Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture.

Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods.

Measles seroepidemiology and decay rate of vaccine-induced measles IgG titers in Taiwan, 1995-1997.

Community-based seroepidemiologic studies were conducted to monitor the effectiveness of measles immunization programmes and to estimate the decay rate of vaccine-induced measles IgG titres. Sera collected from a mountain (792 sera), rural (875 sera) and urban (894 sera) populations in 1995-1997 were available. Measles IgG was quantified using a commercial EIA kit. Measles IgG seroprevalence and geometric mean titre (GMT) were calculated by setting the cut-off titre at 50 mIU/ml. The decay rate of measles IgG titres was estimated by assuming that the measles IgG titres, without exposing to wild measles virus, decay exponentially and constantly after 1 year post vaccination. The half-life of measles IgG titres was calculated from the corresponding decay rate. Measles IgG seroprevalences in these three populations have reached >95% in school children (7-18 years old) and >98% in young adults (19-25 years old) but varied from 87 to 96% in pre-school children (4-6 years old). Two-dose vaccinees, comparing with 1-dose vaccinees, had a significantly higher seroprevalence (98 versus 92%, P<0.01) and a slightly longer half-life of measles IgG titres (61 versus 27 months, P=0.08) but the measles IgG GMT in the two groups did not differ significantly (675 versus 618 mIU/ml, P=0.78).

Antibody to the nonstructural protein NS1 of Japanese encephalitis virus: potential application of mAb-based indirect ELISA to differentiate infection from vaccination.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect and differentiate the antibody responses to Japanese encephalitis (JE) virus nonstructural protein NS1 between infected and vaccinated individuals. The results showed that all convalescent sera from JE patients contained NS1-specific IgG antibodies, while 65 and 40% of these sera showed detectable NS1-specific IgM and IgA antibodies, respectively. Specificity analysis showed that NS1-specific IgM and IgA antibodies from JE patients do not cross-react to dengue virus NS1 glycoprotein, while IgG antibodies from 10% of JE patients showed significant cross-reaction to dengue virus NS1 glycoprotein. To differentiate infection from vaccination, the immune sera from 24 children vaccinated with inactivated JE vaccine were analyzed. The data showed that none of these immune sera had detectable NS1-specific IgG antibodies. The results demonstrated the potential application of JE NS1-specific indirect ELISA to differentiate infection from vaccination.

Dengue NS1-specific antibody responses: isotype distribution and serotyping in patients with Dengue fever and Dengue hemorrhagic fever.

To understand the antibody responses to dengue (DEN) nonstructural 1 (NS1) glycoprotein and their roles in protective immunity or pathogenesis of dengue fever (DF) and dengue hemorrhagic fever (DHF), we have analyzed the NS1-speccific IgM, IgA and IgG antibodies from patients with DF and DHF. An isotype-specific, indirect enzyme-linked immunosorbent assay (ELISA) was established by coating a NS1-specific monoclonal antibody (MAb), D2/8-1, to capture soluble NS1 antigens secreted in the culture supernatants of Vero cells infected with DEN virus. We observed strong anti-NS1 antibody responses in all of the convalescent sera of patients with DF and DHF. Similar NS1-specific isotypic and serotypic antibody responses were found in the sera from DF and DHF patients. The results showed that all DEN infections induced significant NS1-specific IgG, whereas 75% and 60% of primary DF patients vs. 40% and 90% of secondary DF patients produced IgM and IgA antibodies, respectively. Specificity analysis showed that DEN NS1-specific IgG and IgA antibodies cross-react strongly to Japanese encephalitis (JE) virus NS1 glycoprotein, whereas DEN NS1-specific IgM antibodies do not cross-react to JE virus NS1 glycoprotein at all. The serotype specificity of NS1-specific IgM, IgA and IgG were found to be 80%, 67% and 75% for primary infections, and 50%, 22% and 30% for secondary infections in positive samples of DF patients. Similar pattern was found in DHF patients. The results showed that all of the DF and DHF patients produced significant NS1-specific antibodies. We did not observe direct correlation between the anti-NS1 antibody responses and DHF because sera from patients with DF and DHF showed similar anti-NS1 antibody responses.

Efficient diagnosis of dengue infections using patients' peripheral blood leukocytes and serum/plasma.

