Distribution and antifungal susceptibility profile of oropharyngeal Candida species isolated from people living with HIV in the era of universal test and treat policy in Uganda.

Background: Despite the increased frequency of oropharyngeal candidiasis among people living with human immunodeficiency virus (HIV), its management is no longer effective due to empirical treatment and emergence of antifungal resistance (AFR). This study sought to investigate the prevalence of oropharyngeal candidiasis and assess the antifungal susceptibility profile of oropharyngeal Candida species isolated from people living with human immunodeficiency virus. Additionally, we evaluated the correlation between oropharyngeal candidiasis and CD4 T cell as well as viral load counts. Methods: A descriptive cross-sectional study was carried out from April to October 2023 in which 384 people living with HIV underwent clinical examination for oral lesions. Oropharyngeal swabs were collected and cultured on Sabouraud Dextrose agar to isolate Candida species which were identified using the matrix assisted laser desorption ionization time of flight mass spectrometry. Additionally, the antifungal susceptibility profile of Candida isolates to six antifungal drugs was determined using VITEK® (Marcy-l'Étoile, France) compact system. Data on viral load were retrieved from records, and CD4 T cell count test was performed using Becton Dickinson Biosciences fluorescent antibody cell sorter presto. Results: The prevalence of oropharyngeal candidiasis was 7.6%. Oropharyngeal candidiasis was significantly associated with low CD4 T cell count and high viral load. A total of 35 isolates were obtained out of which Candida albicans comprised of 20 (57.1%) while C. tropicalis and C. glabrata comprised 4 (11.4%) each. C. parapsilosis, C. dubliniensis and C. krusei accounted for 2 (5.7%) each. Additionally, 7 (20%) isolates were resistant to fluconazole, 1 (2.9%) to flucytocine and 0.2 (5.7%) isolates were intermediate to caspofungin. However, specific specie isolates like C. albicans showed 20% (4/20), C. glabrata 50% (2/4) and C. krusei 50% (1/2) resistance to fluconazole. Additionally, C. krusei showed 50% resistance to flucytosine. Conclusion: The prevalence of oropharyngeal candidiasis (OPC) among people living with HIV was low, and there was a significant association between OPC and CD4 T cell count as well as viral load. C. albicans was the most frequently isolated oropharyngeal Candida species. C. glabrata and C. krusei exhibited the highest AFR among the non-albicans Candida species. The highest resistance was demonstrated to fluconazole.

In Vitro Evaluation of the Virulence Attributes of Oropharyngeal Candida Species Isolated from People Living with Human Immunodeficiency Virus with Oropharyngeal Candidiasis on Antiretroviral Therapy.

Background: Oropharyngeal Candida species are part commensal microflora in the the oral cavity of health individuals. Commensal Candida species can become opportunist and transition to pathogenic causes of oropharyngeal candidiasis (OPC) in individuals with impaired immunity through ecological cues and expression of virulence factors. Limited studies have evaluated virulence attributes of oropharyngeal Candida species among people living with human immunodeficiency virus (PLHIV) with OPC on antiretroviral therapy (ART) in Uganda. Objective: Evaluation of the Virulence Attributes of Oropharyngeal Candida Species Isolated from People Living with Human Immunodeficiency Virus with Oropharyngeal Candidiasis on Antiretroviral Therapy. Methods: Thirty-five (35) Candida isolates from PLHIV with OPC on ART were retrieved from sample repository and evaluated for phospholipase activity using the egg yolk agar method, proteinase activity using the bovine serum albumin agar method, hemolysin activity using the blood agar plate method, esterase activity using the Tween 80 opacity test medium method, coagulase activity using the classical tube method and biofilm formation using the microtiter plate assay method in vitro. Results: Phospholipase and proteinase activities were detected in 33/35 (94.3%) and 31/35 (88.6%) of the strains, respectively. Up to 25/35 (71.4%) of the strains exhibited biofilm formation while esterase activity was demonstrated in 23/35 (65.7%) of the strains. Fewer isolates 21/35 (60%) of the strains produced hemolysin and coagulase production was the least virulence activity detected in 18/35 (51.4%). Conclusion: Phospholipase and proteinase activities were the strongest virulence attributes of oropharyngeal Candida species.

