Dataset Information

Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

ABSTRACT: The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus surveillance.

SUBMITTER: Dacheux L

PROVIDER: S-EPMC4933377 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

Dacheux Laurent L Larrous Florence F Lavenir Rachel R Lepelletier Anthony A Faouzi Abdellah A Troupin Cécile C Nourlil Jalal J Buchy Philippe P Bourhy Herve H

PLoS neglected tropical diseases 20160705 7

The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge ...[more]

PMID: 27380028

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Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Project description:BackgroundRapid and accurate diagnosis of paediatric tuberculosis (TB) is key to manage the disease and to control and prevent its transmission. Collection of quality sputum samples without invasion methods from paediatrics (age < 16 years) with presumptive pulmonary tuberculosis (PTB) remains a challenge. Thus, the aim of this meta-analysis was to assess the overall accuracy of a real-time polymerase chain reaction (RT-PCR)-based assay, for routine diagnosis of MTB in different samples from paediatrics with active pulmonary and extra-pulmonary tuberculosis using mycobacterial culture as the gold standard in clinical microbiology laboratories.MethodsWe conducted a systematic review and meta-analysis to examine the diagnostic test accuracy of RT-PCR based assay for the detection of MTB in paediatric clinical samples. A systematic literature search was performed for publications in any language. MEDLINE via PubMed, EMBASE, and Web of Science were among 9 bibliographic databases searched from August 2019 until November 2020. Bivariate random-effects model of meta-analysis were performed to generate pooled summary estimates (95% CIs) for overall accuracy of RT-PCR based assay compared to mycobacterial culture as the reference standard.ResultsOf the 1592 candidate studies, twenty-one eligible studies met our inclusion criteria. In total, the review and meta-analysis included 5536 (3209 PTB and 2327 EPTB). Summary estimates for pulmonary TB (11 studies) were as follows: sensitivity 56 (95% CI 51-62), specificity 97 (95% CI 96-98) and summary estimates for extra-pulmonary TB (10 studies) were as follows: sensitivity 87 (95% CI 82-91)) specificity 100 (95% CI 99-100). There was significant heterogeneity in sensitivity and specificity among the enrolled studies (p < 0.001).ConclusionsOur results suggested that the RT-PCR based assay could be a useful test for the diagnosis of paediatrics TB with high sensitivity and specificity in low-income/high-burden and upper medium income/low-burden settings. From the study, RT-PCR assay demonstrated a high degree of sensitivity for extra-pulmonary TB and good sensitivity for pulmonary TB which is an important factor in achieving effective global control and for patient management in terms of initiating early and appropriate anti-tubercular therapy.Systematic review registrationPROSPERO CRD42018104052.

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Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

Publications

Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

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