Evaluation of PCR in the molecular diagnosis of endocarditis

Objective. Infective endocarditis (IE) is diagnosed by the Duke criteria, which can be inconclusive particularly when blood cultures are negative. This study investigated the application of polymerase chain reaction (PCR) to identify bacterial DNA in excised valvular tissue, and its role in establishing the diagnosis of IE. Methods. Ninety-eight patients undergoing valve replacement surgery were studied. Twenty-eight patients were confirmed as definite for endocarditis by the Duke criteria; nine were considered as possible and 61 had no known or previous microbial infection of the endocardium. A broad-range PCR technique was used to amplify prokaryotic 16S rRNA genes present within homogenised heart valve tissue. Subsequent DNA sequencing of the PCR amplicon allowed identification of the infecting microorganism. Results. PCR results demonstrated the presence of bacterial DNA in the heart valves obtained from 14 out of 20 (70%) definite IE patients with positive blood cultures preoperatively. The causative microorganism for one patient with definite culture negative endocarditis was identified by PCR. Two out of nine (22%) of the valves from possible endocarditis patients also had bacterial DNA present converting them into the definite criteria whereas in the valves of seven out of nine (78%) of these patients no bacterial DNA was detected. Conclusion. The application of PCR to the explanted valves in patients with possible or confirmed diagnosis can augment the Duke criteria thereby improving post-surgical antimicrobial therapeutic options. © 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits

Molecular methods are fundamental tools for the diagnosis of viral infections. While interpretation of results is straightforward for unvaccinated animals, where positivity represents ongoing or past infections, the presence of vaccine virus in the tissues of recently vaccinated animals may mislead diagnosis. In this study, we investigated the interference of RHDV2 vaccination in the results of a RT-qPCR for RHDV2 detection, and possible associations between mean Cq values of five animal groups differing in age, vaccination status and origin (domestic/wild). Viral sequences from vaccinated rabbits that died of RHDV2 infection (n = 14) were compared with the sequences from the commercial vaccines used in those animals. Group Cq means were compared through Independent t-test and One-way ANOVA. We proved that RHDV2 vaccine-RNA is not detected by the RT-qPCR as early as 15 days post- vaccination, an important fact in assisting results interpretation for diagnosis. Cq values of vaccinated and non-vaccinated infected domestic adults showed a statistically significant difference (p < 0.05), demonstrating that vaccination-induced immunity reduces viral loads and delays disease progression. Contrarily, in vaccinated young rabbits higher viral loads were registered compared to non-vaccinated kittens. No significant variation (p = 0.3824) was observed between viral loads of non- vaccinated domestic and wild RHDV2-victimised rabbits. Although the reduced number of vaccinated young animals analysed hampered a robust statistical analysis, this occurrence suggests that passively acquired maternal antibodies may inhibit the active immune response to vaccination, delaying protection and favouring disease progression. Our finding emphasises the importance of adapting kitten RHDV2 vaccination schedules to circumvent this interference phenomenon.

Diagnóstico molecular de tristeza parasitária bovina em rhipicephalus microplus da região tropical do méxico / molecular diagnosis of cattle tick fever agents in rhipicephalus microplus from tropical region of mexico

2016

Integrating structural variant calling, annotation and prioritization into whole genome analysis workflows: a practical application in the molecular diagnosis of neurodevelopmental disorders

Background: WGS is increasingly used as a first-line diagnostic test for patients with rare genetic diseases such as neurodevelopmental disorders (NDD). Clinical applications require a robust infrastructure to support processing, storage and analysis of WGS data. The identification and interpretation of SVs from WGS data also needs to be improved. Finally, there is a need for a prioritization system that enables downstream clinical analysis and facilitates data interpretation. Here, we present the results of a clinical application of WGS in a cohort of patients with NDD. Methods: We developed highly portable workflows for processing WGS data, including alignment, quality control, and variant calling of SNVs and SVs. A benchmark analysis of state-of-the-art SV detection tools was performed to select the most accurate combination for SV calling. A gene-based prioritization system was also implemented to support variant interpretation. Results: Using a benchmark analysis, we selected the most accurate combination of tools to improve SV detection from WGS data and build a dedicated pipeline. Our workflows were used to process WGS data from 77 NDD patient-parent families. The prioritization system supported downstream analysis and enabled molecular diagnosis in 32% of patients, 25% of which were SVs and suggested a potential diagnosis in 20% of patients, requiring further investigation to achieve diagnostic certainty. Conclusion: Our data suggest that the integration of SNVs and SVs is a main factor that increases diagnostic yield by WGS and show that the adoption of a dedicated pipeline improves the process of variant detection and interpretation.

