Prevalence and Characteristics of Plasmodium vivax Gametocytes in Duffy-Positive and Duffy-Negative Populations across Ethiopia.

Plasmodium parasites replicate asexually in human hosts. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear which proportion of Plasmodium vivax infections in Duffy-negative populations carries gametocytes. We determined the prevalence and characteristics of P. vivax gametocytes in Duffy-positive and -negative populations across broad regions of Ethiopia. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of Plasmodium species and Duffy blood group genotyping was done using SYBR green and the Taqman quantitative polymerase chain reaction method. Of the 447 febrile patients who were shown to be P. vivax smear positive, 414 (92.6%) were confirmed by molecular screening as P. vivax and 16 (3.9%) of them were from Duffy-negative individuals. Of these, 5 of 16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly greater than that in Duffy-negative samples. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negative individuals were commonly associated with low parasitemia, some of these infections were shown to have relatively high parasitemia and may represent a prominent erythrocyte invasion capability of P. vivax, and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of P. vivax malaria in Africa.

Genetic differentiation of Plasmodium vivax duffy binding protein in Ethiopia and comparison with other geographical isolates.

BACKGROUND: Plasmodium vivax Duffy binding protein (PvDBP) is a merozoite surface protein located in the micronemes of P. vivax. The invasion of human reticulocytes by P. vivax merozoites depends on the parasite DBP binding domain engaging Duffy Antigen Receptor for Chemokine (DARC) on these red blood cells (RBCs). PvDBPII shows high genetic diversity which is a major challenge to its use in the development of a vaccine against vivax malaria. METHODS: A cross-sectional study was conducted from February 2021 to September 2022 in five study sites across Ethiopia. A total of 58 blood samples confirmed positive for P. vivax by polymerase chain reaction (PCR) were included in the study to determine PvDBPII genetic diversity. PvDBPII were amplified using primers designed from reference sequence of P. vivax Sal I strain. Assembling of sequences was done using Geneious Prime version 2023.2.1. Alignment and phylogenetic tree constructions using MEGA version 10.1.1. Nucleotide diversity and haplotype diversity were analysed using DnaSP version 6.12.03, and haplotype network was generated with PopART version 1.7. RESULTS: The mean age of the participants was 25 years, 5 (8.6%) participants were Duffy negatives. From the 58 PvDBPII sequences, seven haplotypes based on nucleotide differences at 8 positions were identified. Nucleotide diversity and haplotype diversity were 0.00267 ± 0.00023 and 0.731 ± 0.036, respectively. Among the five study sites, the highest numbers of haplotypes were identified in Arbaminch with six different haplotypes while only two haplotypes were identified in Gambella. The phylogenetic tree based on PvDBPII revealed that parasites of different study sites shared similar genetic clusters with few exceptions. Globally, a total of 39 haplotypes were identified from 223 PvDBPII sequences representing different geographical isolates obtained from NCBI archive. The nucleotide and haplotype diversity were 0.00373 and 0.845 ± 0.015, respectively. The haplotype prevalence ranged from 0.45% to 27.3%. Two haplotypes were shared among isolates from all geographical areas of the globe. CONCLUSIONS: PvDBPII of the Ethiopian P. vivax isolates showed low nucleotide but high haplotype diversity, this pattern of genetic variability suggests that the population may have undergone a recent expansion. Among the Ethiopian P. vivax isolates, almost half of the sequences were identical to the Sal-I reference sequence. However, there were unique haplotypes observed in the Ethiopian isolates, which does not share with isolates from other geographical areas. There were two haplotypes that were common among populations across the globe. Categorizing population haplotype frequency can help to determine common haplotypes for designing an effective blood-stage vaccine which will have a significant role for the control and elimination of P. vivax.

Potential hidden Plasmodium vivax malaria reservoirs from low parasitemia Duffy-negative Ethiopians: Molecular evidence.

