Diagnosing Clostridioides difficile infections with molecular diagnostics: multicenter evaluation of revogene C. difficile assay.

Clostridioides difficile infections are a significant threat to our healthcare system, and rapid and accurate diagnostics are crucial to implement the necessary infection prevention and control measurements. Nucleic acid amplification tests are such reliable diagnostic tools for the detection of toxigenic Clostridioides difficile strains directly from stool specimens. In this multicenter evaluation, we determined the performance of the revogene C. difficile assay. The analysis was conducted on prospective stool specimens collected from six different sites in Europe. The performance of the revogene C. difficile assay was compared to the different routine diagnostic methods and, for a subset of the specimens, against toxigenic culture. In total, 2621 valid stool specimens were tested, and the revogene C. difficile assay displayed a sensitivity/specificity of 97.1% [93.3-99.0] and 98.9% [98.5-99.3] for identification of Clostridioides difficile infection. Discrepancy analysis using additional methods improved this performance to 98.8% [95.8-99.9] and 99.6% [99.2-99.8], respectively. In comparison to toxigenic culture, the revogene C. difficile assay displayed a sensitivity/specificity of 93.0% [86.1-97.1] and 99.5% [98.7-99.9], respectively. These results indicate that the revogene C. difficile assay is a robust and reliable aid in the diagnosis of Clostridioides difficile infections.

Workflow optimization for syndromic diarrhea diagnosis using the molecular Seegene Allplex™ GI-Bacteria(I) assay.

Syndromic panel-based molecular testing has been suggested to improve and accelerate microbiological diagnosis. We aimed to analyze workflow improvements when using the multiplex Seegene Allplex™ GI-Bacteria(I) assay as a first-line assay for bacterial diarrhea. Technical assay evaluation was done using spiked stool samples and stored patient samples. After implementation of the assay in the routine clinical workflow, an analysis of 5032 clinical samples analyzed by the Seegene assay and 4173 control samples examined by culture in a similar time period 1 year earlier was performed. Sensitivity of the assay was shown to be between 0.4 and 95.9 genome equivalents/PCR. For 159 positive patient samples with a composite reference of culture and/or a molecular assay, the sensitivity of the assay was 100% for Campylobacter, 92% for Salmonella, 89% for Aeromonas, and 83% for Shigella. Sensitivity for C. difficile toxin B detection was 93.9%. The comparison of clinical samples obtained in two 8-month periods showed increased detection rates for Aeromonas (2.90%vs. 0.34%), Campylobacter spp. (2.25% vs. 1.34%), Shigella spp. (0.42% vs. 0.05%) whereas detection of Salmonella was slightly decreased (0.46% vs. 0.67%) when using the Seegene assay. An analysis of the time-to-result showed that the median dropped from 52.7 to 26.4 h when using the molecular panel testing. The Seegene Allplex™ GI-Bacteria(I) assay allows accelerated, reliable detection of major gastrointestinal bacteria roughly within 1 day. Workload is reduced, specifically in a low-prevalence setting.

Diagnosis of whooping cough in Switzerland: differentiating Bordetella pertussis from Bordetella holmesii by polymerase chain reaction.

Bordetella holmesii, an emerging pathogen, can be misidentified as Bordetella pertussis by routine polymerase chain reaction (PCR). In some reports, up to 29% of the patients diagnosed with pertussis have in fact B. holmesii infection and invasive, non-respiratory B. holmesii infections have been reported worldwide. This misdiagnosis undermines the knowledge of pertussis' epidemiology, and may lead to misconceptions on pertussis vaccine's efficacy. Recently, the number of whooping cough cases has increased significantly in several countries. The aim of this retrospective study was to determine whether B. holmesii was contributing to the increase in laboratory-confirmed cases of B. pertussis in Switzerland. A multiplex species-specific quantitative PCR assay was performed on 196 nasopharyngeal samples from Swiss patients with PCR-confirmed Bordetella infection (median age: 6 years-old, minimum 21 days-old, maximum 86 years-old), formerly diagnosed as Bordetella pertussis (IS481+). No B. holmesii (IS481+, IS1001-, hIS1001+) was identified. We discuss whether laboratories should implement specific PCR to recognize different Bordetella species. We conclude that in Switzerland B. holmesii seems to be circulating less than in neighboring countries and that specific diagnostic procedures are not necessary routinely. However, as the epidemiological situation may change rapidly, periodic reevaluation is suggested.

