Comparative evaluation of various commercial assays for diagnosis of dengue fever

Dengue fever (DF) is endemic in India and dengue hemorrhagic fever (DHF) has been reported with increasing frequency in the last decade. We evaluated three commercial assays for detection of antibodies to dengue virus, to assess their performance in a diagnostic laboratory. Sera from 58 patients collected during a febrile outbreak in New Delhi in 1997 were studied. The methods evaluated were MRL Diagnostic Dengue Fever Virus IgM Capture ELISA, Pan Bio Dengue Duo IgM and IgG Capture ELISA and Pan Bio Rapid Immunochromatographic test. The MRL ELISA correctly identified 97.8% (43 of 44) of samples as dengue positive while the Pan Bio Duo ELISA and Pan Bio RIT identified 95.45% (42 of 44). The sensitivities of both Pan Bio Duo ELISA and Pan Bio RIT for primary dengue and secondary dengue were 100% and 93.54% respectively. The specificity of three assays were MRL IgM ELISA 100%, Pan Bio Duo ELISA 92.8% and Pan Bio RIT 85.7%.     

Retrospective analysis of dengue specific IgM reactive serum samples

Objective

To conduct a retrospective analysis of dengue cases in Kolkata, on the basis of presence of anti-dengue IgM in their sera and presence or absence of anti-dengue IgG and dengue specific Non structural 1 (NS1) antigen in each of the serum sample.

Methods

Sample was tested quantitatively employing ELISA technique, using Biorad test kits, with a view to get a more comprehensive picture of dengue in an urban endemic area and also to evaluate individual cases.

Results

This reconstructed study revealed that of those 91 dengue cases, 70.3% (64) and 29.7% (27) were suffering from secondary and primary dengue respectively, showing that number of secondary dengue cases were much more than that of primary dengue cases with a possibility of emergence of DHF. A small proportion of cases 18.7% (17) were reactive for NS1. The duration of fever in NS1 antigen positive cases varied between 5 and 7 days. Of 17 NS1 reactive cases, 10 (10.9%) and 7 (7.7%) were suffering from secondary and primary dengue respectively.

Conclusions

Early detection of primary and secondary dengue cases would be facilitated by utilizing all three parameters (NS1 antigen, anti-dengue IgM and IgG) helping to evaluate, monitor and treat a dengue case effectively.     

感染登革病毒后抗体产生规律及应用于暴发疫情的血清学诊断

目的了解感染登革病毒后特异性抗体产生规律,指导基层实验室应用血清抗体检测方法对暴发疫情的早期病例作出正确诊断,为控制暴发提供实验室依据。方法采集福建省1次暴发登革热疫情中的发热病人和密切接触者血清标本441份,以及恢复期血清22份,用捕获法ELISA检测登革IgG和IgM抗体,并结合病例的流行病学资料进行分析。结果根据本研究规定的病例判定标准,441例中登革热感染者为228例。其中,IgM抗体检出226例,IgG抗体检出119例。226例IgM抗体阳性标本中,IgG抗体同时阳性的为118份;119例IgG阳性标本中,IgM抗体阴性1例;两者均阴性的标本中用RT-PCR方法检测出1例。22份恢复期血清两类抗体检测均为阳性。绝大部分病例集中在10～70岁,病例的职业分布较广,男女比例为1∶1.53。结论登革病毒感染后特异性IgM抗体在发病早期即可检出,并可维持至少一个半月,但个别病例发病早期IgM可能为阴性;IgG抗体随病程的延长,其检出率逐步增高,至发病后50 d,其检出率可达95%左右。本研究结果抗体产生规律同以往研究结果一致,可为基层实验室开展登革热血清学检测和病例诊断提供参考。     

Clinical differences among PCR-proven dengue serotype infections

The objective of this study was to compare the clinical spectra of the dengue serotypes proven by the PCR technique. This retrospective study reviewed the clinical information of dengue-infected patients who were admitted to northeastern provincial hospitals in Thailand from June to September 2002. Dengue infection and viral serotypes were confirmed by polymerase chain reaction (PCR). Paired anti-dengue immunoglobulin G (IgG) and IgM from paired sera were analyzed by enzyme-linked immunosorbent assay (ELISA). Ninety-nine PCR-proven dengue-infected Thai patients were studied. Their ages ranged from 3-30 years. They were infected with DEN1, DEN2, DEN3 and DEN4 in 21, 55, 12, and 12%, respectively. Twenty-two percent had primary and 78% had secondary infections. Dengue fever was the most common presentation for both primary (77.2%) and secondary infections (46.7%). The ratios of dengue fever:dengue hemorrhagic fever (DF:DHF) and non-dengue shock syndrome:dengue shock syndrome (non-DSS:DSS) for DEN2 was the lowest of the dengue serotypes. There was no difference in the duration of fever, percentage of hepatomegaly and bleeding among the serotypes in both DF and DHF. The trends in the white blood cells, lymphocyte and atypical lymphocyte counts in DEN3 were the highest, while those of DEN1 were the lowest of the dengue serotypes.     

