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In Nepal, many infections remain poorly characterized, partly due to limited diagnostic facilities. We studied consecutive febrile adults presenting to a general hospital in Kathmandu, Nepal. Of the 876 patients enrolled, enteric fever and pneumonia were the most common clinical diagnoses. Putative pathogens were identified in 323 (37%) patients, the most common being Salmonella enterica serotype Typhi and S. enterica serotype Paratyphi A (117), Rickettsia typhi (97), Streptococcus pneumoniae (53), Leptospira spp. (36), and Orientia tsutsugamushi (28). Approximately half of the Salmonella isolates were resistant to nalidixic acid. No clinical predictors were identified to reliably distinguish between the different infections. These findings confirm the heavy burden of enteric fever and pneumonia in Kathmandu, and highlight the importance of murine typhus, scrub typhus, and leptospirosis. Given the lack of reliable clinical predictors, the development of cheap and accurate diagnostic tests are likely to be of great clinical utility in this setting.

The geographic distribution of leptospirosis is widespread but no national surveillance program exists in Nepal to establish the incidence of leptospirosis or the disease burden. This study reports the incidence of symptomatic leptospirosis in military personnel participating in an efficacy study of a hepatitis E virus vaccine in Nepal. Among the 1566 study volunteers who completed follow-up, we evaluated 271 illnesses over 2.2 years for the presence of leptospira IgM antibodies by ELISA. Positive ELISA results were confirmed by the microscopic agglutination test. The annual incidence of disease was between 3.5 and 6.1 cases/1000. The prevalence of confirmed leptospirosis was 9% among hepatitis cases and 8% among febrile cases. The most reactive serovars were Bratislava, Autumnalis, Icterohaemorrhagiae, and Sejroe. Leptospirosis should be considered in the differential diagnosis of febrile illnesses and icteric syndromes in Nepal. Additional studies are needed to establish the broader distribution and the spectrum of disease in Nepal.

Diagnosis of acute hepatitis E by detection of hepatitis E virus (HEV)-specific immunoglobulin M (IgM) is an established procedure. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more information than conventional IgM tests that are interpreted as positive or negative. A previously described indirect immunoassay for total Ig against a baculovirus-expressed HEV capsid protein was modified to quantitate HEV-specific IgM in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model. A receiver-operating characteristics curve derived from 197 true-positive specimens and 449 true-negative specimens identified 30 WR units/ml as an optimum cut point. The median HEV IgM level in 36 patients with acute hepatitis E fell from 3,000 to 100 WR units/ml over 6 months, suggesting that 100 WR units/ml would be a more appropriate cut point for distinguishing recent from remote IgM responses. Among three hepatitis E case series, determination of the HEV IgM-to-total-Ig ratio in acute-phase serum revealed that most patients had high ratios consistent with primary infections whereas a few had low ratios, suggesting that they had sustained reinfections that elicited anamnestic antibody responses. The diagnostic utility of the new IgM test was similar to that of a commercially available test that uses different HEV antigens. In conclusion, we found that HEV IgM can be detected specifically in >95% of acute hepatitis E cases defined by detection of the virus genome in serum and that quantitation of HEV IgM and its ratio to total Ig provides insight into infection timing and prior immunity.

We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and intertest coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.

To determine safety and immunogenicity, a single 0.5 ml dose of a monovalent live-attenuated dengue (DEN) 4 (341750 Carib) vaccine was given sc to 3 groups of flavivirus nonimmune volunteers in increasing concentrations. Two recipients received 10(3) plaque forming units (PFU)/dose (1:100 dilution of stock vaccine). One remained asymptomatic, but became viremic between days 12 and 15, experienced a mild elevation of temperature (37.4 degrees C), and developed DEN-4 specific antibody. Neither recipient of the 10(4) PFU became infected. Eight volunteers then received undiluted vaccine (10(5) PFU). Viremia and antibody (neutralizing, hemagglutination inhibition, and IgM) developed in 5 of the 8 (63%). These 5 volunteers also developed a scarcely noticeable macular, blanching rash and minimal temperature elevations (37.3, 38.1, 37, 37.9, and 37.9 degrees C). Clinically insignificant decreases in total white blood cell, lymphocyte, and polymorphonuclear cell counts and an elevation in mononuclear cell counts occurred in association with viremia. This vaccine is safe, reasonably immunogenic, and suitable for further evaluation.

