Dengue

Dengue Bulletin Volume 28 (2004)

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Clinical and Laboratory Observations Associated with the 2000 Dengue Outbreak in Dhaka, Bangladesh

Monira Pervin*#, Shahina Tabassum**, Md. Mobarak Ali***,
Kazi Zulfiquer Mamun* and Md. Nazrul Islam**

*Department of Virology, Dhaka Medical College, Dhaka, Bangladesh
**Department of Virology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh
***Shahid Suhrawardi Hospital, Dhaka Bangladesh

Introduction

Dengue was first reported in Bangladesh in 1964 and the outbreak came to be known as ‘Dacca Fever’^[1]. For a long period after that, dengue cases remained undetected. A few cases were reported in 1999 before a large outbreak occurred in 2000, during which 5,551 cases and 93 dengue-related deaths were reported^[2,3]. Cases reported during the outbreaks were mostly diagnosed clinically, except for a few serologically diagnosed cases^[4].

Several studies have observed that sequential or secondary dengue infections are more likely to produce a severe form of the disease. DHF and DSS occur mostly in persons with pre-existing dengue antibodies acquired actively or passively^[5-7].

Detection of anti-dengue IgM indicates the diagnosis of recent dengue infection in both primary and secondary cases. Anti-dengue IgM develops earlier than IgG in primary infection and is usually detectable by day 5 of illness and wanes after 1-2 months^[8]. The ratio of IgM and IgG antibodies determined by ELISA is useful for distinguishing primary and secondary infections^[8,9]. In primary infection, the IgM/IgG ratio generally exceeds 1.8 OD units in acute or convalescent sera^[8]. Detection of an early and excess of IgG characterizes secondary infection. In the present study, dengue patients were classified into primary and secondary cases on the basis of this concept and it was carried out to confirm the clinical diagnosis, correlate the clinical manifestations with laboratory findings and establish the association of haemorrhagic manifestations with different dengue virus serotypes responsible for the outbreak of 2000.

Materials and methods

The study was conducted on clinically suspected cases of dengue fever attending the inpatient and outpatient departments of Medicine and Paediatrics, Bangabandhu Sheikh Mujib Medical University (BSMMU) Hospital, and patients admitted at the DhakaMedicalCollegeHospital during June – December 2000. Some patients seeking diagnostic facilities at the Department of Virology, BSMMU, were also included in the study.

According to specific inclusion criteria, 105 clinically suspected patients with fever (presenting within 5 days of onset with body temperature above 100 °F at the time of blood sample collection) and fulfilling the case-definition criteria of dengue fever (DF) and dengue haemorrhagic fever (DHF) of WHO^[10] were enrolled in the study. The exclusion criteria defined cases of febrile illness of more than 5 days and/or with definite sources of infection, chronic illnesses like tuberculosis, bronchial asthma, congenital heart disease, renal failure, history of bleeding tendency since birth and patients who refused to give two blood samples.

Clinical data were collected through interviewing the patients or their attendants and meticulous physical examination of the patients conducted by a doctor. The tourniquet test was performed in all patients by conventional method^[11]. Hepatomegaly and ascites were ascertained by physical examination and on reports of ultrasonography and X-rays. 5 ml of venous blood was collected aseptically from all patients during both the early and convalescent stages of fever irrespective of age and sex. This was processed and stored appropriately for virus isolation, antibody assay, platelet count and haematocrit estimation. Detection of IgM and IgG anti-dengue antibody, isolation of dengue viruses by mosquito inoculation technique and serotyping of the isolated viruses were done as described previously^[12,13]. Patients were classified into DF and DHF or DSS according to WHO, 1997^[10].

Statistical analysis

The numerical data obtained from the study were analysed and the significance of the difference was estimated by using statistical methods. The data were expressed in frequency, percentage, mean and standard deviation as applicable. The comparison between groups was done by Student’s ‘t’ test and ‘Chi square’ and ‘Z’ test as applicable. All data were analysed by using the computer-based SPSS programme. Probability less than 0.05 was considered as significant.

Results

Of the 105 clinically suspected dengue patients, 39 (37.1%) were positive for either anti-dengue IgM or IgM and IgG antibodies and 38 (36.2%) patients developed only anti-dengue IgG in acute stage by ELISA. When these patients were tested in the convalescent stage, 21 (20%) were positive for only IgM and 76 (72.4%) were positive for both IgM and IgG antibodies. Eight patients did not develop anti-dengue IgM and IgG in either of the specimens (Table 1) and were also negative on virus isolation. Thus, a total of 97 (92.4%) cases were diagnosed serologically as current dengue infection, while 8 (9.8%) cases were considered as non-dengue febrile illness and were excluded from further analysis. Of the 97 dengue patients, 46 (47.4%) had primary and 51 (52.6%) had secondary infection depending on anti-dengue IgM and IgG antibody level of acute and convalescent stage sera (Table 2). Among them, 17 patients developed DHF and one developed DSS. Of the 18 DHF/DSS patients, 14 had secondary type of infection and 4 had primary infection.

Table 1. IgM and IgG dengue antibody positive cases by ELISA

* Total number of dengue infection cases detected in acute stage serum (21+18) = 39 (37.1%).
**Total number of dengue infected cases detected in convalescence serum (21+76) = 97 (92.4%).
*** Non-dengue febrile illness cases = 8 (7.6%).
Figures in parenthesis indicate percentage

Table 2. Age distribution of dengue patients by primary and secondary dengue infection

P value 0.214 (unpaired student’s ‘t’ test)
* Mean of age
Figures in parenthesis indicate percentage

The involvement of all age groups, especially an adult predominance, was observed. The mean age of the dengue patients was 29.2±12.9 years and most belonged to the 20-29-year age group. The mean age of primary dengue fever patients was 27.5±11.7 years and that of secondary patients was 30.7±14.4 years.

Dengue viruses were isolated from 44 of the 97 dengue patients. The rate of dengue virus isolation was significantly higher (68.2% vs 31.2%) among primary than secondary infection patients (P=0.018). The isolation rate decreased gradually with the increasing duration of fever (see Figure).

Figure. Isolation rate of dengue virus between primary and secondary dengue patients by day of fever

(P=0.018)
P value reached from Z test

The distribution of clinical manifestations in dengue cases is given in Table 3. The mean body temperature of the dengue patients was 101.5±1.4 ºF but there was no significant difference in the mean body temperature between primary and secondary DF patients. Other common symptoms included myalgia (84.5%), headache (82.5%), arthralgia (68%), lethargy (80.4%) and retro-orbital pain (49.5%). Patients with primary dengue infection presented more commonly with headache, arthralgia and retro-orbital pain, whereas lethargy was commonly associated with secondary dengue infection. The most common presenting sign was rash, especially maculopapular type, and its association was significantly higher with primary DF cases (P=0.016). Ascitis was observed in 5 (9.8%) cases; all had secondary DF. Hepatomegaly was present in 13 (13.4%) patients and its association was significantly higher (P=0.002) in secondary DF patients. Abdominal pain (6.2%) and lymphadenopathy (4.1%) was less frequent among our study patients though abdominal pain was more common (7.8%) in secondary DF patients.

Table 3. Distribution of clinical manifestations in dengue patients

Figures in parenthesis indicate percentage
P-value reached from chi-square analysis

Haemorrhagic manifestations in primary and secondary dengue infections are indicated in Table 4. The most common sign of bleeding manifestation, i.e. a positive tourniquet test, was observed in 18 (18.6%) patients. Petechiae 15 (15.5%), purpura 12 (12.4%), gum bleeding 12 (12.4%) and haematemasis/malaena 11 (11.3%) were the other bleeding manifestations. Besides these, conjunctival bleeding, haematuria and per rectal bleeding also occurred in a small number of patients.

Table 4. Haemorrhagic manifestations among primary and secondary dengue cases

Figures in parenthesis indicate percentage

The haematological features in primary and secondary dengue infections are given in Table 5. Platelet counts of <1x10⁵/mm³ was detected in 22 (22.7%) patients and was more frequently 16 (31.4%) associated with secondary DF (Table 5). Haematocrit value of >45% was observed in 16 (16.5%) patients and a significantly higher association of >45% haematocrit level was detected among secondary DF patients (P=0.02).

Haemorrhagic manifestations were mostly associated with DEN-3 and DEN-4 infections; only one patient with DEN-1 infection had per rectal bleeding (Table 6).

Table 5. Haematological features in primary and secondary dengue infection

Figures in parenthesis indicate percentage
P-value reached from chi-square analysis

Table 6. Haemorrhagic manifestations in different serotypes of dengue virus infections

Figures in parenthesis indicate percentage

Discussion

The secondary dengue infection is usually associated with more severe manifestations than the primary infection. In our study, out of the 97 dengue cases, 46 (47.4%) had primary and 51 (52.6%) had secondary dengue infection. The presence of secondary cases indicated that DF was present in the area and perhaps a low-grade transmission was continuing during the previous years. Thus, it may be speculated that a considerable proportion of cases, which were diagnosed as “viral flu” in the past, may have been due to dengue virus infection. On serological test in acute stage, only 39 (37.1%) cases were found to be positive for dengue antibody. However, when the serum from the patients was tested again in the convalescence stage, 97 (92.4%) cases were found to be positive, indicating that they had recent dengue virus infection (Table 1). Thus, the detection of a significant number of cases could have been missed if the tests were not done again at the convalescence stage. A second test should therefore be considered at the convalescence stage to delineate the actual numbers of infection due to dengue.

Our study indicates the involvement of all age groups of the population with adult predominance. This finding is not consistent with the epidemiological data from other endemic countries in Asia where young children were affected^[14,15]. However, adults may also be the major victims of DHF as reported in different epidemics where dengue was endemic^[16,17,18]. No significant age or sex difference among patients was observed between primary and secondary infections in the present study. Similar observations were also reported from an outbreak in Fiji^[11].

Anorexia, nausea, vomiting, abdominal pain and ascitis were associated more with secondary than primary dengue infections (Table 3). In the present study, a statistically significant (P=0.01) association of rash (32.9%) among primary dengue infection cases was demonstrated. The predominant type of rash in primary infection was macular or maculopapular whereas petechial rash was found frequently in secondary infection. Other studies also reported similar association of rash in DF^[11,19]. Petechiae were frequently found in DHF in Cambodian^[20] and Thai children^[21], while it was less frequent in patients in our study. In the present study, statistically significant (P=0.002) hepatomegaly (13.4%) in secondary dengue infection was noted. Hepatomegaly was also a common clinical finding in several other studies^[22-24]. Acute abdominal pain, which is considered as an early sign of shock in DF/DHF^[10], was present in only 6 (6.2%) patients in the present study. This is similar to a study from Lucknow, India, where abdominal pain was found in only 5% of cases in an epidemic of DHF^[19]. In some studies, acute abdominal pain was strongly correlated with DHF and DSS^[10,24,25]. In the present study, ascitis, a sign of plasma leakage, was present in only 5 (9.8%) secondary infection cases and was in agreement with the findings of other studies^[25,26].

The positive tourniquet test, which reflects capillary fragility and is used as a guiding test for detecting dengue illness^[27], was found in only 18.6% cases in the present study. There was no statistically significant difference in test positivity between primary and secondary DF patients although it was more frequently positive in secondary DF. In most studies, comparing DHF with classic DF, either a higher incidence of tourniquet test positivity in DHF^[23,26,28] or no difference between the two groups^[24,29], was reported. These variable findings are probably because the pathogenesis of tourniquet positivity and other bleeding manifestations in dengue cases are different. Moreover, the defects which lead to increase in capillary fragility may also predispose to the development of shock, thus indicating a better correlation of the test to shock rather than to haemorrhage.

In our study, platelet counts of <1´10⁵/mm³ were observed among 22 patients and were associated more with secondary than primary infections (Table 4). Lack of correlation between a moderately low platelet count and bleeding manifestation in dengue patients has been noted by other investigators^[8]. The bleeding in dengue is probably due to other factors with or without the additive effect of thrombocytopenia. Haemoconcentration, the evidence of plasma leakage and shock, was observed in 16 patients with secondary infection and the association was statistically significant (P=0.02). Increased haematocrit was found in 95.5% patients with shock in contrast to 31.7% cases without shock^[21].

In the present study, the virus isolation rate was significantly higher in patients with primary (68.2%) than secondary (31.2%) infections (P=0.018). Similar results have been reported in a study from Fiji^[11]. In Bantal, Indonesia, the isolation rate was 100% among patients with primary dengue infection and 57% with secondary dengue infection^[30]. However, a study from Thailand reported a high rate of virus isolation in both primary (98%) as well as secondary (93%) infections^[31]. The reason for a higher isolation rate in secondary infection was probably because blood samples were collected at an early febrile phase (within 72 hours). In our study, the isolation rate was also high in both primary (100%) and secondary (68%) infections in early febrile period, i.e. on day 1 of fever. Thereafter, the rate of isolation was always higher in primary than in secondary dengue infection in the subsequent days of fever (Figure).

In conclusion, the clinical diagnosis is not very reliable in dengue infection as there is a wide variation in the presence of various symptoms, which may misguide the physician regarding the severity of the disease. Therefore, the use of clinical case-definition results in inaccuracies. Thus, in order to identify the cases accurately, one should take the help of a simple and cheap diagnostic test that is able to diagnose accurately in the laboratory. In this context ELISA is clearly very promising for the confirmation of clinical diagnosis and to differentiate between primary and secondary infections. This will guide the clinician to take prompt and meticulous clinical management and early hospitalization of severe cases. Indeed, it is hoped that accurate laboratory diagnosis will not only reduce the morbidity and mortality but will also reduce the economic burden of the patient and the government.

Acknowledgements

We express our sincere thanks to Dr Vorndam Vance, CDC Dengue Lab, San Juan, Puerto Rico, USA, and Dr Ichiro Kurane, Arbovirus Research Laboratory, NIID, Japan, for their kind support in providing cell line and anti-dengue monoclonal antibodies. We are also grateful to the patients who participated in the study. We would like to thank Mr Tauhid Uddin Ahmed, Ex-PSO, IEDCR, Bangladesh, and Noor-e-Jannat for providing mosquito colony and rearing techniques. We would also like to thank Dr Bijon Kumar Sil, Ex-Senior Scientific Officer, Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh. We also express our gratitude to the Department of Medicine and Paediatrics, BSMMU, and Dhaka Medical College, Bangladesh, for their support in the study.

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