Molecular characterization of a new serovar of Salmonella bongori 13,22:z39:- isolated from a lizard

Three Salmonella strains isolated from a lizard (Gallotia simoni) in the "Isla del Hierro" (Canary Islands, Spain) were serotyped as Salmonella bongori serotype 13,22:z39:-, which has not been described in the Kauffmann-White scheme of Salmonella serovars. In order to shed light on the assignment of those strains to the S. bongori species, several genes were amplified and/or sequenced. The iroB gene has been reported to be present only in S. enterica, while the invA gene has been described as being a helpful tool in distinguishing Salmonella from other bacterial species. Both genes were amplified and, as expected, only invA could be amplified. The fliC gene, encoding the phase 1 flagellin fljB gene, encoding phase 2 flagellin, and the gapA gene, which is believed to present polymorphic alleles among different subspecies, were amplified and sequenced. The sequence obtained from fliC(z39) matched with the sequences fliC(z39) obtained from other serovars. The sequence obtained from gapA clustered into the S. bongori group when it was compared to others previously described. We conclude that these three isolates are members of the S. bongori species representing a new serovar that will be described in the next supplement to the Kauffmann-White scheme.     

Clonal nature of Salmonella typhi and its genetic relatedness to other salmonellae as shown by multilocus enzyme electrophoresis, and proposal of Salmonella bongori comb. nov

Crude cell extracts of 26 isolates of Salmonella serotype typhi (S. typhi) and 48 other Salmonella isolates representing 28 serotypes and seven DNA hybridization subgroups were analyzed for electrophoretic variants of 24 metabolic enzymes by starch gel electrophoresis. All strains of S. typhi had identical isoenzyme patterns, indicating that they were a single clone. All of the enzymes detected in the remaining strains were polymorphic, and the degree of genetic variation was quite high. The average number of alleles per enzyme locus was 4.7, and the mean genetic diversity per locus was 0.556. Thirty-two distinct allele profiles, or electrophoretic types (ETs), were found in these 48 strains of Salmonella serotypes other than S. typhi. Analysis of the genetic relationships of the ETs to each other showed that, with one exception, the ETs formed subgroups that were consistent with the subgroupings based on DNA hybridization studies. ET profiles were not always linked to specific serologic patterns. These data show that multilocus enzyme electrophoresis has a potential application in epidemiologic and taxonomic studies of salmonellae, although it is not differential for S. typhi. We also propose a new species, Salmonella bongori comb. nov., a new combination base on the elevation of Salmonella choleraesuis subsp. bongori to the level of species.     

Occurrence and characterization of Salmonella enterica subspecies enterica serovar 9,12:l,v:- strains from Bulgaria, Denmark, and the United States

In 2006, Salmonella enterica serovar I 9,12:l,v:- emerged in Bulgaria. The aim of this study was to characterize Salmonella serovar I 9,12:l,v:- isolates from Bulgaria, Denmark, and the United States. We compared isolates of Salmonella I 9,12:l,v:- and diphasic serovars with similar antigenic formulas by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility. The phase 2 flagellin gene (fljB) was also sequenced for selected isolates. By PFGE, the Salmonella I 9,12:l,v:- isolates from Bulgaria were indistinguishable from the isolate from the United States and distinct from isolates from Denmark; furthermore, several Salmonella I 9,12:l,v:- were indistinguishable from an isolate of Salmonella serovar Goettingen. Sequence analysis showed 100% sequence identity with known H:e,n,z15 sequences of Salmonella Goettingen, which has the antigenic formula I 9,12:l,v:e,n,z15. The study indicated that Salmonella I 9,12:l,v:- is a monophasic variant of Salmonella Goettingen and is present in different countries and on different continents.     

Application of molecular methods for identification of strains classified as Salmonella enterica serovar 6, 7:-:- by conventional serotyping

