Evaluation of the FluoroType Mycobacteria assay for identification of mycobacteria.

Accurate species identification is a prerequisite for successful management of tuberculosis and non-tuberculous mycobacterial (NTM) diseases. The novel FluoroType Mycobacteria assay combines three established GenoType DNA strip assays (CM, AS, and NTM-DR), allowing detection of Mycobacterium tuberculosis and 32 NTM species/subspecies in a single assay with automatic detection and result analysis. We evaluated the clinical performance of the FluoroType assay and its feasibility in replacing the GenoType Mycobacterium CM assay as the initial method for mycobacterial identification. A total of 191 clinical mycobacterial cultures were analyzed in this study: 180 identified for one mycobacterial species, 6 for multiple, and 5 for no mycobacterial species. Positive percent agreement (PPA) for the FluoroType assay was 87.8% (n = 158), with full agreement for 23/29 species. Weakest PPA was observed for Mycobacterium gordonae (50%, n = 9/18), Mycobacterium interjectum (40%, n = 2/5), and Mycobacterium intracellulare (42%, n = 5/12). Clinical and mixed cultures containing multiple mycobacterial species gave equally single species and genus level identifications (n = 30). No cross-reactivity with non-mycobacterial species was observed (n = 22). In a separate in silico analysis of 2016-2022 HUS area (Finland) register data (n = 2,573), the FluoroType assay was estimated to produce 18.8% (n = 471) inadequate identifications (genus/false species) if used as the primary identification method compared to 14.2% (n = 366) with the GenoType CM assay. The FluoroType assay was significantly more convenient in terms of assay workflow and result interpretation compared to the entirely manual and subjective GenoType CM assay. However, the feasibility of the assay should be critically assessed with respect to the local NTM species distribution. IMPORTANCE: This study is the first clinical evaluation report of the novel FluoroType Mycobacteria assay. The assay has the potential to replace the established GenoType NTM product family in identification of culture-enriched mycobacteria. However, our research results suggest that the assay performs suboptimally and may not be feasible for use in all clinical settings.

Molecular characteristics of Cryptosporidium spp. in human cases in five Finnish hospital districts during 2021: first findings of Cryptosporidium mortiferum (Cryptosporidium chipmunk genotype I) in Finland.

The aims of the study were to characterise the distribution of Cryptosporidium spp. and subtypes causing infections in Finland during 2021. This was carried out with 60 clinical samples from the hospital districts of Helsinki and Uusimaa, Vaasa, Kymenlaakso, South Karelia, and Central Finland, as well as with Finnish Infectious Diseases Register (FIDR) data. Additionally, the study aimed to explore the potential exposures related to Cryptosporidium mortiferum (Cryptosporidium chipmunk genotype I) infections via interview. Species identification was carried out with quantitative real-time PCR (qPCR) and 18S sequencing. Further typing was performed with gp60 subtyping. Over 70% of the samples were identified as Cryptosporidium parvum and 20% as C. mortiferum, which had not been identified in Finland before. Two cases of Cryptosporidium hominis were identified from patients reported to have travelled outside Europe. The C. parvum subtype IIaA15G2R1 and the C. mortiferum subtype XIVaA20G2T1 were the most common subtypes identified. The interviewed C. mortiferum cases did not report shared exposures such as contact with wild rodents. In conclusion, C. parvum and C. mortiferum were the major causes of cryptosporidiosis in the five studied Finnish hospital districts.

Shiga toxin-producing Escherichia coli (STEC) stool multiplex PCR can replace culture for clinical diagnosis and follow-up.

Shiga toxin (stx)-producing Escherichia coli (STEC) causes potentially severe gastrointestinal infections. Due to its public health importance, control measures are required, and carriers may need to refrain from work or daycare when the risk of spread to vulnerable people is high. We evaluated the use of direct stool multiplex PCR compared to culture for primary STEC diagnostics and for follow-up in order to update the national guidelines for STEC monitoring. We analyzed primary and follow-up samples of 236 STEC PCR-positive cases at HUSLAB, Helsinki, Finland in 2016-2017, altogether 858 samples. All STEC PCR-positive samples were inoculated on non-selective chromogenic agar plates. Culture positivity was confirmed from culture sweeps by PCR. 211 (89%) of the cases were culture positive in their primary sample. Of all primary and follow-up samples, 499 were PCR positive and of these 450 (90%) were culture positive. PCR-negative follow-up samples were available from 125 cases. Of these, 88 cases were followed for at least three consecutive PCR-negative samples. Two cases (2%) had culture-positive sample(s) after two consecutive PCR-negative samples. The median time for STEC clearance was 22-23 days. The laboratory-developed multiplex PCR test used in this study is a reliable method for STEC diagnostics and follow-up in a clinical laboratory. When non-selective methodology is used, the majority of PCR-positive samples (90%) are also culture positive. Furthermore, only two cases (2%) in our material had two consecutive PCR-negative samples followed by positive samples. Consequently, to demonstrate the clearance from STEC infection, we consider two PCR-negative follow-up samples sufficient. The Finnish national guidelines for STEC monitoring have been updated accordingly.

Evaluation of the Novel CE-IVD-Marked Multiplex PCR QIAstat-Dx Meningitis/Encephalitis Panel.

Central nervous system (CNS) infections such as meningitis and encephalitis are life-threatening conditions that demand hospital care and prompt identification of the causative agent. Since 2015, there has been only one CE-IVD-marked rapid multiplexed diagnostic assay in cassette format for bacterial and viral detection from cerebrospinal fluid (CSF): the BioFire FilmArray meningitis/encephalitis (ME) panel. In the beginning of 2022, Qiagen introduced the QIAstat-Dx meningitis/encephalitis panel. It is a CE-IVD-marked multiplex PCR cassette test intended for the identification of suspected infectious meningitis, encephalitis, or meningoencephalitis caused by bacterial, viral, or fungal pathogens. In this study, we evaluated patient and quality control samples using the QIAstat-Dx meningitis/encephalitis panel and compared the results to those of the BioFire FilmArray meningitis/encephalitis panel and reference methods (current routine analysis methods in our laboratory, PCR, or cultivation). The combined positive percent agreement between the two panel assays was 100%, and the negative percent agreement was 94%. We further compared specifically herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) dilution series using six commercial herpesvirus assays, including the two cassette tests. The results suggested that real-time PCR methods (with separate extraction) were the most sensitive methods. When comparing the cassette tests, the BioFire FilmArray meningitis/encephalitis panel produced more positive results than the QIAstat-Dx meningitis/encephalitis panel in the herpesvirus analyses. IMPORTANCE The diagnosis of infectious meningitis and encephalitis relies mostly on specific PCR and culturing methods, but commercial syndromic panel assays are bringing a change in diagnostics. With multiplexed analysis, the identification of the pathogen is potentially faster, and less sample material is needed. The novel QIAstat-Dx meningitis/encephalitis panel assay is intended for the rapid identification of pathogens from cerebrospinal fluid for suspected central nervous system (CNS) infection, which is a life-threatening condition and difficult to diagnose. We studied the performance of this panel assay using patient samples and dilution series of selected viruses. The evaluation data for this novel meningitis/encephalitis panel assay are useful for other clinical laboratories and organizations using or considering using this test.

Rapid molecular detection of pathogenic microorganisms and antimicrobial resistance markers in blood cultures: evaluation and utility of the next-generation FilmArray Blood Culture Identification 2 panel.

