Validation and diagnostic efficacy of a TaqMan real-time PCR for the detection of Mycoplasma conjunctivae in the eyes of infected Caprinae

Infectious keratoconjunctivitis (IKC), caused by Mycoplasma conjunctivae, is a highly contagious ocular disease in Caprinae. To detect rapidly and sensitively M. conjunctivae from individual conjunctival swabs of infected domestic and wild animals, a specific real-time PCR was developed using an lppS-directed hydrolysis probe in a TaqMan platform.

A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.

PCR and real-time PCR primers developed for detection and identification of Bifidobacterium thermophilum in faeces

BACKGROUND: Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora. RESULTS: Design of specific primers and probe was achieved based on comparison of 108 published bifidobacterial 16S rDNA sequences with the recently published sequence of the human faecal isolate B. thermophilum RBL67. Specificity of the primer was tested in silico by similarity search against the sequence database and confirmed experimentally by PCR amplification on 17 Bifidobacterium strains, representing 12 different species, and two Lactobacillus strains. The qPCR assay developed was linear for B. thermophilum RBL67 DNA quantities ranging from 0.02 ng/microl to 200 ng/microl and showed a detection limit of 10(5) cells per gram faeces. The application of this new qPCR assay allowed to detect the presence of B. thermophilum in one sample from a 6-month old breast-fed baby among 17 human faecal samples tested. Additionally, the specific qPCR primers in combination with selective plating experiments led to the isolation of F9K9, a faecal isolate from a 4-month old breast-fed baby. The 16S rDNA sequence of this isolate is 99.93% similar to that of B. thermophilum RBL67 and confirmed the applicability of the new qPCR assay in faecal samples. CONCLUSION: A new B. thermophilum-specific qPCR assay was developed based on species-specific target nucleotides in the 16S rDNA. It can be used to further characterize the composition of the bifidobacterial community in the human gastrointestinal tract. Until recently, B. thermophilum was considered as a species of animal origin, but here we confirm with the application of this new PCR assay the presence of B. thermophilum strains in the human gut.

Comparison of multiplex ligation-dependent probe amplification and real-time PCR accuracy for gene copy number quantification using the beta-defensin locus

The reliable quantification of gene copy number variations is a precondition for future investigations regarding their functional relevance. To date, there is no generally accepted gold standard method for copy number quantification, and methods in current use have given inconsistent results in selected cohorts. In this study, we compare two methods for copy number quantification. beta-defensin gene copy numbers were determined in parallel in 80 genomic DNA samples by real-time PCR and multiplex ligation-dependent probe amplification (MLPA). The pyrosequencing-based paralog ratio test (PPRT) was used as a standard of comparison in 79 out of 80 samples. Realtime PCR and MPLA results confirmed concordant DEFB4, DEFB103A, and DEFB104A copy numbers within samples. These two methods showed identical results in 32 out of 80 samples; 29 of these 32 samples comprised four or fewer copies. The coefficient of variation of MLPA is lower compared with PCR. In addition, the consistency between MLPA and PPRT is higher than either PCR/MLPA or PCR/PPRT consistency. In summary, these results suggest that MLPA is superior to real-time PCR in beta-defensin copy number quantification.

Assessment of a real-time PCR test to diagnose syphilis from diverse biological samples

OBJECTIVES: To investigate the contribution of a real-time PCR assay for the detection of Treponema pallidum in various biological specimens with the secondary objective of comparing its value according to HIV status. METHODS: Prospective cohort of incident syphilis cases from three Swiss hospitals (Geneva and Bern University Hospitals, Outpatient Clinic for Dermatology of Triemli, Zurich) diagnosed between January 2006 and September 2008. A case-control study was nested into the cohort. Biological specimens (blood, lesion swab or urine) were taken at diagnosis (as clinical information) and analysed by real-time PCR using the T pallidum 47 kDa gene. RESULTS: 126 specimens were collected from 74 patients with primary (n = 26), secondary (n = 40) and latent (n = 8) syphilis. Among primary syphilis, sensitivity was 80% in lesion swabs, 28% in whole blood, 55% in serum and 29% in urine, whereas among secondary syphilis, it was 20%, 36%, 47% and 44%, respectively. Among secondary syphilis, plasma and cerebrospinal fluid were also tested and provided a sensitivity of 100% and 50%, respectively. The global sensitivity of T pallidum by PCR (irrespective of the compartment tested) was 65% during primary, 53% during secondary and null during latent syphilis. No difference regarding serology or PCR results was observed among HIV-infected patients. Specificity was 100%. CONCLUSIONS: Syphilis PCR provides better sensitivity in lesion swabs from primary syphilis and displays only moderate sensitivity in blood from primary and secondary syphilis. HIV status did not modify the internal validity of PCR for the diagnosis of primary or secondary syphilis.

IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology

BACKGROUND: Control of brucellosis in livestock, wildlife and humans depends on the reliability of the methods used for detection and identification of bacteria. In the present study, we describe the evaluation of the recently established real-time PCR assay based on the Brucella-specific insertion sequence IS711 with blood samples from 199 wild boars (first group of animals) and tissue samples from 53 wild boars (second group of animals) collected in Switzerland. Results from IS711 real-time PCR were compared to those obtained by bacterial isolation, Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and indirect ELISA (i-ELISA). RESULTS: In the first group of animals, IS711 real-time PCR detected infection in 11.1% (16/144) of wild boars that were serologically negative. Serological tests showed different sensitivities [RBT 15.6%, c-ELISA 7.5% and i-ELISA 5.5%] and only 2% of blood samples were positive with all three tests, which makes interpretation of the serological results very difficult. Regarding the second group of animals, the IS711 real-time PCR detected infection in 26% of animals, while Brucella spp. could be isolated from tissues of only 9.4% of the animals. CONCLUSION: The results presented here indicate that IS711 real-time PCR assay is a specific and sensitive tool for detection of Brucella spp. infections in wild boars. For this reason, we propose the employment of IS711 real-time PCR as a complementary tool in brucellosis screening programs and for confirmation of diagnosis in doubtful cases.

