Asymptomatic Plasmodium falciparum carriage and clinical disease: a 5-year community-based longitudinal study in The Gambia.

BACKGROUND: Carriers of persistent asymptomatic Plasmodium falciparum infections constitute an infectious reservoir that maintains malaria transmission. Understanding the extent of carriage and characteristics of carriers specific to endemic areas could guide use of interventions to reduce infectious reservoir. METHODS: In eastern Gambia, an all-age cohort from four villages was followed up from 2012 to 2016. Each year, cross-sectional surveys were conducted at the end of the malaria transmission season (January) and just before the start of the next one (June) to determine asymptomatic P. falciparum carriage. Passive case detection was conducted during each transmission season (August to January) to determine incidence of clinical malaria. Association between carriage at the end of the season and at start of the next one and the risk factors for this were assessed. Effect of carriage before start of the season on risk of clinical malaria during the season was also examined. RESULTS: A total of 1403 individuals-1154 from a semi-urban village and 249 from three rural villages were enrolled; median age was 12 years (interquartile range [IQR] 6, 30) and 12 years (IQR 7, 27) respectively. In adjusted analysis, asymptomatic P. falciparum carriage at the end of a transmission season and carriage just before start of the next one were strongly associated (adjusted odds ratio [aOR] = 19.99; 95% CI 12.57-31.77, p < 0.001). The odds of persistent carriage (i.e. infected both in January and in June) were higher in rural villages (aOR = 13.0; 95% CI 6.33-26.88, p < 0.001) and in children aged 5-15 years (aOR = 5.03; 95% CI 2.47-10.23, p = < 0.001). In the rural villages, carriage before start of the season was associated with a lower risk of clinical malaria during the season (incidence risk ratio [IRR] 0.48, 95% CI 0.27-0.81, p = 0.007). CONCLUSIONS: Asymptomatic P. falciparum carriage at the end of a transmission season strongly predicted carriage just before start of the next one. Interventions that clear persistent asymptomatic infections when targeted at the subpopulation with high risk of carriage may reduce the infectious reservoir responsible for launching seasonal transmission.

Plasmodium falciparum merozoite invasion ligands, linked antimalarial resistance loci and ex vivo responses to antimalarials in The Gambia.

BACKGROUND: Artemether/lumefantrine is the most commonly used artemisinin-based combination treatment (ACT) for malaria in sub-Saharan Africa. Drug resistance to ACT components is a major threat to malaria elimination efforts. Therefore, rigorous monitoring of drug efficacy is required for adequate management of malaria and to sustain the effectiveness of ACTs. OBJECTIVES: This study identified and described genomic loci that correlate with differences in ex vivo responses of natural Plasmodium falciparum isolates from The Gambia to antimalarial drugs. METHODS: Natural P. falciparum isolates from The Gambia were assayed for IC50 responses to four antimalarial drugs (artemether, dihydroartemisinin, amodiaquine and lumefantrine). Genome-wide SNPs from 56 of these P. falciparum isolates were applied to mixed-model regression and network analyses to determine linked loci correlating with drug responses. Genomic regions of shared haplotypes and positive selection within and between Gambian and Cambodian P. falciparum isolates were mapped by identity-by-descent (IBD) analysis of 209 genomes. RESULTS: SNPs in 71 genes, mostly involved in stress and drug resistance mechanisms correlated with drug responses. Additionally, erythrocyte invasion and permeability loci, including merozoite surface proteins (Pfdblmsp, Pfsurfin), and high-molecular-weight rhoptry protein 2 (Pfrhops2) were correlated with responses to multiple drugs. Haplotypes of pfdblmsp2 and known drug resistance loci (pfaat1, pfcrt and pfdhfr) from The Gambia showed high IBD with those from Cambodia, indicating co-ancestry, with significant linkage disequilibrium between their alleles. CONCLUSIONS: Multiple linked genic loci correlating with drug response phenotypes suggest a genomic backbone may be under selection by antimalarials. This calls for further analysis of molecular pathways to drug resistance in African P. falciparum.

Tolerance of Gambian Plasmodium falciparum to Dihydroartemisinin and Lumefantrine Detected by Ex Vivo Parasite Survival Rate Assay.

