clinically suspected DEN. Demographic data and details of clinical history and laboratory investigations were collected.
Blood (serum) samples collected from the patients were processed using IgM and IgG capture ELISA and NS1 ELISA according to manufacturer's instructions. (J. Mitra and Co. Pvt. Ltd.). Secondary infection was indicated when the IgM-to-IgG ratio was <1.2.,
Detection of DEN viral RNA and serotyping was performed on all samples positive by at least one of the above tests (NS1 antigen/IgM/IgG antibody ELISA) and on samples from the rest of the patients with a history of ≤5 days of fever using multiplex real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using a method described earlier. Briefly, RNA was extracted from 140 μl of serum specimens using a QIAamp viral RNA Mini Kit (Qiagen, Germany) following the manufacturer's protocol. A multiplex TaqMan real-time one-step RT-PCR assay for detection and serotyping of all four DEN serotypes was performed using ABI 7500 PCR thermal cycler (Applied Biosystems, USA).
| ~ Results|| |
The study included 285 patients with clinically suspected DEN viral infection. All the patients belonged to areas in and around Mangalore, except one traveller from West Bengal. A male preponderance (61%) was noted. A total of 45 (54%) patients were in the age group of 21–40 years, followed by 16 (19%) patients who were 20 years or below, 12 (14%) in the age group of 41–50 years and 10 (12%) patients were above 50 years. Fever was the most common presenting symptom. 139 (48.8%) patients had a history of ≤5 days of fever, while 146 (51.2%) reported after >5 days of fever. Haematological investigations revealed thrombocytopaenia in 60 (72%) cases. Comorbid conditions such as malignancy, diabetes mellitus and ischaemic heart disease were seen in eight patients and one patient succumbed to the illness.
Out of 285 suspected cases, 83 (29%) cases were positive by at least one of the following tests-IgM ELISA, IgG ELISA, NS1 ELISA; 55 (65%) patients with a history of ≤5 days of fever and 29 (35%) patients with a history of >5 days fever. Secondary infection (IgM-to-IgG ratio <1.2) was observed in 18 of the 83 cases.
[Table 1] and [Table 2] show the clinical and haematological parameters seen in serologically positive cases and correlation between fever and positive serological tests, respectively.
|Table 1: Clinical presentation and haematological parameters of positive cases (n=83)|
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|Table 2: Correlation between number of days of fever and serological tests|
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NS1 antigen was detected in 27 (49%) patients with <5 days of fever. When NS1 was accompanied with IgM or IgG, additional 16 cases with fever <5 days showed positivity. A significant correlation was seen between patients presenting with fever of ≤5 days and positive NS1 ELISA (P = 0.041).
Of the 83 samples positive for NS1 antigen and/or IgM/IgG antibodies, 73 samples were processed by real-time RT-PCR for detection of viral RNA, while the rest (10 samples) could not be processed due to insufficient quantity. Of the 202 cases negative for DEN by NS1 antigen and/or IgM/IgG antibodies, 84 cases had a history of <5 days of fever at presentation. Out of these 84 samples, 47 samples were further processed by real-time RT-PCR for detection of viral RNA, 10 samples had insufficient quantity and rest of the 27 cases were excluded because another infectious aetiological agent was identified in these samples. Therefore, a total of 120 samples were processed for detection of viral RNA and serotyping by multiplex real-time RT-PCR. 35/120 (29%) samples were positive for DEN viral RNA and the serotypes identified were DENV 1, 2 and 3. PCR was positive in 26 of 33 (79%) cases with a history of ≤5 days of fever positive by IgM/IgG ELISA (significant, P = 0.033) and in 29 of 33 (89%) cases positive by NS1 ELISA (significant, P = 0.010). However, PCR was positive in only 2 of the 47 cases with a history of ≤5 days, negative by NS1/IgM/IgG ELISA [Table 3].
