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.

Surveillance of anti-hantavirus antibodies among certain high-risk groups in Taiwan.

Hemorrhagic fever with renal syndrome is a serious health concern in neighboring countries of Taiwan, such as mainland China and Korea. In Taiwan, only two suspected cases were recorded before 1994. The first confirmed case was reported in 1995, but this proved to be imported. To study hantavirus infection in Taiwan, we tested blood collected from garbage collectors, animal handlers, patients with febrile illness of unknown origin, and field rats, the host of hantavirus, for the presence of antibody against hantavirus using an indirect immunofluorescent antibody technique. The positive rates were 1.55% (3/193), 3.45% (1/29), 1.42% (3/211), and 5.56% (9/162), respectively. The subtypes of hantavirus involved were either Hantaan-like or Seoul-like. These results showed that hantavirus may have already invaded Taiwan without our knowledge and physicians should be aware of this.

Post mass-immunization measles outbreak in Taoyuan County, Taiwan: dynamics of transmission, vaccine effectiveness, and herd immunity.

OBJECTIVES: Analysis of national surveillance data and a seroepidemiologic investigation were conducted to elucidate the causes and epidemiologic characteristics of a measles outbreak in Taoyuan, Taiwan, 1994. METHODS: Measles cases were identified through a national surveillance system. Reported cases and their physician or school nurses were interviewed to trace additional suspect cases and were sampled for serologic diagnosis. Measles-specific IgG and IgM were assayed. A confirmed case was defined as being positive for measles IgM test but not having received measles vaccination within the previous 3 months. RESULTS: The outbreak began in Taoyuan City in December 1993 and continued to spread in primary schools and kindergartens, but caused only sporadic cases in neighboring towns. Among 42 confirmed cases, 15 (38%) were primary school children and 16 (38%) were kindergarten children. Among 24 confirmed cases with a vaccination record, 7 had one dose of vaccination, 4 had two doses of vaccination, and 13 (54%) were unvaccinated. The overall measles susceptible proportion at a kindergarten before the outbreak was 8.1% (17/209) and the overall measles cumulative incidence among susceptibles was 0.65 (11/17). CONCLUSIONS: A measles vaccination coverage of 82% with the first dose at 9 months of age and 63% with the second dose (measles, mumps, and rubella) at 15 months was inadequate to block measles virus circulation in Taoyuan City in 1994. The city center, with a growing population, represents a high risk as an epicenter for measles outbreaks. Measles outbreaks may occur in a school population with 92% herd immunity.

[Seroepidemiology of Japanese encephalitis viral infection among 3-6 years old children from mountainous and plains townships located in the northern, central, southern and eastern Taiwan].

In order to evaluate the Japanese encephalitis virus (JEV) vaccination program in rural Taiwan, we conducted a seroepidemiological survey of JEV among rural children 3 to 6 years of age in Taiwan. The children were selected through a systemic sampling following stratification by age of children in 4 selected aboriginal villages and 4 adjacent nonaboriginal villages. The overall vaccine coverage rate for the primary (2 doses) dose was 81.2% (1853/2281) with higher rates (87.7%-87.9%) found among the more recent birth cohort of 3 to 4 years of age. The neutralizing antibody (NT) against JEV was measured with plaque reduction neutralization test (PRNT) using Nakayama strain as the virus. With a positive NT antibody defined as > or = 1:10 dilution of serum yielding more than 50% plaque reduction, the overall JEV NT antibody positive rate among children receiving 3 doses of vaccine was 67%. However, the age-specific positive rates varied significantly with varying ages; the lowest of 47% being among children 4 years of age which was lower than the rates of 68%, 76% and 87% among children of 3, 5 and 6 years of age, respectively. This trend of rising seropositive rates of JEV antibody with increasing age among 4 and 6 years of age was also noted among children who had received no vaccine, suggesting the importance of natural infection among rural Taiwanese children. Despite the high frequency of natural infection, the seropositive rates of JEV antibody still correlated well with the dose of vaccine received, i.e., 67% (1122/1664), 66% (65/97), 33% (4/12) and 40% (19/47) for children receiving 3, 2, 1, and 0 dose of JE vaccines, respectively (P < 0.0001 Chi-square for trend test). When stratified analysis by dose and by type of vaccines was conducted, a significantly higher seropositive rate of JEV NT antibody was noted among children receiving JE vaccine of Beijing type (87%) than children receiving Nakayama type (39%) (p < 0.0001, Chi-square test). Our data indicated that the JEV vaccination, in conjunction with JEV natural infection, has maintained high JEV NT antibody level among rural children of Taiwan.

