Is dengue emerging as a major public health problem?

Indian J Med Res 121, February 2005, pp 100-107 Is dengue emerging as a major public health problem? T.S. Vijayakumar, Sara Chandy, Narayanan Sathish, Mary Abraham, Priya Abraham & Gopalan Sridharan Department of Clinical Virology, Christian Medical College, Vellore, India Received October 21, 2003 Background & objectives: Diagnosis of dengue infection is easily and best accomplished by demonstration of specific IgM antibodies in blood. We analyzed retrospectively the dengue IgM seropositivity available for samples obtained over a period of five year (1999-2003) from patients with suspected dengue fever (DF)-like illness to investigate whether there was an overall increase in the dengue IgM prevalence over this period. Methods: Serum samples from a total of 1426 individuals (suspected dengue cases) obtained over five year were tested for dengue specific IgM antibodies. Of the 1426 patients, 693 were adults (>15 yr) and 694 children (<15 yr) (excluding 39 individuals whose age was not known). There were 807 males and 610 females (excluding 9 individuals whose status on sex was unknown). Results: A total of 423 (29.7%) samples were positive for dengue IgM over the five year period. Overall, there was a significant increase in the percentage of dengue IgM positive individuals over the this period (P<0.001). When the individuals were grouped into children (<15 yr) and adults (>15 yr), a significant increase in the number of dengue IgM positive individuals was noticed only in children (P<0.001) and not in adults. When the individuals were grouped into males and females, a significant increase in the number of dengue IgM positive individuals was noticed in both the sexes (P<0.03). Month-wise analysis of the dengue IgM positivity rates indicated the year-wide occurrence of dengue. A total of 158 (41%) of the dengue IgM positive individuals showed positivity for dengue IgG also suggestive of a secondary heterotypic infection. Interpretation & conclusion: The overall significant increase in dengue IgM seropositivity among the suspected cases indicates an increase in dengue virus activity, raising the question whether dengue is emerging/re-emerging as a major health problem in southern India. Increase in probable secondary infection (as evidenced by dual positivity for dengue IgM and IgG) seen in this study is also a point of concern. Such an increase especially in a country like ours where multiple serotypes are prevalent, raises concern over probable increase in the incidences of the more serious DHF/DSS. As this report could well be an underestimate of true incidence, the alarming increase observed in 2003, may be a warning/indication of epidemics to come soon that merits serious consideration. Key words Dengue - emerging problem - India Dengue is an important mosquito-borne disease in the world in terms of morbidity, mortality and economic costs 1, especially in the tropics, with more than 2/5th of the world s population living in areas at risk for dengue 2-4. From being a sporadic illness, 100 epidemics of dengue have now become a regular occurrence worldwide. The clinical features of dengue virus infection range from nonapparent infection through dengue

fever (DF) and the more severe dengue haemorrhagic fever (DHF) and the dengue shock syndrome (DSS) 5-7. Laboratory diagnosis of a recent dengue viral infection may be done by detection of the virus in patient s blood, either by virus isolation in susceptible cell cultures 3 or by detection of the viral RNA by reverse transcription-polymerase chain reaction (RT-PCR) based techniques 8,9. However, these methods prove to be laborious, time consuming and require specialized laboratory facilities. Further, these procedures prove to be successful only when performed within a few days following the onset of illness as with appearance of antibodies the level of circulating virus in the blood drops 10. Dengue virus specific IgM antibody tends to appear early during the course of disease and allows for a provisional diagnosis of dengue to be made from a single serum sample. Detection of dengue IgM antibodies is an easier method of diagnosing DF as compared to other classical serological methods like haemagglutination inhibition, neutralization and complement fixation tests. There are numerous studies from the Indian subcontinent investigating outbreaks of DF or DHF in various parts of the country 11-17, and on evaluation of different assays to detect dengue IgM antibodies 18,19. There are no studies investigating the overall prevalence of dengue in India on a long term basis. We therefore analyzed data available in our hospital, a tertiary care centre in south India, on dengue IgM seropositivity status on samples obtained over a period of five year (1999-2003) from patients with suspected DF-like illness. Dengue IgM testing was performed prospectively as and when samples were received in the laboratory during the study period but the data were analyzed retrospectively. The data were also analysed to investigate as to whether there was an overall increase in the dengue IgM prevalence over this period. VIJAYAKUMAR et al: DENGUE, AN EMERGING PROBLEM 101 Patients come from in and around Vellore as well as from other parts of the country. Blood samples were collected from patients with DF-like febrile illnesses attending the Paediatric and the Medicine clinics. A small proportion of the samples were referral samples sent by physicians in private practice. The patients were diagnosed as having DF based on standard criteria like presentation of febrile illness of 2-7 days duration with features like headache, myalgia, arthralgia, rash, haemorrhagic manifestations and leucopenia. The samples were collected usually 5-10 days following the onset of illness but the exact date of sampling was not available for most of the patients. Approximately 5 ml blood was collected, serum was separated and dengue specific IgM antibodies were detected. Till November 1999, the samples were tested by dengue IgM and IgG blot tests (Genelabs Diagnostics,USA). From thereon till the latter half of 2002, the samples were tested using the PanBio Duo Rapid Immunochromatographic Card Test (Brisbane, Australia). This test also allowed for the detection of dengue IgG in the same card. From this time period till the end of 2003, samples were tested by the PanBio Dengue Duo IgM and IgG Rapid Strip Test (Brisbane, Australia) detecting both dengue IgM and IgG antibodies on the same strip. For a very brief period of time in 2002, a small proportion of samples were tested with the PanBio Dengue IgM Capture ELISA (Brisbane, Australia). All the tests were carried out as per the manufacturer s instructions and were able to provide information on both dengue IgM and IgG responses. Statistical analysis: The data presented were analyzed using Chi-square test for proportion and the Chisquare test for linear trend using the Epi Info version 6.03 programme. Results were considered statistically significant at P<0.05. Results Material & Methods The Christian Medical College (CMC) is located in Vellore town in the state of Tamil Nadu, India. During the study period (1999-2003), a total of 1426 serum samples were tested for dengue IgM antibodies. This included 693 (48.6 %) samples from adults (>15 yr) and 694 (48.6%) from children (<15 yr). The

