Clinical Stool Specimens Research Articles

Evaluation of the Use of Singleplex and Duplex CerTest VIASURE Real-Time PCR Assays to Detect Common Intestinal Protist Parasites

Cryptosporidium spp., Giardia duodenalis and Entamoeba histolytica are species of protozoa- causing diarrhoea that are common worldwide, while Entamoeba dispar, Dientamoeba fragilis and Blastocystis sp. appear to be commensal parasites whose role in pathogenicity remains controversial. We conducted the clinical evaluation of five singleplex and one duplex CerTest VIASURE Real-Time PCR Assays against a large panel of positive DNA samples (n = 358), and specifically to Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25) E. dispar (n = 11), Blastocystis sp. (n = 42), D. fragilis (n = 37), and related parasitic phylum species such as Apicomplexa, Euglenozoa, Microsporidia and Nematoda. DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference centre. Estimated diagnostic sensitivity and specificity values were 0.94–1 for Cryptosporidium spp., 0.96–0.99 for G. duodenalis, 0.96–1 for E. histolytica, 1–1 for E. dispar, and 1–0.99 for D. fragilis in the evaluated singleplex assays. In the duplex assay for the simultaneous detection of Blastocystis sp. and D. fragilis these values were 1–0.98 and 1–0.99, respectively. Measures of diagnostic precision for repeatability and reproducibility were found to be under acceptable ranges. The assays identified six Cryptosporidium species (C. hominis, C. parvum, C. canis, C. felis, C. scrofarum, and C. ryanae), four G. duodenalis assemblages (A, B, C, and F), and six Blastocystis subtypes (ST1-ST5, and ST8). The evaluated singleplex and duplex VIASURE Real-Time PCR assays provide sensitive, practical, and cost-effective choices to the molecular diagnosis of the main diarrhoea-causing intestinal protists in clinical microbiology and research laboratories.

Prevalence of diagnostically-discrepant Clostridioides difficile clinical specimens: insights from longitudinal surveillance

BackgroundClostridioides difficile Infection (CDI) is a healthcare-associated diarrheal disease prevalent worldwide. A common diagnostic algorithm relies on a two-step protocol that employs stool enzyme immunoassays (EIAs) to detect the pathogen, and its toxins, respectively. Active CDI is deemed less likely when the Toxin EIA result is negative, even if the pathogen-specific EIA is positive for C. difficile. We recently reported, however, that low-toxin-producing C. difficile strains recovered from Toxin-negative (‘discrepant’) clinical stool specimens can be fully pathogenic, and cause lethality in a rodent CDI model. To document frequency of discrepant CDI specimens, and evaluate C. difficile strain diversity, we performed longitudinal surveillance at a Southern Arizona tertiary-care hospital.MethodsDiarrheic stool specimens from patients with clinical suspicion of CDI were obtained over an eight-year period (2015–2022) from all inpatient and outpatient Units of a > 600-bed Medical Center in Southern Arizona. Clinical laboratory EIA testing identified C. difficile-containing specimens, and classified them as Toxin-positive or Toxin-negative. C. difficile isolates recovered from the stool specimens were DNA fingerprinted using an international phylogenetic lineage assignment system (“ribotyping”). For select isolates, toxin abundance in stationary phase supernatants of pure cultures was quantified via EIA.ResultsOf 8,910 diarrheic specimens that underwent diagnostic testing, 1733 (19.4%) harbored C. difficile. Our major findings were that: (1) C. difficile prevalence and phylogenetic diversity was stable over the 8-year period; (2) toxigenic C. difficile was recovered from 69% of clinically Tox-neg (‘discrepant’) specimens; (3) the six most prevalent USA ribotypes were recovered in significant proportions (>60%) from Tox-neg specimens; and (4) toxin–producing C. difficile recovered from discrepant specimens produced less toxin than strains of the same ribotype isolated from non-discrepant specimens.ConclusionOur study highlights the dominance of Toxin EIA-negative CDI specimens in a clinical setting and the high frequency of known virulent ribotypes in these specimens. Therefore, a careful reevaluation of the clinical relevance of diagnostically-discrepant specimens particularly in the context of missed CDI diagnoses and C. difficile persistence, is warranted.