An increased prevalence of Salmonella enterica serovar Tennessee (6, 7: z 29 :-) was observed in broiler flocks in Denmark in 1994 and a parallel increase in the prevalence of Salmonella enterica serovar 6, 7:-:- was demonstrated, albeit at a lower level. Plasmid profiling and ribotyping revealed similar genotypes and it was speculated that serovar 6, 7:-:- could represent a non-motile variant of Salmonella Tennessee. Re-testing of the Salmonella 6, 7:-:- isolates demonstrated the presence of flagella through positive motility. All isolates but one demonstrated motility using both tube tests and light microscopy of overnight broth cultures. Molecular characterization indicated that all but two isolates previously classified as Salmonella 6, 7:-:, were isolates of Salmonella Tennessee and Salmonella Infantis, exhibiting reduced motility. Re-serotyping and multiplex polymerase chain reaction analysis for the phase 2 gene fljB demonstrated variants of Salmonella Infantis (6, 7: r: z 49 ) expressing the R-phase antigen (Rz 49 ) and possessing the gene for normal phase 2 antigen H: 1, 5. One of the two undefined strains demonstrated genotypic identity with a Salmonella Livingstone reference strain. The remaining putative 6, 7:-:- strain could not be identified and was genuinely non-motile. Diagnostic procedures performed initially were thus insufficient to differentiate between the different levels of motility and also resulted in mis-serotyping. As similar observations were made with two of 14 isolates received from a foreign laboratory, this may represent a general diagnostic problem.     

Supplement 1999 (no. 43) to the Kauffmann-White scheme

This supplement reports the characterization of 26 new Salmonella serovars recognized in 1999 by the WHO Collaborating Centre for Reference and Research on Salmonella: 15 were assigned to S. enterica subsp. enterica, seven to subspecies salamae, two to subspecies diarizonae, and one to subsp. houtenae; and one to S. bongori. In addition, the antigenic factor H:z89 is described.     

Inferring the potential success of pneumococcal vaccination in Italy: serotypes and antibiotic resistance of Streptococcus pneumoniae isolates from invasive diseases

To evaluate the potential impact of antipneumococcal vaccination in Italy, Streptococcus pneumoniae isolates from invasive disease were collected from 65 laboratories in the years 1997-2000. Of the 503 isolates examined, 15% were from children <5 years and 34% from adults > or = 65 years. The most frequent serogroups were, in ranking order, 14, 19, 6, and 23. Overall, 93.8% of the isolates belonged to serogroups enclosed in the 23-valent polysaccharide vaccine. Among children isolates, serotypes 14, 6B, and 23F comprised 60% of the isolates; overall, 72% of the isolates belonged to serotypes included in the heptavalent conjugate vaccine. Penicillin nonsusceptible isolates (10%) belonged to a limited number of serogroups, being more common in serogroups 19 and 9 and in the nonvaccine serogroups 24 and 35. Erythromycin-resistant isolates (29%) belonged to several serogroups, more frequently to serogroups 14, 6, and 19. Both vaccines are potentially able to prevent the majority of resistant infections in the respective age groups in Italy.     

Occurrence of extended spectrum beta-lactamases among isolates of Enterobacteriaceae from urinary tract infections in southern Italy

The present investigation was undertaken to assess the prevalence of extended-spectrum beta-lactamases (ESbetaLs) among urinary tract infection (UTI) isolates. During 4 months in 2004, a total of 650 Enterobacteriaceae strains from UTIs was collected by five clinical microbiology laboratories located in southern Italy and the beta-lactamase production was investigated. A total of 50 of the 650 isolates were double-disk positive and suspected of producing an ESbetaL; Escherichia coli (36.0%) and Klebsiella pneumoniae (32.0%) were the most common species among all ESbetaL producers. Characterization of ESbetaL determinants was carried out by the colony blot hybridization method, and polymerase chain reaction (PCR) and DNA sequencing in order to identify the presence of bla (TEM), bla (SHV), bla (PER), and bla (CTX-M) determinants. The ESbetaL variants found in this study were the following: TEM-15, TEM-24, TEM-52, TEM-134, SHV-12, CTX-M-1, CTX-M-3, CTX-M-15, and PER-1. As expected, the majority of the isolates were found to be susceptible to imipenem (94%), cefepime (54%) and piperacillin-tazobactam (54%). The results of this survey show the prevalence of ESbetaL enzymes among enterobacterial pathogens causing UTIs in southern Italy.     

