Detection of human parechovirus in stool samples collected from children with acute gastroenteritis in Japan during 2007-2008

Of 477 stool specimens, which had been screened for rotavirus, adenovirus, norovirus, sapovirus and astrovirus, collected from infants and children with acute gastroenteritis in pediatric clinics encompassing five localities (Sapporo, Tokyo, Maizuru, Osaka, and Saga) in Japan from July 2007 to June 2008, 247 negative samples (51.7%) were subjected to screening for human parechovirus. Human parechovirus (HPeV) was detected by RT‐PCR using a primer pair to amplify 5′UTR region of its genome and was genotyped by sequencing of the VP1 gene. HPeV was detected in 20 of 247 specimens tested, and the detection rate was found to be 8.1%. Seventeen of the 20 strains that tested positive for HPeV were sequenced successfully the VP1 gene. The majority of the HPeV strains (n = 15) could be identified as HPeV1, and the remaining 2 strains could be typed as HPeV3. By phylogenetic and identical matrix analyses of HPeV VP1 sequences, HPeV1 should be divided into two lineages, and all of the Japanese studied HPeV1 strains belong to the lineage 2 accordingly. This is the first report of the circulation of HPeV, especially HPeV1 in Japan. J. Med. Virol. 83:331–336, 2011. © 2010 Wiley‐Liss, Inc.     

Campylobacter, from obscurity to celebrity

After its successful isolation from stools in the 1970s, Campylobacter jejuni has rapidly become the most commonly recognised cause of bacterial gastroenteritis in man. Reported cases of human campylobacteriosis represent only a small fraction of the actual number. In industrialised countries, the incidence of C. jejuni/Campylobacter coli infections peaks during infancy, and again in young adults aged 15-44 years. Acute self-limited gastrointestinal illness, characterised by diarrhoea, fever and abdominal cramps, is the most common presentation of C. jejuni/C. coli infection. The introduction of selective media has made the diagnosis of Campylobacter enteritis a simple procedure. In general, Campylobacter enteritis is a self-limiting disease which seldom requires antimicrobial therapy, although one in 1000 infections may lead to the Guillain-Barré syndrome. In industrialised countries, most infections are acquired through the handling and consumption of poultry meat. In developing countries, where the disease is confined to young children, inadequately treated water and contact with farm animals are the most important risk factors. Many infections are acquired during travel. Fluoroquinolone resistance has been reported in C. jejuni since the late 1980s in Europe and Asia, and since 1995 in the USA. The use of fluoroquinolones to treat animals used for food has accelerated this trend of resistance. In Australia, where fluoroquinolones have not been licensed for use in food production animals, C. jejuni remains susceptible to fluoroquinolones. The public health burden of Campylobacter spp. other than C. jejuni/C. coli remains unmeasured. Better diagnostic methods may reveal the true health burden of these organisms.     

在兰州地区儿童粪便中检测Aichi virus

目的 从兰州腹泻和正常儿童粪便标本中检测 Aichi virus,同时探讨 Aichi virus 与婴幼儿腹泻之间的联系.方法 根据文献发表资料,采用RT-PCR方法扩增 Aichi virus 3CD 片段,阳性产物经测序确定,并与已发表的该病毒序列进行比对分析.结果 在46份腹泻住院患儿粪便标本和299份腹泻门诊就诊患儿标本中各检出1例 Aichi virus,总检出率为0.06%,正常对照儿童中未检测到 Aichi virus.2株病毒3CD区基因与已知参考株核苷酸序列同源性均为97%,系统进化分析显示这2株病毒属于B基因型.结论 我国存在B基因型的 Aichi virus,但要明确我国 Aichi virus 的病原学及流行病学特点需要更多研究.     

Detection and identification of entamoeba species in stool samples by a reverse line hybridization assay

Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba species and genetic variants known to infect humans. The assay was performed after amplification with general Entamoeba-specific primers. The assay could identify four genetic variants of Entamoeba polecki-like cysts as well as E. histolytica, Entamoeba dispar, Entamoeba hartmanni, Entamoeba moshkovskii and Entamoeba coli and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections.     

