This was a nationwide proficiency testing survey for health workers at 80 health centers across 40 districts and six regions, selected as described below, that use mRDTs to diagnose malaria cases representing a subset of all testers within Togo. These 80 health centers represent 9.6% of the total 835 health centers in Togo which use mRDTs and have been accredited by the National Malaria Control Program (NMCP). A cut off of 80 sites for this activity was necessary based on the total number of DTS vials available. The program was performed in Togo in October 2018.
Site selection and organization of PT activity
The PT activities were planned and implemented by the National Quality Assessment Program management (NQAPM) team of the Division of Laboratories of Togo Ministry of Health. Three field teams from the central level conducted the activity in the following health regions: Team 1: Lomé and Maritime health regions; Team 2: Plateaux health region and Team 3: Centrale, Kara and Savanes health regions.
Based on the information from the NMCP and the number of PT samples available for use, a subset, selected as described below, of the total number of health centers using mRDT to diagnose malaria were selected to be included in the malaria PT activity. As sites are unevenly distributed within Togo, facilities in locations with high capacities of microscopy diagnosis (e.g. Lomé) were eliminated to give priority to locations with lowerlevel facilities or without a dedicated laboratory. A second review of the list of sites was undertaken to identify those sites with higher throughput of patients and group them geographically to ensure representation of all six health regions of Togo while maintaining ease of access by the field teams. Finally, after applying these criteria, 80 of these sites were randomly selected as participant sites with all workers present on the day of the field team visit being evaluated. The number of facilities meeting these criteria and selected to participate in the PT program varied across 6 regions, with Centrale Region represented by 10 facilities, Kara Region by 13 facilities, Lomé Region by 7 facilities, Maritime Region by 17 facilities, Plateaux Region by 24 facilities, and Savanes Region by 9 facilities (Figure 1).
Dried tube specimen preparation
DTS were prepared in the USA as previously described. 11 Briefly, a culture-adapted P. falciparum strain 3D7 parasite line was grown in culture and harvested when parasitemia reached 2% (~100,000 parasites/µL). Parasite preparation was then diluted with washed (in incomplete medium; RPMI1640, Gibco™ Thermo Fisher, Grand Island, NY, USA) parasite negative blood at a similar hematocrit to generate preparations with 0, 100, 200, 500 or 1,000 parasites/µL. Parasite negative blood was procured from US resident donors with no reported overseas travel and confirmed by PCR to have no malaria infections. Blood was determined to be HIV and HBV negative. Blood was also confirmed in the CDC malaria laboratory to not produce a positive test on RDTs before being used to produce DTS. The diluted parasite preparations were then tested on a histidine-rich protein 2 (HRP2)-based mRDT brand with performance characteristics that met WHO procurement criteria. 13 After confirmation of reactivity, the diluted parasites were distributed in 50µL aliquots in Sarstedt® Type I microtubes (Sarstedt Inc., Newton, NC, USA) and, with the caps open, air-dried overnight in a biosafety cabinet.
To check that baseline reactivity was not affected by drying, 1 vial each of DTS at each of the parasite densities was re-hydrated with a solution of PBS-Tween20 (Sigma Aldrich, St. Louis, MO, USA) and tested on the same RDTs brands as prior to drying. All DTS vials were from the same batch and stored at +4oC before shipping via commercial air service at ambient temperatures to Togo and, similarly, were stored centrally in Lomé at +4oC prior to field activities. As previously described, short term storage (up to 4 weeks) at ambient temperatures does not impact the reactivity of the DTS11.
mRDT procurement and storage
A batch of SD Bioline® Malaria Ag Pf RDT (lot number 05CD094A, manufactured in Republic of Korea 09/25/2017, expiration date 09/24/2019) used in Togo was obtained from the NMCP for use in preliminary QC of DTS and in the PT. All procured kits were stored centrally until use. This allowed for the same lot number of kits to be used throughout the evaluation and provided an opportunity for baseline assessment prior to using the kits for the PT as well as eliminating the potential impact of variable storage conditions at testing sites. mRDTs and DTS were provided to each health center by the NQAPM team at the time of PT.
Quality control (QC) of Dried tube specimen at Malaria reference laboratory in Togo
Before distributing DTS vials to sites, the DTS panels were evaluated by the NQAPM in collaboration with the NMCP in the malaria reference lab in Lomé. Six aliquots of each parasitemia level (0, 100, 200, 500, and 1,000 parasites/µl), 30 vials in total, were reconstituted in 50µL of PBS-Tween rehydration buffer according to standard procedures provided by the malaria laboratory, CDC. Tubes were allowed to rehydrate for 1.5 hours, gently shaken and 5µL were then tested using SD Bioline® Malaria Ag Pf RDT (Standard Diagnostics, Inc., Abbott, Gyeonggi, Korea) in the same manner as patient blood samples would be tested.
Reconstitution of the specimens
To standardize reconstitution and remove a potential source of error, all DTS were reconstituted by the NQAPM team in the field according to the procedure provided by the CDC malaria laboratory and described in previous paragraph. Each reconstituted DTS contained 50µL of sample and thus provided sufficient sample to evaluate multiple health workers at a given site.
Distribution of samples and site visits
To evaluate the performance of the health workers using mRDTs, PT panels comprised of five reconstituted samples with different parasite densities (0, 100, 200, 500, 1,000 parasites/µl) were distributed to health centers and selected laboratories. One facility received only three samples containing 0, 100, and 1,000 parasites/µl due to an insufficient number of samples with parasite density of 200 and 500 parasites/μl. DTS vials were rehydrated onsite as described above. A total of 235 health workers participated in the PT activity, with three, two and one health workers testing samples in 76, three and one health facility, respectively. The number of participants at each site was a function of the number of staff on duty at the time of the supervisory visit. All personnel with a role in mRDT work and present on the day of the visit participated in the PT. The NQAPM team explained the PT procedure to the health workers prior to providing reconstituted DTS. To record the observations, each evaluated health worker received a results form designed by the NQAPM. Completed forms were collected by the field teams and returned to the NQAPM for analysis.
XLSTAT Basic (version 2019, Addinsoft, France), a statistical application for Microsoft Excel, was used for statistical analysis of data and Tableau Desktop (Version 2019.4, Tableau Software, Inc., USA) was used for data visualization. The difference between proportions was assessed using Chi-square test, followed by Marascuilo procedure to identify proportions responsible for statistically significant differences. Correlation between proportions of qualified testing personnel and regional performance was assessed using Pearson’s correlation coefficient. Paired Student’s t-test was used to compare the means of two paired samples. P-value of <0.05 was considered significant. For the assessment of individual tester performance, PT scores were calculated as percentage of samples identified by a tester correctly (that is, negative aliquot content was tested negative and positive aliquot content was tested positive). For the assessment of health center, district, or regional performance, correct results were aggregated as percentage of tests performed correctly at corresponding level from a total number of tests performed at corresponding level. For this analysis, a level of 80% correct PT result was selected as a value to mark acceptable proficiency. While 80% correct is used as a threshold for malaria microscopy testing and Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) 5-star certification, ideally testers would achieve 100% correct results on PT to offer appropriate patient care when conducting malaria testing.