Temperature effect on the sensitivity of ELISA, PA and WB to detect anti-HIV-1 antibody and infectivity of HIV-1.

BACKGROUND: This study is designed to resolve the problem of whether temperature or freeze/thaw cycle will have any impact on the sensitivity for detection of anti-HIV-1 antibody by particle agglutination (PA), enzyme-linked immunosorbent assay (ELISA) and western blotting (WB). To reduce potential risk for laboratory personnel exposed to HIV-infection, it will be useful to determine the temperature effect on HIV infectivity. METHODS: Testing sera were incubated at different temperatures or treated with several cycles of freeze and thaw. PA, ELISA and WB were used to detect anti-HIV-antibodies, whereas syncytia formation assay and polymerase chain reaction (PCR) were applied to detect HIV-infection. RESULTS: The data showed that certain temperature points (no treatment, 25 degrees C for 1 hr, 2 hrs and 4 hrs, 37 degrees C for 30 minutes and 60 minutes, 56 degrees C for 30 minutes and 60 minutes, 65 degrees C for 15 minutes and 30 minutes) had no impact on the testing results of ELISA, PA and WB in detection of anti-HIV-1 antibody. In addition, testing results of 50 normal human serum samples which had been heated to 56 degrees C for 30 minutes were still negative by ELISA and PA. Only the samples incubated at 65 degrees C for 60 minutes had slight differences in results. Freeze and thaw treatments of the serum did not alter anti-HIV testing results, either. Treatments of supernatant of HTLV-IIIB culture at 56 degrees C for 30 minutes and 60 minutes, 65 degrees C for 15 minutes and 30 minutes could eliminate the syncytia formation caused by HIV-infection. Further analysis of the samples by PCR was able to detect HIV-specific sequences in all the treatments. CONCLUSIONS: Anti-HIV antibody is quite stable in serum, even when it is pre-heated to 56 degrees C for 30 minutes. Freeze and thaw treatment of serum samples up to seven cycles did not change the results, either. In addition, to minimize the potential risk of laboratory personnel exposed to HIV infection, pre-treatment of serum samples with heat at 56 degrees C for 30 minutes or 60 minutes can reduce HIV infectivity. However, laboratories still must emphasize the importance of universal precautions rather than heat-inactivation of serum to prevent occupational transmission of HIV.

Food-borne disease outbreaks due to bacteria in Taiwan, 1986 to 1995.

Between 1986 and 1995, 852 outbreaks of food-borne disease involving 26,173 cases and 20 deaths were reported in Taiwan. About 80% of the outbreaks occurred in the warmer months, i.e., between April and October. Of the 852 reported outbreaks, 555 (65%) were caused by bacterial pathogens. The three most common bacteria involved were Vibrio parahaemolyticus (35%, 197 of 555 outbreaks), Staphylococcus aureus (30%, 169 of 555 outbreaks), and Bacillus cereus (18%, 104 of 555 outbreaks).

[Primary surveillance of spotted fever group antibodies on rats in the Kinmen area].

The positive rate of rickettsial antibodies of 107 rats in the Kinmen area by indirect immunofluorescent antibody (IFA) technique was 0% (0/107) in typhus fever, 38.3% (41/107) in scrub typhus and 66.4% (71/107) in spotted fever group; the positive rate (42.9%) of spotted fever group of 21 rats in Taiwan island also higher than scrub typhus (19.0). It suggests that spotted fever group patients may be present in our country but have not been discovered.

