Children with Plasmodium vivax infection previously observed in Namibia, were Duffy negative and carried a c.136G > A mutation.

BACKGROUND: In a previous study, using a molecular approach, we reported the presence of P. vivax in Namibia. Here, we have extended our investigation to the Duffy antigen genetic profile of individuals of the same cohort with and without Plasmodium infections. METHODS: Participants with P. vivax (n = 3), P. falciparum (n = 23) mono-infections and co-infections of P. vivax/P. falciparum (n = 4), and P. falciparum/P. ovale (n = 3) were selected from seven regions. Participants with similar age but without any Plasmodium infections (n = 47) were also selected from all the regions. Duffy allelic profile was examined using standard PCR followed by sequencing of amplified products. Sequenced samples were also examined for the presence or absence of G125A mutation in codon 42, exon 2. RESULTS: All individuals tested carried the - 67 T > C mutation. However, while all P. vivax infected participants carried the c.G125A mutation, 7/28 P. falciparum infected participants and 9/41 of uninfected participants did not have the c.G125A mutation. The exon 2 region surrounding codon 42, had a c.136G > A mutation that was present in all P. vivax infections. The odds ratio for lack of this mutation with P. vivax infections was (OR 0.015, 95% CI 0.001-0.176; p = 0.001). CONCLUSION: We conclude that P. vivax infections previously reported in Namibia, occurred in Duffy negative participants, carrying the G125A mutation in codon 42. The role of the additional mutation c.136 G > A in exon 2 in P. vivax infections, will require further investigations.

Molecular detection of P. vivax and P. ovale foci of infection in asymptomatic and symptomatic children in Northern Namibia.

BACKGROUND: Knowledge of the foci of Plasmodium species infections is critical for a country with an elimination agenda. Namibia is targeting malaria elimination by 2020. To support decision making regarding targeted intervention, we examined for the first time, the foci of Plasmodium species infections and regional prevalence in northern Namibia, using nested and quantitative polymerase chain reaction (PCR) methods. METHODS: We used cross-sectional multi-staged sampling to select 952 children below 9 years old from schools and clinics in seven districts in northern Namibia, to assess the presence of Plasmodium species. RESULTS: The median participant age was 6 years (25-75%ile 4-8 y). Participants had a median hemoglobin of 12.0 g/dL (25-75%ile 11.1-12.7 g/dL), although 21% of the cohort was anemic, with anemia being severer in the younger population (p<0.002). Most of children with Plasmodium infection were asymptomatic (63.4%), presenting a challenge for elimination. The respective parasite prevalence for Plasmodium falciparum (Pf), Plasmodium vivax (Pv) and Plasmodium ovale curtisi (Po) were (4.41%, 0.84% and 0.31%); with Kavango East and West (10.4%, 6.19%) and Ohangwena (4.5%) having the most prevalence. Pv was localized in Ohangwena, Omusati and Oshana, while Po was found in Kavango. All children with Pv/Pf coinfections in Ohangwena, had previously visited Angola, affirming that perennial migrations are risks for importation of Plasmodium species. The mean hemoglobin was lower in those with Plasmodium infection compared to those without (0.96 g/dL less, 95%CI 0.40-1.52 g/dL less, p = 0.0009) indicating that quasi-endemicity exists in the low transmission setting. CONCLUSIONS: We conclude that Pv and Po species are present in northern Namibia. Additionally, the higher number of asymptomatic infections present challenges to the efforts at elimination for the country. Careful planning, coordination with neighboring Angola and execution of targeted active intervention, will be required for a successful elimination agenda.

Variation of Fungal Metabolites in Sorghum Malts Used to Prepare Namibian Traditional Fermented Beverages Omalodu and Otombo.

Sorghum malts, which are important ingredients in traditional fermented beverages, are commonly infected by mycotoxigenic fungi and mycotoxins may transfer into the beverages, risking consumers' health. Liquid chromatography⁻tandem mass spectrometry was used to determine variation of fungal metabolites in 81 sorghum malts processed for brewing of Namibian beverages, otombo (n = 45) and omalodu (n = 36). Co-occurrence of European Union (EU)-regulated mycotoxins, such as patulin, aflatoxins (B1, B2, and G2), and fumonisins (B1, B2, and B3) was detected in both malts with a prevalence range of 2⁻84%. Aflatoxin B1 was quantified in omalodu (44%) and otombo malts (14%), with 20% of omalodu malts and 40% of otombo malts having levels above the EU allowable limit. Fumonisin B1 was quantified in both omalodu (84%) and otombo (42%) malts. Emerging mycotoxins, aflatoxin precursors, and ergot alkaloids were quantified in both malts. Notably, 102 metabolites were quantified in both malts, with 96% in omalodu malts and 93% in otombo malts. An average of 48 metabolites were quantified in otombo malts while an average of 67 metabolites were quantified in omalodu malts. The study accentuates the need to monitor mycotoxins in sorghum malts intended for brewing and to determine their fate in the beverages.

Evaluation of loop-mediated isothermal amplification as a surveillance tool for malaria in reactive case detection moving towards elimination.