Of the 35 samples which were positive for DEN viral RNA by PCR, 6, 17 and 12 samples could be serotyped into DENV-1, -2 and -3, respectively. Region-wise distribution of DEN serotypes is shown in [Figure 1]. A total of 16 patients were from the rural setting and 19 from the urban setting. In the rural setting, DENV-2 was found to be the predominant serotype (11) followed by DENV-3 (4) and DENV-1 (1) whereas in the urban area, all three serotypes were present (DENV 1 , DENV 2 and DENV 3 ). PCR was positive in seven cases of secondary infection, of which four cases belonged to DENV 3 and three cases to DENV 2.
|Figure 1: Distribution of dengue serotype. White: DEN-1, Blue: DEN-2, Pink: DEN-3. Not shown in map: Two cases of dengue virus-2 from Karwar (situated in Uttar Kannada, near Goa).|
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DENV-4 was not detected in our study. [Table 4] shows the circulating serotypes of DENV over the past decade in India.
|Table 4: Circulating serotypes in different regions of India in the last decade|
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The results were statistically analysed using Chi-square test.
| ~ Discussion|| |
DEN is an emerging vector-borne disease. Rapid urbanisation, globalisation, poor solid waste and water management and increasing population have given rise to new habitats for mosquito breeding thereby increasing the number of cases. Increased awareness among treating physicians and communities has brought down the mortality due to DEN; however, increasing number of cases year after year is an area of concern.
DF is nicknamed 'break-bone fever', and it is therefore not surprising that majority of the patients presented with myalgia or arthralgia along with fever. Fever is the most common presenting symptom followed by headache and musculoskeletal symptoms as also seen in other studies., The association of the DEN positivity with thrombocytopaenia was in concordance with the findings of other recent Indian studies , Majority of the cases in our study belong to the age group 20–40 years, with a male preponderance as seen in other studies., Studies done by Halsey et al. and Kashinkunti et al. to correlate DEN with its clinical manifestations revealed that headache and myalgia/arthralgia were the most common clinical manifestation of DEN infection which was also seen in our study., Clinical manifestation of vomiting and cough with expectoration was present in 22% and 7% of cases, respectively, comparable to the findings of the study done by Halsey et al. Furthermore, hepatomegaly and encephalitis were seen as a complication in 1% cases, almost similar to their study (1.2% and 2.7%, respectively). Skin rash was seen as a manifestation in 5% of ours and 7.5% cases in their study. As seen in the study done by Kashinkunti et al., abdominal pain was present in around 45% cases. A study done in Udupi, a neighbouring city, showed similar presentation of the infection with fever followed by myalgia, vomiting, headache and abdominal pain as the common clinical manifestations.
Majority of our cases (40%) were detected exclusively by the presence of viral NS1 antigen compared to IgM (7%) antibodies in patient's sera. It is known that early detection of DEN cases by NS1 assay helps in diagnostic detection and confirmation of cases., NS1 antigen detection is particularly useful during the first 5 days of illness and significantly more sensitive for primary than secondary DEN infection as was also seen in our study.,,, Five percent of NS1-positive samples were also IgG positive. These patients provided serological evidence of previous exposure. Thirteen patients who were simultaneously positive for NS1 antigen, IgM and IgG antibodies were probably in the late stage of either a primary or a secondary infection., Seven of these 13 patients had serological evidence of secondary infection. It is a known fact that during a primary infection, individuals develop IgM after 5–6 days and IgG antibodies after 7–10 days., Majority of the patients (71%) presenting with fever of >5 days in our study were positive for IgM ELISA as compared to other serological parameters. In 2007, Kumarasamy et al. compared the use of NS1 ELISA with viral isolation in cell cultures and RT-PCR assay and achieved great results in patients in the early stages of infection concluding that NS1 antigen detection may be an appropriate marker of acute DENV infection.
Thus, it can be stated that supporting clinical symptoms along with early detection of viral NS1 antigen can help speedup diagnosis of DEN cases during the first 5 days of fever and fever beyond that can be diagnosed by IgM ELISA alone.