[Diagnosis of Yersinia pestis].

There is no plaque case report in Taiwan since 1952. However, it is necessary to set up a laboratory system to investigate the distribution of Yersinia pestis in the natural environment to implement the public policy for preventing plague. Besides the traditional methods; e.g. culture, microscopic observation, biochemical characteristics, anti-F1 antigen detection by slide agglutination, immunofluorescence, and phage lytic assay, PCR was used as rapid screening test in our study. These laboratory methods were used to examine whether the flea samples harvested in King-Men island carry Y. pestis. The results showed that the flea index per mouse was high but no Y. pestis was detected in the fleas.

[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.

Complete nucleotide sequence of the genome of Japanese encephalitis virus ling strain: the presence of a 25-nucleotide deletion in the 3'-nontranslated region.

The complete sequence of the genome of the Japanese encephalitis virus (JEV) Ling strain isolated from the brain of a patient in Taiwan in 1965 was cloned by using the reverse transcription-polymerase chain reaction method. Seven overlapping cDNA clones that span the entire virus genome were isolated and sequenced to determine the complete nucleotide sequence, which is 10,951 nucleotides in length. As reported for three other JEV strains (Beijing-1, SA-14, and JaOArS982), the Ling strain contains 95 nucleotides in the 5' nontranslated region (NTR), followed by a single open reading frame of 10,296 nucleotides. However, the length of the 3' NTR of JEV Ling is 560 nucleotides, 25 nucleotides shorter than that of other JEV strains sequenced to date. Comparison of nucleotide and amino acid sequences among these four JEV strains showed that nucleotide (amino acid) sequence divergence in the translated region varied from 1.25% to 3.27% (0.49-1.63%). The nucleotide (amino acid) divergences between the Ling and Beijing-1 strains were 1.25% (0.87%) and between the SA-14 and JaOArS982 strains were 1.42% (0.49%). These values are lower than those found between the Ling and SA-14 [2.44% (1.02%)] or the Ling and JaOArS982 strains [2.84% (0.93%)], as well as those between Beijing-1 and SA-14 [3.14% (1.60%)] or Beijing-1 and JaOArS982 [3.27% (1.63%)] strains. Sequence comparisons of subregions of the genomes i.e., structural genes, nonstructural genes, or individual genes, showed divergence similar to that obtained by comparing the entire sequence. It is likely that the JEV sequence divergence between two human isolates or between two mosquito isolates is lower than that between a human isolate and a mosquito isolate.

Large-scale Vero cell culture on microcarriers in a twenty-liter stirred tank fermentor.

In biotechnology, animal cell culture is an important process for the production of many biologicals such as vaccines, monoclonal antibodies, or other recombinant products. Among many established continuous cell lines, Vero cells can be maintained in many passages in cultures without inducing tumorigenicity and have been recommended by World Health Organization for the production of human biologicals. Owing to its anchorage-dependent growth characteristics, Vero cells can be grown on microcarrier in a suspension vessel where microcarrier provides the culture system with a high culture surface to volume ratio. In this paper we compared the growth kinetics of Vero cells on Cytodex 1 microcarrier in a 20-liter fermentor vs. 100 ml spinner flask culture. The kinetics of Vero cell growth in the 20-liter fermentor was similar to the results obtained from small spinner flask culture, as determined by cell specific growth rate or corresponding doubling time. The approximately 150-fold increase in culture vessel volume did not compromise the growth kinetics of Vero cells, suggesting the system is applicable for large stirred-tank fermentor cultures.

[Serotype distribution and antimicrobial susceptibility of group A streptococci (Streptococcus pyogenes) isolated in Taiwan].