Motility of Campylobacter concisus isolated from saliva,feces, and gut mucosal biopsies

Campylobacter concisus is an emerging pathogen associated with gastrointestinal disorders such as gastroenteritis and inflammatory bowel diseases (IBD), but the species is also found in healthy subjects. The heterogeneous genome of C. concisus increases the likelihood of varying virulence between strains. Flagella motility is a crucial virulence factor for the well‐recognized Campylobacter jejuni; therefore, this study aimed to analyze the motility of C. concisus isolated from saliva, gut biopsies, and feces of patients with IBD, gastroenteritis, and healthy subjects. The motility zones of 63 isolates from 52 patients were measured after microaerobic growth in soft‐agar plates for 72 hours. The motility of C. concisus was significantly lower than that of Campylobacter jejuni and Campylobacter fetus subsp. fetus. The motility of C. concisus varied between isolates (4–22 mm), but there was no statistical significant difference between isolates from IBD patients and healthy subjects (p = 0.14). A tendency of a larger motility zones was observed for IBD gut mucosa isolates, although it did not reach statistical significance (p = 0.13), and no difference was found between oral or fecal isolates between groups. In conclusion, the varying motility of C. concisus could not be related to disease outcome or colonization sites.     

High incidence of Campylobacter concisus in gastroenteritis in North Jutland,Denmark: a population-based study

The incidence of non-thermophilic Campylobacter species was assessed in an unselected population-based study in a mixed urban and rural community in North Jutland, Denmark. In a 2-year study period, 11 314 faecal samples from 8302 patients with gastroenteritis were cultured with supplement of the filter method. We recovered a high incidence of Campylobacter concisus (annual incidence 35/100 000 inhabitants), almost as high as the common Campylobacter jejuni/coli. In contrast, there was a very low incidence of other non-thermophilic Campylobacter species, such as Campylobacter upsaliensis. Campylobacter concisus was, unlike C. jejuni/coli, found more frequently among small children (<1 year) and the elderly (≥65 years). Around 10% of the patients with C. consisus had co-infections dominated by Clostridium difficile and Salmonella enterica, whereas co-infections occurred in about 5% of C. jejuni/coli patients. We observed a seasonal variation in C. jejuni/coli with a peak incidence in late summer months and autumn, whereas there was an almost constant monthly prevalence of C. concisus. Among patients participating in a questionnaire sub-study, there was a higher degree of close contacts with animals, especially dogs, as well as a higher travel exposure among C. jejuni/coli patients compared with C. concisus patients. We did not culture any C. concisus in stool samples from a small cohort of healthy individuals. Future studies have to focus on the clinical follow-up and the long-term risk of inflammatory bowel diseases in C. concisus-positive patients. We conclude that there is a high incidence of C. concisus in Denmark.     

Detection of Campylobacter species in foods by indirect competitive ELISA using hen and rabbit antibodies

The competitive enzyme immunoassays for detection of Campylobacter jejuni, C. coli and C. fetus subsp. fetus have been developed. Rabbit and hen immunoglobulins were prepared for these purposes. The working conditions of ELISAs, such as the concentrations of immunoreactants, incubation temperatures and time, and the composition of the substrate have been established. The detection limits were in the range 5.0 10⁴-3.2 10⁶ cfu/ml. The application of chemiluminescent substrates did not result in any significant improvement of the assay's detectability and sensitivity. Prepared antibodies showed rather high specificity and cross-reactivity profiles, and both rabbit and hen immunoglobulins were similar. Only IgY to C. jejuni cross-reacted with seven strains of C. jejuni and two other Campylobacter spp.

A limited number of naturally and artificially contaminated food samples were tested. The results obtained by means of an enzyme immunoassay were compared with those obtained from PCR or commercially available Singlepath® Campylobacter GLISA-Rapid Test. Poultry products were naturally contaminated with Campylobacters. The wild species were identified as C. jejuni and C. coli.     