Rapid detection of pathogens causing bloodstream infections (BSI) directly from positive blood cultures is of highest importance in order to enable an adequate and timely antimicrobial therapy. In this study, the utility and performance of a recently launched next-generation fully automated test system, the Biofire FilmArray® Blood Culture Identification 2 (BCID2) panel, was evaluated using a set of 103 well-characterized microbial isolates including 29 antimicrobial resistance genes and 80 signal-positive and 23 signal-negative clinical blood culture samples. The results were compared to culture-based reference methods, MALDI-TOF, and/or 16S rDNA sequencing. Of the clinical blood culture samples, 68 were monomicrobial (85.0%) and 12 polymicrobial (15.0%). Six samples contained ESBL (blaCTX-M), two MRSA (mecA), and three MRSE (mecA) isolates. In overall, the FilmArray BCID2 panel detected well on-panel targets and resistance markers from mono- and polymicrobial samples. However, one Klebsiella aerogenes and one Bacteroides ovatus were undetected, and the assay falsely reported one Shigella flexneri as Escherichia coli. Hence, the sensitivity and specificity for detecting microbial species were 98.8% (95%CI, 95.8-99.9%) and 99.9% (95%CI, 99.8-99.9%), respectively. The sensitivity and specificity for detecting of resistance gene markers were 100%. The results were available within 70 min from signal-positive blood cultures with minimal hands-on time. In conclusion, the BCID2 test allows reliable and simplified detection of a vast variety of clinically relevant microbes causing BSI and the most common antimicrobial resistance markers present among these isolates.

Comparison of Two Commercial Platforms and a Laboratory-Developed Test for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA.

Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. In this study, the performance of one laboratory-developed test (LDT) and two commercial tests, cobas SARS-CoV-2 (Roche) and Amplidiag COVID-19 (Mobidiag), were evaluated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens. A total of 183 specimens collected from suspected COVID-19 patients were studied with all three methods to compare their performance. In relation to the reference standard, which was established as the result obtained by two of the three studied methods, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the highest value was obtained for the LDT (100%). The dilution series of positive specimens, however, suggests significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. In general, all tested assays performed adequately. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming months to mitigate the pandemic. To ensure no interruption, it is critical that clinical laboratories maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing.

Bacterial, viral and parasitic pathogens analysed by qPCR: Findings from a prospective study of travellers' diarrhoea.

BACKGROUND: The diagnostics of travellers' diarrhoea (TD) has been revolutionised by multiplex qPCR assays. While mostly of bacterial aetiology, viruses and parasites account for the disease among 10-20% of travellers. Despite this, prospective studies applying qPCR assays remain scarce that cover not only bacteria, such as the various diarrhoeagenic Escherichia coli (DEC), but also viral and parasitic pathogens. METHOD: We analysed by qPCR pre- and post-travel stool samples of 146 Finnish travellers for bacterial, viral and parasitic pathogens: enteropathogenic (EPEC), enteroaggregative (EAEC), enterotoxigenic (ETEC), enterohaemorrhagic (EHEC), and enteroinvasive (EIEC) E. coli; Shigella, Campylobacter, Salmonella, Yersinia and Vibrio cholerae; norovirus G1 and G2, rotavirus, enteroviruses, and sapovirus; and Giardia lamblia, Entamoeba histolytica, and Cryptosporidium. Symptoms and medication data during travel were collected by questionnaires. RESULTS: We detected bacterial pathogens in 102/146 samples (69.9%; EAEC, EPEC, ETEC most common), viral ones in 13 (8.9%; norovirus most common), and parasitic ones in one (0.7%; Giardia). Noroviruses were associated with severe symptoms (23.5% versus non-severe 4.9%). In the TD group, 41.7% (5/12) of those with viral pathogens (vs. 13.3%; 11/83 without) took antibiotics. CONCLUSION: Viral pathogens, particularly noroviruses, prevail in severe TD. The symptoms of viral disease are often severe and lead to unwarranted use of antibiotics.

Evaluation of three molecular carbapenemase tests: Eazyplex SuperBug complete B, Novodiag CarbaR+, and Amplidiag CarbaR+MCR.

