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 Malaria External Quality Assessment Rechecking Laboratories in Ethiopia

Background: The Performance of Malaria Microscopists in all health facilities have been raised concerns by many experts. Microscopic diagnosis of Giemsa stained thick and thin blood films by skilled microscopists has remained the standard laboratory method for the diagnosis of malaria. Microscopists who are working at Malaria Rechecking Laboratories have to be competent to cross check blood film slides which are collected from testing sites. Objective: The current study aims to assess the Performance of Malaria Microscopists and Malaria EQA Rechecking Laboratories in Ethiopia from February 1-May 10, 2015. Methods/design:A cross-sectional study design was conducted to assess the performance of 107 Malaria Microscopists who were working at 23 Malaria Rechecking Laboratories in Ethiopia. A set of 12 blood film slides containing Negative and positive (different species, stage, parasite density) results were distributed to each Malaria microscopists and 10 minutes were given for each blood film slides. Then all data were analyzed using SPSS Version 20 and agreement in detection and species identification of malaria parasites between participants and expert microscopists was estimated using the Kappa score. Chi Square was used for categorical data and P value (P<0.05) was considered significant. Result: From a total of 107 study participants, the maximum number of participants 90 (84.1%) were a male and most of them were working at regional reference laboratory 54(50.5%).About 34(31.8%) participants were used unrecommended quantification system. Overall, the sensitivity of participants in detection and species identification of malaria parasites were 96.8% and 56.7%, respectively. The overall agreement on detection and identification of malaria species was 96.8% (Kappa = 0.9) and 64.77% (kappa = 0.33), respectively. The least malaria species which were identified correctly by the participants were P. malerea and P.ovale which was identified correctly 2.8% and 32.7%, respectively. The number of participants who were scored <80% was higher among participants with diploma 25(100%) followed by participants with degree 59(93.6%) and participants with MSc and above 13(68.4%)(P=0.001). Participants at Hospital laboratory had higher percent agreement (72.3 %, Kappa=0.51) compared with participants from other health facilities. Conclusion and Recommendation: Agreement of the participants with expert microscopist in the identification of different malaria species and quantification were very low. Most participants were not identified P. malerea and P.ovale correctly. Therefore, to fill those gaps we have to have a policy for conducting regular competency assessment and training for malaria microscopists

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.</jats:p

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

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.</jats:p

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

BackgroundFederal 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.MethodA 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.ResultIn 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.ConclusionIn 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

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

International audienceBackgroundThe 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.MethodsA 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.ResultIn 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).ConclusionThis 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

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

Abstract 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

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.</jats:p