Detection of Campylobacter species in foods by indirect competitive ELISA using hen and rabbit antibodies

The competitive enzyme immunoassays for detection of Campylobacter jejuni, C. coli and C. fetus subsp. fetus have been developed. Rabbit and hen immunoglobulins were prepared for these purposes. The working conditions of ELISAs, such as the concentrations of immunoreactants, incubation temperatures and time, and the composition of the substrate have been established. The detection limits were in the range 5.0 10⁴–3.2 10⁶ cfu/ml. The application of chemiluminescent substrates did not result in any significant improvement of the assay's detectability and sensitivity. Prepared antibodies showed rather high specificity and cross-reactivity profiles, and both rabbit and hen immunoglobulins were similar. Only IgY to C. jejuni cross-reacted with seven strains of C. jejuni and two other Campylobacter spp.

A limited number of naturally and artificially contaminated food samples were tested. The results obtained by means of an enzyme immunoassay were compared with those obtained from PCR or commercially available Singlepath® Campylobacter GLISA-Rapid Test. Poultry products were naturally contaminated with Campylobacters. The wild species were identified as C. jejuni and C. coli.     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples.

Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.     

The role of Campylobacter jejuni cytolethal distending toxin in gastroenteritis: toxin detection, antibody production, and clinical outcome

The role of Campylobacter jejuni cytolethal distending toxin (CDT) on clinical outcome after gastroenteritis was investigated. Clinical data, blood serum samples, and Campylobacter spp. isolated, from each of 30 patients were collected over a period of 6 months. The CDT encoding genes, cdtABC, characterized by PCR, revealed that all but one of the C. jejuni strains had the wild-type sequence. Sequencing of cdtABC from this strain showed two major deletions. From all of the strains, CDT titers were determined, and toxin neutralizing antibodies were documented using an in vitro assay. Three of the thirty clinical isolates, including the one with the mutant cdtABC coding genes, did not have a detectable CDT activity. Analyzing the relationship between CDT titer, serum neutralization of CDT, and the clinical outcome showed that campylobacteriosis caused by CDT-negative strains was clinically indistinguishable from that of patients infected with an isolate that produced high levels of CDT. These results suggest that CDT does not solely determine severity of infection and clinical outcome.     

Rotavirus epidemiology in Vellore, south India: group, subgroup, serotype, and electrophoretype.

Rotaviruses were detected in 163 of 916 (17.8%) specimens collected from children under 3 years of age with gastroenteritis in Vellore, South India, between August 1983 and July 1985. Rotaviruses were detected throughout the study period, with a peak prevalence in December to February (winter) and June to August (southwest monsoon season). A total of 117 rotavirus strains were tested for subgroup, serotype, and rotavirus double-stranded RNA electrophoretic migration pattern; 24.8% of the strains were subgroup I, 69.2% were subgroup II, and 6.0% were neither subgroup I nor subgroup II. Subgroup I and II strains were circulating concurrently throughout the study. Of the 117 rotavirus strains, 32 (27.4%) were serotyped; 15 were serotype 1, 3 were serotype 2, 2 were serotype 3, and 12 were serotype 4. Three serotypes were circulating concurrently during the periods of peak rotavirus prevalence. In 100 of the 117 strains (85.4%) an RNA pattern was detected. One unusual subgroup I group A rotavirus with a long migration pattern and four atypical rotaviruses serologically related to group C were also detected.     

Evaluation of media for primary isolation of Campylobacter jejuni from faecal samples from children and animals.

Different media were used for primary isolation of Campylobacter. Butzler & Preston medium was found to be more selective compared to Skirows & Blaserwang.     

Genotypic resistance profile of HIV-1 protease gene: a preliminary report from Vellore, south India

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.     

