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.

A bacterial endosymbiont of the fungus Rhizopus microsporus drives phagocyte evasion and opportunistic virulence.

Opportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitous in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this and the evolutionary drivers underlying the acquisition of virulence mechanisms are poorly understood. Here, we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice and that this endosymbiosis enables the secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebas are natural environmental predators of both bacteria and fungi, we propose that this tri-kingdom interaction contributes to establishing endosymbiosis and the acquisition of anti-phagocyte activity. Importantly, we show that this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a new role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals and suggest a mechanism of virulence acquisition through environmental interactions with amoebas.

Bacteriological profile, antibiotic susceptibility and factors associated with neonatal Septicaemia at Kilembe mines hospital, Kasese District Western Uganda.

INTRODUCTION: Neonatal septicaemia is one of the most common leading causes of neonatal morbidity and mortality in developing countries. It is estimated to affect more than 30 million people worldwide annually, potentially leading to 6 million deaths. OBJECTIVE(S): To determine the prevalence, bacteriological profile, antibiotic susceptibility and factors associated with neonatal septicaemia among neonates suspected to sepsis at Kilembe mines hospital. METHODS: We conducted a descriptive cross-sectional study, where purposive sampling technique was used and blood was drawn from 122 neonates suspected to sepsis attending Kilembe Mines Hospital during the period (July to November 2020). Specimens were inoculated in Brain heart infusion broth, transported to Fortportal Regional Referral Hospital, plated daily up to 7 days on blood, chocolate, MacConkey agar and incubated in aerobic and 5% carbondioxide. Pure colonies were identified by Gram stain, biochemical tests and antibiotic sensitivities obtained by Kirby Bauer disc diffusion method. Associations were tested using Chi square with Fisher's exact or Yates correction tests where necessary and statistical significance was set at P < 0.05. Stata (version 14) used for statistical analysis. RESULTS: Blood cultures were positive in 59.0% cases with 55.5% male and 44.4% female. EOS was present in 56.9% and LOS 43.1% of the cases. Gram negative (56.9%) organisms were most implicated with neonatal septicaemia than Gram positives ones (43.1%). Gram positive organisms exhibited better susceptibility to amikacin, linezolid and vancomycin but more resistant to ampicillin and gentamicin. Of the aminoglycosides, amikacin exhibited a verge over netilmicin and gentamicin against Gram negative isolates. Risk factors of neonatal septicaemia were mother's age of ≥25 years, employed mothers, tertiary-level of education, SVD, ANC attendance of ≥4 times, UTI during pregnancy, PROMS, foul Smelling liquor, urban residence, neonatal birth weight of ≥2500 g, Apgar score 1st and 5th min ≥6 and resuscitation. CONCLUSION: Multi-drug resistant organisms were isolated. Therefore caution is required in selection of antibiotic therapy and avoid empirical treatment.

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.

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.

AmpC-BETA Lactamases among Enterobacteriaceae Isolated at a Tertiary Hospital, South Western Uganda.

AIM: To characterize AmpC-beta lactamases among Enterobacteriaceae isolates from clinical samples at Mbarara Regional Referral Hospital. STUDY DESIGN: Laboratory-based descriptive cross-sectional study. PLACE AND DURATION OF STUDY: Microbiology Department, Mbarara Regional Referral Hospital and MBN clinical Laboratories, between May to September 2013. METHODOLOGY: This study included 293 Enterobacteriaceae isolates recovered from clinical specimens that included blood, urine, stool and aspirates. AmpC Beta lactamase production was determined using disc placement method for cefoxitin at a break point of <18mm. Common AmpC plasmid mediated genes were EBC, ACC, FOX, DHA, CIT and MOX were; was determined by Multiplex PCR as described by Hanson and Perez-Perez. RESULTS: Plasmid mediated AmpC phenotype was confirmed in 107 of the 293 (36.5%) cefoxitin resistant isolates with 30 isolates having more than one gene coding for resistance. The commonest source that harbored AmpC beta lactamases was urine and E. coli was the most common AmpC producer (59.5%). The genotypes detected in this study, included EBC (n=36), FOX (n=18), ACC (n=11), CIT (n=10), DHA (n=07) and MOX (n=1). CONCLUSION: Our findings showed that prevalence of AmpC beta-lactamase at MRRH was high (39.6), with EBC as the commonest genotype among Enterobacteriaceae Urine and E. coli were the commonest source and organism respectively that harbored AmpC beta-lactamases. There's rational antimicrobial therapy and antibiotic susceptibility tests should be requested by health workers especially patients presenting with urinary tract infections and bacteraemias.

Clindamycin Resistance among Staphylococcus Aureus Isolated at Mbarara Regional Referral Hospital, in South Western Uganda.

AIMS: The study was conducted to determine the prevalence of Clindamycin (CL) resistance and antimicrobial susceptibility among clinical isolates of Staphylococcus aureus (S. aureus) from Mbarara Regional Referral Hospital (MRRH) in Southwestern Uganda. STUDY DESIGN: Laboratory based cross sectional study. PLACE AND DURATION OF THE STUDY: The study was conducted at the Microbiology department of Mbarara Regional referral hospital between November 2012 and December 2013. METHODOLOGY: In our study, we recruited 300 S. aureus isolates that were stored in the laboratory and were obtained from different clinical samples. The isolates were tested for antimicrobial susceptibility by phenotypic methods and for the genotypic expression of Macrolide Lincosamide StreptograminB (MLSB) resistance genes (ermA, ermB, ermC, and msrA). The D-test was also performed. RESULTS: Phenotypically, a total of 109 (36%) S. aureus isolates were resistant to CL, of which 9 (3%) were constitutively resistant while 100 (33.3%) were inducibly resistant. Genotypicaly, 134/300 (44.7%) isolates possessed at least one of the MLSB resistance genes. 23/300 (7.7%) tested positive for ermB, 98/300 (32.7%) tested positive for the ermC and 43/300 (14.3%) tested positive for the msrA genes with none possessing the ermA gene. Isolates were highly resistant to Sulfamethoxazole/trimethoprim, Erythromycin and Oxacillin with moderate resistance to Vancomycin and Imipenem and least resistance to Linezolid. CONCLUSION: S. aureus resistance to CL was high in this set up. There was also high resistance to Sulfamethoxazole/trimethoprim, Erythromycin and Oxacillin but low resistance to Linezolid.