Gut Microbiota Profiles of Children with Obesity or Metabolic Syndrome: Body Mass Index Is a Lead Actor

The worldwide prevalence of obesity and associated metabolic syndrome (MetS) has increased threefold over the last five decades. Among children, this trend is alarming due to the premature onset of MetS. The data regarding how the structure and composition of gastrointestinal (GIT) microbiota either promote or attenuate obesity and MetS are limited. Objectives: We carried out this study to investigate the relationship between microbial profiles and diagnosis of MetS among children with obesity. Fifty subjects with a diagnosis of obesity or Mets were enrolled. We collected clinical information, demographic data, dietary records, and stool specimens. Overall, there was no significant difference in the diversity of GIT microbiota between the two subgroups of children with obesity or MetS. We also found no differences in the diversity of GIT microbiota between the sexes and blood pressure categories. However, we observed a significant difference between the structure, composition, and diversity of the gut microbiome when the subjects were stratified using a BMI cut-off of 30. Subjects with a BMI ≥ 30 had a lower abundance of Bacteroidetes and a greater abundance of Actinobacteria and Firmicutes compared to those with a BMI value of less than 30. This gut microbiota signature is more like the GIT microbiome profile of adults with obesity and may represent accelerated changes among children. Additional studies are needed to investigate the role of obesity in the maturation of gut microbiota in children with morbid obesity.

Genetic and Biochemical Detection of Salmonella enterica Isolated from Patients Suffering Watery Diarrhea and Typhoid Fever in Babylon Province

Background: Salmonellosis is a severe infectious zoonotic disease, which increases the importance of identifying and controlling the causative strains. Objectives: This study aimed to develop a rapid molecular diagnostic test to determine and purify bacterial isolates based on the specific primer. SE1472298-2 for Salmonella serovar enteritidis; gene STM4497 demonstrated specificity for Salmonella serovar typhimurium and gene O antigen synthesis tyv for Salmonella serovar typhi isolated from patients in Babylon province, Iraq. Materials and Methods: Two hundred clinical stool specimens were collected from patients suffering from watery diarrhea. Blood specimens obtained from patients with typhoid fever who were admitted to three hospitals of Babylon Governorate. Results: There were a total of 200 samples; 34 (17%) were discovered by biochemical tests. The diagnosis of these samples was confirmed by polymerase chain reaction, depending on the target gene, so the number of isolates was 25 (73.53%), Salmonella enteritidis 13 (52%) Salmonella typhimurium 6 (24%), and Salmonella typhi 6 (24%). Conclusion: Molecular techniques, particularly polymerase chain reaction, can rapidly and precisely identify Salmonella isolates.

Evaluation of a Novel Commercial Real-Time PCR Assay for the Simultaneous Detection of Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica.

ABSTRACTCryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica are the most common diarrhea-causing protozoan species globally. Misdiagnosis is a concern for asymptomatic and chronic infections. Multiplexing, i.e., the detection of more than one parasite in a single test by real-time PCR, allows high diagnostic performance with favorable cost-effectiveness. We conducted a clinical evaluation of the VIASURE Cryptosporidium, Giardia, & E. histolytica real-time PCR assay (CerTest Biotec, San Mateo de Gállego, Spain) against a large panel (n = 358) of well-characterized DNA samples positive for Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25), and other parasitic species of the phyla Amoebozoa (n = 11), Apicomplexa (n = 14), Euglenozoa (n = 8), Heterokonta (n = 42), Metamonada (n = 37), Microsporidia (n = 4), and Nematoda (n = 6). DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference center. Estimated sensitivity and specificity were 0.96 and 0.99 for Cryptosporidium spp., 0.94 and 1 for G. duodenalis, and 0.96 and 1 for E. histolytica, respectively. Positive and negative predictive values were calculated as 1 and 0.98 for Cryptosporidium spp., 0.99 and 0.98 for G. duodenalis, and 1 and 0.99 for E. histolytica, respectively. The assay identified six Cryptosporidium species (Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium scrofarum, and Cryptosporidium ryanae) and four G. duodenalis assemblages (A, B, C, and F). The VIASURE assay provides rapid and accurate simultaneous detection and identification of the most commonly occurring species and genetic variants of diarrhea-causing parasitic protozoa in humans.IMPORTANCE Thorough independent assessment of the diagnostic performance of novel diagnostic assays is essential to ascertain their true usefulness and applicability in routine clinical practice. This is particularly true for commercially available kits based on multiplex real-time PCR aimed to detect and differentiate multiple pathogens in a single biological sample. In this study, we conducted a clinical evaluation of the VIASURE Cryptosporidium, Giardia, & E. histolytica real-time PCR assay (CerTest Biotec) for the detection and identification of the diarrhea-causing enteric protozoan parasites Cryptosporidium spp., G. duodenalis, and E. histolytica. A large panel of well-characterized DNA samples from clinical stool specimens or cultured isolates from a reference center was used for this purpose. The VIASURE assay demonstrated good performance for the routine testing of these pathogens in clinical microbiological laboratories.

