Frequent detection of polyomaviruses in stool samples from hospitalized children

BACKGROUND. Infection with BK virus (BKV) generally occurs early during life, but its mode of transmission has not been clearly defined. We tested the hypothesis that polyomavirus shedding in stool may be a source of BKV exposure.METHODS. Pediatric stool and rectal swab samples were tested for the presence of polyomavirus DNA by a polymerase chain reaction (PCR) assay that could detect a conserved region in the large T antigen gene of BKV, JC virus (JCV), and simian virus 40 (SV40). The specific viruses detected by this assay were confirmed by DNA sequence analysis of the PCR amplicons.Results. Of 120 samples collected from 99 patients, 54 (45.0%) were positive for polyomavirus DNA. Of the 99 patients, 46 (46.5%) had at least 1 positive sample, with 38 (38.4%) positive for BKV and 8 (8.1%) positive for SV40. JCV was not detected. There was no association between polyomavirus fecal shedding and age, sex, race/ethnicity, immune status, or symptoms of gastrointestinal disease in the children studied. The BKV strains detected displayed polymorphisms in the T antigen sequence.Conclusions. Polyomaviruses are frequently present in stool samples from hospitalized children. These findings suggest that fecal-oral transmission of BKV may play a role in the ubiquity of infection.     

Molecular detection of Helicobacter pylori antibiotic resistance in stool vs biopsy samples

AIMTo compare (1) demographics in urea breath test (UBT) vs endoscopy patients; and (2) the molecular detection of antibiotic resistance in stool vs biopsy samples.METHODSSix hundred and sixteen adult patients undergoing endoscopy or a UBT were prospectively recruited to the study. The GenoType HelicoDR assay was used to detect Helicobacter pylori (H. pylori) and antibiotic resistance using biopsy and/or stool samples from CLO-positive endoscopy patients and stool samples from UBT-positive patients.RESULTSInfection rates were significantly higher in patients referred for a UBT than endoscopy (overall rates: 33% vs 19%; treatment-naïve patients: 33% vs 14.7%, respectively). H. pylori-infected UBT patients were younger than H. pylori-infected endoscopy patients (41.4 vs 48.4 years, respectively, P < 0.005), with a higher percentage of H. pylori-infected males in the endoscopy-compared to the UBT-cohort (52.6% vs 33.3%, P = 0.03). The GenoType HelicoDR assay was more accurate at detecting H. pylori infection using biopsy samples than stool samples [98.2% (n = 54/55) vs 80.3% (n =53/66), P < 0.005]. Subset analysis using stool and biopsy samples from CLO-positive endoscopy patients revealed a higher detection rate of resistance-associated mutations using stool samples compared to biopsies. The concordance rates between stool and biopsy samples for the detection of H. pylori DNA, clarithromycin and fluoroquinolone resistance were just 85%, 53% and 35%, respectively.CONCLUSIONDifferences between endoscopy and UBT patients provide a rationale for non-invasive detection of H. pylori antibiotic resistance. However, the GenoType HelicoDR assay is an unsuitable approach.     

A survey of pathogenic genes from diarrhea stool samples obtained in Nara prefecture

We conducted a survey of diarrhea stool samples in which no virulent agents had previously been detected at clinical laboratories. DNA extracted directly and purified from the diarrhea stool was tested for bacterial pathogenic genes by polymerase chain reaction. The test results for 85 specimens were as follows: one sample was positive for lt, ipaH, and eae; two were positive for aggR; and eight were positive for astA. Inoculation with the stool specimens led to the isolation of a strain of Escherichia coli possessing eae, three strains of E. coli possessing astA, and a strain of Klebsiella possessing astA.     

实时荧光PCR检测儿童肺结核粪便中Mtb-DNA的效果评价

目的 应用实时荧光PCR快速检测肺结核患儿粪便中Mtb-DNA,并初步评估其临床效果。 方法 收集肺结核住院儿童粪便标本76份,应用实时荧光PCR检测Mtb-DNA,检测结果与20例其他呼吸系统疾病患儿的粪便实时荧光PCR检测Mtb-DNA、76例粪便抗酸杆菌涂片检查以及41例患儿痰标本的涂片和(或)培养、实时荧光PCR检测结果进行比较。 结果 粪便实时荧光PCR检测敏感度达到23.68%(18/76),特异度为100.00％(20/20),肺结核患儿粪便PCR阳性率［23.68%(18/76)］明显高于涂片阳性率［6.58%(5/76)］,41例患儿中痰涂片和(或)培养阳性例数(15例)和痰PCR检测阳性例数(18例)高于粪便PCR检测阳性例数(11例)。 结论 实时荧光PCR检测儿童粪便Mtb-DNA,是一种特异度高、比较敏感、非侵入性且相对安全的儿童肺结核快速诊断方法。     

