Multiplex real-time PCR for diagnosing malaria in a non-endemic setting: a prospective comparison to conventional methods.

Almost a decade ago our diagnostic laboratory implemented an in-house real-time PCR for the detection of Plasmodium DNA to diagnose malaria in parallel with conventional diagnostics, i.e., microscopy (thick and thin smears), quantitative buffy coat microscopy (QBC), and a rapid diagnostic test (RDT). Here we report our experiences and make a comparison between the different diagnostic procedures used in this non-endemic setting. All patients during the period February 2009-December 2017 suspected of malaria were prospectively tested at the moment of sample collection. Both PCR and conventional malaria diagnostics were carried out on a total of 839 specimens from 825 patients. In addition, three Plasmodium falciparum (Pf) patients were closely followed by real-time PCR and microscopy after treatment. Overall, 56 samples (55 patients) tested positive by real-time PCR, of which six were missed by microscopy and seven by QBC. RDT showed fairly good results in detecting Pf, whereas specificity was not optimal. RDT failed to detect 10 of 17 non-Pf PCR positive specimens. One Plasmodium malariae patient would have been missed if only conventional diagnostic tests had been used. The high sensitivity of the PCR was confirmed by the number of PCR positive, microscopy negative post-treatment samples. In conclusion, within our routine diagnostic setting, malaria real-time PCR not only showed a high level of agreement with the conventional methods used, but also showed higher sensitivity and better specificity. Still, for complete replacement of the conventional procedures in a non-endemic setting, the time-to-results of the real-time PCR is currently too long.

Pretransplantation Donor-Recipient Pair Seroreactivity Against BK Polyomavirus Predicts Viremia and Nephropathy After Kidney Transplantation.

Kidney transplant donors are not currently implicated in predicting BK polyomavirus (BKPyV) infection in kidney transplant recipients. It has been postulated, however, that BKPyV infection originates from the kidney allograft. Because BKPyV seroreactivity correlates with BKPyV replication and thus might mirror the infectious load, we investigated whether BKPyV seroreactivity of the donor predicts viremia and BKPyV-associated nephropathy (BKPyVAN) in the recipient. In a retrospective cohort of 407 living kidney donor-recipient pairs, pretransplantation donor and recipient sera were tested for BKPyV IgG levels and correlated with the occurrence of recipient BKPyV viremia and BKPyVAN within 1 year after transplantation. Donor BKPyV IgG level was strongly associated with BKPyV viremia and BKPyVAN (p < 0.001), whereas recipient BKPyV seroreactivity showed a nonsignificant inverse trend. Pairing of high-BKPyV-seroreactive donors with low-seroreactive recipients resulted in a 10-fold increased risk of BKPyV viremia (hazard ratio 10.1, 95% CI 3.5-29.0, p < 0.001). In multivariate analysis, donor BKPyV seroreactivity was the strongest pretransplantation factor associated with viremia (p < 0.001) and BKPyVAN (p = 0.007). The proportional relationship between donor BKPyV seroreactivity and recipient infection suggests that donor BKPyV seroreactivity reflects the infectious load of the kidney allograft and calls for the use of pretransplantation BKPyV serological testing of (potential) donors and recipients.

Comparison of ePlex Respiratory Pathogen Panel with Laboratory-Developed Real-Time PCR Assays for Detection of Respiratory Pathogens.

