Examining 7 years of implementing quality management systems in medical laboratories in sub-Saharan Africa.

BACKGROUND: Achievement of ISO15189 accreditation demonstrates competency of a laboratory to conduct testing. Three programmes were developed to facilitate achievement of accreditation in low- and middle-income countries: Strengthening Laboratory Management Towards Accreditation (SLMTA), Stepwise Laboratory Improvement Process Towards Accreditation (SLIPTA) and Laboratory Quality Stepwise Implementation (LQSI). OBJECTIVE: To determine the level of accreditation and associated barriers and facilitators among medical laboratories in the WHO-AFRO region by 2020. METHODS: A desk review of SLIPTA and SLMTA databases was conducted to identify ISO15189-accredited medical laboratories between January 2013 and December 2020. Data on access to the LQSI tool were extracted from the WHO database. Facility and country characteristics were collected for analysis as possible enablers of accreditation. The chi-square test was used to analyse differences with level of significance set at <0.05. RESULTS: A total of 668 laboratories achieved accreditation by 2020 representing a 75% increase from the number in 2013. Accredited laboratories were mainly in South Africa (n = 396; 55%) and Kenya (n = 106; 16%), two countries with national accreditation bodies. About 16.9% (n = 113) of the accredited laboratories were registered for the SLIPTA programme and 26.6% (n = 178) for SLMTA. Approximately 58,217 LQSI users were registered by December 2020. Countries with a higher UHC index for access to HIV care and treatment, higher WHO JEE scores for laboratory networks, a larger number of registered LQSI users, with national laboratory policy/strategic plans and PEPFAR-priority countries were more likely to have an accredited laboratory. Of the 475 laboratories engaged in the SLIPTA programme, 154 attained ≥4 SLIPTA stars (ready to apply for accreditation) and 113 achieved ISO 15189 accreditation, with 96 enrolled into the SLMTA programme. Lower-tier laboratories were less likely to achieve accreditation than higher-tier laboratories (7.7% vs. 30%) (p < 0.001). The probability of achieving ISO 15189 accreditation (19%) was highest during the first 24 months after enrolment into the SLIPTA programme. CONCLUSION: To sustainably anchor quality improvement initiatives at facility level, national approaches including access to a national accreditation authority, adoption of national quality standards and regulatory frameworks are required.

Genomic surveillance of SARS-COV-2 reveals diverse circulating variant lineages in Nairobi and Kiambu Counties, Kenya.

Genomic surveillance and identification of COVID-19 outbreaks are important in understanding the genetic diversity, phylogeny, and lineages of SARS-CoV-2. Genomic surveillance provides insights into circulating infections, and the robustness and design of vaccines and other infection control approaches. We sequenced 57 SARS-CoV-2 isolates from a Kenyan clinical population, of which 55 passed quality checks using the Ultrafast Sample placement on the Existing tRee (UShER) workflow. Phylo-genome-temporal analyses across two regions in Kenya (Nairobi and Kiambu County) revealed that B.1.1.7 (Alpha; n = 32, 56.1%) and B.1 (n = 9, 15.8%) were the predominant lineages, exhibiting low Ct values (5-31) suggesting high infectivity, and variant mutations across the two regions. Lineages B.1.617.2, B.1.1, A.23.1, A.2.5.1, B.1.596, A, and B.1.405 were also detected across sampling sites within target populations. The lineages and genetic isolates were traced back to China (A), Costa Rica (A.2.5.1), Europe (B.1, B.1.1, A.23.1), the USA (B.1.405, B.1.596), South Africa (B.1.617.2), and the United Kingdom (B.1.1.7), indicating multiple introduction events. This study represents one of the genomic SARS-CoV-2 epidemiology studies in the Nairobi metropolitan area, and describes the importance of continued surveillance for pandemic control.

Potential application of the haematology analyser XN-31 prototype for field malaria surveillance in Kenya.