Disease burden of rotavirus gastroenteritis in children up to 5 years of age in two Swiss cantons: paediatrician- and hospital-based surveillance.

Rotavirus gastroenteritis (RV GE) is a leading cause of diarrhoea in young children. The purpose of this epidemiological surveillance was to measure the disease burden of RV GE among children <5 years of age in two regions of Switzerland, Geneva and Lucerne. One hospital and four paediatricians participated per region. The surveillance lasted from December 2006 to June 2007. The population denominator for calculation of the RV GE incidence rate was the average of the overall study population <5 years of age under surveillance during the surveillance period. At the study sites, 513 children with GE were presented. Stool sample was collected and examined in 341 cases, of which 130 were RV positive (38.1%). Informed consent to participate in the study was obtained for 113 RV positive subjects. The overall RV GE incidence rate was 0.97% in Lucerne [lower incidence interval (LCI), 0.71%; upper incidence interval (UCI), 1.2%] compared with 0.65 and in Geneva (LCI, 0.50%; UCI, 0.81%). Disease severity assessments using the Vescari score showed that the RV GE episodes were more severe in Lucerne than in Geneva (14.05 +/- 3.05 vs 12.85 +/- 2.87), which was confirmed by a higher hospitalisation rate in Lucerne at the study visit (82.9% vs 23.6%). More children had fever in Geneva than in Lucerne (42.9% vs 26.8%), and more children were hospitalised during the follow-up period in Geneva than in Lucerne (14.5% vs 2.5%). Genotyping of RV positive stool samples revealed that both G1 and P8 were the most prevalent types in both regions. There was a statistically significant difference in the distribution frequency of G1 between the two regions (p = 0.039). Assessment of health economic data confirmed the economic burden of RV GE episodes. In conclusion, RV GE episodes are a health burden as well as an economic burden also for the children in a developed country such as Switzerland.

Genotyping reveals a wide heterogeneity of Tropheryma whipplei.

Tropheryma whipplei, the causative agent of Whipple's disease, is associated with various clinical manifestations as well as an asymptomatic carrier status, and it exhibits genetic heterogeneity. However, relationships that may exist between environmental and clinical strains are unknown. Herein, we developed an efficient genotyping system based on four highly variable genomic sequences (HVGSs) selected on the basis of genome comparison. We analysed 39 samples from 39 patients with Whipple's disease and 10 samples from 10 asymptomatic carriers. Twenty-six classic gastrointestinal Whipple's disease associated with additional manifestations, six relapses of classic Whipple's disease (three gastrointestinal and three neurological relapses), and seven isolated infections due to T. whipplei without digestive involvement (five endocarditis, one spondylodiscitis and one neurological infection) were included in the study. We identified 24 HVGS genotypes among 39 T. whipplei DNA samples from the patients and 10 T. whipplei DNA samples from the asymptomatic carriers. No significant correlation between HVGS genotypes and clinical manifestations of Whipple's disease, or asymptomatic carriers, was found for the 49 samples tested. Our observations revealed a high genetic diversity of T. whipplei strains that is apparently independent of geographical distribution and unrelated to bacterial pathogenicity. Genotyping in Whipple's disease may, however, be useful in epidemiological studies.

Identification of moulds in the diagnostic laboratory--an algorithm implementing molecular and phenotypic methods.

Sequence analysis provides a valuable alternative to phenotypic identification of moulds, especially for isolates lacking characteristic morphology. In this comparative prospective study, isolates that could not be identified by standard phenotypic criteria within 5 days (n = 244) were subjected to sequence analysis and further in-depth phenotypic investigations. Comparison of sequence-based with extended phenotypic identification revealed that sequence analysis was more precise in 52.0% of the isolates; in 38.6% of the isolates, both methods gave concordant results. The construction of a database consisting of high-quality sequences allowed improvement of sequence-based identification. Based on these results, we propose a diagnostic algorithm for the effective use of both phenotypic and genetic procedures for identification of moulds in the diagnostic laboratory.

Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification.

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.

Rapid detection and species identification of Mycobacterium spp. using real-time PCR and DNA-microarray.

