Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; ... more Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; however, culture has a long turnaround-time. The TC20 automated cell counter (Bio-Rad) is a benchtop instrument used to count cells in 30 seconds. In vitro studies suggest trypan blue staining can distinguish between viable and dead cryptococcal yeasts. We hypothesized that trypan blue staining with automated cell counting may provide rapid quantification of viable CSF Cryptococcus yeasts. In sum, 96 HIV-infected participants with cryptococcal meningitis were enrolled and provided 194 CSF specimens in Kampala, Uganda. Cryptococcosis was diagnosed by CSF cryptococcal antigen (CRAG). CSF was stained with trypan blue and quantified yeasts with the TC20 cell counter. We compared the log 10 transformed cell counter readings with gating of 4–10 µm versus log 10 quantitative Cryptococcus cultures/ml. TC20 showed more positive results (95.4%) overall than culture (78.4%) with reference to CSF CRAG. TC20 had higher readings compared to culture in most cases with only a 25% level of agreement between the two methods. TC20 had a poor correlation to culture throughout the 14 days of antifungal therapy. The median of log 10 transformed counts were 5.22 (IQR = 4.79–5.44) for the TC20 and 3.99 (IQR = 2.59–5.14) for culture. Overall, a linear regression showed no significant relationship between the TC20 and culture (r = −0.0025; P = .92). TC20 automated cell C
Background Cryptococcus is the most common cause of adult meningitis in Africa. We assessed the s... more Background Cryptococcus is the most common cause of adult meningitis in Africa. We assessed the safety and microbiological efficacy of adjunctive sertraline, previously shown to have in-vitro and in-vivo activity against cryptococcus.
Antimicrobial agents and chemotherapy, Jan 31, 2015
Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV car... more Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with CD4 count <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 2010-2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998/99 reporting an MIC50 of 4 μg/mL and an MIC90 of 8 μg/mL prior to wide-spread human fluconazole and agricultural azole fungicide usage, we report an upward shift in fluconazole MIC50 to 8 μg/mL, and a MIC90 value of 32 μg/mL. We observed an amphotericin B MIC50 of 0.5 μg/mL and an MIC90 of 1 μg/mL, of which 99.5% (197/198) of isolates were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and flucon...
JAIDS Journal of Acquired Immune Deficiency Syndromes, 2015
The effect of tenofovir and amphotericin co-administration on kidney function is poorly character... more The effect of tenofovir and amphotericin co-administration on kidney function is poorly characterized. We measured creatinine during induction therapy and at 4-weeks post-diagnosis in Ugandans undergoing cryptococcal meningitis therapy, and classified as not receiving antiretroviral therapy (ART), receiving non-tenofovir ART, or receiving tenofovir-based ART. Longitudinal creatinine changes and Grade 2-4 creatinine adverse events were evaluated across groups. Creatinine concentrations were similar across ART groups. At 4 weeks post-diagnosis, creatinine was 0.25mg/dL higher than at diagnosis, but similar across groups. Adverse event incidence was also similar across ART groups. Tenofovir and amphotericin co-administration did not increase the risk of kidney dysfunction.