Verification of analytical bacterial spectrum of QIAstat-Dx® GI V2 and Novodiag® Bacterial GE+ V2-0 diagnostic panels.

BackgroundImplementing multiplex PCR or syndromic panel-based testing platforms to detect microbial species that cause acute diarrhoea may guide patient management more effectively and efficiently.ObjectivesTo assess and compare the performance of two syndromic panel-based testing systems, QIAstat-Dx® Gastrointestinal Panel V2 (QGI) and the Novodiag® Bacterial GE+ V2-0 (NGE).MethodsThe QGI and NGE panels include 16 and 14 bacterial gastrointestinal pathogens, respectively. The performance of the panels was tested retrospectively using 141 positive clinical stool specimens, External Quality Assessment (EQA) panels and spiked faecal specimens.ResultsFor Campylobacter jejuni and coli (n = 20), Salmonella (n = 24), Shigella (n = 13), Yersinia enterocolitica (non-1A biotypes) (n = 8), Clostridioides difficile (n = 24) and Vibrio parahaemolyticus (n = 2), QGI correctly verified 19/20, 20/24, 13/13, 8/8, 23/24 and 2/2, whereas NGE correctly verified 20/20, 17/24, 13/13, 8/8, 14/24 and 1/2. Among diarrhoeagenic Escherichia coli (n = 29), QGI reported one Shiga toxin-producing E. coli (STEC) stx1a O26:H11 as STEC serotype O157:H7 and NGE failed on one enteropathogenic E. coli, one enteroaggregative E. coli and one STEC (stx2e). Y. enterocolitica biotype 1A (non-pathogenic) (n = 6) were all positive in QGI, but negative in NGE.ConclusionsBoth QGI and NGE testing panels can improve laboratory workflow and patient management by providing user-friendly platforms that can rapidly detect a number of targets with one specimen. QGI was significantly more sensitive in identifying C. difficile. Both methods had suboptimal detection of Salmonella and this needs to be examined further. The short hands-on time and turnaround time are of value for on-demand testing and use in a high-throughput setting.

Performance of molecular methods for the detection of Salmonella in human stool specimens.

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture and the lack of validated molecular diagnostic tests for the detection of Salmonella in the stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimised an in-house monoplex real-time polymerase chain reaction (PCR) for the detection of Salmonella ttr and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same ttr and InvA genes and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: Ttr and InvA primers were both able to detect all the different Salmonella serovars tested and had superior limits of detection when DNA was extracted after selenite pre-culture. T tr sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99%, respectively. Culture showed the highest PPV (99.73%), and monoplex- ttr had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance, although stool culture and monoplexed ttr primers had superior specificity and sensitivity, respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events.

A method for detection of SARS-CoV-2 RNA in healthy human stool: a validation study

BackgroundFaecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool.MethodsIn this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at −80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing.FindingsThe lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at −80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at −80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2.InterpretationThis is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic.FundingNational Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.