Infections with mycobacteria are an important issue in public health care. Here we present a "proof-of-principle" concept for the identification of 37 different Mycobacterium species using 5' exonuclease real-time PCR and DNA microarray based on the region upstream of the 65 kDa heat shock protein. With our two PCR probes, one complementary to all mycobacteria species, the other specific for the M. tbc-complex, 34 species were properly classified by real-time PCR. After reamplification and hybridization to a DNA microarray, all species showed a specific pattern. All 10 blindly tested positive cultures revealed a positive real-time PCR signal with the genus probe. After reamplification and hybridization, six samples could unambiguously be identified. One sample showed a mixture of presumably three species-specific patterns and sequencing the 16S rRNA confirmed the presence of a mixture. The hybridization results of three specimens could not be interpreted because the signal to background ratio was not sufficient. Two samples considered as negative controls (LAL Reagent Water (Cambrex) and DNA of Candida albicans) gave neither a genus nor a M. tbc-complex positive PCR signal. Based on these results we consider our method to be a promising tool for the rapid identification of different mycobacteria species, with the advantage of possible identification of mixed infections or contaminations.

Immunohistostaining assays for detection of Chlamydia pneumoniae in atherosclerotic arteries indicate cross-reactions with nonchlamydial plaque constituents.

Detection of Chlamydia pneumoniae antigens in PCR-negative atheromata by immunohistochemistry assays has given rise to controversies regarding a link between the bacterium and atherosclerosis. One hundred ninety-seven human arterial segments removed surgically were examined for C. pneumoniae DNA by conventional PCR with three different primer pairs and by real-time PCR in two different laboratories. No C. pneumoniae DNA was detected. Eighty atherosclerotic lesions were studied by immunohistochemistry assays. Immunoreactivity for C. pneumoniae was frequently present but was not related to the extent of atherosclerosis. Mammary arteries showed immunoreactivity. Serial sections of 17 atheromata were analyzed by Western blotting, histological staining, and UV fluorescence microscopy. Chlamydial proteins were not detected. The sites with positive results by C. pneumoniae immunohistostaining assays precisely matched the sites with autofluorescent ceroid deposits. Immunoblotting and antigenic staining for C. pneumoniae were negative in tests with fetal aortas. The absence of C. pneumoniae DNA in human atherosclerotic lesions, together with negative results for C. pneumoniae proteins by Western blotting analysis, and the perfect matching of C. pneumoniae immunoreactive sites with sites with autofluorescent ceroid deposits suggest a nonspecific reactivity of antichlamydial antibodies with plaque constituents. On the basis of the results of the present study, there are no arguments for an etiologic role of C. pneumoniae in atherosclerosis.

Cloning and sequencing an unknown gene of Tropheryma whipplei and development of two LightCycler PCR assays.

Whipple's disease is a rare infectious illness that can affect any organ system in the body. It is caused by Tropheryma whipplei, a Gram-positive rod-shaped bacterium with a high G + C content, classified within the actinobacteria. For decades, laboratory detection has been based on microscopy and the periodic acid-Schiff (PAS) staining of biopsies. Recently, PCR has become a useful tool to detect T. whipplei DNA in various clinical specimens. However, a positive PCR result does not confirm Whipple's disease as it has been shown that asymptomatic persons can harbor T. whipplei DNA. Since there is not yet much known about the genome of T. whipplei, genome-walking represents a convenient method to determine unknown gene sequences. Starting from a RAPD fragment we have sequenced and cloned an open reading frame (ORF) of 843 bp. Two real-time PCR assays targeting the ORF fragment and the 16S rRNA gene, respectively, were developed. Compared to a conventional 16S rRNA PCR system the ORF LightCycler assay proved to be very specific (100%) but not sufficiently sensitive (62.4%). In contrast, the 16S rRNA LightCycler assay showed a sensitivity of 95.7% and a specificity of 97.8%. Thus, the 16S rRNA gene assay but not that targeting the new ORF is a suitable alternative to conventional PCR methods.

Etiologic diagnosis of infective endocarditis by broad-range polymerase chain reaction: a 3-year experience.

