Stool Specimens Of Patients Research Articles

High Frequency and Diversity of Species C Enteroviruses in Cameroon and Neighboring Countries

Human enteroviruses (HEVs) are endemic worldwide and among the most common viruses infecting humans. Nevertheless, there are very limited data on the circulation and genetic diversity of HEVs in developing countries and sub-Saharan Africa in particular. We investigated the circulation and genetic diversity of HEVs among 436 healthy children in a limited area of the far north region of Cameroon in 2008 and 2009. We also characterized the genetic biodiversity of 146 nonpolio enterovirus (NPEV) isolates obtained throughout the year 2008 from stool specimens of patients with acute flaccid paralysis (AFP) in Cameroon, Chad, and Gabon. We found a high rate of NPEV infections (36.9%) among healthy children in the far north region of Cameroon. Overall, 45 different HEV types were found among healthy children and AFP patients. Interestingly, this study uncovered a high rate of HEVs of species C (HEV-C) among all typed NPEVs: 63.1% (94/149) and 39.5% (49/124) in healthy children and AFP cases, respectively. Besides extensive circulation, the most prevalent HEV-C type, coxsackievirus A-13, featured a tremendous intratypic diversity. Africa-specific HEV lineages were discovered, including HEV-C lineages and the recently reported EV-A71 "genogroup E." Virtually all pathogenic circulating vaccine-derived polioviruses (cVDPVs) that have been fully characterized were recombinants between oral poliovaccine (OPV) strains and cocirculating HEV-C strains. The extensive circulation of diverse HEV-C types and lineages in countries where OPV is massively used constitutes a major viral factor that could promote the emergence of recombinant cVDPVs in the Central African subregion.

Stool submission by general practitioners in SW England - when, why and how? A qualitative study

BackgroundWe know little about when and why general practitioners (GPs) submit stool specimens in patients with diarrhoea. The recent UK-wide intestinal infectious disease (IID2) study found ten GP consultations for every case reported to national surveillance. We aimed to explore what factors influence GP’s decisions to send stool specimens for laboratory investigation, and what guidance, if any, informs them.MethodsWe used qualitative methods that enabled us to explore opinions and ask open questions through 20 telephone interviews with GPs with a range of stool submission rates in England, and a discussion group with 24 GPs. Interviews were transcribed and subjected to content analysis.ResultsInterviews: GPs only sent stool specimens to microbiology if diarrhoea persisted for over one week, after recent travel, or the patient was very unwell. Very few had a systematic approach to determine the clinical or public health need for a stool specimen. Only two GPs specifically asked patients about blood in their stool; only half asked about recent antibiotics, or potential food poisoning, and few asked about patients’ occupations. Few GPs gave patients advice on how to collect specimens.Results from interviews and discussion group in relation to guidance: All reported that the HPA stool guidance and patient collection instructions would be useful in their clinical work, but only one GP (an interviewee) had previously accessed them. The majority of GPs would value links to guidance on electronic requests. Most GPs were surprised that a negative stool report did not exclude all the common causes of IID.ConclusionsGPs value stool culture and laboratories should continue to provide it. Patient instructions on how to collect stool specimens should be within stool collection kits. Through readily accessible guidance and education, GPs need to be encouraged to develop a more systematic approach to eliciting and recording details in the patient’s history that indicate greater risk of severe infection or public health consequences. Mild or short duration IID (under one week) due to any cause is less likely to be picked up in national surveillance as GPs do not routinely submit specimens in these cases.

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Detection of <i>Campylobacter</i> spp. in Stool Specimens and Comparison with Culture Method

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).

Immunochromatography Method Was Useful in Prompt Diagnosis of Potentially Fatal Norovirus Gastroenteritis After Hematopoietic Stem Cell Transplantation