Fatal dengue hemorrhagic fever in Cuba, 1997.

OBJECTIVES: After more than 15 years without dengue activity, a dengue II epidemic was reported in Cuba in 1997. Three thousand and twelve serologically confirmed cases were reported, with 205 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) cases and 12 fatalities. This report presents the clinical, serologic, and virologic findings in the 12 fatal DHF/DSS cases. METHODS: Serum and necropsy samples were studied by viral isolation in C636 cell line and polymerase chain reaction. Serum samples were tested by IgM capture enzyme-linked immunoassay (ELISA) and ELISA inhibition method (EIM). RESULTS: All 12 cases were classified as DHF/DSS according to the Pan American Health Organization Guidelines for Control and Prevention of Dengue and Dengue Hemorrhagic Fever in the Americas. All patients were older than 15 years. Women were more frequently affected. The symptoms and signs presented by these patients were similar to those previously described in DHF/DSS cases. Clinical deterioration occurred on average at day 3.75. Abdominal pain and persistent vomiting were the earliest and most frequent warning signs. Dengue infection was confirmed in all cases. IgM antibodies were detected in 11 of 12 cases, all of them with a secondary infection. Dengue II virus was detected by viral isolation in 12 samples and by polymerase chain reaction in 17. Virus or RNA was detected in various tissues, including kidney, heart, lung, and brain. CONCLUSION: The clinical, pathologic, and laboratory features of 12 cases of fatal dengue hemorrhagic fever were reviewed. The results obtained demonstrate that adults with a primary dengue infection are at risk of developing the severe disease (DHF) if they are infected with a different serotype.     

Fatal dengue hemorrhagic fever in adults during a dengue epidemic in Singapore.

BACKGROUND: Dengue fever has seen a significant re-emergence in Southeast Asia. Associated with the rise of dengue has been the increase in dengue-associated mortality. To better understand the predictors of mortality, we conducted a review of hospitalized adult dengue infections within our institution. METHODS: This was a retrospective case-control study of dengue-associated deaths at a large tertiary care hospital. RESULTS: In 2004, of 3186 cases of dengue fever (DF)/hemorrhagic dengue fever (DHF) admitted to our institution, there were 130 cases of DHF and seven dengue-associated deaths (case-fatality rate 5.4%). At least three of the seven fatal cases had serological evidence of primary dengue infection. All dengue-mortality cases had rapidly progressive clinical deterioration at an average of day 4 of fever with intensive care admission occurring on a mean of 5.6 days of fever. Adult respiratory distress syndrome, disseminated intravascular coagulopathy, and multi-organ failure were the most common causes of death despite early hospitalization, intravenous fluid, and blood-product support. CONCLUSION: Dengue is associated with severe disease, and deaths do occur despite current supportive management. Fatal DHF/dengue shock syndrome (DSS) does occur in adults and in primary dengue infection. Better early predictors of disease severity and clinical interventions are needed.     

Kinetics of antibodies in sera, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infections.

OBJECTIVES: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue infection were studied. DESIGN: Serum, saliva, and urine samples were collected from 22 patients with clinical and confirmed dengue 3 virus infection during the outbreak in Havana City in 2001. They were tested by capture IgM (MAC-ELISA), IgA (AAC-ELISA), and IgE (EAC-ELISA) and IgG ELISA inhibition method (EIM) to detect specific dengue antibodies. RESULTS: Similar kinetics were observed in IgM, IgA, and IgG antibodies in saliva and IgA and IgG in urine samples from secondary cases compared with kinetics in serum samples, although the values were lower. No IgG antibody was detected in saliva and urine samples in primary cases and IgM antibody was not detected in urine samples from either primary or secondary infection. All secondary cases were positive for IgG in saliva and urine samples at day 7. The kinetics of specific IgE antibodies in primary and secondary cases were different. CONCLUSIONS: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infection were studied for the first time, showing its behavior and usefulness in dengue virus diagnosis. The specific IgE could play a role as a serological marker in secondary infections.     