804. Use of a Trained Canine to Detect Clostridioides difficile in the Hospital Environment

Background Clostridioides difficile infection (CDI) is the most common nosocomial infection in this country. The organism forms spores, which likely contribute to spread of infection in the hospital setting. Current guidelines call for special cleaning of rooms following discharge of patients who tested positive for CDI. There are currently no means to validate that cleaning has eradicated all of the spores. Canines (dogs) have been used to detect a variety of odors because of their superior olfactory abilities. A beagle was trained to detect the odor of C. difficile in the hospital setting.Harley the Clostridium difficile sniffing canine. Methods C. difficile was grown at the microbiology department at Vidant Medical Center (VMC). Culture plates containing the organism were used by a professional trainer to train a two-year old female beagle dog. The canine was taught to sit when she identified the odor. The canine was given positive reinforcement with oral treats each time she identified the odor. Once the dog was proficient in identifying the organism, the trainer used clinical stool specimens that tested either positive or negative for C. difficile. Prior to coming into the hospital setting, the canine was able to identify positive stool samples with near 100% accuracy. The dog was brought to search rooms occupied or previously occupied by patients who had CDI. The study was approved by the Animal Use and Care Committee at East Carolina University and took place between October 2019 and March 2020 at VMC.ResultsThe canine detected the presence of C. difficile in 51% of 47 rooms inhabited by patients currently being treated for CDI and in 46% of 28 rooms previously inhabited by patients that tested positive for CDI. In all positive rooms, the organism was detected on the bedrails. She did not detect C. difficile in any positive areas after they had been cleaned with bleach wipes.Rooms found to be positive by canine. ConclusionTraining a canine to detect C. difficile is an effective means of detecting the organism in the hospital environment. Use of a trained dog appears to be effective in validating the cleaning process of rooms that have been previously occupied by patients with CDI.Disclosures Paul P. Cook, MD, Contrafect (Grant/Research Support, Scientific Research Study Investigator)Gilead (Grant/Research Support, Scientific Research Study Investigator)Leonard-Meron (Grant/Research Support, Scientific Research Study Investigator)Lilly (Grant/Research Support, Scientific Research Study Investigator)

Circulation of classic and recombinant human astroviruses detected in South Africa: 2009 to 2014.

Astroviruses (AstVs) are associated with diarrhoeal and extra-intestinal infections in human, animal and avian species. A prevalence of 7% was reported in selected regions in SA while AstVs detected from clinical stool specimens were almost identical phylogenetically to strains identified in environmental and water samples. This study investigated the molecular diversity of astroviruses circulating between 2009 and 2014 in South Africa (SA). Astroviruses detected in stool specimens collected from hospitalised children were investigated retrospectively. Astroviruses were characterised using type-specific RT-PCR, partial nucleotide sequence analyses in ORF1 and ORF2 and whole genome sequencing. Different genotypes were compared with clinical features to investigate genotype-related associations. The Vesikari severity scale (VSS) was evaluated for scoring astrovirus diarrhoeal infections. Of 405 astroviruses detected, 49.9 % (202/405) were characterised into 32 genotypes comprising 66.3 % (134/202) putative-recombinants and 33.7 % (68/202) classic strains. No trends by year of collection, age or site were observed. Whole genome analysis in eight strains revealed that genotypes assigned by partial nucleotide sequence analyses to five astroviruses were incorrect. Bivariate analyses showed there were no significant associations between genotypes and clinical symptoms or severity of infection. A comparison of Vesikari parameters with astrovirus-positive proxy values demonstrated that Vesikari scores for duration of diarrhoea and admission temperatures would result in a milder infection rating in astrovirus-positive cases. Diverse genotypes co-circulated with putative-recombinants predominating. Astrovirus classification was complicated by the lack of a consistent characterisation system and reliable reference database. The VSS should be used cautiously to rate astrovirus diarrhoea. While surveillance in communities and out-patient clinics must be continued, screening for human astroviruses in alternate hosts is needed to determine the reservoir species.

Performance of molecular methods for the detection of Salmonella in human stool specimens

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture, and the lack of validated molecular diagnostic tests for the detection of Salmonella in stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimized an in-house monoplex real time polymerase chain reaction (PCR) for the detection of Salmonella TTR and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction, and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same TTR and InvA genes, and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over a period of 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: TTR and InvA primers were both able to detect all the different Salmonella serovars tested, and had superior limits of detection if DNA was extracted after selenite pre-culture. TTR sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99% respectively. Culture showed the highest PPV (99.73%) and mono-TTR had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance although stool culture and monoplexed TTR primers had superior specificity and sensitivity respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events.

Coronavirus disease 2019 among pregnant Chinese women: case series data on the safety of vaginal birth and breastfeeding.