应用FTA/PCR检测粪便中隐孢子虫的实验研究

目的 建立用FTA/PCR检测粪便中隐孢子虫的技术,并与常用的商业化的粪便DNA抽提试剂盒进行比较.方法 选择经病原学检测已经确诊的隐孢子虫阳性和阴性粪便,分别采用FTA卡和商业化DNA抽提试剂盒抽提粪便样本中DNA,进行巢式PCR扩增,对PCR阳性产物进行测序并利用BLAST在NCBI上与GenBank数据库进行比对,做同源性分析,确定虫种.结果 应用商业化DNA抽提试剂盒抽提DNA,无论是否反复冻融破壁,2个阳性对照样本扩增出目的 片段,另3个阳性样本未扩增出特异性条带.样本纯化后应用FTA卡抽提DNA进行PCR检测,所有阳性粪样均扩增出830 bp左右的目的 片段,通过测序和比对,其中2个为贝氏隐孢子虫,1个为火鸡隐孢子虫.结论 应用FTA进行粪便样本中隐孢子虫DNA的抽提方法具有简单有效、快速灵敏、准确可靠等特点,可以应用于粪便中隐孢子虫的检测.     

Evaluation of usefulness of different methods for detection of Cryptosporidium in human and animal stool samples

There are many methods for detection of Cryptosporidium oocysts. Most of them (more than 20) enable the microscopic detection of Cryptosporidium oocysts in faecal smears. Such a great variability of diagnostic methods may lead to confusion as far as the choice of an appropriate technique by a given laboratory is concerned. This study evaluated the diagnostic usefulness of Cryptosporidium oocysts and coproantigen detection methods in the diagnosis of cryptosporidiosis in human (266 stool specimen) and animals (205 from cattle, 160 from sheep, 30 from horses, 80 from cats, 227 from dogs and 11 from wild animals). The total number of human and animal stool specimens processed was 266 and 713, respectively. In this study the usefulness of several diagnostic methods was compared. The following techniques were taken into account: wet mounts, hematoxylin staining, four different specific methods (modified Zeihl-Neelsen, Kinyoun's, safranin-methylene blue, as well as carbol-methyl violet and tartrazyne) and commercially available kit based on enzyme-linked immunoassay (ProspecT(r) Cryptosporidium Microplate Assay). The final number of positive specimens was 123. Out of them 77 were positive in all specific methods. The oocysts found in stool specimens were measured. Humans were infected with C. parvum and animals with C. parvum, C. andersoni or C. felis. The statistical analysis has shown that EIA test was a better than microscopy method for identification of Cryptosporidium in faecal samples in human and wild animal. Sensitivity and specificity are important factors for the choice of a proper diagnostic method for Cryptosporidium detection, however other factors such as cost, simplicity and ease of interpretation of results are also important considerations.     

Usefulness of immunological detection of both toxin A and toxin B in stool samples for rapid diagnosis of Clostridium difficile-associated diarrhea

Toxin detection from stool specimens is critical for the diagnosis of Clostridium difficile-associated diarrhea (CDAD). In Japan, only two toxin detection kits targeting toxin A alone are commercially available. We evaluated ImmunoCard Toxin A & B (ImmunoCard), based on enzyme immunoassay for the rapid detection of both C. difficile toxins A and B in stool specimens, compared to a toxin A detection kit (Uniquick) and cytotoxin assay. C. difficile was also cultured from stool specimens and the toxin production type of C. difficile isolates was identified by PCR analysis. Compared to cytotoxin assay, ImmunoCard sensitivity was 86.2%, specificity 93.8%, positive predictive value 91.8%, and negative predictive value 89.4% (n = 146). Sensitivity was significantly higher than that of Uniquick (60.0%, p = 0.0016). ImmunoCard detected 90.6% of cytotoxin positive specimens with isolated toxin A-positive, toxin B-positive C. difficile strains (Uniquick; 67.9%, p = 0.008) and 70.0% of these with isolated toxin A-negative, toxin B-positive C. difficile strains. Although ImmunoCard was slightly less sensitive than cytotoxin assay, it requires no special equipment and completes the entire test in up to 20 min. ImmunoCard thus appears very useful in the rapid diagnosis of CDAD in the clinical laboratory. Kits for the detection of both C. difficile toxins A and B should be immediately introduced into Japan to ensure the correct diagnosis of CDAD and infection control.     