ABSTRACT Background: (1-3)-β-D-glucan (BDG) is a helpful diagnostic marker for many invasive fung... more ABSTRACT Background: (1-3)-β-D-glucan (BDG) is a helpful diagnostic marker for many invasive fungal infections. However, BDG is not thought to be useful in diagnosing cryptococcosis. We evaluated the utility of cerebrospinal fluid (CSF) and serum BDG as an adjunct diagnostic tool for HIV-infected cryptococcal meningitis patients. Methods: BDG concentrations were measured on cryopreserved CSF (n=177) and serum (n=109) of HIV-infected patients with suspected meningitis in Uganda and South Africa using the Fungitell® assay. Correlations between BDG concentrations and quantitative CSF cryptococcal cultures, CSF cryptococcal antigen (CRAG) titers, and 18 different CSF cytokine concentrations were assessed using non-parametric tests. Mixed models evaluated longitudinal changes in CSF BDG concentrations. Survival analyses evaluated BDG’s relationship with mortality. Results: The Fungitell® BDG assay provided 90% sensitivity (69/77) and 85% specificity (34/40) in CSF when compared to cryptococcal meningitis diagnosed by CSF culture or cryptococcal antigen at diagnosis (n=117, 66% with Cryptococcus neoformans). Sensitivity in CSF improved to 98% (57/58) when initial fungal burdens were ≥10,000 CFU/mL. Median (IQR) CSF BDG concentrations at diagnosis were 346 (202-597) pg/mL in cryptococcal patients and 37 (20-46) pg/mL in patients without cryptococcosis. Baseline BDG concentrations correlated with CSF fungal burden (rho=0.820, P&lt;.001) and CRAG LFA titers (rho=0.780, P&lt;.001). BDG normalized rapidly after initiating antifungal therapy [-0.25 (95%CI: -0.31, -0.20) average change in log2BDG level per day of follow-up]. Baseline BDG concentrations correlated with MIP-1β and MCP-1 levels in CSF. Among cryptococcal meningitis patients, a BDG concentration ≥500 pg/mL was associated with increased 10-week mortality. The diagnostic performance of the BDG assay for cryptococcal meningitis was not as good in serum, where we observed 79% (37/47) sensitivity and 61% (38/62) specificity. Conclusion: BDG is detectable in the CSF of HIV-infected patients with Cryptococcus, and may provide useful diagnostic and prognostic information. Further research is needed to clarify the role of BDG in the immunology and management of cryptococcal disease.
Mortality due to AIDS-related Cryptococcal meningitis (CM) is often >50% in low-middle income ... more Mortality due to AIDS-related Cryptococcal meningitis (CM) is often >50% in low-middle income countries. Dissemination of CM can result in intracranial mass lesions known as cryptococcoma. Patients who develop cryptococcomas often have worse outcomes when compared to patients with cryptococcosis without cryptococcoma. We describe a cryptococcoma in the central nervous system (CNS) in a Ugandan patient with AIDS, and review the diagnosis and management with special focus on difficulties encountered in low or middle-income countries.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2015
Cryptococcus neoformans is the most common cause of adult meningitis in sub-Saharan Africa. The ... more Cryptococcus neoformans is the most common cause of adult meningitis in sub-Saharan Africa. The cryptococcal antigen lateral flow assay (CRAG 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 in Kampala, Uganda with suspected meningitis. Venous blood was also collected and centrifuged to obtain serum and/or plasma. CSF was tested after lumbar puncture. Of 207 participants, 149 (72%) were fingerstick CRAG-positive. 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%...
Earlier antiretroviral therapy (ART) initiation in cryptococcal meningitis resulted in higher mo... more Earlier antiretroviral therapy (ART) initiation in cryptococcal meningitis resulted in higher mortality compared with deferred ART initiation (1-2 weeks vs 5 weeks postmeningitis diagnosis). We hypothesized this was due to ART-associated immune pathology, without clinically recognized immune reconstitution inflammatory syndrome. Three macrophage activation markers and 19 cytokines/chemokines were measured from cryopreserved cerebrospinal fluid (CSF) and serum during the Cryptococcal Optimal ART Timing (COAT) trial. Comparisons were made between trial arms (early vs deferred) at 1, 8, 14, and 21 days following meningitis diagnosis. More participants with early ART initiation had CSF white cell count (WCC) ≥5/µL at day 14 (58% vs 40%; P = .047), after a median of 6-days ART. Differences were mainly driven by participants with CSF WCC <5/µL at meningitis diagnosis: 28% (10/36) of such persons in the early ART group had CSF WCC ≥5/µL by day 14, compared with 0% (0/27) in the defer...
