Genetic determinants of macrolide and tetracycline resistance in penicillin non-susceptible Streptococcus pneumoniae isolates from people living with HIV in Dar es Salaam, Tanzania

Background: Over one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing. Methods: We assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP. Results: Fifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLSB phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic correlates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes. Conclusion: The erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLSB in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon.publishedVersio

Predominance of PVL-negative community-associated methicillin-resistant Staphylococcus aureus sequence type 8 in newly diagnosed HIV-infected adults, Tanzania

Difficult-to-treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are of concern in people living with HIV infection as they are more vulnerable to infection. We aimed to identify molecular characteristics of MRSA colonizing newly diagnosed HIV-infected adults in Tanzania. Individuals newly diagnosed with HIV infection were recruited in Dar es Salaam, Tanzania, from April 2017 to May 2018, as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890). Nasal/nasopharyngeal isolates of Staphylococcus aureus were susceptibility tested by disk diffusion method, and cefoxitin-resistant isolates were characterized by short-reads whole genome sequencing. Four percent (22/537) of patients carried MRSA in the nose/nasopharynx. MRSA isolates were frequently resistant towards gentamicin (95%), ciprofloxacin (91%), and erythromycin (82%) but less often towards trimethoprim-sulfamethoxazole (9%). Seventy-three percent had inducible clindamycin resistance. Erythromycin-resistant isolates harbored ermC (15/18) and LmrS (3/18) resistance genes. Ciprofloxacin resistance was mediated by mutations of the quinolone resistance-determining region (QRDR) sequence in the gyrA (S84L) and parC (S80Y) genes. All isolates belonged to the CC8 and ST8-SCCmecIV MRSA clone. Ninety-five percent of the MRSA isolates were spa-type t1476, and one exhibited spa-type t064. All isolates were negative for Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) type 1. All ST8-SCCmecIV-spa-t1476 MRSA clones from Tanzania were unrelated to the globally successful USA300 clone. Carriage of ST8 MRSA (non-USA300) was common among newly diagnosed HIV-infected adults in Tanzania. Frequent co-resistance to non-beta lactam antibiotics limits therapeutic options when infection occurs.publishedVersio

High rate of antimicrobial resistance and multiple mutations in the dihydrofolate reductase gene among Streptococcus pneumoniae isolated from HIV-infected adults in a community setting in Tanzania

Objectives The aim of this study was to characterize molecular mechanisms of resistance to trimethoprim and other antibiotics in Streptococcus pneumoniae isolates from HIV-infected adults in Dar es Salaam, Tanzania. Methods A total of 1877 nasopharyngeal swabs were collected and screened for pneumococcal colonization from 537 newly diagnosed individuals with HIV at four clinic visits during a 1-year follow-up from 2017–2018 as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov ID: NCT03087890). Results A total of 76 pneumococcal isolates were obtained. Of the 70 isolates that could be serotyped, 42 (60.0%) were vaccine serotypes included in pneumococcal conjugate vaccine 23 (PCV23). The majority of isolates (73.7%; 56/76) were non-susceptible to penicillin (MICs of 0.06–2 μg/mL). Isolates were frequently resistant to co-trimoxazole (trimethoprim/sulfamethoxazole) (71.1%) but less so to azithromycin (22.4%), erythromycin (21.1%), chloramphenicol (18.4%), tetracycline (14.5%), clindamycin (10.5%) and levofloxacin (0%). Moreover, 26.3% were multidrug-resistant (resistant to ≥3 antibiotic classes). Vaccine-type pneumococci were resistant to more classes of antibiotics, were more frequently resistant to erythromycin, azithromycin, clindamycin and tetracycline, and had higher MICs to penicillin (median, 0.19 μg/mL; range, 0.002–1.5 μg/mL) compared with non-vaccine serotypes (median, 0.125 μg/mL; range, 0.012–0.25 μg/mL) (P = 0.003). Co-trimoxazole-resistant isolates carried from 1 to 11 different mutations in the dihydrofolate reductase (DHFR) gene, most commonly Ile100Leu (100%), Glu20Asp (91.8%), Glu94Asp (61.2%), Leu135Phe (57.1%), His26Tyr (53.1%), Asp92Ala (53.1%) and His120Gln (53.1%). Conclusion Streptococcus pneumoniae isolated from HIV-diagnosed patients were frequently non-susceptible to penicillin and co-trimoxazole. Most isolates carried multiple mutations in DHFR.publishedVersio

High prevalence of fecal carriage of extended spectrum β-lactamase-producing enterobacteriaceae among newly HIV-diagnosed adults in a community setting in Tanzania