We analyzed surgically resected endocardial specimens from 49 patients by broad-range PCR. PCR results were compared with (1) results of previous blood cultures, (2) results of culture and Gram staining of resected specimens, and (3) clinical data (Duke criteria). Molecular analyses of resected specimens and previous blood cultures showed good overall agreement. However, in 18% of patients with sterile blood cultures, bacterial DNA was found in the resected materials. When data from patients with definite or rejected cases of infective endocarditis (IE) were included, the sensitivity, specificity, and positive and negative predictive values of broad-range PCR were 82.6%, 100%, 100%, and 76.5%, respectively, overall, and 94.1%, 100%, 100%, and 90%, for cases of native valve endocarditis. The sensitivity, specificity, and positive and negative predictive values of culture of resected specimens from patients with native valve endocarditis were 17.6%, 88.9%, 75%, and 36.4%. We recommend broad-range PCR of surgically resected endocardial material in cases of possible IE, in cases of suspected IE in which blood cultures are sterile, and in cases in which organisms grow in blood cultures but only Duke minor criteria are met. We propose to add molecular techniques to the pathologic criteria of the Duke classification scheme.

Prevalence of enteroaggregative Escherichia coli among children with and without diarrhea in Switzerland.

In a prospective study between July 1999 and September 2000, stool specimens of children below the age of 16 years with (n = 187) and without (n = 137) diarrhea were tested for the presence of enterovirulent bacteria by standard culture methods and by PCR. Targets for the PCR were the plasmid pCVD432 for enteroaggregative Escherichia coli (EAEC), the verotoxin 1 and verotoxin 2 genes for enterohemorrhagic E. coli, ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., genes coding for heat-stable and heat-labile toxins for enterotoxigenic E. coli (ETEC), and the eaeA gene for enteropathogenic E. coli. The following bacteria could be associated with diarrhea: Salmonella enterica (P = 0.001), Campylobacter spp. (P = 0.036), ETEC (P = 0.012), and EAEC (P = 0.006). The detection of EAEC, ETEC, and S. enterica was strongly associated with a history of recent travel outside of Switzerland. EAEC isolates were found in the specimens of 19 (10.2%) of 187 children with diarrhea and in those of 3 (2.2%) of 137 children without diarrhea (P = 0.006) and were the most frequently detected bacteria associated with diarrhea. Among the children below the age of 5 years, the specimens of 18 (11.9%) of 151 with diarrhea were positive for EAEC, while this agent was found in the specimens of 2 (2.2%) of 91 controls (P = 0.007). Enteropathogenic E. coli isolates were found in the specimens of 30 (16.4%) of the patients and in those of 15 (10.9%) of the controls, with similar frequencies in all age groups (P > 0.05). We conclude that EAEC bacteria are involved in a significant proportion of diarrhea cases among children. Children younger than 5 years of age are more often affected by EAEC than older children.

Monitoring intracellular replication of Chlamydophila (Chlamydia) pneumoniae in cell cultures and comparing clinical samples by real-time PCR.

Strains of Chlamydophila pneumoniae may be associated with respiratory disease or atherosclerosis. Two real-time quantitative PCR assays targeting the species-specific genes Cpn0278 and ArgR were developed to compare the in vitro growth of respiratory strains AR39 and K6 with that of atherosclerotic strain A03 and to quantify C. pneumoniae in clinical samples. A third real-time PCR assay was designed to assess contamination with Mycoplasma spp. The assays targeting C. pneumoniae detected DNA concentrations corresponding to 10(4) to 10(-4) inclusion-forming units (IFU)/reaction and were highly specific. AR39 exhibited the longest lag phase and period of exponential growth; K6 augmented growth rates at higher inocula; and A03 grew at highest rates. Contamination with Mycoplasma spp. of AR39 and A03 unlikely accounted for growth differences between them. Numbers of IFU in C. pneumoniae-positive respiratory secretions varied within 4 to 5 orders of magnitude. The assays described may prove valuable for pathogenicity studies.

Quantitative detection of Moraxella catarrhalis in nasopharyngeal secretions by real-time PCR.

