A nitrocellulose membrane based IgM capture enzyme immunoassay for etiological diagnosis of dengue virus infections.

A nitrocellulose membrane based immunoassay for the detection of dengue virus specific IgM suitable for use in field situations or in peripheral laboratories would be useful for disease surveillance and control. This paper describes such an assay in an IgM capture format (MAC DOT) similar to the microplate based MAC ELISAs currently in use in several research and reference laboratories around the world. The MAC DOT was tested on several sample sets including a retrospective study of 119 patients from Children's Hospital, Bangkok, Thailand, with confirmed dengue infection. The sensitivity of the test was shown to be 94% taking only admission sera into consideration but rising to 99% when both an admission and a discharge specimen were considered. Other sample sets confirmed the high sensitivity and a study of 494 unselected febrile children showed that the specificity of the MAC DOT was 98%.

Applications of polymerase chain reaction for identification of dengue viruses isolated from patient sera.

A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT-PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single-step procedure. In this study we compared the RT-PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty-six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti-dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type-specific monoclonal antibodies (MAb) and by RT-PCR. Thirty-two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN-1), 25 were type 2 (DEN-2) and 2 contained both DEN-1 and DEN-2. All cultures that were positive by PAP method were also positive by RT-PCR and vice versa. Thus, the results obtained by RT-PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN-1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN-1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type-specific MAb for serotyping of dengue viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiology and characterization of leptospirosis at an urban and provincial site in Thailand.

Patients with FUOs at the Children's Hospital in Bangkok and the Chao Phya Abhai Bhu Bejhr Hospital in Prachinburi were screened for leptospirosis by blood and urine culture in addition to microagglutination testing of their serum. Animal populations in urban and periurban areas of Bangkok were surveyed for evidence of leptospira infection. Three rural sites near the Prachinburi Provincial Hospital were also surveyed. The rodents' and domestic animals' blood, urine, and/or kidney cell samples were cultured for leptospira. Sera from these animals were also tested for leptospira antibody. The bataviae serovar was the most commonly detected leptospiral agent in both man and animals. Presenting symptoms varied with age with children showing primarily fever, vomiting, headache, abdominal and generalized muscle pain and diarrhea whereas adults had fever, headache, anorexia, muscle pain and constipation. Blood samples from patients suspected of having leptospirosis were tested for antibody by the MAT and cultured in EMJH media. The following serogroups were identified: bataviae, autumanalis, javanica, hebdomadis, and pyrogens. Leptospirosis incidence in humans was much higher in the rainy/flooding year of 1983 compared to the relatively dry year of 1984. Results of our animal surveillance studies indicate that in addition to rats, which have previously been mentioned, dogs, bandicoots, cattle and pigs could be the source of human leptospirosis infection in both urban and provincial locations in Thailand.

Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants.

To establish the role of maternal dengue-specific antibodies in the development of dengue hemorrhagic fever and dengue shock syndrome caused by dengue 2 virus in infants, we examined sera from mothers of infants and toddlers with dengue hemorrhagic fever or dengue shock syndrome and mothers of infants with pyrexia of unknown origin. The mean titers of hemagglutination inhibition, neutralization, and infection-enhancing activities against dengue 2 virus were not statistically different among the three groups. However, among infants who developed dengue hemorrhagic fever/dengue shock syndrome there was a strong correlation between the mothers' dengue 2 neutralizing titers and infant age at the time of onset of severe illness, where no such correlation was found among the other two groups. Furthermore, the actual age at which dengue hemorrhagic fever/dengue shock syndrome occurred in each infant correlated with the age at which maximum enhancing activity for dengue 2 infection in mononuclear phagocytes was predicted. This critical time for the occurrence of dengue hemorrhagic fever/dengue shock syndrome was observed to be approximately 2 months after the time calculated for maternal dengue 2 neutralizing antibodies to degrade below a protective level. In addition, sera of mothers of infants with dengue hemorrhagic fever/dengue shock syndrome enhanced dengue 2 virus infection to a slightly greater degree than did sera from mothers of infants with pyrexia of unknown origin and toddlers with dengue hemorrhagic fever/dengue shock syndrome. These data are consistent with the hypothesis that maternal dengue antibodies play a dual role by first protecting and later increasing the risk of development of dengue hemorrhagic fever/dengue shock syndrome in infants who become infected by dengue 2 virus.

The effect of interferon-alpha A on two cases of Japanese encephalitis in Thailand.

Two Japanese encephalitis cases with serious comatous symptoms were treated with the Human Recombinant Interferon-alpha A. The clinical responses to IFN were found to be satisfactory. The first case showed improvement on the 5th day of IFN treatment and the general condition slowly improved. The second case recovered from the comatous stage on the 6th day of IFN, followed by quick improvement of general symptoms in the 2nd week and complete recovery without any mental sequelae. Leukopenia and neutropenia occurred during the first week of administration of IFN, but were only temporary. Slight elevation of SGOT and SGPT was observed in the first case. No other side effects including general toxicity, neurotoxicity or allergy, or any abnormal hematological and blood chemistry changes were observed in these 2 cases. Two other JE cases (the 3rd and 4th consecutive JE cases) were not treated with IFN, but received the usual regimens of symptomatic and supportive drugs. Both patients died on the 7th-9th day of illness. This study suggests that the Human Recombinant Leukocyte A Interferon possibly is an effective and promising agent in the treatment of Japanese encephalitis in Thailand. More studies to treat JE cases with this IFN are being performed in order to assess the efficacy, tolerance and safety of rIFN-alpha A on Japanese encephalitis in Thailand.

The pattern and nature of the lymphocyte population response in dengue hemorrhagic fever.

Eighty-eight specimens consisting of lymphocytes separated from peripheral blood samples from 76 patients with dengue hemorrhagic fever (DHF) were studied. The results revealed a significant increase in numbers of atypical lymphocytes and B lymphocytes, and a decrease in T lymphocytes by comparison with normal controls. Anti-T lymphocyte antibody was also detected in the serum of all patients with DHF. The greatest increments and decrements of the above were noted on the day of shock of subsidence of fever.

Demonstration of dengue antibody complexes on the surface of platelets from patients with dengue hemorrhagic fever.

By the direct immunofluorescent technic, dengue antigen, human immunoglobulins, and beta 1C globulin were detectable on the surfaces of platelet suspensions from 48% of patients with dengue hemorrhagic fever. The percentages of positive-staining platelets were not related to the severity of thrombocytopenia, which was marked on the day after the patient developed shock or subsidence of fever. It is suggested that an immunologic mechanism is one of the factors associated with the thrombocytopenia caused by increased platelet destruction.

Circulating immune complexes in serum from patients with dengue haemorrhagic fever.

Circulating immune complexes were detectable in 80% of serum from patients with dengue haemorrhagic fever. The immune complexes were detected for the first time on day two after the onset of the fever. The amount of complexes reached the maximum value on day 4 or 5 after onset, or when the patients developed shock or subsidence of fever, after which the complexes decreased in number. The number of complexes also correlated well with the clinical grading (severity) of the disease, i.e. the maximum amount was shown in grade III. These complexes may play a part in the pathogenesis of this disease.