Emergence of dengue virus type-3 in northern India

During the last few decades dengue has reemerged in several parts of Southeast Asia, including India. A major outbreak of dengue infection occurred in northern India during October to December 2003. To determine the etiology, we carried out serological, virological and molecular investigations of this outbreak. A total of 76 dengue suspected patient blood samples were collected from Gwalior, Madhya Pradesh and Delhi, India. Serological investigations carried out using an in-house Dipstick ELISA protocol revealed the presence of anti-dengue antibodies in 53 patients. Twelve of them (22%) had a positive IgM response, indicative of primary infection, and 22 of them (42%) revealed only IgG antibodies, indicative of secondary infection. RT-PCR analysis employing dengue group specific amplimer revealed the presence of dengue specific RNA in four acute phase samples. These four RT-PCR positive samples were further processed for virus isolation in C6/36 cells and suckling mice, yielding four dengue virus isolates. The Nested PCR analysis employing serotype specific amplimer revealed the presence of dengue-3 specific 389 bp amplicon. This study confirmed the reemergence of dengue virus type-3 in a dominant form in India after a gap of nine years. Earlier, dengue virus type-2 was implicated as the etiology of a major dengue epidemic in Delhi in 1996 and Gwalior in 2001. The implication of dengue type-3 as etiology of a DHF epidemic in neighboring Sri Lanka and Bangladesh recently confirms the reemergence of dengue type-3 as the dominant form on the Indian subcontinent.     

Isolation and partial characterization of dengue virus type 2 and 4 strains from dengue fever and dengue haemorrhagic fever patients from Mindanao, Republic of the Philippines

OBJECTIVE: Isolation of dengue virus from dengue fever and dengue haemorrhagic fever cases from Mindanao, Republic of the Philippines. METHODS: 12 patients with clinically suspected dengue fever (DF) or dengue haemorrhagic fever (DHF) presenting in four regional hospitals between August and September 1995 on Minadano were enrolled in the study. Dengue virus was isolated by inoculation of Vero/E6 or C6/36 cells with patient serum. IgM antibodies were measured using a commercial test system. Up to 454 bp of the capsid region and 240 bp of the E/NS1 gene junction of different viral isolates were sequenced and phylogenetically analyzed. RESULTS: Virus could be isolated from seven patients, five isolates were typed as dengue virus type 2 and two as dengue virus type 4 by immunostaining with monoclonal antibodies or by RT/PCR. Phylogenetic analysis confirmed a close relationship of the dengue virus type 2 isolates with viruses isolated in the Philippines in 1983 and 1988. CONCLUSION: As observed in studies from other parts of South East Asia, dengue virus type 2 was readily isolated from dengue haemorrhagic fever cases. Dengue virus type 2 and 4 circulate in Mindanao, Philippines, with dengue type 2 being responsible for most of our severe DF or DHF cases.     

An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate

The diagnostic sensitivity and specificity of detection of anti-dengue IgM by antibody capture enzyme-linked immunosorbent assay (ELISA) was investigated in dengue infections in a variety of clinical settings. Sera from uninfected controls were uniformly negative. Serial specimens from experimental and natural infections showed that viremia and fever terminated as anti-dengue IgM became detectable. Anti-dengue IgM appeared in most cases by the 3rd afebrile day of illness and declined to undetectable levels after 30-60 days. Assay sensitivity was 78% in admission sera (924/1,183; 95% CI = 75-81%) and 97% in paired sera (1,030/1,062; 95% CI = 96-98%) thus exceeding or matching the performance of the hemagglutination-inhibition assay. Measurement of the anti-dengue IgM to anti-Japanese encephalitis IgM ratio correctly identified all sera from 112 patients with strictly defined Japanese encephalitis and 98% (307/312; 95% CI = 96-99%) of sera from patients whose dengue infections were confirmed by virus isolation. Dengue infections could be classified as primary or secondary by determining the ratio of units of dengue IgM to IgG antibody. We propose that measurement of dengue and Japanese encephalitis IgM and IgG antibodies upon admission and discharge from hospital care should replace the hemagglutination inhibition assay as the standard dengue serologic technique in regions where these 2 viruses co-circulate.     

