European and Developing Countries Clinical Trials Partnership.

BACKGROUND. Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) — a resistant subpopulation within a susceptible strain — is a recently described phenomenon in Cryptococcus neoformans ( Cn ) and Cryptococcus gattii ( Cg ), the significance of which has not previously been studied in humans. METHODS. A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp . were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. RESULTS. Heteroresistance was detectable in Cryptococcus spp . in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11 , which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. CONCLUSION. Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. FUNDING. This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship.

DBS, in combination with the NucliSENS EasyQ HIV-1 v1.2 asay, performed well in monitoring HIV viral loads in patients who received ART in rural Tanzania, although the sensitivity was reduced when viral burden was low. The use of DBS can simplify virological monitoring in resource-limited settings.

The Haydom, Tanzania, site (TZH) of The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) Study is in north-central Tanzania, 300 km from the nearest urban center. TZH is in a remote rural district where most of the population are agropastoralists and grow maize as the staple food. The average household size is 7. The average woman achieves a parity of 6 and has 1 child death. Socioeconomic indicators are poor, with essentially no household having access to electricity, piped water, or improved sanitary facilities (compared with 14%, 7%, and 12%, respectively, reported nationally). The Demographic Health Survey Tanzania 2004 indicated that the region had high rates of stunting and underweight (40% and 31% of children aged <5 years had a height-for-age z score and weight-for-age z score, respectively, of <-2 ) and an under-5 child mortality rate of 5.8%. Human immunodeficiency virus prevalence among 18-month-old children is <0.5%. TZH represents a remote rural African population with profound poverty and malnutrition, but a strong community-based research infrastructure.