Molecular analysis of strains of Shigella boydii isolated in northern and southern Italy

In the years 1981-1988, Shigella boydii played a very limited role in the aetiology of diarrhoeal disease in Italy. However, between September and November, 1985, 19 isolates of serotype 2 were recovered in northern Italy from a dysentery outbreak which occurred in a geriatrics hospital in Abbiategrasso (Milan, Lombardy) and seven were identified in southern Italy during the period January-July, 1986 from apparently unrelated infection cases occurring in Brindisi (Apulia). These isolates were compared by molecular methods to seven strains of S. boydii of serotype 2 isolated since 1981 from the same geographic areas. Plasmid DNA analysis showed a large variety of patterns, whereas hybridization of chromosomal DNA with E. coli rRNA identified only two different profiles, one of which was exclusively found in all isolates from the hospital outbreak. No differences were detected among rDNA patterns of the remaining strains of S. boydii, irrespective of their geographic origin. These findings are consistent with the hypothesis that the infrequent cases of infection from S. boydii of serotype 2 which occurred during the years under study could probably be attributed to two different bacterial clones. Hybridization procedure and detection of hybrids were simplified by replacement of radioactive labelling of rRNA by the use of photobiotin.     

SmaI macrorestriction analysis of Italian isolates of erythromycin-resistant Streptococcus pyogenes and correlations with macrolide-resistance phenotypes

High rates of erythromycin resistance among Streptococcus pyogenes strains have been reported in Italy in the last few years. In this study, 370 erythromycin-resistant (MIC, > or = 1 microg/mL) Italian isolates of this species obtained in 1997-1998 from throat swabs from symptomatic patients were typed by analyzing SmaI macrorestriction fragment patterns by pulsed-field gel electrophoresis (PFGE). Among the typable isolates (n = 341; the genomic DNA of the remaining 29 isolates was not restricted by SmaI), 48 distinct PFGE types were recognized, of which 31 were recorded in only one isolate (one-strain types). Fifty-two percent of typable isolates fell into three type clusters and 75% into six, suggesting that erythromycin-resistant group A streptococci circulating in Italy are polyclonal, but the majority of them probably derives from the spread of a limited number of clones. In parallel experiments, the 370 test strains were characterized for the macrolide resistance phenotype: 80 were assigned to phenotype cMLS, 89 to phenotype iMLS-A, 33 to phenotype iMLS-B, 11 to phenotype iMLS-C, and 157 to phenotype M. There was a close correlation between these phenotypic data and the genotypic results of PFGE analysis, the vast majority of the isolates assigned to individual PFGE classes belonging usually to a single phenotype of macrolide resistance. All of the 29 untypable isolates belonged to the M phenotype. Further correlations were observed with tetracycline resistance.     

Molecular epidemiology and origin of cholera reemergence in Italy and Albania in the 1990s

In 1994 a cholera epidemic occurred in Italy and Albania after more than a decade of case absence. To investigate genotypic characteristics and the origin of the epidemic strains, 110 Vibrio cholerae O1 El Tor isolates from Italy and Albania were studied by randomly amplified polymorphic DNA analysis (RAPD), BglI ribotyping, and pulsed-field gel electrophoresis (PFGE) of genomic DNA. The Italian and Albanian strains were all ribotype 6 and their RAPD and PFGE patterns were identical as well. These findings indicated that the 1994 isolates belonged to the same clone and that the clone was part of the larger global spread of epidemic ribotype 6 strains, which started in southern Asia in 1990.     

Group B streptococcus prevalence in pregnant women from North-Eastern Italy: advantages of a screening strategy based on direct plating plus broth enrichment

AIMS: To assess the sensitivity of a combined selective broth enrichment technique plus selective plating for the detection of group B streptococcus (GBS) colonisation in a large cohort of pregnant women from North-Eastern Italy. METHODS: During 2002-2005, 5020 pregnant women were screened between the 35th and the 37th week of gestation. A lower vaginal sample and a rectal sample were collected and inoculated onto LIM broth and a selective colistin aztreonam blood agar plate (CAP). Direct agar plates were examined after 18-24 hours and, if negative, after 48 hours. LIM broth was subcultured after 18-24 hours onto a Columbia blood agar plate. All colonies suggestive for GBS were submitted to phenotypic identification. RESULTS: 901 Women (17.9%) were positive for GBS. On 728 positive samples, corresponding to patients enrolled between 2003 and 2005, the results of selective direct plating and selective broth enrichment were compared. A total of 561 (77.1% of positive samples, corresponding to 13.9% of patients) were positive on direct selective agar; an additional 167 isolates (22.9% of samples, 4.1% of patients) were recovered from the LIM broth subculture. CONCLUSIONS: The prevalence of GBS carriage in this population-based study is a reliable estimate considering the sensitivity of the microbiological methods used, the rate of attendance of pregnant women to clinical and laboratory settings and the compliance to the protocol. Results confirm that the combination of selective enrichment broth and selective direct plating is a time-saving and sensitive method.     

