Detecção do herpes vírus felino tipo 1 (HVF-1) pela técnica de PCR em tempo real e sua associação com sinais oculares em uma poipulação de gatos domésticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

First detection of Leishmania spp. DNA in Brazilian bats captured strictly in urban areas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Validation of absolute quantitative real-time PCR for the diagnosis of streptococcus agalactiae in fish

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Seasonality of tuberculosis in India: is it real and what does it tell us?

India has a third of the world’s tuberculosis cases. Large-scale expansion of a national program in 1998 has allowed for population-based analyses of data from tuberculosis registries. We assessed seasonal trends using quarterly reports from districts with stable tuberculosis control programs (population 115 million). In northern India, tuberculosis diagnoses peaked between April and June, and reached a nadir between October and December, whereas no seasonality was reported in the south. Overall, rates of new smear-positive tuberculosis cases were 57 per 100 000 population in peak seasons versus 46 per 100 000 in trough seasons. General health-seeking behavior artifact was ruled out. Seasonality was highest in paediatric cases, suggesting variation in recent transmission.

A relaxed constant positive linear dependence constraint qualification and applications

In this work we introduce a relaxed version of the constant positive linear dependence constraint qualification (CPLD) that we call RCPLD. This development is inspired by a recent generalization of the constant rank constraint qualification by Minchenko and Stakhovski that was called RCRCQ. We show that RCPLD is enough to ensure the convergence of an augmented Lagrangian algorithm and that it asserts the validity of an error bound. We also provide proofs and counter-examples that show the relations of RCRCQ and RCPLD with other known constraint qualifications. In particular, RCPLD is strictly weaker than CPLD and RCRCQ, while still stronger than Abadie's constraint qualification. We also verify that the second order necessary optimality condition holds under RCRCQ.

Brazilian spotted fever: Real-time PCR for diagnosis of fatal cases

Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cutoff IgG and/or IgM >= 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (Citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n = 86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis. (C) 2012 Elsevier GmbH. All rights reserved.

Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses

Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K=0.812, 95% CI = 0.786-0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation. Published by Elsevier B.V.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Cryptosporidium parvum infection is very important with respect to public health, owing to foodborne and waterborne outbreaks and gastrointestinal illness in immunocompetent and immunocompromised persons. In cattle, infection with this species manifests either as a subclinical disease or with diarrheal illness, which occurs more often in the presence of other infectious agents than when alone. The aim of this study was to develop a real-time polymerase chain reaction (PCR) assay for the detection of C. parvum in calf fecal samples and to compare the results of this assay with those of the method routinely used for the diagnosis of Cryptosporidium spp., nested PCR targeting the 18S rRNA gene. Two hundred and nine fecal samples from calves ranging in age from 1 day to 6 months were examined using real-time PCR specific for the actin gene of C. parvum and by a nested PCR targeting the 18S rRNA gene of Cryptosporidium spp. Using real-time PCR detection, 73.2% (153 out of 209) of the samples were positive for C. parvum, while 56.5% (118 out of 209) of the samples were positive for Cryptosporidium spp. when the nested PCR amplification method was used for the detection. The analytical sensitivity of the real-time PCR was approximately one C. parvum oocyst. There was no significant nonspecific DNA amplification of any of the following species and genotype: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium galli, Cryptosporidium ryanae, Cryptosporidium serpentis, or avian genotype II. Thus, we conclude that real-time PCR targeting the actin gene is a sensitive and specific method for the detection of C. parvum in calf fecal samples.

Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT

1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA. Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G). A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed. Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values. Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR. Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous. 2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy. Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy. Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV. Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.

Rapid qualitative urinary tract infection pathogen identification by SeptiFast real-time PCR

Background Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical. Aim To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast®) and to compare the results with dipslide and microbiological culture. Design of study Pilot study with prospectively collected urine samples. Setting University hospital. Methods 82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast®) was performed in all samples. Results 61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results. Conclusion The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods.

Comparison of real-time PCR assays for detection, quantification, and differentiation of campylobacter jejuni and campylobacter coli in broiler neck skin samples

We tested the use of multiplex real-time PCR for detection and quantification of Campylobacter jejuni and Campylobacter coli on broiler carcass neck skin samples collected during 2008 from slaughterhouses in Switzerland. Results from an established TaqMan assay based on two different targets (hipO and ceuE for C. jejuni and C. coli, respectively) were corroborated with data from a newly developed assay based on a single-nucleotide polymorphism in the fusA gene, which allows differentiation between C. jejuni and C. coli. Both multiplex real-time PCRs were applied simultaneously for direct detection, differentiation, and quantification of Campylobacter from 351 neck skin samples and compared with culture methods. There was good correlation in detection and enumeration between real-time PCR results and quantitative culture, with real-time PCR being more sensitive. Overall, 251 (71.5%) of the samples were PCR positive for Campylobacter, with 211 (60.1%) in the hipO-ceuE assays, 244 (69.5%) in the fusA assay, and 204 (58.1%) of them being positive in both PCR assays. Thus, the fusA assay was similarly sensitive to the enrichment culture (72.4% positive); however, it is faster and allows for quantification. In addition, real-time PCR allowed for species differentiation; roughly 60% of positive samples contained C. jejuni, less than 10% C. coli, and more than 30% contained both species. Real-time PCR proved to be a suitable method for direct detection, quantification, and differentiation of Campylobacter from carcasses, and could permit time-efficient surveillance of these zoonotic agents.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

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.

Low prevalence of SV40 in Swiss mesothelioma patients after elimination of false-positive PCR results

The association of simian virus 40 (SV40) with malignant pleural mesothelioma is currently under debate. In some malignancies of viral aetiology, viral DNA can be detected in the patients' serum or plasma. To characterize the prevalence of SV40 in Swiss mesothelioma patients, we optimized a real-time PCR for quantitative detection of SV40 DNA in plasma, and used a monoclonal antibody for immunohistochemical detection of SV40 in mesothelioma tissue microarrays. Real-time PCR was linear over five orders of magnitude, and sensitive to a single gene copy. Repeat PCR determinations showed excellent reproducibility. However, SV40 status varied for independent DNA isolates of single samples. We noted that SV40 detection rates by PCR were drastically reduced by the implementation of strict room compartmentalization and decontamination procedures. Therefore, we systematically addressed common sources of contamination and found no cross-reactivity with DNA of other polyomaviruses. Contamination during PCR was rare and plasmid contamination was infrequent. SV40 DNA was reproducibly detected in only 4 of 78 (5.1%) plasma samples. SV40 DNA levels were low and not consistently observed in paired plasma and tumour samples from the same patient. Immunohistochemical analysis revealed a weak but reproducible SV40 staining in 16 of 341 (4.7%) mesotheliomas. Our data support the occurrence of non-reproducible SV40 PCR amplifications and underscore the importance of proper sample handling and analysis. SV40 DNA and protein were found at low prevalence (5%) in plasma and tumour tissue, respectively. This suggests that SV40 does not appear to play a major role in the development of mesothelioma.