Search

Background: Prompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT)., Methods: This study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction., Results: The prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15. 4-23.1). Microscopy-based prevalence of P. falciparum infection was 3.7% (54/1,453) while the prevalence was 6.9% (100/1,453) using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10-11.9 g/dl)., Conclusions: Asymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy.

Background: Artemether–lumefantrine (AL) has been the primary anti-malarial drug used to treat uncomplicated Plasmodium falciparum malaria in Ethiopia since 2004. However, there have been recent reports of AL resistance mutations in different African countries, including Ethiopia. This is concerning and requires periodic monitoring of anti-malarial drug resistance. Therefore, the current study aimed to evaluate the therapeutic efficacy of AL in treating uncomplicated P. falciparum malaria in the Arba Minch Zuria District, Gamo Zone, Southwest Ethiopia. Methods: A single-arm prospective study with a 28-day follow-up period was conducted from July to October 2022. Capillary blood samples were collected for RDT and microscopic examination. The study enrolled monoinfected P. falciparum patients aged ≥ 18 years at Ganta Sira Health Post. Sociodemographic and clinical data were recorded, and a dried blood spot (DBS) was prepared for each participant. Nested polymerase chain reaction (nPCR) genotyping of the msp-1 and msp-2 genes was only performed for recurrent cases to distinguish between recurrence and reinfection. Data entry and analysis were performed using the WHO Excel spreadsheet and SPSS version 26. Results: A total of 89 patients were enrolled, and 67 adequately completed the 28-day follow-up period. AL showed a 100% clearance rate for fever on day 2 and asexual parasites on day 3. Gametocytes were detected in 13.5% (12/89) of the participants. The gametocyte clearance rate was 58.3% (7/12) until day 7 and 100% (12/12) until day 14. Five participants developed recurrent malaria, three of whom experienced relapse and two of whom experienced reinfection. Based on the Kaplan–Meier survival analysis, the PCR-uncorrected and PCR-corrected cumulative incidence of success were 93.7% (95% CI 85.5–97.3) and 96.2% (95% CI 85.5–98.7), respectively. Conclusion: AL was efficacious in treating uncomplicated P. falciparum malaria in the study area. However, the detection of recurrent patients highlights the need for continuous efficacy studies in this area. [ABSTRACT FROM AUTHOR]

Asymptomatic malaria can impact existing malaria control and elimination efforts around the world, particularly in Africa, where the majority of malaria cases and death occurs. This is a cross-sectional study aimed to determine the prevalence and predictors of asymptomatic malaria among migrant farmworkers from June to July 2020 in the Upper Awash Agro-industry, East Shewa zone, Oromia Regional State, Ethiopia. A total of 254 migrant farmworkers without signs and symptoms of malaria were enrolled. Data on socio-demographic characteristics and malaria prevention practices were obtained through a structured questionnaire. Venous blood samples were collected and diagnosed using microscopy, rapid diagnostic tests, and polymerase chain reaction (PCR). Data were coded, entered, and analyzed using SPSS version-21 statistical software. Multivariable logistic regression was used to assess associated factors. A p < 0.05 was considered statistically significant. The overall prevalence of asymptomatic malaria among farmworkers in this study was 5.1% [95% CI 1.6, 6.7]. The proportions of Plasmodium falciparum was 90.0% (9/10) while it was 10.0% (1/10) for Plasmodium vivax. Out of the microscopy and/or RDT-confirmed malaria cases, (n = 9; 100%) were confirmed to be P. falciparum by nested PCR, while (n = 3/122; 2.46%) were found to be P. falciparum among 50% negative cases with the microscopy and/or RDT. The gametocyte stage was detected in 40% of microscopically positive cases out of which 44.4% belongs to P. falciparum. Home area/origin of migrant laborers [AOR = 6.08, (95% CI 1.08, 34.66)], family history of malaria [AOR = 8.15, (95% CI 1.43, 46.44)], and outdoor sleeping [AOR = 10.14, (95% CI 1.15, 89.14)] were significantly associated with asymptomatic malaria. In conclusion, asymptomatic malaria was detected among farmworkers in the study area and it was significantly associated with outdoor sleeping, home area, and family history of malaria. Prevention tools and control strategies, particularly focusing on migrant farmworkers, should be considered to support the ongoing malaria control and elimination effort in Ethiopia. [ABSTRACT FROM AUTHOR]