Base molecular da hemocromatose hereditária não-clássica em Portugal

A Hemocromatose Hereditária (HH) é uma doença autossómica recessiva caracterizada pela absorção excessiva de ferro a nível intestinal e sua acumulação em órgãos vitais, podendo originar cardiomiopatia, cirrose e carcinoma hepatocelular. O correspondente diagnóstico molecular é obtido pela associação com genótipos específicos no gene HFE (homozigotia para p.Cys282Tyr ou heterozigotia composta p.Cys282Tyr/p.His63Asp). Contudo, nos países do sul da Europa, cerca de um terço dos doentes com diagnóstico clínico de HH não apresenta os referidos genótipos. Para identificar a base molecular da HH não-clássica em Portugal usaram-se metodologias de pesquisa geral de variantes genéticas (SSCP e dHPLC), Next-Generation Sequencing (NGS) e sequenciação de Sanger, cobrindo seis genes relacionados com o metabolismo do ferro em 303 doentes. Identificaram-se 69 variantes diferentes e de vários tipos, por ex. missense, nonsense, de splicing, que perturbam a transcrição do gene ou a regulação da tradução do mRNA. Seguidamente, realizaram-se estudos in silico e in vitro para esclarecer o significado etiológico de algumas das novas variantes. Concluiu-se que a base molecular desta patologia é bastante heterogénea e que a NGS é uma ferramenta adequada para efetuar a análise simultânea dos vários genes num grande número de amostras. Contudo, o estabelecimento da relevância clínica de algumas variantes requer a realização de estudos funcionais.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%).

Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection

The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens. However, the application to the detection of Schistosoma mansoni is rare, despite a recommendation of the World Health Organization that a major focus of research on schistosomiasis should be on the development and evaluation of new strategies and tools for control of the disease. In this context, a few studies were published for the detection of the parasite in snails, monitoring of cercariae in water bodies, and diagnosis of human infection. The present minireview describes sensitive and specific PCR based systems to detect S. mansoni, indicating possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection.

IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies.

The molecular diagnosis of retinal dystrophies is difficult because of the very important number of genes implicated and is rarely helped by genotype-phenotype correlations. This prompted us to develop IROme, a custom designed in solution-based targeted exon capture assay (SeqCap EZ Choice library, Roche NimbleGen) for 60 retinitis pigmentosa-linked genes and three candidate genes (942 exons). Pyrosequencing was performed on a Roche 454 GS Junior benchtop high-throughput sequencing platform. In total, 23 patients affected by retinitis pigmentosa were analyzed. Per patient, 39.6 Mb were generated, and 1111 sequence variants were detected on average, at a median coverage of 17-fold. After data filtering and sequence variant prioritization, disease-causing mutations were identified in ABCA4, CNGB1, GUCY2D, PROM1, PRPF8, PRPF31, PRPH2, RHO, RP2, and TULP1 for twelve patients (55%), ten mutations having never been reported previously. Potential mutations were identified in 5 additional patients, and in only 6 patients no molecular diagnosis could be established (26%). In conclusion, targeted exon capture and next-generation sequencing are a valuable and efficient approach to identify disease-causing sequence variants in retinal dystrophies.

IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies-A Price Comparison with Sanger Sequencing.

The molecular diagnosis of retinal dystrophies (RD) is difficult because of genetic and clinical heterogeneity. Previously, the molecular screening of genes was done one by one, sometimes in a scheme based on the frequency of sequence variants and the number of exons/length of the candidate genes. Payment for these procedures was complicated and the sequential billing of several genes created endless paperwork. We therefore evaluated the costs of generating and sequencing a hybridization-based DNA library enriched for the 64 most frequently mutated genes in RD, called IROme, and compared them to the costs of amplifying and sequencing these genes by the Sanger method. The production cost generated by the high-throughput (HT) sequencing of IROme was established at CHF 2,875.75 per case. Sanger sequencing of the same exons cost CHF 69,399.02. Turnaround time of the analysis was 3 days for IROme. For Sanger sequencing, it could only be estimated, as we never sequenced all 64 genes in one single patient. Sale cost for IROme calculated on the basis of the sale cost of one exon by Sanger sequencing is CHF 8,445.88, which corresponds to the sale price of 40 exons. In conclusion, IROme is cheaper and faster than Sanger sequencing and therefore represents a sound approach for the diagnosis of RD, both scientifically and economically. As a drop in the costs of HT sequencing is anticipated, target resequencing might become the new gold standard in the molecular diagnosis of RD.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (â^ž), NLR (0.017), and Ef (99%).