Infections of the respiratory tract can be caused by a diversity of pathogens, both viral and bacterial. Rapid microbiological diagnosis ensures appropriate antimicrobial therapy as well as effective implementation of isolation precautions. The ePlex respiratory pathogen panel (RP panel) is a novel molecular biology-based assay, developed by GenMark Diagnostics, Inc. (Carlsbad, CA), to be performed within a single cartridge for the diagnosis of 25 respiratory pathogens (viral and bacterial). The objective of this study was to compare the performance of the RP panel with those of laboratory-developed real-time PCR assays, using a variety of previously collected clinical respiratory specimens. A total of 343 clinical specimens as well as 29 external quality assessment (EQA) specimens and 2 different Middle East respiratory syndrome coronavirus isolates have been assessed in this study. The RP panel showed an agreement of 97.4% with the real-time PCR assay regarding 464 pathogens found in the clinical specimens. All pathogens present in clinical samples and EQA samples with a threshold cycle (CT ) value of <30 were detected correctly using the RP panel. The RP panel detected 17 additional pathogens, 7 of which could be confirmed by discrepant testing. In conclusion, this study shows excellent performance of the RP panel in comparison to real-time PCR assays for the detection of respiratory pathogens. The ePlex system provided a large amount of useful diagnostic data within a short time frame, with minimal hands-on time, and can therefore potentially be used for rapid diagnostic sample-to-answer testing, in either a laboratory or a decentralized setting.

[The prevalence of the human rhinoviruses and coronaviruses circulating in the Moscow region during 2007-2012].

The rhinoviruses and coronaviruses are the most common causative agents of the acute upper respiratory tract infection in humans. They include several species that vary in the pathogenicity, some causing severe respiratory tract diseases. In this work, the species prevalence of rhinoviruses and coronaviruses was studied in 92 virus-positive clinical patients that were collected at the area of the Moscow region during the period from 2007 to 2012. Using the real-time PCR the virus circulation has been established for all species common in humans, including three rhinoviruses, HRV A, HRV B, and HRV C, and four coronaviruses, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. For eight patients, the identity of the rhinoviruses, including 4 cases of HRV-C, 3 cases of HRV-A, and a single case of HRV-B, was corroborated using partial sequencing of the 5 non-coding regions and phylogenetic analysis. The viruses of HRV-C, HCoV-NL63, and HCoV-OC43 were prevalent in children with severe respiratory diseases.

Clinical performance of two new, fully integrated molecular platforms used for HIV-1, HBV and HCV viral load analysis, the NeuMoDx 288 and the Alinity m.

BACKGROUND: Viral load (VL) determination in patients with human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) is essential for proper patient management and follow-up. New molecular platforms have been developed to fully automate these diagnostic assays. OBJECTIVE: Evaluation of the clinical performance of HIV-1, HBV and HCV VL assays on the Alinity m (Abbott) and NeuMoDx (Qiagen) molecular platforms. METHOD: Test panels of the three viruses have been compiled of 100 plasma and/or serum samples per target containing non-detectable, non-quantifiable and quantifiable VLs. All samples were retrospectively tested on the Alinity m and NeuMoDx platforms according to manufacturers' instructions. RESULTS: A total of 74, 86 and 66 samples with valid results for both platforms were included in the HIV-1, HBV and HCV analysis respectively. Overall qualitative agreement of the assays on both platforms was 78% for HIV-1, 93% for HBV and 100% for HCV. Quantitative agreement (less than 0.5 log difference) was shown to be 68% for HIV-1, 68% for HBV and 94% for HCV. CONCLUSION: The Alinity m and NeuMoDx HCV assay have a comparable performance. Quantification differences in the HIV-1 assay were mostly apparent in the lower VLs and under-quantification of the NeuMoDx HBV assay was observed.

Evaluation of the sample-to-result, random access NeuMoDx platform for viral load testing of Cytomegalovirus and Epstein Barr virus in clinical specimens.