102 INDIAN J MED RES, FEBRUARY 2005 age of the remaining 39 patients was not known, as they were mostly referral samples. The age range of the patients was from 0 to 89 yr (mean ± SD; 19±17 yr). Of the 1426 patients tested, 807 (56.6%) were males and 610 (42.8%) were females. The status on sex was not known for 9 patients. Of the 1426 samples, 423 (29.7%) were positive for dengue specific IgM. There was an increase in the total number of samples received in the laboratory for dengue serology in 2003 as compared to the previous years (Table I). The overall increase seen in the percentage of samples positive for dengue IgM over the five year period was statistically significant (P<0.001). Among children there was a steady and significant (P<0.001) rise in the number of individuals found dengue IgM positive increasing from 21 per cent in 1999 to 42 per cent in 2003 (Table II). Among adults also there was a steady rise in the dengue IgM positivity increasing from 11 per cent in 2000 to 32 per cent in 2003, but was not statistically significant. The total number of males with suspected dengue fever was higher than the number of females in all the years (Table III). From 2000 onwards, the number of individuals with dengue IgM positivity continued to rise in both sexes (with a rise from 21 to 32% in males and a rise from 19 to 42% in females). The overall increase in the dengue IgM positivity seen over the five year period in both the sexes was statistically significant (P<0.02 in males and <0.01 in females). The annual difference in dengue incidence (as evidenced by dengue IgM positivity) between the sexes was statistically significant only in 1999 and 2003 (P = 0.024 and P = 0.026 respectively). Data on the 9 individuals, whose sex was not indicated in the lab request, were not subject to statistical analysis. The observed dengue-igm seropositivity percentage month-wise is illustrated in Fig.1. Dengue IgM positive cases were prevalent throughout with the exception of few occasional months. In 2003, though showing variation, the dengue IgM positivity percentage consistently remained above 20 per cent throughout the year. The observed dengue IgM seropositivity percentage in comparison to the average Table I. Year-wise distribution of suspected cases of dengue fever and dengue IgM positive cases over a five-year period (1999-2003) Year Total no. of suspected Total no. positive dengue cases for dengue IgM (%) 1999 135 46 (34) 2000 321 64 (20) 2001 282 73 (26) 2002 235 76 (32) 2003 453 164 (36) Table II. Age-wise distribution of suspected cases of dengue and dengue IgM positive cases over a five-year period (1999-2003) Year Category Children Adults Total no. of No. (%) Total no. of No. (%) suspected positive suspected positive dengue for dengue dengue for dengue cases IgM cases IgM 1999 33 7 (21) 100 39 (39) 2000 187 49 (26) 125 14 (11) 2001 159 48 (30) 115 23 (20) 2002 137 49 (36) 92 26 (28) 2003 178 75 (42) 261 84 (32) Dengue IgM seropositivity status is not shown for 39 individuals whose age was not known Table III. Sex-wise distribution of suspected cases of dengue and dengue IgM positive cases over a five-year period (1999-2003) Year Category Children Adults Total no. of No. (%) Total no. of No. (%) suspected positive suspected positive dengue for dengue dengue for dengue cases IgM cases IgM 1999 71 18 (25) 64 28 (44) 2000 176 37 (21) 137 26 (19) 2001 157 39 (25) 125 33 (26) 2002 146 44 (30) 89 32 (36) 2003 257 82 (32) 195 82 (42) Dengue IgM seropositivity status is not shown for 9 individuals for whom the sex was not known