Abstract 4542 Background:Norovirus-gastroenteritis (NV-GE) is considered as a highly transmittable disease that can lead to fatal outcomes in vulnerable populations. Therefore, prompt detection of norovirus in stool specimens is important for patients who undergo hematopoietic stem cell transplantation (HSCT). The commercially available immunochromatography kit (Denka Seiken, Tokyo, Japan) is a diagnostic tool that can easily and rapidly detect norovirus antigens with high specificity and relatively less sensitivity compared with reverse transcription polymerase chain reaction (RT-PCR). Although previous studies used RT-PCR to detect norovirus in stool, this method is not necessarily a standardized technique for the clinical use, and only limited information is available about clinical significance of norovirus infection among the HSCT recipients. Here, we report an analysis of patients with NV-GE in our HSCT unit using the immunochromatography method. Patients and Methods:We prospectively examined stool specimens to detect norovirus antigens in patients who developed diarrhea in our HSCT unit between December 2008 and June 2011. We also retrospectively examined stool specimens which had been collected for Clostridium difficile (CD) toxin test and frozen at –40°C between January 2007 and November 2008. During the study period, a total of 468 patients underwent HSCT (autologous 83, allogeneic 385) at our center and we tested stool specimens from 355 patients who developed diarrhea. Results:Norovirus was detected by the immunochromatography method in 11 patients among the HSCT recipients, and in 1 patient who died before HSCT because of disease progression. Among the 12 patients with NV-GE, 3 were retrospectively diagnosed using the frozen specimens. The CD toxin test was also positive in 1 of the 12 patients with NV-GE. The median age of the 12 patients was 56 years (range, 29–66). The median duration of symptoms was 30 days (2–134). Among the 11 patients who developed NV-GE after HSCT, primary disease included lymphoma (6 patients), acute leukemia (4 patients), and multiple myeloma (1 patient), and 9 of them were not in remission at HSCT. One patient developed NV-GE after autologous HSCT, and 10 patients after allogeneic HSCT. Among the 10 allo-HSCT recipients, 6 received grafts from unrelated bone marrow donor and 4 received grafts from related donor. Five patients received myeloablative conditioning and 5 received reduced-intensity conditioning before allo-HSCT. The median time from HSCT to the onset of symptoms was 36 days (4–93). The median time from HSCT to diagnosis of NV-GE was 37 days (11–101). At diagnosis of NV-GE, all allo-HSCT recipients were given immunosuppressive agents, and 2 of them received corticosteroids for intestinal graft-versus host disease (GVHD). The volume of diarrhea was more than 500 ml per day in 4 patients. Among 4 patients who underwent endoscopy of the lower gastrointestinal tract, intestinal GVHD was diagnosed in 2 patients by histopathology findings, whereas the other 2 patients had no evidence of intestinal GVHD, which resulted in no need for an intensification of immunosuppression. Of the 12 patients with NV-GE, 6 were alive with a median follow-up of 826 days (17–1168) after the diagnosis of NV-GE. No patients died of NV-GE, and 6 died of other causes (disease progression, 4; GVHD, 1; multiple organ failure, 1). There was no outbreak of NV-GE as we promptly implemented isolation of infected patients and enhanced hygiene strategy within an hour of collection of stool specimens in patients with diarrhea. Conclusions:In this study, we detected 11 patients who developed NV-GE after HSCT using the immunochromatography method. Our results suggested that this method is helpful in the differential diagnosis of patients with diarrhea after HSCT and enable us to take an appropriate and prompt preventive measure. In the future study, validation with RT-PCR and immunochromatography method is warranted among the immune-compromised populations. Disclosures:No relevant conflicts of interest to declare.

Screening for colorectal cancer: established and emerging modalities

It has been estimated that >95% of cases of colorectal cancer (CRC) would benefit from curative surgery if diagnosis was made at an early or premalignant polyp stage of disease. Over the past 10 years, most developed nation states have implemented mass population screening programs, which are typically targeted at the older (at-risk) age group (>50-60 years old). Conventional screening largely relies on periodic patient-centric investigation, particularly involving colonoscopy and flexible sigmoidoscopy, or else on the fecal occult blood test. These methods are compromised by either low cost-effectiveness or limited diagnostic accuracy. Advances in the development of diagnostic molecular markers for CRC have yielded an expanding list of potential new screening modalities based on investigations of patient stool (for colonocyte DNA mutations, epigenetic changes or microRNA expression) or blood specimens (for plasma DNA mutations, epigenetic changes, heteroplasmic mitochondrial DNA mutations, leukocyte transcriptome profile, plasma microRNA expression or protein and autoantibody expression). In this Review, we present a critical evaluation of the performance data and relative merits of these various new potential methods. None of these molecular diagnostic methods have yet been evaluated beyond the proof-of-principle and pilot-scale study stage and it could be some years before they replace existing methods for population screening in CRC.