BACKGROUND: The interaction between the Plasmodium vivax Duffy-binding protein and the corresponding Duffy Antigen Receptor for Chemokines (DARC) is primarily responsible for the invasion of reticulocytes by P. vivax. The Duffy-negative host phenotype, highly prevalent in sub-Saharan Africa, is caused by a single point mutation in the GATA-1 transcription factor binding site of the DARC gene promoter. The aim of this study was to assess the Duffy status of patients with P. vivax infection from different study sites in Ethiopia. METHODS: A cross-sectional study was conducted from February 2021 to September 2022 at five varying eco-epidemiological malaria endemic sites in Ethiopia. Outpatients who were diagnosed with P. vivax infection (pure and mixed P. vivax/P. falciparum) by microscopy and Rapid Diagnostic Test (RDT) were subjected to PCR genotyping at the DARC promoter. The associations between P. vivax infection, host genotypes and other factors were evaluated. RESULT: In total, 361 patients with P. vivax infection were included in the study. Patients with pure P. vivax infections accounted for 89.8% (324/361), while the remaining 10.2% (37/361) had mixed P. vivax/P. falciparum infections. About 95.6% (345/361) of the participants were Duffy-positives (21.2% homozygous and 78.8%, heterozygous) and 4.4% (16/361) were Duffy-negatives. The mean asexual parasite density in homozygous and heterozygous Duffy-positives was 12,165 p/µl (IQR25-75: 1,640-24,234 p/µl) and11,655 p/µl (IQR25-75: 1,676-14,065 p/µl), respectively, significantly higher than that in Duffy-negatives (1,227p/µl; IQR25-75: 539-1,732p/µl). CONCLUSION: This study confirms that Duffy-negativity does not provide complete protection against P. vivax infection. The development of P. vivax-specific elimination strategies, including alternative antimalarial vaccines should be facilitated by a better understanding of the epidemiological landscape of vivax malaria in Africa. More importantly, low parasitemia associated with P. vivax infections in Duffy-negative patients may represent hidden reservoirs of transmission in Ethiopia.

Significant number of Plasmodium vivax mono-infections by PCR misidentified as mixed infections (P. vivax/P. falciparum) by microscopy and rapid diagnostic tests: malaria diagnostic challenges in Ethiopia.

BACKGROUND: Plasmodium vivax malaria is now recognized as a cause of severe morbidity and mortality, resulting in a substantial negative effect on health especially in endemic countries. Accurate and prompt diagnosis and treatment of P. vivax malaria is vital for the control and elimination of the disease. METHODS: A cross-sectional study was conducted from February 2021 to September 2022 at five malaria endemic sites in Ethiopia including Aribaminch, Shewarobit, Metehara, Gambella, and Dubti. A total of 365 samples that were diagnosed positive for P. vivax (mono and mixed infection) using RDT, site level microscopists and expert microscopists were selected for PCR. Statistical analyses were performed to calculate the proportions, agreement (k), frequencies, and ranges among different diagnostic methods. Fisher's exact tests and correlation test were used to detect associations and relationship between different variables. RESULTS: Of the 365 samples, 324 (88.8%), 37(10.1%), 2 (0.5%), and 2 (0.5%) were P. vivax (mono), P. vivax/Plasmodium falciparum (mixed), P. falciparum (mono) and negative by PCR, respectively. The overall agreement of rapid diagnostic test (RDT), site level microscopy and expert microscopists result with PCR was 90.41% (k: 0.49), 90.96% (k: 0.53), and 80.27% (k: 0.24). The overall prevalence of sexual (gametocyte) stage P. vivax in the study population was 215/361 (59.6%). The majority of these 215 samples (180; 83.7%) had below 1000 parasites/µl, with only four samples (1.9%) had ≥ 5000 parasites/µl. The gametocyte density was found to be weakly positive but statically significant with asexual parasitaemia (r = 0.31; p < 0.001). CONCLUSION: Both microscopy and RDT showed moderate agreement with PCR in the detection and identification of P. vivax (mono) and P. vivax/P. falciparum (mixed) infections. Therefore, to achieve malaria elimination goals, strengthening routine malaria diagnostic methods by implementing diagnostic tools with a good performance in detecting and accurately identifying malaria species in clinical settings is recommended.

Prevalence and characteristics of Plasmodium vivax Gametocytes in Duffy-positive and Duffy-negative populations across Ethiopia.

