Cholera rapid diagnostic tests recycled for PCR confirmation
Authors: Amanda K Debes, Wensheng Luo, Ethel Waswa, Waqo Boru, David A Sack
Journal: The Lancet Global Health (2021)

Detecting and confirming a diagnosis of cholera is challenging for remote areas where culture methods are not readily available. Rapid diagnostic tests are available in which a dipstick placed into a diluted stool sample provides results within 15 min;1Debes AK Murt KN Waswa E et al.Laboratory and field evaluation of the Crystal VC-O1 cholera rapid diagnostic test.Am J Trop Med Hyg. 2021; 104: 2017-2023Google Scholar, 2Mwaba J Ferreras E Chizema-Kawesa E et al.Evaluation of the SD bioline cholera rapid diagnostic test during the 2016 cholera outbreak in Lusaka, Zambia.Trop Med Int Health. 2018; 23: 834-840Google Scholar however, because of the performance of rapid diagnostic tests, particularly in terms of specificity, positive tests might need confirmation by culture or PCR. Faecal samples that are spotted on filter paper and dried can be used for PCR confirmation.1Debes AK Murt KN Waswa E et al.Laboratory and field evaluation of the Crystal VC-O1 cholera rapid diagnostic test.Am J Trop Med Hyg. 2021; 104: 2017-2023Google Scholar, 3Debes AK Ateudjieu J Guenou E et al.Clinical and environmental surveillance for Vibrio cholerae in resource constrained areas: application during a 1-year surveillance in the far north region of Cameroon.Am J Trop Med Hyg. 2016; 94: 537-543Google Scholar Such filter paper samples are not biohazardous, do not require refrigeration, and DNA is stable even after extended storage. Furthermore, such samples can be used for advanced molecular characterisation.4Bénard AHM Guenou E Fookes M et al.Whole genome sequence of Vibrio cholerae directly from dried spotted filter paper.PLoS Negl Trop Dis. 2019; 13e0007330Google Scholar However, filter paper is an additional laboratory supply that adds to budget and logistical considerations. We explored whether DNA from residual faecal matter on rapid diagnostic tests could be extracted for PCR confirmation. We reasoned that, after the rapid diagnostic test is read, the dipstick could be stored like filter paper until PCR confirmation and could replace the need for the filter paper sample. We conducted two independent studies to optimise this method. The first study included laboratory-based experiments. We grew a broth culture of Vibrio cholerae O1 overnight and prepared serial 10-fold dilutions of the culture in Luria broth (Fisher Scientific) with bacterial concentrations from 107 to 103 colony-forming units (CFU) per mL. We tested each dilution using the rapid diagnostic test Crystal VC O1 (Arkray, Gujarat, India) per manufacturer's instructions. Subsequently, we spotted two drops of each dilution onto filter paper (Whatman 903, Sigma-Aldrich, St Louis, MO, USA) and allowed the paper to dry. We extracted DNA from the filter paper and then assessed two separate portions of the dipsticks for optimal DNA extraction: the tip and the shaft above the tip (appendix p 1). We then analysed the extracted DNA by PCR as previously described.5Nandi B Nandy RK Mukhopadhyay S Nair GB Shimada T Ghose AC Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW.J Clin Microbiol. 2000; 38: 4145-4151Google Scholar A positive line on the rapid diagnostic test was visible with the 107 CFU per mL sample, but not with more dilute samples. Using PCR analysis, the filter-paper extract was positive with samples from 107 to 104 CFU per mL. The extract from the rapid diagnostic test tip was PCR positive with samples from 107 to 105 CFU per mL. The extract from the rapid diagnostic test shaft was PCR positive for samples from 107 to 106 CFU per mL. In our second study, we assessed 74 paired filter paper and rapid diagnostic test samples from individuals in Kenya, who were suspected to have cholera in 2019, findings from whom have been reported previously.1Debes AK Murt KN Waswa E et al.Laboratory and field evaluation of the Crystal VC-O1 cholera rapid diagnostic test.Am J Trop Med Hyg. 2021; 104: 2017-2023Google Scholar PCR from filter paper extracts was more sensitive than that from rapid diagnostic test extracts (table). Of the rapid diagnostic test extracts, PCR was more sensitive from tip extracts than shaft extracts, but showed high specificity on both parts of the dipstick tested.TablePCR performance on tip-extracted and shaft-extracted DNA from a rapid diagnostic test dipstick compared to filter paper-extracted DNAFilter paper PCR resultsTotalSensitivitySpecificityPositiveNegativeTip of dipstick PCR result......100% (54·07–100)100% (89·77–100)Positive606....Negative02626....Total62632....Shaft of dipstick PCR result......87·10% (70·17–96·37)100% (91·78–100)Positive27027....Negative44347....Total314374....Data in parentheses are 95% CIs. Open table in a new tab Data in parentheses are 95% CIs. We suggest extracting half of the tip to reserve samples for repeat testing (appendix p 1). Use of rapid diagnostic tests for cholera PCR confirmation would simplify the logistics and reduce the cost of sample collection. We thank members of the Kenyan Ministry of Health for the assistance in conducting this study. Patients suspected of having cholera gave informed consent for participation in the previous study. Funding for the study was provided by The Bill & Melinda Gates Foundation (OPP1148763), which provided financial support through the John Hopkins University Delivery of Oral Cholera Vaccine Effectively (DOVE) project, and from a grant from the US National institute of Allergy and Infectious Disease (5R01AI123422). The funding agencies had no role in collecting, analysing, or interpreting the results. The rapid diagnostic tests were provided by Arkray Healthcare, Gujarat, India. The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health. We declare no competing interests. Download .pdf (.12 MB) Help with pdf files Supplementary appendix