Browsing by Author "Kwizera, Richard"
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Item Acridine Orange Fluorescent Microscopy is more Sensitive than India Ink Light Microscopy in the Rapid Detection of Cryptococcosis among Crag Positive HIV Patients(PLoS One, 2017) Akampurira, Andrew; Kwizera, Richard; Williams, Darlisha; Boulware, David R.; Meya, David B.; on behalf of the ASTRO-CM Study TeamIndia ink microscopy on cerebrospinal fluid is still utilized in resource limited settings for the diagnosis of cryptococcal meningitis despite its poor sensitivity. We hypothesized that staining fungal nucleic acids with fluorescent dyes instead of the capsule with India ink might improve sensitivity for the diagnosis of cryptococcal meningitis.We enrolled 96 HIV-infected participants with cryptococcal meningitis who provided 194 CSF specimens at serial time points in Kampala, Uganda. Cryptococcosis was diagnosed by cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) test and only positive samples were included. We stained CSF with India ink and acridine orange. We cultured the same samples on standard fungal media. We compared acridine orange to CrAg, India ink and CSF culture.Acridine orange was more sensitive (96%) than India ink (79%) with reference to CSF CrAg. Acridine orange and India ink had a statistically significant difference (P<0.001) with a 25% correlation for detection of Cryptococcus yeasts. India ink had more negative results (22%) than acridine orange (4%). The sensitivity for India ink increased (86%) while that of acridine orange did not change (97%) when compared to CSF culture. However, both India ink and acridine orange had poor predictive values with reference to culture. Acridine orange is a better alternative to India ink in the rapid detection of cryptococcosis among CrAg positive HIV patients.Item Algorithm-Aided Diagnosis of Chronic Pulmonary Aspergillosis in Low- And Middle-Income Countries by Use of a Lateral Flow Device(European Journal of Clinical Microbiology & Infectious Diseases, 2020) Kwizera, Richard; Katende, Andrew; Teu, Anneth; Apolot, Denise; Worodria, William; Kirenga, Bruce J.; Bongomin, FelixChronic pulmonary aspergillosis (CPA) is a slowly progressive parenchymal lung disease typically caused by Aspergillus fumigatus. CPA affects immunocompetent or subtly immunocompromised patients with underlying structural lung diseases and is estimated to affect approximately three million people per year worldwide. It can co-exist with pulmonary tuberculosis (PTB), has both pulmonary and systemic symptoms that are clinically indistinguishable from that of PTB, and is often misdiagnosed and managed as smear-negative PTB. According to the Infectious Diseases Society of America (IDSA), the European Society for Clinical Microbiology and Infectious Diseases (ESCMID), the European Confederation of Medical Mycology (ECMM), and the European Respiratory Society (ERS) Guidelines, the diagnosis of CPA should be based on characteristic symptoms and radiologic features present or presumed to have been present for at least 3 months in a patient with no or minimal immunosuppression and a prior or current lung condition with microbiological or immunological evidence of Aspergillus spp. infection. This definition is consistent with the original definition of CPA proposed by Denning and colleagues . Still, CPA is under- and mis-diagnosed in resource-constrained settings where adequate diagnostics are unavailable. Previously treated PTB is the most common risk factor for the development of CPA even in the developed world . The global burden of CPA attributed to healed TB lesions alone has been estimated to over 1.2 million cases annually globally. On the other hand, active PTB is the number one differential diagnosis for CPA and CPA is the number one differential diagnosis for patients previously treated for microbiologically confirmed PTB who are currently sputum smear-negative. Recent evidence has shown that the annual rate of new CPA development following completion of PTB treatment is about 6.5% in those with chest radiography cavitation and 0.2% in those without.Item Building clinical pharmacology laboratory capacity in low- and middle-income countries: Experience from Uganda(African Journal of Laboratory Medicine, 2023) Omali, Denis; Buzibye, Allan; Kwizera, Richard; Byakika-Kibwika, Pauline; Namakula, Rhoda; Matovu, Joshua; Mbabazi, Olive; Mande, Emmanuel; Sekaggya-Wiltshire, Christine; Nakanjako, Damalie; Gutteck, Ursula; McAdam, Keith; Easterbrook, Philippa; Kambugu, Andrew; Fehr, Jan; Castelnuovo, Barbara; Manabe, Yukari C.; Lamorde, Mohammed; Mueller, Daniel; Merry, ConceptaResearch and clinical use of clinical pharmacology laboratories are limited in low- and middle-income countries. We describe our experience in building and sustaining laboratory capacity for clinical pharmacology at the Infectious Diseases Institute, Kampala, Uganda. Intervention: Existing laboratory infrastructure was repurposed, and