Pulmonary dimorphic fungal infections among HIV/AIDS non-TB patients with chronic cough in Kampala, Uganda.

INTRODUCTION: Dimorphic fungi cause infection following the inhalation of spores into the pulmonary system. In the lower respiratory tract, the conidia transform into yeasts, which are engulfed by alveolar macrophages and may be destroyed without disease manifestation. However, in some immunocompromised individuals, they may persist and cause active fungal disease characterized by formation of granulomas in the infected tissues, which may mimic Mycobacterium tuberculosis (MTB). OBJECTIVE: To determine the prevalence of pulmonary dimorphic fungal infections among HIV/AIDS patients with non-TB chronic cough at Mulago National Referral and Teaching Hospital in Kampala, Uganda. METHODS: Sputum samples were collected from 175 consented HIV/AIDS patients attending the immuno-suppression syndrome (ISS) clinic at the hospital. Upon Xpert MTB/RIF sputum testing, 21 patients tested positive for MTB, and these were excluded from further analysis. The other 154 sputum negative samples were then subjected to PCR for dimorphic fungi at MBN Clinical Laboratories. Singleplex PCR was used to detect the target sequences in selected respective genes of each dimorphic fungal species of interest. DNA amplicons were detected based on gel electrophoresis. RESULTS: Dimorphic fungi were detected in 16.2% (25/154) of the studied population. Of these 9.1% (14/154) had Blastomyces dermatitidis and 7.1% (11/154) had Talaromyces marneffei. The remaining 84% of the studied participants had no dimorphic fungi. Histoplasma capsulatum, Coccidioides immitis and Paracoccidioides brasiliensis were not detected in any of the participants. CONCLUSION: Dimorphic fungi (B. dermatitidis and T. marneffei) were found in 16.2% of the HIV/AIDS patients with non-TB chronic cough in Kampala, Uganda. We recommend routine testing for these pathogens among HIV/AIDS patients with chronic cough.

Effect of mixed Mycobacterium tuberculosis infection on rapid molecular diagnostics among patients starting MDR-TB treatment in Uganda.

BACKGROUND: Mixed M. tuberculosis (MTB) infection occurs when one is infected with more than one clonally distinct MTB strain. This form of infection can assist MTB strains to acquire additional mutations, facilitate the spread of drug-resistant strains, and boost the rate of treatment failure. Hence, the presence of mixed MTB infection could affect the performance of some rapid molecular diagnostic tests such as Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays. METHODS: This was a cross-sectional study that used sputum specimens collected from participants screened for STREAM 2 clinical trial between October 2017 and October 2019. Samples from 62 MTB smear-positive patients and rifampicin-resistant patients from peripheral health facilities were processed for Xpert and LPA as screening tests for eligibility in the trial. From November 2020, processed stored sputum samples were retrieved and genotyped to determine the presence of mixed-MTB strain infection using a standard 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem-Repeat (MIRU-VNTR). Samples with at least 20/24 MIRU-VNTR loci amplified were considered for analysis. Agar proportional Drug Susceptibility Test (DST) was performed on culture isolates of samples that had discordant results between LPA and Xpert. The impact of the presence of mixed-MTB strain on Xpert and LPA test interpretation was analyzed. RESULTS: A total of 53/62 (85%) samples had analyzable results from MIRU-VNTR. The overall prevalence of mixed-MTB infection was 5/53 (9.4%). The prevalence was highest among male's 3/31 (9.7%) and among middle-aged adults, 4/30 (33.3%). Lineage 4 of MTB contributed 3/5 (60.0%) of the mixed-MTB infection prevalence. Having mixed MTB strain infection increased the odds of false susceptible Xpert test results (OR 7.556, 95% CI 0.88-64.44) but not for LPA. Being HIV-positive (P = 0.04) independently predicted the presence of mixed MTB infection. CONCLUSIONS: The presence of mixed-MTB strain infection may affect the performance of the GeneXpert test but not for LPA. For patients with high pre-test probability of rifampicin resistance, an alternative rapid method such as LPA should be considered.

A systematic review reveals that African children of 15-17 years demonstrate low hepatitis B vaccine seroprotection rates.