BACKGROUND: The detection and follow up of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) viral loads (VL) are crucial in the management of immunocompromised patients. Recently, molecular CE-IVD assays for detection and quantification of CMV and EBV have been launched for use on the random-access and sample-to-result NeuMoDx 96 and 288 platforms (Qiagen). OBJECTIVE: Evaluating the qualitative and quantitative performance of the NeuMoDx CMV and EBV assays in clinical specimens compared to a lab developed tests (LDT) and the CE-IVD assays on the Abbott m2000 system. METHOD: Both a prospective and a retrospective panel, compiled of non-detectable (ND), non-quantifiable (NQ) and quantifiable VLs in plasma samples have been evaluated for both CMV and EBV: NeuMoDx versus LDT and NeuMoDx versus Abbott m2000. Quantitative agreement was determined for samples with a quantifiable VL on both systems. RESULTS: Qualitative and quantitative agreement between the NeuMoDx and LUMC's LDT CMV assays was 88%. Qualitative agreement between the NeuMoDx and Abbott m2000 CMV assays was 92% and quantitative agreement was 87%. Qualitative and quantitative agreement between the NeuMoDx and the LDT EBV assays was 87%. Qualitative agreement between the NeuMoDx and Abbott m2000 EBV assays was 91% and quantitative agreement was 0%. CONCLUSION: These data show that the NeuMoDx assays are suitable for both detection and quantification of CMV and EBV in a medium- to high throughput diagnostic setting, but that differences in sensitivity and quantification (for EBV, NeuMoDx versus Abbott m2000) warrant an extensive transition period when using the respective assays for following VL in patient samples.

Lower respiratory tract infection in the community: associations between viral aetiology and illness course.

OBJECTIVES: This study determined associations between respiratory viruses and subsequent illness course in primary care adult patients presenting with acute cough and/or suspected lower respiratory tract infection. METHODS: A prospective European primary care study recruited adults with symptoms of lower respiratory tract infection between November 2007 and April 2010. Real-time in-house polymerase chain reaction (PCR) was performed to test for six common respiratory viruses. In this secondary analysis, symptom severity (scored 1 = no problem, 2 = mild, 3 = moderate, 4 = severe) and symptom duration were compared between groups with different viral aetiologies using regression and Cox proportional hazard models, respectively. Additionally, associations between baseline viral load (cycle threshold (Ct) value) and illness course were assessed. RESULTS: The PCR tested positive for a common respiratory virus in 1354 of the 2957 (45.8%) included patients. The overall mean symptom score at presentation was 2.09 (95% confidence interval (CI) 2.07-2.11) and the median duration until resolution of moderately bad or severe symptoms was 8.70 days (interquartile range 4.50-11.00). Patients with influenza virus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), coronavirus (CoV) or rhinovirus had a significantly higher symptom score than patients with no virus isolated (0.07-0.25 points or 2.3-8.3% higher symptom score). Time to symptom resolution was longer in RSV infections (adjusted hazard ratio (AHR) 0.80, 95% CI 0.65-0.96) and hMPV infections (AHR 0.77, 95% CI 0.62-0.94) than in infections with no virus isolated. Overall, baseline viral load was associated with symptom severity (difference 0.11, 95% CI 0.06-0.16 per 10 cycles decrease in Ct value), but not with symptom duration. CONCLUSIONS: In healthy, working adults from the general community presenting at the general practitioner with acute cough and/or suspected lower respiratory tract infection other than influenza impose an illness burden comparable to influenza. Hence, the public health focus for viral respiratory tract infections should be broadened.

High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.

Potential influence of more-sensitive HIV-1 load detection by the new Roche Cobas AmpliPrep/Cobas TaqMan version 2.0 assay on clinical management of HIV-positive pregnant women.

With the introduction of the new Roche Cobas AmpliPrep/Cobas TaqMan version 2.0 assay, HIV-1 viral loads will be detected more frequently during the peripartum period in pregnant HIV-positive women. The implications for the clinical management of these patients are discussed in this paper.

Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method.