An analysis on results of monitoring nontyphoidal Salmonella in diarrhea patients in Huizhou

Objective To investigate prevalence baseline of nontyphoidal Salnonella in Huizhou city,to identify an epidemic outbreak earlier,and to analyze epidemic factors.To explore the serotypes,distribution,and changes in drug resistance of nontyphoidal Salmonella isolated from diarrhea patients,in order to instruct clinical use of medication.Methods Stool specimens of diarrhea patients collected from 2008 to 2009 in the surveillance hospitals in Huizhou were detected for nontyphoidal Salmonella.The isolated strains were further serotyped; drug sensitivity testing and pulse field gel electrophoresis were performed.Results Of 430 diarrhea patients,19 were infected by Salmonella,with the positive rate of 4.42％.The 19 strains had five serotypes,mainly Salmonella typhimurium ( 47.37％ ) and Salmonella enteritidis ( 26.32％ ).Most strains of Salmonellas were sensitive to common antibiotics including cephems and quinolones.Conclusions Salmonella is an important type of enteric pathogen and it causes disease by oral pathway.We should strengthen food safety surveillance and of health education on personal protection,eventually prevent infection with Salmonella. Key words: Diarrhea; Salmonella; Food safety surveillance

Etiological surveillance and analysis of infectious diarrhea in Beijing in year 2010

To explore the pathogenic form, epidemic features and serotype distribution of the pathogenic bacteria causing infectious diarrhea in Beijing. A total of 2118 samples of rectal swabs and stool specimens of diarrheal patients were collected from 6 surveillant intestinal tract clinics during the period between April and October, 2010. Enteric multiple pathogens including Vibrio cholerae, Vibrio parahaemolyticus, Salmonella, Shigella and diarrheagenic Escherichia coli were detected by the isolation culture, biochemical identification and serotyping methods. The population distribution, temporal distribution and serotype distribution of the above pathogenic bacteria were analyzed by descriptive statistical methods. 478 strains isolated from the total 2118 specimens were positive for pathogen detection, accounting to 22.6%. Among the 478 strains of pathogenic bacteria, Shigella accounting for 40.8% (195/478) was the most frequent pathogen, followed by Vibrio parahaemolyticus accouting for 23.8% (114/478), Salmonella accounting for 19.0% (91/478) and diarrheagenic Escherichia coli accounting for 4.8% (23/478). Enteric pathogenic bacteria spread mainly among adults aging between 20 and 39; and the distribution was different among different age groups, while the highest detected rate was in 30 - 39 age group, accounting for 27.2% (92/338). The detected rate of pathogenic bacteria showed evident seasonal variations, with a peak from July to October, whose detected rates were 23.5% (114/486), 32.8% (176/536), 36.1% (90/249) and 25.9% (29/112) respectively. The detected rates in other months were all under 16.0%. Shigella Sonnei was the dominant serotype, accounting for 83.1% (162/195). O3:K6 was the dominant serotype among Vibrio parahaemolyticus, accounting for 63.2% (72/114). Salmonella Enteritidis and Salmonella Typhimurium were dominant serotypes among Salmonella, accounting for 13.2% (12/91) and 12.1% (11/91) separately. Enterpathogenic Escherichia coli and enterotoxigenic Escherichia coli were the dominant serotypes among Diarrheagenic Escherichia coli, accounting for 69.6% (16/23) and 30.4% (7/23) respectively. The three main pathogenic bacteria causing infectious diarrhea in Beijing are Shigella, Vibrio parahaemolyticus, Salmonella; and there are obvious changes in the serotype distribution of Shigella and Samonella compared to previous years.

Norovirus gastroenteritis causes severe and lethal complications after chemotherapy and hematopoietic stem cell transplantation

AbstractNorovirus (NV) infections are a frequent cause of gastroenteritis (GE), but data on this disease in immunocompromised patients are limited. We analyzed an NV outbreak, which affected immunosuppressed patients in the context of chemotherapy or HSCT. On recognition, 7 days after admission of the index patient, preventive measures were implemented. Attack rates were only 3% (11/334) and 10% (11/105) among patients and staff members, respectively. The median duration of symptoms was 7 days in patients compared with only 3 days in staff members (P = .02). Three patients died of the NV infection. Commonly used clinical diagnostic criteria (Kaplan-criteria) were unsuitable because they applied to 11 patients with proven NV-GE but also to 15 patients without NV-GE. With respect to the therapeutic management, it is important to differentiate intestinal GVHD from NV-GE. Therefore, we analyzed the histopathologic patterns in duodenal biopsies, which were distinctive in both conditions. Stool specimens in patients remained positive for NV-RNA for a median of 30 days, but no transmission was observed beyond an asymptomatic interval of 48 hours. NV-GE is a major threat to patients with chemotherapy or HSCT, and meticulous measures are warranted to prevent transmission of NV to these patients.

