Investigating Metabolic and Molecular Ecological Evolution of Opportunistic Pulmonary Fungal Coinfections: Protocol for a Laboratory-Based Cross-Sectional Study.

BACKGROUND: Fungal-bacterial cocolonization and coinfections pose an emerging challenge among patients suspected of having pulmonary tuberculosis (PTB); however, the underlying pathogenic mechanisms and microbiome interactions are poorly understood. Understanding how environmental microbes, such as fungi and bacteria, coevolve and develop traits to evade host immune responses and resist treatment is critical to controlling opportunistic pulmonary fungal coinfections. In this project, we propose to study the coexistence of fungal and bacterial microbial communities during chronic pulmonary diseases, with a keen interest in underpinning fungal etiological evolution and the predominating interactions that may exist between fungi and bacteria. OBJECTIVE: This is a protocol for a study aimed at investigating the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections through determining and characterizing the burden, etiological profiles, microbial communities, and interactions established between fungi and bacteria as implicated among patients with presumptive PTB. METHODS: This will be a laboratory-based cross-sectional study, with a sample size of 406 participants. From each participant, 2 sputa samples (one on-spot and one early morning) will be collected. These samples will then be analyzed for both fungal and bacterial etiology using conventional metabolic and molecular (intergenic transcribed spacer and 16S ribosomal DNA-based polymerase chain reaction) approaches. We will also attempt to design a genome-scale metabolic model for pulmonary microbial communities to analyze the composition of the entire microbiome (ie, fungi and bacteria) and investigate host-microbial interactions under different patient conditions. This analysis will be based on the interplays of genes (identified by metagenomics) and inferred from amplicon data and metabolites (identified by metabolomics) by analyzing the full data set and using specific computational tools. We will also collect baseline data, including demographic and clinical history, using a patient-reported questionnaire. Altogether, this approach will contribute to a diagnostic-based observational study. The primary outcome will be the overall fungal and bacterial diagnostic profile of the study participants. Other diagnostic factors associated with the etiological profile, such as incidence and prevalence, will also be analyzed using univariate and multivariate schemes. Odds ratios with 95% CIs will be presented with a statistical significance set at P<.05. RESULTS: The study has been approved by the Mbarara University Research Ethic Committee (MUREC1/7-07/09/20) and the Uganda National Council of Science and Technology (HS1233ES). Following careful scrutiny, the protocol was designed to enable patient enrollment, which began in March 2022 at Mbarara University Teaching Hospital. Data collection is ongoing and is expected to be completed by August 2023, and manuscripts will be submitted for publication thereafter. CONCLUSIONS: Through this protocol, we will explore the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections among patients with presumptive PTB. Establishing key fungal-bacterial cross-kingdom synergistic relationships is crucial for instituting fungal bacterial coinfecting etiology. TRIAL REGISTRATION: ISRCTN Registry ISRCTN33572982; https://tinyurl.com/caa2nw69. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/48014.

Women's support for voluntary medical male circumcision in fishing communities on the shores of Lake Victoria, Uganda.

BACKGROUND: Women's support can improve uptake of voluntary medical male circumcision (VMMC). We assessed the level of women's support for VMMC and associated factors in fishing settlements on the shores of Lake Victoria in Uganda, to inform interventions aimed at increasing the uptake of safe male circumcision services in such high-risk populations. METHODS: We conducted a cross-sectional study, employing mixed methods of data collection, at Kasenyi and Kigungu landing sites in April 2018. We included women aged 18-49 years, who had stayed at the landing sites for ≥3 months. We obtained qualitative data using focus group discussions (FGDs), and interviewer-administered semi-structured questionnaires for quantitative data. The tool captured demographic characteristics, community factors including cultural norms and beliefs, women's experiences, and health facility-related factors. The dependent variable was derived from the response to the question: "Would you encourage your partner/husband to go for VMMC?", and used as a proxy for support of VMMC. We used modified Poisson regression to identify factors associated with women's support for VMMC. Qualitative data were analysed using thematic content analysis. RESULTS: We enrolled 313 women with a mean age of 28 (SD±6.8) years. Of the 313 women, 230 (73.5%) supported VMMC. Belief that VMMC increases penile hygiene (Adjusted prevalence ratio [aPR]=1.9; CI: 1.8-3.2), performing VMMC for religious reasons (aPR=1.9; CI: 1.8-2.9), preference for a circumcised man (aPR=1.3; CI: 1.2-1.5), belief that vaginal fluids facilitate wound healing (aPR=1.9; CI: 1.3-2.7), and knowledge about when a man can resume sex (4 weeks) after circumcision (aPR=2.1; CI: 1.8-3.3) were associated with women's support for VMMC. FGDs revealed that women were not adequately involved in VMMC activities for decision making. CONCLUSION: The support for VMMC was high among women in the fishing communities. However, women perceived they were not involved in decision-making for VMMC and had several misconceptions, including a belief that vaginal fluids facilitate wound healing. The Ministry of Health and VMMC implementing partners should devise strategies to increase sensitization and involvement of women in VMMC decision-making without slowing service uptake.

