Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus.

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60 degrees C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Evaluation of a commercially available immunoglobulin M capture enzyme-linked immunosorbent assay kit for diagnosing acute dengue infections.

Recently, commercially available kits for the detection of anti-dengue virus (anti-DEN) immunoglobulin M (IgM) antibodies have been developed. These standardized assays have greatly enhanced our ability to effectively diagnose DEN infections. We conducted an evaluation of a test kit manufactured by MRL Diagnostics Inc. that is designed to detect anti-DEN IgM antibodies. Eighty paired samples from DEN-infected individuals were tested by the MRL DEN Fever Virus IgM Capture enzyme-linked immunosorbent assay (ELISA), the PanBio Duo ELISA, the PanBio Rapid Immunochromatographic Test (PRIT), and the IgM-IgG antibody capture (MAC/GAC) ELISA. All infections were confirmed by either PCR-assisted detection of DEN transcripts or by DEN isolation in C6/36 cells. Seventeen paired samples from individuals with no evidence of acute DEN infection were used as negative controls. The PRIT had the best sensitivity (100%), whereas the MAC/GAC ELISA and the PanBio Duo assay had the highest levels of specificity. The MRL ELISA and the PanBio Duo assay were the top performers when taking into consideration both sensitivity and specificity. All assays were able to detect DEN-specific antibodies in samples from patients with either primary or secondary infections, regardless of the infecting DEN serotype.

Complex-specific immunoglobulin M antibody patterns in humans infected with alphaviruses.

Sera from humans with serologically confirmed eastern equine encephalitis, western equine encephalitis, Pogosta (Ockelbo), Mayaro, Ross River, and chikungunya virus infections were tested by immunoglobulin M (IgM) antibody capture enzyme immunoassay. Diagnostically useful IgM antibody titers were detected, and selected sera with high IgM antibody titers were tested for IgM antibody with nine heterologous alphaviruses. The results provide evidence for the complex specificity of IgM antibody and indicate the usefulness of this test in both individual cases and epidemic situations.

Seroepidemiological survey on dengue and Japanese encephalitis virus infections in Asian monkeys.

To investigate the ecology of dengue and Japanese encephalitis (JE) viruses in the forest in Asia, a seroepidemiological survey was carried out on 358 Southeast Asian cynomolgus (Macaca iris), 33 Indian bonnet (Macaca radiata) and 37 Japanese (Macaca fuscata) monkey sera by a plaque reduction neutralization test. The results indicated that Southeast Asian monkeys were naturally infected with these viruses but the frequency of antibody to them varied considerably according to the geographical origin of the monkeys. The frequency of antibody to one or more types of dengue virus were 87.2, 49.5, 34.3, 34.2 and 14.9% in Malaysian, Vietnamese, Cambodian, Indonesian and Filipino cynomolgus monkey sera, respectively. None of the Indian bonnet monkey sera neutralized type 1 dengue virus which was the only virus type examined with this monkey species. Monkey sera collected in Japan where dengue virus infection had not been known since 1944 did not significantly neutralize dengue viruses. JE virus antibody was detected at 29.7, 9.0, 8.6, 2.7, 1.4 and 0% in Japanese, Cambodian, Vietnamese, Indonesian, Filipino and Malaysian monkey sera respectively.

Clinical observations on virologically confirmed fatal dengue infections in Jakarta, Indonesia.

Thirty virologically confirmed cases of dengue infection with a fatal outcome were studied clinically in Jakarta, Indonesia, from 1975 to 1978. All 4 dengue virus serotypes were isolated from fatal cases, but dengue type 3 was responsible for 21 (70%) of these isolates, compared to only 47% of isolates from all cases of dengue infection. The majority (60%) of these 30 cases were males in the 5-9-year age group. Nonspecific signs and symptoms in the fatal cases were no different from those in patients who survived dengue infection, but 70% of the patients with fatal outcome had one or more signs of encephalitis, primarily convulsions and somnolence; 3 of them developed spastic tetraparesis before death and 2 died of an illness clinically compatible with viral encephalitis. Other unexpected observations were that only 63% of the patients had classical dengue shock syndrome with haemoconcentration, thrombocytopenia and shock. A high percentage (80%) had gastrointestinal haemorrhage, and in 9 patients (30%) this was severe enough to cause shock and death. In these 9 cases, the gastrointestinal haemorrhage and haematemesis began before the onset of shock and there was no evidence of haemoconcentration or pleural effusion at any time during hospitalization. According to certain widely accepted criteria, these patients would not be diagnosed as dengue haemorrhagic fever (DHF). But as they made up nearly one-third of the confirmed fatal dengue infections in this study and had massive gastrointestinal haemorrhages with thrombocytopenia, the definition of DHF should be changed to include this type of patient. It is proposed that the disease should be more realistically classified as dengue fever with or without haemorrhage and dengue shock syndrome.