new equipment was acquired. Laboratory personnel were hired and trained to optimise, validate, and develop in-house methods for testing antiretroviral, anti-tuberculosis and other drugs, including 10 high-performance liquid chromatography methods and four mass spectrometry methods. We reviewed all research collaborations and projects for which samples were assayed in the laboratory from January 2006 to November 2020. We assessed laboratory staff mentorship from collaborative relationships and the contribution of research projects towards human resource development, assay development, and equipment and maintenance costs. We further assessed the quality of testing and use of the laboratory for research and clinical care. Lessons learnt: Fourteen years post inception, the clinical pharmacology laboratory had contributed significantly to the overall research output at the institute by supporting 26 pharmacokinetic studies. The laboratory has actively participated in an international external quality assurance programme for the last four years. For clinical care, a therapeutic drug monitoring service is accessible to patients living with HIV at the Adult Infectious Diseases clinic in Kampala, Uganda. Recommendations: Driven primarily by research projects, clinical pharmacology laboratory capacity was successfully established in Uganda, resulting in sustained research output and clinical support. Strategies implemented in building capacity for this laboratory may guide similar processes in other low- and middle-income countries.Item Burden of Fungal Asthma in Africa: A Systematic Review and Meta-Analysis(PloS one, 2019) Kwizera, Richard; Musaazi, Joseph; Meya, David B.; Worodria, William; Bwanga, Freddie; Kajumbula, Henry; Fowler, Stephen J.; Kirenga, Bruce J.; Gore, Robin; Denning, David W.Asthma is one of the neglected diseases in Africa with a high prevalence. Allergic fungal diseases have been reported to complicate asthma progression and treatment outcomes. However, data about fungal asthma and its associated complications are limited in Africa. We aimed to estimate the burden of fungal asthma among adults and children in Africa using a systematic review.We first engaged the Institute for Health Metrics and Evaluation (IHME) to highlight the trend in morbidity and mortality attributed to asthma in Africa. We then searched PubMed, HINARI and Google Scholar for all studies of any design focusing on fungal asthma in any African country. Languages were restricted to English and French, but not year of publication. We estimated the weighted prevalence of allergic fungal infections among asthmatics with a 95% CI and pooled the results using a random effects model. This study is registered with PROSPERO, number CRD42019117319.The IHME data showed that there has been a gradual increase in morbidity and mortality due to asthma in African adults with a prevalence of 4%. Our search retrieved 5233 citations. We retained 20 studies that met our selection criteria. These were from 13 African countries published between 1967 and 2018. There were eight cross-sectional studies and twelve review articles. The average asthma prevalence in Africa was 6% from these studies. The prevalence of fungal sensitisation was relatively high (3–52%) in the asthmatic population with an average of 28% and a pooled estimate of 23.3%, mostly due to Aspergillus species. Prevalence of Allergic bronchopulmonary apsergillosis was estimated at 1.6–21.2%. Diagnosis of fungal allergy was mostly made by skin prick tests. There was no data on the use of medication to manage fungal asthma. None of the studies evaluated the association between fungal allergy and asthma severity. Data were lacking in children.There is a high prevalence of fungal sensitization among Africans with asthma. Fungal asthma is a significant problem in Africa but there remains a paucity of data on the epidemiology and associated complications. There is urgent need for national epidemiological studies to estimate the actual burden of fungal asthma in Africa.Item Comment on: Susceptibility Breakpoints and Target Values for Therapeutic Drug Monitoring of Voriconazole and Aspergillus Fumigatus in an in Vitro Pharmacokinetic/Pharmacodynamic Model(Journal of Antimicrobial Chemotherapy, 2015) Denning, David W.; Ghnan, Nesrin; Kwizera, Richard; Osmanov, AliSiopi et al.1 are to be congratulated on a superb paper with convincing results using a combination of resistant isolates and sophisticated pharmacodynamic modelling to demonstrate the strong relationship between voriconazole serum concentrations and outcome. Three different MIC determinations yielded similar although numerically slightly different results. The authors propose slightly different breakpoints for resistance using the EUCAST method and breakpoints can now be set for Aspergillus fumigatus for the other two methods.Item Elevated Aspergillus-specific antibody levels among HIV infected Ugandans with pulmonary tuberculosis(BMC Pulmonary