JCM Accepts, published online ahead of print on 24 December 2014 J. Clin. Microbiol. doi:10.1128/JCM.03218-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Stability of Chlamydia trachomatis on storage of dry swabs for accurate detection by nucleic
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acid amplification tests.
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L. Dize1
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C.A. Gaydos1
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T.C. Quinn1,3
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S.K. West2
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International Sexually Transmitted Diseases Research Laboratory,. Division of Infectious
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Diseases, Johns Hopkins University, Baltimore, MD, USA. 2
Dana Center for Preventive Ophthalmology, Wilmer Eye Institute, Johns Hopkins University,
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Baltimore, MD, USA. 3
Division of Intramural Research, National Institute for Allergy and Infectious Diseases,
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National Institutes of Health, Bethesda, MD, USA.
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Corresponding Author:
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Laura Dize
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855 North Wolfe St., 530 Rangos Building, Baltimore, MD 21205
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Ph: 410-614-0924
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FAX: 410-614-9775
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[email protected]
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Trachoma, caused by repeated ocular infections with Chlamydia trachomatis (CT) is the primary
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cause for infectious blindness throughout the world and predominately affects individuals in
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resource poor countries. (Harding-Esch 2009) Operational research on trachoma involves not
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only clinical trachoma grading methods, but also the collection of ocular swabs for the detection
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of CT by nucleic acid amplification tests (NAAT) assays. In our studies, after the ocular swab is
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collected in Tanzania, it is stored refrigerated at 2-8 degrees Celsius and shipped dry on cold
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packs within 30 days of collection to the International Sexually Transmitted Diseases Laboratory
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at Johns Hopkins University (JHU) in Baltimore, MD for CT detection. (Stare D 2011,). Upon
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arrival they are stored at -80 degrees C until processing, years of previous experiments using
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frozen ocular samples have indicated that ocular secretions do not interfere with detection of
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chlamydia DNA by NAAT assays. Due to potential delays in international shipping, it is
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important to know the length of time CT remains stable on dry ocular swabs at 4 degrees C. This
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study was conducted to determine those parameters by spiking dry ocular swabs with cultured
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CT at concentrations of 106 IFU/ml, 105 IFU/ml, 104 IFU/mL and 103 IFU/mL. The serial
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dilutions of the CT inoculum were made with DEPC water by putting 50uL of organism into
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450uL of DEPC water (Quality Biological, Gaithersburg, MD). Seven swabs were spiked with
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50uL of each concentration, placed in a refrigerator at 4 degrees C and analyzed by a NAAT
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assay (Genprobe Aptima Combo2 (AC2) assay, Hologic/Gen-Probe, San Diego, CA) for the
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detection of CT at baseline (day 0), 30 days, 40 days, 50 days, 60 days, 75 days, and 90 days.
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One hour prior to analysis, the dry swab was rehydrated with 1mL of DEPC water, vortexed for
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one minute and 200uL of the rehydrated sample was added to an APTIMA Specimen Transfer
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Tube for processing by the AC2 assay. All samples were tested according to the manufacturer’s
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protocol. We determined that all CT concentrations of 106 IFU/ml and 105 IFU/ml were
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detectable at each time point of the study, baseline through day 90. A concentration of 104
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IFU/mL was fully detectable from baseline through day 75; however, an equivocal result was
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obtained on day 90 of the study. The lowest concentration of 103 IFU/mL was only detected at
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baseline, day 30 and day 40, while it was not detected on the remaining days, 50, 60, 75, and 90
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(Table 1).
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Having to ship specimens frequently or ship on dry ice greatly increases the costs of research on
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trachoma, where there is no laboratory capacity in country. We demonstrated that CT remains
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detectable by NAATs on dry swabs at 4 degrees beyond 30 days, and that the detection of CT
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remains reliable even to day 40 using the lowest concentration.. NAAT tests can be helpful in
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the determination of infection in trachoma endemic areas. Our findings that all concentrations
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tested were detectable at day 40 ensure that the current protocol of storing ocular specimens at 4
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degrees C post collection and shipping dry on ice packs for testing within 30 days is sufficient,
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this is assuming that testing occurs immediately after samples are delivered to the laboratory
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without other storage conditions such as freezing.
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ACKNOWLEDGEMENTS
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This work was supported in part by the Division of Intramural Research, National Institute of
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Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH).
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References: 1.) Harding-Esch E, Edwards T, Sillah A, Sarr I, Roberts C, Snell P, Aryee E, Molina S,
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Holland M, Mabey D, and Bailey R. Active Trachoma and Ocular Chlamydia
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trachomatis Infection in Two Gambian Regions: On Course for Elimination by 2020.
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PLoS Neglected Tropical diseases. 2009; 3(12): e573.
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2.) Stare D, Harding-Esch E, Munoz B, Bailey R, Mabey D, Holland M, Gaydos C, and
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West S. Design and Baseline Data of a Randomized Trial to Evaluate Coverage and
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Frequency of Mass Treatment with Azithromycin: The partnership for Rapid Elimination
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of Trachoma (PRET) in Tanzania and The Gambia. Opthalmic Eidemiology. 2011;
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18(1): 20-29.
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Table 1. Stability study of Chlamydia trachomatis at refrigerator temperature (40 C) for dry
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swabs. Concentration Concentration Baseline 30 of CT culture of CT days (per 50 ul) 106 IFU/ml 50,000 + + IFU/swab 105 IFU/ml 5,000 + + 104 IFU/ml 500 + + 103 IFU/ml 50 + +
98 99 100 101 102 103
40 days
50 days
60 days
75 days
90 days
+
+
+
+
+
+ + +
+ + -
+ + -
+ + -
+ Equivocal -
