Characterization of Shigella spp. by antimicrobial resistance and PCR detection of ipa genes in an infantile population from Porto Velho (Western Amazon region), Brazil

The incidence of Shigella spp. was assessed in 877 infants from the public hospital in Rondônia (Western Amazon region, Brazil) where Shigella represents the fourth cause of diarrhea. Twenty-five isolates were identified: 18 were Shigella flexneri, three Shigella sonnei, three Shigella boydii and one Shigella dysenteriae. With the exception of S. dysenteriae, all Shigella spp. isolated from children with diarrhea acquired multiple antibiotic resistances. PCR detection of ipa virulence genes and invasion assays of bloody diarrhea and fever (colitis) were compared among 25 patients testing positive for Shigella. The ipaH and ipaBCD genes were detected in almost all isolates and, unsurprisingly, all Shigella isolates associated with colitis were able to invade HeLa cells. This work alerts for multiple antibiotic resistant Shigella in the region and characterizes presence of ipa virulence genes and invasion phenotypesin dysenteric shigellosis.

Primary endemic Cryptococcosis gattii by molecular type VGII in the state of Pará, Brazil

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Pará, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50%), Cryptococcus gattii VGII (25/56, 44.64%) and C. gattii VGI (3/56, 5.26%). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95% 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Pará, Eastern Amazon.

Multiplex-PCR serotyping of Listeria monocytogenes isolated from human clinical specimens

The genus Listeria is composed of six species of which Listeria monocytogenes is considered the single pathogenic species that causes listeriosis in humans. Of the 13 serovars of L. monocytogenes, 1/2a, 1/2b and 4b are responsible for the majority of clinical cases. The aim of this work was to detect L. monocytogenes in the cerebrospinal fluid sample of premature newborns and to characterize this sample using biotyping, serotyping and molecular typing. The results indicated the presence of L. monocytogenesin the clinical sample studied. Moreover, the isolate was identified as the 4b serovar that was characterized by the presence of a unique 691 bp band after analysis using the Multiplex-PCR technique. The results of repeated Multiplex-PCR and sequencing have indicated that the L. monocytogenes isolate was an atypical 4b serovar, which is the first time this finding has been reported.

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.

Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies

Background. During the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR (Mycobacterial interspersed repetitive units- variable number of tandem repeats) (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format. Results. We evaluated the new MIRU-15 tool in two different samples. First, we analyzed the same convenience sample that had been used to evaluate MIRU-12 in a previous study, and the new 15-loci version offered higher discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.995 vs 0.978; 34.4% of clustered cases vs 57.5%) and better correlation (full or high correlation with RFLP for 82% of the clusters vs 47%). Second, we evaluated MIRU-15 on a population-based sample and, once again, good correlation with the RFLP clustering data was observed (for 83% of the RFLP clusters). To understand the meaning of the discrepancies still found between MIRU-15 and RFLP, we analyzed the epidemiological data for the clustered patients. In most cases, splitting of RFLP-clustered patients by MIRU-15 occurred for those without epidemiological links, and RFLP-clustered patients with epidemiological links were also clustered by MIRU-15, suggesting a good epidemiological background for clustering defined by MIRU-15. Conclusion. The data obtained by MIRU-15 suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. If we add this to the speed with which it provides results, MIRU-15 could be considered a suitable tool for real-time genotyping.

RFLP clusters of Mycobacterium tuberculosis strains from the Indian Ocean Region: local and South Asian characteristics

This is the first study describing the genetic polymorphism of Mycobacterium tuberculosis strains in the Indian Ocean Region. Using IS6110 RFLP analysis, 475 M. tuberculosis isolates from Madagascar, Comoros, Mauritius, Mozambique and La Reunion were compared. Of the 332 IS6110 profiles found, 43 were shared by clusters containing 2-65 strains. Six clusters were common to at least two countries. Of 52 families of strains with similar IS6110 profiles, 10 were common to at least two countries. Interestingly, another characteristic was the frequency (16.8%) of IS6110 single-copy strains. These strains could be distinguished using the DR marker. This preliminary evaluation suggests genetic similarity between the strains of the Indian Ocean Region. However, additional markers would be useful for epidemiological studies and to assess the ancient transmission of strains between countries of this region.

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.

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.

Prospective universal application of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping to characterize Mycobacterium tuberculosis isolates for fast identification of clustered and orphan cases.

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.