Genetic diversity and population structure of plasmodium falciparum from four epidemiological locations in Malawi

In malaria-endemic regions, Plasmodium falciparum (P. falciparum) infection is characterized by extensive genetic/antigenic diversity. Describing this diversity provides important information about the local molecular epidemiology of infecting P. falciparum parasites. Intriguingly, one of the major obstacles to the development of an effective malaria vaccine has been the genetic polymorphisms exhibited by P. falciparum genes encoding targets of human immune system. This situation has necessitated the development of polyvalent vaccines with wide antigenic coverage that would increase the likelihood of vaccine efficacy that covers wide geographical areas of malaria endemic countries. Limited reports are available on the population genetic diversity and structure of P. falciparum in Malawi, and this is of particular concern as the country has put in place several interventions to combat the disease. The primary aim of the research project was to determine the genetic diversity and population structure of P. falciparum isolates and comparing complexity from four different epidemiological settings in Malawi using msp-2 gene polymorphisms. Samples were collected from four epidemiological locations in the north, centre and southern regions of Malawi. The diversity and genetic differentiation of P. falciparum populations were analyzed based on the highly polymorphic block 3 msp-2 gene. One hundred and twenty patient samples who presented with signs and symptoms of malaria and who had microscopically confirmed P. falciparum infection were enrolled in the study after they had satisfied the inclusion criteria. Parasite DNA was extracted from the blood spot on to filter paper and analyzed by genotyping the msp-2 gene using allele-specific nested PCR. A total of 28 msp-2 block 3 fragments, defined by the size and the allelic types were detected in the 102 patients. The length variants of the PCR product ranged from 240basepairs (bp) to 450bp for the K1/FC and 410bp to 780bp for the 3D7/IC allelic families. Isolates of the 3D7 alleles were predominant in the population (59 percent), compared to isolates of the K1/ FC27 alleles (41 percent) and for 3D7 and K1 most of the isolates were monoclonal infections. In comparisons between the sites, we observed the highest prevalence of mixed infection in Mwanza (46.7 percent) followed by Dwangwa (23.3 percent) compared to Bolero (16.7 percent) and Mitundu (16.7 percent). The difference in prevalence of mixed infections between Mwanza and the other sites was statistically significant (p=0.041). There was also a non-significant trend towards a higher mean genotype number per isolate in the children aged >5 years compared to those below 5 years of age. These data suggest differences in prevalence rates of mixed infections in different geographical/epidemiological settings in Malawi. Further studies are needed to confirm, with larger sample sizes, the observation of a non-significant trend towards higher multiclonality of infection in older children in malaria endemic areas of Malawi

Dry matter intake, dry matter digestibility and growth performance in goats fed grass-based diet (Brachiaria or Cenchrus) compared with a concentrate-based diet

Dry matter digestibility (DMD), dry matter intake (DMI) and growth performance of concentrate- and forage-based diets were investigated using 24 growing Blended goats (12 males, 12 females) weighing 16.5±1.2 kg (mean±sd). Goats were blocked by sex and assigned randomly to three dietary treatments that were fed for 10 weeks, after two weeks of adaptation. Dietary treatments were MRG (40% maize bran, 40% rice bran, 20% Gliricidia leaf meal), BG (80% Brachiaria brizantha cv. Piatã, 20% Gliricidia leaf meal) and CG (80% Cenchrus ciliaris, 20% Gliricidia leaf meal). All diets contained sufficient nutrients to meet the requirements of growing goats. The MRG diet had lower DMI than CG, likely because MRG had small particle size and goats adapted slowly to that diet. Average daily weight gain was 10.7, 9.9 and 22.8 g/day for MRG, BG, and CG respectively, and did not differ among the diets. Goats fed MRG had lower overall live weight change than goats fed CG, while goats fed BG were intermediate. These findings indicate that B. brizantha cv. Piatã and C. ciliaris are equally useful as supplementary feedstuffs for growing goats during the dry season

Rapid emergence of multidrug resistant, H58-lineage Salmonella typhi in Blantyre, Malawi.

INTRODUCTION: Between 1998 and 2010, S. Typhi was an uncommon cause of bloodstream infection (BSI) in Blantyre, Malawi and it was usually susceptible to first-line antimicrobial therapy. In 2011 an increase in a multidrug resistant (MDR) strain was detected through routine bacteriological surveillance conducted at Queen Elizabeth Central Hospital (QECH). METHODS: Longitudinal trends in culture-confirmed Typhoid admissions at QECH were described between 1998-2014. A retrospective review of patient cases notes was conducted, focusing on clinical presentation, prevalence of HIV and case-fatality. Isolates of S. Typhi were sequenced and the phylogeny of Typhoid in Blantyre was reconstructed and placed in a global context. RESULTS: Between 1998-2010, there were a mean of 14 microbiological diagnoses of Typhoid/year at QECH, of which 6.8% were MDR. This increased to 67 in 2011 and 782 in 2014 at which time 97% were MDR. The disease predominantly affected children and young adults (median age 11 [IQR 6-21] in 2014). The prevalence of HIV in adult patients was 16.7% [8/48], similar to that of the general population (17.8%). Overall, the case fatality rate was 2.5% (3/94). Complications included anaemia, myocarditis, pneumonia and intestinal perforation. 112 isolates were sequenced and the phylogeny demonstrated the introduction and clonal expansion of the H58 lineage of S. Typhi. CONCLUSIONS: Since 2011, there has been a rapid increase in the incidence of multidrug resistant, H58-lineage Typhoid in Blantyre. This is one of a number of reports of the re-emergence of Typhoid in Southern and Eastern Africa. There is an urgent need to understand the reservoirs and transmission of disease and how to arrest this regional increase