Medicine, 2017) Kwizera, Richard; Parkes-Ratanshi, Rosalind; Wiltshire, Christine Sekaggya; Musaazi, Joseph; Castelnuovo, Barbara; Kambugu, Andrew; Denning, David W.The incidence of tuberculosis (TB) is high among human immunodeficiency virus (HIV) infected Ugandans. Recent evidence suggests that Chronic Pulmonary Aspergillosis and Aspergillus sensitisation might be responsible for significant mortality in patients treated for tuberculosis in Uganda. We retrieved and tested paired serum aliquots for 101 HIV-TB co-infected patients at the beginning and week 24 of TB treatment. We tested samples for Aspergillus-specific immunoglobulin G (IgG) and immunoglobulin E (IgE) using ImmunoCAP®; and Aspergillus-specific IgG and total serum IgE using Immulite® immunoassays. We compared antibody levels between baseline and week 24, relating them to selected baseline characteristics.10% of the patients had elevated Aspergillus-specific IgE (Aspergillus sensitization) and Aspergillus-specific IgG antibodies were elevated in 9% of the patients at the end of TB treatment. There was a significant fall in the Aspergillus-specific IgG antibody levels between baseline and week 24 (P = 0.02). Patients with cluster of differentiation 4 (CD4) T-cell count <100 cells/μl and those who were not on anti-retroviral therapy at baseline had more elevated Aspergillus-specific IgG antibodies (P = 0.01, P = 0.03). The ImmunoCAP® Aspergillus-specific IgG antibody titres were higher at week 24 than baseline with more positives at week 24; even though the difference in means was small. However, this difference was statistically significant (P = 0.02). Pulmonary infiltrates were the commonest x-ray abnormality and only 5% of the patients had pulmonary cavities on chest x-ray at week 24.These results suggest that Aspergillus infection may complicate active pulmonary TB and further studies including fungal culture and thoracic imaging may now be indicated to measure the prevalence of pulmonary aspergillosis complicating tuberculosis.Item Estimation of the Burden of Tinea Capitis Among Children in 10 Africa(Mycoses, 2021) Bongomin, Felix; Olum, Ronald; Nsenga, Lauryn; Namusobya, Martha; Russell, Laura; Sousa, Emma de; Iyabo Osaigbovo, Iriagbonse; Kwizera, Richard; Baruch Baluku, JosephTinea capitis is a common and endemic dermatophytosis among school age children in Africa. However, the true burden of the disease is unknown in Africa. Objective: We aimed to estimate the burden of tinea capitis among children less than 18 years of age in Africa. Methods: A systematic review was performed using Embase, Medline and the Cochrane Library of Systematic Reviews to identify articles on tinea capitis among children in Africa published between January 1990 and October 2020. The United Nation’s Population data (2019) was used to identify the number of children at risk of tinea capitis in each African country. Using the pooled prevalence, the country-specific and total burden of tinea capitis was calculated. Results: Forty studies involving a total of 229,086 children from 17/54 African countries were identified and included in the analysis. The pooled prevalence of tinea capitis was 23% (95% CI, 17%-29%) mostly caused by Trichophyton species. With a population of 600 million (46%) children, the total number of cases of tinea capitis in Africa was estimated at 138.1 (95% CI, 102.0 – 174.1) million cases. Over 96% (132.6 million) cases occur in Sub-Saharan Africa alone. Nigeria and Ethiopia with the highest population of children contributed 16.4% (n=98.7 million) and 8.5% (n=52.2 million) of cases, respectively. Majority of the participants were primary school children with a mean age of 10 years. Cases are mostly diagnosed clinically. There was a large discrepancy between the clinical and mycological diagnosis. Conclusions: About one in every 5 children in Africa has tinea capitis making it one of the most common childhood conditions in the region. A precise quantification of the burden of this neglected tropical disease is required to inform clinical and public health intervention strategies.Item Evaluation of a Novel Point-of-Care Lateral Flow Assay To Detect Cryptococcal Antigen in Plasma and CSF(Opportunistic Infections, 2012) Rolfes, Melissa; Butler, Elissa; Nabeta, Henry; Kwizera, Richard; Meya, David; Boulware, DavidCurrent diagnostics for Cryptococcal meningitis (CM) rely on India ink, CSF culture, or cryptococcal antigen (CRAG) latex agglutination test. India ink is relatively insensitive. Culture and CRAG require lab infrastructure. Recently, a novel point-of-care lateral flow assay (LFA) has been FDA-approved for diagnosis of C. neoformans in serum samples. The aim of this study was to evaluate the LFA performance in plasma and CSF samples. ART-naïve subjects with and without cryptococcosis were enrolled in two prospective cohorts in Kampala, Uganda. Initial CM diagnosis was by CRAG, India ink, and quantitative CSF culture. Plasma and CSF were frozen at - 