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Comparison of Babesia microti Real-Time Polymerase Chain Reaction Assays for Confirmatory Diagnosis of Babesiosis.

ABSTRACT: Babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia Most human infections in the United States are caused by Babesia microti, but other infection-causing Babesia parasites have been documented as well. Polymerase chain reaction (PCR)-based methods can be used to identify this parasite to the species level. In this study, published real-time PCR assays for the specific detection of B. microti were evaluated against conventional PCR for their analytical performance. All evaluated real-time PCR assays had comparable dynamic range and amplification efficiency, but the sensitivity and specificity varied. The best performing test, a TaqMan assay targeting the 18S ribosomal RNA gene, was further evaluated for diagnostic performance using blood specimens submitted to the Centers for Disease Control and Prevention for parasite detection and was found to have 100% sensitivity and specificity. In conclusion, the 18S TaqMan real-time PCR assay is a sensitive, specific, and rapid method for identification of B. microti among cases of babesiosis in the United States.

SUBMITTER: Souza SS

PROVIDER: S-EPMC5154459 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Comparison of Babesia microti Real-Time Polymerase Chain Reaction Assays for Confirmatory Diagnosis of Babesiosis.

Souza Samaly S SS Bishop Henry S HS Sprinkle Patrick P Qvarnstrom Yvonne Y

The American journal of tropical medicine and hygiene 20161017 6

Babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia Most human infections in the United States are caused by Babesia microti, but other infection-causing Babesia parasites have been documented as well. Polymerase chain reaction (PCR)-based methods can be used to identify this parasite to the species level. In this study, published real-time PCR assays for the specific detection of B. microti were evaluated against conventional PCR for their analytic ...[more]

PMID: 27928088

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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:ContextReliable methods to screen newborns for congenital cytomegalovirus (CMV) infection are needed for identification of infants at increased risk of hearing loss. Since dried blood spots (DBS) are routinely collected for metabolic screening from all newborns in the United States, there has been interest in using DBS polymerase chain reaction (PCR)-based methods for newborn CMV screening.ObjectiveTo determine the diagnostic accuracy of DBS real-time PCR assays for newborn CMV screening.Design, setting, and participantsBetween March 2007 and May 2008, infants born at 7 US medical centers had saliva specimens tested by rapid culture for early antigen fluorescent foci. Results of saliva rapid culture were compared with a single-primer (March 2007-December 2007) and a 2-primer DBS real-time PCR (January 2008-May 2008). Infants whose specimens screened positive on rapid culture or PCR had congenital infection confirmed by the reference standard method with rapid culture testing on saliva or urine.Main outcome measuresSensitivity, specificity, and positive and negative likelihood ratios (LRs) of single-primer and 2-primer DBS real-time PCR assays for identifying infants with confirmed congenital CMV infection.ResultsCongenital CMV infection was confirmed in 92 of 20,448 (0.45%; 95% confidence interval [CI], 0.36%-0.55%) infants. Ninety-one of 92 infants had positive results on saliva rapid culture. Of the 11,422 infants screened using the single-primer DBS PCR, 17 of 60 (28%) infants had positive results with this assay, whereas, among the 9026 infants screened using the 2-primer DBS PCR, 11 of 32 (34%) screened positive. The single-primer DBS PCR identified congenital CMV infection with a sensitivity of 28.3% (95% CI, 17.4%-41.4%), specificity of 99.9% (95% CI, 99.9%-100%), positive LR of 803.7 (95% CI, 278.7-2317.9), and negative LR of 0.7 (95% CI, 0.6-0.8). The positive and negative predictive values of the single-primer DBS PCR were 80.9% (95% CI, 58.1%-94.5%) and 99.6% (95% CI, 99.5%-99.7%), respectively. The 2-primer DBS PCR assay identified infants with congenital CMV infection with a sensitivity of 34.4% (95% CI, 18.6%-53.2%), specificity of 99.9% (95% CI, 99.9%-100.0%), positive LR of 3088.9 (95% CI, 410.8-23 226.7), and negative LR of 0.7 (95% CI, 0.5-0.8). The positive and negative predictive values of the 2-primer DBS PCR were 91.7% (95% CI, 61.5%-99.8%) and 99.8% (95% CI, 99.6%-99.9%), respectively.ConclusionAmong newborns, CMV testing with DBS real-time PCR compared with saliva rapid culture had low sensitivity, limiting its value as a screening test.

Project description:BackgroundReal-time polymerase chain reaction (RT-PCR) testing for the identification of viral nucleic acid is the current standard for the diagnosis of SARS-CoV-2 infection, but technical issues limit its utilization for large-scale screening. Serological immunoglobulin M (IgM)/IgG testing has been proposed as a useful tool for detecting SARS-CoV-2 exposure.ObjectiveThe objective of our study was to compare the results of the rapid serological VivaDiag test for SARS-CoV-2-related IgM/IgG detection with those of the standard RT-PCR laboratory test for identifying SARS-CoV-2 nucleic acid.MethodsWe simultaneously performed both serological and molecular tests with a consecutive series of 191 symptomatic patients. The results provided by a new rapid serological colorimetric test for analyzing IgM/IgG expression were compared with those of RT-PCR testing for SARS-CoV-2 detection.ResultsOf the 191 subjects, 70 (36.6%) tested positive for SARS-CoV-2 based on RT-PCR results, while 34 (17.3%) tested positive based on serological IgM/IgG expression. Additionally, 13 (6.8%) subjects tested positive based on serological test results, but also tested negative based on RT-PCR results. The rapid serological test had a sensitivity of 30% and a specificity of 89% compared to the standard RT-PCR assay. Interestingly, the performance of both assays improved 8 days after symptom appearance. After 10 days had passed since symptom appearance, the predictive value of the rapid serological test was higher than that of the standard molecular assay (proportion of positive results: 40% vs 20%). Multivariate analysis showed that age >58 years (P<.01) and period of >15 days after symptom onset (P<.02) were significant and independent factors associated with serological test positivity.ConclusionsThe rapid serological test analyzed in this study seems limited in terms of usefulness when diagnosing SARS-CoV-2 infection. However, it may be useful for providing relevant information on people's immunoreaction to COVID-19 exposure.

Project description: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.

Dataset Information

Comparison of Babesia microti Real-Time Polymerase Chain Reaction Assays for Confirmatory Diagnosis of Babesiosis.

Publications

Comparison of Babesia microti Real-Time Polymerase Chain Reaction Assays for Confirmatory Diagnosis of Babesiosis.

Similar Datasets

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