The recognition of Moraxella catarrhalis as an important cause of respiratory tract infections has been protracted, mainly because it is a frequent commensal organism of the upper respiratory tract and the diagnostic sensitivity of blood or pleural fluid culture is low. Given that the amount of M. catarrhalis bacteria in the upper respiratory tract may change during infection, quantification of these bacteria in nasopharyngeal secretions (NPSs) by real-time PCR may offer a suitable diagnostic approach. Using primers and a fluorescent probe specific for the copB outer membrane protein gene, we detected DNA from serial dilutions of M. catarrhalis cells corresponding to 1 to 10(6) cells. Importantly, there was no difference in the amplification efficiency when the same DNA was mixed with DNA from NPSs devoid of M. catarrhalis. The specificity of the reaction was further confirmed by the lack of amplification of DNAs from other Moraxella species, nontypeable Haemophilus influenzae, H. influenzae type b, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus pyogenes, Bordetella pertussis, Corynebacterium diphtheriae, and various Neisseria species. The assay applied to NPSs from 184 patients with respiratory tract infections performed with a sensitivity of 100% and a specificity of up to 98% compared to the culture results. The numbers of M. catarrhalis organisms detected by real-time PCR correlated with the numbers detected by semiquantitative culture. This real-time PCR assay targeting the copB outer membrane protein gene provided a sensitive and reliable means for the rapid detection and quantification of M. catarrhalis in NPSs; may serve as a tool to study changes in the amounts of M. catarrhalis during lower respiratory tract infections or following vaccination against S. pneumoniae, H. influenzae, or N. meningitidis; and may be applied to other clinical samples.

Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions.

Atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae are an important cause of community-acquired pneumonia. The available detection methods (culture and serology) either lack sensitivity or give only a retrospective diagnosis. In order to improve their detection and quantification in respiratory samples, a real-time multiplex PCR, performed in two separate reactions, was developed for these three pathogens. The comparison of multiplex real-time and conventional PCR assay on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8%, 100% and 100% agreement for C. pneumoniae, L. pneumophila and M. pneumoniae, respectively. Clinical application of this multiplex real-time PCR was done on 40 respiratory samples from 38 patients with respiratory tract infections. Of 19 serology-positive patients, 14 were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative with the multiplex real-time PCR.

Use of simple heuristics to target macrolide prescription in children with community-acquired pneumonia.

BACKGROUND: Macrolides are the first-line antibiotic treatment of community-acquired pneumonia (CAP). Owing to alarming resistance rates among invasive Streptococcus pneumoniae isolates, particularly in young children, macrolide use should be restricted to patients infected with susceptible pathogens, eg, Mycoplasma pneumoniae. OBJECTIVE: To develop a simple clinical prediction rule for identifying M pneumoniae as the cause of CAP in children. DESIGN AND SETTING: Prospective cohort study in 253 children with radiologically confirmed CAP in a walk-in clinic of a tertiary care hospital. MAIN OUTCOME MEASURES: Mycoplasma infection, proven by results of antibody testing of paired serum samples (gold standard). We compared the area under the receiver operating characteristic curve (c statistic) of the following 2 prediction models: a scoring system derived from logistic regression analysis and a fast-and-frugal decision tree. RESULTS: Mycoplasma pneumoniae infection was confirmed in 32 (13%) of 253 children. A scoring system based on duration of fever and patient age yielded a c statistic of 0.84 (95% confidence interval [CI], 0.77-0.91), compared with that of the decision tree (c = 0.76 [95% CI, 0.70-0.83]). The scoring system identified 75% of all cases as being at high or very high risk for M pneumoniae infection; the decision tree, 72% at high risk. The scoring system would curtail macrolide prescriptions by 75%; the decision tree, by 68%. CONCLUSIONS: In children with CAP, simple clinical decision rules identify patients at risk for M pneumoniae infection. At present US macrolide resistance rates among invasive S pneumoniae isolates, both rules increase the chance of prescribing effective first-line antibiotics compared with general macrolide administration.

Turicibacter sanguinis gen. nov., sp. nov., a novel anaerobic, Gram-positive bacterium.

An unknown, strictly anaerobic, Gram-positive, rod-shaped bacterium (strain MOL361T) was isolated from a blood culture of a febrile patient with acute appendicitis and characterized using phenotypic and molecular methods. Fatty acid analysis and biochemical examination indicated that the isolate most closely resembles members of the Gram-positive bacteria with low DNA G+C content. 16S rDNA sequencing revealed a relatively high overall similarity (97%) to an uncultured bacterium, but these two strains both exhibit low (<87%) 16S rDNA similarity to other bacteria. Phylogenetic analysis with different treeing methods showed that this strain forms a novel line of descent within the Gram-positive bacteria with low G+C content. Strain MOL361T is described as the type strain of a novel species within a new genus, Turicibacter sanguinis gen. nov., sp. nov.