Status of pulmonary fungal pathogens among individuals with clinical features of pulmonary tuberculosis at Mbarara University Teaching Hospital in Southwestern Uganda.

BACKGROUND: Pulmonary mycoses are important diseases of the respiratory tract caused by pulmonary fungal pathogens. These pathogens are responsible for significant morbidity and mortality rates worldwide; however, less attention has been paid to them. In this study we determined the prevalence of pulmonary fungal pathogens among individuals with clinical features of pulmonary tuberculosis at Mbarara Regional Referral Hospital. METHOD: This was a hospital based cross sectional survey. Sputum samples were collected from each study participant. For each sample, the following tests were performed: Sabouraud dextrose agar for fungal culture, GeneXpert for Mycobacteria tuberculosis (MTB) and potassium hydroxide for fungal screening. Filamentous fungal growth and yeasts were further examined with lactophenol cotton blue staining and germ tube respectively. RESULTS: Out of 113 study participants, 80 (70.7%) had pulmonary fungal pathogens whilst those with pulmonary tuberculosis numbered five (4.4%). Candida albicans [21 (22.58%)] and Aspergillus species [16 (17.20%)] were the pathogens most identified among others. Two (1.7%) TB GeneXpert positive participants had fungal pathogens isolated from their sputum samples. We established a prevalence of 57 (71.3%) for pulmonary fungal pathogen (PFP) isolates, three (60.0%) for MTB in HIV positive patients and 18 (22.5%) for PFP, and zero (0.0%) for MTB in HIV negative patients. On the other hand, two (100%) HIV positive patients had both PFP isolates and MTB. CONCLUSION: Our findings highlight the diversity of neglected pulmonary fungal pathogens whose known medical importance in causing pulmonary mycoses cannot be overemphasised. Therefore this presents a need for routine diagnosis for pulmonary mycoses among TB suspects and set-up of antimicrobial profile for pulmonary fungal isolates to support clinical management of these cases.

Antimicrobial resistance in pathogenic aerobic bacteria causing surgical site infections in Mbarara regional referral hospital, Southwestern Uganda.

Surgical site infections (SSI) remain a common postoperative complication despite use of prophylactic antibiotics and other preventive measures, mainly due to increasing antimicrobial resistance. Here, we present antimicrobial resistance rate of bacteria isolated in clinical cases of SSI. A hospital based descriptive cross sectional study was conducted on 83 consented postoperative patients with clinical SSI. Data on patients was obtained using structured data collection form. Two swabs were collected aseptically from each patient. Bacteriological culture examination and identification was done following standard microbiological techniques. Antibiotic susceptibility test was done by Kirby-Bauer disc diffusion method. Gram negative bacteria (GNB) were predominant (65.59%) with the dominant being Klebsiella species (29.03%). Overall 86% of aerobic bacteria isolated were multidrug resistant (MDR) where 65.63% and 96.72% of Gram positive and Gram negative isolates were MDR respectively. All the isolates with exception of Enterococci species were resistant to ampicillin. GNB showed high resistance to ceftriaxone, sulfamethoxazole/trimethoprim and gentamicin. All the isolated Klebsiella spp were MDR. S. aureus were all resistant to oxacillin. The isolation rate was higher in emergency, males and dirty wounds in relation to nature of surgery, gender and class of surgical wound respectively. These findings necessitate judicious antibiotic use and calls for surveillance of SSIs periodically as well as strict adherence to good sanitation practice to reduce spread of drug-resistant pathogens.

Phenotypic prevalence of extended spectrum beta-lactamases among enterobacteriaceae isolated at Mulago National Referral Hospital: Uganda.

OBJECTIVE: Enterobacteriaceae, common causes of health care associated and community acquired infections are mainly treated with beta-lactam agents. Our study objective was to determine the prevalence and common enterobacteriaceae pathogen producing extended spectrum beta lactamases (ESBLs). The isolates were recovered from various clinical specimens. This was cross sectional study conducted between July 2016 and September 2016 at Mulago National Referral Hospital, Uganda. We used ChromID™ ESBL agar (Biomerieux SA, Lyon, France) and Vitek2 compact system GN83 card (BioMerieux Inc, Hazelwood, Missouri, USA) to detect and confirm presence of phenotypic extended spectrum beta lactamases producing pathogens respectively. RESULTS: Of the 261 tested clinical isolates, 35 (13.4%) were identified as ESBLs producing bacteria. Escherichia coli predominated in the samples [18 (51.4%)], presenting the highest frequency of ESBLs producing, followed by Klebsiella pneumonia [10 (28.5%)], Proteus mirabilis [4 (11.4%)], Enterobacter sp. [2 (5.7%)] and least among Acinetobacter baumanii [1 (2.8%)].