Plasmodium parasites replicate asexually in the human host. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear what proportion of P. vivax infections in Duffy-negatives carries gametocytes. This study aims to determine the prevalence of P. vivax in Duffy-negatives across broad regions of Ethiopia and characterize parasite stages. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of plasmodium species and Duffy blood group genotyping was done using SYBR green and Taqman qPCR method. Among the total 447 samples, 414 (92.6%) were P. vivax confirmed and, 16 (3.9%) of them were from Duffy-negatives. Of these, 5/16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly higher than that in Duffy-negatives. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negatives are commonly associated with low parasitemia, some of these infections were shown with relatively high parasitemia and may represent better erythrocyte invasion capability of P. vivax and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of vivax malaria in Africa.

Baseline malaria prevalence at the targeted pre-elimination districts in Ethiopia.

BACKGROUND: Encouraged by the previous success in malaria control and prevention strategies, the Ethiopian ministry of health launched malaria elimination with a stepwise approach by primarily targeting the low-transmission Districts and their adjacent areas/zones in order to shrink the country's malaria map progressively. Hence, this community survey was conducted to establish baseline malaria information at the preliminary phase of elimination at targeted settings. METHODS: A community-based cross-sectional survey was conducted at 20 malaria-elimination targeted Districts selected from five Regional states and one city administration in Ethiopia. The GPS-enabled smartphones programmed with Open Data Kit were used to enumerate 9326 study households and collect data from 29,993 residents. CareStart™ Malaria PAN (pLDH) Rapid Diagnostic Tests (RDTs) were used for blood testing at the field level. Armpit digital thermometers were used to measure axillary temperature. RESULT: Overall malaria prevalence by RDTs was 1.17% (339/28973). The prevalence at District levels ranged from 0.0 to 4.7%. The proportion of symptomatic cases (axillary temperature > 37.5oc) in the survey was 9.2% (2760/29993). Among the 2510 symptomatic individuals tested with RDTs, only 3.35% (84/2510) were malaria positive. The 75.2% (255/339) of all malaria positives were asymptomatic. Of the total asymptomatic malaria cases, 10.2% (26/255) were under-five children and 89.8% (229/255) were above 5 years of age. CONCLUSION: The study shows a decrease in malaria prevalence compared to the reports of previous malaria indicator surveys in the country. The finding can be used as a baseline for measuring the achievement of ongoing malaria elimination efforts. Particularly, the high prevalence of asymptomatic individuals (0.88%) in these transmission settings indicates there may be sustaining hidden transmission. Therefore, active case detection with more sensitive diagnostic techniques is suggested to know more real magnitude of residual malaria in the elimination-targeted areas.

Mentorship on malaria microscopy diagnostic service in Ethiopia: baseline competency of microscopists and performance of health facilities.

BACKGROUND: In Ethiopia, malaria cases are declining as a result of proven interventions, and in 2017 the country launched a malaria elimination strategy in targeted settings. Accurate malaria diagnosis and prompt treatment are the key components of the strategy to prevent morbidity and stop the continuation of transmission. However, the quality of microscopic diagnosis in general is deteriorating as malaria burden declines. This study was carried out to evaluate the competency of microscopists and the performance of health facilities on malaria microscopic diagnosis. METHODS: A cross-sectional study was conducted from 1 August to 30 September, 2019 in 9 regional states and one city administration. A standard checklist was used for on-site evaluation, archived patient slides were re-checked and proficiency of microscopists was tested using a WHO-certified set of slides from the national slide bank at the Ethiopian Public Health Institute (EPHI). The strength of agreement, sensitivity, specificity, and positive and negative predictive values were calculated. RESULTS: In this study, 102 health facilities (84 health centres and 18 hospitals) were included, from which 202 laboratory professionals participated. In slide re-checking, moderate agreement (agreement (A): 76.0%; Kappa (K): 0.41) was observed between experts and microscopists on malaria detection in all health facilities. The sensitivity and specificity of routine slide reading and the re-checking results were 78.1 and 80.7%, respectively. Likewise, positive predictive value of 65.1% and negative predictive value of 88.8% were scored in the routine diagnosis. By panel testing, a substantial overall agreement (A: 91.8%; K: 0.79) was observed between microscopists and experts in detecting malaria parasites. The sensitivity and specificity in the detection of malaria parasites was 92.7 and 89.1%, respectively. In identifying species, a slight agreement (A: 57%; K: 0.18) was observed between microscopists and experts. CONCLUSION: The study found significant false positive and false negative results in routine microscopy on slide re-checking of Plasmodium parasites. Moreover, reduced grade in parasite species identification was reported on the panel tests. Implementing comprehensive malaria microscopy mentorship, in-service training and supportive supervision are key strategies to improve the overall performance of health facilities in malaria microscopy.