Four human coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) are associated with a range of respiratory outcomes, including bronchiolitis and pneumonia. Their epidemiologies and clinical characteristics are poorly described and are often reliant on case reports. To address these problems, we conducted a large-scale comprehensive screening for all four coronaviruses by analysis of 11,661 diagnostic respiratory samples collected in Edinburgh, United Kingdom, over 3 years between July 2006 and June 2009 using a novel four-way multiplex real-time reverse transcription-PCR (RT-PCR) assay. Coronaviruses were detected in 0.3 to 0.85% of samples in all age groups. Generally, coronaviruses displayed marked winter seasonality between the months of December and April and were not detected in summer months, which is comparable to the pattern seen with influenza viruses. HCoV-229E was the exception; detection was confined to the winter of 2008 and was sporadic in the following year. There were additional longer-term differences in detection frequencies between seasons, with HCoV-OC43 predominant in the first and third seasons and HCoV-HKU1 dominating in the second (see Results for definitions of seasons). A total of 11 to 41% of coronaviruses detected were in samples testing positive for other respiratory viruses, although clinical presentations of coronavirus monoinfections were comparable to those of viruses which have an established role in respiratory disease, such as respiratory syncytial virus, influenza virus, and parainfluenza viruses. The novel multiplex assay for real-time pan-coronavirus detection enhances respiratory virus diagnosis, overcomes potential diagnostic problems arising through seasonal variation in coronavirus frequency, and provides novel insights into the epidemiology and clinical implications of coronaviruses.

Campylobacter jejuni bacteremia and Helicobacter pylori in a patient with X-linked agammaglobulinemia.

We describe a 15-year-old patient with X-linked agammaglobulinemia who developed malabsorption and bacteremia due to infection of Helicobacter pylori and Campylobacter jejuni. The Campylobacter bacteremia was only recognized after subculturing of blood culture bottles that failed to signal in the automated system. After 2 weeks of treatment with meropenem and erythromycin for 4 weeks, the patient developed a relapse of bacteremia 10 months later with a high level erythromycin resistant C. jejuni. Sequencing revealed an A2058C mutation in the 23 S rRNA gene associated with this resistance. Treatment with doxycycline for 4 weeks finally resulted in complete eradication. This case report illustrates the importance for physicians to use adapted culture methods and adequate prolonged therapy in patients with an immunodeficiency. A summary of published case reports and series of patients with hypogammaglobulinemia or agammaglobulinemia with Campylobacter or Helicobacter bacteremia is given.

Rapid molecular detection of influenza outbreaks in nursing homes.

BACKGROUND: Nursing home influenza outbreaks occur in spite of established vaccination programs, and require rapid and sensitive laboratory confirmation for timely intervention. OBJECTIVES: To evaluate diagnostic approaches for rapid confirmation of nursing home influenza outbreaks. STUDY DESIGN: Influenza virus real-time PCR and Directigen Flu A+B enzyme immunoassay were performed on nasopharyngeal swabs, nasopharyngeal washes and throat swabs collected from residents with clinical suspicion of influenza during seven probable nursing home outbreaks in 2004-2005 and 2005-2006. The efficacy of specimen sampling and transport management by Public Health Service outbreak team was evaluated. RESULTS: PCR detected influenza RNA in 80% (68/85) of specimens from 81% (38/47) residents, confirming six suspected outbreaks. Immunoassay sensitivity was highest on nasopharyngeal swabs (38%; 11/29) with a positive predictive value of 100% compared to PCR. Nasopharyngeal swabs were equally sensitive to nasopharyngeal washes by PCR. Nasopharyngeal wash sampling appeared unpractical due to common underlying disability of residents. Outbreak team support was associated with a shorter time to PCR diagnosis compared to outbreaks with no logistical support (mean, 28.2h vs. 84h; P=0.05). CONCLUSIONS: Influenza real-time PCR on nasopharyngeal swabs, obtained by Public Health Service outbreak teams, enabled rapid and sensitive confirmation of nursing home influenza outbreaks.

PCR assays for detection of human astroviruses: In silico evaluation and design, and in vitro application to samples collected from patients in the Netherlands.

