Conventional and molecular identification of Giardia intestinalis in human stool samples in Baghdad Province, Iraq.

A Cross-Sectional Study of Helicobacter Infection Among Laboratory Animals and Animal Research Workers

BackgroundIn Brazil, schistosomiasis is a parasitic disease of public health relevance, mainly in poor areas where Schistosoma mansoni is the only human species encountered and Biomphalaria straminea is one of the intermediate host snails. A nested-PCR based on a specific mitochondrial S. mansoni minisatellite DNA region has been successfully developed and applied as a reference method in Brazil for S. mansoni detection, mainly in host snails for epidemiological studies. The amplification efficiency of LAMP is known to be higher than PCR. The present work aimed to assess the utility of our previously described SmMIT-LAMP assay for S. mansoni detection in human stool and snail samples in a low-transmission area of schistosomiasis in the municipality of Umbuzeiro, Paraíba State, Northeast Region of Brazil.Methodology/Principal findingsA total of 427 human stool samples were collected during June-July 2016 in the municipality of Umbuzeiro and an overall prevalence of 3.04% (13/427) resulted positive by duplicate Kato-Katz thick smear. A total of 1,175 snails identified as Biomphalaria straminea were collected from 14 breeding sites along the Paraíba riverbank and distributed in 46 pools. DNA from human stool samples and pooled snails was extracted using the phenol/chloroform method. When performing the SmMIT-LAMP assay a total of 49/162 (30.24%) stool samples resulted positive, including 12/13 (92.31%) that were Kato-Katz positive and 37/149 (24.83%) previously Kato-Katz negative. By nested-PCR, only 1/46 pooled DNA snail samples was positive. By SmMIT-LAMP assay, the same sample also resulted positive and an additional one was positive from a different breeding site. Data of human and snail surveys were used to build risk maps of schistosomiasis incidence using kernel density analysis.Conclusions/SignificanceThis is the first study in which a LAMP assay was evaluated in both human stool and snail samples from a low-transmission schistosomiasis-endemic area. Our SmMIT-LAMP proved to be much more efficient in detection of S. mansoni in comparison to the 'gold standard' Kato-Katz method in human stool samples and the reference molecular nested-PCR in snails. The SmMIT-LAMP has demonstrated to be a useful molecular tool to identify potential foci of transmission in order to build risk maps of schistosomiasis.

Background: Detection of fastidious enteropathogenic bacteria in fecal samples of patients with gastroenteritis is a challenge in clinical microbiological laboratories. Objectives: The aim of this study was to compare the detection limits of the PCR and culture methods for the diagnosis of Campylobacter spp., Yersinia spp., Clostridium perfringens, and Clostridium difficile in human stool samples. Methods: Healthy human stool and sterile phosphate-buffered saline (PBS) samples were separately spiked with 10-fold dilutions of C. jejuni, C. difficile, Y. enterocolitica, and C. perfringens reference strains to obtain final concentrations of 101 - 108 colony forming units (CFU) per gram. Dilutions of each suspension were inoculated onto specific culture media and colony counts were determined. Polymerase chain reaction (PCR) was carried out on DNA extracts of each dilution using specific primers. All of the assays were performed in two separate replicas. Results: In the cases of the culture and PCR assays, detection limits of 101 and 102 CFU/g for C. difficile, 2 × 104 and 2 × 104 CFU/g for C. perfringens, 104 and 102 CFU/g for C. jejuni, and 102 and 104 CFU/g for Y. enterocolitica, respectively, were obtained. In the cases of the spiked PBS samples, a detection limit of 101 for C. jejuni and Y. enterocolitica was obtained using the culture method. While 102 -fold higher sensitivity was observed for C. jejuni via PCR compared with the culture assay, equal (C. perfringens) or lower sensitivity limits (C. difficile and Y. enterocolitica) were detected for the spiked stool samples with other bacteria. Conclusions: These results showed differences in the bacterial culture and PCR methods for quantitative detection of fastidious bacteria in human stool samples. However, a bacterial load of 104 CFU per gram of stool was measured as a sufficient amount for detection of the fastidious bacteria by either culture or PCR assays. More suitable PCR methods could be used for rapid diagnosis of the slow-growing bacteria in the patients' stool samples.