Monitoring of Plasmodium falciparum sensitivity to antimalarial drugs in Africa is vital for malaria elimination. However, the commonly used ex vivo/in vitro 50% inhibitory concentration (IC50) test gives inconsistent results for several antimalarials, while the alternative ring-stage survival assay (RSA) for artemisinin derivatives has not been widely adopted. Here, we applied an alternative two-color flow cytometry-based parasite survival rate assay (PSRA) to detect ex vivo antimalarial tolerance in P. falciparum isolates from The Gambia. The PSRA infers parasite viability by quantifying reinvasion of uninfected cells following 3 consecutive days of drug exposure (10-fold the IC50 of drug for field isolates). The drug survival rate is obtained for each isolate from the slope of the growth/death curve. We obtained parasite survival rates of 41 isolates for dihydroartemisinin (DHA) and lumefantrine (LUM) out of 51 infections tested by ring-stage survival assay (RSA) against DHA. We also determined the genotypes for known drug resistance genetic loci in the P. falciparum genes Pfdhfr, Pfdhps, Pfmdr, Pfcrt, and Pfk13 The PSRA results determined for 41 Gambian isolates showed faster killing and lower variance after treatment with DHA than after treatment with LUM, despite a strong correlation between the two drugs. Four and three isolates were tolerant to DHA and LUM, respectively, with continuous growth during drug exposure. Isolates with the PfMDR1-Y184F mutant variant showed increased LUM survival, though the results were not statistically significant. Sulfadoxine/pyrimethamine (SP) resistance markers were fixed, while all other antimalarial variants were prevalent in more than 50% of the population. The PSRA detected ex vivo antimalarial tolerance in Gambian P. falciparum This calls for its wider application and for increased vigilance against resistance to artemisinin combination therapies (ACTs) in this population.

Mass Drug Administration With Dihydroartemisinin-piperaquine and Malaria Transmission Dynamics in The Gambia: A Prospective Cohort Study.

BACKGROUND: Mass drug administration (MDA) may further reduce malaria transmission in low-transmission areas. The impact of MDA on the dynamics of malaria transmission was determined in a prospective cohort study. METHODS: Annual rounds of MDA with dihydroartemisinin-piperaquine (DP) were implemented were implemented in 2014 and 2015 in six village pairs before the malaria transmission season. Blood samples were collected from residents between July and December for microscopy and nested PCR. Incidence and prevalence of infection, clinical disease, and risk of malaria reinfection post-MDA were determined. RESULTS: Coverage of three DP doses was 68.2% (2014) and 65.6% (2015), compliance was greater than 80%. Incidence of infection was significantly lower in 2014 (incidence rate [IR] = 0.2 per person year [PPY]) than in 2013 (IR = 1.1 PPY; P < .01); monthly infection prevalence declined in the first three months post-MDA. Clinical malaria incidence was lower in 2014 (IR = 0.1 PPY) and 2015 (IR = 0.2 PPY) than in 2013 (IR = 0.4 PPY; P < .01), but remained higher in eastern Gambia. Individuals infected before MDA had a 2-fold higher odds of reinfection post-MDA (adjusted odds ratio = 2.5, 95% confidence interval 1.5-4.3; P < .01). CONCLUSIONS: MDA reduced malaria infection and clinical disease during the first months. The reduction was maintained in low-transmission areas, but not in eastern Gambia. Annual MDA could be followed by focal MDA targeting individuals infected during the dry season. Repeated MDA rounds, some during the dry season over larger geographical areas, may result in a more marked and sustained decrease of malaria transmission.

Hospital-based Surveillance for Pediatric Bacterial Meningitis in the Era of the 13-Valent Pneumococcal Conjugate Vaccine in Ghana.