Childhood HBV immunization remains globally fundamental to the elimination of hepatitis B virus (HBV). However, monitoring proportions of HBV vaccine seroprotection and their determinants among African Pediatric recipients is crucial. This study sought to verify extent of immune protection accorded by the HBV vaccine in African children of up to 17 years of age by pooling the prevalence of seroprotection reported by primary studies conducted in the Northern, Western, and Southern African regions. We included 19 eligible articles out of the 197 initially downloaded, published from 1999 to 2021 from African Journals Online (AJOL), EMBASE, Scopus, and PubMed. The study protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO), University of York Centre for Reviews and Dissemination, under the registration number CRD42022361277. Significantly higher (p < 0.0001) proportion of HBV vaccine seroprotection (69.07%) was found among children under 15 years of age than children 15-17 years (32.368%), 95% CI [34.2454-39.0847%]. Whereas successful integration of the HBV vaccine on the extended programs on immunizations (EPI) has been a major achievement in the reduction of HBV infection in Africa, markedly reduced HBV vaccine seroprotection is persistently demonstrated among adolescent children 15-17 years of age. Future studies are required to clarify the need for booster dose vaccination in most at risk populations and age groups.

Effect of mixed Mycobacterium tuberculosis infection on rapid molecular diagnostics among patients starting MDR-TB treatment in Uganda.

Background: We evaluated the effect of mixed-MTB strain infection on the performance of Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays among patients initiating MDR-TB treatment in Uganda. Methods: This was a cross-sectional study using sputum specimens collected from participants screened for STREAM 2 clinical trial between October 2017 and October 2019. Samples from 62 MTB smear-positive patients and rifampicin-resistant patients from the peripheral health facilities were processed for Xpert and LPA as screening tests for eligibility in the trial. From November 2020, processed stored sputum samples were retrieved and genotyped to determine the presence of mixed-MTB strain infection using a standard 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem-Repeat (MIRU-VNTR). Samples with at least 20/24 MIRU-VNTR loci amplified were considered for analysis. Agar proportional Drug Susceptibility Test (DST) was performed on culture isolates of samples that had discordant results between LPA and Xpert. The impact of the presence of mixed-MTB strain on Xpert and LPA test interpretation was analyzed. Results: A total of 53/62 (85%) samples had analyzable results from MIRU-VNTR. The overall prevalence of mixed-MTB infection was 5/53 (9.4%). The prevalence was highest among males 3/33 (9.7%) and among middle-aged adults, 4/30 (13.3%). Lineage 4 of MTB contributed 3/33 (9.1%) of the mixed-MTB infection prevalence. Having mixed MTB strain infection increased the odds of false susceptible Xpert test results (OR 7.556, 95% CI 0.88-64.44) but not for LPA. Being HIV-positive (P=0.04) independently predicted the presence of mixed MTB infection. Conclusions: The presence of mixed-MTB strain infection may affect the performance of the GeneXpert test but not for LPA. For patients with high pre-test probability of rifampicin resistance, an alternative rapid method such as LPA should be considered.

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.

Dimorphic Fungal Infections in HIV/AIDS Patients with non-TB Chronic Cough at Mulago Hospital, Kampala, Uganda.

Introduction: Dimorphic fungi cause infection following inhalation of spores into the pulmonary system. In the lower respiratory tract, the conidia transform into the yeast phase which are engulfed by alveolar macrophages and may be destroyed without disease manifestation. However, in some cases they may persist and cause fungal disease characterized by formation of granulomas in the infected tissues, which may mimic MTB. Objective: To explore if dimorphic fungi play any role in pulmonary disease among XpertTB/RIF Negative HIV Patients with chronic cough attending ISS Clinic at Mulago hospital Uganda. Methods: Sputum samples were collected from 175 consented HIV infected patients attending ISS Clinic. Upon Xpert/RIF test at ISS Clinic 21 of these tested positive, the 154 negative sputum samples were then subjected to PCR for dimorphic fungi at MBN Clinical Laboratories. Singleplex PCR using specific primers was used to detect a target sequency in the gene of each dimorphic fungi of interest, the resulting amplicons were electrophoresed on a 2% gel then visualized under UV light. Results: Blastomyces dermatitidis and Tarolomyces marneffei were detected in 16.4% of the studied participants, with 9.1% and 7.1% respectively and 83.8% of the participant sample had no dimorphic fungi. Coccidiodes immitis, Paracoccidiodes brasiliensis and Histoplasma capsulatum were not detected in any of the participants. Conclusion: Dimorphic fungi play a role in pulmonary disease among the HIV/AIDS with non- TB chronic in Uganda.

