Differences in PPD- and mitogen-induced T-cell activation marker expression characterize immunopathology in acute tuberculosis patients.

Impaired T-cell responses to mitogens and high T-cell activation marker (TAM) expression on Mycobacterium tuberculosis-specific T-cells characterize immunopathology in patients with tuberculosis (TB). In a study of patients with TB (n = 60) and asymptomatic contacts (controls, n = 37), we found that TB patients had higher CD38+ T-cell proportions specific for M. tuberculosis protein (PPDMtb), yet total proportions of PPDMtb-specific T-cells were comparable. Notably, both activated (CD38+) and total IFN-γ+ T-cells from TB patients had lower mitogen (phytohemagglutinin, PHA)-induced responses. This impaired mitogen response improved the classification efficacy of the TAM-TB assay, especially employing the PPD/PHA-induced T-cell ratio.

Doxycycline Treatment of Mansonella perstans-Infected Individuals Affects Immune Cell Activation and Causes Long-term T-cell Polarization.

BACKGROUND: Doxycycline is used for treatment of Mansonella perstans infection. Immune modulatory effects of both M. perstans and doxycycline have been described but long-term implications on host immune response are not defined. Here we determined multiple immune parameters of M. perstans-infected individuals before and after doxycycline treatment to characterize doxycycline effects on host T-cell immunity. METHODS: Immune characterization of doxycycline-treated M. perstans-infected individuals was performed as part of an open-label randomized clinical trial. Immune cell population phenotyping by flow cytometry and functional in vitro T-cell assays were performed at baseline, 6 months, and "long term" (18-24 months) after treatment start. Treatment efficacy, based on peripheral blood microfilaria (mf) burden, was correlated with immune parameters and effects on immune response against concomitant Mycobacterium tuberculosis infection were determined. RESULTS: Immune population phenotyping indicated changes in functional T-cell responses after doxycycline treatment. Constitutive and superantigen-induced T-cell activation and polarization towards T-helper type (TH) 1 phenotype at baseline declined after doxycycline treatment, whereas low proportions of TH17 and TH1* cells at baseline increased significantly at follow-up. In accordance, long-term decline in antigen-specific TH1 responses against concomitant M. tuberculosis infection was seen. Notably, only TH17 and TH1* changes after 6 months and TH17 at baseline were negatively correlated with M. perstans microfilaria burden or reduction, whereas long-term changes were not associated with treatment efficacy. CONCLUSIONS: We found long-term immune modulatory effects of doxycycline treatment leading to decreased constitutive T-cell activation, polarization towards TH17/TH1*, and impaired immune response against concomitant M. tuberculosis infection.

Monocyte pathology in human tuberculosis is due to plasma milieu changes and aberrant STAT signalling.

Monocyte-derived macrophages contribute centrally to immune protection in Mycobacterium tuberculosis infection and changes in monocyte phenotype characterize immunopathology in tuberculosis patients. Recent studies highlighted an important role of the plasma milieu in tuberculosis immunopathology. Here, we investigated monocyte pathology in patients with acute tuberculosis and determined tuberculosis plasma milieu effects on phenotype as well as cytokine signalling of reference monocytes. Patients with tuberculosis (n = 37) and asymptomatic contacts (controls n = 35) were recruited as part of a hospital-based study in the Ashanti region of Ghana. Multiplex flow cytometry phenotyping of monocyte immunopathology was performed and effects of individual blood plasma samples on reference monocytes prior to and during treatment were characterized. Concomitantly, cell signalling pathways were analysed to elucidate underlying mechanisms of plasma effects on monocytes. Multiplex flow cytometry visualization characterized changes in monocyte subpopulations and detected higher expression of CD40, CD64 and PD-L1 in monocytes from tuberculosis patients as compared to controls. Aberrant expression normalized during anti-mycobacterial treatment and also CD33 expression decreased markedly. Notably, higher CD33, CD40 and CD64 expression was induced in reference monocytes when cultured in the presence of plasma samples from tuberculosis patients as compared to controls. STAT signalling pathways were affected by the aberrant plasma milieu and higher levels of STAT3 and STAT5 phosphorylation was found in tuberculosis plasma-treated reference monocytes. Importantly, high pSTAT3 levels were associated with high CD33 expression and pSTAT5 correlated with CD40 as well as CD64 expression. These results suggested plasma milieu effects with potential implications on monocyte phenotype and function in acute tuberculosis.

Cytokine-induced transient monocyte IL-7Ra expression and the serum milieu in tuberculosis.

Bacterial components and cytokines induce IL-7 receptor (IL-7Rα) expression in monocytes. Aberrant low IL-7Rα expression of monocytes has been identified as a feature of tuberculosis immunopathology. Here, we investigated the mechanisms underlying IL-7Rα regulation of monocytes and tuberculosis serum effects on IL-7Rα expression. Serum samples from tuberculosis patients and healthy controls, cytokine candidates, and mycobacterial components were analyzed for in vitro effects on IL-7Rα expression of primary monocytes, monocyte-derived macrophages (MDM), and monocyte cell lines. IL-7Rα regulation during culture and the role of FoxO1 were characterized. In vitro activation-induced IL-7Rα expression in human monocytes and serum samples from tuberculosis patients boosted IL-7Rα expression. Although pathognomonic tuberculosis cytokines were not associated with serum effects, we identified cytokines (i.e., GM-CSF, IL-1ß, TNF-α, IFN-γ) that induced IL-7Rα expression in monocytes and/or MDM comparable to mycobacterial components. Blocking of cytokine subsets (i.e., IL-1ß/TNF-α in monocytes, GM-CSF in MDM) largely diminished IL-7Rα expression induced by mycobacterial components. Finally, we showed that in vitro-induced IL-7Rα expression was transient and dependent on constitutive FoxO1 expression in primary monocytes and monocyte cell lines. This study demonstrated the crucial roles of cytokines and constitutive FoxO1 expression for transient IL-7Rα expression in monocytes.

Plasma cytokine levels characterize disease pathogenesis and treatment response in tuberculosis patients.

BACKGROUND: Mycobacterium (M.) tuberculosis-caused immunopathology is characterized by aberrant expression of plasma cytokines in human tuberculosis. Disease severity and long-term anti-mycobacterial treatment are potentially influenced by immunopathology and normalization of plasma cytokine levels during therapy may indicate treatment efficacy and recovery. STUDY DESIGN AND METHODS: In this study, we analyzed the concentrations of selected plasma cytokines (i.e., IL-6, IP-10, IL-10, IL-22, IFNγ, GM-CSF, IL-8) and M. tuberculosis sputum burden in patients with tuberculosis (n = 76). Cytokine levels were compared to healthy contacts (n = 40) and changes under treatment were monitored (i.e., 6 and 16 weeks after treatment start). According to differences in M. tuberculosis sputum burden and conversion, tuberculosis patients were classified as paucibacillary as well as 'rapid' or 'slow' treatment responders. A subgroup of tuberculosis patients had fatal disease courses. RESULTS: Six of seven cytokines were significantly higher in tuberculosis patients as compared to contacts and four of these (i.e., IL-6, IP-10, IL-10, and IL-22) were detectable in the majority of tuberculosis patients. IL-6 showed the strongest discriminating capacity for tuberculosis disease and in combination with IL-10 concentrations efficiently classified paucibacillary tuberculosis cases as well as those with fatal disease outcome. In addition, IL-6 and IP-10 levels decreased significantly after 6 weeks of treatment and analyses of subgroups with differential treatment response showed delayed decline of IL-6 levels in slow treatment responders. CONCLUSIONS: Combinations of different plasma cytokine (namely, IL-6, IL-10, and IP-10) efficiently classified tuberculosis patients with differential mycobacterial burden and especially IL-6 qualified as a biomarker candidate for early treatment response.

Impaired T-cell response to phytohemagglutinin (PHA) in tuberculosis patients is associated with high IL-6 plasma levels and normalizes early during anti-mycobacterial treatment.

PURPOSE: Human tuberculosis is characterized by immunopathology that affects T-cell phenotype and functions. Previous studies found impaired T-cell response to phytohemagglutinin (PHA) in patients with acute tuberculosis. However, the influence of disease severity, affected T-cell subsets, and underlying mechanisms remain elusive. METHODS: Here we investigated PHA-induced and antigen-specific T-cell effector cytokines in tuberculosis patients (n = 55) as well as in healthy asymptomatic contacts (n = 32) from Ghana. Effects of Mycobacterium (M.) tuberculosis sputum burden and treatment response were analyzed and compared during follow-up. Finally, cytokine characteristics of the aberrant plasma milieu in tuberculosis were analyzed as a potential cause for impaired PHA response. RESULTS: PHA-induced IFN-γ expression was significantly lower in sputum-positive tuberculosis patients as compared to both, contacts and paucibacillary cases, and efficiently discriminated the study groups. T-cell responses to PHA increased significantly early during treatment and this was more pronounced in tuberculosis patients with rapid treatment response. Analysis of alternative cytokines revealed distinct patterns and IL-22, as well as IL-10, showed comparable expression to IFN-γ in response to PHA. Finally, we found that high IL-6 plasma levels were strongly associated with impaired IFN-γ and IL-22 response to PHA. CONCLUSION: We conclude that impaired T-cell response to PHA stimulation in acute tuberculosis patients (i) was potentially caused by the aberrant plasma milieu, (ii) affected differentially polarized T-cell subsets, (iii) normalized early during treatment. This study shed light on the mechanisms of impaired T-cell functions in tuberculosis and yielded promising biomarker candidates for diagnosis and monitoring of treatment response.

Lower IL-7 Receptor Expression of Monocytes Impairs Antimycobacterial Effector Functions in Patients with Tuberculosis.

Altered monocyte differentiation and effector functions characterize immune pathogenesis of tuberculosis. IL-7 is an important factor for proliferation of T cells and impaired IL-7 sensitivity due to decreased IL-7 receptor α-chain (IL-7Rα) expression was found in patients with acute tuberculosis. Peripheral blood monocytes have moderate IL-7Rα expression and increased IL-7Rα levels were described for inflammatory diseases. In this study, we investigated a potential role of IL-7 and IL-7Rα expression for monocyte functions in tuberculosis. We analyzed the phenotype of monocytes in the blood from tuberculosis patients (n = 33), asymptomatic contacts of tuberculosis patients (contacts; n = 30), and healthy controls (n = 20) from Ghana by multicolor flow cytometry. Mycobacterial components were analyzed for their capacity to induce IL-7Rα expression in monocytes. Functional effects of monocyte to IL-7 were measured during signaling and by using an antimycobacterial in vitro kill assay. Monocytes were more frequent in peripheral blood from patients with tuberculosis and especially higher proportions of CD14+/CD16+ (M1/2) monocytes with increased PD-L1 expression characterized acute tuberculosis. IL-7Rα expression was decreased particularly on M1/2 monocytes from patients with tuberculosis and aberrant low expression IL-7Rα correlated with high PD-L1 levels. Constitutive low pSTAT5 levels of monocytes ex vivo and impaired IL-7 response confirmed functionally decreased monocyte IL-7 sensitivity of patients with tuberculosis. Mycobacteria and mycobacterial cell wall components induced IL-7 receptor expression in monocytes and IL-7 boosted mycobacterial killing by monocyte-derived macrophages in vitro. We demonstrated impaired monocyte IL-7 receptor expression as well as IL-7 sensitivity in tuberculosis with potential effects on antimycobacterial effector functions.

Rifampicin and clarithromycin (extended release) versus rifampicin and streptomycin for limited Buruli ulcer lesions: a randomised, open-label, non-inferiority phase 3 trial.

BACKGROUND: Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans infection that damages the skin and subcutis. It is most prevalent in western and central Africa and Australia. Standard antimicrobial treatment with oral rifampicin 10 mg/kg plus intramuscular streptomycin 15 mg/kg once daily for 8 weeks (RS8) is highly effective, but streptomycin injections are painful and potentially harmful. We aimed to compare the efficacy and tolerability of fully oral rifampicin 10 mg/kg plus clarithromycin 15 mg/kg extended release once daily for 8 weeks (RC8) with that of RS8 for treatment of early Buruli ulcer lesions. METHODS: We did an open-label, non-inferiority, randomised (1:1 with blocks of six), multicentre, phase 3 clinical trial comparing fully oral RC8 with RS8 in patients with early, limited Buruli ulcer lesions. There were four trial sites in hospitals in Ghana (Agogo, Tepa, Nkawie, Dunkwa) and one in Benin (Pobè). Participants were included if they were aged 5 years or older and had typical Buruli ulcer with no more than one lesion (caterories I and II) no larger than 10 cm in diameter. The trial was open label, and neither the investigators who took measurements of the lesions nor the attending doctors were masked to treatment assignment. The primary clinical endpoint was lesion healing (ie, full epithelialisation or stable scar) without recurrence at 52 weeks after start of antimicrobial therapy. The primary endpoint and safety were assessed in the intention-to-treat population. A sample size of 332 participants was calculated to detect inferiority of RC8 by a margin of 12%. This study was registered with ClinicalTrials.gov, NCT01659437. FINDINGS: Between Jan 1, 2013, and Dec 31, 2017, participants were recruited to the trial. We stopped recruitment after 310 participants. Median age of participants was 14 years (IQR 10-29) and 153 (52%) were female. 297 patients had PCR-confirmed Buruli ulcer; 151 (51%) were assigned to RS8 treatment, and 146 (49%) received oral RC8 treatment. In the RS8 group, lesions healed in 144 (95%, 95% CI 91 to 98) of 151 patients, whereas lesions healed in 140 (96%, 91 to 99) of 146 patients in the RC8 group. The difference in proportion, -0·5% (-5·2 to 4·2), was not significantly greater than zero (p=0·59), showing that RC8 treatment is non-inferior to RS8 treatment for lesion healing at 52 weeks. Treatment-related adverse events were recorded in 20 (13%) patients receiving RS8 and in nine (7%) patients receiving RC8. Most adverse events were grade 1-2, but one (1%) patient receiving RS8 developed serious ototoxicity and ended treatment after 6 weeks. No patients needed surgical resection. Four patients (two in each study group) had skin grafts. INTERPRETATION: Fully oral RC8 regimen was non-inferior to RS8 for treatment of early, limited Buruli ulcer and was associated with fewer adverse events. Therefore, we propose that fully oral RC8 should be the preferred therapy for early, limited lesions of Buruli ulcer. FUNDING: WHO with additional support from MAP International, American Leprosy Missions, Fondation Raoul Follereau France, Buruli ulcer Groningen Foundation, Sanofi-Pasteur, and BuruliVac.

High prevalence of asymptomatic malaria infections in adults, Ashanti Region, Ghana, 2018.

BACKGROUND: Ghana is among the high-burden countries for malaria infections and recently reported a notable increase in malaria cases. While asymptomatic parasitaemia is increasingly recognized as a hurdle for malaria elimination, studies on asymptomatic malaria are scarce, and usually focus on children and on non-falciparum species. The present study aims to assess the prevalence of asymptomatic Plasmodium falciparum and non-falciparum infections in Ghanaian adults in the Ashanti region during the high transmission season. METHODS: Asymptomatic adult residents from five villages in the Ashanti Region, Ghana, were screened for Plasmodium species by rapid diagnostic test (RDT) and polymerase chain reaction (PCR) during the rainy season. Samples tested positive were subtyped using species-specific real-time PCR. For all Plasmodium ovale infections additional sub-species identification was performed. RESULTS: Molecular prevalence of asymptomatic Plasmodium infection was 284/391 (73%); only 126 (32%) infections were detected by RDT. While 266 (68%) participants were infected with Plasmodium falciparum, 33 (8%) were infected with Plasmodium malariae and 34 (9%) with P. ovale. The sub-species P. ovale curtisi and P. ovale wallikeri were identified to similar proportions. Non-falciparum infections usually presented as mixed infections with P. falciparum. CONCLUSIONS: Most adult residents in the Ghanaian forest zone are asymptomatic Plasmodium carriers. The high Plasmodium prevalence not detected by RDT in adults highlights that malaria eradication efforts must target all members of the population. Beneath Plasmodium falciparum, screening and treatment must also include infections with P. malariae, P. o. curtisi and P. o. wallikeri.

An IL7RA exon 5 polymorphism is associated with impaired IL-7Rα splicing and protection against tuberculosis in Ghana.

Functional interleukin-7 receptor α-chain (IL-7Rα) genetic variants, which affect alternative splicing and expression of the soluble IL-7Rα, are associated with susceptibility to autoimmunity. We previously described aberrant IL-7Rα expression and impaired IL-7-mediated T-cell functions in tuberculosis patients. In the present study, we investigated a possible role of IL7RA gene variants. Six exonic IL7RA polymorphisms were genotyped and two minor alleles were found at lower frequencies in tuberculosis patients as compared to healthy contacts from Ghana (rs11567764, p = 0.002; rs1494558, p = 0.01). The rs11567764 polymorphism tags an IL7RA haplotype exclusively found in African populations and was predicted to affect splicing of exon 5. Reduced mRNA expression of the Δexon_5-6 variant was found in T-cells from carriers of the protective rs11567764 allele. Although we were not able to demonstrate the causative effect of rs11567764, our findings suggested functional implications of genetic variants on IL-7Rα splicing and with potential impact on T-cell protection against tuberculosis.

Drug resistance outcomes of long-term ART with tenofovir disoproxil fumarate in the absence of virological monitoring.

Objectives: The resistance profiles of patients receiving long-term ART in sub-Saharan Africa have been poorly described. This study obtained a sensitive assessment of the resistance patterns associated with long-term tenofovir-based ART in a programmatic setting where virological monitoring is yet to become part of routine care. Methods: We studied subjects who, after a median of 4.2 years of ART, replaced zidovudine or stavudine with tenofovir disoproxil fumarate while continuing lamivudine and an NNRTI. Using deep sequencing, resistance-associated mutations (RAMs) were detected in stored samples collected at tenofovir introduction (T0) and after a median of 4.0 years (T1). Results: At T0, 19/87 (21.8%) subjects showed a detectable viral load and 8/87 (9.2%) had one or more major NNRTI RAMs, whereas 82/87 (94.3%) retained full tenofovir susceptibility. At T1, 79/87 (90.8%) subjects remained on NNRTI-based ART, 5/87 (5.7%) had introduced lopinavir/ritonavir due to immunological failure, and 3/87 (3.4%) had interrupted ART. Whilst 68/87 (78.2%) subjects maintained or achieved virological suppression between T0 and T1, a detectable viral load with NNRTI RAMs at T0 predicted lack of virological suppression at T1. Each treatment interruption, usually reflecting unavailability of the dispensary, doubled the risk of T1 viraemia. Tenofovir, lamivudine and efavirenz selected for K65R, K70E/T, L74I/V and Y115F, alongside M184V and multiple NNRTI RAMs; this resistance profile was accompanied by high viral loads and low CD4 cell counts. Conclusions: Viraemia on tenofovir, lamivudine and efavirenz led to complex resistance patterns with implications for continued drug activity and risk of onward transmission.

High frequency of active HCV infection among seropositive cases in west Africa and evidence for multiple transmission pathways.

BACKGROUND: Sub-Saharan Africa (SSA) has one of the highest global hepatitis C virus (HCV) prevalence estimates. However, reports that suggest high rates of serologic false positives and low levels of viremia have led to uncertainty regarding the burden of active infection in this region. Additionally, little is known about the predominant transmission risk factors in SSA. METHODS: We prospectively recalled 363 past blood donors (180 who were rapid screen assay [RSA] positive and 183 who were RSA negative at time of donation) to identify the level of active infection and risk factors for infection at a teaching hospital in Kumasi, Ghana. Participants had repeat blood testing and were administered a questionnaire on risk factors. RESULTS: The frequency of HCV active infection ranged from 74.4% to 88% depending on the criteria used to define serologically positive cases. Individuals with active disease had biochemical evidence of liver inflammation and median viral loads of 5.7 log copies/mL. Individuals from the northern and upper regions of Ghana had greater risks of infection compared with participants from other areas. Additional risk factors included traditional circumcision, home birth, tribal scarring, and hepatitis B virus coinfection. CONCLUSIONS: Viremic infection was common among serologically confirmed cases. Attention to testing algorithms is needed in order to define the true HCV burden in SSA. These data also suggest that several transmission modes are likely contributing to the current HCV epidemic in Ghana and that the distribution of these practices may result in substantial regional variation in prevalence.

A re-evaluation of the origin of hepatitis C virus genotype 2 in West Africa.

Hepatitis C virus (HCV) is classified into seven genotypes based on genetic diversity, and most genotypes have been found in Africa. Infections with HCV genotype 2 (HCV2) are most prevalent in West Africa and it was suggested that HCV2 originated in West Africa. To better understand the evolutionary epidemiology of HCV2 in Africa, we examined new NS5B sequences of HCV2 strains obtained from Côte d'Ivoire, Ghana and Nigeria sequenced at the Centers for Disease Control and Prevention with those available from West, North and Central Africa. Bayesian phylogeographic analysis using a discrete trait model showed that Ghana was the most likely geographical region for the origin of HCV2. Spread of HCV2 from Ghana did not appear to be through diffusion to adjacent countries along the coast. Rather, it was transmitted from Ghana to many distant countries in Africa, suggesting that certain routes of geographical dissemination were historically more efficient than mere proximity and that the HCV2 epidemic history in West Africa is extremely complex.

Infection with Mansonella perstans Nematodes in Buruli Ulcer Patients, Ghana.

During August 2010-December 2012, we conducted a study of patients in Ghana who had Buruli ulcer, caused by Mycobacterium ulcerans, and found that 23% were co-infected with Mansonella perstans nematodes; 13% of controls also had M. perstans infection. M. perstans co-infection should be considered in the diagnosis and treatment of Buruli ulcer.

Clinical and bacteriological efficacy of rifampin-streptomycin combination for two weeks followed by rifampin and clarithromycin for six weeks for treatment of Mycobacterium ulcerans disease.

Buruli ulcer, an ulcerating skin disease caused by Mycobacterium ulcerans infection, is common in tropical areas of western Africa. We determined the clinical and microbiological responses to administration of rifampin and streptomycin for 2 weeks followed by administration of rifampin and clarithromycin for 6 weeks in 43 patients with small laboratory-confirmed Buruli lesions and monitored for recurrence-free healing. Bacterial load in tissue samples before and after treatment for 6 and 12 weeks was monitored by semiquantitative culture. The success rate was 93%, and there was no recurrence after a 12-month follow-up. Eight percent had a positive culture 4 weeks after antibiotic treatment, but their lesions went on to heal. The findings indicate that rifampin and clarithromycin can replace rifampin and streptomycin for the continuation phase after rifampin and streptomycin administration for 2 weeks without any apparent loss of efficacy.

Kinetics of mycolactone in human subcutaneous tissue during antibiotic therapy for Mycobacterium ulcerans disease.

BACKGROUND: Mycobacterium ulcerans (M. ulcerans) causes a devastating necrotising infection of skin tissue leading to progressive ulceration. M. ulcerans is the only human pathogen that secretes mycolactone, a polyketide molecule with potent cytotoxic and immunomodulatory properties. These unique features make mycolactone an attractive biomarker for M. ulcerans disease. We sought to measure the concentration of mycolactone within lesions of patients with Buruli ulcer before, during and after antibiotic treatment to evaluate its association with the clinical and bacteriological response to therapy. METHODS: Biopsies of M. ulcerans infected skin lesions were obtained from patients before, during and after antibiotic therapy. Lipids were extracted from the biopsies and concentration of mycolactone was assayed by mass spectrometry and a cytotoxicity assay and correlated with clinical and bacteriological response to therapy. RESULTS: Baseline concentration of mycolactone measured by mass spectrometry predicted time to complete healing of small nodules and ulcers. Even though intra-lesional concentrations of mycolactone declined with antibiotic treatment, the toxin was still present after antibiotic treatment for 6 weeks and also 4 weeks after the end of treatment for 8 weeks in a subgroup of patients with slowly healing lesions. Additionally viable bacilli were detected in a proportion of these slowly healing lesions during and after treatment. CONCLUSIONS: Our findings indicate that baseline intra-lesional mycolactone concentration and its kinetics with antibiotic therapy are important prognostic determinants of clinical and bacteriological response to antibiotic treatment for Mycobacterium ulcerans disease. Mycolactone may be a useful biomarker with potential utility in optimising antibiotic therapy.

Diversity of Culicoides in the middle belt of Ghana with Implications on the transmission of Mansonella perstans; a molecular approach.

BACKGROUND: Culicoides, also known as biting midges, carry pathogens which include Mansonella perstans. Mansonella perstans is a nematode parasite implicated in a number of disease outcomes. Even though a high prevalence of about 75% M. perstans infection has been recorded in some communities in the middle belt of Ghana, and a wide diversity of Culicoides species has been identified, the exact Culicoides species transmitting M. perstans in Ghana has not yet been deciphered. This study therefore aimed at assessing the species diversity of Culicoides and their role in the transmission of M. perstans in the middle belt of Ghana. METHODS: Culicoides species were sampled from 11 communities in the Asante-Akim North and Sene West districts in the middle belt of Ghana. Centre for Disease Control (CDC) UV light traps, as well as human bait (i.e. human landing catch and engorged catch) methods were used to assess the species abundance and diversity of Culicoides in the study communities in the wet and dry season. A colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay was performed to assess the vector competence of the various Culicoides species. RESULTS: A total of 4810 Culicoides from 6 species were sampled. These included Culicoides inornatipennis, C. milnei, C. schultzei, C. grahamii, C. neavei, and C. imicola. Culicoides imicola was the most abundant species (56%) followed by C. grahamii (16%). Light traps sampled the most diverse species (6 species). Human landing catch and engorged catch methods identified three anthropophilic species, C. grahamii, C. milnei, and C. inornatipennis, with C. grahamii being the most anthropophilic with a peak biting time between the hours of 5 p.m. to 6 p.m. Generally, there was relatively higher species abundance in the wet than dry season. LAMP assay identified C. grahamii as the potential vector for M. perstans transmission in the middle belt of Ghana. CONCLUSIONS: For the first time, we have demonstrated that C. grahamii is the potential competent vector for M. perstans transmission in the middle belt of Ghana. It is more abundant in the rainy season and has a peak biting time between the hours of 5 and 6 p.m.

BCG Vaccination-Associated Lower HbA1c and Increased CD25 Expression on CD8+ T Cells in Patients with Type 1 Diabetes in Ghana.

BCG vaccination affects other diseases beyond tuberculosis by unknown-potentially immunomodulatory-mechanisms. Recent studies have shown that BCG vaccination administered during overt type 1 diabetes (T1D) improved glycemic control and affected immune and metabolic parameters. Here, we comprehensively characterized Ghanaian T1D patients with or without routine neonatal BCG vaccination to identify vaccine-associated alterations. Ghanaian long-term T1D patients (n = 108) and matched healthy controls (n = 214) were evaluated for disease-related clinical, metabolic, and immunophenotypic parameters and compared based on their neonatal BCG vaccination status. The majority of study participants were BCG-vaccinated at birth and no differences in vaccination rates were detected between the study groups. Notably, glycemic control metrics, i.e., HbA1c and IDAA1c, showed significantly lower levels in BCG-vaccinated as compared to unvaccinated patients. Immunophenotype comparisons identified higher expression of the T cell activation marker CD25 on CD8+ T cells from BCG-vaccinated T1D patients. Correlation analysis identified a negative correlation between HbA1c levels and CD25 expression on CD8+ T cells. In addition, we observed fractional increases in glycolysis metabolites (phosphoenolpyruvate and 2/3-phosphoglycerate) in BCG-vaccinated T1D patients. These results suggest that neonatal BCG vaccination is associated with better glycemic control and increased activation of CD8+ T cells in T1D patients.

Impact of antibiotic intake on the incidence of extended-spectrum ß-lactamase-producing Enterobacterales in sub-Saharan Africa: results from a community-based longitudinal study.

OBJECTIVES: The global prevalence of intestinal extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) is approximately 17% in communities, with significant variations among regions. This longitudinal study aimed to assess the impact of antibiotic intake on the incidence of intestinal ESBL-PE in Ghanaian pharmacy customers outside of hospitals. METHODS: Screening for ESBL-PE was performed in four independent pharmacies in Kumasi, Ghana, using rectal swabs and an ESBL-PE-selective medium. Pharmacy customers purchasing antibiotics were recruited, and those buying non-antibiotic drugs served as controls. Participants who were negative for ESBL-PE provided follow-up swabs for up to 28 days. RESULTS: At baseline, 302 (75%) of 404 participants were colonized with ESBL-PE. Sixty-three participants who were negative for ESBL-PE at baseline received per-protocol follow-up, including 28 individuals who took antibiotics and 35 controls. The cumulative proportions of ESBL-PE in the antibiotics and control groups were 71% (20/28) and 54% (19/35) at the first follow-up (p 0.258), 86% (24/28) and 80% (28/35) at the second follow-up (p 0.741) and 86% (24/28) and 94% (33/35) at the third follow-up (p 0.393), respectively. DISCUSSION: The rate of intestinal ESBL-PE carriage among pharmacy customers outside of hospitals was higher than expected at baseline and further increased during the 28 days of follow-up, irrespective of antibiotic intake. This alarming finding needs to be considered in the antibiotic treatment of outpatients and emphasizes the urgent need for improved prevention strategies, development of new antibiotic drugs and potential future elimination strategies. Further longitudinal studies on ESBL-PE in African communities, also outside of pharmacy settings, are required.

Diminished Interleukin-7 receptor expression on T-cell subsets in tuberculosis patients.

Immunopathology in human tuberculosis affects T-cell phenotype and functions. Previous studies identified impaired T-cell sensitivity to Interleukin (IL)-7 accompanied by lower IL-7 receptor α-chain (IL-7Rα) expression in patients with acute tuberculosis. In the present study, we characterized affected T-cell subsets and determined the influence of tuberculosis disease severity and treatment response. Tuberculosis patients (n = 89) as well as age- and gender-matched asymptomatic contacts (controls, n = 47) were recruited in Ghana. Mycobacterium (M.) tuberculosis sputum burden was monitored prior to and during treatment. Blood samples from all patients and controls were analyzed for IL-7Rα expression and T-cell markers by multi-colour flow cytometry. CD4+ and CD8+ T-cells of tuberculosis patients showed generally lower IL-7Rα expression as compared to controls. Concomitantly, tuberculosis patients had higher proportions of naïve and lower proportions of memory CD4+ T-cells. Notably, a subset of CD27 positive central memory T-cells (Tcm), which lacked IL-7Rα expression was enriched in tuberculosis patients as compared to controls. M. tuberculosis sputum burden was not associated with differences in IL-7Rα expression. Treatment duration and response showed no clear effects although IL-7Rα expression patterns were highly variable. These results suggested generally impaired generation of memory CD4+ T-cells and enrichment of a Tcm subset without IL-7Rα expression in patients with tuberculosis.