BACKGROUND: Global surveillance for vaccine preventable invasive bacterial diseases has been set up by the World Health Organization to provide disease burden data to support decisions on introducing pneumococcal conjugate vaccine (PCV). We present data from 2010 to 2016 collected at the 2 sentinel sites in Ghana. METHODS: Data were collected from children <5 years of age presenting at the 2 major teaching hospitals with clinical signs of meningitis. Cerebrospinal fluid specimens were collected and tested first at the sentinel site laboratory with conventional microbiology methods and subsequently with molecular analysis, at the World Health Organization Regional Reference Laboratory housed at the Medical Research Council Unit The Gambia, for identification of Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, the 3 most common bacteria causing meningitis. RESULTS: There were 4008 suspected cases of meningitis during the surveillance period, of which 31 (0.8%) were laboratory confirmed. Suspected meningitis cases decreased from 923 in 2010 to 219 in 2016. Of 3817 patients with available outcome data, 226 (5.9%) died. S. pneumoniae was the most common bacterial pathogen, accounting for 68.5% of confirmed cases (50 of 73). H. influenzae and N. meningitidis accounted for 6.8% (5 of 73) and 21.9% (16 of 73), respectively. The proportion of pneumococcal vaccine serotypes causing meningitis decreased from 81.3% (13 of 16) before the introduction of 13-valent PCV (2010-2012) to 40.0% (8 of 20) after its introduction (2013-2016). CONCLUSIONS: Cases of suspected meningitis decreased among children <5 years of age between 2010 and 2016, with declines in the proportion of vaccine-type pneumococcal meningitis after the introduction of 13-valent PCV in Ghana.

Iron homeostasis in full-term, normal birthweight Gambian neonates over the first week of life.

Human neonates elicit a profound hypoferremia which may protect against bacterial sepsis. We examined the transience of this hypoferremia by measuring iron and its chaperone proteins, inflammatory and haematological parameters over the first post-partum week. We prospectively studied term, normal weight Gambian newborns. Umbilical cord vein and artery, and serial venous blood samples up to day 7 were collected. Hepcidin, serum iron, transferrin, transferrin saturation, haptoglobin, c-reactive protein, α1-acid-glycoprotein, soluble transferrin receptor, ferritin, unbound iron-binding capacity and full blood count were assayed. In 278 neonates we confirmed the profound early postnatal decrease in serum iron (22.7 ± 7.0 µmol/L at birth to 7.3 ± 4.6 µmol/L during the first 6-24 h after birth) and transferrin saturation (50.2 ± 16.7% to 14.4 ± 6.1%). Both variables increased steadily to reach 16.5 ± 3.9 µmol/L and 36.6 ± 9.2% at day 7. Hepcidin increased rapidly during the first 24 h of life (19.4 ± 14.4 ng/ml to 38.9 ± 23.9 ng/ml) and then dipped (32.7 ± 18.4 ng/ml) before rising again at one week after birth (45.2 ± 19.1 ng/ml). Inflammatory markers increased during the first week of life. The acute postnatal hypoferremia in human neonates on the first day of life is highly reproducible but transient. The rise in serum iron during the first week of life occurs despite very high hepcidin levels indicating partial hepcidin resistance.Trial Registration: clinicaltrials.gov (NCT03353051). Registration date: November 27, 2017.

Comparison of DNA concentration and bacterial pathogen PCR detection when using two DNA extraction kits for nasopharyngeal/oropharyngeal samples.

INTRODUCTION: Several important human pathogens that cause life-threatening infections are asymptomatically carried in the Nasopharynx/Oropharynx (NP/OP). DNA extraction is a prerequisite for most culture-independent techniques used to identify pathogens in the NP/OP. However, components of DNA extraction kits differ thereby giving rise to differences in performance. We compared the DNA concentration and the detection of three pathogens in the NP/OP using the discontinued DNeasy PowerSoil Kit (Kit DP) and the DNeasy PowerLyzer PowerSoil Kit (Kit DPP). METHODS: DNA was extracted from the same set of 103 NP/OP samples using the two kits. DNA concentration was measured using the Qubit 2.0 Fluorometer. Real-time Polymerase Chain reaction (RT-PCR) was done using the QuantStudio 7-flex system to detect three pathogens: S. pneumoniae, H. influenzae, and N. meningitidis. Bland-Altman statistics and plots were used to determine the threshold cycle (Ct) value agreement for the two kits. RESULTS: The average DNA concentration from kit DPP was higher than Kit DP; 1235.6 ng/ml (SD = 1368.3) vs 884.9 ng/ml (SD = 1095.3), p = 0.002. Using a Ct value cutoff of 40 for positivity, the concordance for the presence of S. pneumoniae was 82% (84/102); 94%(96/103) for N. meningitidis and 92%(95/103) for H. influenzae. Kit DP proportionately resulted in higher Ct values than Kit DPP for all pathogens. The Ct value bias of measurement for S. pneumoniae was +2.4 (95% CI, 1.9-3.0), +1.4 (95% CI, 0.9-1.9) for N. meningitidis and +1.4 (95% CI, 0.2-2.5) for H. influenzae. CONCLUSION: The higher DNA concentration obtained using kit DPP could increase the chances of recovering low abundant bacteria. The PCR results were reproducible for more than 90% of the samples for the gram-negative H. influenzae and N. meningitidis. Ct value variations of the kits must be taken into consideration when comparing studies that have used the two kits.