BACKGROUND: Human astroviruses (HAstV) comprise three phylogenetically compact and non-adjacent groups of species including classical HAstV (HAstV-C) and the novel ones (HAstV-VA/HMO and HAstV-MLB). Of these, HAstV-C is known to be responsible for gastroenteritis while the novel HAstV are associated with cases of neurological disorders. Accurate detection of all known variants by (real-time) PCR is challenging because of the high intra- and intergroup genetic divergence of HAstV. OBJECTIVES: To evaluate published HAstV PCR assays in silico, design de novo real-time PCR assays that can detect and discriminate three groups of HAstV, and apply those to patient samples to analyse the prevalence of HAstV in stool and cerebrospinal fluid (CSF) specimens. STUDY DESIGN: In silico evaluation of published PCR assays and design of real-time PCR assays for detection of different subsets of HAstV was conducted within a common computational framework that used all astrovirus full genome sequences from GenBank. The newly designed real-time PCR assays were evaluated in vitro and applied to faecal samples (collected in January-May 2016) and cerebrospinal fluid specimens (2010-2016) from patients in the Netherlands. RESULTS: Quantitative in silico evaluation of published PCRs is provided. The newly designed real-time PCR assays can reliably assign all available HAstV genome sequences to one of the three phylogenetic groups in silico, and differentiate among HAstV-specific controls in vitro. A total of 556 samples were tested using these PCR assays. Fourteen fecal samples (2.5%) tested positive for HAstV, 3 of which could be identified as the novel HAstV-MLB variants. No novel HAstV were found in CSF specimens. CONCLUSION: Newly designed real-time PCR assays with improved detection of all known HAstV allowed the first-time identification of novel astroviruses from stool samples in the Netherlands.

Aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 11 European countries.

OBJECTIVES: To describe the role of bacteria (including bacterial resistance), viruses (including those recently described) and mixed bacterial-viral infections in adults presenting to primary care with lower respiratory tract infection (LRTI). METHODS: In all, 3104 adults with LRTI were enrolled, of whom 141 (4.5%) had community-acquired pneumonia (CAP), and 2985 matched controls in a prospective study in 16 primary care networks in Europe, and followed patients up at 28-35 days. We detected Streptococcus pneumoniae and Haemophilus influenzae and assessed susceptibility, atypical bacteria and viruses. RESULTS: A potential pathogen was detected in 1844 (59%) (in 350 (11%) bacterial pathogens only, in 1190 (38%) viral pathogens only, and in 304 (10%) both bacterial and viral pathogens). The most common bacterial pathogens isolated were S. pneumoniae (5.5% overall, 9.2% in CAP patients) and H. influenzae (5.4% overall, 14.2% in CAP patients). Less than 1% of S. pneumoniae were highly resistant to penicillin and 12.6% of H. influenzae were ß-lactamase positive. The most common viral pathogens detected were human rhinovirus (20.1%), influenza viruses (9.9%), and human coronavirus (7.4%). Influenza virus, human parainfluenza viruses and human respiratory syncytial virus as well as human rhinovirus, human coronavirus and human metapneumovirus were detected significantly more frequently in LRTI patients than in controls. CONCLUSIONS: A bacterial pathogen is identified in approximately one in five adult patients with LRTI in primary care, and a viral pathogen in just under half, with mixed infections in one in ten. Penicillin-resistant pneumococci and ß-lactamase-producing H. influenzae are uncommon. These new findings support a restrictive approach to antibiotic prescribing for LRTI and the use of first-line, narrow-spectrum agents in primary care.

Comparable incidence and severity of cytomegalovirus infections following T cell-depleted allogeneic stem cell transplantation preceded by reduced intensity or myeloablative conditioning.

Reports on infectious complications following reduced intensity conditioning (RIC) before allogeneic stem cell transplantation (allo-SCT) are equivocal. This prospective follow-up study compared the impact of cytomegalovirus (CMV) infections following RIC with fludarabine, ATG and busulphan or conventional myeloablative conditioning (MAC). Forty-eight RIC and 59 MAC patients were enrolled. The occurrence and severity of CMV infections within 100 days following allo-SCT were assessed, using plasma CMV DNA load kinetics. CMV DNAemia was observed in 21 RIC (60%) and in 19 MAC (44%) patients at risk for CMV. The mean CMV DNAemia free survival time was comparable following RIC and MAC: 70 days (95% (confidence interval) CI: 59-80 days) and 77 days (95% CI: 68-86 days), respectively (P=0.24). Parameters indicative for the level of CMV reactivation, including the area under the curve of CMV DNA load over time as well as the onset, the peak values and duration of CMV infection episodes, the numbers and duration of CMV treatment episodes and recurrent infections, were not different in both groups. During follow-up, none of the patients developed CMV disease. RIC with fludarabine, ATG and busulphan demonstrated safety comparable to conventional MAC with regard to frequency and severity of CMV infections within 100 days following T cell-depleted allo-SCT.