A study was conducted to determine the prevalence of Clostridium difficile and characterize C. difficile isolates from human stool and retail grocery meat samples. Human stool samples (n=317) were obtained from a clinical laboratory and meat samples (n=303) were collected from 8 retail grocery stores from October 2011 through September 2012 from Centre County of Pennsylvania and were examined for C. difficile. C. difficile was isolated from 16.7% of stool samples (n=317) and 6.9%, 11.5%, 14.5%, and 7.8% of beef (n=72), pork (n=78), turkey (n=76), and chicken (n=77) samples, respectively. Six different toxin gene profiles were detected in all human and meat isolates of C. difficile based on the presence or absence of toxin genes tcdA, tcdB, and cdtA and cdtB. Interestingly, 75.6% of the human C. difficile isolates lacked any deletion in the tcdC gene (139-bp), whereas a 39-bp deletion was observed in 61.3% of the C. difficile strains isolated from meat samples. C. difficile from meat samples were more susceptible to clindamycin, moxifloxacin, vancomycin, and metronidazole than C. difficile isolates from human samples. Twenty-five different ribotypes were identified in human and meat C. difficile isolates. In conclusion, significant genotypic and phenotypic differences were observed between human and meat isolates of C. difficile; however, a few C. difficile isolates from meat-in particular ribotypes 078, PA01, PA05, PA16, and PA22 with unique profiles (toxin gene, tcdC gene size and antimicrobial resistance profiles)-were similar to human C. difficile isolates.

Introduction. Bacterial dysentery is one of the greatest causes of morbidity and mortality worldwide. Campylobacter spp. and diarrhoeagenic Escherichia coli (DEC) are recognised as the most common causes of bacterial enteritis in developing countries including India.Hypothesis/Gap statement. Rapid and accurate identification of dysentery causing organisms using molecular methods is essential for better disease management, epidemiology and outbreak investigations.Aim. In view of the limited information available on the dysentery causing agents like Campylobacter spp., enterohemorrhagic E. coli (EHEC)/enteropathogenic E. coli (EPEC) and enteroinvasive E. coli (EIEC)/Shigella in India, this study was undertaken to investigate the presence of these pathogens in human and poultry stool samples by molecular methods.Methodology. In total, 400 human stool samples and 128 poultry samples were studied. Microaerophilic culture along with real-time multiplex PCR with the targets specific to the genus Campylobacter, Campylobacter jejuni, Campylobacter coli, EHEC, EPEC and EIEC/Shigella was performed. Further species confirmation was done using MALDI-TOF MS.Results. On microaerophilic culture, C. coli was isolated in one human sample and two C. jejuni and one C. fetus in poultry samples. On PCR analysis, among human stool samples, typical EPEC (42%) was predominantly seen followed by Campylobacter spp. (19%) and EIEC/Shigella (10%). In contrast, Campylobacter spp. (41%) was predominant in poultry samples, followed by typical EPEC (26%) and EIEC/Shigella (9%). Poly-infections with Campylobacter spp. and DEC were also observed among both sources.Conclusion. The present study documented the increased prevalence of Campylobacter spp. in humans compared with the results of previous studies from India. Typical EPEC was found to be predominant in children less than 5 years of age in this study. The high prevalence of coinfections in the current study indicates that a multiple aetiology of diarrhoea is common in our settings.

Background and Aim:Vibrio parahaemolyticus is a marine bacterium commonly associated with foodborne illnesses due to the consumption of contaminated seafood. Understanding its prevalence in both fish meat and human infections is crucial for public health. This study aimed to estimate the occurrence of V. parahaemolyticus in human stool and fish meat samples while analyzing seasonal and species-specific variations in the Al-Hodeidah governorate.Materials and Methods:A total of 225 samples were collected, including 75 human stool samples from patients with gastrointestinal symptoms and 150 fish meat samples from five fish species commonly consumed in the region. Standard microbiological methods were used for the isolation and identification of V. parahaemolyticus, including culture on Thiosulfate–Citrate–Bile Salts–Sucrose (TCBS) agar, biochemical tests, and growth analysis in varying NaCl concentrations. Data were statistically analyzed using SPSS version 12, applying the Chi-square test for group comparisons with a significance level of p ≤ 0.05.Results:The overall occurrence of V. parahaemolyticus was 7.1%. Human stool samples had a occurrence of 6.7%, while fish meat samples had a slightly higher occurrence of 7.3%. The highest monthly occurrence in human samples was recorded in July (15.0%), while the highest fish contamination was detected in September (12.0%). Among fish species, Rastrelliger kanagurta (Bagah) had the highest contamination rate (20.0%), followed by Scomberomorus commerson (Dairak) at 13.3%, whereas no V. parahaemolyticus isolates were found in Dasyatis kuhlii (Safon) and Rachycentron canadum (Sakalah).Conclusion:The findings confirm the presence of V. parahaemolyticus in both human and fish meat samples, highlighting seasonal variations and species-specific differences. The peak occurrence in fish during warm months suggests a potential link between higher temperatures and bacterial prevalence. Improved seafood handling, monitoring, and public health awareness are essential to mitigate the risk of foodborne infections. Further research is needed to explore genetic determinants of virulence and antimicrobial resistance in local isolates.