Background: Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize ... more Background: Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. Delay in diagnosis leads to excess morbidity, mortality and healthcare costs related to unnecessary empiric treatment and isolation procedures. Methods: From January-April 2014, cerebrospinal fluid (CSF) from 39 HIV-infected persons with suspected meningitis in Kampala, Uganda was collected at time of diagnosis (n=18) and among persons with cryptococcal meningitis at therapeutic lumbar punctures (n=53). Standard bacterial, mycobacterial and fungal CSF diagnostics were performed on site. Cryopreserved CSF specimens (200 mcL) were then analyzed on the FilmArray™ System using a Meningitis/Encephalitis PCR panel (BioFire Diagnostics, Salt Lake City, UT; research use only). The panel targets 16 common pathogens: 6 bacterial, 8 viral, and Cryptococcus neoformans/gattii speciation. Operators were blinded to microbiology results. We assessed the diagnostic performance of the panel. Res...
Despite improvements in the antifungal regimens and the roll out of antiretroviral therapy (ART) ... more Despite improvements in the antifungal regimens and the roll out of antiretroviral therapy (ART) in sub-Saharan Africa, mortality due to cryptococcal meningitis remains high. Relapse of an initially successfully treated infection contributes to this mortality and is often a clinical dilemma in differentiating between paradoxical immune reconstitution inflammatory syndrome (IRIS) and culture-positive relapse or treatment failure. Herein, we present a clinical case scenario and review the case definitions, differential diagnosis, and management of relapse with an emphasis on the current diagnostic and management strategies. We also highlight the challenges of resistance testing and management of refractory relapse cases. The risk of relapse is influenced by: 1) the choice of induction therapy, with higher mortality risk with fluconazole monotherapy which can select for resistance; 2) non-adherence to or lack of secondary prophylaxis; 3) failure of linkage-to-care or retention-in-care of HIV ART programs.
Despite improvements in the antifungal regimens and the roll out of antiretroviral therapy (ART) ... more Despite improvements in the antifungal regimens and the roll out of antiretroviral therapy (ART) in sub-Saharan Africa, mortality due to cryptococcal meningitis remains high. Relapse of an initially successfully treated infection contributes to this mortality and is often a clinical dilemma in differentiating between paradoxical immune reconstitution inflammatory syndrome (IRIS) and culture-positive relapse or treatment failure. Herein, we present a clinical case scenario and review the case definitions, differential diagnosis, and management of relapse with an emphasis on the current diagnostic and management strategies. We also highlight the challenges of resistance testing and management of refractory relapse cases. The risk of relapse is influenced by: 1) the choice of induction therapy, with higher mortality risk with fluconazole monotherapy which can select for resistance; 2) non-adherence to or lack of secondary prophylaxis; 3) failure of linkage-to-care or retention-in-care of HIV ART programs.
Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; ... more Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; however, culture has a long turnaround-time. The TC20 automated cell counter (Bio-Rad) is a benchtop instrument used to count cells in 30 seconds. In vitro studies suggest trypan blue staining can distinguish between viable and dead cryptococcal yeasts. We hypothesized that trypan blue staining with automated cell counting may provide rapid quantification of viable CSF Cryptococcus yeasts. In sum, 96 HIV-infected participants with cryptococcal meningitis were enrolled and provided 194 CSF specimens in Kampala, Uganda. Cryptococcosis was diagnosed by CSF cryptococcal antigen (CRAG). CSF was stained with trypan blue and quantified yeasts with the TC20 cell counter. We compared the log 10 transformed cell counter readings with gating of 4–10 µm versus log 10 quantitative Cryptococcus cultures/ml. TC20 showed more positive results (95.4%) overall than culture (78.4%) with reference to CSF CRAG. TC20 had higher readings compared to culture in most cases with only a 25% level of agreement between the two methods. TC20 had a poor correlation to culture throughout the 14 days of antifungal therapy. The median of log 10 transformed counts were 5.22 (IQR = 4.79–5.44) for the TC20 and 3.99 (IQR = 2.59–5.14) for culture. Overall, a linear regression showed no significant relationship between the TC20 and culture (r = −0.0025; P = .92). TC20 automated cell C
Background Cryptococcus is the most common cause of adult meningitis in Africa. We assessed the s... more Background Cryptococcus is the most common cause of adult meningitis in Africa. We assessed the safety and microbiological efficacy of adjunctive sertraline, previously shown to have in-vitro and in-vivo activity against cryptococcus.