OBJECTIVE: Dengue fever has been one of the most important health problems in Taiwan since a large outbreak during 1987 and 1988. It is critically necessary to have a diagnostic approach that can detect early infections in an outbreak or even find infections existing in silent transmission of the disease. METHODS: To develop an efficient diagnostic protocol, 105 plasma/serum and 35 peripheral blood leukocyte (PBL) specimens from the 1994 outbreak in southern Taiwan were collected for assessment by various diagnostic techniques in this study. RESULTS: In acute blood samples, dengue viruses were isolated from 19.4% (14/72) and 33.3% (14/42) of reported and confirmed cases, respectively. Viral RNA in serum/plasma was detected from 20.0% (12/60) of acute samples, which was significantly higher than that from convalescent samples (3/44; 6.8%). However, viral RNA in PBLs, detected by reverse transcription polymerase chain reaction (PBL-RT-PCR), could be observed in 73.2% (19/26) and 66.7% (6/9) of acute and convalescent samples, respectively. The persistence of dengue viruses in PBLs was also evidenced by the presence of viral antigens in 42.9% (4/7) of confirmed convalescent samples by the immunofluorescence antibody test. In addition, IgM antibodies were detected in 43.8% (46/105) of reported cases and 85.2% (46/54) of confirmed cases by the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). CONCLUSIONS: Although IgM antibody detection achieved the highest detection rate among techniques assessed in this study, no individual test can actually reach full efficiency for early diagnosis of dengue infections. Here, we propose a protocol which applies MAC-ELISA and PBL-RT-PCR in sequence, by which 22 confirmed cases were definitely proved as dengue positive. High levels of both sensitivity and specificity were shown in this protocol.

Genetic variation of Japanese encephalitis virus in Taiwan.

A 280-nucleotide sequence from the capsid-premembrane (C/preM) gene region of 44 Japanese encephalitis (JE) virus strains isolated in Taiwan from mosquitoes from 1983 to 1994, and 3 strains, (Ling [1965], Chang [1965], and HV1 [1958]) isolated from human brain were analyzed by direct sequencing of reverse transcription-polymerase chain reaction (RT-PCR) amplified products and compared with the corresponding sequences of reference strains. The overall sequence homology of the 47 isolates was > or = 93.3%. Taking 12% nucleotide divergence as a cut-off value, all isolates fell into genotype 3, which included strains from Japan, China, the Philippines, Sri Lanka, India, and Nepal. High nucleotide homology was observed among isolates from different regions of Taiwan and different time periods; on the other hand, high variation existed among isolates from the same region and time period. Phylogenetic analysis showed that the 47 Taiwan isolates fell into three clusters. Twenty-five isolates formed cluster 1, 18 isolates cluster 2, and four isolates cluster 3. Isolates in cluster 1 showed greater (< or = 2.9%) intragroup divergence compared to those in cluster 2 (< or = 1.1%) or cluster 3 (< or = 0.7%). The majority of isolates from northern (73.3%) and central (60%) Taiwan belonged to cluster 1, whereas most isolates (66.7%) from southern Taiwan belonged to cluster 2. Comparison with other Asian JE virus strains showed that isolates of cluster 1 were more specific to Taiwan than isolates of cluster 2 and cluster 3.

The epidemiology of Japanese encephalitis on Taiwan during 1966-1997.

Japanese encephalitis (JE) is an endemic disease in Taiwan. A mass vaccination program of children against JE was first implemented in 1968. Along with general improvements in various aspects of living conditions over the years, the program has brought JE well under control. The main characteristics of JE epidemiology in Taiwan in the past 3 decades are as follows. The transmission mode remains unchanged-that is, the amplification stage of the virus in pigs is followed by a human epidemic each year. The frequency of JE incidence has dropped significantly. The incidence rate of confirmed cases was 2.05 per 100,000 in 1967, the highest in record, and merely 0.03 per 100,000 in 1997. Confirmed cases occur sporadically all over the island. The peak of the epidemic season has shifted from August in the 1960s to June since the 1980s. The age distribution of confirmed cases has shifted gradually from mainly children to adults. Vaccine efficacy for those having received more than 2 doses of the vaccine is estimated to be about 85%.

[Immunity against diphtheria in Taiwan].

The vaccination of combined diphtheria, pertussis, and tetanus (DPT) vaccine provides good immunity against childhood diphtheria in Taiwan. However, a waning protective antibody level has been observed with an increase in age. Therefore, we assessed diphtheria immunoglobulin (DIG) level in Taipei City as representatives of urban dwellers and in King-Shan County as representatives of rural dwellers to evaluate the status of immunity against diphtheria in the population of Taiwan. In total, 1239 serum samples collected from the resident population, age 0-91 years, were detected by toxin neutralization test with VERO cells. The DIG level > or = 0.01 IU/mL was considered to be seropositive and > or = 0.1 IU/mL was considered to be fully protective. The positive rate and fully protective rate for all persons were 79.9% and 36.6%, respectively. The age specific positive rate and fully protective rate for children under 14 years of age were 97.0% and 78.0%, for 20-29 years of age group, were 38.0% and 8.3%, respectively, to be the lowest record among tested age groups. Then, both rates increased proportionately with age. Among those birth cohorts born in the diphtheria immunization era (since 1955), the antibody levels were inversely correlated with age, suggesting a decreased opportunity for exposure to natural diphtheria infection in the recent years and the duration of the vaccine inoculation. This finding indicates the need of a booster vaccination for young adults to ensure a full term protection and a long lasting diphtheria control.