80C. Quantitative CRAG titers and LFA testing with titers were conducted on stored CSF, plasma, and serum. Plasma samples were tested in 112 subjects (n=61 with CM, n=51 with no clinical OI). CSF was tested in 161 subjects with suspected meningitis (105 with CM) from 2006-2009 and on 102 (58 with CM) in 2011. LFA results were compared for concordance with CRAG latex agglutination, India ink, and CSF culture.The LFA was highly sensitive and specific with CSF and plasma samples compared to both culture and CRAG. CRAG and LFA assays had high Kappa concordance in plasma and CSF samples, 0.93 and 0.91, respectively. LFA titers were 2.5 – 10-fold higher than CRAG titers in CSF and in plasma, respectively; however high correlation existed between the two assays (Spearman’s ρ=0.90 for plasma and 0.93 for CSF; p<0.001 for each). Culture and India ink had much lower concordance (Kappa 0.69 and 0.72, respectively) with LFA results (due to better LFA sensitivity), and titers were less correlated with quantitative culture results (ρ=0.71, p<0.001). Similar low concordance with culture and India ink were also seen with CRAG titers; suggesting the LFA results were not false-positives but more sensitive in detecting cryptococcal infection. The LFA assay has excellent concordance with CRAG latex agglutination and titers in both plasma and CSF samples and is more sensitive than standard India ink and CSF culture. Because of the wide availability of plasma samples for other clinical assessments (e.g. CD4 testing), the excellent agreement with the established CRAG assay, and the ease of use, the cryptococcal LFA holds great promise for rapid diagnosis of CM as well as the potential for cryptococcal screening prior to ART initiation, particularly in resource-limited areas.Item Evaluation of an Aspergillus IgG/IgM lateral flow assay for serodiagnosis of fungal asthma in Uganda(Plos one, 2019) Kwizera, Richard; Cresswell, Fiona V.; Mugumya, Gerald; Okirwoth, Micheal; Kagimu, Enock; Bangdiwala, Ananta S.; Williams, Darlisha A.; Rhein, Joshua; Boulware, David R.; Meya, David B.The diagnostic utility of the Mycobacteria tuberculosis lipoarabinomannan (TB-LAM) antigen lateral flow assay on cerebrospinal fluid (CSF) for the diagnosis of tuberculous meningitis (TBM) has not been extensively studied and the few published studies have conflicting results. Lumbar CSF from 59 HIV-positive patients with suspected TBM was tested with TB-LAM and Xpert MTB/Rif Ultra. The diagnostic performance of CSF TB-LAM was compared to positive CSF Xpert MTB/Rif Ultra (definite TBM) and a composite reference of probable or definite TBM according to the uniform case definition. Of 59 subjects, 12 (20%) had definite TBM and five (9%) had probable TBM. With reference to definite TBM, CSF TB-LAM assay had a diagnostic sensitivity of 33% and specificity of 96%. When compared to a composite reference of definite or probable TBM, the sensitivity was 24% and specificity was 95%. There were two false positive tests with TB-LAM (3+ grade). In-hospital mortality in CSF TB-LAM positive patients was 17% compared to 0% in those with definite TBM by Xpert MTB/Rif Ultra but negative LAM. Lumbar CSF TB-LAM has a poor performance in diagnosing TBM. Both urine TB-LAM and Xpert Ultra should be further investigated in the diagnosis of TBM.Item Evaluation of Fingerstick Cryptococcal Antigen Lateral Flow Assay in HIV-Infected Persons: A Diagnostic Accuracy Study(Clinical Infectious Diseases, 2015) Williams, Darlisha A.; Kiiza, Tadeo; Kwizera, Richard; Kiggundu, Reuben; Velamakanni, Sruti; Meya, David B.; Rhein, Joshua; Boulware, David R.Cryptococcus neoformans is the most common cause of adult meningitis in sub-Saharan Africa. The cryptococcal antigen (CRAG) lateral flow assay (LFA) has simplified diagnosis as a point-of-care test approved for serum or cerebrospinal fluid (CSF). We evaluated the accuracy of the CRAG LFA using fingerstick whole blood compared with serum/plasma and CSF for diagnosing meningitis.From August 2013 to August 2014, CRAG LFA (IMMY, Norman, Oklahoma) tests were performed on fingerstick whole blood, plasma/serum, and CSF in 207 HIV-infected adults with suspected meningitis in Kampala, Uganda. Venous blood was also collected and centrifuged to obtain serum and/or plasma. CSF was tested after lumbar puncture.Of 207 participants, 149 (72%) had fingerstick CRAG-positive results. There was 100% agreement between fingerstick whole blood and serum/plasma. Of the 149 fingerstick CRAG-positive participants, 138 (93%) had evidence of cryptococcal meningitis with a positive CSF CRAG. Eleven participants (5%) had isolated cryptococcal antigenemia with a negative CSF CRAG and culture, of whom 8 had CSF abnormalities (n = 3 lymphocytic pleocytosis, n = 5 elevated protein, n = 4 increased opening pressure). No persons with cryptococcal meningitis had negative fingersticks.The 100% agreement between whole blood, serum, and plasma CRAG LFA results demonstrates that fingerstick CRAG is a reliable