Development of new molecular methods for the diagnosis and the study of viral diseases of fish

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

Molecular and serological diagnosis of Bartonella infection in 61 dogs from the United States

Molecular diagnosis of canine bartonellosis can be extremely challenging and often requires the use of an enrichment culture approach followed by PCR amplification of bacterial DNA. HYPOTHESES: (1) The use of enrichment culture with PCR will increase molecular detection of bacteremia and will expand the diversity of Bartonella species detected. (2) Serological testing for Bartonella henselae and Bartonella vinsonii subsp. berkhoffii does not correlate with documentation of bacteremia. ANIMALS: Between 2003 and 2009, 924 samples from 663 dogs were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Diseases Diagnostic Laboratory for diagnostic testing with the Bartonella α-Proteobacteria growth medium (BAPGM) platform. Test results and medical records of those dogs were retrospectively reviewed. METHODS: PCR amplification of Bartonella sp. DNA after extraction from patient samples was compared with PCR after BAPGM enrichment culture. Indirect immunofluorescent antibody assays, used to detect B. henselae and B. vinsonii subsp. berkhoffii antibodies, were compared with PCR. RESULTS: Sixty-one of 663 dogs were culture positive or had Bartonella DNA detected by PCR, including B. henselae (30/61), B. vinsonii subsp. berkhoffii (17/61), Bartonella koehlerae (7/61), Bartonella volans-like (2/61), and Bartonella bovis (2/61). Coinfection with more than 1 Bartonella sp. was documented in 9/61 dogs. BAPGM culture was required for PCR detection in 32/61 cases. Only 7/19 and 4/10 infected dogs tested by IFA were B. henselae and B. vinsonii subsp. berkhoffii seroreactive, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs were most often infected with B. henselae or B. vinsonii subsp. berkhoffii based on PCR and enrichment culture, coinfection was documented, and various Bartonella species were identified. Most infected dogs did not have detectable Bartonella antibodies.

Diagnosis, Treatment, And Follow-up Of Medullary Thyroid Carcinoma: Recommendations By The Thyroid Department Of The Brazilian Society Of Endocrinology And Metabolism.

Medullary thyroid carcinoma (MTC) originates in the thyroid parafollicular cells and represents 3-4% of the malignant neoplasms that affect this gland. Approximately 25% of these cases are hereditary due to activating mutations in the REarranged during Transfection (RET) proto-oncogene. The course of MTC is indolent, and survival rates depend on the tumor stage at diagnosis. The present article describes clinical evidence-based guidelines for the diagnosis, treatment, and follow-up of MTC. The aim of the consensus described herein, which was elaborated by Brazilian experts and sponsored by the Thyroid Department of the Brazilian Society of Endocrinology and Metabolism, was to discuss the diagnosis, treatment, and follow-up of individuals with MTC in accordance with the latest evidence reported in the literature. After clinical questions were elaborated, the available literature was initially surveyed for evidence in the MedLine-PubMed database, followed by the Embase and Scientific Electronic Library Online/Latin American and Caribbean Health Science Literature (SciELO/Lilacs) databases. The strength of evidence was assessed according to the Oxford classification of evidence levels, which is based on study design, and the best evidence available for each question was selected. Eleven questions corresponded to MTC diagnosis, 8 corresponded to its surgical treatment, and 13 corresponded to follow-up, for a total of 32 recommendations. The present article discusses the clinical and molecular diagnosis, initial surgical treatment, and postoperative management of MTC, as well as the therapeutic options for metastatic disease. MTC should be suspected in individuals who present with thyroid nodules and family histories of MTC, associations with pheochromocytoma and hyperparathyroidism, and/or typical phenotypic characteristics such as ganglioneuromatosis and Marfanoid habitus. Fine-needle nodule aspiration, serum calcitonin measurements, and anatomical-pathological examinations are useful for diagnostic confirmation. Surgery represents the only curative therapeutic strategy. The therapeutic options for metastatic disease remain limited and are restricted to disease control. Judicious postoperative assessments that focus on the identification of residual or recurrent disease are of paramount importance when defining the follow-up and later therapeutic management strategies.

Molecular and morphological markers for rapid distinction between 2 Colletotrichum species

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia (espinheira-santa) in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense. Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Coll and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.