Multiplex real-time RT-PCR assay was carried out for detection of viral RNA and serotyping; only 35 of 120 cases (29%) were positive. Although a significant association of PCR was seen in cases positive for NS1 and/or IgM ELISA and/or IgG ELISA with fever of <5 days (P = 0.033) and NS1 ELISA (P = 0.01), only 55% (29 of 53) of NS1-positive cases were positive by RT-PCR. PCR was positive in only 2 of 47 (4.2%) cases negative for NS1 antigen and IgM/IgG antibodies. Thus, NS1 antigen detection was found to have better sensitivity than detection of viral RNA by PCR. Similarly, in a study done by Khan et al., 39.3% among NS1-positive cases showed the presence of viral RNA. DENV NS1 antigen detection in travellers on arrival at airports in Taiwan was important in detection of 19 RT-PCR-negative travellers who would have been labelled DENV negative. Furthermore, in Malaysia, 42 of 55 patients with a diagnosis of acute DEN were positive for NS1 but negative by both RT-PCR and virus isolation. However, molecular assays have an advantage in determining the circulating serotype which is the main limitation of serological tests, especially in cases of outbreaks.
DEN was first isolated in India in 1945. After a quiescent period of 18 years, in 1963–1964, an initial epidemic of DF was reported on the Eastern Coast of India; it spread northwards and reached North India. Simultaneously, it also involved the southern part of the country, and gradually, the whole country was involved with widespread epidemics followed by endemic prevalence of all the four serotypes of DEN. Intermittent reports of DEN and its sequelae have come from various parts of the country [Table 4]. These include reports from Punjab, Delhi,, Lucknow, Kolkata, Chennai,, Mangalore, Assam/Nagaland  and Vellore.,, Post-1970, DEN-4 has rarely been reported from the country.,
In our study, Mangalore showed three circulating serotypes with DEN-2 being the predominant serotype in both urban and rural areas. A study done by Padbidri et al. in 1993 also showed that DEN-2 was the circulating serotype during the epidemic. DEN-1 was only isolated from patients from Mangalore; 5 from the urban area and 1 from rural area of Mangalore. Apart from Mangalore, DEN-2 was also isolated from other surrounding rural areas. Three patients from rural areas around Mangalore were infected with DEN-3. However, more data are required to determine the predominant circulating serotype from areas around Mangalore. DEN-4 was not isolated from any of the cases.
DEN-2 was the most common serotype in our study which is in concordance to the study done by Vinodkumar et al. which concluded that DEN-2 was the predominant circulating serotype in central Karnataka followed by DEN-3.
Rapid growths of population and urbanisation along with a change in climate have contributed significantly towards the cases of DF/dengue haemorrhagic fever (DHF) in India. Therefore, it can be stated that DEN is no more an urban area infection but is extending to rural areas also as seen in our study.
Infection with one serotype (primary infection) results in immunity to that serotype but does not confer immunity against other serotypes. Thus, infection can occur with heterologous serotypes (secondary infection). Secondary DENV infection has been shown to be a significant risk factor for the development of severe disease, including DHF and dengue shock syndrome (DSS)., DEN-2 has been implicated as the causative agent in most of the outbreaks of DHF and DSS the incidence of which have increased in the last two decades.,, Hence, rapid detection and differentiation between primary and secondary DENV infections and determination of the infecting serotypes in past and current infections are vital for patient management.
| ~ Conclusion|| |
Three serotypes (DEN-1, 2 and 3) were found circulating in this part of the country. In addition to primary infection, DEN-2 and 3 serotypes were also responsible for secondary DEN infection. NS1 antigen detection was found to have a better sensitivity than viral RNA detection by PCR for early detection of DEN infection. NS1 antigen when combined with IgM capture ELISA increased the diagnostic efficacy. Periodic monitoring of circulating DEN viral serotypes is essential for epidemiological purposes and for the patient management.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]