bedside diagnostic test. Using point-of-care CRAG testing simplifies screening large numbers of patients and enables physicians to prioritize on whom to measure CSF opening pressure using manometers.Item Evaluation of the Bio Fire Film Array Meningitis/Encephalitis panel in an adult and pediatric Ugandan population(Journal of Medical Mycology, 2021) Bridge, Sarah; Hullsiek, Kathy Huppler; Nerima, Carol; Evansa, Emily E.; Nuwagira, Edwin; Stadelmana, Anna M.; Kiiza, K. Tadeo; Kwizera, Richard; Mwesigye, James; Meya, David B.; Muzoora, Conrad; Boulware, David R.; Rhein, JoshuaMeningitis causes significant mortality in sub-Saharan Africa and limited diagnostics exist. We evaluated the utility of the BioFire® FilmArray® Meningitis/Encephalitis multiplex PCR panel (BioFire ME) in HIV-infected adults and HIV-infected and uninfected children presenting with suspected meningitis in Uganda. We tested cerebrospinal fluid (CSF) using a stepwise meningitis diagnostic algorithm including BioFire ME. We determined the diagnostic performance of BioFire ME for cryptococcal meningitis, using cryptococcal antigen (CrAg) and CSF culture as reference standards, and assessed other central nervous system (CNS) pathogens identified by the panel. We evaluated 328 adult and 42 pediatric CSF specimens using BioFire ME. Of the adult CSF samples tested, 258 were obtained at baseline, and 70 were obtained from repeat lumbar punctures in cryptococcal meningitis. For Cryptococcus, sensitivity was 82%, specificity was 98%, PPV was 98%, and NPV was 79% in baseline specimens using CSF CrAg as the reference standard. Among follow-up specimens, a negative BioFire ME for Cryptococcus predicted CSF culture sterility with 84% NPV. Overall sensitivity was decreased at low fungal burdens: 29% for 0–99 Cryptococcus CFU/mL compared to 94% for ≥100 CFU/mL in baseline specimens. Other pathogens detected included E. Coli, H. influenzae, S. pneumoniae, CMV, enterovirus, HSV, HHV-6, and VZV. Two specimens tested positive for S. pneumoniae and one for Cryptococcus in the pediatric population. Multiplex PCR is a promising rapid diagnostic test for meningitis in adults and children in resource-limited settings. Cryptococcus at low fungal burdens in CSF may be missed by BioFire ME.Item Evaluation of the Diagnostic Performance of a Semiquantitative Cryptococcal Antigen Point-of-Care Assay among HIV-Infected Persons with Cryptococcal Meningitis(Journal of clinical microbiology, 2021) Tadeo, Kiiza Kandole; Nimwesiga, Audrey; Kwizera, Richard; Apeduno, Lucy; Martyn, Emily; Okirwoth, Michael; Nalintya, Elizabeth; Rajasingham, Radha; Williams, Darlisha A.; Rhein, Joshua; Meya, David B.; Kafufu, Bosco; Boulware, David R.; Skippera, Caleb P.A newly developed cryptococcal antigen (CrAg) semiquantitative (SQ) lateral flow assay (LFA) provides a semiquantitative result in a rapid one-step test instead of performing serial dilutions to determine CrAg titer. We prospectively compared the diagnostic performance of the CrAgSQ assay (IMMY) with the CrAg LFA (IMMY) on cerebrospinal fluid (CSF) samples collected from persons with HIV-associated meningitis. The CrAgSQ grades (1+ to 5+) were compared with CrAg LFA titers and quantitative CSF fungal cultures. Among 87 participants screened for HIV-associated meningitis, 60 had cryptococcal meningitis (59 CrAg positive [CrAg+] by LFA and 1 false negative due to prozone with CrAg LFA titer of 1:1,310,000 and culture positivity), and 27 had no cryptococcal meningitis by CrAg LFA or culture. The CrAgSQ on CSF had 100% (60/60) sensitivity and 100% specificity (27/27). CSF CrAg titers ranged from 1:5 to 1:42 million. CrAgSQ grades of 1+, 2+, 3+, 4+, and 5+ corresponded to median CrAg LFA titers of 1:<10, 1:60, 1:7,680, 1:81,920, and 1:1,474,000, respectively. CSF CrAgSQ grades 3+ or higher were always CSF culture positive. Mortality at 14 days for those with low CrAgSQ grade (1+ to 3+) was 5% (1/22) versus 21% (8/38) with high CrAgSQ grades (4+ to 5+) (P = 0.084). The CrAgSQ demonstrates excellent diagnostic performance, maintaining both the sensitivity and specificity of the CrAg LFA, and counters false-negative prozone effects. The CrAgSQ assay reading is more complex but does provide useful clinical information about disease burden and probability of culture positivity in a single rapid diagnostic test.Item Evaluation of the Dynamiker Cryptococcal Antigen Lateral Flow Assay for the Diagnosis of HIV-Associated Cryptococcosis(Journal of clinical microbiology, 2021) Kwizera, Richard; Omali, Denis; Tade, Kiiza; Kasibante, John; Rutakingirwa, Morris K.; Kagimu, Enock; Ssebambulidde, Kenneth; Williams, Darlisha A.; Rhein, Joshua; Boulware, David; Meya, David B.Cryptococcal meningitis is a leading cause of meningitis in sub-Saharan Africa. Given the need for rapid point-of-care testing, we evaluated the diagnostic performance of the Dynamiker cryptococcal antigen (CrAg) lateral flow assay (LFA). We assessed the diagnostic performance of the Dynamiker CrAg