Microsatellites reveal high polymorphism and high potential for use in anti-malarial efficacy studies in areas with different transmission intensities in mainland Tanzania.

BACKGROUND: Tanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of anti-malarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes, merozoite surface proteins 1 and 2 (msp1 and msp2), and replacing the glutamate-rich protein (glurp) gene with one of the highly polymorphic microsatellites in Plasmodium falciparum to adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which is routinely performed in Tanzania. METHODS: Plasmodium falciparum samples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro) and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was done using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and M2490) by capillary method, and the data were analysed to determine the extent of their polymorphisms and genetic diversity at the four sites. RESULTS: Overall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥ 50.0% of the markers), and > 50.0% of the samples (range = 47.6-59.1%) were polyclonal, with a mean multiplicity of infection (MOI) ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (RS = 7.48, range = 7.27-8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (He = 0.83, range = 0.80-0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (FST) did not reveal population structure or clustering of parasites according to geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while M2490 was the least polymorphic. CONCLUSION: Microsatellite genotyping revealed high polyclonality and genetic diversity but no significant population structure. Poly-α, C2M34, TA1 and C3M69 were the most polymorphic markers, and Poly-α alone or with any of the other three markers could be adopted for use in TES in Tanzania.

Discovery medicine - the HVTN's iterative approach to developing an HIV-1 broadly neutralizing vaccine.

PURPOSE OF REVIEW: In the past two decades, there has been an explosion in the discovery of HIV-1 broadly neutralizing antibodies (bnAbs) and associated vaccine strategies to induce them. This abundance of approaches necessitates a system that accurately and expeditiously identifies the most promising regimens. We herein briefly review the background science of bnAbs, provide a description of the first round of phase 1 discovery medicine studies, and suggest an approach to integrate these into a comprehensive HIV-1-neutralizing vaccine. RECENT FINDINGS: With recent preclinical success including induction of early stage bnAbs in mouse knockin models and rhesus macaques, successful priming of VRC01-class bnAbs with eOD-GT8 in a recent study in humans, and proof-of-concept that intravenous infusion of VRC01 prevents sexual transmission of virus in humans, the stage is set for a broad and comprehensive bnAb vaccine program. Leveraging significant advances in protein nanoparticle science, mRNA technology, adjuvant development, and B-cell and antibody analyses, the HVTN has reconfigured its HIV-1 vaccine strategy by developing the Discovery Medicine Program to test promising vaccine candidates targeting six key epitopes. SUMMARY: The HVTN Discovery Medicine program is testing multiple HIV-1-neutralizing vaccine candidates.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated malaria and prevalence of Pfk13 and Pfmdr1 polymorphisms after a decade of using artemisinin-based combination therapy in mainland Tanzania.

BACKGROUND: The World Health Organization recommends regular therapeutic efficacy studies (TES) to monitor the performance of first and second-line anti-malarials. In 2016, efficacy and safety of artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria were assessed through a TES conducted between April and October 2016 at four sentinel sites of Kibaha, Mkuzi, Mlimba, and Ujiji in Tanzania. The study also assessed molecular markers of artemisinin and lumefantrine (partner drug) resistance. METHODS: Eligible patients were enrolled at the four sites, treated with standard doses of AL, and monitored for 28 days with clinical and laboratory assessments. The main outcomes were PCR corrected cure rates, day 3 positivity rates, safety of AL, and prevalence of single nucleotide polymorphisms in Plasmodium falciparum kelch 13 (Pfk13) (codon positions: 440-600) and P. falciparum multi-drug resistance 1 (Pfmdr1) genes (codons: N86Y, Y184F and D1246Y), markers of artemisinin and lumefantrine resistance, respectively. RESULTS: Of 344 patients enrolled, three withdrew, six were lost to follow-up; and results were analysed for 335 (97.4%) patients. Two patients had treatment failure (one early treatment failure and one recrudescent infection) after PCR correction, yielding an adequate clinical and parasitological response of > 98%. Day 3 positivity rates ranged from 0 to 5.7%. Common adverse events included cough, abdominal pain, vomiting, and diarrhoea. Two patients had serious adverse events; one died after the first dose of AL and another required hospitalization after the second dose of AL (on day 0) but recovered completely. Of 344 samples collected at enrolment (day 0), 92.7% and 100% were successfully sequenced for Pfk13 and Pfmdr1 genes, respectively. Six (1.9%) had non-synonymous mutations in Pfk13, none of which had been previously associated with artemisinin resistance. For Pfmdr1, the NFD haplotype (codons N86, 184F and D1246) was detected in 134 (39.0%) samples; ranging from 33.0% in Mlimba to 45.5% at Mkuzi. The difference among the four sites was not significant (p = 0.578). All samples had a single copy of the Pfmdr1 gene. CONCLUSION: The study indicated high efficacy of AL and the safety profile was consistent with previous reports. There were no known artemisinin-resistance Pfk13 mutations, but there was a high prevalence of a Pfmdr1 haplotype associated with reduced sensitivity to lumefantrine (but no reduced efficacy was observed in the subjects). Continued TES and monitoring of markers of resistance to artemisinin and partner drugs is critical for early detection of resistant parasites and to inform evidence-based malaria treatment policies. Trial Registration ClinicalTrials.gov NCT03387631.

Effect of Supportive Supervision on Malaria Microscopy Competencies in Sub-Saharan Africa.