Gene Polymorphisms Among Plasmodium vivax Geographical Isolates and the Potential as New Biomarkers for Gametocyte Detection.

The unique biological features of Plasmodium vivax not only make it difficult to control but also to eliminate. For the transmission of the malaria parasite from infected human to the vector, gametocytes play a major role. The transmission potential of a malarial infection is inferred based on microscopic detection of gametocytes and molecular screening of genes in the female gametocytes. Microscopy-based detection methods could grossly underestimate the reservoirs of infection as gametocytes may occur as submicroscopic or as micro- or macro-gametocytes. The identification of genes that are highly expressed and polymorphic in male and female gametocytes is critical for monitoring changes not only in their relative proportions but also the composition of gametocyte clones contributing to transmission over time. Recent transcriptomic study revealed two distinct clusters of highly correlated genes expressed in the P. vivax gametocytes, indicating that the male and female terminal gametocytogeneses are independently regulated. However, the detective power of these genes is unclear. In this study, we compared genetic variations of 15 and 11 genes expressed, respectively, in the female and male gametocytes among P. vivax isolates from Southeast Asia, Africa, and South America. Further, we constructed phylogenetic trees to determine the resolution power and clustering patterns of gametocyte clones. As expected, Pvs25 (PVP01_0616100) and Pvs16 (PVP01_0305600) expressed in the female gametocytes were highly conserved in all geographical isolates. In contrast, genes including 6-cysteine protein Pvs230 (PVP01_0415800) and upregulated in late gametocytes ULG8 (PVP01_1452800) expressed in the female gametocytes, as well as two CPW-WPC family proteins (PVP01_1215900 and PVP01_1320100) expressed in the male gametocytes indicated considerably high nucleotide and haplotype diversity among isolates. Parasite samples expressed in male and female gametocyte genes were observed in separate phylogenetic clusters and likely represented distinct gametocyte clones. Compared to Pvs25, Pvs230 (PVP01_0415800) and a CPW-WPC family protein (PVP01_0904300) showed higher expression in a subset of Ethiopian P. vivax samples. Thus, Pvs230, ULG8, and CPW-WPC family proteins including PVP01_0904300, PVP01_1215900, and PVP01_1320100 could potentially be used as novel biomarkers for detecting both sexes of P. vivax gametocytes in low-density infections and estimating transmission reservoirs.

Alternative Invasion Mechanisms and Host Immune Response to Plasmodium vivax Malaria: Trends and Future Directions.

Plasmodium vivax malaria is a neglected tropical disease, despite being more geographically widespread than any other form of malaria. The documentation of P. vivax infections in different parts of Africa where Duffy-negative individuals are predominant suggested that there are alternative pathways for P. vivax to invade human erythrocytes. Duffy-negative individuals may be just as fit as Duffy-positive individuals and are no longer resistant to P.vivax malaria. In this review, we describe the complexity of P. vivax malaria, characterize pathogenesis and candidate invasion genes of P. vivax, and host immune responses to P. vivax infections. We provide a comprehensive review on parasite ligands in several Plasmodium species that further justify candidate genes in P. vivax. We also summarize previous genomic and transcriptomic studies related to the identification of ligand and receptor proteins in P. vivax erythrocyte invasion. Finally, we identify topics that remain unclear and propose future studies that will greatly contribute to our knowledge of P. vivax.

Establishment of COVID-19 testing laboratory in resource-limited settings: challenges and prospects reported from Ethiopia.

The Coronavirus pandemic is recording unprecedented deaths worldwide. The temporal distribution and burden of the disease varies from setting to setting based on economic status, demography and geographic location. A rapid increase in the number of COVID-19 cases is being reported in Africa as of June 2020. Ethiopia reported the first COVID-19 case on 13 March 2020. Limited molecular laboratory capacity in resource constrained settings is a challenge in the diagnosis of the ever-increasing cases and the overall management of the disease. In this article, the Ethiopian Public Health Institute (EPHI) shares the experience, challenges and prospects in the rapid establishment of one of its COVID-19 testing laboratories from available resources. The first steps in establishing the COVID-19 molecular testing laboratory were i) identifying a suitable space ii) renovating it and iii) mobilizing materials including consumables, mainly from the Malaria and Neglected Tropical Diseases (NTDs) research team at the EPHI. A chain of experimental design was set up with distinct laboratories to standardize the extraction of samples, preparation of the master mix and detection. At the commencement of sample reception and testing, laboratory contamination was among the primary challenges faced. The source of the contamination was identified in the master mix room and resolved. In summary, the established COVID-19 testing lab has tested more than 40,000 samples (August 2020) and is the preferred setting for research and training. The lessons learned may benefit the further establishment of emergency testing laboratories for COVID-19 and/or other epidemic/pandemic diseases in resource-limited settings.