Table IV. Year-wise distribution of dengue IgG positive cases among the dengue IgM positive samples over a five- year period (1999-2003) Year Total no. of No. of samples samples positive positive for dengue for dengue IgM* IgG among the dengue IgM positive samples (%) 1999 46 20 (43.5) 2000 54 15 (27.8) 2001 70 19 (27.1) 2002 60 17 (28.3) 2003 158 87 (55.1) *Does not include 34 samples tested by PanBio ELISA VIJAYAKUMAR et al: DENGUE, AN EMERGING PROBLEM 103 monthly rainfall did not reveal a definitive temporal association with the exception of the year 2002 (Fig.2). The observed dengue IgM seropositivity percentage in comparison to the monthly average temperature (maximum and minimum) showed no association (Fig.3). Of the 423 samples positive for dengue IgM, 34 were tested by the PanBio ELISA and hence only the IgM status was known. Of the remaining 389 samples, 158 (41%) were positive for dengue-specific IgG (thereby suggestive of secondary dengue infection). The percentage of samples positive for both dengue IgM and IgG was found to be unusually high in 1999 (43.5%) and in 2003 (55.1%) whereas in the intervening years the percentage was just above Table V. Age-wise distribution of the dengue IgG positive samples over a five-year time period (1999-2003) Year Category Children Adults Total no. of samples No. of samples positive for Total no. of samples No. of samples positive for positive for dengue dengue IgG among the dengue positive for dengue dengue IgG among the dengue IgM IgM positive samples (%) IgM IgM positive samples (%) 1999 7 3 (42.9) 39 17 (43.6) 2000 44 10 (22.7) 9 5 (55.6) 2001 46 10 (21.7) 23 9 (39.1) 2002 37 7 (18.9) 23 10 (43.5) 2003 71 28 (39.4) 81 54 (66.7) Five individuals with positivity for dengue IgM and IgG were excluded from analysis as age was not known Year Table VI. Sex-wise distribution of the dengue IgG positive samples over a five-year time period (1999-2003) Category Children Adults Total no. of samples No. of samples positive for Total no. of samples No. of samples positive for positive for dengue dengue IgG among the dengue positive for dengue dengue IgG among the dengue IgM IgM positive samples (%) IgM IgM positive samples (%) 1999 18 11 (61.1) 28 9 (32.1) 2000 31 8 (25.8) 23 7 (30.4) 2001 38 4 (10.5) 32 15 (46.9) 2002 31 10 (32.3) 29 7 (24.1) 2003 77 40 (51.9) 80 46 (57.5) One individual with positivity for dengue IgM and IgG was excluded from analysis as sex was not known

104 INDIAN J MED RES, FEBRUARY 2005 Fig.1. Month-wise distribution of dengue IgM seropositivity among suspected cases of dengue fever for the period 1999-2003. Fig.2. Relationship between month-wise dengue IgM seropositivity and average monthly rainfall (in mm) during the period 1999-2003.

VIJAYAKUMAR et al: DENGUE, AN EMERGING PROBLEM 105 Fig.3. Relationship between month-wise dengue IgM seropositivity and monthly maximum and minimum temperature (in o C) during 1999-2003. 25 per cent (Table IV). The observed rise in the number of samples positive for both dengue IgM and IgG over the five year period was statistically significant (P <0.01). Overall 8.35 per cent of children were dengue IgM and IgG positive in comparison to 13.7 per cent of adults. The increase in percentage positivity seen in adults as compared to children was statistically significant (P<0.01). There was a statistically significant increase (P<0.02) in the overall percentage positivity rates of dengue IgM and IgG positive cases only in adults (Table V). Overall 9.04 per cent of males were dengue IgM and IgG positive in comparison to 14.09 per cent of females. The increase in percentage positivity seen in females as compared to males was statistically significant (P<0.03). There was a statistically significant increase (P<0.01) in the total number of samples positive for both dengue IgM and IgG over the study period only in females (Table VI). Discussion The first isolation of dengue virus serotypes 1 and 4 was reported from India in 1964 20, 21, and dengue virus serotype 3 in 1968 12. Ever since, intermittent reports of dengue and its sequelae have come from various parts of the country. These include reports from Ludhiana 16, Delhi 11, 17, 22, Lucknow 23, Calcutta 14,24, Chennai 25, Mangalore 26, Assam/ Nagaland 27 and Vellore 15. Dengue epidemics have been earlier reported from Vellore 12,15,28. The present results showed an unusual increase in dengue virus activity (as evidenced by dengue IgM positivity) in this region during 2003 (similar to that noticed in 1999). Though there was a decrease in the number of cases with suspected dengue infection in 2001 and 2002, there was a steady increase in the percentage of samples positive for dengue IgM in these years compared to 2000 providing evidence