[Detection of neutralizing antibodies to Japanese encephalitis virus by enzyme-linked immunosorbent assay (ELISA)].

Competitive ELISA was used for the detection of neutralizing antibody to JE. Based on the principle that human serum JE antibody competed with JE monoclonal antibody (MAb) for JE antigen, it was found that 3 JE MAbs (E3-3, NPF-5 and NNN-5) were suitable for competitive ELISA for the detection of JE neutralizing antibody. The sensitivity of cometitive ELISA for 29 JE confirmed serum specimens with titer of plaque reduction neutralization test (PRNT) was checked to be 82.1% (23/28). The specificity of E3-3 MAb to JE used in competitive ELISA was 100%. Correlation coefficient of JE confirmed cases of 57 hemagglutination inhibition (HI) titers in 1995 and 37 PRNT titers in 1994 compared with competitive ELISA were 0.744 and 0.732, respectively. Compared the competitive ELISA titers of 154 sera of healthy people with PRNT titers, the results showed that 70% of the sera could be detected by competitive ELISA which saved a lot of time and manpower.

Detection of IgM antibody to Japanese encephalitis virus infection by enzyme-linked immunosorbent assay (ELISA).

BACKGROUND: Japanese encephalitis (JE) is an important infectious disease in Taiwan, with reported cases observed all the year around. Laboratory tests for JE consist mainly of hemagglutination inhibition (HI) test and neutralization test (NT). Commercialized enzyme-linked immunosorbent assay (ELISA) kits for detection of JE-IgM are still not available. Therefore an attempt has been made to develop a sensitive and rapid ELISA for detection of the IgM antibody to JE to serve as an indication of recent Japanese encephalitis virus infection. METHODS: Both positive and negative JE serum specimens, confirmed by HI test, were checked for IgM antibody to JE by ELISA. The optimum concentration of biotin-IgG and avidin horse-radish peroxidase conjugate used in MAC-ELISA were 1/2000 and 1/ 15000, respectively. RESULTS: From 1987 to 1989, 118 paired serum specimens of HI-confirmed JE patients were tested for JE-IgM by ELISA. The positive rate of JE-IgM was 65.7% (25/38), 73.9% (17/23), 93.5% (29/31) and 88.8% (8/9) for the consecutive first to fourth weeks after onset of the disease. The JE-IgM antibody of 17 serum specimens collected from the 5th to the 10th week after onset of the disease were 100% detected. In addition, among the 13 HI-confirmed JE cases occurring in 1994, 84.6% of the acute phase serum specimens demonstrated the JE-IgM antibody. CONCLUSIONS: About 65.7% of the JE-IgM of the acute serum specimens collected within one week after onset of the disease were detected. The JE-IgM positive rate elevated as the days from disease onset increased. Therefore the appearance of JE-IgM could be used as an indication of recent JEV infection to serve as a rapid laboratory diagnostic tool.

Homologous and heterologous neutralization antibody responses after immunization with Japanese encephalitis vaccine among Taiwan children.

Because 21 immunized children (13%) among the 162 confirmed Japanese encephalitis (JE) cases during 1986-1991 occurred in Taiwan, we collected 320 serum samples from Taiwan children aged 15-31 and 27-44 months immediately before the 1st dose (n = 41) and 1-3 months after the 2nd dose (n = 78, 27 pairs), and immediately before (n = 58) and 1-3 months after the 3rd dose (n = 143, 44 pairs) to determine neutralization antibody (Nt Ab) against the Nakayama (N) and Beijing-1 (B) strains and two Taiwan wild type JE viruses (JEV): CC-27 and CH-1392. Our Nt results showed that (1) B vaccine stimulated a better homologous Ab response than N vaccine for Nt Ab seropositivity rate (NASR), produced a higher level of Nt titer after the primary immunization [2 doses = 100% vs. 91%, geometric mean titer (GMT) = 115 vs. 22], had a greater booster effect (3 doses: 100% vs. 95%; GMT = 320 vs 33), and showed a better capability to neutralize two local Taiwan JEV strains, particularly only after 3 doses (ave. NASR for B vs. N = 90% vs. 10%; and GMT for B vs. N = 154 vs. 1); (2) the two wild type JEV strains had different plaque morphology and antigenic variation and the CC-27 strain was not neutralized as well as the CH-1392 strain after 3 doses of vaccine (BBB or NNN or NNB); and (3) 30% of the children had lost JEV Nt Ab one year after the 2nd dose of N vaccine and natural infection with JE virus did occur among those children after immunization. In conclusion, (1) three doses of mouse-brain vaccine are the minimum requirement to protect children against the local Taiwan JEV-, (2) the best strain for a JE vaccine depends on level of Nt Ab it induced, the molecular epidemiology and antigenic variation of the JEV in each local area; and (3) future vaccine must produce better B- and T-cell memory.