A strain of Langat virus (LGT), T-1674, was isolated from a pool of Haemaphysalis papuana Thorell collected in the forest of Khao Yai National Park in Central Thailand. The isolated virus was found to have growth and antigenic similarity to the prototype Langat strain (TP-21). This is the first report of a natural infection of Haemaphysalis ticks with LGT and of the isolation of this arbovirus in Thailand. The wide distribution of known vectors of LGT in Thailand suggests that the virus may be present in other areas of the country.

Sera from patients with pemphigus. other bullous dermatoses, and various control groups were examined to determine whether paramyxoviruses, particularly SVS and DU, could be isolated. Indirect immunofluorescent tests and virus neutralization tests were employed to determine the incidence of SV5 virus antibody in sera from patients with various dermatoses, and to determine whether antibody to SV5 was related to the antiepithelial antibody associated with pemphigus. The frequency of antibody to SV5 (9%) and DU (11%) virus in sera from patients with pemphigus was not higher than the frequency ot antibodies to SV5 (14%) and DU (17%) in sera of various control groups. The absence of antibodies to 8V5 and DU virus in sera from most patients with pemphigus, the absence of intercellular antiepithelial antibody in sets from dogs and most humans with antibody to SV5 and DU virus, and the absence of correlation of titers in a patient with both types of antibody indicate that SV5 antibody is not functionally related to “pemphigus antibody.” In addition, no SV5, DU, or other hemadsorbing paramyxovirus was isolated from sera of patients with pemphigus. Isolation from blister fluid was not attempted. Infection with SV5, DU, or similar paramyxovirus is probably not usually, if at all, related to the etiology of non-Brazilian pemphigus.

Dengue viruses were isolated by a plaque technique in LLC-MK2 cells from washed peripheral blood leukocytes of patients with hemorrhagic fever. In comparison with plasma, the use of peripheral blood leukocytes permitted greater than three times the recovery rate of viruses, allowed for the isolation of strains of virus from patients with high serum levels of antibody to dengue virus, and extended the period of detectable viremia. The use of peripheral blood leukocytes was especially useful for isolation of viruses from patients with hemorrhagic fever, in whom antibody titers were generally quite high during the acute phase of the disease. This method is recommended for use by laboratories with access to the appropriate acute-phase specimens. Of the peripheral blood leukocytes, adherent monocytes appeared to be the cells most likely affected; however, involvement of atypical lymphocytes, which are commonly found in patients with hemorrhagic fever, or polymorphonuclear leukocytes could not be excluded by this study. Only a small number of infected centers could be identified by infectious-center assays, but the marked augmentation of recovery of virus with the use of peripheral blood leukocytes indicated the presence in patients of a subpopulation of cells that permit dengue virus infection.

Patients with primary dengue infection developed dengue 2 virus (D2V) permissive peripheral blood leukocytes (PBL) 2–3 weeks after infection. PBL from healthy individuals with dengue antibody were permissive to D2V in vitro, suggesting that immunologically mediated in vitro D2V permissiveness persists for a relatively long time after recovery from dengue infection. However, PBL obtained from second infection dengue hemorrhagic fever patients did not support D2V growth during the acute phase of illness but did so during convalescence. Leukocytes from dengue-immune patients with typhoid fever or non-dengue viral illness were permissive throughout both acute and convalescent phases of illness although there was a tendency for increased permissiveness during convalescence. Acute phase PBL from DHF patients synthesized and secreted dengue neutralizing antibody in culture. Absence of D2V replication in these cultures was strongly, but not completely, correlated with antibody production. Other immunological mechanisms, in addition to antibody, may be operating in vitro or in vivo during acute phase dengue hemorrhagic fever to alter the permissiveness of PBL to D2V infection.

Counterimmunoelectrophoresis (CIE) was applied to detect dengue viral antigen in an experimental system and in clinical situations. In the experimental system, all four types of dengue viruses (dengue 1 to 4) were detected when tested against rabbit hyperimmune sera. With homologous antibody, antigen could be detected when hemagglutinating titer was as low as 1:20. In clinical situations, 67% of the sera of patients with dengue hemorrhagic fever collected on day 4 after onset of fever were positive by CIE. Antigens could be detected most frequently from day 4 to day 7. In 11% of the cases, antigens were found to persist as long as 38 days. Validity of this detection was confirmed in some cases by immunologic methods. Virtues of CIE are its technical simplicity, rapidity, and economy.