Distribution of secretory inhibitor of platelet microbiddal protein among anaerobic bacteria isolated from stool of children with diarrhea

AIM: To study the secretory inhibitor of platelet microbicidal protein (SIPHP) phenotypes of faecal anaerobic isolates from patients with diarrhea.METHODS: Faecal isolates of anaerobic bacteria(B.fragiliS,n=42; B.longum,n=70;A.israelii,n=21;E.lentum,n=12) from children with diarrhea were tested.SlPHP production was tested by inhibition of platelet microbicidal protein (PHP) bioactivity against B.subtilis and was expressed as percentage of inhibition of PMP bactericidal activity.RESULTS: Among anaerobic isolates 80% of B.Iongum strains,85.7% of A.israelii strains,50%of E.lentum strains and 92.86% of B.fragilis strains were SIPMP-positive.The isolated anaerobic organisms demonstrated SIPHP production at a mean level of 13.8%±0.7%,14.7%±1.8%,3.9%±0.9% (P＜0.05) and 26.8%±7.5% (P＜0.05) for bifidobacteria,A.israelii,E.lentum and B.fragilis,respectively.CONCLUSION: Data from the present study may have significant implications in understanding the pathogenesis of microecological disorders in the intestine,as well as for future improvement in the prevention and therapy of anaerobe-associated infections.     

Evaluation of a multiplex PCR assay for detection of cytomegalovirus in stool samples from patients with ulcerative colitis

AIM: To evaluate a multiplex PCR assay for the detection of bacterial and viral enteropathogens in stool samples from patients with ulcerative colitis (UC).METHODS: We prospectively analyzed 300 individuals, including immunocompetent patients, immunocompromised patients, and patients with UC. Stool samples were collected from the recto-sigmoid region of the colon by endoscopy. The samples were qualitatively analyzed for bacterial and viral enteropathogens with a multiplex PCR assay using a Seeplex^® Kit. Additional clinical and laboratory data were collected from the medical records.RESULTS: A multiplex PCR assay detected 397 pathogens (191 bacteria and 206 viruses) in 215 samples (71.7%). The most frequently detected bacteria were Escherichia coli H7, 85 (28.3%); followed by Aeromonas spp., 43 (14.3%); and Clostridium perfringens, 36 (12.0%) samples. The most prevalent viruses were Epstein-Barr virus (EBV), 90 (30.0%); followed by human herpes virus-6 (HHV-6), 53 (17.7%); and cytomegalovirus (CMV), 37 (12.3%) samples. The prevalence rate of CMV infection was significantly higher in the immunocompromised group than in the immunocompetent group (P < 0.01). CMV infection was more common in patients with UC (26/71; 36.6%) than in the immunocompetent patients excluding UC (6/188; 3.2%) (P < 0.01). CMV infection was more prevalent in UC active patients (25/58; 43.1%) than in UC inactive patients (1/13; 7.7%) (P < 0.05). Among 4 groups which defined by the UC activity and immunosuppressive drugs, the prevalence rate of CMV infection was highest in the UC active patients with immunosuppressive drugs (19/34; 55.8%). Epstein-Barr virus (EBV) infection was more common in the immunocompromised patients excluding UC (18/41; 43.9%) than in the immunocompetent patients excluding UC (47/188; 25.0%) (P < 0.05). The simultaneous presence of CMV and EBV and/or HHV6 in UC active patients (14/58; 24.1%) was greater than in immunocompromised patients excluding UC (5/41; 12.2%) (P < 0.05).CONCLUSION: The multiplex PCR assay that was used to analyze the stool samples in this study may serve as a non-invasive approach that can be used to exclude the possibility of CMV infection in patients with active UC who are treated with immunosuppressive therapy.     