BACKGROUND: Simple and accurate diagnosis is a key component of malaria control programmes. Microscopy is the current gold standard, however it requires extensive training and the results largely rely on the skill of the microscopists. Malaria rapid diagnostic tests (RDT) can be performed with minimal training and offer timely diagnosis, but results are not quantitative. Moreover, some Plasmodium falciparum parasites have evolved and can no longer be detected by existing RDT. Developed by the Sysmex Corporation, the XN-31 prototype (XN-31p) is an automated haematology analyser capable of detecting Plasmodium-infected erythrocytes and providing species differentiation and stage specific parasite counts in venous blood samples without any preparation in approximately one minute. However, factors such as stable electricity supply in a temperature-controlled room, cost of the instrument and its initial set-up, and need for proprietary reagents limit the utility of the XN-31p across rural settings. To overcome some of these limitations, a hub and spoke diagnosis model was designed, in which peripheral health facilities were linked to a central hospital where detection of Plasmodium infections by the XN-31p would take place. To explore the feasibility of this concept, the applicability of capillary blood samples with the XN-31p was evaluated with respect to the effect of sample storage time and temperature on the stability of results. METHODS: Paired capillary and venous blood samples were collected from 169 malaria-suspected outpatients in Homa Bay County Referral Hospital, Kenya. Malaria infections were diagnosed with the XN-31p, microscopy, RDT, and PCR. Capillary blood samples were remeasured on the XN-31p after 24 h of storage at either room (15-25 °C) or chilled temperatures (2-8 °C). RESULTS: Identical results in malaria diagnosis were observed between venous and capillary blood samples processed immediately after collection with the XN-31p. Relative to PCR, the sensitivity and specificity of the XN-31p with capillary blood samples were 0.857 and 1.000, respectively. Short-term storage of capillary blood samples at chilled temperatures had no adverse impact on parasitaemia and complete blood counts (CBC) measured by the XN-31p. CONCLUSION: These results demonstrate the potential of the XN-31p to improve routine malaria diagnosis across remote settings using a hub and spoke model.

Presence of Plasmodium falciparum strains with artemisinin-resistant K13 mutation C469Y in Busia County, Western Kenya.

Malaria remains a key health and economic problem, particularly in sub-Saharan Africa. The emergence of artemisinin drug resistance (ART-R) parasite strains poses a serious threat to the control and elimination of this scourge. This is because artemisinin-based combination therapies (ACTs) remain the first-line treatment in the majority of malaria-endemic regions in Sub-Saharan Africa. Certain single-nucleotide polymorphisms in the propeller domains of Plasmodium falciparum Kelch 13 protein (K13) have been associated with delayed parasite clearance in vivo and in vitro. These mutations serve as vital molecular markers for tracking the emergence and dispersion of resistance. Recently, there have been increasing reports of the emergence and spread of P. falciparum ART-R parasites in the Eastern Africa region. This necessitates continued surveillance to best inform mitigation efforts. This study investigated the presence of all reported mutations of K13 propeller domains in the parasite population in Busia County, Kenya, a known malaria-endemic region. Two hundred twenty-six participants with microscopically confirmed uncomplicated malaria were recruited for this study. They were treated with artemether-lumefantrine under observation for the first dose, and microscopic examination was repeated 1 day later after ensuring the participants had taken the second and third doses. P. falciparum DNA from all samples underwent targeted amplification of the K13 gene using a semi-nested PCR approach, followed by Sanger sequencing. The recently validated ART-R K13 mutation C469Y was identified in three samples. These three samples were among 63 samples with a low reduction in parasitemia on day 1, suggesting day 1 parasitemia reduction rate is a useful parameter to enrich the ART-R parasites for further analysis. Our findings highlight the need for continuous surveillance of ART-R in western Kenya and the region to determine the spread of ART-R and inform containment.

Multi-repeat sequences identification using genome mining techniques for developing highly sensitive molecular diagnostic assay for the detection of Chlamydia trachomatis.

Chlamydia trachomatis ( C. trachomatis) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed C. trachomatis infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the C. trachomatis genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/µL to 1×10 -3pg/µL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, C. trachomatis IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting C. trachomatis DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.

Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae.