T-protein serotypes and antimicrobial susceptibility of a total of 139 group A streptococci (GAS) strains isolated in Taiwan area in 1993 and during the outbreak of scarlet fever in 1994 were analyzed. All strains were T-typable, and T12 (42.46%) and T4 (38.85%) were the dominant T types. According to the results of analysis of antimicrobial susceptibility, all GAS strains were divided into 9 resistotypes, A (all susceptible), B (resistant to tetracycline), C (resistant to erythromycin and tetracycline), D (resistant to chloramphenicol and tetracycline), E (resistant to chloramphenicol and clindamycin), F (resistant to chloramphenicol, clindamycin and tetracycline), G (resistant to clindamycin, erythromycin and tetracycline), H (resistant to chloramphenicol, clindamycin, erythromycin and tetracycline), and I (resistant to chloramphenicol, clindamycin, erythromycin, tetracycline and vancomycin). Type B (37.42%) was the dominant type. Type A (25.91%), and type H (26.63%) also appered with high incidence. Most of strains isolated from Mid-Taiwan were type H. Only one strain, that was isolated in I-lan, was resistant to vancomycin, in addition to resistant to chloramphenicol, clindamycin, erythromycin, and tetracycline. All strains were susceptible to penicillin G, ampicillin, and ceftriaxone. Some strains were resistant to chloramphenicol (32.38%), clindamycin (30.22%), erythromycin (31.66%), tetracycline (73.39%), and vancomycin (0.70%). During the outbreak of scarlet fever in 1994, the dominant T types of strains isolated in North-Taiwan and Mid-Taiwan were T4 and T12, respectively, and the major resistotypes of those strains were B and H types, respectively. These clues suggested that the outbreaks occurring in North-Taiwan and Mid-Taiwan may have no epidemiological linkage between each other.

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.

Legionella pneumophila infection in Taiwan: a preliminary report.

Legionella pneumophila infection has been recognized as an important cause of pneumonia. However, epidemiologic baseline data about the occurrence of legionella-related pneumonia and the distribution of legionellae in aquatic environments are still not available in Taiwan. A survey conducted by the National Institute of Preventive Medicine found that 8.6% of 487 pneumonia patients studied were positive for the L. pneumophila serologic test (indirect fluorescent test, IFA) and 32.0% of the 25 water samples were positive for isolation of legionellae. In conclusion, this survey supported the existence of Legionnaires' disease in Taiwan, indicated a preliminary prevalence of L. pneumophila antibodies among pneumonia patients and identified the prevalence of legionellae in water environments.

Identification of serotypes of Rickettsia tsutsugamushi by nested polymerase chain reaction.

Nested polymerase chain reaction (PCR) was applied to identify the serotypes of Rickettsia tsutsugamushi isolated from patients. The primers used for PCR were based on the nucleotide sequences encoding a 56 kDa antigen of rickettsiae. Comparing to the conventional immunofluorescence assay (IFA), which displays a considerable degree of cross-reactivity among different species, the result obtained suggests that the polymerase chain reaction method is much more reliable than IFA.

Use of the ProSpecT microplate enzyme immunoassay for the detection of pathogenic and non-pathogenic Entamoeba histolytica in faecal specimens.

A commercial microplate enzyme immunoassay (ProSpecT EIA; Alexon Inc., Sunnyvale, CA 94089, USA) was compared with conventional microscopy for the diagnosis of Entamoeba histolytica infection. Using specimens known to be infected, the sensitivity of the ProSpecT EIA was 78% and its specificity was 99%. No cross reaction with other intestinal parasites was observed. The ProSpecT EIA and conventional microscopy (using merthiolate-iodine-formaldehyde direct wet mounts and concentration techniques) were then used to detect E. histolytica infections in 431 patients in a mental hospital in Taiwan. Using single stool specimens, microscopy detected infection in 10.9% of the patients, compared with 16.9% detected by ProSpecT EIA. The latter method was simple and quick, but more expensive, and could be used to complement microscopy if a prompt diagnosis is desired clinically. However, ProSpecT EIA cannot differentiate between pathogenic E. histolytica and non-pathogenic strains (= E. dispar), which limits its usefulness.