Comprehensive competency assessment of malaria microscopists and laboratory diagnostic service capacity in districts stratified for malaria elimination in Ethiopia​.

BACKGROUND: Federal Ministry of Health (FMoH) Ethiopia achieved significant declines in malaria mortality and incidence and has recently launched malaria elimination in selected low transmission settings. Successful malaria elimination calls for rapid and accurate diagnosis of cases so that the patients can promptly be treated before the occurrence of transmission. Therefore, this study assessed the competency of malaria microscopists using panal slides, and laboratory service availability and readiness in terms of supplies and equipments in malaria elimination targeted districts in Ethiopia. METHOD: A cross-sectional study was conducted from February to June 2018 in all hospitals, health centers and private clinics in 20 malaria elimination targeted districts, selected out of the 6 regional states in Ethiopia. All malaria microscopists available in the study health facilities during the study period were included in the study. Questionnaires were used for interviewing sociodemography of personnel and laboratory supplies. Per World Health Organization (WHO) criteria set for proficiency testing, 10 Giemsa stained malaria slide panels (8 positive low/high density pf/pv/Mixed and 2 negative slides) were administered to each study participant for performance assessment on malaria parasite detection, species identification and parasite count using light microscopy. The slide panels are PCR confirmed and WHO approved ones, which have been stored in the slide banks at the national reference laboratory in Ethiopian Public Health Institute. RESULT: In this assessment, 17(16%) district hospitals, 71(67%) health centers (HCs) and 18(17%) private clinics (PCs) were included. Of the 18 PCs, only 10(55.6%) had license certificate. Of the study facilities, 91.5%(97) use light microscopy, 2.83%(3) use RDTs and 2.9%(3) use both microscopy and RDT to detect malaria. Accessible and appropriate storage of Giemsa was reported by 58.8%(10) hospitals, 81.7%(58) HCs & 72.2%(13) private clinics. Of the 1896 malaria positive & 474 negative slides administered to 237 study participants, 318(16.8%) slides reported falsely negative & 47(9.9%) reported falsely positive. The participants achieved "good" grade [Agreement(A): 84.6%, Kappa(K): 0.6] on parasite detection and "poor" agreement (A: 43.8%; K: 0.11) on every species identification. No or slight agreement seen on differentiation of P. falciparum from other species (A: 28.41%; K:0.29). Above 95%(201) of participants, did not count or used plus system of parasite estimation which is the least accurate and unreccomended method per WHO guideline. CONCLUSION: In the current study, low performance of malaria microscopists particularly in species identification & poor to moderate capacity of laboratories observed. This is really a great obstacle to malaria elimination strategy of the country. Therefore, national malaria control and elimination program in collaboration with partners is supposed to provide comprehensive training for professionals giving laboratory service and to fulfill laboratory supplies to have the gold standard service.

Performance evaluation of tuberculosis smear microscopists working at rechecking laboratories in Ethiopia.

