A Hybrid of Amodiaquine and Primaquine Linked by Gold(I) Is a Multistage Antimalarial Agent Targeting Heme Detoxification and Thiol Redox Homeostasis.

Hybrid-based drugs linked through a transition metal constitute an emerging concept for Plasmodium intervention. To advance the drug design concept and enhance the therapeutic potential of this class of drugs, we developed a novel hybrid composed of quinolinic ligands amodiaquine (AQ) and primaquine (PQ) linked by gold(I), named [AuAQPQ]PF6. This compound demonstrated potent and efficacious antiplasmodial activity against multiple stages of the Plasmodium life cycle. The source of this activity was thoroughly investigated by comparing parasite susceptibility to the hybrid's components, the annotation of structure-activity relationships and studies of the mechanism of action. The activity of [AuAQPQ]PF6 for the parasite's asexual blood stages was influenced by the presence of AQ, while its activity against gametocytes and pre-erythrocytic parasites was influenced by both quinolinic components. Moreover, the coordination of ligands to gold(I) was found to be essential for the enhancement of potency, as suggested by the observation that a combination of quinolinic ligands does not reproduce the antimalarial potency and efficacy as observed for the metallic hybrid. Our results indicate that this gold(I) hybrid compound presents a dual mechanism of action by inhibiting the beta-hematin formation and enzymatic activity of thioredoxin reductases. Overall, our findings support the potential of transition metals as a dual chemical linker and an antiplasmodial payload for the development of hybrid-based drugs.

Traditional Kenyan herbal medicine: exploring natural products' therapeutics against schistosomiasis.

Praziquantel (PZQ) remains the only drug of choice for the treatment of schistosomiasis, caused by parasitic flatworms. The widespread use of PZQ in schistosomiasis endemic areas for about four decades raises concerns about the emergence of resistance of Schistosoma spp. to PZQ under drug selection pressure. This reinforces the urgency in finding alternative therapeutic options that could replace or complement PZQ. We explored the potential of medicinal plants commonly used by indigenes in Kenya for the treatment of various ailments including malaria, pneumonia, and diarrhoea for their antischistosomal properties. Employing the Soxhlet extraction method with different solvents, seven medicinal plants Artemisia annua, Ajuga remota, Bredilia micranta, Cordia africana, Physalis peruviana, Prunus africana and Senna didymobotrya were extracted. Qualitative phytochemical screening was performed to determine the presence of various phytochemicals in the plant extracts. Extracts were tested against Schistosoma mansoni newly transformed schistosomula (NTS) and adult worms and the schistosomicidal activity was determined by using the adenosine triphosphate quantitation assay. Phytochemical analysis of the extracts showed different classes of compounds such as alkaloids, tannins, terpenes, etc., in plant extracts active against S. mansoni worms. Seven extracts out of 22 resulted in <20% viability against NTS in 24 h at 100 µg/ml. Five of the extracts with inhibitory activity against NTS showed >69.7% and ≥72.4% reduction in viability against adult worms after exposure for 24 and 48 h, respectively. This study provides encouraging preliminary evidence that extracts of Kenyan medicinal plants deserve further study as potential alternative therapeutics that may form the basis for the development of the new treatments for schistosomiasis.

First In Silico Screening of Insect Molecules for Identification of Novel Anti-Parasitic Compounds.

Schistosomiasis is a neglected tropical disease caused by blood flukes of the genus Schistosoma. In silico screenings of compounds for the identification of novel anti-parasitic drug candidates have received considerable attention in recent years, including the screening of natural compounds. For the first time, we investigated molecules from insects, a rather neglected source in drug discovery, in an in silico screening approach to find novel antischistosomal compounds. Based on the Dictionary of Natural Products (DNP), we created a library of 1327 insect compounds suitable for molecular docking. A structure-based virtual screening against the crystal structure of a known druggable target in Schistosoma mansoni, the thioredoxin glutathione reductase (SmTGR), was performed. The top ten compounds predominantly originated from beetles and were predicted to interact particularly with amino acids in the doorstop pocket of SmTGR. For one compound from a jewel beetle, buprestin H, we tested and confirmed antischistosomal activity against adult and juvenile parasites in vitro. At concentrations with anti-parasitic activity, we could also exclude any unspecific cytotoxic activity against human HepG2 cells. This study highlights the potential of insect molecules for the identification of novel antischistosomal compounds. Our library of insect-derived molecules could serve not only as basis for future in silico screenings against additional target proteins of schistosomes, but also of other parasites.

Analysis of drug resistance among difficult-to-treat tuberculosis patients in Ghana identifies several pre-XDR TB cases.

Background: Resistance to tuberculosis (TB) drugs has become a major threat to global control efforts. Early case detection and drug susceptibility profiling of the infecting bacteria are essential for appropriate case management. The objective of this study was to determine the drug susceptibility profiles of difficult-to-treat (DTT) TB patients in Ghana. Methods: Sputum samples obtained from DTT-TB cases from health facilities across Ghana were processed for rapid diagnosis and detection of drug resistance using the Genotype MTBDRplus and Genotype MTBDRsl.v2 from Hain Life science. Results: A total of 298 (90%) out of 331 sputum samples processed gave interpretable bands out of which 175 (58.7%) were resistant to at least one drug (ANYr); 16.8% (50/298) were isoniazid-mono-resistant (INHr), 16.8% (50/298) were rifampicin-mono-resistant (RIFr), and 25.2% (75/298) were MDR. 24 (13.7%) of the ANYr were additionally resistant to at least one second line drug: 7.4% (2 RIFr, 1 INHr, and 10 MDR samples) resistant to only FQs and 2.3% (2 RIFr, 1 INHr, and 1 MDR samples) resistant to AMG drugs kanamycin (KAN), amikacin (AMK), capreomycin (CAP), and viomycin (VIO). Additionally, there were 4.0% (5 RIFr and 2 MDR samples) resistant to both FQs and AMGs. 81 (65.6%) out of 125 INH-resistant samples including INHr and MDR had katG-mutations (MT) whereas 15 (12%) had inhApro-MT. The remaining 28 (22.4%) had both katG and inhA MT. All the 19 FQ-resistant samples were gyrA mutants whereas the 10 AMGs were rrs (3), eis (3) as well as rrs, and eis co-mutants (4). Except for the seven pre-XDR samples, no sample had eis MT. Conclusion: The detection of several pre-XDR TB cases in Ghana calls for intensified drug resistance surveillance and monitoring of TB patients to, respectively, ensure early diagnosis and treatment compliance.

Gut microbiota signature of pathogen-dependent dysbiosis in viral gastroenteritis.

Acute gastroenteritis associated with diarrhea is considered a serious disease in Africa and South Asia. In this study, we examined the trends in the causative pathogens of diarrhea and the corresponding gut microbiota in Ghana using microbiome analysis performed on diarrheic stools via 16S rRNA sequencing. In total, 80 patients with diarrhea and 34 healthy adults as controls, from 2017 to 2018, were enrolled in the study. Among the patients with diarrhea, 39 were norovirus-positive and 18 were rotavirus-positive. The analysis of species richness (Chao1) was lower in patients with diarrhea than that in controls. Beta-diversity analysis revealed significant differences between the two groups. Several diarrhea-related pathogens (e.g., Escherichia-Shigella, Klebsiella and Campylobacter) were detected in patients with diarrhea. Furthermore, co-infection with these pathogens and enteroviruses (e.g., norovirus and rotavirus) was observed in several cases. Levels of both Erysipelotrichaceae and Staphylococcaceae family markedly differed between norovirus-positive and -negative diarrheic stools, and the 10 predicted metabolic pathways, including the carbohydrate metabolism pathway, showed significant differences between rotavirus-positive patients with diarrhea and controls. This comparative study of diarrheal pathogens in Ghana revealed specific trends in the gut microbiota signature associated with diarrhea and that pathogen-dependent dysbiosis occurred in viral gastroenteritis.

In vitro Anti-Mycobacterium ulcerans and cytotoxic activities of some selected medicinal plants and an indoloquinoline alkaloid.

Background: Buruli ulcer (BU) is a neglected tropical disease caused by the Mycobacterium ulcerans. BU is an endemic disease in many communities in sub-Saharan Africa where population have long history of using medicinal plants for treatment. Indeed, several medicinal plants have been documented against BU and related conditions. The present study was undertaken to prove the efficacy of seven medicinal plants documented for the treatment of mycobacterial infections and related symptoms in Ghana. Method: Antimycobacterial activity of the stem bark extracts and reference control drugs were conducted using the resazurin microtiter assay (REMA) assay method in clear round bottom 96-well microtiter plates. The extracts that showed anti-mycobacterium ulcerans activity were assessed for cytotoxicity using the Alamar blue assay. Results: Overall, The Cryptolepis sanguinolenta root aqueous extract exhibited the highest antimycobacterial activity (MIC=64 µg/mL) followed by Cleistopholis patens (MIC=256 µg/mL). Based on the marked activity of the Cryptolepis sanguinolenta extracts, pure cryptolepine, its major metabolite recorded a MIC value of 32 µg/mL. These extracts with considerable antimycobacterial activity showed 50% cytotoxic concentration (CC50) ranging from 94 to 384 µg/mL. Conclusions: Thus, Cleistopholis patens and Cryptolepis sanguinolenta are primed for further studies and could afford novel drugs for the mitigation of buruli ulcer disease.

Molecular epidemiology of Mycobacterium tuberculosis complex in the Volta Region of Ghana.

CONTEXT: Available molecular epidemiological data from recent studies suggest significant genetic variation between the different lineages of Mycobacterium tuberculosis complex (MTBC) and the MTBC lineages might have adapted to different human populations. AIM: This study sought to determine the population structure of clinical MTBC isolates from the Volta Region of Ghana. METHODS: The MTBC isolates obtained from collected sputum samples were identified by PCR detecting of IS6110 and genotyped using spoligotyping. Non-tuberculous mycobacterial isolates were characterized by amplification of the heat shock protein 65 (hsp65) gene and sequencing. The drug susceptibility profiles of the MTBCs determined using GenoType MTBDRplus. RESULTS: One hundred and seventeen (117, 93.6%) out of 125 mycobacterial positive isolates were characterized as members of the MTBC of which M. tuberculosis sensu stricto (MTBss) and M. africanum (MAF) were respectively 94 (80.3%) and 23 (19.7%). In all, 39 distinct spoligotype patterns were obtained; 26 for MTBss and 13 for MAF lineages. Spoligotyping identified 89 (76%) Lineage 4, 16 (13.6%) Lineage 5, 7 (6.0%) Lineage 6, 3 (2.6%) Lineage 2, 1(0.9%) Lineage 3 and 1 (0.9%) Lineage 1. Among the Lineage 4 isolates, 62/89 (69.7%) belonged to Cameroon sub-lineage, 13 (14.7%) Ghana, 8 (9.0%) Haarlem, 2 (2.2%) LAM, 1 (1.1%) Uganda I, 1 (1.1%) X and the remaining two (2.2%) were orphan. Significant localization of MAF was found within the Ho municipality (n = 13, 29.5%) compared to the more cosmopolitan Ketu-South/Aflao (n = 3, 8.3%) (p-value = 0.017). Eight (8) non-tuberculous mycobacteria were characterized as M. abscessus (7) and M. fortuitum (1). CONCLUSION: We confirmed the importance of M. africanum lineages as a cause of TB in the Volta region of Ghana.

Genetic Analysis of TB Susceptibility Variants in Ghana Reveals Candidate Protective Loci in SORBS2 and SCL11A1 Genes.

Despite advancements made toward diagnostics, tuberculosis caused by Mycobacterium africanum (Maf) and Mycobacterium tuberculosis sensu stricto (Mtbss) remains a major public health issue. Human host factors are key players in tuberculosis (TB) outcomes and treatment. Research is required to probe the interplay between host and bacterial genomes. Here, we explored the association between selected human/host genomic variants and TB disease in Ghana. Paired host genotype datum and infecting bacterial isolate information were analyzed for associations using a multinomial logistic regression. Mycobacterium tuberculosis complex (MTBC) isolates were obtained from 191 TB patients and genotyped into different phylogenetic lineages by standard methods. Two hundred and thirty-five (235) nondisease participants were used as healthy controls. A selection of 29 SNPs from TB disease-associated genes with high frequency among African populations was assayed using a TaqMan® SNP Genotyping Assay and iPLEX Gold Sequenom Mass Genotyping Array. Using 26 high-quality SNPs across 326 case-control samples in an association analysis, we found a protective variant, rs955263, in the SORBS2 gene against both Maf and Mtb infections (P BH  = 0.05; OR = 0.33; 95% CI = 0.32-0.34). A relatively uncommon variant, rs17235409 in the SLC11A1 gene was observed with an even stronger protective effect against Mtb infection (MAF = 0.06; PBH = 0.04; OR = 0.05; 95% CI = 0.04-0.05). These findings suggest SLC11A1 and SORBS2 as a potential protective gene of substantial interest for TB, which is an important pathogen in West Africa, and highlight the need for in-depth host-pathogen studies in West Africa.

A molecular and epidemiological study of Vibrio cholerae isolates from cholera outbreaks in southern Ghana.

Cholera remains a major global public health threat and continuous emergence of new Vibrio cholerae strains is of major concern. We conducted a molecular epidemiological study to detect virulence markers and antimicrobial resistance patterns of V. cholerae isolates obtained from the 2012-2015 cholera outbreaks in Ghana. Archived clinical isolates obtained from the 2012, 2014 and 2015 cholera outbreaks in Ghana were revived by culture and subjected to microscopy, biochemical identification, serotyping, antibiotic susceptibility testing, molecular detection of distinct virulence factors and Multi-Locus Variable-Number of Tandem-Repeat Analysis (MLVA). Of 277 isolates analysed, 168 (60.6%) were confirmed to be V. cholerae and 109 (39.4%) isolates constituted other bacteria (Escherichia coli, Aeromonas sobria, Pseudomonas aeruginosa, Enterobacter cloacae and Enterococci faecalis). Serotyping the V. cholerae isolates identified 151 (89.9%) as Ogawa, 3 (1.8%) as Inaba and 14 (8.3%) as non-O1/O139 serogroup. The O1 serogroup isolates (154/168, 91.7%) carried the cholera toxin ctxB gene as detected by PCR. Additional virulence genes detected include zot, tcpA, ace, rtxC, toxR, rtxA, tcpP, hlyA and tagA. The most common and rare virulence factors detected among the isolates were rtxC (165 isolates) and tcpP (50 isolates) respectively. All isolates from 2014 and 2015 were multidrug resistant against the selected antibiotics. MLVA differentiated the isolates into 2 large unique clones A and B, with each predominating in a particular year. Spatial analysis showed clustering of most isolates at Ablekuma sub-district. Identification of several virulence genes among the two different genotypes of V. cholerae isolates and resistance to first- and second-line antibiotics, calls for scaleup of preventive strategies to reduce transmission, and strengthening of public health laboratories for rapid antimicrobial susceptibility testing to guide accurate treatment. Our findings support the current WHO licensed cholera vaccines which include both O1 Inaba and Ogawa serotypes.

Molecular epidemiology and whole genome sequencing analysis of clinical Mycobacterium bovis from Ghana.

BACKGROUND: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is a re-emerging problem in both livestock and humans. The association of some M. bovis strains with hyper-virulence, MDR-TB and disseminated disease makes it imperative to understand the biology of the pathogen. METHODS: Mycobacterium bovis (15) among 1755 M. tuberculosis complex (MTBC) isolated between 2012 and 2014 were characterized and analyzed for associated patient demography and other risk factors. Five of the M. bovis isolates were whole-genome sequenced and comparatively analyzed against a global collection of published M. bovis genomes. RESULTS: Mycobacterium bovis was isolated from 3/560(0.5%) females and 12/1195(1.0%) males with pulmonary TB. The average age of M. bovis infected cases was 46.8 years (7-72years). TB patients from the Northern region of Ghana (1.9%;4/212) had a higher rate of infection with M. bovis (OR = 2.7,p = 0.0968) compared to those from the Greater Accra region (0.7%;11/1543). Among TB patients with available HIV status, the odds of isolating M. bovis from HIV patients (2/119) was 3.3 higher relative to non-HIV patients (4/774). Direct contact with livestock or their unpasteurized products was significantly associated with bTB (p<0.0001, OR = 124.4,95% CI = 30.1-508.3). Two (13.3%) of the M. bovis isolates were INH resistant due to the S315T mutation in katG whereas one (6.7%) was RIF resistant with Q432P and I1491S mutations in rpoB. M. bovis from Ghana resolved as mono-phyletic branch among mostly M. bovis from Africa irrespective of the host and were closest to the root of the global M. bovis phylogeny. M. bovis-specific amino acid mutations were detected among MTBC core genes such as mce1A, mmpL1, pks6, phoT, pstB, glgP and Rv2955c. Additional mutations P6T in chaA, G187E in mgtC, T35A in Rv1979c, S387A in narK1, L400F in fas and A563T in eccA1 were restricted to the 5 clinical M. bovis from Ghana. CONCLUSION: Our data indicate potential zoonotic transmission of bTB in Ghana and hence calls for intensified public education on bTB, especially among risk groups.

Wood-decaying fungi found in Southern Ghana: A potential source of new anti-infective compounds.

Background: Discovery of bioactive natural products are instrumental for development of novel antibiotics. The discovery and development of natural products such as penicillin represented a major milestone in the treatment of bacterial infections. Currently, many antibiotics have lost their relevance in clinics due to the emergence of drug-resistant microbial pathogens. Hence, there is the need for continuous search of new compounds endowed with potent antimicrobial activity. Methods: In this study, wood-decaying fungi (WDF) from Ghana were explored for their potential as sources of novel antimicrobial compounds with intent of expanding the effort into a drug discovery programme in the near future. Extracts from cultures of 54 morphologically distinct WDF isolates were analyzed for the presence of antimicrobial agents. Results: The extracts from 40 out of the 54 WDF isolates exhibited significant antimicrobial activity against either Staphylococcus aureus, Escherichia coli or Candida albicans. Fractionation of these bioactive extracts, followed by bioassay of the organic fractions obtained, indicate that extracts exhibiting antimicrobial activity against more than one of the three test organisms could be attributed to the presence of different bioactive compounds. Analysis of the composition of the extracts revealed that terpenes were predominant. Conclusions: This study suggests that a significant proportion of WDF in Ghana produce antimicrobial compounds which could be potential sources of novel anti-infective agents and support the plans of developing a drug discovery programme in Ghana based on the fermentation of WDF.

Comparative genomics of Mycobacterium africanum Lineage 5 and Lineage 6 from Ghana suggests distinct ecological niches.

Mycobacterium africanum (Maf) causes a substantial proportion of human tuberculosis in some countries of West Africa, but little is known on this pathogen. We compared the genomes of 253 Maf clinical isolates from Ghana, including N = 175 Lineage 5 (L5) and N = 78 Lineage 6 (L6). We found that the genomic diversity of L6 was higher than in L5 despite the smaller sample size. Regulatory proteins appeared to evolve neutrally in L5 but under purifying selection in L6. Even though over 90% of the human T cell epitopes were conserved in both lineages, L6 showed a higher ratio of non-synonymous to synonymous single nucleotide variation in these epitopes overall compared to L5. Of the 10% human T cell epitopes that were variable, most carried mutations that were lineage-specific. Our findings indicate that Maf L5 and L6 differ in some of their population genomic characteristics, possibly reflecting different selection pressures linked to distinct ecological niches.

Susceptibility to Mycobacterium ulcerans Disease (Buruli ulcer) Is Associated with IFNG and iNOS Gene Polymorphisms.

Buruli ulcer (BU) is a chronic necrotizing disease of the skin and subcutaneous fat tissue. The causative agent, Mycobacterium ulcerans, produces mycolactone, a macrolide toxin, which causes apoptosis of mammalian cells. Only a small proportion of individuals exposed to M. ulcerans develop clinical disease, as surrounding macrophages may control the infection by bacterial killing at an early stage, while mycolactone concentration is still low. Otherwise, bacterial multiplication leads to in higher concentrations of mycolactone, with formation of necrotizing lesions that are no more accessible to immune cells. By typing a cohort of 96 Ghanaian BU patients and 384 endemic controls without BU, we show an association between BU and single nucleotide polymorphisms (SNPs) in iNOS (rs9282799) and IFNG (rs2069705). Both polymorphisms influence promoter activity in vitro. A previously reported SNP in SLC11A1 (NRAMP, rs17235409) tended to be associated with BU. Altogether, these data reflect the importance of IFNG signaling in early defense against M. ulcerans infection.

Environmental and Behavioral Drivers of Buruli Ulcer Disease in Selected Communities Along the Densu River Basin of Ghana: A Case-Control Study.

AbstractThe exact route of transmission of Mycobacterium ulcerans (MU) (causative agent of Buruli ulcer [BU]), risk factors, and reservoir hosts are not clearly known, although it has been identified as an environmental pathogen. This study assessed potential environmental and behavioral risk factors that influence BU infections. We conducted a case-control study where cases were matched by their demographic characteristics and place of residence. A structured questionnaire was administered to solicit information on the environmental and behavioral factors of participants that may expose them to infection. A total of 176 cases and 176 controls were enrolled into the study. Multivariate conditional logistic regression analysis identified farming in swampy areas (odds ratio [OR] = 4.10, 95% confidence interval [CI] = 3.82-7.18), farming while wearing short clothing (OR = 1,734.1, 95% CI = 68.1-44,120.9), insect bite (OR = 988.3, 95% CI = 31.4-31,115.6), and application of leaves on wounds (OR = 6.23, 95% CI = 4.74-18.11) as potential risk factors. Farming in long clothing (OR = 0.000, 95% CI = 0.00-0.14), washing wound with water and soap (OR = 0.37, 95% CI = 0.29-0.98), and application of adhesive bandage on wounds (OR = 0.31, 95% CI = 0.15-0.82) were found to be protective against BU infection. In the absence of the exact MU transmission mechanisms, education of public in BU-endemic zones on the use of protective clothing during farming activities to limit exposure of the skin and proper wound care management would be essential in the fight against BU.

Genomic analysis of ST88 community-acquired methicillin resistant Staphylococcus aureus in Ghana.

BACKGROUND: The emergence and evolution of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as an "African" CA-MRSA clone. In this study, we employed whole genome sequencing to provide more information on the genetic background of ST88 CA-MRSA isolates from Ghana and to describe in detail ST88 CA-MRSA isolates in comparison with other MRSA lineages worldwide. METHODS: We first established a complete ST88 reference genome (AUS0325) using PacBio SMRT sequencing. We then used comparative genomics to assess relatedness among 17 ST88 CA-MRSA isolates recovered from patients attending Buruli ulcer treatment centres in Ghana, three non-African ST88s and 15 other MRSA lineages. RESULTS: We show that Ghanaian ST88 forms a discrete MRSA lineage (harbouring SCCmec-IV [2B]). Gene content analysis identified five distinct genomic regions enriched among ST88 isolates compared with the other S. aureus lineages. The Ghanaian ST88 isolates had only 658 core genome SNPs and there was no correlation between phylogeny and geography, suggesting the recent spread of this clone. The lineage was also resistant to multiple classes of antibiotics including ß-lactams, tetracycline and chloramphenicol. DISCUSSION: This study reveals that S. aureus ST88-IV is a recently emerging and rapidly spreading CA-MRSA clone in Ghana. The study highlights the capacity of small snapshot genomic studies to provide actionable public health information in resource limited settings. To our knowledge this is the first genomic assessment of the ST88 CA-MRSA clone.

Assessing and managing wounds of Buruli ulcer patients at the primary and secondary health care levels in Ghana.

BACKGROUND: Beyond Mycobacterium ulcerans-specific therapy, sound general wound management is required for successful management of Buruli ulcer (BU) patients which places them among the large and diverse group of patients in poor countries with a broken skin barrier. METHODS: Clinically BU suspicious patients were enrolled between October 2013 and August 2015 at a primary health care (PHC) center and a municipal hospital, secondary health care (SHC) center in Ghana. All patients were IS2404 PCR tested and divided into IS2404 PCR positive and negative groups. The course of wound healing was prospectively investigated including predictors of wound closure and assessment of infrastructure, supply and health staff performance. RESULTS: 53 IS2404 PCR positive patients-31 at the PHC center and 22 at the SHC center were enrolled-and additionally, 80 clinically BU suspicious, IS2404 PCR negative patients at the PHC center. The majority of the skin ulcers at the PHC center closed, without the need for surgical intervention (86.7%) compared to 40% at the SHC center, where the majority required split-skin grafting (75%) or excision (12.5%). Only 9% of wounds at the PHC center, but 50% at the SHC center were complicated by bacterial infection. The majority of patients, 54.8% at the PHC center and 68.4% at the SHC center, experienced wound pain, mostly severe and associated with wound dressing. Failure of ulcers to heal was reliably predicted by wound area reduction between week 2 and 4 after initiation of treatment in 75% at the PHC center, and 90% at the SHC center. Obvious reasons for arrested wound healing or deterioration of wound were missed additional severe pathology; at the PHC center (chronic osteomyelitis, chronic lymphedema, squamous cell carcinoma) and at the SHC center (malignant ulceration, chronic lymphedema) in addition to hygiene and wound care deficiencies. When clinically suspicious, but IS2404 PCR negative patients were recaptured in the community, 76/77 (98.7%) of analyzed wounds were either completely closed (85.7%) or almost closed (13%). Five percent were found to have important missed severe pathology (chronic osteomyelitis, ossified fibroma and suspected malignancy). CONCLUSION: The wounds of most BU patients attending the primary health care level can be adequately managed. Additionally, the patients are closer to their families and means of livelihood. Non-healing wounds can be predicted by wound area reduction between 2 to 4 weeks after initiation of treatment. Patients with clinically BU suspicious, but PCR negative ulcers need to be followed up to capture missed diagnoses.

Isolation and characterization of nontuberculous mycobacteria from patients with pulmonary tuberculosis in Ghana.

OBJECTIVE/BACKGROUND: Nontuberculous mycobacterial (NTM) species are assuming public health importance in pulmonary diseases; they are increasingly being isolated, and importantly, most NTMs do not respond to routine tuberculosis (TB) drugs. This study aimed to identify NTMs isolated from pulmonary TB cases and also determine their susceptibility to streptomycin (STR), isoniazid (INH), and rifampicin (RIF). METHODS: A total of 1755 mycobacterial isolates, obtained between August 2012 and July 2014, from 2036 smear-positive pulmonary cases were identified using polymerase chain reaction amplification of IS6110, and hsp65 gene sequencing analysis. Drug susceptibility testing (DST) was then performed for the identified NTMs against STR, INH, and RIF using microplate Alamar blue assay. The results were analyzed against patients' biodata for statistical associations. RESULTS: Of the 1755 analyzed isolates, we identified 43 (2.5%) NTMs, which included 18 (41.9%) Mycobacterium intracellulare, 13 (30.2%) Mycobacterium avium subs. paratuberculosis, 5 (11.3%) Mycobacterium abscessus, 3 (7.0%) each of Mycobacterium mucogenicum and Mycobacterium colombiense, and 1 (2.3%) Mycobacterium simiae. Patients infected with NTMs (52.0%) were more likely to be human immunodeficiency virus-positive (P = 0.001, odds ratio = 6.6, 95% confidence interval = 2.7-16.2) than those infected with M. tuberculosis complex (5.8%). All the 43 (100%) NTMs were resistant to INH, whereas 32 (74%) and 19 (44%) were resistant to RIF and STR, respectively. Furthermore, 16 (37.2%) NTMs were resistant to all three drugs, 20 were resistant to INH and RIF, and 3 were resistant to STR and INH. All the M. abscessus isolates were resistant to all the three drugs, whereas all the M. avium isolates were resistant to INH and RIF, but only three were resistant to STR. Among the M. intracellulare isolates, 8, 18, and 15 isolates were resistant to STR, INH, and RIF, respectively. CONCLUSION: The observed high-resistance level to INH and RIF supports the need for rapid species identification and DST of nonresponding TB cases before retreatment.

Seasonal Pattern of Mycobacterium ulcerans, the Causative Agent of Buruli Ulcer, in the Environment in Ghana.

This study aimed to contribute to the understanding of Mycobacterium ulcerans (MU) ecology by analysing both clinical and environmental samples collected from ten communities along two major river basins (Offin and Densu) associated with Buruli ulcer (BU) at different seasons. We collected clinical samples from presumptive BU cases and environmental samples from ten communities. Following DNA extraction, clinical samples were confirmed by IS2404 PCR and environmental samples were confirmed by targeting MU-specific genes, IS2404, IS2606 and the ketoreductase (KR) using real-time PCR. Environmental samples were first analysed for IS2404; after which, IS2404-positive samples were multiplexed for the IS2606 and KR gene. Our findings indicate an overall decline in BU incidence along both river basins, although incidence at Densu outweighs that of Offin. Overall, 1600 environmental samples were screened along Densu (434, 27 %) and Offin (1166, 73 %) and MU was detected in 139 (9 %) of the combined samples. The positivity of MU along the Densu River basin was 89/434 (20.5 %), whilst that of the Offin River basin was 50/1166 (4.3 %). The DNA was detected mainly in snails (5/6, 83 %), moss (8/40, 20 %), soil (55/586, 9 %) and vegetation (55/675, 8 %). The proportion of MU positive samples recorded was higher during the months with higher rainfall levels (126/1175, 11 %) than during the dry season months (13/425, 3 %). This study indicates for the first time that there is a seasonal pattern in the presence of MU in the environment, which may be related to recent rainfall or water in the soil.

RNA Aptamer That Specifically Binds to Mycolactone and Serves as a Diagnostic Tool for Diagnosis of Buruli Ulcer.

BACKGROUND: Buruli ulcer (BU) is a subcutaneous skin disease listed among the neglected tropical diseases by the World Health Organization (WHO). Early case detection and management is very important to reduce morbidity and the accompanied characteristic disfiguring nature of BU. Since diagnosis based on clinical evidence can lead to misdiagnosis, microbiological confirmation is essential to reduce abuse of drugs; since the anti-mycobacterial drugs are also used for TB treatment. The current WHO gold standard PCR method is expensive, requires infrastructure and expertise are usually not available at the peripheral centers where BU cases are managed. Thus one of the main research agendas is to develop methods that can be applied at the point of care. In this study we selected aptamers, which are emerging novel class of detection molecules, for detecting mycolactone, the first to be conducted in a BUD endemic country. METHODS: Aptamers that bind to mycolactone were isolated by the SELEX process. To measure their affinity and specificity to mycolactone, the selected aptamers were screened by means of isothermal titration calorimetry (ITC) and an enzyme-linked oligonucleotide assay (ELONA). Selected aptamers were assessed by ELONA using swab samples from forty-one suspected BU patients with IS2404 PCR and culture as standard methods. ROC analysis was used to evaluate their accuracy and cutoff-points. RESULTS: Five out of the nine selected aptamers bound significantly (p< 0.05) to mycolactone, of these, three were able to distinguish between mycolactone producing mycobacteria, M. marinum (CC240299, Israel) and other bacteria whilst two others also bounded significantly to Mycobacterium smegmatis. Their dissociation constants were in the micro-molar range. At 95% confidence interval, the ROC curve analysis among the aptamers at OD450 ranged from 0.5-0.7. Using this cut-off for the ELONA assay, the aptamers had 100% specificity and sensitivity between 0.0% and 50.0%. The most promising aptamer, Apt-3683 showed a discernible cleavage difference relative to the non-specific autocatalysis over a 3-minute time course. CONCLUSION: This preliminary proof-of-concept indicates that diagnosis of BUD with RNA aptamers is feasible and can be used as point of care upon incorporation into a diagnostic platform.

Clinical and Laboratory Diagnosis of Buruli Ulcer Disease: A Systematic Review.

Background. Buruli ulcer (BU) is a necrotizing cutaneous infection caused by Mycobacterium ulcerans. Early diagnosis is crucial to prevent morbid effects and misuse of drugs. We review developments in laboratory diagnosis of BU, discuss limitations of available diagnostic methods, and give a perspective on the potential of using aptamers as point-of-care. Methods. Information for this review was searched through PubMed, web of knowledge, and identified data up to December 2015. References from relevant articles and reports from WHO Annual Meeting of the Global Buruli Ulcer initiative were also used. Finally, 59 articles were used. Results. The main laboratory methods for BU diagnosis are microscopy, culture, PCR, and histopathology. Microscopy and PCR are used routinely for diagnosis. PCR targeting IS2404 is the gold standard for laboratory confirmation. Culture remains the only method that detects viable bacilli, used for diagnosing relapse and accrued isolates for epidemiological investigation as well as monitoring drug resistance. Laboratory confirmation is done at centers distant from endemic communities reducing confirmation to a quality assurance. Conclusions. Current efforts aimed at developing point-of-care diagnostics are saddled with major drawbacks; we, however, postulate that selection of aptamers against MU target can be used as point of care.