Microbial characteristics of dental caries in HIV positive individuals.

Background: Dental caries is a multifactorial disease that affects many people. Even though microorganisms play a crucial role in causing dental caries, diagnosis is routinely macroscopic. In order to improve early detection especially in HIV patients who are disproportionately affected, there is need to reconcile the macroscopic and microscopic characteristics of dental caries. Therefore, the aim of this study was to characterize the oral microbiota profile along the decayed, missing, filled teeth (DMFT) index using amplicon sequencing data. Methods: Amplicon sequencing of the V6-V8 region of the 16S rRNA gene was done on DNA recovered from whole unstimulated saliva of 59 HIV positive and 29 HIV negative individuals. The microbial structure, composition and co-occurrence networks were characterized using QIIME-2, Phyloseq, Microbiome-1.9.2 and Metacoder in R. Results: We characterized the oral microbiota into 2,093 operational taxonomic units (OTUs), 21 phyla and 239 genera from 2.6 million high quality sequence reads. While oral microbiota did not cluster participants into distinct groups that track with the DMFT index, we observed the following: (a) The proportion of accessory microbiota was highest in the high DMFT category while the core size (∼50% of richness) remained relatively stable across all categories. (b) The abundance of core genera such as Stomatobaculum, Peptostreptococcus and Campylobacter was high at onset of dental caries, (c) A general difference in oral microbial biomass. (d) The onset of dental caries (low DMFT) was associated with significantly lower oral microbial entropy. Conclusions: Although oral microbial shifts along the DMFT index were not distinct, we demonstrated the potential utility of microbiota dynamics to characterize oral disease. Therefore, we propose a microbial framework using the DMFT index to better understand dental caries among HIV positive people in resource limited settings.

Prevalence of Cryptococcus gattii in Ugandan HIV-infected patients presenting with cryptococcal meningitis.

INTRODUCTION: Cryptococcal meningitis (CM) is a life threatening disease and leading cause of opportunistic fungal-related mortality in HIV/AIDS. Most CM infections are caused by C. neoformans species complexes but the prevalence of Cryptococcus gattii species complexes in Uganda is unknown however, it is known in a few other parts of Africa. We estimated the prevalence of C. gattii in patients living with HIV and a diagnosis of cryptococcal meningitis in Uganda. METHODS: Cryptococcus isolates (n = 200) obtained from cerebrospinal fluid of patients with CM recruited at the Infectious Diseases Institute, Kampala, Uganda, were tested by phenotypic methods. The Cryptococcus isolates were sub-cultured on Sabouraud Dextrose Agar plates for 48 hours. The yeast colonies were examined by India ink stain, urea hydrolysis, and C. gattii was identified by blue pigmentation on CGB agar. The results were analyzed for frequency of C. gattii. Patient demographic characteristics were collected from the case record forms. RESULTS: From the 200 patients' case record forms, 87 (43.5%) were female and 113 (56.5%) were male. The median age was 35 (19-64) years. Most patients, 93% (187/200) were from Central Uganda in the districts of Kampala and Wakiso. 97.51% (157/161) of the patients had absolute CD4 lymphocyte counts of less than 200 cells per cubic millimeter; 1.86% (3/161) 200-350 cells per cubic millimeter and 0.62% (1/161) above 500 cells per cubic millimeter. 45.4% (74/163) were not yet on HAART and 54.6% (89/163) were on HAART. 66.7% (58/87) had poor adherence to HAART treatment and 33.3% (29/87) had reported good adherence to HAART treatment. A total of 200 clinical isolates of Cryptococcus isolates were tested. No (0% (0/200) C. gattii was identified among the Cryptococcus isolates. CONCLUSION: In this study among patients living with HIV and a diagnosis of cryptococcal meningitis in Uganda, we found no C. gattii infections.

Medical Mycology dissertation topics require prioritisation among Postgraduate Microbiology trainees of Makerere University, Uganda.