Secondary dengue virus type 4 infections in Vietnam

This study was designated to describe clinical and biological features of patients with a suspected diagnosis of dengue fever/dengue hemorrhagic fever during an outbreak in Central Vietnam. One hundred and twenty-five consecutive patients hospitalized at Khanh Hoa and Binh Thuan Provincial hospitals between November 2001 and January 2002 with a diagnosis of suspected dengue infection were included in the present study.Viruses were isolated in C6/36 and VERO E6 cell cultures or detected by RT-PCR. A hemagglutination-inhibition test (HI) was done on each paired sera using dengue antigens type 1-4, Japanese encephalitis (JE) virus antigen, Chickungunya virus antigen and Sindbis virus antigen. Anti-dengue and anti-JE virus IgM were measured by a capture enzyme-linked immunosorbent assay (MAC-ELISA). Anti-dengue and anti-JE virus IgG were measured by an ELISA test. Dengue viruses were isolated in cell culture and/or detected by RT-PCR in 20.8% of blood samples. DEN-4 and DEN-2 serotypes were found in 18.4% and 2.4% of the patients, respectively. A total of 86.4% of individuals had a diagnosis of acute dengue fever by using the HI test and/or dengue virus-specific IgM capture-ELISA and/or virus isolation and/or RT-PCR. The prevalence of primary and secondary acute dengue infection was 4% and 78.4%, respectively. Anti-dengue IgG ELISA test was positive in 88.8% of the patients. In 5 cases (4%), Japanese encephalitis virus infection was positive by serology but the cell culture was negative. No Chickungunya virus or Sindbis virus infection was detected by the HI test. In patients with acute dengue virus infection, the most common presenting symptom was headache, followed by conjunctivitis, petechial rash, muscle and joint pain, nausea and abdominal pain. Four percent of hospitalized patients were classified as dengue hemorrhagic fever. The clinical presentation and blood cell counts were similar between patients hospitalized with acute dengue fever and patients with other febrile illnesses.     

Changing clinical and biological manifestations of dengue during the dengue-2 epidemic in French Polynesia in 1996/97 – description and analysis in a prospective study

In August 1996 dengue-2 virus was detected in French Polynesia for the first time since 1976. A prospective study was conducted from November 1996 to April 1997. Each time one of 7 physicians suspected dengue, the patient was enrolled and epidemiological, clinical and biological data were recorded. Dengue diagnosis was confirmed by virus isolation and IgM detection. The aims of this study were to find clinical and biological predictive factors constituting a specific profile of dengue (DF) and dengue haemorrhagic fever (DHF/DSS) and to assess the possibility of diagnosing dengue at primary health care level using clinical criteria and basic laboratory parameters. Of 298 clinically suspect cases, 196 (66%) were confirmed as dengue. The association of macular rash, pruritis, low platelet count and leukopenia was statistically predictive of dengue but not clinically, since these four signs occur in many other viral infections. As the prevalence of clinical and biological manifestations varied over time in our study, a specific profile useful for dengue diagnosis cannot be defined. With six cases of DHF, the morbidity of this dengue-2 outbreak was very low despite the sequential infection scheme DEN-3/DEN-2. The clinical expression of dengue could depend on a specific virus strain circulating in a specific population in a particular place, with varying virulence over time.     

Influence of the dengue serotype, previous dengue infection, and plasma viral load on clinical presentation and outcome during a dengue-2 and dengue-4 co-epidemic

Martinique experienced a dengue outbreak with co-circulation of DENV-2 and DENV-4. In an emergency department-based study, we analyzed whether the clinical presentation and outcome of adult patients were related to serotype, immune status, or plasma viral load. Of the 146 adult patients who had confirmed dengue infection, 91 (62.3%) were classified as having classic dengue fever, 11 (7.5%) fulfilled World Health Organization criteria for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), 21 other patients (14.4%) presented with at least one typical feature of DHF/DSS [i.e., internal hemorrhage, plasma leakage, marked thrombocytopenia (platelet count < or = 50,000 platelets/mm(3)) and/or shock], and 23 further patients (15.8%) had unusual manifestations. Four patients died. Severe illness was more frequent in patients with secondary dengue infection (odds ratio, 7.18; 95% confidence interval, 3.1-16.7; P < 0.001). Multivariate regression analysis showed that gastrointestinal symptoms and other unusual manifestations were independently associated with DENV-2 infection, whereas cough and DHF/DSS features were independently associated with secondary immune response. A high plasma viral load was associated with DENV-2 infection, increased serum liver enzymes, and with DHF/DSS features in patients presenting after the third day of illness. The most severe cases of dengue resulted from the combined effects of DENV-2 and secondary infection.     

