Functional role of aspartate-31 and leucine-32 in Mycobacterium avium dihydrofolate reductase

Scope and Method of Study: Dihydrofolate reductase (DHFR: 1.5.1.3) has long been a drug target in antibacterial therapy. However, DHFR of Mycobacterium avium and other mycobacteria are naturally resistant to trimethoprim and other antituberculous drugs. Recent reports show that a new class of drugs: 2,4-diaminodeazapteridine (DMDPs) are showing increased selectivity for Mycobacterium avium. Better understanding of the binding sites of M. avium DHFR, will contribute towards developing better and more effective drugs. Based on sequence alignments and X-ray crystal structures of other DHFR's, aspartic acid 31 (D31) and leucine 32 (L32) were identified as functionally important residues in interactions with the substrate dihydrofolate and inhibitors, respectively. D31 and L32 of M. avium DHFR were modified by site-directed mutagenesis (GeneEditor, Promega). to D31A, D31E, D31Q, D31N, D31L,. L32A, L32F and L32D. Mutations were verified by full length gene sequencing. These mutants were then expressed in E. coli BL21(DE3)pLysS and the recombinant mutant protein purified using HisBind-Resin (Novangen). Functionality of the mutants was assessed in comparison with the recombinant wild type by a standard enzyme assay as well as by growth complementation. Kinetic parameters were determined and computed using the non-linear curve fit program Enzfitter (BioSoft, UK).Findings and Conclusions: All D31 mutations rendered the enzyme severely dysfunctional. Enzyme activity of the mutants D31E, D31Q and D31N were reduced by between 80 and 90%. Functionality of the mutants D31A and D31L were reduced by over 90% compared to the wild type.All D31 mutants show differences in kinetics compared to the wild type. Of the L32 mutants, only L32D reduced the enzyme's activity by two-thirds and showed differences in kinetic behavior compared to the wild type. L32F and L32A did not show selectivity for trimethoprim, while L32D did. The DMDP inhibitors were highly effective against the wild type. The mutants showed differences in selectivity to the DMDPs. The findings support the hypotheses that D31 plays a functional role with the substrate and L32 plays a functional role with inhibitors. All D31 mutations studies resulted in a dysfunctional enzyme, regardless of changes in side chain size or charge. Modification of L32 led to increased selectivity for trimethoprim, but decreased selectivity for the DMDPs

Methicillin-resistant staphylococci among school children in Mariental, Namibia

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to beta-lactam antibiotics, while some strains are multi-drug resistant and may produce disease-causing toxins. Drug resistant strains are often responsible for chronic, persistent and recurrent infections, which pose a challenge for healthcare practitioners. Our study aimed to determine the prevalence of nasal MRSA and methicillin-resistant coagulase-negative staphylococci (MRCoNS) among school children in the Mariental community, southern Namibia. This was a cross-sectional study in the Mariental District. Nasal specimens (swabs) were collected from 272 randomly selected learners aged 6–14 years attending school in the area during the months of March, September and October 2016. Isolation and identification of staphylococci were performed using standard microbiological methods. Methicillin-resistant isolates were identified by their resistance towards cefoxitin (30 μg) using the Kirby-Bauer disk diffusion assay. Enterotoxin production among multi-drug resistant MRSA isolates was detected with a SET-RPLA toxin detection kit. Methicillin-resistant S. aureus was isolated from 48 (17.6%) learners and MRCoNS from only seven (2.6%). Methicillin-resistant S. aureus colonization was significantly higher (P = 0.003) in the age group 11–14 years than in the group 6–10 years. Among the 433 staphylococcal isolates screened for cefoxitin resistance, 51 (11.8%) were MRSA and seven (1.6%) were MRCoNS. From the 51 MRSA isolates, 22 (43.1%) were multi-drug resistant of which six were enterotoxigenic. This is the first report on MRSA and MRCoNS among school children in Namibia. The presence of multidrug resistant and potentially virulent staphylococci among school children in Mariental, Namibia, is of concern. Self-infection by these bacteria poses various health risks for the children. It is recommended that school health programmes improve current hygiene practices. Frequent handwashing can prevent staphylococcal disease and spread of resistant strains among learners and the wider community.http://www.elsevier.com/locate/sciafdm2022Biochemistr

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

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.Peer reviewedEntomology and Plant Patholog

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Ronnie Earl

https://egrove.olemiss.edu/bock_photo/1531/thumbnail.jp

Ronnie Earl

https://egrove.olemiss.edu/bock_photo/1530/thumbnail.jp

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&rsquo; health. Liquid chromatography&ndash;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&ndash;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

Children with Plasmodium vivax infection previously observed in Namibia, were Duffy negative and carried a c.136G &gt; 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