A multi-centre pilot proficiency programme to assess the quality of molecular detection of respiratory viruses.

OBJECTIVES: To assess the quality of molecular detection of respiratory viruses in clinical diagnostic laboratories. STUDY DESIGN: Respiratory virus proficiency panels were produced from diluted stocks of respiratory viruses provided and tested by four reference laboratories. The panels consisted of strong positive, positive, low positive and negative samples for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1 and 3, adenovirus serotypes 4 and 7, human rhinovirus serotypes 16, 72 and 90, human coronaviruses OC43 and 229E. The panels were sent to 17 participants; results and information on methodology was collected. RESULTS: All laboratories returned results, of which five submitted complete data sets. So, for analysis all results were combined. Samples were correctly identified by participants in 93.75%, 76.75% and 47.03% for the high positive, positive and low positive samples, respectively. One false positive was reported for all data sets (1.1%). The overall score for all assays using different methodologies was 78.8%. Laboratory performance was not dependant on methodology as all in-house methodologies could achieve optimal results, but dependant on careful optimisation and procedures specific to the laboratory. CONCLUSIONS: The first proficiency panel showed that in general all participants performed well. Although, it also highlights areas for improvement for all participants in order to generate robust results for use in clinical diagnostics.

Clinical relevance of quantitative varicella-zoster virus (VZV) DNA detection in plasma after stem cell transplantation.

Detection of Varicella-Zoster virus (VZV) DNA in plasma can facilitate the early recognition of complicated VZV-infection in immunocompromised hosts. The correlation of VZV-DNA in plasma with clinical presentations of VZV-infection and subsequent aciclovir treatment in allogeneic stem cell transplant (allo-SCT) recipients was studied. In 81 consecutive VZV-IgG positive allo-SCT recipients, VZV-DNA was measured at regular time points (1, 2 and 4 months) following allo-SCT and patient records were screened for VZV-related symptoms and aciclovir treatment. Subsequently, possible VZV-cases were studied in detail for the course of VZV-DNA and treatment effects. During the initial screening, VZV-DNA was detectable in seven patients. The survey of VZV-related symptoms revealed five additional possible VZV-cases. In cases where suitable plasma samples were available (10 out of 12), VZV-DNA was present almost simultaneously with the first clinical manifestations. No evidence of a preceding phase detectable by VZV-DNA only could be observed. Treatment with aciclovir was associated with a prompt reduction of VZV-DNA load. Detection of VZV-DNA in plasma in allo-SCT recipients accurately reflected the clinical presentation of VZV-infection and treatment with aciclovir. VZV-DNA detection in plasma of allo-SCT recipients appears clinically relevant as this may support early recognition and therapeutic management of VZV-infections following allo-SCT.

Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR.

A real-time PCR assay for Clostridium difficile was developed, based on the tcdB gene, which detected all known toxinogenic reference strains (n = 45), within 30 serogroups and 24 toxinotypes. The analytical sensitivity was 1 x 10(3) CFU/mL, and the detection limit in faeces was 1 x 10(5) CFU/g. The optimal protocol for DNA extraction from faecal samples involved use of the MagnaPure system with a Stool Transport and Recovery (STAR) buffer pre-treatment. In a 1-month prospective study of 85 patients with diarrhoea, the sensitivity, specificity and positive and negative predictive values of the assay were 100%, 94%, 55% and 100%, respectively, compared with the standard cell cytotoxicity assay.