Duplex-PCR assay for the detection of adenovirus and respiratory syncytial virus in nasopharyngeal samples

Human adenovirus (HAdV) and human respiratory syncytial virus (HRSV) are important etiologic agents of acute respiratory infections. In this study, a duplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of HAdV and HRSV in clinical samples. Sixty previously screened nasopharyngeal aspirates were used: 20 HAdV-positive, 20 HRSV-positive and 20 double-negative controls. Eight samples were positive for both viruses. The duplex PCR assay proved to be as sensitive and specific as single-target assays and also detected the mixed infections with certainty. The identification of both viruses in a single reaction offers a reduction in both cost and laboratory diagnostic time.

Association between pre-diagnostic circulating vitamin D concentration and risk of colorectal cancer in European populations:a nested case-control study

Objective. To examine the association between pre-diagnostic circulating vitamin D concentration, dietary intake of vitamin D and calcium, and the risk of colorectal cancer in European populations. Design Nested case-control study. Setting. The study was conducted within the EPIC study, a cohort of more than 520 000 participants from 10 western European countries. Participants: 1248 cases of incident colorectal cancer, which developed after enrolment into the cohort, were matched to 1248 controls. Main outcome measures. Circulating vitamin D concentration (25-hydroxy-vitamin-D, 25-(OH)D) was measured by enzyme immunoassay. Dietary and lifestyle data were obtained from questionnaires. Incidence rate ratios and 95% confidence intervals for the risk of colorectal cancer by 25-(OH)D concentration and levels of dietary calcium and vitamin D intake were estimated from multivariate conditional logistic regression models, with adjustment for potential dietary and other confounders. Results. 25-(OH)D concentration showed a strong inverse linear dose-response association with risk of colorectal cancer (P for trend <0.001). Compared with a pre-defined mid-level concentration of 25-(OH)D (50.0-75.0 nmol/l), lower levels were associated with higher colorectal cancer risk (<25.0 nmol/l: incidence rate ratio 1.32 (95% confidence interval 0.87 to 2.01); 25.0-49.9 nmol/l: 1.28 (1.05 to 1.56), and higher concentrations associated with lower risk (75.0-99.9 nmol/l: 0.88 (0.68 to 1.13); ≥100.0 nmol/l: 0.77 (0.56 to 1.06)). In analyses by quintile of 25-(OH)D concentration, patients in the highest quintile had a 40% lower risk of colorectal cancer than did those in the lowest quintile (P<0.001). Subgroup analyses showed a strong association for colon but not rectal cancer (P for heterogeneity=0.048). Greater dietary intake of calcium was associated with a lower colorectal cancer risk. Dietary vitamin D was not associated with disease risk. Findings did not vary by sex and were not altered by corrections for season or month of blood donation. Conclusions The results of this large observational study indicate a strong inverse association between levels of pre-diagnostic 25-(OH)D concentration and risk of colorectal cancer in western European populations. Further randomised trials are needed to assess whether increases in circulating 25-(OH)D concentration can effectively decrease the risk of colorectal cancer.

Genus-specific kinetoplast-DNA PCR and parasite culture for the diagnosis of localised cutaneous leishmaniasis: applications for clinical trials under field conditions in Brazil

The positivities of two methods for the diagnosis of localised cutaneous leishmaniasis (CL) were estimated in 280 patients enrolled in a clinical trial. The trial was conducted in an endemic area of Leishmania (Viannia) braziliensis and trial participants were patients with skin ulcers and positive leishmanin skin tests. Patients underwent aspirative skin punctures of the ulcerated lesions and lymph nodes for in vitro cultures, which were processed under field conditions at the local health centre. Skin lesion biopsies were tested at a reference laboratory using kinetoplastid DNA (kDNA)-PCR to detect DNA. The median time required to obtain a positive culture from the skin samples was seven days and the contamination rate of the samples was 1.8%. The positivities of the cultures from skin lesions, kDNA-PCR and the combination of the two methods were 78.2% (95% CI: 73-82.6%), 89.3% (95% CI: 85.1-92.4%) and 97.1% (95% CI: 94.5-98.5%). We conclude that parasite culture is a feasible method for the detection of Leishmania in field conditions and that the combination of culture and PCR has a potential role for the diagnosis of CL in candidates for clinical trials.

RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene as a tool to identify Enterococcus strains

Restriction fragment length polymorphism (RFLP) analysis of a PCR-amplified fragment of the 16S rRNA gene was performed on reference strains belonging to 21 different enterococcal species and on 75 Enterococcus isolates recovered from poultry meat, pasteurised milk and fresh cheese. PCR amplification generated a 275 bp fragment, which was digested with three restriction endonucleases (DdeI, HaeIII, HinfI). The strains were divided into five groups (groups A-E) on the basis of their restriction patterns. Five biochemical tests (arabinose, arginine, manitol, methyl-β-D-glucopyranoside and raffinose) were then performed in addition to RFLP analysis to narrow the identification of enterococcal strains to the species level. PCR-RFLP, in conjunction with the selected biochemical tests, allowed the precise identification of the 21 species of Enterococcus included in the present study. This proposed method is relatively simple and rapid and can be useful as an adjunct tool for accurate identification of Enterococcus.

Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples

A previously reported sensitive PCR assay for the detection of Schistosoma mansoni DNA was updated and evaluated. Changes in the DNA extraction method, including the use of a worldwide available commercial kit and the inclusion of additional quality control measures, increased the robustness of the test, as confirmed by the analysis of 67 faecal samples from an endemic area in Brazil. The PCR assay is at hand as a proven, reliable diagnostic test for the control of schistosomiasis in specific settings.

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

One major goal of research on Chagas disease is the development of effective chemotherapy to eliminate the infection from individuals who have not yet developed cardiac and/or digestive disease manifestations. Cure evaluation is the more complex aspect of its treatment, often leading to diverse and controversial results. The absence of reliable methods or a diagnostic gold standard to assess etiologic treatment efficacy still constitutes a major challenge. In an effort to develop more sensitive tools, polymerase chain reaction (PCR)-based assays were introduced to detect low amounts of Trypanosoma cruzi DNA in blood samples from chagasic patients, thus improving the diagnosis and follow-up evaluation after chemotherapy. In this article, I review the main problems concerning drug efficacy and criteria used for cure estimation in treated chagasic patients, and the work conducted by different groups on developing PCR methodologies to monitor treatment outcome of congenital infections as well as recent and late chronic T. cruzi infections.

Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats

More sensitive methodologies are necessary to improve strongyloidiasis diagnosis. This study compared the sensitivities of the McMaster modified technique and polymerase chain reaction (PCR) assays, both performed in faecal samples. Lewis rats were subcutaneously infected with 4,000, 400 or 40 infective third-stage larvae, considered as high, moderate or low infection, respectively. Seven days later, they were euthanized to count adult nematodes recovered from the small intestine. Stool samples were used to count the number of eggs per gram (EPG) of faeces and to detect parasite DNA by PCR performed with a species and a genus primer pair. The sensitivity of these assays depended upon parasite burden and the primer specificity. All assays presented 100% sensitivity at the highest parasite load. In the moderate infection, EPG and PCR with the genus primer maintained 100% specificity, whereas PCR sensitivity with the species primer decreased to 77.7%. In low infection, the sensitivity was 60% for EPG, 0% for PCR with the species primer and 90% for PCR done with the genus primer. Together, these results suggest that PCR with a genus primer can be a very sensitive methodology to detect Strongyloides venezuelensisin faeces of Lewis rats infected with very low parasite burden.

Comparison of HPV genotyping by type-specific PCR and sequencing

Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide and there is a strong link between certain high-risk viral types and cervical carcinogenesis. Although there are several typing methods, it is still unclear which test is the best. This study compared the effectiveness of type-specific PCR (TS-PCR) and sequencing, with a focus on their clinical application. A total of 260 cervical samples from HPV-positive patients were tested for types 6, 11, 16, 18, 31, 33 and 35 using TS-PCR and sequencing. The genotype was identified in 36% of cases by TS-PCR and in 75% by sequencing. Sequencing was four times more likely to identify the viral type in positive samples than TS-PCR (p = 0.00). Despite being more effective for virus genotyping, sequencing was unable to identify viral types in multiple infections. Combining both techniques resulted in highly sensitive detection (87% of cases), showing that they are complementary methods. HPV genotyping is an important step in HPV management, helping to identify patients with a higher risk of developing cervical cancer and contributing to the development of type-specific vaccines.

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.

Effective empirical treatment is of paramount importance to improve the outcome of patients with Staphylococcus aureus bacteraemia. We aimed to evaluate a PCR-based rapid diagnosis of methicillin resistance (GeneXpert MRSA) after early detection of S. aureus bacteraemia using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients with a first episode of S. aureus bacteraemia identified using MALDI-TOF MS were randomized in a prospective interventional open study between October 2010 and August 2012. In the control group, antibiotic susceptibility testing was performed after MALDI-TOF MS identification on blood culture pellets. In the intervention group, a GeneXpert MRSA was performed after S. aureus identification. The primary outcome was the performance of GeneXpert MRSA directly on blood cultures. We then assessed the impact of early diagnosis of methicillin resistance on the empirical treatment. In all, 197 episodes of S. aureus bacteraemia were included in the study, of which 106 were included in the intervention group. Median time from MALDI-TOF MS identification to GeneXpert MRSA result was 97 min (range 25-250). Detection of methicillin resistance using GeneXpert MRSA had a sensitivity of 99% and a specificity of 100%. There was less unnecessary coverage of MRSA in the intervention group (17.1% versus 29.2%, p 0.09). GeneXpert MRSA was highly reliable in diagnosing methicillin resistance when performed directly on positive blood cultures. This could help to avoid unnecessary prescriptions of anti-MRSA agents and promote the introduction of earlier adequate coverage in unsuspected cases.

Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement

The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies.

Helicobacter pylori transiently in the mouth may participate in the transmission of infection

Helicobacter pylori infection is associated with peptic ulcer and gastric carcinoma. The oral cavity may be a reservoir for H. pylori; however, the results of studies on this subject are controversial. We employed single-step and nested polymerase chain reactions (PCR) to detect the presence of the vacA, ureA and 16S rDNA genes of H. pylori in the stomach, saliva and dental plaque of 30 subjects. The results were confirmed by sequencing. Nested 16S rDNA and ureA amplification was achieved in 80% of gastric, 30% of saliva and 20% of dental plaque specimens. Sequencing of 10, seven and four 16S rDNA products from stomach, saliva and dental plaque, respectively, showed > 99% identity with H. pylori. Sequencing of the other four oral cavity PCR products showed similarity with Campylobacter and Wolinella species. Additionally, the vacA genotype identified in the samples of different sites was the same within a given subject.H. pylori may be found in the oral cavity of patients with gastric infection, thus it could be a source of transmission. However, results obtained with detection methods based only on PCR should be interpreted with caution because other microorganisms that are phylogenetically very close to H. pylori are also present in the mouth.

Human Exposure to Endocrine-Disrupting Chemicals and Prenatal Risk Factors for Cryptorchidism and Hypospadias: A Nested Case-Control Study

BACKGROUND. Exposure to xenoestrogens during pregnancy may disturb the development and function of male sexual organs. OBJECTIVE. In this study we aimed to determine whether the combined effect of environmental estrogens measured as total effective xenoestrogen burden (TEXB) is a risk factor for male urogenital malformations. METHODS. In a case-control study, nested in a mother-child cohort (n = 702) established at Granada University Hospital, we compared 50 newborns with diagnosis of cryptorchidism and/or hypospadias with 114 boys without malformations matched by gestational age, date of birth, and parity. Controls did not differ from the total cohort in confounding variables. TEXB and levels of 16 organochlorine pesticides were measured in placenta tissues. Characteristics of parents, pregnancy, and birth were gathered by questionnaire. We used conditional and unconditional regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS. TEXB from organohalogenated compounds was detectable in 72% and 54% of case and control placentas, respectively. Compared with controls, cases had an OR for detectable versus non-detectable TEXB of 2.82 (95% CI, 1.10-7.24). More pesticides were detected in cases than in controls (9.34 +/- 3.19 vs. 6.97 +/- 3.93). ORs for cases with detectable levels of pesticides, after adjusting for potential confounders in the conditional regression analysis, were o,p'-DDT (OR = 2.25; 95% CI, 1.03-4.89), p,p'-DDT (OR = 2.63; 95% CI, 1.21-5.72), lindane (OR = 3.38; 95% CI, 1.36-8.38), mirex (OR = 2.85; 95% CI, 1.22-6.66), and endosulfan alpha (OR = 2.19; 95% CI, 0.99-4.82). Engagement of mothers in agriculture (OR = 3.47; 95% CI, 1.33-9.03), fathers' occupational exposure to xenoestrogens (OR = 2.98; 95% CI, 1.11-8.01), and history of previous stillbirths (OR = 4.20; 95% CI, 1.11-16.66) were also associated with risk of malformations. CONCLUSIONS We found an increased risk for male urogenital malformations related to the combined effect of environmental estrogens in placenta.

Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay

In this study, a genotypification of Leishmaniawas performed using polimerase chain reaction-restriction fragment length polymorfism (PCR-RFLP) and sequencing techniques to identify species of Leishmaniaparasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpiswere grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantumby PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpisamong the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantumin the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

Alternative PCR protocol using a single primer set for assessing DNA quality in several tissues from a large variety of mammalian species living in areas endemic for leishmaniasis

The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100%, 55% and 22% of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.