Distribution of secretory inhibitor of platelet microbicidal protein among anaerobic bacteria isolated from stool of children with diarrhea

AIM： To study the secretory inhibitor of platelet microbicidal protein （SIPMP） phenotypes of faecal anaerobic isolates from patients with diarrhea.
METHODS： Faecal isolates of anaerobic bacteria （B. fragilis, n = 42; B. longum, n = 70; A. israelii, n = 21; E. lentum, n = 12） from children with diarrhea were tested. SIPMP production was tested by inhibition of platelet microbicidal protein （PMP） bioactivity against B. subtilis and was expressed as percentage of inhibition of PMP bactericidal activity.
RESULTS： Among anaerobic isolates 80% of B. longum strains, 85.7% of A. israeli/strains, 50% of E. lentum strains and 92.86% of B. fragilis strains were SIPMP-positive. The isolated anaerobic organisms demonstrated SIPMP production at a mean level of 13.8% ± 0.7%, 14.7% ± 1.8%, 3.9% ± 0.9% （P 〈 0.05） and 26.8% ± 7.5% （P 〈 0.05） for bifidobacteria, A. israelii, E. lentum and B. fragilis, respectively.
CONCLUSION： Data from the present study may have significant implications in understanding the pathogenesis of microecological disorders in the intestine, as well as for future improvement in the prevention and therapy of anaerobe-associated infections.     

Enhanced surveillance of non-O157 verotoxin-producing Escherichia coli in human stool samples from Manitoba.

BACKGROUND: Relatively few enhanced surveillance studies have been undertaken to investigate the extent to which verotoxin-producing non-O157 serotypes of Escherichia coli occur in stool samples received for the detection of verotoxin-producing organisms. OBJECTIVES: To describe the prevalence, molecular and epidemiological characteristics, and geographical patterns associated with non-O157 verotoxin-producing E coli (VTEC) in Manitoba. RESULTS: Thirty-two VTEC isolates consisting of 10 serogroups and 13 different serotypes were isolated over a 22-month period. Twenty-three isolates (71.8%) possessed verotoxin-encoding gene stx1 only, five isolates (15.6%) possessed stx2 only, two isolates (6.3%) possessed both stx1 and stx2, and two isolates (6.3%) possessed stx2c. Only three instances of indistinguishable pulsed-field gel electrophoresis patterns were identified. The age of the individuals from whom non-O157 VTEC were isolated ranged from eight months to 87 years. Mean and median ages were 30 and 22 years of age, respectively. Some areas of the province appeared to experience a higher than expected number of non-O157 E coli in comparison with the number of stools that were received from these areas. CONCLUSIONS: The present study demonstrated a large number of infections associated with non-O157 VTEC in Manitoba. Most non-O157 cases appear to result from sporadic infections, and these occur typically in rural areas. Continued enhanced surveillance is necessary to understand the temporal patterns of non-O157 VTEC and the underlying epidemiological factors driving these patterns.     

Parasites detected from diarrheal stool samples collected in Nepal

Intestinal parasites were investigated in 396 diarrheal stool samples collected from individuals aged 1 to 68 years (males: 239 and females: 157) in Nepal. Samples were collected at different medical centers located in Kathmandu and from two public schools in a village setting in Kathmandu Valley and outside, during October 1999 to January 2001. The stool samples were mixed with 2% dichromate solution and transported to Japan for investigations. Parasites were detected by employing the formal-ether sedimentation technique. Of a total of 396 fecal samples investigated, 193 (49%) were positive for some kind of parasite. Altogether, 15 species of parasites were detected. Giardia intestinalis topped the list of protozoa, whereas Trichuris trichiura was the most frequently detected among helminth parasites. Of the 193 positive samples, 109 (56%) had single parasite infections, whereas 84 (43%) had multiple infections with a maximum of five species. Of the total positive, 45 (23%) had both protozoa and helminths whereas 37 (19%) had only protozoa. Females (52%) and children (15 years and under) (52%) had a marginally higher prevalence compared with males (46%) and adults (45%), respectively (p > 0.05). Samples collected from two public schools in a village setting inside Kathmandu Valley and outside had a significantly higher positive rate compared with those observed in individuals visiting different medical centers in the city and suburban areas in Kathmandu (p < 0.05).     

First molecular identification of Entamoeba moshkovskii in human stool samples in Tunisia

We report the first intestinal infections in Tunisia with Entamoeba moshkovskii in two healthy adults. Entamoeba moshkovskii cysts were distinguished from those of the morphologically identical parasites Entamoeba histolytica and Entamoeba dispar by specific nested polymerase chain reaction and sequencing.