E N D O (3 A R D I T I S m a y be caused by highly pathogenic microorganisms such as Staphylococcus aureus, Streptococcus pyogenes, or Neisseria gonorrhoaeae, or it may be caused by bacteria of low virulence such as Streptococcus viridans or Streptococcus faecalis (enterococcus). Among the less common agents are the nonpathogenic Neisseria which normally inhabi t the h u m a n upper respiratory tract. The following is a report of a case caused by such an organism.

During 1974, 114 patients with dengue hemorrhagic fever were studied at the Bankok Children's Hospital. Over 40% of the patients had dengue shock syndrome. Five fatal cases were included in the study. Primary dengue infections were identified by absent or low titered antibodies in acute sera and the sequential development of IgM antibodies followed by IgG antibodies during convalescence. Three patients, aged 4,8, and 12 years, had primary dengue infections with shock. Although no convalescent sera could be tested two other patients, aged 7 to 12 years, with fatal disease also appeared to have primary infections. At the time of shock patients with primary infections had subnormal concentrations of complement factor 3. The data show that in older children dengue shock syndrome associated with complement depression can occur during primary as well as secondary infections.

A pool of whole-mouth saliva collected from five human carriers of hepatitis B surface antigen, subtype adr, was found to contain antigen particles with mean diameters of 23.3 and 41.8 nm as seen by immune electron microscopy. Two gibbons received subcutaneous injections of the pooled saliva and developed serological and, in at least one animal, biochemical evidence of hepatitis B virus infection at 12 and 22 weeks, respectively. Although none of eight other gibbons that were exposed by the nasal or oral routes were infected, the experiment demonstrated that human saliva can serve as a vehicle for the transmission of hepatitis B virus.

Histocompatibility antigen (HLA) A and B typing on lymphocytes from 87 unrelated Thai children who had been hospitalized with dengue shock syndrome (DSS) and/or dengue hemorrhagic fever (DHF) was compared with that found in 138 controls who had not been hospitalized with clinical dengue infection. These data are presented as descriptive information; however, a statistical analysis was performed to identify potentially important relationships for future study. Several deviations (P less than 0.05) were detected in the distribution of four HLA-A and three HLA-B antigens. The prevalence of one HLA-A antigen and two HLA-B antigens appeared to relate to the development of DSS, with a positive association seen for HLA-A2 and HLA-B blank and a negative relationship for HLA-B13. These findings require confirmation, but they do suggest that genetic susceptibility may be important in the development of DHF/DSS and indicate that further broader studies of genetic markers might be rewarding.

Studies of the immunologic responses following administration of a live, enteric-coated adenovirus (ADV) type 21 vaccine showed that nine of ten vaccinees and none of five controls developed neutralizing antibody. Antibody activity of serum and secretory immunoglobulins was assayed by using a 14 C-labeled ADV-21 antigen in a radioimmunodiffusion system. Increases in immunoglobulin M, A and G (IgM, IgA, IgG) activity were detected in sera from vaccinees but not in those from controls. IgA copro antibody activity was also shown in vaccinees but not in controls. Nasal secretions showed no detectable IgA antibody responses by this method. These studies show marked differences in serum and local IgA antibody activity in induced enteric ADV infection compared to previously reported responses after natural infection. The protective role of secretory IgA in adenovirus infections is obscure. However, absence of nasal IgA responses may indicate that protection against disease with enteric ADV vaccines depends primarily upon humoral antibody.

Sera obtained during a dengue-2 epidemic from individuals with primary or secondary group B arbovirus infections contained cross-reactive antibody against several viruses in this group. Hemagglutination-inhibition antibody titers of the immunoglobulin M fractions from these sera revealed a rise in antibody specific for dengue-2. The acute and convalescent sera in over half of the secondary infection group also had low-level unchanging immunoglobulin M antibody titers to yellow fever which implicated yellow fever immunization as the primary infection.