BACKGROUND: Carbapenemase-producing Gram-negative bacilli, i.e., Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter, are of increased concern for the public health around the world. There is urgent need for rapid and accurate tests in order to provide correct treatment and to prevent bacterial spread in healthcare settings. METHODS: The aim of this study was to evaluate three commercial multiplex carbapenemase tests with CE-IVD marking: Eazyplex SuperBug complete B (AmplexDiagnostics), Novodiag CarbaR+ (Mobidiag), and Amplidiag CarbaR+MCR (Mobidiag). All these tests recognize KPC, NDM, OXA-48/181 group, VIM, OXA-23 group, and OXA-24/40 group, and Novodiag CarbaR+ and Amplidiag CarbaR+MCR additionally recognize IMP, OXA-51 group (with promoter located within ISAbaI), OXA-58 group, and MCR, and Amplidiag CarbaR+MCR further recognizes GES (carbapenemase-type only). RESULTS: The sensitivities and specificities of these tests with bacterial isolates were 100%. The sensitivity directly from clinical samples was 100%, but the specificity was lower, which is simply explained by the higher sensitivity of the molecular methods compared with culture method. CONCLUSIONS: Overall, these CE-IVD marked tests provide a good alternative in the detection of carbapenemase-producing organisms.

Clinical aspects of heat-labile and heat-stable toxin-producing enterotoxigenic Escherichia coli: A prospective study among Finnish travellers.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major pathogen causing travellers' diarrhoea (TD) among visitors to low- and middle-income countries (LMIC). Scant data are available on rates of travel-acquired ETEC producing heat-labile (LT) and/or heat-stable (ST) toxin or its subtypes, STh (human) and STp (porcine) in various geographic regions, and on clinical pictures associated with each toxin. METHODS: Using qPCR, we analysed LT, STh, and STp in stools positive for ETEC in a prospective study among 103 Finnish travellers visiting LMIC. They filled in questionnaires and provided stool samples before and after travel. We scrutinized geographic distribution of LT, STh, and STp ETEC findings, and association between these different ETEC subtypes and moderate/severe TD. RESULTS: Among the 103 stool samples positive for ETEC toxins, the rate for LT was 76%, for STh 26%, and STp 41%. The rate for LT-only was 44%, for STh-only 6%, STp-only 16%, LT + STh 10%, LT + STp 15%, STh + STp 3%, and LT + STh + STp 8%. Findings varied by destination; the rates of LT, STh, and STp were 79%, 21%, and 57%, respectively, in Southern Asia (n = 14); 85%, 10%, and 20% in South-eastern Asia (n = 20); 84%, 13%, and 29% in Eastern Africa (n = 31); and 56%, 50%, and 63% in Western Africa (n = 32), respectively. Of travellers positive for LT, STh, and STp, 83%, 100%, and 88%, encountered TD; 35%, 55%, and 41% reported moderate/severe TD. STh was associated with moderate/severe TD. CONCLUSIONS: Toxin findings varied by destination; multiple toxins were commonly detected. Moderate/severe TD was reported most frequently by subjects with STh-ETEC.

Impact of pre-operative antimicrobial treatment on microbiological findings from endocardial specimens in infective endocarditis.

Treatment of infective endocarditis (IE) should be initiated promptly. This might hamper the chances to identify the causative organism in blood cultures. Microbiological sampling of infected valve in patients undergoing surgery might identify the causative organism. The impact of pre-operative antimicrobial treatment on the yield of valve samples is not known. This study evaluated the impact of the duration of the pre-operative antibiotic treatment on valve culture and 16S rRNA PCR findings from resected endocardial samples. Patients meeting the modified Duke criteria of definite or possible IE and undergoing valve surgery due to IE during 2011-2016 were included from Southern Finland. Eighty-seven patients were included. In patients with shorter than 2 weeks of pre-operative antimicrobial treatment, PCR was positive in 91% (n = 42/46) and valve culture in 41% (n = 19/46) of cases. However, in patients who had 2 weeks or longer therapy before operation, PCR was positive in 53% (n = 18/34) and all valve cultures were negative. In 14% of patients, PCR had a diagnostic impact. In blood-culture negative cases (n = 13), PCR could detect the causative organism in ten patients (77%). These included five cases of Bartonella quintana, one Tropheryma whipplei, and one Coxiella burnetii. Long pre-operative antimicrobial treatment was shown to have a negative impact on microbiological tests done on resected endocardial material. After 2 weeks of therapy, all valve cultures were negative, but PCR was positive in half of the cases. PCR aided in diagnostic work-up, especially in blood culture negative cases.