Background: As elsewhere worldwide, there is an increasing burden of fungal diseases in Uganda. However, expertise in medical mycology (the study of fungal diseases of medical importance) among clinicians and laboratory personnel remains low. Objective: This study sought to determine the proportion of dissertations on medical mycology among postgraduate medical microbiology trainees at the College of Health Sciences, Makerere University, Uganda. Methods: We retrospectively reviewed the topics of dissertations submitted to the Departments of Medical Microbiology and Immunology & Molecular Biology from 2011 through 2018. The proportion of dissertation topics on medical mycology was analysed using descriptive statistics. Results: A total of 152 dissertations were retrieved. Of these, only 5 (3.3%) were on medical mycology compared to bacteriology (50.7%, n = 77), virology (27.6%, n = 42), parasitology (14.5%, n = 22) and immunology (4.0%, n = 6). Of the 5 dissertations on fungal diseases, the distribution was as follows: cryptococcal meningitis (40%, n = 2), Candidiasis (20%, n = 1), superficial mycoses (20%, n = 1) and other invasive fungal diseases (20%, n = 1). The most common method that was used for studying the fungal diseases was culture 60%, n = 3. Conclusion: There is limited research on medical mycology among the postgraduate medical microbiology trainees of Makerere University, Uganda.

Aspergillus-specific IgM/IgG antibody serostatus of patients hospitalized with moderate-critical COVID-19 in Uganda.

Invasive pulmonary aspergillosis is known to complicate the coronavirus diseases-2019 (COVID-19), especially those with critical illness. We investigated the baseline anti-Aspergillus antibody serostatus of patients with moderate-critical COVID-19 hospitalized at 3 COVID-19 Treatment Units in Uganda. All 46 tested patients, mean age 30, and 11% with underlying respiratory disease had a negative serum anti-Aspergillus IgM/IgG antibody immunochromatographic test on day 3 (mean) of symptom onset (range 1-26), but follow up specimens to assess seroconversion were not available.

Aspergillus-specific IgM/IgG antibody serostatus of patients hospitalized with moderate-critical COVID-19 in Uganda

Invasive pulmonary aspergillosis is known to complicate the coronavirus diseases-2019 (COVID-19), especially those with critical illness. We investigated the baseline anti Aspergillus antibody serostatus of patients with moderate-critical COVID-19 hospitalized at 3 COVID-19 Treatment Units in Uganda. All 46 tested patients, mean age 30, and 11% with underlying respiratory disease had a negative serum anti-Aspergillus IgM/IgG antibody immunochromatographic test on day 3 (mean) of symptom onset (range 1-26), but follow up specimens to assess seroconversion were not available

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.

The simple direct slide method is comparable to indirect Lowenstein Jensen proportion culture for detecting rifampicin resistant tuberculosis.

Introduction. Drug resistant tuberculosis remains a worldwide problem that requires prompt diagnosis.Hypothesis/Gap statement. The WHO recommended direct, rapid Xpert MTB/RIF is prohibitively costly, therefore, there is a need to validate a rapid, affordable DST for use in low- and middle-income settings.Aim. The technical performance and time to results of a simple, direct microscopy-based slide DST (SDST) assay for diagnosis of rifampicin-resistant TB was evaluated in Uganda.Methodology. Sputum samples from 122 smear-positive re-treatment TB patients presenting to the TB treatment centre at Uganda's National Referral Hospital, Mulago, Kampala, Uganda were examined. The sputum samples were tested by the direct SDST which was compared to the indirect Lowenstein Jensen Proportion Method (LJDST) method as the gold standard. The time to results was defined as the time from DST setting to results interpretation. The results were further analysed for sensitivity and specificity as well as agreement between LJDST and SDST for rifampicin resistance determination.Results. A total of 117 smear positive sputum samples with valid results for both tests were compared. The median time to results for SDST was 14 days with an interquartile range (IQR) of 10-14 days compared to 60 days with IQR of 60-75 days for LJDST. The number for rifampicin resistance by the gold standard LJDST was 26. The SDST had a sensitivity of 96 % (95 %; CI 81-99 %) and a specificity of 97.8 % (95 %; CI 93-100 %). The Positive Predictive and Negative Predictive values for SDST were 92.3 % (95 %; CI 76.8-99 %) and 98.9 % (95 %; CI 94-100 %), respectively. The kappa agreement between SDST and LJDST was 92.3 %.Conclusion. The SDST was found to be a rapid and accurate direct test for the detection of rifampicin resistance among retreatment TB cases in low-income settings.

