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.

MinION Whole-Genome Sequencing in Resource-Limited Settings: Challenges and Opportunities.

Purpose of Review: The introduction of MinION whole-genome sequencing technology greatly increased and simplified complete genome sequencing in various fields of science across the globe. Sequences have been generated from complex organisms to microorganisms and are stored in genome databases that are readily accessible by researchers. Various new software for genome analysis, along with upgrades to older software packages, are being generated. New protocols are also being validated that enable WGS technology to be rapidly and increasingly used for sequencing in field settings. Recent Findings: MinION WGS technology has been implemented in developed countries due to its advantages: portability, real-time analysis, and lower cost compared to other sequencing technologies. While these same advantages are critical in developing countries, MinION WGS technology is still under-utilized in resource-limited settings. Summary: In this review, we look at the applications, advantages, challenges, and opportunities of using MinION WGS in resource-limited settings.

Cryptococcus neoformans Genotypic Diversity and Disease Outcome among HIV Patients in Africa.

Cryptococcal meningoencephalitis, a disease with poor patient outcomes, remains the most prevalent invasive fungal infection worldwide, accounting for approximately 180,000 deaths each year. In several areas of sub-Saharan Africa with the highest HIV prevalence, cryptococcal meningitis is the leading cause of community-acquired meningitis, with a high mortality among HIV-infected individuals. Recent studies show that patient disease outcomes are impacted by the genetics of the infecting isolate. Yet, there is still limited knowledge of how these genotypic variations contribute to clinical disease outcome. Further, it is unclear how the genetic heterogeneity of C. neoformans and the extensive phenotypic variation observed between and within isolates affects infection and disease. In this review, we discuss current knowledge of how various genotypes impact disease progression and patient outcome in HIV-positive populations in sub-Saharan African, a setting with a high burden of cryptococcosis.

Detection of Mycobacterium tuberculosis multiple strains in sputum samples from patients with pulmonary tuberculosis in south western Uganda using MIRU-VNTR.

Infections with multiple strains of Mycobacterium tuberculosis are now widely recognized as a common occurrence. Identification of patients infected with multiple strains provides both insight into the disease dynamics and the epidemiology of tuberculosis. Analysis of Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeats (MIRU-VNTR) has been shown to be highly sensitive in detecting multiple M. tuberculosis strains even in sputum. The goal of this study was to identify cases of multiple M. tuberculosis strain infections among patients diagnosed with pulmonary tuberculosis in Southwestern Uganda and assessment of factors associated with multiple strain infections. DNA extracted directly from 78 sputum samples, each from an individual patient, was analyzed using the standard 24 loci MIRU-VNTR typing. Five (6.4%) of the 78 patients were infected with multiple strains of M. tuberculosis with all of them being the newly diagnosed cases while two-thirds of them were co-infected with HIV. Exact regression analysis projected that the natives were more likely to harbor multiple strains (OR; 0.981, 95% CI 0-7.926) as well as those with a high microbial load (OR; 0.390, 95% CI 0-3.8167). Despite these findings being not statistically significant due to the small sample size, this points to a critical component of disease dynamics that has clinical implications and emphasizes a need for a study using a larger cohort. It is also essential to study the potential factors associated with higher risk of exposure to newly diagnosed and HIV positive patients at the community level. In addition, our ability to detect multiple M. tuberculosis strains using the standard 24 loci MIRU-VNTR typing especially with allelic diversity in loci 2059 and 3171, which are excluded from the 15-locus MIRU-VNTR, lead us to recommend the use of this genotyping technique, especially in areas with tuberculosis endemicity similar to this study.

Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda.

BACKGROUND: The evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda. METHODS: The blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile. RESULTS: Of the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months-81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected. CONCLUSION: The community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

Rifampicin and isoniazid drug resistance among patients diagnosed with pulmonary tuberculosis in southwestern Uganda.

Multidrug-resistant tuberculosis (MDR-TB) has become a major threat to the control of tuberculosis globally. Uganda is among the countries with a relatively high prevalence of tuberculosis despite significant control efforts. In this study, the drug resistance of Mycobacterium tuberculosis to rifampicin (RIF) and isoniazid (INH) was investigated among patients diagnosed with pulmonary tuberculosis in Southwestern Uganda. A total of 283 sputum samples (266 from newly diagnosed and 17 from previously treated patients), collected between May 2018 and April 2019 at four different TB diagnostic centres, were assessed for RIF and INH resistance using high-resolution melt curve analysis. The overall prevalence of monoresistance to INH and RIF was 8.5% and 11% respectively, while the prevalence of MDR-TB was 6.7%. Bivariate analysis showed that patients aged 25 to 44 years were at a higher risk of developing MDR-TB (cOR 0.253). Furthermore, among the newly diagnosed patients, the prevalence of monoresistance to INH, RIF and MDR-TB was 8.6%, 10.2% and 6.4% respectively; while among the previously treated cases, these prevalence rates were 5.9%, 23.5% and 11.8%. These rates are higher than those reported previously indicating a rise in MTB drug resistance and may call for measures used to prevent a further rise in drug resistance. There is also a need to conduct frequent drug resistance surveys, to monitor and curtail the development and spread of drug-resistant TB.

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.

Diversity of Mycobacterium tuberculosis Complex Lineages Associated with Pulmonary Tuberculosis in Southwestern, Uganda.