Capnocytophaga canimorsus Capsular Serovar and Disease Severity, Helsinki Hospital District, Finland, 2000-2017.

We assembled a collection of 73 Capnocytophaga canimorsus isolates obtained from blood cultures taken from patients treated at Helsinki University Hospital (Helsinki, Finland) during 2000-2017. We serotyped these isolates by PCR and Western blot and attempted to correlate pathogen serovar with patient characteristics. Our analyses showed, in agreement with previous research, that 3 C. canimorsus serovars (A-C) caused most (91.8%) human infections, despite constituting only 7.6% of isolates found in dogs. The 3 fatalities that occurred in our cohort were equally represented by these serovars. We found 2 untypeable isolates, which we designated serovars J and K. We did not detect an association between serovar and disease severity, immune status, alcohol abuse, or smoking status, but dog bites occurred more frequently among patients infected with non-A-C serovars. Future research is needed to confirm serovar virulence and develop strategies to reduce risk for these infections in humans.

Multiplex PCR detection of Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica directly from dried stool samples from Guinea-Bissauan children with diarrhoea.

BACKGROUND: In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. METHODS: A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea. RESULTS: The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. CONCLUSIONS: Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.

Antimicrobials increase travelers' risk of colonization by extended-spectrum betalactamase-producing Enterobacteriaceae.

BACKGROUND: More than 300 million travelers visit regions with poor hygiene annually. A significant percentage of them become colonized by resistant intestinal bacteria such as extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) and may transmit the strains to others and to medical care settings when they return home. Despite the threats to global healthcare caused by an upsurge in antimicrobial resistance, no effort has been centered on prevention of colonization while traveling. METHODS: Stool samples were collected from 430 Finns before and after traveling outside Scandinavia. All specimens were analyzed for ESBL- and carbapenemase-producing Enterobacteriaceae (CPE). Questionnaires were used to survey volunteers about use of antimicrobials as well as other potential risk factors. The results were subjected to multivariable analysis. RESULTS: Twenty-one percent (90/430) of the travelers became colonized by ESBL-PE and none by CPE. Geographic region, occurrence of travelers' diarrhea (TD), age, and use of antimicrobial (AB) for TD were identified as independent risk factors predisposing to contracting ESBL-PE. Eleven percent of those in subgroup TD-AB-, 21% in TD+AB-, and 37% in TD+AB+ acquired ESBL-PE. The risk proved to be highest in South Asia (46%); 23% became colonized in subgroup TD-AB-, 47% in TD+AB-, and 80% in TD+AB+. In Southeast Asia, the rates were 14%, 37%, and 69%, respectively. CONCLUSIONS: TD and antimicrobials for TD proved to be independent risk factors, with up to 80% of TD+AB+ travelers contracting ESBL-PE. In modern pre-travel counseling for those visiting high-risk regions, travelers should be advised against taking antibiotics for mild or moderate TD.

Novel microbiological and spatial statistical methods to improve strength of epidemiological evidence in a community-wide waterborne outbreak.