High genetic complexity but low relatedness in Plasmodium falciparum infections from Western Savannah Highlands and coastal equatorial Lowlands of Cameroon.

To determine the diversity and connectivity of infections in Northwestern and Southwestern Cameroon, 232 Plasmodium falciparum infections, collected in 2018 from the Ndop Health District (NHD) in the western savannah highlands in the Northwest and the Limbe Health District (LHD) in the coastal lowland forests in the Southwest of Cameroon were genotyped for nine neutral microsatellite markers. Overall infection complexity and genetic diversity was significantly (p < 0.05) lower in NHD than LHD, (Mean MOI = 2.45 vs. 2.97; Fws = 0.42 vs. 0.47; Mean He = 0.84 vs. 0.89, respectively). Multi-locus linkage disequilibrium was generally low but significantly higher in the NHD than LHD population (mean ISA= 0.376 vs 0.093). Consequently, highly related pairs of isolates were observed in NHD (mean IBS = 0.086) compared to those from the LHD (mean IBS = 0.059). Infections from the two regions were mostly unrelated (mean IBS = 0.059), though the overall genetic differentiation across the geographical range was low. Indices of differentiation between the populations were however significant (overall pairwise Fst = 0.048, Jost's D = 0.133, p < 0.01). Despite the high human migration across the 270km separating the study sites, these results suggest significant restrictions to gene flow against contiguous geospatial transmission of malaria in west Cameroon. Clonal infections in the highland sites could be driven by lower levels of malaria prevalence and seasonal transmission. How these differences in genetic diversity and complexity affect responses to interventions such as drugs will require further investigations from broader community sampling.

Exposure to household air pollution from solid cookfuels and childhood stunting: a population-based, cross-sectional study of half a million children in low- and middle-income countries.

BACKGROUND: Household air pollution from the incomplete combustion of solid cookfuels in low- and middle-income countries (LMICs) has been largely ignored as a potentially important correlate of stunting. Our objective was to examine the association between solid cookfuel use and stunting in children aged <5 y. METHODS: We used data from 59 LMICs' population-based cross-sectional demographic and health surveys; 557 098 children aged <5 y were included in our analytical sample. Multilevel logistic regression was used to examine the association between exposure to solid cookfuel use and childhood stunting, adjusting for child sex, age, maternal education and number of children living in the household. We explored the association across key subgroups. RESULTS: Solid cookfuel use was associated with child stunting (adjusted OR 1.58, 95% CI 1.55 to 1.61). Children living in households using solid cookfuels were more likely to be stunted if they lived in rural areas, the poorest households, had a mother who smoked tobacco or were from the Americas. CONCLUSIONS: Focused strategies to reduce solid cookfuel exposure might contribute to reductions in childhood stunting in LMICs. Trial evidence to assess the effect of reducing solid cookfuel exposure on childhood stunting is urgently needed.

Risk factors of infant mortality in rural The Gambia: a retrospective cohort study.