106 INDIAN J MED RES, FEBRUARY 2005 that there was an overall increase in dengue virus activity over these years. The vulnerability of children to dengue infection 15, was re-established again in our study. The seasonality of transmission of dengue with increased activity in the post monsoon season 29 was seen in the present study also. This shows that presence of stagnating water sources following heavy rainfall could favour breeding of the mosquito vector resulting in an increased incidence of dengue. These findings also indicate that preventive measures against dengue infection should probably come into full-swing during the post-monsoon months. Presence of some dengue IgM positive cases even during dry months as seen in this study could probably be reflective of the year-round activity of the mosquito vector. Even minimal collections of water sources (like stagnating water within indoor plants) can favour breeding of the vector thereby helping in the maintenance of the vector population throughout the year. The year-round occurrence of dengue infection, with peak in rainy season was concordant with reported patterns of dengue transmission 29. There was a significant overall increase in the dengue IgM seropositivity in children and not in adults. This is an important observation that needs to be taken notice of. Curtailment of outdoor playing activities (especially during the post-monsoon months) by children could be done. As the mosquito vector exhibits activity during the dusk, this could reduce probably to a great extent the chances of children getting exposed to the vector. Sporadic reports of DHF/DSS have appeared from various parts of India 16,17,23, including Vellore 15. In our study, there was a statistically significant increase in the percentage of samples positive for dengue IgM and IgG (suggestive of secondary infection) only in adults and not in children. This could probably be a favourable point to be considered as DHF and DSS prove to have a high mortality in children as compared to adults 15. This scenario on the dengue status over the five year period reported from Vellore may be no different from other parts of India, the climatic and demographic features being the same and so is the risk. From the high incidence of dengue IgM seropositivity seen in 1999 and 2003, it appears that dengue could probably be fast emerging as a major health concern in this part of India. Molecular studies on the circulating serotypes and their genotypes may be of help in addressing the probabilities of DSS/DHF incidence in future. Involvement of many laboratories in diagnosis of dengue coupled with general awareness among the public and constant vigilance by the health care officials could go a long way in combating dengue. Acknowledgment Authors thank Shri Achary of the Indian Meteorological Department, Chennai, Shri I. Suresh and Ms. Kavitha in the Department of Clinical Virology, Christian Medical College, Vellore for obtaining the meteorological and other data from the records. References 1. Monath TP. Dengue: the risk to developed and developing countries. Proc Natl Acad Sci USA 1994; 91 : 2395-400. 2. McBride WJ, Bielefeldt-Ohmann H. Dengue viral infections, pathogenesis and epidemiology. Microbes Infection 2000; 2 : 1041-50. 3. Henchal EA, Putnak JR. The dengue viruses. Clin Microbiol Rev 1990; 3 : 376-96. 4. Solomon T, Mallewa M. Dengue and other emerging flaviviruses. J Infection 2001; 42 : 104-15. 5. Gregson A, Edelman R. Dengue virus infection. Pediatr Infect Dis J 2003; 22 : 179-81. 6. Dengue hemorrhagic fever: Diagnosis, treatment and control. 2nd ed. 1997 Geneva, WHO. 7. Nimmannitya S. Dengue and dengue hemorrhagic fever. In: Cook GC, Zumla AI, editors. Manson s tropical diseases. 21st ed. Philadelphia, USA: Saunders; 2003 p. 765-72. 8. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992; 30 : 545-51. 9. Henchal EA, Polo SL, Vorndam V, Yaemsiri C, Innis BL, Hoke CH. Sensitivity and specificity of a universal primer set for the rapid diagnosis of dengue virus infections by polymerase chain reaction and nucleic acid hybridization. Am J Trop Med Hyg 1991; 45 : 418-28.

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