LFA compared to the IMMY CrAg LFA as the reference standard. We tested 150 serum, 115 plasma, and 100 cerebrospinal fluid (CSF) samples from HIV patients with symptomatic meningitis and 113 serum samples from patients with suspected asymptomatic cryptococcal antigenemia. Compared to the IMMY CrAg LFA, sensitivity of Dynamiker CrAg LFA was 98% in serum, 100% in plasma, 100% in CSF from symptomatic patients and 96% in serum from asymptomatic patients. Specificity was 66% in serum, 61% in plasma, and 91% in CSF from symptomatic patients, and 86% in serum from asymptomatic patients. The positive predictive value was 85% in serum, 82% in plasma, and 96% in CSF from symptomatic patients, and 69% in serum from asymptomatic patients. The negative predictive value was 94% in serum, 100% in plasma, and 100% in CSF from symptomatic patients, and 99% in serum from asymptomatic patients. The interassay reproducibility was 100% across the four sample types with no observed discordant results when Dynamiker CrAg LFA was tested in duplicate. However, a high number of false positives were observed on serum of symptomatic patients (11%), serum of asymptomatic patients (11%) and plasma of symptomatic patients (14%). The Dynamiker CrAg LFA had excellent sensitivity but poor specificity, particularly when tested on serum and plasma.Item Evaluation of the Initial 12months of a Routine Cryptococcal Antigen Screening Program in Reduction of HIV-Associated Cryptococcal Meningitis in Uganda(BMC Health Services Research, 2022) Enock, Kagimu; Kiwanuka, Julius; Abila, Derrick Bary; Rutakingirwa, Morris K.; Kasibante, John; Kiiza, Tadeo Kandole; Kwizera, Richard; Semeere, Aggrey; Meya, David B.Asymptomatic Cryptococcal Antigenemia (CrAg) patients develop meningitis within a month of testing positive. Pre-emptive antifungal therapy can prevent progression to Cryptococcal meningitis (CM). In April 2016, a national CrAg screening program was initiated in 206 high-volume health facilities that provide antiretroviral therapy in Uganda. We report the evaluation of the CrAg screening cascade focusing on linkage to care, fluconazole therapy for 10 weeks and 6 months follow up, and ART initiation in a subset of facilities. We conducted a retrospective, cross-sectional survey of patients with CD4 < 100 at seven urban and seven rural facilities after 1 year of program implementation. We quantified the number of patients who transitioned through the steps of the CrAg screening cascade over six-months follow-up. We defined cascade completion as a pre-emptive fluconazole prescription for the first 10 weeks. We conducted semi-structured interviews with lab personnel and clinic staff to assess functionality of the CrAg screening program. Data was collected using REDCap. We evaluated 359 patient records between April 2016 to March 2017; the majority (358/359, 99.7%) were from government owned health facilities and just over half (193/359, 53.8%) had a median baseline CD4 cell count of < 50 cell/μL. Overall, CrAg screening had been performed in 255/359 (71.0, 95% CI, 66.0–75.7) of patients’ records reviewed, with a higher proportion among urban facilities (170/209 (81.3, 95% CI, 75.4–86.4)) than rural facilities (85/150 (56.7, 95% CI, 48.3–64.7)). Among those who were CrAg screened, 56/255 (22.0, 95% CI, 17.0–27.5%) had cryptococcal antigenemia, of whom 47/56 (83.9, 95% CI, 71.7–92.4%) were initiated on pre-emptive therapy with fluconazole and 8/47 (17.0, 95% CI, 7.6–30.8%) of these were still receiving antifungal therapy at 6 months follow up. At least one CNS symptom was present in 70% (39/56) of those with antigenemia. In patients who had started ART, almost 40% initiated ART prior to CrAg screening. Inadequacy of equipment/supplies was reported by 15/26 (58%) of personnel as a program barrier, while 13/26 (50%) reported a need for training about CM and CrAg screening. There was a critical gap in the follow-up of patients after initiation on fluconazole therapy. ART had been initiated in almost 40% of patients prior to CrAg screening.. Higher antigenemia patients presenting with CNS symptoms could be related to late presentation. There is need to address these gaps after a more thorough evaluation.Item Evaluation of Trypan Blue Stain in a Haemocytometer for Rapid Detection of Cerebrospinal Fluid Sterility in HIV Patients with Cryptococcal Meningitis(BMC microbiology, 2017) Kwizera, Richard; Akampurira, Andrew; Kandole, Tadeo K.; Kambugu, Andrew; Kambugu, Andrew; Meya, David B.; Boulware, David R.; Rhein, Joshua; on behalf of the ASTRO-CM Study TeamQuantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis. A major drawback of cultures is a long turnaround-time. Recent evidence demonstrates that live and dead Cryptococcus yeasts can be distinguished using trypan blue staining. We hypothesized that trypan blue staining combined with haemocytometer counting may provide a rapid estimation of quantitative culture count and detection of CSF sterility. To test this, we