Objective: The main objective was to assess the risk factors for infant mortality among children living in the Health and Demographic Surveillance System (HDSS) in Farafenni, The Gambia. Our secondary objective was to assess these risks separately in the neonatal and postneonatal (>28 days) period. Design: Retrospective cohort study. Setting: HDSS in an urban centre and surrounding area in The Gambia. Patients: 7365 infants (47% female) born between 2014 and 2018, of which 126 (1.71%) died in the first year. Main outcome measures: Infant mortality. Results: Risk factors for mortality were death of any sibling (HR 2.78, 95% CI 1.54 to 5.00), having a twin (HR 1.96, 95% CI 1.01 to 3.80), being born in the harvest season (HR 1.55, 95% CI 1.07 to 2.24), living in a rural village (HR 4.34, 95% CI 2.03 to 9.29) and longer distance to the nearest village with a public health centre (HR 1.33, 95% CI 1.11 to 1.59). In addition, no breast feeding (HR 10.73, 95% CI 6.83 to 16.86) and no BCG vaccination in the first week of life (HR 3.47, 95% CI 1.07 to 11.24) were associated with infant mortality. Similar risk factors were found in the neonatal and postneonatal periods. Conclusion: Most risk factors associated with infant mortality (neonatal and postneonatal) are not easily modifiable at the individual level and would require programmatic approaches to target vulnerable infants and facilitate access to health services.

Ethnicity-specific BMI cutoffs for obesity based on type 2 diabetes risk in England: a population-based cohort study.

BACKGROUND: National and global recommendations for BMI cutoffs to trigger action to prevent obesity-related complications like type 2 diabetes among non-White populations are questionable. We aimed to prospectively identify ethnicity-specific BMI cutoffs for obesity based on the risk of type 2 diabetes that are risk-equivalent to the BMI cutoff for obesity among White populations (≥30 kg/m2). METHODS: In this population-based cohort study, we used electronic health records across primary care (Clinical Practice Research Datalink) linked to secondary care records (Hospital Episodes Statistics) from a network of general practitioner practices in England. Eligible participants were aged 18 years or older, without any past or current diagnosis of type 2 diabetes, had a BMI of 15·0-50·0 kg/m2 and complete ethnicity data, were registered with a general practitioner practice in England at any point between Sept 1, 1990, and Dec 1, 2018, and had at least 1 year of follow-up data. Patients with type 2 diabetes were identified by use of a CALIBER phenotyping algorithm. Self-reported ethnicity was collapsed into five main categories. Age-adjusted and sex-adjusted negative binomial regression models, with fractional polynomials for BMI, were fitted with incident type 2 diabetes and ethnicity data. FINDINGS: 1 472 819 people were included in our study, of whom 1 333 816 (90·6%) were White, 75 956 (5·2%) were south Asian, 49 349 (3·4%) were Black, 10 934 (0·7%) were Chinese, and 2764 (0·2%) were Arab. After a median follow-up of 6·5 years (IQR 3·2-11·2), 97 823 (6·6%) of 1 472 819 individuals were diagnosed with type 2 diabetes. For the equivalent age-adjusted and sex-adjusted incidence of type 2 diabetes at a BMI of 30·0 kg/m2 in White populations, the BMI cutoffs were 23·9 kg/m2 (95% CI 23·6-24·0) in south Asian populations, 28·1 kg/m2 (28·0-28·4) in Black populations, 26·9 kg/m2 (26·7-27·2) in Chinese populations, and 26·6 kg/m2 (26·5-27·0) in Arab populations. INTERPRETATION: Revisions of ethnicity-specific BMI cutoffs are needed to ensure that minority ethnic populations are provided with appropriate clinical surveillance to optimise the prevention, early diagnosis, and timely management of type 2 diabetes. FUNDING: National Institute for Health Research.

Tracking the uptake and trajectory of COVID-19 vaccination coverage in 15 West African countries: an interim analysis.

