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Pooled-testing procedures for screening high volume clinical specimens in heterogeneous populations.

ABSTRACT: Pooled testing is a procedure commonly used to reduce the cost of screening a large number of individuals for infectious diseases. In its simplest form, pooled testing works by compositing a set of individual specimens (e.g., blood or urine) into a common pool. If the pool tests negative, all individuals within it are diagnosed as negative. If the pool tests positive, retesting is needed to decode the positive individuals from the negative individuals. Traditionally, pooled testing has assumed that each individual has the same probability of being positive. However, this assumption is often unrealistic, especially when known risk factors can be used to measure distinct probabilities of positivity for each individual. In this paper, we investigate new pooled-testing algorithms that exploit the heterogeneity among individual probabilities and subsequently reduce the total number of tests needed, while maintaining accuracy levels similar to standard algorithms that do not account for heterogeneity. We apply these algorithms to data from the Infertility Prevention Project, a nationally implemented program supported by the Centers for Disease Control and Prevention.

SUBMITTER: Bilder CR

PROVIDER: S-EPMC3500568 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Pooled-testing procedures for screening high volume clinical specimens in heterogeneous populations.

Bilder Christopher R CR Tebbs Joshua M JM

Statistics in medicine 20120313 27

Pooled testing is a procedure commonly used to reduce the cost of screening a large number of individuals for infectious diseases. In its simplest form, pooled testing works by compositing a set of individual specimens (e.g., blood or urine) into a common pool. If the pool tests negative, all individuals within it are diagnosed as negative. If the pool tests positive, retesting is needed to decode the positive individuals from the negative individuals. Traditionally, pooled testing has assumed t ...[more]

PMID: 22415972

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Project description:BackgroundReal-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens.Methodology/principal findingsThe specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing.Conclusion/significanceTaken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

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Pooled-testing procedures for screening high volume clinical specimens in heterogeneous populations.

Publications

Pooled-testing procedures for screening high volume clinical specimens in heterogeneous populations.

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