Benefits of enhanced infection prophylaxis at antiretroviral therapy initiation by cryptococcal antigen status

OBJECTIVES: To assess baseline prevalence of cryptococcal antigen (CrAg) positivity; and its contribution to reductions in all-cause mortality, deaths from cryptococcus and unknown causes, and new cryptococcal disease in the REALITY trial. DESIGN: Retrospective CrAg testing of baseline and week-4 plasma samples in all 1805 African adults/children with CD4(+) cell count less than 100 cells/μl starting antiretroviral therapy who were randomized to receive 12-week enhanced-prophylaxis (fluconazole 100 mg/day, azithromycin, isoniazid, cotrimoxazole) vs. standard-prophylaxis (cotrimoxazole). METHODS: Proportional hazards models were used to estimate the relative impact of enhanced-prophylaxis vs. standard-cotrimoxazole on all, cryptococcal and unknown deaths, and new cryptococcal disease, through 24 weeks, by baseline CrAg positivity. RESULTS: Excluding 24 (1.4%) participants with active/prior cryptococcal disease at enrolment (all treated for cryptococcal disease), 133/1781 (7.5%) participants were CrAg-positive. By 24 weeks, 105 standard-cotrimoxazole vs. 78 enhanced-prophylaxis participants died. Of nine standard-cotrimoxazole and three enhanced-prophylaxis cryptococcal deaths, seven and two, respectively, were CrAg-positive at baseline. Among deaths of unknown cause, only 1/46 standard-cotrimoxazole and 1/28 enhanced-prophylaxis were CrAg-positive at baseline. There was no evidence that relative reductions in new cryptococcal disease associated with enhanced-prophylaxis varied between baseline CrAg-positives [hazard-ratio =0.36 (95% confidence interval 0.13−0.98), incidence 19.5 vs. 56.5/100 person-years] and CrAg-negatives [hazardratio =0.33 (0.03−3.14), incidence 0.3 vs. 0.9/100 person-years; P(heterogeneity) =0.95]; nor for all deaths, cryptococcal deaths or unknown deaths (P(heterogeneity) > 0.3). CONCLUSION: Relative reductions in cryptococcal disease/death did not depend on CrAg status. Deaths of unknown cause were unlikely to be cryptococcus-related; plausibly azithromycin contributed to their reduction. Findings support including 100 mg fluconazole in an enhanced-prophylaxis package at antiretroviral therapy initiation where CrAg screening is unavailable/impractical

Proportion of sequenced <i>S</i>.Typhi isolates from MLW collection belonging to each haplotype, the number above each bar represents the total number sequenced for each year.

<p>Proportion of sequenced <i>S</i>.Typhi isolates from MLW collection belonging to each haplotype, the number above each bar represents the total number sequenced for each year.</p

Age distribution of Typhoid in Blantyre 2011–14.

<p>2A reflects the total age distribution frequency and 2B reflects the median age each year with interquartile range.</p

Monthly trends in bloodstream invasive <i>Salmonella</i> diagnosed at QECH from November 2010-October 2014.

<p>Monthly trends in bloodstream invasive <i>Salmonella</i> diagnosed at QECH from November 2010-October 2014.</p

Temporal trends in <i>S</i>. Typhi isolation and antimicrobial resistance at QECH, Blantyre 1998–2013.

<p>*MDR: Multidrug resistant to amoxicillin, chloramphenicol, cotrimoxazole</p><p>Temporal trends in <i>S</i>. Typhi isolation and antimicrobial resistance at QECH, Blantyre 1998–2013.</p

Maximum-likelihood tree of 112 isolates of <i>S</i>.Typhi from Malawi, placed in the context of 24 isolates representative of the global diversity of <i>S</i>. Typhi and highlighting the previous diversity of Typhi isolates and the recent clonal expansion of the H58 haplotype.

<p>The left column depicts lineage, the right column depicts time category. Scale bar reveals indicates substitutions/variable site.</p

Phenotypic antimicrobial resistance patterns (n,%) of different clades.

<p>^† Susceptible to amoxicillin, chloramphenicol, cotrimoxazole, ciprofloxacin, ceftriaxone.</p><p>*MDR: Multidrug resistant to amoxicillin, chloramphenicol, cotrimoxazole</p><p>Phenotypic antimicrobial resistance patterns (n,%) of different clades.</p