The DTS were prepared as described . Briefly, culture-grown P. falciparum parasites (strain 3D7) were diluted using haematocrit-matched parasite negative blood (0 parasites/μL) washed in RPMI1640 culture medium (Life Technologies, Grand Island, NY, USA) to concentrations of 500 or 1,000 parasites/μL. After testing for reactivity on replicate RDTs, 50 μL aliquots of each dilution (0, 500 and 1,000 parasites/μL) were transferred into open skirted base, conical-bottomed Sarstedt® Type I micro tubes (Sarstedt Inc, Newton, NC, USA) and air dried in a biosafety cabinet. To test for retention of reactivity after drying, one tube each of 0, 500 and 1,000 parasites/μL DTS was rehydrated in PBS-Tween solution as previously described  and tested on the same RDT brand as that used prior to drying. After verifying baseline RDT reactivity, the DTS were capped and stored in a 4°C refrigerator until transported to Addis Ababa, Ethiopia in November 2012.
Study setting (site selection)
The regional reference laboratory, Adama Regional Reference Laboratory (ARRL), about 110 km southeast of Addis Ababa within the Oromia Regional State, was selected as the reference laboratory due to the availability of controlled temperature environments to store DTS at the recommended 4°C. The criteria for choosing the two peripheral health centres were the routine use of RDTs and the feasibility of RDT storage at ambient temperature. To simplify study logistics, the two health centres were selected in order for both to be visited on the same day and within eight hours of DTS reconstitution at the reference laboratory. Accordingly, Wanji Health Centre, located 10 km and Walanchiti Health Centre, located 27 km from ARRL and within the Great Rift Valley in the central part of Ethiopia were selected as the health centres for this study.
The malaria RDT brand chosen for the study was the CareStart malaria pLDH/HRP2 combo test. These RDTs, with an expiry date of February 2015, were obtained from a recent shipment to the UNICEF central warehouse in Addis Ababa procured through the PMI and meant for distribution throughout Ethiopia. The CareStart malaria pLDH/HRP2 combo test contains a membrane strip pre-coated with two monoclonal antibodies, designed for the diagnosis of P. falciparum and/or other Plasmodium species, including Plasmodium vivax.
DTS accession and field monitoring
DTS were prepared at CDC, Atlanta in advance, with aliquots of 0 parasites (negative), 500 and 1,000 parasites/μL and shipped on ice packs to Ethiopia. Because DTS were initially tested at baseline in Atlanta with different RDT brands from those used in Ethiopia, RDT reactivity was confirmed on the CareStart malaria pLDH/HRP2 combo RDT brand immediately upon arrival in Ethiopia. Following confirmation of reactivity, DTS were stored at 4°C at ARRL and under site-specific conditions at the two health centres. The CareStart Combo RDTs from the same lot as mentioned above were also distributed to these three study sites. According to the product insert supplied by the manufacturer, the CareStart Combo RDT has sensitivity and specificity for P. falciparum of 98 and 97.5%, respectively. However, the parasite density of the samples used in defining these test characteristics and the lower limit of detection of the test were not provided.
At time week zero and at four-week intervals over a period of six months, RDTs stored at ARRL under manufacturer-recommended storage conditions and the same lot/batch of RDTs stored at room temperature in the two health centres were tested for performance using DTS stored at 4°C at the ARRL and at room temperature in the peripheral health centres. DTS were rehydrated with the same volume of PBS/Tween 20 solution as initial blood volume and tested on RDTs within eight hours of rehydration.
DTS field monitoring
Adama regional reference laboratory (ARRL) testing
On the day of testing, one aliquot each of DTS at 0, 500 and 1,000 parasites/μL stored at 4°C at the ARRL was rehydrated over one hour with two drops of PBS/Tween using a plastic Pasteur pipette (i.e., approximately 50 μL) and tested on duplicate RDTs stored under manufacturer-recommended storage conditions (≤30°C) at the reference laboratory. Field testing at the health centres was done on the same day. The ARRL site served as the gold standard for DTS and RDT optimal storage conditions and RDT test performance in Ethiopia while the two health centres represented peripheral facility level ambient conditions for comparison.
On the same day as ARRL testing, the rehydrated DTS and RDTs from ARRL were transported at ~4°C and manufacturer-recommended storage conditions (i.e., optimal conditions), respectively, to the first health centre. At the health centre, rehydrated DTS from ARRL and rehydrated replicate health centre-stored (room temperature) DTS were each tested on both RDTs brought from ARRL and health centre-stored RDTs. For the purposes of this evaluation, DTS were tested within eight hours of rehydration.
To avoid errors that could be attributed to the competence of health-worker performance and interpretation of RDT results, regional laboratory staff with experience in performing malaria RDTs were selected and provided with additional training. To rule out interpersonal variability, the same person was appointed for the whole study period. In each month the collected data were checked by the investigators for any transcriptional error resulting either while interpreting or summarizing the data from the facility reports.
Four DTS tubes stored at 4°C at ARRL were assembled into blinded panels each consisting of one 0 parasites/μL (negative control), two 500 parasites/μL and one 1,000 parasites/μL DTS tubes for PT. During health centre visits at weeks 12 and 24, health workers at the facility were asked to perform RDT tests using this panel on RDTs stored under manufacturer-suggested conditions brought from ARRL. Using a checklist and without intervention, ARRL staff observed and scored performance of nine critical steps in RDT testing and result interpretation by health centre staff. Errors in test performance by health centre staff were corrected by ARRL staff after the proficiency-testing was completed.
Data collection procedures and management
The testing site name, week and test results of the three study sites were collected on a standard form every four weeks and then transferred to a single form. Proficiency panel test results were recorded on a separate form. The results were subsequently transferred into an electronic database, Microsoft Excel.
The results obtained from ARRL were considered the gold standard by which to assess the DTS and RDT stored under field conditions in the two health centres. The concordance of ARRL results with the result from health centre-DTS tested on ARRL RDT was used to show the stability of health centre-stored DTS. The concordance of results of ARRL-stored DTS on the health centre-stored RDTs with ARRL-stored DTS on ARRL-stored RDTs determined the stability of health centre-stored RDTs.
The protocol for this study was reviewed and approved by the Human Subjects Coordinators for the Division of Parasitic Diseases and Malaria and the Center for Global Health, Centers for Disease Control and Prevention, Atlanta. A full Institutional Review Board assessment was not required because no human or animal subjects were involved in the study.