evaluated 194 CSF specimens from 96 HIV-infected participants with cryptococcal meningitis in Kampala, Uganda. Cryptococcal meningitis was diagnosed by CSF cryptococcal antigen (CRAG). We stained CSF with trypan blue and quantified yeasts using a haemocytometer. We compared the haemocytometer readings versus quantitative Cryptococcus CSF cultures. Haemocytometer counting with trypan blue staining had a sensitivity of 98% (64/65), while CSF cultures had a sensitivity of 95% (62/65) with reference to CSF CRAG for diagnostic CSF specimens. For samples that were positive in both tests, the haemocytometer had higher readings compared to culture. For diagnostic specimens, the median of log10 transformed counts were 5.59 (n = 64, IQR = 5.09 to 6.05) for haemocytometer and 4.98 (n = 62, IQR = 3.75 to 5.79) for culture; while the overall median counts were 5.35 (n = 189, IQR = 4.78–5.84) for haemocytometer and 3.99 (n = 151, IQR = 2.59–5.14) for cultures. The percentage agreement with culture sterility was 2.4% (1/42). Counts among non-sterile follow-up specimens had a median of 5.38 (n = 86, IQR = 4.74 to 6.03) for haemocytometer and 2.89 (n = 89, IQR = 2.11 to 4.38) for culture. At diagnosis, CSF quantitative cultures correlated with haemocytometer counts (R2 = 0.59, P < 0.001). At 7–14 days, quantitative cultures did not correlate with haemocytometer counts (R2 = 0.43, P = 0.4). Despite a positive correlation, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative cultures in patients receiving antifungal therapy.Item Getting Histoplasmosis on the Map of International Recommendations for Patients with Advanced HIV Disease(Journal of Fungi, 2019) Bongomin, Felix; Kwizera, Richard; Denning, David W.Progressive disseminated histoplasmosis, caused by H. capsulatum, is a life-threatening illness and is an AIDS-defining opportunistic infection. It is neglected, worryingly under-diagnosed, and often misdiagnosed as cancer or tuberculosis with fatal consequences. Globally, over 100,000 cases of disseminated histoplasmosis have been estimated. In 2017, the World Health Organization (WHO) noted that disseminated histoplasmosis is a significant cause of mortality in AIDS patients. Through the rigorous efforts of the Global Action Fund for Fungal Infections (GAFFI) and partners, in 2019, the Histoplasma antigen test was included on the 2nd Edition of the WHO List of Essential Diagnostics. The drugs used in the treatment of histoplasmosis (amphotericin B and itraconazole) are on the WHO Essential Medicine List. The Manaus Declaration on histoplasmosis in the Americas and the Caribbean, where histoplasmosis kills more people with HIV than tuberculosis, advocates for universal access to rapid testing for histoplasmosis and availability of essential drugs for the treatment of histoplasmosis in every country by 2025. Hyperendemic areas are present in the Americas, Caribbean, Southeast Asia, and Latin America. In conclusion, histoplasmosis remains an important clinical and public health problem. To reduce HIV-associated mortality, disseminated histoplasmosis must be addressed through advocacy, increased awareness, and universal access to essential diagnostics and antifungal agentsItem Histoplasmosis Overlapping with HIV and Tuberculosis in Sub-Saharan Africa: Challenges and Research Priorities(Therapeutic Advances in Infectious Disease, 2021) Kuate, Marius Paulin Ngouanom; Ekeng, Bassey Ewa; Kwizera, Richard; Mandengue, Christine; Bongomin, FelixHistoplasmosis, tuberculosis and HIV are all highly prevalent in sub-Saharan Africa (SSA). Co-occurrence of two or more of these infections has been reported in several populations of patients, especially those with advanced HIV infection where these opportunistic infections contribute to a significant morbidity and mortality. With a high burden of pulmonary tuberculosis (PTB) secondary to HIV in SSA, histoplasmosis is commonly misdiagnosed as smear-negative PTB in HIV patients due to similar clinical and radiological presentations. This is also partly the result of the lack of trained clinical and laboratory personnel to make a definite diagnosis of histoplasmosis. There is a low index of clinical suspicion for histoplasmosis, and cases are mostly discovered accidently and documented through case reports and case series. Similarly, the high cost and lack of fungal diagnostics in most SSA countries makes it difficult to make a diagnosis. There is a need to build local capacity for mycology so that patients are managed to improve on the index of clinical suspicion and diagnostic capabilities. Moreover, simple accurate point-of-care diagnostic tests and first-line antifungal treatment for histoplasmosis are not available in many SSA countries. This review describes the existence of co-infections of histoplasmosis, tuberculosis and HIV in SSA, highlighting the challenges and research priorities.Item Impact of Community Engagement and Social Support on The Outcomes of HIV Related Meningitis Clinical Trials in