The African Union Bureau of Heads of State and Government endorsed the COVID-19 Vaccine Development and Access Strategy to vaccinate at least 60% of each country's population with a safe and efficacious vaccine by 2022, to achieve the population-level immunity needed to bring the pandemic under control. Using publicly available, country-level population estimates and COVID-19 vaccination data, we provide unique insights into the uptake trends of COVID-19 vaccinations in the 15 countries that comprise the Economic Community of West Africa States (ECOWAS). Based on the vaccination rates in the ECOWAS region after three months of commencing COVID-19 vaccinations, we provide a projection of the trajectory and speed of vaccination needed to achieve a COVID-19 vaccination coverage rate of at least 60% of the total ECOWAS population. After three months of the deployment of COVID-19 vaccines across the ECOWAS countries, only 0.27% of the region's total population had been fully vaccinated. If ECOWAS countries follow this trajectory, the sub-region will have less than 1.6% of the total population fully vaccinated after 18 months of vaccine deployment. Our projection shows that to achieve a COVID-19 vaccination coverage of at least 60% of the total population in the ECOWAS sub-region after 9, 12 and 18 months of vaccine deployment; the speed of vaccination must be increased to 10, 7 and 4 times the current trajectory, respectively. West African governments must deploy contextually relevant and culturally acceptable strategies for COVID-19 vaccine procurements, distributions and implementations in order to achieve reasonable coverage and save lives, sooner rather than later.

Early postnatal hypoferremia in low birthweight and preterm babies: A prospective cohort study in hospital-delivered Gambian neonates.

BACKGROUND: Neonates, particularly those born preterm (PTB) and with low birthweight (LBW), are especially susceptible to bacterial and fungal infections that cause an estimated 225,000 deaths annually. Iron is a vital nutrient for the most common organisms causing septicaemia. Full-term babies elicit an immediate postnatal hypoferremia assumed to have evolved as an innate defence. We tested whether PTB and LBW babies are capable of the same response. METHODS: We conducted an observational study of 152 babies who were either PTB (born ≥32 to <37 weeks gestational age) and/or LBW (<2500 g) (PTB/LBW) and 278 term, normal-weight babies (FTB/NBW). Blood was sampled from the umbilical cord vein and artery, and matched venous blood samples were taken from all neonates between 6-24 h after delivery. We measured haematological, iron and inflammatory markers. FINDINGS: In both PTB/LBW and FTB/NBW babies, serum iron decreased 3-fold within 12 h of delivery compared to umbilical blood (7·5 ± 4·5 vs 23·3 ± 7·1 ng/ml, P < 0·001, n = 425). Transferrin saturation showed a similar decline with a consequent increase in unsaturated iron-binding capacity. C-reactive protein levels increased over 10-fold (P < 0·001) and hepcidin levels doubled (P < 0·001). There was no difference in any of these responses between PTB/LBW and FTB/NBW babies. INTERPRETATION: Premature or low birthweight babies are able to mount a very rapid hypoferremia that is indistinguishable from that in normal term babies. The data suggest that this is a hepcidin-mediated response triggered by acute inflammation at birth, and likely to have evolved as an innate immune response against bacterial and fungal septicaemia. TRIAL REGISTRATION: clinicaltrials.gov (NCT03353051). Registration date: November 27, 2017. FUNDING: Bill & Melinda Gates Foundation (OPP1152353).

Neonatal iron distribution and infection susceptibility in full term, preterm and low birthweight babies in urban Gambia: study protocol for an observational study.

Background: Neonatal infection is the third largest cause of death in children under five worldwide.  Nutritional immunity is the process by which the host innate immune system limits nutrient availability to invading organisms. Iron is an essential micronutrient for both microbial pathogens and their mammalian hosts. Changes in iron availability and distribution have significant effects on pathogen virulence and on the immune response to infection. Our previously published data shows that, during the first 24 hours of life, full-term neonates have reduced overall serum iron. Transferrin saturation decreases rapidly from 45% in cord blood to ~20% by six hours post-delivery. Methods: To study neonatal nutritional immunity and its role in neonatal susceptibility to infection, we will conduct an observational study on 300 full-term normal birth weight (FTB+NBW), 50 preterm normal birth weight (PTB+NBW), 50 preterm low birth weight (PTB+LBW) and 50 full-term low birth weight (FTB+LBW), vaginally-delivered neonates born at Kanifing General Hospital, The Gambia. We will characterize and quantify iron-related nutritional immunity during the early neonatal period and use ex vivo sentinel bacterial growth assays to assess how differences in serum iron affect bacterial growth. Blood samples will be collected from the umbilical cord (arterial and venous) and at serial time points from the neonates over the first week of life. Discussion: Currently, little is known about nutritional immunity in neonates. In this study, we will increase understanding of how nutritional immunity may protect neonates from infection during the first critical days of life by limiting the pathogenicity and virulence of neonatal sepsis causing organisms by reducing the availability of iron. Additionally, we will investigate the hypothesis that this protective mechanism may not be activated in preterm and low birth weight neonates, potentially putting these babies at an enhanced risk of neonatal infection. Trial registration: clinicaltrials.gov ( NCT03353051) 27/11/2017.