16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal Bacteroidales: A Bayesian approach

Objective: Investigate the type of pathogenic Vibrio strains from water and stool samples collected from Migori, Sondu-Miriu, Nyando and Yala regions in Western Kenya. Methods: A total of 811 samples (596 water and 215 stool samples) were collected during the study periods of May to December 2013 and August to September 2014. Pathogenic Vibrio strains were identified through culturing in TCBS Agar, followed by oxidation, string and serological (polyvalent) tests, respectively. The PCR analysis was done using combined primers targeting Vibrionaceae 16SrRNA and species specific primers for V. vulnificus and V. cholerae. Results: The results showed the presence of V. vulnificus and V. cholerae. However, V. parahaemolyticus was not found in any of the samples. The PCR results for 16SrRNA, Vib 1, and Vib 2 showed polymorphism in the genes, this was an indication of cross combination of genes from more than one strain in one isolate. Conclusion: The study showed the presence of V. cholerae (Ogawa and Inaba) in water and human stool samples. Type B V. vulnificus was detected in the water sample collected from River Migori. This information is of essence in controlling and managing cholera in the western part of Kenya. J Microbiol Infect Dis 2016;6(1): 1-7 Key words: V. cholerae, V. vulnificus, V. parahaemolyticus, PCR

Objective: Investigate the type of pathogenic Vibrio strains from water and stool samples collected from Migori, SonduMiriu,
\nNyando and Yala regions in Western Kenya.
\nMethods: A total of 811 samples (596 water and 215 stool samples) were collected during the study periods of May
\nto December 2013 and August to September 2014. Pathogenic Vibrio strains were identified through culturing in TCBS
\nAgar, followed by oxidation, string and serological (polyvalent) tests, respectively. The PCR analysis was done using
\ncombined primers targeting Vibrionaceae 16SrRNA and species specific primers for V. vulnificus and V. cholerae.
\nResults: The results showed the presence of V. vulnificus and V. cholerae. However, V. parahaemolyticus was not found
\nin any of the samples. The PCR results for 16SrRNA, Vib 1, and Vib 2 showed polymorphism in the genes, this was an
\nindication of cross combination of genes from more than one strain in one isolate.
\nConclusion: The study showed the presence of V. cholerae (Ogawa and Inaba) in water and human stool samples. Type
\nB V. vulnificus was detected in the water sample collected from River Migori. This information is of essence in controlling
\nand managing cholera in the western part of Kenya. J Microbiol Infect Dis 2016;6(1): 1-7
\n

Comparison of Cape Town and Skirrow's protocols used in isolation of Campylobacter in humans and broilers was carried out in a cross-sectional study in Morogoro, Tanzania. A total of 176 and 158 human stool and broiler intestinal samples were collected, respectively. While human stool samples were collected from selected health centers, broiler intestinal samples were obtained from selected farms and chicken markets. Samples were inoculated and cultured in duplicate using two protocols and prevalence of Campylobacter were established. In humans, the prevalence of Campylobacter isolates was significantly higher (P < 0.001) (21.6%) with Cape Town protocol than Skirrow's method (9.1%). Similarly, a higher prevalence (P < 0.05) in broilers was recorded in Cape Town protocol (77.8%) than Skirrow's method (66.5%). There was a moderate (0.53) Kappa test of agreement between Skirrow's and Cape Town protocols for human samples and substantial agreement (0.72) for broiler samples. This demonstrates that Cape Town protocol is superior over the Skirrow's protocol in Campylobacter isolation. Campylobacter jejuni, Campylobacter coli and Campylobacter lari were the Campylobacter spp. isolated. In humans, C. jejuni accounted for 92.1% and 87.5% of all positive samples with Cape Town and Skirrow's protocols, respectively. In broilers, C. jejuni was isolated at 91.1% and 92.5% of all species obtained with Cape Town and Skirrow's protocols, respectively. This shows that C. jejuni is the common species that may be circulating from either broilers to humans or other animals and vice versa. The present study has introduced Cape Town protocol in Tanzania for Campylobacter isolation from human and animal samples, which is expected to improve the isolation of Campylobacter species. Cape Town protocol may also be a good alternative for use in routine isolation of Campylobacter.

BackgroundFish-borne zoonotic clonorchiasis, caused by Clonorchis sinensis, is an emerging public health problem in several countries with more than 15 million people infected globally. However, a lack of accurate point-of-care (POC) diagnostic tests in resource-limited areas is still a critical barrier to effective treatment and control of clonorchiasis. The development of the recombinase polymerase amplification(RPA) assay, a POC diagnostic test based on the amplification of pathogen DNA, has provided a new, simple and inexpensive tool for disease detection with high sensitivity and specificity. MethodsA novel RPA method was developed based on specific primers and probes, and combined with the dipstick, to allow for the rapid and intuitive detection of C. sinensis through the amplification of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. The lower limit of detection for the combined RPA/lateral flow dipstick (RPA-LFD) assay was evaluated using dilutions of the target DNA sequence. Cross-reactivity was evaluated using genomic DNA from 10 additional control parasites. Forty human clinical stool samples were tested to verify its performance.ResultsThe evaluated primers designed from the C. sinensisCOX1 region can be used to detect adult worms, metacercariae, and eggs at 39 °C within 20 min, and the results can be visually observed using the LFD. The detection limit of pathogen genomic DNA was as low as 10 fg, and the number of metacercaria(e) in fish and egg(s) in faeces were both as low as one. This improved the sensitivity of low-infection detection tremendously. The test is species-specific, and no other related control parasites were detected. In human stool samples with eggs per gram (EPG) > 50, the RPA-LFD assay was performed consistent with conventional Kato-Katz (KK) and PCR methods.ConclusionThe established RPA-LFD assay provides a powerful tool for the diagnosis and epidemiological survey of C. sinensis from human and animal samples, and has important implications for the effective control of clonorchiasis.Graphical

Aim: To establish and evaluate a quick and accurate DNA microarray method to detect intestinal pathogens directly from human diarrheal stool samples as an alternative to traditional culture methods. Method: Primers and 21 oligonucleotide probes based on sequences of the bacterial 16SrRNA gene were arrayed on microarray slides. Hybridization between probes and amplicons was performed. To determine the consistency of DNA microarray and culture method, 1,500 samples of clinical diarrheal stool and 200 samples of normal stool from healthy individuals were examined in a double-blind fashion. Basic information from patients was collected and analyzed. Result: Our data showed that the probes of the assay were successful in discriminating 14 genera or species of intestinal pathogens. The limit of detection was approximately 10³ CFU/ml for one species of pathogen. Of the 1,500 clinical cases, 32.7% of the patient stools were positive for bacteria. Using stool culture as a control, gene-chip sensitivity was 100%, specificity 95.2%, and index of accurate diagnosis 0.952. Conclusion: Our data suggested that DNA microarray with its high efficiency and accuracy could be used as an alternative to the culture method.

Aim: This study aimed to investigate the occurrence of some virulence genes distributed in Listeria monocytogenes isolated from ready-to-eat (RTE) meat products and consumers in Cairo province, Egypt. Materials and Methods: A total of 120 beef luncheon, chicken luncheon and frankfurter beef (40 samples, each) were collected from 10 different local shops situated in Al-salam city, Cairo province, Egypt. Stool samples were collected from 40 people who had the habit of consuming RTE meat. The suspected L. monocytogenes isolates were subjected to a multiplex polymerase chain reaction (PCR) for rapid speciation and virulence determination using primers specific for inIA, inIC, and inIJ genes. Results: Culture examination of all samples on Oxford media revealed presence of colonies characteristic to L. monocytogenes in 6 beef luncheon (15%), 4 chicken luncheon (10%), 1 frankfurter beef (2.5%) and 1 human stool (2.5%) samples. Species identity of L. monocytogenes was verified through the amplification of a 800 bp fragment with inIA primers in 2 out of 6 culture isolates from beef luncheon (5%), and 1 out 4 culture isolates from chicken luncheon (2.5%) samples. Statistical analysis revealed no significant difference between the occurrence of L. monocytogenes in different food samples examined (p>0.05). The virulence of these strains was ascertained by the presence of 517 bp and 238 bp fragments of inIC and inIJ genes, respectively in the isolates that contained the 800 bp fragment. The culture isolates obtained from one frankfurter beef sample, and one human stool sample were found negative by multiplex PCR for the presence of L. monocytogenes and its virulence specific genes. Conclusion: It could be concluded that L. monocytogenes are circulating in beef and chicken luncheon sold in Cairo, Egypt. Multiplex PCR is reliable for confirmation of L. monocytogenes. This study suggests the implementation of hygienic measures at all levels from production to consumption in order to improve food safety. Furthermore, authors recommended consumption of beef frankfurter or any RTE meat sold in their original intact packing due to low level of contamination.

Prevalence and genetic characteristics of Cronobacter spp. from food and human clinical stool samples in Wenzhou, China 2008–2018

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes