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Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis.

ABSTRACT: Specific identification of Entamoeba spp. in clinical specimens is an important confirmatory diagnostic step in the management of patients who may be infected with Entamoeba histolytica, the species that causes clinical amebiasis. Distinct real-time PCR protocols have recently been published for identification of E. histolytica and differentiation from the morphologically identical nonpathogenic Entamoeba dispar. In this study, we compared three E. histolytica real-time PCR techniques published by December 2004. The limits of detection and efficiency of each real-time PCR assay were determined using DNA extracted from stool samples spiked with serially diluted cultured E. histolytica trophozoites. The ability of each assay to correctly distinguish E. histolytica from E. dispar was evaluated with DNA extracted from patients' stools and liver aspirates submitted for confirmatory diagnosis. Real-time PCR allowed quantitative analysis of the spiked stool samples, but major differences in detection limits and assay performance were observed among the evaluated tests. These results illustrate the usefulness of comparative evaluations of diagnostic assays.

SUBMITTER: Qvarnstrom Y

PROVIDER: S-EPMC1287814 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis.

Qvarnstrom Yvonne Y James Cleve C Xayavong Maniphet M Holloway Brian P BP Visvesvara Govinda S GS Sriram Rama R da Silva Alexandre J AJ

Journal of clinical microbiology 20051101 11

Specific identification of Entamoeba spp. in clinical specimens is an important confirmatory diagnostic step in the management of patients who may be infected with Entamoeba histolytica, the species that causes clinical amebiasis. Distinct real-time PCR protocols have recently been published for identification of E. histolytica and differentiation from the morphologically identical nonpathogenic Entamoeba dispar. In this study, we compared three E. histolytica real-time PCR techniques published ...[more]

PMID: 16272475

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Project description:Orientia tsutsugamushi is the causative agent of scrub typhus. For the diagnosis of scrub typhus, we investigated the performances of conventional PCR (C-PCR), nested PCR (N-PCR), and real-time quantitative PCR (Q-PCR) targeting the O. tsutsugamushi-specific 47-kDa gene. To compare the detection sensitivities of the three techniques, we used two template systems that used plasmid DNA (plasmid detection sensitivity), including a partial region of the 47-kDa gene, and genomic DNA (genomic detection sensitivity) from a buffy coat sample of a single patient. The plasmid detection sensitivities of C-PCR, N-PCR, and Q-PCR were 5 × 10(4) copies/μl, 5 copies/μl, and 50 copies/μl, respectively. The results of C-PCR, N-PCR, and Q-PCR performed with undiluted genomic DNA were negative, positive, and positive, respectively. The genomic detection sensitivities of N-PCR and Q-PCR were 64-fold and 16-fold (crossing point [Cp], 37.7; 426 copies/μl), respectively. For relative quantification of O. tsutsugamushi bacteria per volume of whole blood, we performed real-time DNA PCR analysis of the human GAPDH gene, along with the O. tsutsugamushi 47-kDa gene. At a 16-fold dilution, the copy number and genomic equivalent (GE) of GAPDH were 1.1 × 10(5) copies/μl (Cp, 22.64) and 5.5 × 10(4) GEs/μl, respectively. Therefore, the relative concentration of O. tsutsugamushi at a 16-fold dilution was 0.0078 organism/one white blood cell (WBC) and 117 organisms/μl of whole blood, because the WBC count of the patient was 1.5 × 10(4) cells/μl of whole blood. The sensitivities of C-PCR, N-PCR, and Q-PCR performed with blood samples taken from patients within 4 weeks of onset of fever were 7.3% (95% confidence interval [CI], 1.6 to 19.9), 85.4% (95% CI, 70.8 to 94.4), and 82.9% (95% CI, 67.9 to 92.8), respectively. All evaluated assays were 100% specific for O. tsutsugamushi. In conclusion, given its combined sensitivity, specificity, and speed, Q-PCR is the preferred assay for the diagnosis of scrub typhus.

Dataset Information

Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis.

Publications

Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis.

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