Colonization in HIV-infected populations with extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) is particularly worrisome in low-income settings. This study describes the prevalence of ESBL-PE carriage and associated risk factors among newly HIV-diagnosed adults in a community setting in Tanzania. A total of 595 newly diagnosed HIV-positive adults with a median age of 35 years with interquartile range (IQR) 29–42 years and a median CD4 count of 492 cells/μL (IQR 390–666 cells/μL) were recruited. Among these, 194/595 (32.6%, 95% confidence interval [CI] 28.9–36.6) were ESBL-PE carriers. Participants with low CD4 count (<350 cells/μL) had significantly higher prevalence of ESBL-PE carriage compared with those with CD4 count ≥350 cells/μL (26/58, 44.8%, vs. 168/537, 31.3%, p = 0.04). Antibiotic use in last 4 weeks (odds ratio [OR] 1.55, 95% CI 1.08–2.22, p = 0.02) and CD4 count <350 cells/μL (OR 1.78, 95% CI 1.03–3.09, p = 0.04) were independent risk factors for fecal carriage of ESBL-PE. In total, 244 isolates of ESBL-PE were isolated from 194 participants. Of these, 238/244 (97.5%) harbored blaCTX-M genes, with blaCTX-M-15 being predominant (219/238 (92%), followed by blaCTX-M-27 (9/238 (3.8%), blaCTX-M-14 (8/238 (3.4%), blaCTX-M-55 (1/238), and blaCTX-M 211/3 (1/238). blaSHV-2a genes were detected in four isolates, whereas the blaSHV-12 gene was detected in one isolate. Phenotypic carbapenemase-producing Enterobacteriaceae was detected in one HIV-positive person with CD4 count 132 cells/μL. In conclusion prevalence of ESBL-PE carriage is high among newly diagnosed HIV adults in Dar es Salaam, and is significantly associated antibiotic use and low CD4 count.publishedVersio

Genetic diversity of circulating rotavirus strains in Tanzania prior to the introduction of vaccination

Background: Tanzania currently rolls out vaccination against rotavirus-diarrhea, a major cause of child illness and death. As the vaccine covers a limited number of rotavirus variants, this study describes the molecular epidemiology of rotavirus among children under two years in Dar es Salaam, Tanzania, prior to implementation of vaccination. Methods: Stool specimens, demographic and clinical information, were collected from 690 children admitted to hospital due to diarrhea (cases) and 545 children without diarrhea (controls) during one year. Controls were inpatient or children attending child health clinics. Rotavirus antigen was detected using ELISA and positive samples were typed by multiplex semi-nested PCR and sequencing. Results: The prevalence of rotavirus was higher in cases (32.5%) than in controls (7.7%, P,0.001). The most common G genotypes were G1 followed by G8, G12, and G4 in cases and G1, G12 and G8 in controls. The Tanzanian G1 variants displayed 94% similarity with the Rotarix vaccine G1 variant. The commonest P genotypes were P[8], P[4] and P[6], and the commonest G/P combination G1 P[8] (n = 123), G8 P[4] and G12 P[6]. Overall, rotavirus prevalence was higher in cool (23.9%) than hot months (17.1%) of the year (P = 0.012). We also observed significant seasonal variation of G genotypes. Rotavirus was most frequently found in the age group of four to six months. The prevalence of rotavirus in cases was lower in stunted children (28.9%) than in non-stunted children (40.1%, P = 0.003) and lower in HIV-infected (15.4%, 4/26) than in HIVuninfected children (55.3%, 42/76, P,0.001). Conclusion: This pre-vaccination study shows predominance of genotype G1 in Tanzania, which is phylogenetically distantly related to the vaccine strains. We confirm the emergence of genotype G8 and G12. Rotavirus infection and circulating genotypes showed seasonal variation. This study also suggests that rotavirus may not be an opportunistic pathogen in children infected with HIV

Horizontal plasmid transfer among klebsiella pneumoniae isolates is the key factor for dissemination of extended-spectrum β-lactamases among children in Tanzania

Increased knowledge about the role of horizontal gene transfer is key to improve our understanding of the spread of antimicrobial resistance (AMR) in human populations. We therefore studied the dissemination of the blaCTX-M-15 extended-spectrum-β-lactamase (ESBL) gene in Klebsiella pneumoniae isolates obtained from stool samples from hospitalized children and healthy controls below 2 years of age in Dar es Salaam, Tanzania, from August 2010 to July 2011. We performed Illumina whole-genome sequencing (WGS) to characterize resistance genes, multilocus sequence type (MLST), plasmid incompatibility group (Inc), and plasmid MLST of 128 isolates of K. pneumoniae with blaCTX-M-15 recovered from both healthy and hospitalized children. We assessed the phylogenetic relationship using single nucleotide polymorphism (SNP)-based analysis and resolved the sequences of five reference plasmids by Oxford Nanopore technology to investigate plasmid dissemination. The WGS analyses revealed the presence of a blaCTX-M-15-positive IncFIIK5/IncR plasmid with a highly conserved backbone in 70% (90/128) of the isolates. This plasmid, harboring genes encoding resistance to most β-lactams, aminoglycosides, trimethoprim-sulfamethoxazole, and chloramphenicol, was present in phylogenetically very diverse K. pneumoniae strains (48 different MLSTs) carried by both hospitalized and healthy children. Our data strongly suggest widespread horizontal transfer of this ESBL-carrying plasmid both in hospitals and in the general population. IMPORTANCE - Horizontal spread of plasmids carrying multiple resistance genes is considered an important mechanism behind the global health problem caused by multidrug-resistant bacteria. Nevertheless, knowledge about spread of plasmids in a community is limited. Our detailed molecular analyses of K. pneumoniae isolated from hospitalized and healthy children in Tanzania disclosed an epidemic spread of a resistance plasmid. In this study population, we revealed horizontal plasmid transfer among K. pneumoniae as the key factor for dissemination of ESBLs. Traditional outbreak investigation and surveillance focus on the spread of bacterial clones, and short-read sequencing can result in erroneous plasmid composition. Our approach using long-read sequencing reveals horizontal gene transfer of antimicrobial resistance, and therefore has a potential impact on outbreak investigations and approaches to limit spread of AMR

Oocyst Shedding Dynamics in Children with Cryptosporidiosis: a Prospective Clinical Case Series in Ethiopia

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Oocyst Shedding Dynamics in Children with Cryptosporidiosis: a Prospective Clinical Case Series in Ethiopia

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A comparison of risk factors for cryptosporidiosis and non-cryptosporidiosis diarrhoea: A case-case-control study in Ethiopian children

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Effect of Micronutrient Supplementation on Disease Progression in Asymptomatic, Antiretroviral-Naive, HIV-Infected Adults in Botswana

IMPORTANCE Micronutrient deficiencies occur early in human immunodeficiency virus (HIV) infection, and supplementation with micronutrients may be beneficial; however, its effectiveness has not been investigated early in HIV disease among adults who are antiretroviral therapy (ART) naive. OBJECTIVE To investigate whether long-term micronutrient supplementation is effective and safe in delaying disease progression when implemented early in adults infected with HIV subtype C who are ART-naive. DESIGN, SETTING, AND PARTICIPANTS Randomized clinical trial of supplementation with either daily multivitamins (B vitamins and vitamins C and E), seleniumalone, or multivitamins with selenium vs placebo inafactorial design for 24 months. The study was conducted in 878 patients infected with HIV subtype C with a CD4 cell count greater than 350/μL who were not receiving ART at Princess Marina Hospital in Gaborone, Botswana, between December 2004 and July 2009. INTERVENTIONS Daily oral supplements of B vitamins and vitamins C and E, selenium alone, or multivitamins plus selenium, compared with placebo. MAIN OUTCOMES AND MEASURES Reaching a CD4 cell count less than 200/μL until May 2008; after this date, reaching a CD4 cell count of 250/μL or less, consistent with the standard of care in Botswana for initiation of ART at the time of the study. RESULTS There were 878 participants enrolled and randomized into the study. All participants were ART-naive throughout the study. In intent-to-treat analysis, participants receiving the combined supplement of multivitamins plus selenium had a significantly lower risk vs placebo of reaching CD4 cell count 250/μL or less (adjusted hazard ratio [HR], 0.46; 95% CI, 0.25-0.85; P = .01; absolute event rate [AER], 4.79/100 person-years; censoring rate, 0.92; 17 events; placebo AER, 9.22/100 person-years; censoring rate, 0.85; 32 events). Multivitamins plus selenium in a single supplement, vs placebo, also reduced the risk of secondary events of combined outcomes for disease progression (CD4 cell count ≤250/μL, AIDS-defining conditions, or AIDS-related death, whichever occurred earlier [adjusted HR, 0.56; 95% CI, 0.33-0.95; P = .03; AER, 6.48/100 person-years; censoring rate, 0.90; 23 events]). There was no effect of supplementation on HIV viral load. Multivitamins alone and selenium supplementation alone were not statistically different from placebo for any end point. Reported adverse events were adjudicated as unlikely to be related to the intervention, and there were no notable differences in incidence of HIV-related and health-related events among study groups. CONCLUSIONS AND RELEVANCE In ART-naive HIV-infected adults, 24-month supplementation with a single supplement containing multivitamins and selenium was safe and significantl