Dengue viruses pose a considerable global public health problem with an estimated 100 million cases of illness every year. This illustrates the need for rapid and reliable diagnostic methods for proper patient management and disease control. Currently, laboratory diagnosis depends on serology or virus isolation, with both methods having certain drawbacks. Alternatively, reverse transcription and polymerase chain reaction (RT-PCR) offers the potential for the rapid, highly sensitive and specific detection of dengue viruses. Since we occasionally encounter the problem of insufficient amounts of patient serum for the direct detection of dengue viruses, a method was developed for the extraction of viral RNA after biological amplification in mosquito larvae. Using this method, 15 of 19 clinical samples tested were correctly identified using RT-PCR.
This study describes the use of polymerase chain reaction as a diagnostic tool for detecting and typing of dengue virus. PCR was compared against virus isolation. First RT-PCR was done using dengue consensus primers after which positive samples were subjected to RT-PCR using type-specific primers. This study shows that the local strains of the dengue virus could be detected using the chosen primers. Furthermore, RT-PCR was found to be more sensitive than virus isolation in identifying the dengue positive samples.
The highly sensitive AFRIMS format IgM capture ELISA for the diagnosis of dengue virus infections requires the use of mouse brain derived hemagglutinins and consequently also the use of 20% acetone extracted normal human serum to eliminate high background. These reagents are not always easily available and we have thus compared the AFRIMS format with another published format which uses cell culture derived antigens (culture fluid, CF, format) in order to determine if it is reasonable to use cell culture derived antigens in situations where hemagglutinins and normal human serum are difficult to obtain. The study shows that using AFRIMS results as the reference point, the CF format described here has a sensitivity of 90% and a specificity of 96%.
The first major Malaysian epidemic of dengue hemorrhagic fever with severe manifestations occurred in 1973, with 969 reported cases and 54 deaths. In a detailed study of 138 clinically diagnosed and laboratory confirmed cases at the General Hospital in Kuala Lumpur, hemorrhagic manifestations were observed in 68.7% and shock in 18.1% of the patients. The cases occurred mainly from May to September, largely in urban and suburban areas of the majority of the states in the country. A main focus of infection was Jinjang, a heavily populated outlying district of Kuala Lumpur, where unusually high incidences of morbidity, severe disease and mortality were seen. Severe disease was seen mostly in children under the age of 15 years, although a significant number of adults suffered milder illnesses. The Chinese population was chiefly affected, due to their living in crowded, low-income housing where the vector, Aedes aegypti, occurred in the greatest numbers. All four dengue types were recovered during the epidemic period, although dengue 3 (DEN-3) was incriminated as the major epidemic type. Entomological data revealed high indices of A. aegypti throughout the country and left little doubt that this epidemic was aegypti transmitted. Spraying and fogging operations were carried out in attempts to control vector populations.
A modification of the IgM-capture ELISA which can provide an early diagnosis for dengue infection is presented. The test is technically simple compared to HI and appears to be more sensitive. It has the advantage over HIT for the detection of specific IgM in that it is more sensitive and the reading of the result is not subjective. There is the possibility of the test being able to replace HI and HIT in the future.
In 1982, Malaysia experienced the worst dengue/dengue haemorrhagic fever outbreak in its history. All states in Peninsular and East Malaysia were similarly affected. There was a total of 3,005 cases with 35 deaths, with the majority of cases occurring between the months of July to October. There was a total of 1,001 laboratory confirmed cases. Most of the cases were in patients over the age of 15 years. The Chinese population was mainly affected, although a much higher proportion of Malays was noted in comparison to previous years. The main serotypes involved were dengue-1 and dengue-3. No dengue-4 serotype were isolated.
A dot enzyme immunoassay (DEIA) was used to determine the levels of antibody to dengue 3 virus in the acute and convalescent sera of febrile patients with a clinical diagnosis of dengue fever or dengue haemorrhagic fever. The antibody titres were compared with titres determined by the haemagglutination inhibition (HI) test. The results of the study showed that, besides being more simple to perform, the DEIA is in order of magnitude more sensitive than the HI test. Furthermore, the data suggest that it is possible to use a single dilution as a cutoff point to predict with reasonable accuracy, if a patient has had a recent dengue infection. The DEIA test for antibodies to dengue virus is an appropriate technology highly suitable for rapid diagnosis and surveillance in developing countries.