Efficiency of diagnostic methods for correlation between prevalence of enteric protozoan parasites and HIV/AIDS status––an experience of a tertiary care hospital in East Delhi

Since the beginning of the AIDS pandemic, opportunistic infections have been recognized as common complications of HIV infection. Enteric protozoan parasitic infections are one of the leading causes of morbidity and mortality in HIV infected patients. The present study is, therefore, aimed to determine the prevalence of these parasites and study their association with immune status in HIV patients with emphasis on the correlation between various diagnostic techniques to give an accurate diagnosis to avoid empirical treatment. This prospective study, carried out between November 2009 and May 2010 included all HIV seropositive patients presenting with diarrhea to the ART center. A total of 64 stool samples were analyzed by wet mount examination, three different staining techniques, and antigen detection by ELISA for various enteric protozoan infections. Total prevalence of enteric protozoan parasites was 30%. Among the total cases, Cryptosporidium was seen in 12% cases followed by Giardia, E. histolytica and Isospora belli. The maximum diagnostic yield for coccidian parasites was with safranin-methylene blue staining technique. Parasitic burden contributes towards early morbidity in HIV infection. This study provides important information about prevalence of intestinal protozoan parasites in HIV infection. A combination of procedures should be carried out for the screening of stool specimens of HIV patients for better diagnosis and management.

Genetic Similarity ofCampylobacterIsolates in Humans, Food, and Water Sources in Central Poland

Campylobacter spp. is an important cause of gastroenteritis in humans throughout the world. However, sources of these infections are often difficult to identify. Therefore, this study aimed at analyzing the genetic relatedness of Campylobacter isolates from environmental and food samples as well as stool specimens of diarrheal patients obtained in a single geographic region in Poland. Only a few Campylobacter jejuni isolates (4/18, 22%) could be assigned to one cluster, whereas the majority were unrelated. In contrast, the majority of Campylobacter coli strains (25/35, 71%) belonged to three pulsed-field gel electrophoresis (PFGE) clusters containing isolates of various origins (i.e., water samples, chicken carcasses, and humans). Isolates belonging to the clusters showed also similar antibiotic resistance patterns and similar genotypes with respect to the occurrence of the virB11 and iam genes. This suggests that Campylobacter strains may circulate between humans, poultry, and recreational water sources in the rural region in central Poland.

A multiattribute utility evaluation of different methods for the detection of enteric protozoa causing diarrhea in AIDS patients

BackgroundEnteric protozoa and sporozoa have emerged as important opportunistic parasites and can cause fatal infections in AIDS patients. The line of treatment being different for them necessitates an accurate and prompt identification of these to avoid empirical treatment. In this study which is the first of its kind from India we did a comprehensive evaluation of different techniques, comparing them on the basis of the attributes like yield, cost, time taken, expertise and infrastructure. For the first time combination of Calcoflour White and DAPI, a nuclear stain, were used to identify Microsporidia spp. Thus, a diagnostic protocol was devised for rapid, sensitive and cost effective identification of the opportunistic enteric protozoa.ResultsThe organisms isolated from the stool samples of the cases (450 HIV patients) were predominantly Cryptosporidium spp., Microsporidia spp. and Cyclospora spp. Interestingly, the control group (200 relatives of the patients who were HIV negative) showed a high incidence (21%) of Cryptosporidium spp. We found a significant increase in the sensitivity of microscopy in detecting Cryptosporidium spp. and Cyclospora spp. after formol ether concentration. Kinyoun's staining was better compared to Modified safranin staining for Cryptosporidium spp. identification. Although ELISA had a sensitivity of 93.25% and specificity of 97% for Cryptosporidium spp. detection, we ranked Kinyoun's staining better than ELISA because it is not affordable to most of our patients. For detecting Cyclospora cayetanensis, autoflourescence was the easiest and most cost effective method followed by Safranin technique. Combination of Calcoflour White stain and DAPI gave good results for the identification of Microsporidia spp. We assessed the above techniques and graded the attributes in the following descending order: cost effectiveness, sensitivity, ease of use and interpretation, time taken for the procedure and batch testing.ConclusionThus, we conclude that a combination of minimum three procedures should be carried out for the screening of stool specimens of HIV positive patients. Kinyoun's staining should be made mandatory for every diarrheal stool sample from HIV patients. Also every laboratory should assign its own value to the attributes and apply Multiattribute utility theory or the Analytical hierarchy process to decide the most appropriate methodology.