Epidemic dengue 3 in central Java, associated with low viremia in man.

An outbreak of dengue type 3 was studied in Central Java, Indonesia, in 1978. In contrast to previous dengue 3 epidemics in Central and East Java, this outbreak was less explosive, associated with mild illness, and low viremia. The dengue virus isolation rate from serologically confirmed patients was only 32% compared to 65% for an epidemic in Bantul a year earlier. Neither dengue hemagglutination-inhibition antibody titers nor day of illness on which specimens were collected accounted for this difference. These data suggest that some naturally occurring strains of dengue virus (endemic strains) are associated with low viremia and generally cause only mild illness in man.

Viraemia in patients with naturally acquired dengue infection.

The magnitude and duration of dengue viraemia were studied in 153 patients with naturally acquired dengue infection in Jakarta, Indonesia. The duration of viraemia ranged from 2 to 12 days, but most patients had detectable circulating virus for 4-5 days. Accurate measurement of peak virus titres was not possible for many patients because of late admission to the hospital. Composite pictures of viraemia for each serotype, however, showed that many patients infected with dengue 1, 2, or 3 had circulating virus titres ranging from barely detectable to over 10(8) MID(50) per ml for 3-5 days. Virus titres in patients infected with dengue 4 were about 100-fold lower. Dengue haemagglutination-inhibition antibody titres of 80 or less had little effect on viraemia, but antibody titres of 160 or greater were associated with a decrease in virus isolation rate and in virus titre. The duration and magnitude of viraemia did not vary significantly with the severity of the disease and was only slightly higher in patients classified as primary dengue infections than in those classified as secondary infections. Measurement of viraemia in fatal dengue haemorrhagic fever (DHF) cases showed that these patients had significant quantities of circulating virus at the time of death.

Epidemic dengue hemorrhagic fever in rural Indonesia. I. Virological and epidemiological studies.

Virological studies were carried out during an epidemic of dengue hemorrhagic fever in Central Java, Indonesia in 1976. Dengue virus was isolated from the acute sera of 45 of 69 patients (65%). The isolation rate was higher in primary than secondary cases. Dengue 3 was the predominant serotype being transmitted (27 isolates), but both dengue 1 (8 isolates) and dengue 4 (10 isolates) were also being transmitted. A composite picture of magnitude and duration of viremia showed that many patients were circulating over 10(8) MID50 per milliliter dengue 3 virus for the first 3 days of illness and that viremia persisted for 5-6 days in some persons. If all shock cases were considered, there was no relationship between dengue serotype and severity of disease. All three confirmed fatal cases, however, were associated with dengue type 3 infections.

Dengue virus isolation in Indonesia, 1975-1978.

Virus isolations from dengue hemorrhagic fever patients in Indonesia are reported from 1975 to 1978. All 4 dengue serotypes were endemic in Jakarta, but dengue 3 was the predominant virus isolated. This type was also the most frequently isolated virus from patients outside Jakarta and had the widest distribution in Indonesia. The sensitivity of the mosquito inoculation technique for isolation of dengue viruses is discussed.

Virological surveillance for dengue haemorrhagic fever in Indonesia using the mosquito inoculation technique.

A dengue haemorrhagic fever surveillance system in Indonesia, based on virological and clinical observations, is described. The system uses the mosquito inoculation technique for virus isolation and is simple, economical, and well suited for endemic areas where support and facilities are limited. The data suggest that with good cooperation between the hospital and the virology laboratory, new serotypes and possibly even new strains of virus can be identified before the onset of epidemic activity. This type of virological surveillance may make it possible to prevent major epidemics in the future.