Detection of adenoviruses in stool specimens by nucleic acid spot hybridization

Nucleic acid hybridization was used for the detection of adenovirus DNA in stool specimens, and the results were compared with those obtained by a radioimmunoassay (RIA) for adenovirus hexon antigen. DNA from 40 specimens, 18 of which were positive by RIA, were spotted onto nitrocellulose filters and analyzed by hybridization using radioactively labeled adenovirus-2 DNA or a cloned DNA fragment from enteric adenovirus-41 as probes. With the adenovirus-2 DNA probe, 15 of the 18 RIA-positive specimens were also positive in the hybridization assay, and one of the RIA negative specimens was also scored as positive. The cloned adenovirus-41 fragment gave a positive signal with five specimens, all of which were also detected with the adenovirus-2 DNA probe. The results show that hybridization is an alternative method for detection of adenovirus in stool specimens. The sensitivity of the assay is comparable to that of the RIA.     

Prevalence of neutralising antibodies to Berne virus in animals and humans in Vellore,South India

Summary In Southern India the prevalence of neutralising antibody to Berne virus was high in sera obtained from cattle (49%), horses (38%), and sheep (36%). Neutralising antibody was not detected in sera from humans and monkeys.     

Detection of Giardia duodenalis assemblage A and B isolates by immunochromatography in stool samples from Rwandan children

We evaluated the performance of an immunochromatographic assay (ICA) in comparison with light microscopy and PCR for the detection of Giardia duodenalis in stool samples from 558 Rwandan children. The association of infection with clinical symptoms was similar for the three diagnostic tools. The ICA equally detected parasites of assemblages A and B and was more sensitive than light microscopy (50.4 versus 29.5% of PCR-positive samples considered true positive; p <0.0001). Hence, the ICA shows superior sensitivity compared with microscopy but still misses half of the G. duodenalis infections detected by PCR in this hyperendemic area.     

Detection of adenovirus types 40 and 41 in stool specimens by immune electron microscopy

An immune electron microscope (IEM) test was developed that allowed the direct detection of adenovirus type 40 (ad 40) or ad 41 in stools specimens. The polyclonal rabbit antisera used differentiated ad 40 and 41 from other ad serotypes but not from each other. The method was evaluated in a 13 month prospective study of stools from children with gastroenteritis. Seventy-two specimens found to contain ad by conventional electron microscope screening were retested by IEM. Results were typically obtained within 2 hr and showed that 55 (76%) viruses typed as ad 40/41. No ads were recovered from conventional virus isolation attempts on these specimens. Additionally, 39 of these 55 viruses were tested by restriction endonuclease analysis (REA) after growth in 293 cells, and results showed that all produced digest patterns typical of ad 40 (seven cases) or ad 41 (32 cases). Twenty-four percent (17/72) of viruses could not be typed by IEM; 9/17 (53%) yielded ads [ad 1 (1), ad 2 (4), ad 5 (1), ad 6 (1), ad 7 (2)] in routine culture, whereas REA identified the other eight as ad 2 (6), ad 1 (1), and ad 41 (1). The concordance between IEM and the reference methods was therefore 100% specificity and 97.5% sensitivity. The method described allows the clinically useful diagnosis of ad 40/41 infection to be rapidly made and will be a particularly useful technique in laboratories screening faeces by electron microscopy.     

Detection of Campylobacter species in faecal samples by direct Gram stain microscopy

AIMS: To evaluate the Gram stain with carbol-fuchsin counterstain for the rapid detection of Campylobacter species in faecal samples. METHODS: In total, 842 consecutive diarrhoeic faecal samples were prospectively examined for Campylobacter species by Gram stain and culture. RESULTS: Campylobacter species were isolated from 84 faecal samples (all Campylobacter jejuni). Compared with culture, Gram stain microscopy had a sensitivity of 89%, specificity of 99.7%, positive predictive value of 97%, and negative predictive value of 99% for detecting Campylobacter species. CONCLUSIONS: The direct Gram stain method can provide a presumptive result within 30 minutes of receipt of a faecal sample in the laboratory with relatively high sensitivity, and at low cost. Laboratories in areas where Campylobacter enteritis is common and/or with limited resources for Campylobacter culture should consider adopting this as a routine method.