Azithromycin versus penicillin V for treatment of acute group A streptococcal pharyngitis.

OBJECTIVE: To compare a 3-day azithromycin vs. a 10-day penicillin V regimen for treatment of acute group A streptococcal (GAS) pharyngitis in children and to determine whether viral infection and/or pharyngeal GAS carriage in patients and adult contacts affect clinical and bacteriologic efficacy. METHODS: This multicenter, randomized, comparative, open label study compared 3-day, once daily 10 mg/kg azithromycin oral suspension with a 10-day regimen of 100,000 IU/kg/day penicillin V oral suspension in three divided doses in children with acute GAS pharyngitis. Clinical and bacteriologic efficacy and tolerability of the antibiotics were evaluated. Recurrence of symptoms and infection was monitored for 6 months. RESULTS: In total, 292 children (age range, 2 to 12 years) received at least one dose of study medication. Clinical success (cure/improvement) with either antibiotic was similar at the end of therapy (Day 14; azithromycin, 95%; penicillin V, 97%) and at Day 28 (azithromycin, 94%; penicillin V, 95%). Bacteriologic eradication was significantly less with azithromycin than with penicillin V at Day 14 (azithromycin, 38%; penicillin V, 81%; P < 0.001) and at Day 28 (azithromycin, 31%; penicillin V, 68%; P < 0.001). There was no associated increase in GAS-related sequelae. The lower incidence of bacteriologic eradication with azithromycin was not the result of possible concomitant viral infections in the patients, GAS carriage in one parent/guardian or any reduced susceptibility in pretreatment GAS isolates. Both antibiotics were equally well-tolerated. CONCLUSIONS: Treatment with 3-day, once daily 10 mg/kg azithromycin for GAS pharyngitis is associated with similar high levels of clinical efficacy, but lower levels of bacteriologic eradication, than with 10-day 100,000 IU/kg/day penicillin V.

Characterization of Salmonella serovars by PCR-single-strand conformation polymorphism analysis.

PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb groEL gene from 41 strains of 10 different Salmonella serovars. Three HaeIII RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the groEL genes of various Salmonella serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different Salmonella serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11 Salmonella enterica serovar Enteritidis strains, and two SSCP profiles were generated for the 7 S. enterica serovar Infantis and five S. enterica serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of Salmonella serovars due to its rapidity, simplicity, and typeability.

Detection of Tropheryma whipplei DNA in feces by PCR using a target capture method.

Whipple's disease is a rare multisystemic bacterial infection with variable clinical manifestations. For decades, the laboratory diagnosis was based on the demonstration of periodic acid Schiff-positive inclusions in macrophages of gastrointestinal biopsies. PCR has improved the diagnosis of Whipple's disease due to its increased sensitivity compared to histopathological analysis. To avoid invasive procedures for taking specimens, we have investigated the possibility of detecting Tropheryma whipplei DNA in feces rather than in biopsies or gastric aspirate of patients with and without Whipple's disease. Total bacterial DNA was isolated from stool specimens using Qiagen columns followed by a T. whipplei-specific hybridization step with a biotinylated capture probe and streptavidin-coated magnetic particles. The captured DNA was then amplified using the same seminested PCR targeting the 16S rRNA gene of the organism that had been applied to other specimens without capturing. For five of eight patients with Whipple's disease, duodenal biopsies and stool samples were PCR positive, whereas for the three other patients, both specimens were PCR negative. Of 84 patients without Whipple's disease, 75 tested negative in the duodenal biopsy and in the stool sample. For four, both specimens were positive. Five patients tested positive in the stool sample but not in the biopsy. However, for three of these five patients, the gastric aspirate had been PCR positive, indicating that the stool PCR result was true rather than false positive. Compared to PCR from duodenal biopsies, stool PCR has a sensitivity of 100% and a specificity of 97.3%. Additionally, 15 PCR-positive and 22 PCR-negative stool samples were extracted using the Invisorb Spin Stool DNA kit. The simplified stool extraction showed 93.3% sensitivity and 95.5% specificity compared to the target capture method. We conclude that PCR with stool specimens with either extraction method is a sensitive and specific diagnostic tool for the detection of T. whipplei DNA and one not requiring invasive sampling procedures.