Failures in the drinking water distribution system cause gastrointestinal outbreaks with multiple pathogens. A water distribution pipe breakage caused a community-wide waterborne outbreak in Vuorela, Finland, July 2012. We investigated this outbreak with advanced epidemiological and microbiological methods. A total of 473/2931 inhabitants (16%) responded to a web-based questionnaire. Water and patient samples were subjected to analysis of multiple microbial targets, molecular typing and microbial community analysis. Spatial analysis on the water distribution network was done and we applied a spatial logistic regression model. The course of the illness was mild. Drinking untreated tap water from the defined outbreak area was significantly associated with illness (RR 5.6, 95% CI 1.9-16.4) increasing in a dose response manner. The closer a person lived to the water distribution breakage point, the higher the risk of becoming ill. Sapovirus, enterovirus, single Campylobacter jejuni and EHEC O157:H7 findings as well as virulence genes for EPEC, EAEC and EHEC pathogroups were detected by molecular or culture methods from the faecal samples of the patients. EPEC, EAEC and EHEC virulence genes and faecal indicator bacteria were also detected in water samples. Microbial community sequencing of contaminated tap water revealed abundance of Arcobacter species. The polyphasic approach improved the understanding of the source of the infections, and aided to define the extent and magnitude of this outbreak.

High number of diarrhoeal co-infections in travellers to Benin, West Africa.

BACKGROUND: Travellers' diarrhoea (TD) is the most frequent health problem among travellers to the tropics. Using routine techniques, the aetiology mostly remains unresolved, whereas modern molecular methods enable reducing the number of equivocal cases considerably. While many studies address the aetiology of TD in Asian, Central American and North African tourist resorts, only few focus on Western Africa. METHODS: Stool samples from 45 travellers travelling in Benin, West Africa, were analyzed by a new multiplex qPCR assay for Salmonella, Yersinia, Campylobacter, Vibrio cholerae, Shigella or enteroinvasive (EIEC), enterohaemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), and enteropathogenic Escherichia coli (EPEC). RESULTS: All 18 pre-travel samples proved negative for bacterial pathogens. Of the 39/45 (87%) travellers having had TD, EPEC was detected in post-travel samples in 30 (77%) cases, EAEC in 23 (59%), ETEC in 22 (56%), Shigella or EIEC in 7 (18%), EHEC in two (5%), and Salmonella in one (3%). In 31(79%) of the TD cases two or more bacterial pathogens were identified. Two (8%) samples remained negative: both patients had taken antimicrobials for TD. CONCLUSIONS: EPEC, EAEC and ETEC were the most common findings. 79% of the cases had a co-infection. As modern diagnostics reveals in most patients a multitude of pathogens, the role of each pathogen should be re-evaluated.

A quantitative polymerase chain reaction assay for rapid detection of 9 pathogens directly from stools of travelers with diarrhea.

BACKGROUND & AIMS: Every year, 80 million tourists traveling to tropical and subtropical areas contract traveler's diarrhea (TD). Forty percent to 80% of cases are caused by bacteria, yet clinical diagnostic tests are available to identify only a few of the strains that cause TD. We aimed to develop a quantitative polymerase chain reaction (qPCR) assay to identify all major pathogens in stool samples. METHODS: We developed a low-cost, high-throughput, multiplex qPCR assay for simultaneous detection of 9 bacterial pathogens in stool samples: Salmonella, Yersinia, Campylobacter, and Vibrio cholerae, as well as Shigella or enteroinvasive Escherichia coli, enterohemorrhagic E coli, enterotoxigenic E coli (ETEC), enteroaggregative E coli (EAEC), and enteropathogenic E coli (EPEC). The assay was validated using positive (n = 245) and negative (n = 243) control strains, as well as preselected positive and negative stool samples. In addition, stool samples were collected from 96 returning travelers with TD. The findings were compared with those from routine diagnostic tests. RESULTS: The assay detected the bacterial strains with 100% sensitivity and specificity, compared with results from the reference tests. Of all stool samples collected from travelers with TD, EPEC was found in 47%, EAEC in 46%, ETEC in 22%, enterohemorrhagic E coli in 7%, Campylobacter in 6%, Shigella or enteroinvasive E coli in 2%, and Salmonella in 2%. Multiple pathogens were found in 37% of all samples. CONCLUSIONS: We developed a low-cost, high-throughput qPCR assay for use in routine diagnostic analysis and research. It detects the pathogenic bacteria most commonly associated with TD in stool samples with 100% sensitivity and specificity, compared with reference methods. The assay requires 4 hours, whereas current detection methods require 1 to 7 days. At least 1 TD pathogen was identified in stool samples from 76% of returning travelers, whereas conventional methods found a pathogen in only 17%. The most commonly detected bacteria were EPEC, EAEC, and ETEC.