ObjectiveTo assess whether vaginal secretions and breast milk of women with coronavirus disease 2019 (COVID‐19) contain severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2).DesignSingle centre cohort study.SettingRenmin Hospital of Wuhan University, Wuhan, Hubei province, China.Population We studied 13 SARS‐CoV‐2‐infected pregnant women diagnosed between 31 January and 9 March 2020.MethodsWe collected clinical data, vaginal secretions, stool specimens and breast milk from SARS‐CoV‐2‐infected women during different stages of pregnancy and collected neonatal throat and anal swabs.Main outcomes and measuresWe assessed viral presence in different biosamples.ResultsOf the 13 women with COVID‐19, five were in their first trimester, three in their second trimester and five in their third trimester. Of the five women in their third trimester who gave birth, all delivered live newborns. Among these five deliveries, the primary adverse perinatal outcomes included premature delivery (n = 2) and neonatal pneumonia (n = 2). One of nine stool samples was positive; all 13 vaginal secretion samples, and five throat swabs and four anal swabs collected from neonates, were negative for the novel coronavirus. However, one of three samples of breast milk was positive by viral nucleic acid testing.ConclusionsIn this case series of 13 pregnant women with COVID‐19, we observed negative viral test results in vaginal secretion specimens, suggesting that a vaginal delivery may be a safe delivery option. However, additional research is urgently needed to examine breast milk and the potential risk for viral contamination.Tweetable abstractNew evidence for the safety of vaginal delivery and breastfeeding in pregnant women infected with SARS‐CoV‐2, positive viral result in a breast‐milk sample.

Viral Shedding of COVID-19 in Pregnant Women

Background: Since there is little awareness of the consequences of coronavirus disease 2019 (COVID-19) in pregnancy, data on the safety of vaginal delivery and breastfeeding in pregnant women with COVID-19 are urgently needed. Methods: We studied COVID-19- infected pregnant women diagnosed at the Renmin Hospital of Wuhan University between January 31 and March 9, 2020, and collected clinical data, vaginal secretion, and stool specimens during different stages of pregnancy. We also collected breast milk samples, as well as throat and anal swabs from the corresponding infants to detect the presence of the virus. Results: Of the 13 women with COVID-19, 5 were in their first trimester, 3 in their second trimester, and 5 in their third trimester. Clinical manifestations observed among the 13 women included fever (n = 8), cough (n = 5), increased leukocyte count (n = 3), lymphopenia (n = 2), impaired liver function (n = 3), dyspnea (n = 1), myalgia (n = 1), and diarrhea (n = 1). Of the 5 women during their third trimester who gave birth, all delivered live newborns. Among these 5 deliveries, the primary adverse perinatal outcomes included premature delivery (n = 2) and neonatal pneumonia (n = 2). One of the 5 women followed from the first trimester experienced a biochemical pregnancy. One of 9 stool samples was positive; and all 13 vaginal secretion samples, and 5 throat swabs and 4 anal swabs collected from newborns were negative for the novel coronavirus. However, 1 of 3 samples of breast milk was positive by viral nucleic acid testing. Conclusions: In this case series of 13 pregnant women with COVID-19, we observed negative viral test results in vaginal secretion specimens, suggesting that a vaginal delivery may be a safe delivery option. However, additional research is urgently needed to examine breast milk and the potential risk for viral contamination.

Molecular characteristics and genotypic diversity of enterohaemorrhagic Escherichia coli O157:H7 isolates in Gauteng region, South Africa.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is one of the major foodborne and waterborne pathogens causing severe diseases and outbreaks worldwide. There is scarcity of EHEC O157:H7 data in South Africa. This study was carried out to determine the molecular characteristics and genotypic diversity of EHEC O157:H7 isolates in the Gauteng region, South Africa. Samples were cultured on selective chromogenic media. Antibiotic susceptibility profile of isolates was determined using the VITEK®-2 automated system. Isolates were characterised using multiplex PCR assays and the genetic diversity was determined using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 520 samples of which 270 environmental water samples and 250 stool specimens were collected and analysed. Overall, EHEC O157:H7 was recovered from 2.3% (12/520) of samples collected. Environmental water samples and clinical stool specimens showed a prevalence of 4.07% (11/270) and 0.4% (1/250) respectively. Antibiotic susceptibility profile varied from isolates with full susceptibility to isolates with resistance to multiple antibiotics. Most resistance was detected to the penicillins, specifically ampicillin (7/12), amoxicillin (3/12) and piperacillin/Tazobactam (3/12) followed by one of the folate inhibitors, trimethoprim (3/12) and the carbapenems, imipenem and meropenem (2/12) each. Three isolates harboured a combination of Shiga-toxins (Stx)-2, intimin (eae) and enterohaemolysin (hlyA) genes, while two isolates harboured the Stx-1, Stx-2 and hlyA genes. The PFGE performed showed that EHEC O157:H7 isolates were genetically diverse, with two minor pulsotypes and eight singletons. The MLST analysis identified three sequence types (STs) (ST10, ST11 and ST1204) that have been previously reported associated with outbreaks. The STs identified in this study pose a potential public health risk to consumers of untreated environmental water and closed human contacts. There is necessity to enhance surveillance in reducing the propagation of this bacterium which is a public health problem.