Apport de la PCR dans la recherche et l’identification des microsporidies intestinales chez les sujets infectés par le VIH

AimIntestinal microsporidiosis are among the most frequent opportunistic diseases in immunocompromised subjects. This study aimed to evaluate the contribution of PCR for a better detection and species identification of microsporidia in stool specimens of HIV-infected patients. Patients and methodsStool samples obtained from 119 HIV-infected Tunisian subjects were screened for intestinal microsporidiosis by light microscopy using Weber's modified Trichrome stain and by a PCR method using universal primers V1/PMP2 which amplified a common fragment of the small subunit rRNA gene of microsporidia. The obtained PCR products were then sequenced using an ABI PRISM 377 DNA sequencer. ResultsThe results showed a better sensitivity of PCR in the detection of microsporidia with an infection rate of 14.3% significantly higher than that of 6.7% obtained by light microscopy (p=0.03). As previously described, intestinal microsporidiosis was associated with low CD4 cell counts; 23.9% infection rate in patients having CD4 cell count under 200/mm3 against 5.6% in patients with higher CD4 cell count (p=0.008). The sequencing of 15 out of the 17 positive PCR products has confirmed in all cases the species identified based on the PCR fragment size i.e., 250pb for Enterocytozoon bieneusi (seven cases) and about 270pb for Encephalitozoon intestinalis (nine cases); one case revealed a double infection. ConclusionPCR proved to be more effective than classical Trichrome stain for the diagnosis of intestinal microsporidiosis. Moreover, the ability of PCR to identify the species involved could also be useful for cases management.

Typing and susceptibility of bacterial isolates from the fidaxomicin (OPT-80) phase II study for C. difficile infection

Background Clostridium difficile infection (CDI) has been increasing in incidence and severity in recent years, coincident with the spread of a “hypervirulent” strain, REA type BI (ribotype 027, PFGE NAP 1). Exacerbating the problem has been the observation that metronidazole may be showing decreased effectiveness, particularly in the more severe cases. Fidaxomicin is an 18-membered macrocycle currently in phase 3 trials for the treatment of C. difficile infection (CDI). An open-label, phase II study in CDI patients has been completed and the clinical results published. C. difficile organisms were isolated from patient stool specimens and typed by restriction endonuclease analysis (REA) in order to determine the frequency and susceptibility of the C. difficile isolates and their response to treatment. Methods Fecal samples were plated on CCFA agar for isolation of C. difficile. These isolates were tested for susceptibility to fidaxomicin, vancomycin, and metronidazole using CLSI agar dilution methods and were typed by REA. Results C. difficile was isolated from 38 of 49 subjects and 16 (42%) were the epidemic C. difficile BI group. The BI strain was distributed approximately equally in the three dosing groups. Overall antibiotic susceptibilities were consistent with the previously reported MIC 90 values for the three antibiotics tested, but the MIC 90 of BI strains was two dilutions higher than non-BI strains for metronidazole and vancomycin (for both antibiotics, MIC 90 was 2 μg/mL vs. 0.5 μg/mL, P < 0.01 for metronidazole, P = NS for vancomycin). Clinical cure for BI isolates (11/14, 79%) was not significantly different from non-BI isolates (21/22, 95%). Conclusion These results underscore the high prevalence of the BI epidemic strain and demonstrate that mild to moderate CDI infection as well as severe disease can be caused by these strains. Fidaxomicin cure rates for subjects with BI and with non-BI strains are similar, although the small numbers of subjects preclude a robust statistical comparison.

Etiological analysis of enteric infectious diseases during Beijing Olympic Games

To understand pathogen patterns of enteric infectious diseases and its impact on this pattern due to aggregation of a great deal of foreign visitors during Beijing Olympic Games. The diarrheal patient's rectal swabs and stool specimens were collected from Olympic stadium and hospitals of four districts, including Dongcheng, Xicheng, Haidian and Chaoyang. Enteric multiple pathogens were detected from the total 45 specimens. The culture method was used for the enteric bacteria, ELISA and RT-PCR for the enteric viruses. Molecular typing of Salmonella Enteritidis isolation was completed by PFGE. It was found that 26 out of 45 cases were positive with 57.8 percent for pathogen detection, and 24 were identified as enteric pathogenic bacteria, including Vibrio parahaemolyticus, Salmonella, diarrheagenic Escherichia coli and Campylobacter jejuni, two as norovirus. There were mixed infections of two pathogenic bacteria for three cases. Ten kinds of pathogens were detected from foreign cases, while five kinds from Chinese cases. A total of 5 PFGE patterns were identified in 10 Salmonella Enteritidis isolates from national and foreign diarrheal cases, which were concentrative in some extent. Vibrio parahaemolyticus, Salmonella, diarrheagenic Escherichia coli and Campylobacter jejuni were found to be the primary bacterial pathogens during the Olympic Games. Enteric virus infection existed in summer diarrhea.

Fecal detection of influenza A virus in patients with concurrent respiratory and gastrointestinal symptoms

BackgroundIn seasonal influenza, gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain are sometimes observed, especially among young children. However, fecal excretion of seasonal influenza virus has seldom been studied. ObjectiveTo investigate the presence of human influenza A virus in stool specimens of patients presented with symptoms of gastroenteritis. Study designA retrospective study on 651 stool specimens collected from 627 patients of all age groups presented with symptoms of gastroenteritis during a 12-month period (December 2004–November 2005). Influenza A viral RNA was detected by conventional polymerase chain reaction (PCR) targeting the matrix gene. Virus subtyping was performed by multiplexed H1- and H3-specific PCR. Fecal viral concentration was estimated by TaqMan®-based real-time PCR. Clinical records of positive cases were reviewed. ResultsSeasonal influenza A (H3N2) viral RNA was detected in 7 stool specimens collected from 6 patients. Their time of presentation coincided with local influenza season. All patients were at the extreme of ages (<5 or >60 years) or had underlying comorbidities, and had concurrent respiratory and gastrointestinal symptoms. All required hospitalization and 1 patient died. Two patients with underlying lymphoma had the highest viral concentrations in their stool specimens. ConclusionsInfluenza A viral RNA can be detected in stool specimens of 6 high-risk influenza patients with concurrent respiratory and gastrointestinal symptoms. Further investigation on the gastrointestinal involvement of seasonal influenza is required.

Rapid Molecular Characterization of Clostridium difficile and Assessment of Populations of C. difficile in Stool Specimens

Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.

Prolonged norovirus outbreak in a Finnish tertiary care hospital caused by GII.4-2006b subvariants

Norovirus outbreaks are difficult to control in hospitals. Cohorting and contact isolation, disinfective surface cleaning and hand hygiene are key elements in outbreak control. A new norovirus variant, GII.4.-2006b, spreading across many continents, caused an exceptionally long epidemic period in Finland, from November 2006 to June 2007. Here, we describe the clinical and molecular characteristics of a norovirus outbreak in a large tertiary care hospital in Finland. Altogether 240 (18%) patients and 205 (19%) healthcare workers fell ill in the 504 bedded main building of Helsinki University Central Hospital during December 2006 to May 2007. The epidemic curve had three peaks in January, February and April, and different wards were affected each time. During the outbreak, 502 patient stool specimens were tested for norovirus RNA, 181 (36%) of which were positive. Molecular analysis of 48 positive specimens revealed three main subvariants of GII.4.-2006b circulating temporally within distinct wards. Of all microbiologically confirmed cases, 121 (67%) were nosocomial and nine (5%) died within 30 days of diagnosis. Molecular analysis suggested that the three main GII.4-2006b subvariants entered the hospital with gastroenteritis patients, and the nosocomial spread within wards coincided with the epidemic peaks. Active control measures, including temporary closure of the wards, ultimately confined the single-ward outbreaks. A prolonged outbreak in the community was probably the source for the prolonged outbreak period in the hospital.

Identification of Shiga Toxin-producing E. coli Isolated from Diarrhea Patients and Cattle in Gwangju Area, Korea

Shiga toxin-producing Escherichia coli (STEC) strains are commensal bacteria in cattle and cause food borne disease in human. We analyzed the isolation rate of STEC in stool specimens of patients with diarrhea and in fecal samples of cattle in Gwangju, Korea. STEC strains were detected from 33 (0.19%) out of 17,148 patients with diarrhea while there has been a progressive increase in the incidence rate from 0.07% in 2004 to 0.33% in 2008. We investigated serotypes, shiga toxin genes, and antimicrobial resistance patterns of the 44 STEC isolates from human and cattle sources. The 33 STEC isolates from human belonged to 14 O serotypes including O157, O26 and O111. The 11 isolates from cattle belonged to 11 O serotypes. PCR detection for stx genes showed that 12 (27.3%) isolates carried stx₁ genes, 20 (45.5%) possessed stx₂ genes, and 12 (27.3%) carried both stx₁ and stx₂. Of the 33 STEC isolates from human, 25 strains (76%) were resistant to one or more antibiotics. High level of resistance to tetracycline (73%) was most common, followed by ticarcillin and ampicillin (64%). But none of the 33 isolates from human were resistant to amikacin, cefazolin, cefepime, cefotetan, cefotaxime, ciprofloxacin, or imipenem. The 5 strains (45%) of the 11 isolates from cattle were resistant to at least one or three antibiotics but most of the isolates were sensitive to the 16 antibiotics employed in this survey. In conclusion, toxin types and serotypes of STEC isolated from human and cattle were diverse, and non-O157 STEC was also observed to be a greater proportion of STEC isolates. According to a specific comparison solely based on the toxin types and serotypes, most of the STEC strains isolated from cattle feces in Gwangju, Korea showed characteristics different from those isolated from patients. Therefore, laboratory surveillance is required to detect and carefully monitor the potentially hypervirulent STEC not only in human and cattle but also in other animals.

A novel DNA microarray for rapid diagnosis of enteropathogenic bacteria in stool specimens of patients with diarrhea

A microarray technique for the detection and identification of enteropathogenic bacteria at the species and subspecies levels was developed in this study, and the target bacteria included pathogenic Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella enterica, Campylobacter jejuni, Shigellae, Yersinia enterocolitica, and Listeria monocytogenes. The virulence gene of each pathogen was chosen as the amplification target, labeled with a fluorescence dye by multiplex polymerase chain reaction (PCR), and hybridized to the specific virulence gene probes that had been immobilized on a microchip. Stool specimens from 34 patients with diarrhea were tested in this study. Five were positive for multiple genera. Nested PCRs and sequencing were used to amplify and identify the related genes, which were found to share 95.8% to 100% of the nucleotide identity with the corresponding regions in the Genbank database. Real-time PCR was used to determine the number of gene copies to determine the sensitivity of this technique, which was shown to be 58 copies/μl. The results indicated that the microarray technique which targets multiple virulence genes of enteropathogenic bacteria at the species and subspecies levels is an attractive diagnostic tool for rapidly and simultaneously identifying multiple enteropathogenic pathogens in clinical practice, especially in patients with infectious diarrhea.

Detection of Astrovirus in Premature Infants With Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a major cause of mortality and morbidity in very low birth weight infants (<1500 g birth weight). Although the etiology remains unknown, infectious agents could play a key role. The aim of this analysis was to examine the role of human astrovirus (HAstV) in infants with NEC. All patients admitted during a 5-year period at a tertiary neonatal intensive care unit with NEC (Bell stage I-III) who had examination of stool specimens for bacterial and for viral infections were included. Clinical data were reviewed and compared between infants with NEC and astrovirus detection (NEC + HAstV) and infants with NEC without astrovirus detection (NEC - HAstV) in stool specimens. Forty infants with NEC were identified between 2002 and 2006 and 8 patients were excluded from statistical evaluation because of incomplete viral examinations. HAstV was detected in stool specimens of 6 (19%) of the remaining 32 patients with NEC. Double infection with rotavirus was identified in 1 patient. No other viruses were detected. Significant differences in patients with NEC - HAstV and NEC + HAstV were only shown for age at onset of illness (P < 0.001) but not for severity of illness, need for surgical intervention, or mortality. This study demonstrates that HAstV may be associated with the development of NEC in a subgroup of patients and provides further evidence for the important role of gastrointestinal viral infections in this most common gastrointestinal emergency in premature infants. HAstV should be included in microbiological examination of stool specimens in patients with NEC.