Rapid detection of Salmonella enterica with primers specific for iroB.

The iroB gene of Salmonella enterica is absent from the chromosome of the related organism Escherichia coli. We determined the distribution of this gene among 150 bacterial isolates, representing 51 serotypes of different Salmonella species and subspecies and 8 other bacterial species which are frequent contaminants during routine enrichment procedures by Southern hybridization. An iroB-specific DNA probe detected homologous sequences in all strains of S. enterica, including serotypes of S. enterica subsp. enterica (I), salamae (II), diarizonae (IIIb), and houtenae (IV). No hybridization signal was obtained with strains of Salmonella bongori or other bacterial species. In contrast, hybridization with a DNA probe specific for purD, a purine biosynthesis gene, detected homologs in all bacterial species tested. Primers specific for iroB were used to amplify this gene from 197 bacterial isolates by PCR. The iroB gene could be PCR amplified from S. enterica subsp. enterica (I), salamae (II), diarizonae (IIIb), houtenae (IV), arizonae (IIIa), and indica (VI), but not from S. bongori or other bacterial species. Thus, PCR amplification of iroB can be used to distinguish between S. enterica and other bacterial species, including S. bongori. A combination of preenrichment in buffered peptone water supplemented with ferrioxamine E and amplification of iroB by magnetic immuno-PCR allowed detection of S. enterica in albumen within 24 h. In conclusion, PCR amplification of iroB is a new sensitive and selective method which has the potential to rapidly detect S. enterica serotypes.     

Antigenic and molecular characterization of isolates of the Italy 02 infectious bronchitis virus genotype.

As part of an epidemiological surveillance of infectious bronchitis virus (IBV) in Spain, four Spanish field isolates showed high S1 spike sequence similarities with an IBV sequence from the GenBank database named Italy 02. Given that little was known about this new emergent IBV strain we have characterized the four isolates by sequencing the entire S1 part of the spike protein gene and have compared them with many reference IBV serotypes. In addition, cross-virus neutralization assays were conducted with the main IBV serotypes present in Europe. The four Spanish field strains and the Italy 02 S1 sequence from the NCBI database were established as a new genotype that showed maximum amino acid identities with the 4/91 serotype (81.7% to 83.7%), the D274 group that included D207, D274 and D3896 strains (79.8% to 81.7%), and the B1648 serotype (79.3% to 80%). Furthermore, on the basis of these results, it was demonstrated that the Italy 02 genotype had been circulating in Spain since as early as 1997. Based on the average ratio of synonymous:non-synonymous (dS/dN) amino acid substitutions within Italy 02 sequences, no positive selection pressures were related with changes observed in the S1 gene. Moreover, phylogenetic analysis of the S1 gene suggested that the Italy 02 genotype has undergone a recombination event. Virus neutralization assays demonstrated that little antigenic relatedness (less than 35%) exists between Italy 02 and some of the reference IBV serotypes, and indicated that Italy 02 is likely to be a new serotype.     

Comparison of Susceptibility Testing Methods with mecA Gene Analysis for Determining Oxacillin (Methicillin) Resistance in Clinical Isolates of Staphylococcus aureus and Coagulase-Negative Staphylococcus spp.

Ninety-nine clinical staphylococcal isolates (58 coagulase-negative Staphylococcus spp. [CoNS] and 41 Staphylococcus aureus isolates) were evaluated for susceptibility to oxacillin. The following susceptibility testing methods, media, and incubation conditions were studied: agar dilution by using Mueller-Hinton (MH) medium (Difco) supplemented with either 0, 2, or 4% NaCl and incubation at 30 or 35 degrees C in ambient air for 24 or 48 h; disk diffusion by using commercially prepared MH medium (Difco) and MH II agar (BBL) and incubation at 35 degrees C in ambient air for 24 or 48 h; and agar screen (spot or swab inoculation) by using commercially prepared agar (Remel) or MH agar (Difco) prepared in-house, each containing 4% NaCl and 6 microg of oxacillin/ml (0.6-microg/ml oxacillin was also studied with MH agar prepared in-house for the agar swab method and CoNS isolates) and incubation at 35 degrees C in ambient air for 24 or 48 h for swab inoculation and at 30 or 35 degrees C in ambient air for 24 or 48 h for spot inoculation. The results for these methods were compared to the results for mecA gene detection by a PCR method. Given the ability to support growth and the results for susceptibility testing (the breakpoint for susceptible isolates was 相似文献   

Comparative analysis of antimicrobial susceptibility among organisms from France, Germany, Italy, Spain and the UK as part of the tigecycline evaluation and surveillance trial.