Broadly reactive real-time reverse transcription-polymerase chain reaction assay for the detection of human sapovirus genotypes.

Sapoviruses are associated with acute gastroenteritis. Human sapoviruses are classified into four distinct genogroups (GI, GII, GIV, and GV) based on their capsid gene sequences. A TaqMan probe-based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay that detects the representative strains of these four genogroups is widely used for screening fecal specimens, shellfish, and environmental water samples. However, since the development of this test, more genetically diverse sapovirus strains have been reported, which are not detectable by the previously established assays. In this study, we report the development of a broader-range sapovirus real-time RT-PCR assay. The assay can detect 2.5 × 107 and 2.5 × 10 1 copies of sapovirusand therefore is as sensitive as the previous test. Analysis using clinical stool specimens or synthetic DNA revealed that the new system detected strains representative of all the 18 human sapovirus genotypes: GI.1-7, GII.1-8, GIV.1, and GV.1, 2. No cross-reactivity was observed against other representative common enteric viruses (norovirus, rotavirus, astrovirus, and adenovirus). This new assay will be useful as an improved, broadly reactive, and specific screening tool for human sapoviruses.

A Study of Immunochromatographic Fecal Occult Blood Test Methods.

The sensitivity of the commonly used immunochromatographic fecal occult blood test (iFOBT) is critical in the routine screening of digestive tract cancers. To determine whether the performance of the iFOBT might be improved by optimizing detergents and buffers in stool solution. We tested different buffers and detergents in stool solution. The specificity of the new solution condition was confirmed by dot immunoblotting. A total of 1122 clinical stool specimens were collected to evaluate the clinical application of this refined stool solution. The different salts exerted minor effects; also, sodium dodecyl sulfate (SDS) prevented the test from working as designed. However, 1% Triton X-100 improved the sensitivity significantly, as confirmed by dot immunoblot testing. With 1% Triton X-100 in the stool solution, the rate of positive iFOBT results in patients with digestive tract cancers increased, from 40.4% to 49.4%. Use of 1% Triton X-100 improves the sensitivity of the iFOBT and thus, this substance should be routinely included in iFOBT for screening of digestive tract cancers.

Simultaneous detection and quantification of 19 diarrhea-related pathogens with a quantitative real-time PCR panel assay

Acute diarrheal diseases are causes of global public health concern, especially in developing countries. A variety of diarrhea-associated microbial species, including bacteria, viruses, and protozoa, have been recognized. Simplified methods for detecting a wide range of diarrheagenic enteric microbes can clarify the etiology and aid in the diagnosis of diarrheal diseases. Here, we report a quantitative real-time (q)PCR-based method for simultaneous detection of 24 targets from 19 microbes suspected of causing diarrhea in stool specimens. We first selected the 24 oligonucleotide primer sets and hydrolysis probes conjugated with the fluorescent reporter dyes FAM, NED, or ABY, along with an internal control, and the passive reference dye ROX to establish a single-plate panel assay. The 12-duplex qPCR panel showed high linearity, with R2 values of 0.981–1.0 and limits of detection ranging from 1 to 103 fg for bacterial DNA (1–200 cells), 10–102 copies for viral DNA/RNA, and 10 fg for parasitic DNA (equivalent to approximately 1 parasite) per reaction. The accuracy and robustness of the assay was demonstrated in experiments using clinical stool specimens. This platform is low cost and easily customizable, and can be applied to various types of qPCR instruments and experimental designs for surveillance of acute diarrhea.

Immunological Stability of Clostridium difficile Toxins in Clinical Specimens.