Background. The genetic variation of Plasmodium falciparum has been studied to assess local malaria transmission genetic profile using evidence-based intervention measures. However, there are no known previous reports of P. falciparum polymorphism in Badewacho and Boset districts, Southern Ethiopia. The purpose of this study was to determine the genetic diversity of the merozoite surface protein-1 and -2 (msp-1 and msp-2) allelic families in P. falciparum isolates from an asymptomatic populations. Methods. This study was conducted from finger-prick blood samples spotted on 3 mm Whatman filter paper collected during a community-based cross-sectional study. Nested polymerase chain reaction amplification was used to type the allelic variants of msp-1 and msp-2. Results. From 669 asymptomatic study participants, a total of 50 samples positive for P. falciparum were included for molecular analysis. Of 50 positive samples, 43 P. falciparum isolates were successfully amplified for the msp-1 and msp-2 allelic families. A total of twelve different allele sizes (75–250 bp) were identified within the three allelic families of msp-1, whereas ten different allele sizes (250–500 bp) were detected within the two allelic families of msp-2. MAD20 had a higher allelic proportion, 65% among allelic families of msp-1, whereas the 3D7 allelic family 90.7% was higher in msp-2. A slightly higher frequency of polyclonal infection 53.5% was found in msp-2 allelic family, whereas a low proportion polyclonal infection 46.5% was found in msp-1 allelic family. The overall mean multiplicity of infection (MOI) for msp-1 and msp-2 was identical (MOI = 1.56). Correspondingly, the expected heterozygosity (He) value for msp-1 (He = 0.23) and msp-2 (He = 0.22) was almost similar. Conclusions. The findings of this study revealed low genetic diversity of the msp-1 and msp-2 allelic families in P. falciparum isolates. However, continued monitoring status of the local genetic diversity profile in the P. falciparum population is required to support current malaria control and elimination strategies. [ABSTRACT FROM AUTHOR]

Background: The increase in detections of Plasmodium vivax infection in Duffy-negative individuals in Africa has challenged the dogma establishing the unique P. vivax Duffy Binding Protein-Duffy antigen receptor for chemokines (PvDBP-DARC) pathway used by P. vivax merozoites to invade reticulocytes. Information on the impact of Duffy antigen polymorphisms on the epidemiology of P. vivax malaria remains elusive. The objective of this study was to determine the distribution of asexual parasitaemia of P. vivax according to the Duffy antigen polymorphisms in Ethiopia. Methods: DNA was extracted from dried blood spots (DBS) collected from prospectively recruited 138 P. vivax-infected patients from health centres. The identification and estimation of P. vivax asexual parasitaemia were performed by microscopic examination and quantitative real-time polymerase chain reaction (PCR). Duffy genotyping was conducted by DNA sequencing in a total of 138 P.vivax infected samples. Results: The proportion of Duffy-negatives (FY*BES/FY*BES) in P. vivax infected patients was 2.9% (4/138). Duffy genotype FY*B/FY*BES (48.6%) was the most common, followed by FY*A/FY*BES genotype (25.4%). In one patient, the FY*02 W.01/FY*02 N.01 genotype conferring a weak expression of the Fyb antigen was observed. All P.vivax infected Duffy-negative patients showed low asexual parasitaemia (≤ 110 parasites/µL). The median P. vivax parasitaemia in Duffy-negative patients (53 parasites/µL) was significantly lower than those found in homozygous and heterozygous individuals (P < 0.0001). Conclusion: Plasmodium vivax in Duffy-negative patients shows invariably low asexual parasitaemia. This finding suggests that the pathway used by P. vivax to invade Duffy-negative reticulocytes is much less efficient than that used in Duffy-positives. Moreover, the low asexual parasitaemia observed in Duffy-negative individuals could constitute an 'undetected silent reservoir', thus likely delaying the elimination of vivax malaria in Ethiopia. [ABSTRACT FROM AUTHOR]