BACKGROUND: Tuberculosis is an infectious disease caused by the bacillus Mycobacterium tuberculosis. According to the Ethiopian Federal Ministry of Health's 2013-2014 report, the tuberculosis case detection rate was 53.7%, which was below the target of 81% set for that year. OBJECTIVE: This study assessed the performance of tuberculosis smear microscopists at external quality assessment rechecking laboratories in Ethiopia. METHODS: A cross-sectional study was conducted at 81 laboratories from April to July 2015. Panel slides were prepared and validated at the National Tuberculosis Reference Laboratory. The validated panel slides were used to evaluate the performance of microscopists at these laboratories compared with readers from the reference laboratory. RESULTS: A total of 389 external quality assessment rechecking laboratory microscopists participated in the study, of which 268 (68.9%) worked at hospitals, 241 (62%) had more than five years of work experience, 201 (51.7%) held Bachelors degrees, and 319 (82%) reported tuberculosis smear microscopy training. Overall, 324 (83.3%) participants scored ≥ 80%. Sensitivity for detecting tuberculosis bacilli was 84.5% and specificity was 93.1%. The overall percent agreement between participants and reference readers was 87.1 (kappa=0.72). All 10 slides were correctly read (i.e., scored 100%) by 80 (20.6%) participants, 156 (40.1%) scored 90% - 95%, 88 (22.6%) scored 80% - 85% and 65 (16.7%) scored below 80%. There were 806 (20.7%) total errors, with 143 (3.7%) major and 663 (17%) minor errors. CONCLUSION: The overall performance of participants in reading the slides showed good agreement with the reference readers. Most errors were minor, and the ability to detect tuberculosis bacilli can be improved through building the capacity of professionals.

Performance evaluation of malaria microscopists working at rechecking laboratories in Ethiopia.

BACKGROUND: Microscopic diagnosis of Giemsa-stained thick and thin blood films has remained the standard laboratory method for diagnosing malaria. High quality performance of microscopists that examine blood slides in health facilities remains critically important. MATERIALS AND METHODS: A cross-sectional study was conducted to assess the performance of 107 malaria microscopists working at 23 malaria rechecking laboratories in Ethiopia. A set of 12 blood film slides was distributed to each microscopist. Data was collected and exported to SPSS version 20 for analysis. Chi-square, sensitivity, specificity, percent agreement, and kappa scores were calculated to assess performance in detecting and identification of Plasmodium species. RESULTS: The mean age of the participants was 30 ± 5 yrs and most of them (54; 50.5%) were working at regional reference laboratories. Overall, the sensitivity of participants in detecting and identifying malaria parasite species was 96.8% and 56.7%, respectively. The overall agreement on detection and identification of malaria species was 96.8% (Kappa = 0.9) and 64.8% (Kappa = 0.33), respectively. The least accurately identified malaria parasite species was P. malariae (3/107; 2.8%) followed by P. ovale (35/107; 32.7%). Participants working at hospital laboratories had the highest percentage (72.3 %, Kappa=0.51) of accurate species identification. Study participants that had participated in malaria microscopy and quality assurance trainings were significantly better at quantifying parasite densities (P<0.001). CONCLUSION: The accuracy of parasite identification and quantification differed strongly between participants and expert microscopists. Therefore, regular competency assessment and training for malaria microscopists should be mandatory to assure proper diagnosis and management of malaria in Ethiopia.

Perceptions and attitudes toward SLMTA amongst laboratory and hospital professionals in Ethiopia.

BACKGROUND: Strengthening Laboratory Management Toward Accreditation (SLMTA) is a competency-based management training programme. Assessing health professionals' views of SLMTA provides feedback to inform program planning, implementation and evaluation of SLMTA's training, communication and mentorship components. OBJECTIVES: To assess laboratory professionals' and hospital chief executive officers' (CEOs) perceptions and attitudes toward the SLMTA programme in Ethiopia. METHODS: A cross-sectional descriptive survey was conducted in March 2013 using a structured questionnaire to collect qualitative data from 72 laboratory professionals and hospital CEOs from 17 health facilities, representing all regions and two city administrations in Ethiopia. Focus groups were conducted with laboratory professionals and hospital administration to gain insight into the strengths and challenges of the SLMTA programme so as to guide future planning and implementation. RESULTS: Ethiopian laboratory professionals at all levels had a supportive attitude toward the SLMTA programme. They believed that SLMTA substantially improved laboratory services and acted as a catalyst for total healthcare reform and improvement. They also noted that the SLMTA programme achieved marked progress in laboratory supply chain, sample referral, instrument maintenance and data management systems. In contrast, nearly half of the participating hospital CEOs, especially those associated with low-scoring laboratories, were sceptical about the SLMTA programme, believing that the benefits of SLMTA were outweighed by the level of human resources and time commitment required. They also voiced concerns about the cost and sustainability of SLMTA. CONCLUSION: This study highlights the need for stronger engagement and advocacy with hospital administration and the importance of addressing concerns about the cost and sustainability of the SLMTA programme.