Factors associated with community-acquired urinary tract infections among adults attending assessment centre, Mulago Hospital Uganda.

BACKGROUND: Urinary tract infections (UTI) are a common medical problem affecting the general population and thus commonly encountered in medical practice, with the global burden of UTIs at about 150 million people. Because uropathogens largely originate from colonic flora, they are easy to predict, and this is the rationale for empirical treatment in Community Acquired-UTI (CA-UTIs). With the increasing prevalence of drug-resistant bacteria among adults with CA-UTI in Uganda, it is no longer adequate to manage CA-UTIs on empiric regimen without revising the susceptibility patterns of common CA-UTI causative agents. Thus in this study we set out to identify: The factors associated with CA-UTIs, the common uropathogens and the drug sensitivity patterns of the common uropathogens cultured. METHODOLOGY: This was a cross-sectional study that was conducted in adults who presented with symptoms of a UTI at Mulago Hospital, assessment center. There were 139 patients who consented to the study and were recruited, an interviewer administered questionnaire was used to collect information from the study participants as regards demographic, social and clinical characteristics and Mid Stream Urine (MSU) samples were collected for urinalysis, culture and antibiotic susceptibility testing using the Kirby-Bauer disc diffusion technique was applied to the isolates.Numeric data were summarized using measures of central tendency while the categorical data was summarized using proportions and percentages. RESULTS: Age, female sex and marital status were factors that were significantly associated with CA-UTIs. Fifty four (54) cultures were positive for UTI with 26 giving pure growths. The commonest uropathogen isolated was Escherichia coli at 50%, this was followed by Staphylococcus aureus at 15.4%. The sensitivity of Escherichia coli to Ampicillin and Nitrofurantoin were78.6%, 64.3% respectively, and the sensitivity of Staphylococcus aureus to ciprofloxacin, Nitrofurantoin and gentamycin were 100%, 66.7% and 66.7% respectively. CONCLUSION: There are known factors associated with CA-UTIs such as age, female sex. There was generally high sensitivity to nitrofurantoin and gentamycin by most of the uropathogens isolated, and high resistance to the common antibiotics such as nalidixic acid and erythromycin thus a need for a bigger study that can be used to effect the change of the current recommendations in the Uganda Clinical Guidelines as regards empirical management of CA-UTIs.

A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala.

BACKGROUND: Accurate and high-throughput genotyping of Mycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients. METHOD: Genotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing. RESULTS: The LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage. CONCLUSION: The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.

Increased Antifungal Drug Resistance in Clinical Isolates of Cryptococcus neoformans in Uganda.

Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/µl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC50 of 4 µg/ml and an MIC90 of 8 µg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC50 to 8 µg/ml and an MIC90 value of 32 µg/ml, with 31% of isolates with a fluconazole MIC of ≥ 16 µg/ml. We observed an amphotericin B MIC50 of 0.5 µg/ml and an MIC90 of 1 µg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC50 of 4 µg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P = 0.52).

Oxidative stress responses in the human fungal pathogen, Candida albicans.

Candida albicans is a major fungal pathogen of humans, causing approximately 400,000 life-threatening systemic infections world-wide each year in severely immunocompromised patients. An important fungicidal mechanism employed by innate immune cells involves the generation of toxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Consequently, there is much interest in the strategies employed by C. albicans to evade the oxidative killing by macrophages and neutrophils. Our understanding of how C. albicans senses and responds to ROS has significantly increased in recent years. Key findings include the observations that hydrogen peroxide triggers the filamentation of this polymorphic fungus and that a superoxide dismutase enzyme with a novel mode of action is expressed at the cell surface of C. albicans. Furthermore, recent studies have indicated that combinations of the chemical stresses generated by phagocytes can actively prevent C. albicans oxidative stress responses through a mechanism termed the stress pathway interference. In this review, we present an up-date of our current understanding of the role and regulation of oxidative stress responses in this important human fungal pathogen.

AIDS-related mycoses: the way forward.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries.