Cross-contamination during processing of dried blood spots used for rapid diagnosis of HIV-1 infection of infants is rare and avoidable

Abstract

Summary Dried blood spot (DBS) samples are a convenient way to collect infant blood for HIV-1 diagnostic testing. Minimizing the risk of false positives is critical for diagnostic tests. A protocol for processing and testing DBS for infant HIV-1 diagnosis was evaluated to identify the rate and source of false positive results. DBS were created on Flinders Technology Associates (FTA) filter paper with 500 copies/punch (high) or 5000 copies/punch (very high) concentrations of HIV-1 DNA. Blank discs of filter paper punched after DBS samples were tested for carry-over of HIV-1 DNA using nested PCR for the pol region. No false positives were detected in the 40 series using high concentration DBS. In series with very high concentrations of HIV-1, 8/246 (3%) reactions were falsely positive. When tubes were spun prior to opening, contact with caps minimized, and spaces left between lanes of the gel, repeat second round PCR of five false positives resulted in only one repeat false positive PCR. This study outlines procedures that minimize false positive results for nested PCR of HIV-1 DNA from DBS.

Performance evaluation of an in-house human immunodeficiency virus type-1 protease-reverse transcriptase genotyping assay in Cameroon

“…Moreover, despite the high risk of cross-contamination with nested PCR [44], results obtained in this study showed no event of this nature. Therefore, this finding proves that compliance with these laboratory criteria is a sufficient approach to ensure valid and reliable results in a standard molecular biology laboratory [45].…”

Prospective Evaluation of Diagnostic Accuracy of Dried Blood Spots from Finger Prick Samples for Determination of HIV-1 Load with the NucliSENS Easy-Q HIV-1 Version 2.0 Assay in Malawi

“…Another explanation could be that there was cross-contamination during testing; however, we think this is unlikely because steps to mitigate crosscontamination were taken during the manual excision of filter paper, such as the disinfection of each pair of scissors for every new specimen; in addition, all negative controls run during viral load testing were also negative. DBS-to-DBS carryover has been reported to be minimal in previous studies using either manual or automated cutting approaches (27,28). Perforated filter paper is now commercially available and eliminates the need for manual cutting with scissors and therefore greatly reduces the likelihood of cross-contamination.…”

Antiviral Resistance and Correlates of Virologic Failure in the first Cohort of HIV-Infected Children Gaining Access to Structured Antiretroviral Therapy in Lima, Peru: A Cross-Sectional Analysis

“…To save cost, alternative testing methodologies and transportation modalities were sought, such as the Oligonucleotide Ligation Assay (OLA) [19-21] and filter cards for the transportation of blood samples as dried spots [22-26]. …”