Applicability of DiversiLab repetitive sequence-based PCR method in epidemiological typing of enterohemorrhagic Escherichia coli (EHEC).

Enterohemorrhagic Escherichia coli (EHEC) causes diarrhea, often with severe complications. Rapid and discriminatory typing of EHEC using advanced molecular methods is needed for determination of the genetic relatedness of clones responsible for foodborne outbreaks and for finding out the transmission sources of the outbreaks. This study evaluated the potential of DiversiLab, a semiautomated repetitive sequence-based polymerase chain reaction method for the genotyping of EHEC strains. A set of 52 EHEC strains belonging to 15 O:H serotypes was clustered into 10 DiversiLab groups. All of the O157 strains and one O55 strain were classified into the same group based on a 90% similarity threshold. The other serotypes were classified to their own DiversiLab group, with the exception of one R:H(-) strain that was grouped with O5:H(-) strains. In addition, O26 and O111 strains were grouped together but ultimately subdivided according to their O-serotypes based on a 95% similarity threshold. The O104 strain, which was associated with a major outbreak of hemolytic uremic syndrome in Germany in May 2011, was also classified independently. The DiversiLab performed well in identifying isolates, but the discriminatory power of the repetitive sequence-based polymerase chain reaction method was lower than that of pulsed-field gel electrophoresis. Analysis of 15 enteropathogenic E. coli (EPEC) strains revealed that some EPEC strains clustered together with EHEC strains. Therefore, the DiversiLab system cannot be used to discriminate between these pathogroups. In conclusion, DiversiLab is a rapid and easy system for the primary exclusion of unrelated EHEC strains based on their serotypes, but more discriminatory methods, such as pulsed-field gel electrophoresis, are needed for accurate typing of the EHEC strains.

Identification of a high-molecular-mass Lactobacillus epithelium adhesin (LEA) of Lactobacillus crispatus ST1 that binds to stratified squamous epithelium.

Lactobacilli belong to the normal gastrointestinal and genital tract microbiota of human and animal hosts. Adhesion is important for bacterial colonization; however, only a few Lactobacillus adhesins have been identified so far. We studied extracted surface proteins from an adhesive Lactobacillus crispatus strain, ST1, which efficiently colonizes the chicken alimentary tract, for their binding to tissue sections of the chicken crop, and identified a novel high-molecular-mass repetitive surface protein that shows specific binding to stratified squamous epithelium. The adhesin binds to both crop epithelium and epithelial cells from human vagina, and was named Lactobacillus epithelium adhesin (LEA). Expression of LEA is strain-specific among L. crispatus strains and corresponds directly to in vitro bacterial adhesion ability. The partial sequence of the lea gene predicts that the LEA protein carries an N-terminal YSIRK signal sequence and a C-terminal LPxTG anchoring motif, as well as a highly repetitive region harbouring 82 aa long repeats with non-identical sequences that show similarity to Lactobacillus Rib/alpha-like repeats. LEA-mediated epithelial adherence may improve bacterial colonization in the chicken crop and the human vagina, which are the natural environments for L. crispatus.

Glutamine synthetase and glucose-6-phosphate isomerase are adhesive moonlighting proteins of Lactobacillus crispatus released by epithelial cathelicidin LL-37.

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.