[Field validation of a real-time PCR test for the detection of Mycoplasma hyopneumoniae in nasal swabs of live pigs]

Enzootic pneumonia (EP) of pigs, caused by Mycoplasma hyopneumoniae has been a notifiable disease in Switzerland since May 2003. The diagnosis of EP has been based on multiple methods, including clinical, bacteriological and epidemiological findings as well as pathological examination of lungs (mosaic diagnosis). With the recent development of a real-time PCR (rtPCR) assay with 2 target sequences a new detection method for M. hyopneumoniae became available. This assay was tested for its applicability to nasal swab material from live animals. Pigs from 74 herds (average 10 pigs per herd) were tested. Using the mosaic diagnosis, 22 herds were classified as EP positive and 52 as EP negative. From the 730 collected swab samples we were able to demonstrate that the rtPCR test was 100% specific. In cases of cough the sensitivity on herd level of the rtPCR is 100%. On single animal level and in herds without cough the sensitivity was lower. In such cases, only a positive result would be proof for an infection with M. hyopneumoniae. Our study shows that the rtPCR on nasal swabs from live pigs allows a fast and accurate diagnosis in cases of suspected EP.

Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

In order to improve the diagnosis of enzootic pneumonia (EP) in pigs two real-time polymerase chain reaction (rtPCR) assays for the detection of Mycoplasma hyopneumoniae in bronchial swabs from lung necropsies were established and validated in parallel. As a gold standard, the current "mosaic diagnosis" was taken, including epidemiological tracing, clinical signs, macro- and histopathological lesions of the lungs and immunofluorescence. One rtPCR is targeting a repeated DNA element of the M. hyopneumoniae genome (REP assay), the other a putative ABC transporter gene (ABC assay). Both assays were shown to be specific for M. hyopneumoniae and did not cross react with other bacteria and mollicutes from pig. With material from pigs of defined EP-negative farms the two assays showed to be 100% specific. When testing lungs from pig farms with EP, the REP assay detected 50% and the ABC assay 90% of the farms as positive. Both tests together detected all positive farms. Within a positive herd the two assays tested similarly with on average over 90% of the lung samples analysed from a single farm showing positive scores. A series of samples with suspicion of EP and samples from pigs with diseases other than respiratory taken from current routine diagnostic was assayed. None of the assays showed false positive results. The sensitivities in this sample group were 50% for the REP and 70% for the ABC assays and for both assays together 85%. The two assays run in parallel are therefore a valuable tool for the improvement of the current diagnosis of EP.

Simultaneous detection and discrimination of virulent and benign Dichelobacter nodosus in sheep of flocks affected by foot rot and in clinically healthy flocks by competitive real-time PCR.

Ovine foot rot caused by Dichelobacter nodosus is affecting sheep worldwide. The current diagnostic methods are difficult and cumbersome. Here, we present a competitive real-time PCR based on allelic discrimination of the protease genes aprV2 and aprB2. This method allows direct detection and differentiation of virulent and benign D. nodosus from interdigital skin swabs in a single test. Clinically affected sheep harbored high loads of only virulent strains, whereas healthy sheep had lower loads of predominantly benign strains.

Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

BACKGROUND Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. METHODS In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. RESULTS Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. CONCLUSIONS The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment.

A Multiplex Real-Time PCR with High Resolution Melting Analysis for the Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) gDNA copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity 100%, specificity 90%), cefixime (sensitivity 92%, specificity 94%), azithromycin and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens but this method can be used to screen collections of gonococcal isolates for AMR more quickly than with current culture-based AMR testing.

A new confirmatory Neisseria gonorrhoeae real-time PCR assay targeting the porA pseudogene

The Roche Cobas Amplicor system is widely used for the detection of Neisseria gonorrhoeae but is known to cross react with some commensal Neisseria spp. Therefore, a confirmatory test is required. The most common target for confirmatory tests is the cppB gene of N. gonorrhoeae. However, the cppB gene is also present in other Neisseria spp. and is absent in some N. gonorrhoeae isolates. As a result, laboratories targeting this gene run the risk of obtaining both false-positive and false-negative results. In the study presented here, a newly developed N. gonorrhoeae LightCycler assay (NGpapLC) targeting the N. gonorrhoeae porA pseudogene was tested. The NGpapLC assay was used to test 282 clinical samples, and the results were compared to those obtained using a testing algorithm combining the Cobas Amplicor System (Roche Diagnostics, Sydney, Australia) and an in-house LightCycler assay targeting the cppB gene (cppB-LC). In addition, the specificity of the NGpapLC assay was investigated by testing a broad panel of bacteria including isolates of several Neisseria spp. The NGpapLC assay proved to have comparable clinical sensitivity to the cppB-LC assay. In addition; testing of the bacterial panel showed the NGpapLC assay to be highly specific for N. gonorrhoeae DNA. The results of this study show the NGpapLC assay is a suitable alternative to the cppB-LC assay for confirmation of N. gonorrhoeae-positive results obtained with Cobas Amplicor.