Humoral immune response of dengue hemorrhagic fever cases in children from Tegucigalpa, Honduras

The humoral immune response in Honduran dengue hemorrhagic fever (DHF) hospitalized pediatric cases from the epidemics of 2004 and 2005 was studied in sera collected from 5 to 7 days of fever onset. A total of 145 cases were included in the study: 40 classified as primary with DHF Grade I or II and 86 classified as secondary; from them, 73 were DHF Grade I or II and 13 were dengue shock syndrome (DSS) Grade III or IV. The highest number of primary cases was found in children < 1 year of age. The highest number of secondary cases was observed in children between 5 and 10 years of age. The IgA values showed a statistically significant difference between primary and secondary groups. The relationship between antibody responses and severity grade is discussed. This is the first study related to the humoral immune response and severity grade in DHF cases in Honduran children.     

The use of intravenous gammaglobulin in dengue fever, a case report

We managed a serologically documented case of dengue fever with thrombocytopenia with IV IgG. Acute and convalescent ELISA and HI titers indicated an acute dengue infection with a secondary response that clinically, and by laboratory parameters dramatically improved after IV IgG administration. The use of IV IgG in cases of thrombocytopenia associated with dengue has both theoretical advantages and disadvantages. IV IgG may have a role in the management of DHF/DSS because of the significant morbidity and mortality associated with DHF/DSS. A prospective randomized study should be developed to determine if IV IgG does indeed effect the course of thrombocytopenia in dengue infections.     

Association between sex, nutritional status, severity of dengue hemorrhagic fever, and immune status in infants with dengue hemorrhagic fever

The association between sex, nutritional status, and the severity of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), and immune status was investigated in 245 Vietnamese infants with predominantly primary infections with dengue virus. Male and female infants were at equal risk of developing DHF/DSS. However, infants of low height and weight for age were under-represented among DHF/DSS cases compared with 533 healthy baby clinic infant controls. Acute illness phase blood levels of selected cytokines (interferon-gamma and tumor necrosis factor-alpha) and serum levels of antibodies to dengue virus were elevated in the same range in male and female infants with DHF/DSS, as well as in infants with and without malnutrition.     

云南西部边境地区健康人群登革热血清学调查

目的了解云南西部边境地区健康人群登革热抗体水平状况,为制定有效登革热防治对策提供依据。方法在具有登革热本地感染病例的不同地区采集健康人群血清,并采用间接酶联免疫吸附试验分别检测登革病毒IgG和IgM抗体。结果采集的740份中国籍血清标本登革病毒IgG和IgM抗体阳性率分别为10.9%（81/740）和5.7%（42/740）,两种抗体同为阳性的阳性率为0.81%（6/740）;缅甸籍学生这两种登革抗体阳性率分别为12.5%（13/104）和2.9%（3/104）。登革IgG/IgM抗体阳性率在不同年龄组、性别、民族、不同国籍学生中的分布无统计学差异（P〉0.05）;务农人群IgG抗体阳性率高于非农人群（P〈0.05）;盈江县IgG抗体阳性率较高。结论云南省边境地区人群广泛存在登革病毒的既往和新近隐性感染,建议加强对边境口岸和边境地区的登革热疫情监测,开展登革热防治知识的宣传和提高当地医疗机构的诊疗水平。     

Haiti: absence of dengue hemorrhagic fever despite hyperendemic dengue virus transmission

In 1994-1996, 185 strains of dengue (DEN) virus types 1, 2, and 4 were recovered from febrile United States and other United Nations military personnel in Haiti. We wondered whether risk factors for dengue hemorrhagic fever (DHF) existed and, if so, were DHF cases occurring among Haitian children. Dengue transmission rates were studied in 210 school children (6-13 years old) resident in Carrefour Borough, Port-au-Prince, Haiti. When sera were tested for plaque-reduction neutralizing antibodies to DEN 1-4 viruses, nearly 85% had antibodies to two or more DEN serotypes. The annual transmission rate was estimated at 30%, a rate observed in countries endemic for DHE Haitian DEN 2 isolates were genotype I, which are repeatedly associated with DHF cases in Southeast Asia and American regions. Despite positive virologic pre-conditions, DHF cases were not recorded by experienced Port-au-Prince pediatricians. These observations, which are reminiscent of those in Africa, provide further evidence of a dengue resistance gene in black populations.