Background: Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. Methods: We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/µL to 1 × 10-3 pg/µL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. Results: Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/µL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/µL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/µL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/µL. Conclusion: Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.

Limited genetic variations of the Rh5-CyRPA-Ripr invasion complex in Plasmodium falciparum parasite population in selected malaria-endemic regions, Kenya.

The invasion of human erythrocytes by Plasmodium falciparum merozoites requires interaction between parasite ligands and host receptors. Interaction of PfRh5-CyRPA-Ripr protein complex with basigin, an erythrocyte surface receptor, via PfRh5 is essential for erythrocyte invasion. Antibodies raised against each antigen component of the complex have demonstrated erythrocyte invasion inhibition, making these proteins potential blood-stage vaccine candidates. Genetic polymorphisms present a significant challenge in developing efficacious vaccines, leading to variant-specific immune responses. This study investigated the genetic variations of the PfRh5 complex proteins in P. falciparum isolates from Lake Victoria islands, Western Kenya. Here, twenty-nine microscopically confirmed P. falciparum field samples collected from islands in Lake Victoria between July 2014 and July 2016 were genotyped by whole genome sequencing, and results compared to sequences mined from the GenBank database, from a study conducted in Kilifi, as well as other sequences from the MalariaGEN repository. We analyzed the frequency of polymorphisms in the PfRh5 protein complex proteins, PfRh5, PfCyRPA, PfRipr, and PfP113, and their location mapped on the 3D protein complex structure. We identified a total of 58 variants in the PfRh5 protein complex. PfRh5 protein was the most polymorphic with 30 SNPs, while PfCyRPA was relatively conserved with 3 SNPs. The minor allele frequency of the SNPs ranged between 1.9% and 21.2%. Ten high-frequency alleles (>5%) were observed in PfRh5 at codons 147, 148, 277, 410, and 429 and in PfRipr at codons 190, 255, 259, and 1003. A SNP was located in protein-protein interaction region C203Y and F292V of PfRh5 and PfCyRPA, respectively. Put together, this study revealed low polymorphisms in the PfRh5 invasion complex in the Lake Victoria parasite population. However, the two mutations identified on the protein interaction regions prompts for investigation on their impacts on parasite invasion process to support the consideration of PfRh5 components as potential malaria vaccine candidates.

Contexts for developing of national essential diagnostics lists. Lessons from a mixed-methods study of existing documents, stakeholders and decision making on tier-specific essential in-vitro diagnostics in African countries.

Since 2019, the WHO recommends the development and implementation of National Essential Diagnostics List (NEDL) to facilitate availability of In-Vitro Diagnostics (IVDs) across the various tiers of the healthcare pyramid, facilities with or without a laboratory on-site. To be effective, the development of NEDL should take into consideration the challenges and opportunities associated with current modalities for organization of tier specific testing services in-country. We conducted a mixed-methods analysis set out to explore available national policies, guidelines and decision-making processes that affect accessibility of diagnostics in African countries; 307 documents from 48 African countries were reviewed and 28 in-depth (group) interviews with 43 key-informants in seven countries were conducted between June and July 2022. Of the 48 countries, Nigeria was the only one with formal NEDL. Twenty-five countries had national test menus (63% outdated, from 2015 or earlier) all specifying tests by laboratory tier (5 including the "community tier"), with additional details on equipment (20), consumables (12), and personnel requirements (11). The most popular criteria to select essential IVDs in the quantitative analysis relate to specificities of the tests, whereas in the qualitative study most mentioned were health care and laboratory contextual factors. Quality assurance and waste management for tests at "community tier" were highlighted as concerns by all the respondents. Additional barriers to implementation included the low decision-making power of Laboratory Directorates within the Ministry of Health, as well as the chronic budgetary gaps for clinical laboratory services and policy and strategic plan development outside of vertical programmes. Four countries out of seven would rather revise their test menus by updating them and add ''community tier", than developing a separate NEDL, the former being considered more operational. This study provides a unique set of practical recommendations to the process of development and effective implementation on NEDL in Africa.