BACKGROUND: As malaria transmission decreases, the proportion of infections that are asymptomatic at any given time increases. This poses a challenge for diagnosis as routinely used rapid diagnostic tests (RDTs) miss asymptomatic malaria cases with low parasite densities due to poor sensitivity. Yet, asymptomatic infections can contribute to onward transmission of malaria and therefore act as infectious reservoirs and perpetuate malaria transmission. This study compared the performance of RDTs to loop-mediated isothermal amplification (LAMP) in the diagnosis of malaria during reactive active case detection surveillance. METHODS: All reported malaria cases in the Engela Health District of Namibia were traced back to their place of residence and persons living within the four closest neighbouring houses to the index case (neighbourhood) were tested for malaria infection with RDTs and dried blood spots (DBS) were collected. LAMP and nested PCR (nPCR) were carried out on all RDTs and DBS. The same procedure was followed in randomly selected control neighbourhoods. RESULTS: Some 3151 individuals were tested by RDT, LAMP and nPCR. Sensitivity of RDTs and LAMP were 9.30 and 95.50%, respectively, and specificities were 99.27 and 99.92%, respectively, compared to nPCR. LAMP carried out on collected RDTs showed a sensitivity and specificity of 95.35 and 99.85% compared to nPCR carried out on DBS. There were 2 RDT samples that were negative by LAMP but the corresponding DBS samples were positive by PCR. CONCLUSION: The study showed that LAMP had the equivalent performance as nPCR for the identification of Plasmodium falciparum infection. Given its relative simplicity to implement over more complex and time-consuming methods, such as PCR, LAMP is particularly useful in elimination settings where high sensitivity and ease of operation are important.

Characterization of Acanthosicyos horridus and Citrullus lanatus seed oils: two melon seed oils from Namibia used in food and cosmetics applications.

The physicochemical characteristics, fatty acid, tocopherol, stigmasterol, ß-sitosterol, and 1H NMR profiles of Citrullus lanatus and Acanthosicyos horridus melon seed oils were determined and compared among different extraction methods (cold pressing, traditional, and Soxhlet). The oil content was 40.2 ± 3.45 and 37.8 ± 7.26% for C. lanatus and A. horridus, respectively. Significant differences (p < 0.05) were observed among the different extraction methods in the characteristics studied. Physicochemical characteristics of the melon seed oils were saponification value, 180.48-189.86 mg KOH/g oil; iodine value, 108.27-118.62 g I2/100 g oil; acid value, 0.643-1.63 mg KOH/g oil; peroxide value; 1.69-2.98 mequiv/kg oil; specific gravity, 0.901-0.922; and refractive indices, 1.4676-1.4726. The dominant tocopherol was γ-tocopherol with total tocopherol in the range 27.61-74.39 mg/100 g. The dominant fatty acid was linoleic acid in the range 52.57-56.96%. The favorable oil yield, physicochemical characteristics, tocopherol, and fatty acid composition have the potential to replace or improve major commercial vegetable oils and to be used for various applications in the food industry and nutritive medicines.

Mapping of Schistosomiasis and Soil-Transmitted Helminths in Namibia: The First Large-Scale Protocol to Formally Include Rapid Diagnostic Tests.

BACKGROUND: Namibia is now ready to begin mass drug administration of praziquantel and albendazole against schistosomiasis and soil-transmitted helminths, respectively. Although historical data identifies areas of transmission of these neglected tropical diseases (NTDs), there is a need to update epidemiological data. For this reason, Namibia adopted a new protocol for mapping of schistosomiasis and geohelminths, formally integrating rapid diagnostic tests (RDTs) for infections and morbidity. In this article, we explain the protocol in detail, and introduce the concept of 'mapping resolution', as well as present results and treatment recommendations for northern Namibia. METHODS/FINDINGS/INTERPRETATION: This new protocol allowed a large sample to be surveyed (N = 17,896 children from 299 schools) at relatively low cost (7 USD per person mapped) and very quickly (28 working days). All children were analysed by RDTs, but only a sub-sample was also diagnosed by light microscopy. Overall prevalence of schistosomiasis in the surveyed areas was 9.0%, highly associated with poorer access to potable water (OR = 1.5, P<0.001) and defective (OR = 1.2, P<0.001) or absent sanitation infrastructure (OR = 2.0, P<0.001). Overall prevalence of geohelminths, more particularly hookworm infection, was 12.2%, highly associated with presence of faecal occult blood (OR = 1.9, P<0.001). Prevalence maps were produced and hot spots identified to better guide the national programme in drug administration, as well as targeted improvements in water, sanitation and hygiene. The RDTs employed (circulating cathodic antigen and microhaematuria for Schistosoma mansoni and S. haematobium, respectively) performed well, with sensitivities above 80% and specificities above 95%. CONCLUSION/SIGNIFICANCE: This protocol is cost-effective and sensitive to budget limitations and the potential economic and logistical strains placed on the national Ministries of Health. Here we present a high resolution map of disease prevalence levels, and treatment regimens are recommended.

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase.

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.