Addressing challenges in tuberculosis adherence via performance-based payments for integrated case management: protocol for a cluster randomized controlled trial in Georgia.

BACKGROUND: Tuberculosis is one of the greatest global health concerns and disease management is challenging particularly in low- and middle-income countries. Despite improvements in addressing this epidemic in Georgia, tuberculosis remains a significant public health concern due to sub-optimal patient management. Low remuneration for specialists, limited private-sector interest in provision of infectious disease care and incomplete integration in primary care are at the core of this problem. METHODS: This protocol sets out the methods of a two-arm cluster randomized control trial which aims to generate evidence on the effectiveness of a performance-based financing and integrated care intervention on tuberculosis loss to follow-up and treatment adherence. The trial will be implemented in health facilities (clusters) under-performing in tuberculosis management. Eligible and consenting facilities will be randomly assigned to either intervention or control (standard care). Health providers within intervention sites will form a case management team and be trained in the delivery of integrated tuberculosis care; performance-related payments based on monthly records of patients adhering to treatment and quality of care assessments will be disbursed to health providers in these facilities. The primary outcomes include loss to follow-up among adult pulmonary drug-sensitive and drug-resistant tuberculosis patients. Secondary outcomes are adherence to treatment among drug-sensitive and drug-resistant tuberculosis patients and treatment success among drug-sensitive tuberculosis patients. Data on socio-demographic characteristics, tuberculosis diagnosis and treatment regimen will also be collected. The required sample size to detect a 6% reduction in loss to follow-up among drug-sensitive tuberculosis patients and a 20% reduction in loss to follow-up among drug-resistant tuberculosis patients is 948 and 136 patients, respectively. DISCUSSION: The trial contributes to a limited body of rigorous evidence and literature on the effectiveness of supply-side performance-based financing interventions on tuberculosis patient outcomes. Realist and health economic evaluations will be conducted in parallel with the trial, and associated composite findings will serve as a resource for the Georgian and wider regional Ministries of Health in relation to future tuberculosis and wider health policies. The trial and complementing evaluations are part of Results4TB, a multidisciplinary collaboration engaging researchers and Georgian policy and practice stakeholders in the design and evaluation of a context-sensitive tuberculosis management intervention. TRIAL REGISTRATION: ISRCTN, ISRCTN14667607 . Registered on 14 January 2019.

Prevalence of Panton-Valentine Leukocidin (PVL) and Antimicrobial Resistance in Community-Acquired Clinical Staphylococcus aureus in an Urban Gambian Hospital: A 11-Year Period Retrospective Pilot Study.

Background:Staphylococcus aureus is a major human pathogen. Panton-Valentine leukocidin (PVL) is a virulence factor produced by some strains that causes leukocyte lysis and tissue necrosis. PVL-associated S. aureus (PVL-SA) predominantly causes skin and soft-tissue infections (SSTIs) but can also cause invasive infections such as necrotizing pneumonia. It is carried by both community-associated methicillin susceptible S. aureus (CA-MSSA) and methicillin resistant S. aureus (CA-MRSA). This study aims to determine the prevalence of PVL-SA among patients seen at an urban Gambian hospital and associated antibiotic resistance. Methods: Archived clinical S. aureus (70 invasive bacteraemia and 223 non-invasive SSTIs) from 293 patients were retrieved as well as relevant data from clinical records where available. Antibiotic susceptibility was assessed using disc diffusion according to Clinical Laboratory Standards Institute (CLSI) guidelines. Genomic DNA was extracted and the presence of lukF and lukS PVL genes was detected by conventional gel-based PCR. Result: PVL-SA strains accounted for 61.4% (180/293) of S. aureus isolates. PVL prevalence was high in both Gambian bacteraemia and SSTIs S. aureus strains. Antimicrobial resistance was low and included chloramphenicol (4.8%), cefoxitin (2.4%), ciprofloxacin (3.8%), erythromycin (8.9%), gentamicin (5.5%) penicillin (92.5%), tetracycline (41.0%), and sulfamethoxazole-trimethoprim (24.2%). There was no association of PVL with antimicrobial resistance. Conclusion: PVL expression is high among clinical S. aureus strains among Gambian patients. Reporting of PVL-SA clinical infections is necessary to enable the monitoring of the clinical impact of these strains in the population and guide prevention of the spread of virulent PVL-positive CA-MRSA strains. SUMMARY  Staphylococcus aureus (S. aureus) is a major human pathogen with several virulence factors. We performed a retrospective analysis to investigate the prevalence of one such virulence factor (PVL) amongst clinical S. aureus samples. We found a high prevalence in our setting but antimicrobial resistance including methicillin resistance was low.