As part of the tigecycline evaluation and surveillance trial (TEST), bacterial isolates were collected from 39 centres in France, Germany, Italy, Spain and the UK between January 2004 and August 2006. Antimicrobial susceptibilities were determined according to CLSI guidelines. Italy had the highest rate of methicillin-resistant Staphylococcus aureus (36.4%), and was the only country to report vancomycin-resistant Enterococcus faecalis (8.6%). Tigecycline was the only agent to which all Gram-positive isolates were susceptible. For many of the Gram-negative organisms collected, antimicrobial susceptibilities were lowest among isolates from Italy and highest among isolates from Spain. The notable exception was Acinetobacter baumannii, where the poorest susceptibility profile was among isolates from Spain. For A. baumannii, MIC(90)s of imipenem varied from 1 mg/L for isolates in France and Germany to > or =32 mg/L for isolates from Italy and Spain. Tigecycline was the only agent to maintain an MIC(90) of < or =1 mg/L against isolates from all five countries. The in-vitro activity of tigecycline against both Gram-positive and Gram-negative isolates may make it valuable in the treatment of hospital infections, including those caused by otherwise antimicrobial-resistant organisms.     

Population structure, antimicrobial resistance, and mutation frequencies of Streptococcus pneumoniae isolates from cystic fibrosis patients

Forty-eight Streptococcus pneumoniae isolates recovered from sputum samples from 26 cystic fibrosis (CF) patients attending our CF unit (1995 to 2003) were studied. Mean yearly incidence of isolation was 5.5%, and all were strains recovered from young patients (< or = 12 years). The isolation was linked to clinical exacerbation in 35% of the cases, but only 27% of these were not accompanied by other CF pathogens. Fifty percent of the patients presented with two to four isolates over the studied period. Pulsed-field gel electrophoresis-SmaI digestion revealed a high heterogeneity (32 pulsotypes among 48 isolates) and the persistence over a 6-month period of a single clone (clone A) in two patients. This clone, presenting a varied multiresistance phenotype, was identified as the Spain23F-1 clone and was also recognized in six other patients, including two out of nine patients from the CF unit of Sant Joan de Deu Hospital, Barcelona, Spain. In our isolates, 16 different serotypes were recognized, the most frequent being 23F (33.3%), 19F (18.8%), 6A (6.2%), and 6B (6.2%). High overall resistance rates were observed: to penicillin, 73%; to cefotaxime, 33%; to erythromycin, 42%; to tetracycline, 58%; to chloramphenicol, 48%; and to trimethoprim-sulfamethoxazole, 67%. Resistance to fluoroquinolones was not detected. Multiresistance was a common feature (60%). The percentage of S. pneumoniae strains with increased frequencies of mutation to rifampin resistance (> or = 7.5 x 10(-8)) was significantly higher (P = 0.02) in CF (60%) than among non-CF (37%) isolates in the same institution (M. I. Morosini et al., Antimicrob. Agents Chemother. 47:1464-1467, 2003). Even though a clear association with acute exacerbations could not be observed, long-term clonal persistence and variability, high frequency of antibiotic resistance, and hypermutability indicate the plasticity for adaptation of S. pneumoniae to the CF lung environment.     

Carriage meningococcal isolates with capsule null locus dominate among high school students in a non-endemic period,Italy, 2012–2013

Meningococcal disease incidence in Italy remains quite low in the overall population except for infants. Within a study on carriage isolates among high school students we aimed to define: i) the prevalence of carriage isolates, ii) the phenotypic and iii) the molecular features of meningococci by Whole Genome Sequencing (WGS).A total of 1697 pharyngeal samples from undergraduate students (age range 14–19 years) were collected from 2012 to 2013 from six larger cities in Italy. One hundred and twenty culture positive meningococci (7%) were analyzed. Carriage isolates were sent to the National Reference Laboratory for invasive meningococcal disease (IMD) for PCR-based serogroup identification, Multilocus Sequence Typing, PorA and FetA typing. Moreover, factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) were typed. Core genome MLST (cgMLST) was performed on a subsample of 75 carriage isolates.Capsule null locus (cnl) predominated (47%), followed by serogroup B (27%). The antimicrobial susceptibility profile revealed an high prevalence of reduced susceptibility to penicillin G (54%) and a full susceptibility to ceftriaxone, ciprofloxacin and rifampicin. Carriage isolates presented a high genetic diversity: the clonal complexes (cc_s) cc1136, cc198 and cc41/44, were the predominant. An high heterogeneity was also observed for PorA and FetA types. The fhbp and nhba genes were identified in all the carriage isolates; only 5% of the carriage isolates presented the nadA gene. The core genome MLST analysis revealed that the majority of the cnl isolates clustered in a distinct group.The evidence gathered during this study provides the estimate of carriage isolates in high school students in a non-epidemic period in Italy that was lower than expected. Moreover, the highest proportion of carriage isolates were cnl and, overall, they were molecular heterogeneous.     