ELISA-based colorimetric detection of Rickettsia tsutsugamushi DNA from patient sera by nested polymerase chain reaction.

A rapid diagnostic system for scrub typhus was established using colorimetric detection of nested polymerase chain reaction (PCR). This system relied on binding the amplified DNA via a sequence in one of oligodeoxyribonucleotide to the DNA-binding protein GCN4 coated on the well of a micotiter dish. The primer pairs used for the nested PCR were designed on the basis of the homologous nucleotide sequence of the gene that encodes the 56 kDa antigen of serovariants. With this colorimetric PCR, diagnosis can be performed easily from serum samples of patients before the antibody titer increases or in the early stage of the disease. Furthermore, these positive results are able to be confirmed by pathogenic isolation.

Differentiation of Salmonella typhi strains by ribosomal RNA gene restriction patterns.

Owing to the limited value of phage typing to determine the epidemiological association of Salmonella typhi (S. typhi) strains isolated from the source of typhoid fever, we analyzed ribosomal RNA (rRNA) gene restriction patterns to differentiate the independently isolated strains of identical phage type. The data showed that the restriction patterns of PstI was most polymorphic among four enzymes (BamHI, EcoRI, PstI, and SmaI) used, which revealed 13 types among 25 strains belonged to 4 phage types, 1 untypable and 2 not-determined strains. Total 25 strains of S. typhi were divided into 15 combination types by the rRNA restriction patterns with three enzymes (BamHI, PstI, and SmaI).

[Isolation of Rickettsia tsutsugamushi from the blood samples of patients in Taiwan].

Scrub typhus, an acute febrile eruptive disease caused by Rickettsia tsutsugamushi, is one of the most commonly reported communicable diseases in Taiwan. However, information about the epidemic trends of scrub typhus in Taiwan is still very limited. Therefore, this study was performed to isolate the causative agent and determine the prevalence of three different serotypes. From June 1992 to December 1994, lymphocyte cell cultures were grown from different regions of Taiwan. Cell cultures were assayed for R. tsutsugamuhi via immuno-fluorescence, using antisera against Gillian, Karp and Kato strains. Thirty-three of the specimens were positive for R. tsutsugamushi, including one from Keelung, 23 from Taitung, 2 from Kinmen and 7 from Lienjan. Of these, 15 were classified as Gillian-related types, 10 were Karp-related types and 8 were not positively classified. These results indicate Gilliam-and Karp-related serotype are more prevalent than the Kato-related serotype in Taiwan.

[Influenza virus infections in Taiwan from 1979 to 1994].

In order to understand the epidemic trends of influenza virus infection in Taiwan, 5,677 throat-swab specimens were collected from June 1979 to December 1994. The influenza virus was detected in 300 specimens including samples collected at Taichung and Tainan from 1981 to 1982. Among them, 209 isolates (67.9%) were identified as influenza virus type A and 99 isolates (32.1%) were type B influenza virus. Influenza virus infection can be identified, based on the frequency of detection of the virus, throughout the year in Taiwan. Some strains were referred to as intermediate strains due to antigenic drift. About 80% of the isolates identified in this laboratory were from the children under 12 years old. The rate of isolation of the virus was about 47% during the epidemic season. The influenza virus strain A/Taiwan/1/86(H1N1) used as the standard strain for the CDC reagent kit was first isolated by this laboratory in April 1986. We isolated a new strain of influenza virus on December 16, 1991 and later the virus was identified as A/Beijing/32/92-like, which was first isolated on January 31, 1992 at Beijing. Prior to the identification of B/Panama/45/90, we isolated a strain similar to B/Panama/45/90 on January 3, 1990. Owing to delays in shipping of the isolated viruses to the CDC for further confirmation, however, the two new isolates missed the chance to be named as Taiwan strains and otherwise would be listed as standard Strains by the CDC.