Antimicrobial resistance and virulence genes of non-typhoidal Salmonella isolates in The Gambia and Senegal.

INTRODUCTION: The prevalence of virulence genes in non-typhoidal Salmonella (NTS) and its association with commonly used antibiotics in West Africa is unknown. METHODOLOGY: We tested 185 NTS isolates from children, animals, and food products for the presence of twelve virulence genes by PCR. Ten of the virulence genes tested belonged to the five Salmonella pathogenicity islands implicated in its pathogenesis. RESULTS: Ten of twelve virulence genes except sopE and pefA were present in at least 70% of the isolates tested; sopE and pefA were observed in 33% and 44% of the isolates, respectively. The most prevalent gene was invA (99.5%), which is an invasion gene conserved within the Salmonella enterica. pipD and sopB genes, which were associated with serovar Enteritidis, were detected in 92.4% and 94.1% of isolates respectively. S. Istanbul and S. Javiana, which were isolated from chicken-serving restaurants, carried all the virulence genes of the five pathogenicity islands. There was significant association between sopB, sitC, orfLC, pipD and pefA virulence genes and resistance to commonly used antibiotics in Senegal and The Gambia, namely amoxicillin, ticarcillin, trimethoprim plus sulfamethoxazole, tetracycline, trimethoprim, spectinomycin, streptomycin, sulfonamides and nitrofurantoin. CONCLUSIONS: This study shows that virulence genes are present in NTS strains isolated from various sources. The significant association between some virulence genes and antibiotic resistance may have important implications with regard to the spread and persistence of resistance and virulence genes in Salmonella and to the prudent use of antimicrobial agents in humans and animals in West Africa.

Epstein-Barr virus but not cytomegalovirus is associated with reduced vaccine antibody responses in Gambian infants.

BACKGROUND: Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are persistent herpesviruses that have various immunomodulatory effects on their hosts. Both viruses are usually acquired in infancy in Sub-Saharan Africa, a region where childhood vaccines are less effective than in high income settings. To establish whether there is an association between these two observations, we tested the hypothesis that infection with one or both viruses modulate antibody responses to the T-cell independent meningococcal polysaccharide vaccine and the T-cell dependent measles vaccines. METHODOLOGY/PRINCIPAL FINDINGS: Infection with EBV and CMV was diagnosed by the presence of virus-specific IgM in the peripheral blood or by the presence of IgG at higher levels than that found in umbilical cord blood. Anti-meningococcus IgG and IgM were quantified by ELISA. Anti-measles antibody responses were quantified by haemagglutinin antibody inhibition assay. Infants infected with EBV had reduced IgG and IgM antibody responses to meningococcal polysaccharides and to measles vaccine. Infection with CMV alone predicted no changes in the response to meningococcal polysaccharide. While CMV alone had no discernable effect on the antibody response to measles, the response of infants infected with both CMV and EBV was similar to that of infants infected with neither, suggesting that the effects of CMV infection countered the effects of EBV on measles antibody responses. CONCLUSIONS: The results of this exploratory study indicate that infection with EBV is associated with reduced antibody responses to polysaccharides and to measles vaccine, but suggest that the response to T-cell dependent antigens such as measles haemagglutinin may be restored by infection with CMV.