a Resource-Limited Setting(Research Involvement and Engagement, 2020) Kwizera, Richard; Sadiq, Alisat; Ndyetukira, Jane Frances; Nalintya, Elizabeth; Boulware, David R.; Meya, David B.; on behalf of the COAT and ASTRO trial teamsClinical trials remain the cornerstone of improving outcomes for HIV-infected individuals with cryptococcal meningitis. Community engagement aims at involving participants and their advocates as partners in research rather than merely trial subjects. Community engagement can help to build trust in communities where these trials are conducted and ensure lasting mutually beneficial relationships between researchers and the community. Similarly, different studies have reported the positive effects of social support on patient’s outcomes. We aimed to describe our approach to community engagement in Uganda while highlighting the benefits of community engagement and social support in clinical trials managing patients co-infected with HIV and cryptococcal meningitis. We carried out community engagement using home visits, health talks, posters, music and drama. In addition, social support was given through study staff individually contributing to provide funds for participants’ food, wheel chairs, imaging studies, adult diapers, and other extra investigations or drugs that were not covered by the study budget or protocol. The benefits of this community engagement and social support were assessed during two multi-site, randomized cryptococcal meningitis clinical trials in Uganda. We screened 1739 HIV-infected adults and enrolled 934 with cryptococcal meningitis into the COAT and ASTRO-CM trials during the period October 2010 to July 2017. Lumbar puncture refusal rates decreased from 31% in 2010 to less than 1% in 2017. In our opinion, community engagement and social support played an important role in improving: drug adherence, acceptance of lumbar punctures, data completeness, rate of screening/referrals, reduction of missed visits, and loss to follow-up. Community engagement and social support are important aspects of clinical research and should be incorporated into clinical trial design and conduct.Item Intra-cavitary Pulmonary Cryptococcoma in Poorly Controlled Diabetes Mellitus(Medical mycology case reports, 2020) Kalyango, Najibu; Kwizera, Richard; Baluku, Joseph B.; Bongomin, FelixA 59-year-old HIV-negative Ugandan man presented with a long-standing history of respiratory symptoms and was found to have an intra-cavitary pulmonary cryptococoma by chest imaging and sputum culture. The serum cryptococcal antigen was negative. The sputum Xpert® MTB RIF Ultra assay was negative. He was previously treated for cavitary pulmonary tuberculosis. The patient had poorly controlled diabetes (HbA1c, 9.3%). The patient was successfully treated with oral fluconazole.Item Invasive Fungal Diseases in Africa: A Critical Literature Review(Journal of Fungi,, 2022) Bongomin, Felix; Ekeng, Bassey E.; Kibone, Winnie; Nsenga, Lauryn; Olum, Ronald; Itam-Eyo, Asa; Ngouanom Kuate, Marius Paulin; Pebalo Pebolo, Francis; Davies, Adeyinka A.; Manga, Musa; Ocansey, Bright; Kwizera, Richard; Baruch Baluku, JosephInvasive fungal diseases (IFDs) are of huge concern in resource-limited settings, particularly in Africa, due to the unavailability of diagnostic armamentarium for IFDs, thus making definitive diagnosis challenging. IFDs have non-specific systemic manifestations overlapping with more frequent illnesses, such as tuberculosis, HIV, and HIV-related opportunistic infections and malignancies. Consequently, IFDs are often undiagnosed or misdiagnosed. We critically reviewed the available literature on IFDs in Africa to provide a better understanding of their epidemiology, disease burden to guide future research and interventions. Cryptococcosis is the most encountered IFD in Africa, accounting for most of the HIV-related deaths in sub-Saharan Africa. Invasive aspergillosis, though somewhat underdiagnosed and/or misdiagnosed as tuberculosis, is increasingly being reported with a similar predilection towards people living with HIV. More cases of histoplasmosis are also being reported with recent epidemiological studies, particularly fromWestern Africa, showing high prevalence rates amongst presumptive tuberculosis patients and patients living with HIV. The burden of pneumocystis pneumonia has reduced significantly probably due to increased uptake of anti-retroviral therapy among people living with HIV both in Africa, and globally. Mucormycosis, talaromycosis, emergomycosis, blastomycosis, and coccidiomycosis have also been reported but with very few studies from the literature. The emergence of resistance to most of the available antifungal drugs in Africa is yet of huge concern as reported in other regions. IFDs in Africa is much more common than it appears and contributes significantly to morbidity and mortality. Huge investment is needed to drive awareness and fungi related research especially in diagnostics and antifungal therapy.