BACKGROUND: The control groups in two phase 3 trials of dengue vaccine efficacy included two large regional cohorts that were followed up for dengue infection. These cohorts provided a sample for epidemiologic analyses of symptomatic dengue in children across 10 countries in Southeast Asia and Latin America in which dengue is endemic.
METHODS: We monitored acute febrile illness and virologically confirmed dengue (VCD) in 3424 healthy children, 2 to 16 years of age, in Asia (Indonesia, Malaysia, the Philippines, Thailand, and Vietnam) from June 2011 through December 2013 and in 6939 children, 9 to 18 years of age, in Latin America (Brazil, Colombia, Honduras, Mexico, and Puerto Rico) from June 2011 through April 2014. Acute febrile episodes were determined to be VCD by means of a nonstructural protein 1 antigen immunoassay and reverse-transcriptase-polymerase-chain-reaction assays. Dengue hemorrhagic fever was defined according to 1997 World Health Organization criteria.
RESULTS: Approximately 10% of the febrile episodes in each cohort were confirmed to be VCD, with 319 VCD episodes (4.6 episodes per 100 person-years) occurring in the Asian cohort and 389 VCD episodes (2.9 episodes per 100 person-years) occurring in the Latin American cohort; no trend according to age group was observed. The incidence of dengue hemorrhagic fever was less than 0.3 episodes per 100 person-years in each cohort. The percentage of VCD episodes requiring hospitalization was 19.1% in the Asian cohort and 11.1% in the Latin American cohort. In comparable age groups (9 to 12 years and 13 to 16 years), the burden of dengue was higher in Asia than in Latin America.
CONCLUSIONS: The burdens of dengue were substantial in the two regions and in all age groups. Burdens varied widely according to country, but the rates were generally higher and the disease more frequently severe in Asian countries than in Latin American countries. (Funded by Sanofi Pasteur; CYD14 and CYD15 ClinicalTrials.gov numbers, NCT01373281 and NCT01374516.).
Note: Malaysia is a study site (CYD14 Primary Study Group authors: HIHJMI, Pediatric Institute, Kuala Lumpur Hospital, Kuala Lumpur; RN, Department of Paediatrics, Hospital Pulau Pinang, Pulau Pinang, Malaysia)
BACKGROUND: A candidate tetravalent dengue vaccine is being assessed in three clinical trials involving more than 35,000 children between the ages of 2 and 16 years in Asian-Pacific and Latin American countries. We report the results of long-term follow-up interim analyses and integrated efficacy analyses.
METHODS: We are assessing the incidence of hospitalization for virologically confirmed dengue as a surrogate safety end point during follow-up in years 3 to 6 of two phase 3 trials, CYD14 and CYD15, and a phase 2b trial, CYD23/57. We estimated vaccine efficacy using pooled data from the first 25 months of CYD14 and CYD15.
RESULTS: Follow-up data were available for 10,165 of 10,275 participants (99%) in CYD14 and 19,898 of 20,869 participants (95%) in CYD15. Data were available for 3203 of the 4002 participants (80%) in the CYD23 trial included in CYD57. During year 3 in the CYD14, CYD15, and CYD57 trials combined, hospitalization for virologically confirmed dengue occurred in 65 of 22,177 participants in the vaccine group and 39 of 11,089 participants in the control group. Pooled relative risks of hospitalization for dengue were 0.84 (95% confidence interval [CI], 0.56 to 1.24) among all participants, 1.58 (95% CI, 0.83 to 3.02) among those under the age of 9 years, and 0.50 (95% CI, 0.29 to 0.86) among those 9 years of age or older. During year 3, hospitalization for severe dengue, as defined by the independent data monitoring committee criteria, occurred in 18 of 22,177 participants in the vaccine group and 6 of 11,089 participants in the control group. Pooled rates of efficacy for symptomatic dengue during the first 25 months were 60.3% (95% CI, 55.7 to 64.5) for all participants, 65.6% (95% CI, 60.7 to 69.9) for those 9 years of age or older, and 44.6% (95% CI, 31.6 to 55.0) for those younger than 9 years of age.
CONCLUSIONS: Although the unexplained higher incidence of hospitalization for dengue in year 3 among children younger than 9 years of age needs to be carefully monitored during long-term follow-up, the risk among children 2 to 16 years of age was lower in the vaccine group than in the control group. (Funded by Sanofi Pasteur; ClinicalTrials.gov numbers, NCT00842530, NCT01983553, NCT01373281, and NCT01374516.).
During an outbreak of chikungunya in a dengue hyperendemic area within the Kinta district of Perak, two patients with acute febrile illness were laboratory confirmed to have co-infection of both dengue and chikungunya viruses in their blood. The concomitant presence of two types of viruses transmitted by the same vector in a susceptible population contributed to the resultant event. A good understanding of virus vector ecology in association with population dynamics and wider application of improved laboratory techniques by using different cell-lines suited for optimal replication of each type of virus and the correct utilization of powerful molecular techniques will enhance accurate diagnosis of these infectious diseases.
The performance of a commercial rapid immunochromatographic dengue IgG/IgM assay device was evaluated against an in-place dengue IgM-capture ELISA in the National Public Health laboratory. Of the 239 serum samples from patients with clinical diagnosis of acute dengue illness, 140 and 99 samples were tested positive and negative respectively for anti-dengue IgM by the in-placed ELISA. Comparatively, 72 and 76 samples were tested positive and negative respectively, and 91 samples gave equivocal results by the rapid dengue test device. The rapid immunochromatographic assay device gave a relative sensitivity of 49.3% and a relative specificity of 62.6%. Though the rapid immunochromatographic assay device has the advantages of rapid testing which simultaneously detects both IgG and IgM and can also be performed with whole blood, serum or plasma, the user has to exercise extreme caution with the interpretation of the test result.
A previously well 13-year-old boy presented with a short history of fever and altered mental status. His mother was admitted for dengue fever and there had been a recent dengue outbreak in their neighbourhood. He was diagnosed with dengue encephalitis as both his dengue non-structural protein 1 (NS-1) antigen and cerebrospinal fluid (CSF) dengue polymerase chain reaction (PCR) were positive. He did not have haemoconcentration, thrombocytopenia or any warning signs associated with severe dengue. He recovered fully with supportive treatment. This case highlights the importance of considering the diagnosis of dengue encephalitis in patients from dengue endemic areas presenting with an acute febrile illness and neurological symptoms.
A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confirmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient's demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on single-acute serum samples, 190 of the 558 patients (34.1%) were laboratory-confirmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confirmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was significantly more sensitive than the conventional RT-PCR.
At least three major antigenic dengue 2 virus proteins were recognized by pooled dengue fever patients' sera in infected Aedes albopictus (C6/36) mosquito cells. Dengue virus envelope (E), premembrane (PrM) and non-structural protein 1 (NS 1) dimer were detected beginning on day 3 postinfection in both the cell membrane and cytosolic fractions. Using the patients' sera, the presence of antigenic intermediate core protein (C)-PrM and NS1-non-structural protein 2a (NS2a) in the cytoplasmic fraction of dengue 2 virus infected cells was revealed. The presence of a approximately 92 and approximately 84 kDa NS 1 dimer in the membrane (NS 1m) and cytosolic (NS 1c) fractions of C6/36 cells, respectively, was also recognized. Using individual patient's serum, it was further confirmed that all patients' sera contained antibodies that specifically recognized E, NS 1 and PrM present in the dengue 2 virus-infected cell membrane fractions, suggesting that these glycosylated virus proteins were the main antigenic proteins recognized in vivo. Detection of dengue 2 virus C antibody in some patients further suggested that C could be antigenic if presented in vivo.