Although light microscopy is the reference standard for diagnosing malaria, maintaining skills over time can be challenging. Between 2015 and 2017, the U.S. President's Malaria Initiative-funded MalariaCare project supported outreach training and supportive supervision (OTSS) visits at 1,037 health facilities in seven African countries to improve performance in microscopy slide preparation, staining, and reading. During these visits, supervisors observed and provided feedback to health-care workers (HCWs) performing malaria microscopy using a 30-step checklist. Of the steps observed in facilities with at least three visits, the proportion of HCWs that performed each step correctly at baseline ranged from 63.2% to 94.2%. The change in the proportion of HCWs performing steps correctly by the third visit ranged from 16.7 to 23.6 percentage points (n = 916 observations). To assess the overall improvement, facility scores were calculated based on the steps performed correctly during each visit. The mean score at baseline was 85.7%, demonstrating a high level of performance before OTSS. Regression analysis predicted an improvement in facility scores of 3.6 percentage points (P < 0.001) after three visits across all countries. In reference-level facilities with consistently high performance on microscopy procedures and parasite detection, quality assurance (QA) mechanisms could prioritize more advanced skills, such as proficiency testing for parasite counting and species identification. However, in settings with high staff turnover and declining use of microscopy in favor of rapid diagnostic tests, additional supervision visits and/or additional QA measures may be required to improve and maintain performance.

Introduction and Evaluation of an Electronic Tool for Improved Data Quality and Data Use during Malaria Case Management Supportive Supervision.

Although on-site supervision programs are implemented in many countries to assess and improve the quality of care, few publications have described the use of electronic tools during health facility supervision. The President's Malaria Initiative-funded MalariaCare project developed the MalariaCare Electronic Data System (EDS), a custom-built, open-source, Java-based, Android application that links to District Health Information Software 2, for data storage and visualization. The EDS was used during supervision visits at 4,951 health facilities across seven countries in Africa. The introduction of the EDS led to dramatic improvements in both completeness and timeliness of data on the quality of care provided for febrile patients. The EDS improved data completeness by 47 percentage points (42-89%) on average when compared with paper-based data collection. The average time from data submission to a final data analysis product dropped from over 5 months to 1 month. With more complete and timely data available, the Ministry of Health and the National Malaria Control Program (NMCP) staff could more effectively plan corrective actions and promptly allocate resources, ultimately leading to several improvements in the quality of malaria case management. Although government staff used supervision data during MalariaCare-supported lessons learned workshops to develop plans that led to improvements in quality of care, data use outside of these workshops has been limited. Additional efforts are required to institutionalize the use of supervision data within ministries of health and NMCPs.

Effect of Supportive Supervision on Competency of Febrile Clinical Case Management in Sub-Saharan Africa.

Since 2010, the WHO has recommended that clinical decision-making for malaria case management be performed based on the results of a parasitological test result. Between 2015 and 2017, the U.S. President's Malaria Initiative-funded MalariaCare project supported the implementation of this practice in eight sub-Saharan African countries through 5,382 outreach training and supportive supervision visits to 3,563 health facilities. During these visits, trained government supervisors used a 25-point checklist to observe clinicians' performance in outpatient departments, and then provided structured mentoring and action planning. At baseline, more than 90% of facilities demonstrated a good understanding of WHO recommendations-when tests should be ordered, using test results to develop an accurate final diagnosis, severity assessment, and providing the correct prescription. However, significant deficits were found in history taking, conducting a physical examination, and communicating with patients and their caregivers. After three visits, worker performance demonstrated steady improvement-in particular, with checking for factors associated with increased morbidity and mortality: one sign of severe malaria (72.9-85.5%), pregnancy (81.1-87.4%), and anemia (77.2-86.4%). A regression analysis predicted an overall improvement in clinical performance of 6.3% (P < 0.001) by the third visit. These findings indicate that in most health facilities, there is good baseline knowledge on the processes of quality clinical management, but further training and on-site mentoring are needed to improve the clinical interaction that focuses on second-order decision-making, such as severity of illness, management of non-malarial fever, and completing the patient-provider communication loop.

Determination of lopinavir cerebral spinal fluid and plasma ultrafiltrate concentrations by liquid chromatography coupled to tandem mass spectrometry.

A method for the determination of lopinavir (LPV) concentrations in cerebral spinal fluid (CSF) and plasma ultrafiltrate (UF) was developed and validated to analyze clinical specimens from patients receiving antiretroviral treatment with lopinavir/ritonavir. The CSF (400 microL sample volume) final calibration range for LPV was 0.313-25.0 ng/mL. The final calibration range for UF (50 microL sample volume) was 1.25-100 ng/mL. The samples were prepared using liquid-liquid extraction, concentrated, and analyzed using a reversed phase isocratic separation. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer. Isolation of LPV through chromatographic separation and proper selection of calibration matrix were important factors in achieving accurate results. Plasma UF was found to be an equivalent calibration matrix to CSF whereas plasma matrix produced a positive bias in samples with unknown concentrations. Artificial CSF media prepared chemically were biased and less superior than UF. Sources of plasma for the UF did not affect accuracy. Several CSF sources were tested for specificity of the method and LPV concentrations were accurately produced with atmospheric pressure chemical ionization source producing more accurate results than the electrospray source. The method successfully measured LPV concentrations in CSF that were previously undetectable by HPLC as well as UF from protein binding studies.

Development of an interdisciplinary case management program for combat veterans.

The terrorist attack on September 11, 2001, prompted a major mobilization of Alabama active military reservists and Alabama National Guardsmen to serve in the Middle East. Health problems related to geographic relocation, environmental threats, combat and other traumatic events, and the stress associated with serving in an active capacity have resulted in an increase in the number of patients and the variety of illnesses being seen at Department of Veterans Affairs Medical Centers. The Tuscaloosa Veterans Affairs Medical Center developed a combat veterans care coordination program to efficiently and effectively manage the care of returning combat veterans. The development and first-year outcomes of the program are described.