OBJECTIVE The impact of storage on stability and detection of Clostridium difficile toxins in feces is poorly understood. The objective of this study was to investigate the immunological stability of C. difficile toxins in clinical stool specimens under different storage conditions by evaluating this stability using toxin detection by enzyme immunoassay (EIA). METHODS Stool specimens positive for C. difficile infection (CDI) by quantitative polymerase chain reaction (qPCR) were used for EIA testing with the C. difficile Tox A/B II kit. The EIA-positive specimens were stored aerobically under refrigerated (4-10°C) and frozen (-30°C and -80°C) conditions. Measurement of toxin quantity was conducting using optical density (OD) on days 0, 14, 30, 60, 90, and 120 of storage. RESULTS Clostridium difficile toxins demonstrated good detection in undiluted stool specimens by EIA up to 120 days of storage. Good detection of the toxins was observed in diluted samples at refrigerated and -80°C temperatures. Dilution detrimentally affected toxin detection at -30°C. CONCLUSION Storage of undiluted clinical stool specimens at refrigerated, -30°C, and -80°C temperatures for up to 120 days has no discernible effect on the immunological stability of C. difficile cytotoxins. However, storage at -30°C has a detrimental effect on C. difficile toxin stability in diluted specimens. Infect Control Hosp Epidemiol 2018;39:434-438.

Detection of Blastocystis in clinical stool specimens using three different methods and morphological examination in Jones' medium.

Background:Blastocystis was identified almost a century ago, yet its biology and pathogenicity status in humans is obscure. Studies on Blastocystis in India are scanty and are mostly microscopy based. This study compared three detection modalities to determine their efficiency in the identification of Blastocystis in human feces.Materials and Methods:A total of 279 stool samples were screened using microscopy, culture (Jones' medium), and polymerase chain reaction (PCR)-based methods. Among the three, PCR is considered the gold standard test for detection of Blastocystis, as it helps to authenticate the sensitivity, specificity, and kappa agreement obtained by the other two tests. The morphological features of Blastocystis were recorded at 24, 48, and 72 h. After positive morphological identification, ten samples were cultured on Löwenstein–Jensen (LJ) medium and Locke's egg slant medium.Results:The sensitivity and specificity determined on the basis of microscopy were 36.2% and 99.4%, respectively. On the other hand, Jones' medium showed 67.6% sensitivity and 100% specificity. Further, we documented various morphological and reproductive features of Blastocystis using various staining techniques on cultures positive in Jones' medium. In addition, we also found that LJ medium was not equally efficacious as Jones' medium in assisting the growth of Blastocystis.Conclusions:Although molecular diagnosis is a necessary tool for understanding the true epidemiology of Blastocystis, in laboratories devoid of molecular detection facilities, stool microscopy in conjunction with stool culture on Jones' medium could serve as the best alternative tool for the detection of Blastocystis.

Equivalent Performance of the Cobas® Cdiff Test for Use on the Cobas® Liat® System and the Cobas® 4800 System.

Clostridium difficile infection is a significant health burden, and innovative solutions are needed to shorten time to diagnosis and improve infection control. We evaluated the performance of the cobas® Cdiff test for use on the cobas® Liat® System (cobas® Liat® Cdiff), a single-sample, on-demand, and automated molecular solution with a 20-min turnaround time. The limit of detection was 45–90 colony-forming units (CFUs)/swab for toxigenic strains that covered the most prevalent toxinotypes, including the hyper-virulent epidemic 027/BI/NAP1 strain. Using 442 prospectively collected clinical stool specimens, we compared the performance of the cobas® Liat® Cdiff to direct culture and to the cobas® Cdiff test on the cobas® 4800 System (cobas® 4800 Cdiff) – a medium-throughput molecular platform. The sensitivity and specificity of the cobas® Liat® Cdiff compared to direct culture were 93.1% and 95.1%, respectively, and this performance did not statistically differ from the cobas® 4800 Cdiff (P < 0.05). Direct correlation of the cobas® Liat® and cobas® 4800 Cdiff tests yielded overall percent agreement of 98.6%. The test performance, automation, and turnaround time of the cobas® Liat® Cdiff enable its use for on-demand and out-of-hours testing as a complement to existing batch testing solutions like the cobas® 4800 Cdiff.