Assessment of three medical and research laboratories using WHO AFRO_SLIPTA Quality Standards in Southwestern Uganda: a long way to go.

INTRODUCTION: While the laboratory represents more than 70% of clinical diagnosis and patient management, access to reliable and quality laboratory diagnostics in sub-Saharan Africa remains a challenge. To gain knowledge and suggest evidence based interventions towards laboratory improvement in Southwestern Uganda, we assessed the baseline laboratory quality standards in three medical and research laboratories in Southwestern Uganda. METHODS: We conducted a cross sectional survey from October, 2013 to April, 2014. Selected laboratories, including one private research, one private for profit and one public laboratory, were assessed using the WHO AFRO_SLIPTA checklist and baseline scores were determined. RESULTS: The three laboratories assessed met basic facility requirements, had trained personnel, and safety measures in place. Sample reception was properly designed and executed with a well designated chain of custody. All laboratories had sufficient equipment for the nature of work they were involved in. However, we found that standard operating procedures were incomplete in all three laboratories, lack of quality audit schemes by two laboratories and only one laboratory enrolled into external quality assurance schemes. The SLIPTA scores were one star for the research laboratory and no star for both the public and private-for-profit laboratories. CONCLUSION: While most of the laboratory systems were in place, the low scores obtained by the assessed laboratories reflect the need for improvement to reach standards of quality assured diagnostics in the region. Therefore, routine mentorship and regional supportive supervision are necessary to increase the quality of laboratory services.

A review of antimicrobial resistance in East Africa.

BACKGROUND AND OBJECTIVES: Knowledge of local and regional antimicrobial resistance (AMR) is important for clinical decision making. However, surveillance capacity for AMR is lacking throughout East Africa, and current AMR data are sparse. We sought to address this gap by summarising all available high-quality data on AMR in the East Africa region. METHOD: We searched the PubMed database and African Journals Online archives in April and May 2015 using the search term 'antimicrobial resistance AND sub-Saharan Africa' to find articles published from 2005 onwards. Only full-text articles in English were included. RESULTS: We included 12 published articles in our analysis. Most articles were on bloodstream infections, hospital-based and cross-sectional in design; a majority described either community- or hospital-acquired infections. High levels of AMR to commonly-used antibiotics were reported, including 50% - 100% resistance to ampicillin and cotrimoxazole infections, emerging resistance to gentamicin (20% - 47%) and relatively high levels of resistance to ceftriaxone (46% - 69%) among Gram-negative infections. Much of the resistance was reported to be in Klebsiella species and Escherichia coli. Among Gram-positive infections, extensive resistance was reported to ampicillin (100%), gentamicin and ceftriaxone (50% - 100%), with methicillin-resistant Staphylococcus aureus prevalence ranging from 2.6% - 4.0%. CONCLUSION: Overall, bacterial resistance was reported among commonly-used antibiotics (ampicillin, gentamicin and ceftriaxone), raising concern that these antibiotics may no longer be useful for treating moderate or severe bacterial infections in East Africa. Thus, empirical treatment of bacterial infections needs to be reconsidered and guided by local assessment of AMR. Improvements in the limited amount of quality data and lack of harmonisation in assessing the burden of AMR are also needed.

Epidemiology of Carbapenem Resistance among Multi-drug Resistant Enterobacteriaceae in Uganda.

BACKGROUND: Multi-drug resistant (MDR) Enterobacteriaceae are on the increase worldwide and their spread has become a global challenge. Escalating the challenge is the possibility that many of these are Carbapenemase-producing Enterobacteriaceae (CPE). This further complicates patient management. The magnitude of MDR-CPE in many developed settings has been reported, however, there is paucity of data from resource limited settings. We evaluated the epidemiology of MDR-CPE of clinical origin in South Western Uganda. METHODS: From September 2013 to June 2014, all Enterobacteriaceae isolated from diverse specimens obtained from patients attending Mbarara Regional Referral Hospital, South-western Uganda, were screened for MDR in a laboratory-based cross sectional study. Isolates found to be MDR were screened for carbapenem susceptibility/resistance phenotypically by Kirby Bauer disc diffusion method following CLSI guidelines and genetically using the multiplex real-time Polymerase Chain Reaction (RT-PCR). RESULTS: Of the 658 strains isolated, 183 (27.8%) were MDR and 68 (37.15%) of those MDR exhibited at least one form of carbapenem resistance with 23 (12.57%) and 56 (30.60%) isolates expressing phenotypic and genetic resistance, respectively. Eleven MDR-CPE (6.01%) isolates exhibited both phenotypic and genotypic resistance to carbapenems. Only blaVIM and blaOXA-48 genes were detected among the genetically resistant isolates. CONCLUSION: The high prevalence of MDR-CPE calls for aggressive infection control and prevention strategies, including reinforcement of hand hygiene, using contact precautions and early detection of CPE through use of targeted surveillance and molecular techniques in resource limited settings.