Background. Despite significant progress achieved globally in reducing malaria burden, still it is one of the major public health and economic problems in Ethiopia. Investigation of the local genetic polymorphism of P. falciparum, the most virulent and predominant malaria parasite primarily targeted in malaria control and elimination program, is paramount to assess intensity of parasite transmission. Analysis of the block 3 region of the msp-2 gene of P. falciparum provides strong molecular evidence to evaluate the real picture of malaria epidemiology to fine-tune the ongoing control and elimination programs in the region. Thus, this study was aimed at examining the status of such polymorphic gene and its implications in Adama and its surroundings. Methods. 148 isolates from patients with uncomplicated falciparum malaria were collected in the study from September 2019 to August 2020. Tween® 20 and the Chelex method were employed for parasite DNA extraction. msp-2 allelic families were genotyped by using nested polymerase chain reaction targeting its 3D7 and FC27 allelic variants followed by gel electrophoresis for fragment analysis. Results. Seventeen different polymorphic forms of msp-2 allelic fragments were detected in the study area. Moreover, 47 (31.8%) and 41(27.7%) were detected for 3D7 and FC27 allelic families, respectively. Furthermore, the multiclonal allele type accounted for 60 (40.5%). The mean MOI was 1.4, and the heterogeneity index (He) is 0.49 indicating nearly intermediate malaria transmission in the study area. Conclusions. The study revealed nearly intermediate genetic diversity and mean MOI of P. falciparum in the study area, demanding further scale up of the ongoing control and elimination efforts. [ABSTRACT FROM AUTHOR]

Deletions in Plasmodium falciparum histidine rich protein 2(pfhrp2) gene threaten the usefulness of the most widely used HRP2-based malaria rapid diagnostic tests (mRDTs) that cross react with its structural homologue, PfHRP3. Parasites with deleted pfhrp2/3 genes remain undetected and untreated due to 'false-negative' RDT results. As Ethiopia recently launched malaria elimination by 2030 in certain selected areas, the availability of RDTs and the scale of their use have rapidly increased in recent years. Thus, it is important to explore the presence and prevalence of deletion in the target genes, pfhrp2 and pfhrp3. From a total of 189 febrile patients visited Adama Malaria Diagnostic centre, sixty-four microscopically-and polymerase chain reaction (PCR)-confirmed P. falciparum clinical isolates were used to determine the frequency of pfhrp2/3 gene deletions. Established PCR assays were applied to DNA extracted from blood spotted onto filter papers to amplify across pfhrp2/3 exons and flanking regions. However, analysis of deletions in pfhrp2, pfhrp3 and flanking genomic regions was successful for 50 of the samples. The pfhrp2 gene deletion was fixed in the population with all 50(100%) isolates presenting a deletion variant. This deletion extended downstream towards the Pf3D7 0831900 (MAL7PI.230) gene in 11/50 (22%) cases. In contrast, only 2/50 (4%) of samples had deletions for the Pf3D7 0831700 (MALPI.228) gene, upstream of pfhrp2. Similarly, the pfhrp3 gene was deleted in all isolates (100%), while 40% of the isolates had an extension of the deletion to the downstream flanking region that codes for Pf3D7 13272400 (MAL13PI.485).The pfhrp3 deletion also extended upstream to Pf3D7 081372100 (MAL13PI.475) region in 49/50 (95%) of the isolates, exhibiting complete absence of the locus. Although all samples showed deletions of pfhrp2 exon regions, amplification of an intron region was successful in five cases. Two different repeat motifs in the intron regions were observed in the samples tested. Pfhrp2/3 gene deletions are fixed in Ethiopia and this will likely reduce the effectiveness of PfHRP2-based mRDTs. It will be important to determine the sensitivity PfHRP 2/3-based RDTs in these populations and conduct a countrywide survey to determine the extent of these deletions and its effect on routine RDT-based malaria diagnosis. [ABSTRACT FROM AUTHOR]