EcoRI-RFLP of the Apo B gene: a study in a sample group from South Italy

Eco RI restriction analysis at codon 4154 of the Apo B gene was performed in a sample of 90 subjects from southern Italy (sample S), using total blood cell DNA amplified by PCR. A group of 46 subjects from northern Italy (sample N) was also investigated for comparison. Southern Italians showed an incidence of the R2 allele (absence of the cutting site) twice as high as that found in northern Italians (48 v. 21 %). By ASPCR the mutation which abolishes the restriction site was confirmed as being G→A at the first base of the 4154 codon of the Apo B gene (Glu→Lys) in both S and N samples. By studying the variability of cholesterolaemia among different Eco RI genotypes in the S sample, it was estimated that the average effect of the R2 allele is to lower serum cholesterol by 8-5 mg/dl.     

Gene variations of Epstein-Barr virus nuclear antigen 3A in nasopharyngeal carcinomas, gastric carcinomas and healthy carriers in northern China

The Epstein-Barr virus (EBV) nuclear antigen protein 3A (EBNA-3A), a protein of 944 amino acids, is one of five EBNAs (EBNA-1, -2, -LP, -3A and -3C) essential for conversion of primary B lymphocytes to lymphoblastoid cell lines. To characterize the variations of the EBNA-3A gene and explore the association between EBNA-3A gene variations and EBV-associated diseases, we sequenced the key regions of EBNA-3A in the isolates of 30 EBV-associated gastric carcinomas (EBVaGCs), 44 nasopharyngeal carcinomas (NPCs) and 48 samples from healthy donors in northern China. We found that EBNA-3A shares a common evolutionary origin with isolates from southern China and Japan but has the character of a geographical variant. Based on a phylogenetic tree, all of the samples can be subdivided into three patterns, named 3A-8, 3A-5 and B95-8-like. The distribution of EBNA-3A subtypes among EBVaGC, NPC and healthy donors is not significantly different. The subtype 3A-8 is predominant not only in northern China but also in southern China; it is a geographically associated polymorphism in China.     

Molecular investigation of hepatitis E virus infection in patients with acute hepatitis in Kathmandu,Nepal

One hundred fifty-four consecutive patients with sporadic acute hepatitis, who were seen at a city hospital in the Kathmandu valley of Nepal in 1997, were studied. IgM antibodies to hepatitis A virus were detected in four patients (3%), IgM antibodies to hepatitis B core in four patients (3%), hepatitis B surface antigen in 20 (13%), and hepatitis C virus RNA in four patients (3%). IgM antibodies to hepatitis E virus (HEV) (anti-HEV IgM) and HEV RNA were detected in 77 (50%) and 48 (31%), respectively. Consequently, 86 patients (56%) including nine HEV-viremic patients without anti-HEV IgM, were diagnosed with hepatitis E. The cause of hepatitis was not known in 53 patients (34%). All 48 HEV RNA-positive samples were genotyped as 1, and subtyped further as 1a in 17 (35%), 1c in 29 (60%), and mixed infection of 1a and 1c in 2 (4%). A seasonal difference in the prevalence of HEV subtypes was recognized. Before the rainy season (January to July), both 1a and 1c isolates were found: the intrasubtypic difference was up to 9.0% and 1.7%, respectively, in the 412-nucleotide sequence of open reading frame 2. During the rainy season (August), only 1c isolates (n = 17) with 99.5-100% identity were found; 13 of 17 isolates had the same sequence, being identical to the 3 isolates that emerged at the end of July. These results suggest that a particular HEV 1c strain spread widely during the rainy season and was implicated in a small epidemic in the Kathmandu valley in August 1997.