Uganda is among the 22 countries in the world with a high burden of tuberculosis. The southwestern region of the country has consistently registered a high TB/HIV incidence rate. This study is aimed at characterizing the Mycobacterium tuberculosis complex (MTBC) genotypic diversity in southwestern Uganda. A total of 283 sputum samples from patients with pulmonary tuberculosis were genotyped using specific single nucleotide polymorphism markers for lineages 3 and 4. Most of the patients were males with a mean age of 34. The lineage 4 Ugandan family was found to be the most dominant strains accounting for 59.7% of all cases followed by lineage 3 at 15.2%. The lineage 4 non-Ugandan family accounted for 14.5% of all cases while 4.2% showed amplification for both lineage 4 and lineage 3. Eighteen samples (6.4%) of the strains remained unclassified since they could not be matched to any lineage based on the genotyping technique used. This study demonstrates that a wide diversity of strains is causing pulmonary tuberculosis in this region with those belonging to the lineage 4 Ugandan family being more predominant. However, to confirm this, further studies using more discriminative genotyping methods are necessary.

Surveillance of Plasmodium falciparum pfcrt haplotypes in southwestern uganda by high-resolution melt analysis.

BACKGROUND: Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries. METHODS: Quantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda. RESULTS: The sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10- 5 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2). CONCLUSIONS: The HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.

Detection of Plasmodium Species by High-Resolution Melt Analysis of DNA from Blood Smears Acquired in Southwestern Uganda.

Microscopic diagnosis of malaria using Giemsa-stained blood smears is the standard of care in resource-limited settings. These smears represent a potential source of DNA for PCR testing to confirm Plasmodium infections or for epidemiological studies of archived samples. Therefore, we assessed the use of DNA extracts from stained blood smears for the detection of Plasmodium species using real-time PCR. We extracted DNA from archived blood smears and corresponding red blood cell pellets collected from asymptomatic children in southwestern Uganda in 2010. We then performed real-time PCR followed by high-resolution melting (HRM) to identify Plasmodium species, and we compared our results to those of microscopy. We analyzed a total of 367 blood smears and corresponding red blood cell pellets, including 185 smears (50.4%) that were positive by microscopy. Compared to microscopy, PCR-HRM analysis of smear DNA had a sensitivity of 93.0% (95% confidence interval [CI], 88.2 to 96.2%) and a specificity of 96.7% (95% CI, 93.0 to 98.8%), and PCR-HRM analysis of pellet DNA had a sensitivity of 100.0% (95% CI, 98.0 to 100.0%) and a specificity of 94.0% (95% CI, 89.4 to 96.9%). Identification of positive PCR-HRM results to the species level revealed Plasmodium falciparum (92.0%), Plasmodium ovale (5.6%), and Plasmodium malariae (2.4%). PCR-HRM analysis of DNA extracts from Giemsa-stained thick blood smears or corresponding blood pellets had high sensitivity and specificity for malaria diagnosis, compared to microscopy. Therefore, blood smears can provide an adequate source of DNA for confirmation of Plasmodium species infections and can be used for retrospective genetic studies.

Evaluation of the SD Bioline TB Ag MPT64 test for identification of Mycobacterium tuberculosis complex from liquid cultures in Southwestern Uganda.

BACKGROUND: To confirm presence of Mycobacterium tuberculosis complex, some tuberculosis culture laboratories still rely on para-nitrobenzoic acid (PNB), a traditional technique that requires sub-culturing of clinical isolates and two to three weeks to give results. Rapid identification tests have improved turnaround times for mycobacterial culture results. Considering the challenges of the PNB method, we assessed the performance of the SD Bioline TB Ag MPT64 assay by using PNB as gold standard to detect M. tuberculosis complex from acid-fast bacilli (AFB) positive cultures. OBJECTIVES: The aim of this study was to determine the sensitivity, specificity and turnaround time of the SD MPT64 assay for identification of M. tuberculosis complex, in a setting with high prevalence of tuberculosis and HIV. METHODS: A convenience sample of 690 patients, with tuberculosis symptoms, was enrolled at Epicentre Mbarara Research Centre between April 2010 and June 2011. The samples were decontaminated using NALC-NaOH and re-suspended sediments inoculated in Mycobacterium Growth Indicator Tubes (MGIT) media, then incubated at 37 °C for a maximum of eight weeks. A random sample of 50 known negative cultures and 50 non-tuberculous mycobacteria isolates were tested for specificity, while sensitivity was based on AFB positivity. The time required from positive culture to reporting of results was also assessed with PNB used as the gold standard. RESULTS: Of the 138 cultures that were AFB-positive, the sensitivity of the SD MPT64 assay was 100.0% [95% CI: 97.3 - 100] and specificity was 100.0% (95% CI, 96.4 - 100). The median time from a specimen receipt to confirmation of strain was 10 days [IQR: 8-12] with SD MPT64 and 24 days [IQR: 22-26] with PNB. CONCLUSION: The SD MPT64 assay is comparable to PNB for identification of M. tuberculosis complex and reduces the time to detection.

Lowenstein-Jensen selective medium for reducing contamination in Mycobacterium tuberculosis culture.

We compared Mycobacterium tuberculosis sputum culture recovery and contamination rates between Lowenstein-Jensen medium (LJ) containing the following decontaminants and LJ alone: (i) PANTA (n = 299), (ii) Selectatab-MB (n = 299), and (iii) penicillin G (n = 234). The contamination rate for LJ alone was approximately 31%, versus 5.0% for PANTA-containing, 2% for Selectatab-containing, and 9% for penicillin-containing media (P < 0.001). M. tuberculosis isolation rates were 9.8%, 17%, 18%, and 12% for standard LJ, PANTA, Selectatab, and penicillin cultures, respectively.