Background As the global public-health objectives for malaria evolve from malaria control towards malaria elimination, there is increasing interest in the significance of asymptomatic infections and the optimal diagnostic test to identify them. Method We conducted a cross-sectional study of asymptomatic individuals (N = 562) to determine the epidemiological characteristics associated with asymptomatic malaria. Participants were tested by rapid diagnostic tests (CareStart, Standard Diagnostics [SD] Bioline, and Alere ultrasensitive RDT [uRDT]), loop-mediated isothermal amplification (LAMP), and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine malaria positivity. Hemoglobin values were recorded, and anemia was defined as a binary variable, according to World Health Organization guidelines. Results Compared to reference qRT-PCR, LAMP had the highest sensitivity (92.6%, 95% confidence interval [CI] 86.4–96.5), followed by uRDT Alere Malaria (33.9%, 95% CI 25.5–43.1), CareStart Malaria (14.1%, 95% CI 8.4–21.5), microscopy (5.0%, 95% CI 1.8–10.5), and SD Bioline (5.0%, 95% CI 1.8–10.5). For Plasmodium falciparum specimens only, the sensitivity for uRDT Alere Malaria was 50.0% (95% CI 38.8–61.3) and SD Bioline was 7.3% (95% CI 2.7–15.3). Based on multivariate regression analysis with qRT-PCR as the gold standard, for every 3.2% increase in the prevalence of asymptomatic malaria, hemoglobin decreased by 1 gram per deciliter (prevalence ratio 0.968, 95% CI 0.940–0.997; P =.032). Deletions (4.8%) in hrp2 were noted. Conclusions While uRDT Alere Malaria has superior sensitivity to rapid diagnostic tests and microscopy in detecting asymptomatic malaria, LAMP is superior still. Ultrasensitive diagnostics provide the accurate prevalence estimates of asymptomatic malaria required for elimination. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum resistance to anti-malarials is a major drawback in effective malaria control and elimination globally. Artemisinin-combination therapy (ACT) is currently the key first-line treatment for uncomplicated falciparum malaria. Plasmodium falciparum genetic signatures at pfmdr-1, pfcrt, and pfubp-1 loci are known to modulate in vivo and in vitro parasite response to ACT. The objective of this study was to assess the distribution of these resistance gene markers in isolates collected from different malaria transmission intensity in Ethiopia and Tanzania. Methods: Plasmodium falciparum clinical isolates were collected from different regions of Ethiopia and Tanzania. Genetic polymorphisms in the genes pfcrt, pfmdr-1 and pfubp-1 were analysed by PCR and sequencing. Frequencies of the different alleles in the three genes were compared within and between regions, and between the two countries. Results: The majority of the isolates from Ethiopia were mutant for the pfcrt 76 and wild-type for pfmdr-1 86. In contrast, the majority of the Tanzanian samples were wild-type for both pfcrt and pfmdr-1 loci. Analysis of a variable linker region in pfmdr-1 showed substantial variation in isolates from Tanzania as compared to Ethiopian isolates that had minimal variation. Direct sequencing of the pfubp-1 region showed that 92.8% (26/28) of the Ethiopian isolates had identical genome sequence with the wild type reference P. falciparum strain 3D7. Of 42 isolates from Tanzania, only 13 (30.9%) had identical genome sequences with 3D7. In the Tanzanian samples, 10 variant haplotypes were identified. Conclusion: The majority of Ethiopian isolates carried the main marker for chloroquine (CQ) resistance, while the majority of the samples from Tanzania carried markers for CQ susceptibility. Polymorphic genes showed substantially more variation in Tanzanian isolates. The low variability in the polymorphic region of pfmdr-1 in Ethiopia may be a consequence of low transmission intensity as compared to high transmission intensity and large variations in Tanzania. [ABSTRACT FROM AUTHOR]

Background: Prompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT). Methods: This study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction. Results: The prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15. 4-23.1). Microscopy-based prevalence of P. falciparum infection was 3.7% (54/1,453) while the prevalence was 6.9% (100/1,453) using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10-11.9 g/dl). Conclusions: Asymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy. [ABSTRACT FROM AUTHOR]