Antimicrobial agents and chemotherapy, Jan 31, 2015
Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV car... more Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with CD4 count <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 2010-2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998/99 reporting an MIC50 of 4 μg/mL and an MIC90 of 8 μg/mL prior to wide-spread human fluconazole and agricultural azole fungicide usage, we report an upward shift in fluconazole MIC50 to 8 μg/mL, and a MIC90 value of 32 μg/mL. We observed an amphotericin B MIC50 of 0.5 μg/mL and an MIC90 of 1 μg/mL, of which 99.5% (197/198) of isolates were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and flucon...
JAIDS Journal of Acquired Immune Deficiency Syndromes, 2015
The effect of tenofovir and amphotericin co-administration on kidney function is poorly character... more The effect of tenofovir and amphotericin co-administration on kidney function is poorly characterized. We measured creatinine during induction therapy and at 4-weeks post-diagnosis in Ugandans undergoing cryptococcal meningitis therapy, and classified as not receiving antiretroviral therapy (ART), receiving non-tenofovir ART, or receiving tenofovir-based ART. Longitudinal creatinine changes and Grade 2-4 creatinine adverse events were evaluated across groups. Creatinine concentrations were similar across ART groups. At 4 weeks post-diagnosis, creatinine was 0.25mg/dL higher than at diagnosis, but similar across groups. Adverse event incidence was also similar across ART groups. Tenofovir and amphotericin co-administration did not increase the risk of kidney dysfunction.
ABSTRACT Background: (1-3)-β-D-glucan (BDG) is a helpful diagnostic marker for many invasive fung... more ABSTRACT Background: (1-3)-β-D-glucan (BDG) is a helpful diagnostic marker for many invasive fungal infections. However, BDG is not thought to be useful in diagnosing cryptococcosis. We evaluated the utility of cerebrospinal fluid (CSF) and serum BDG as an adjunct diagnostic tool for HIV-infected cryptococcal meningitis patients. Methods: BDG concentrations were measured on cryopreserved CSF (n=177) and serum (n=109) of HIV-infected patients with suspected meningitis in Uganda and South Africa using the Fungitell® assay. Correlations between BDG concentrations and quantitative CSF cryptococcal cultures, CSF cryptococcal antigen (CRAG) titers, and 18 different CSF cytokine concentrations were assessed using non-parametric tests. Mixed models evaluated longitudinal changes in CSF BDG concentrations. Survival analyses evaluated BDG’s relationship with mortality. Results: The Fungitell® BDG assay provided 90% sensitivity (69/77) and 85% specificity (34/40) in CSF when compared to cryptococcal meningitis diagnosed by CSF culture or cryptococcal antigen at diagnosis (n=117, 66% with Cryptococcus neoformans). Sensitivity in CSF improved to 98% (57/58) when initial fungal burdens were ≥10,000 CFU/mL. Median (IQR) CSF BDG concentrations at diagnosis were 346 (202-597) pg/mL in cryptococcal patients and 37 (20-46) pg/mL in patients without cryptococcosis. Baseline BDG concentrations correlated with CSF fungal burden (rho=0.820, P&lt;.001) and CRAG LFA titers (rho=0.780, P&lt;.001). BDG normalized rapidly after initiating antifungal therapy [-0.25 (95%CI: -0.31, -0.20) average change in log2BDG level per day of follow-up]. Baseline BDG concentrations correlated with MIP-1β and MCP-1 levels in CSF. Among cryptococcal meningitis patients, a BDG concentration ≥500 pg/mL was associated with increased 10-week mortality. The diagnostic performance of the BDG assay for cryptococcal meningitis was not as good in serum, where we observed 79% (37/47) sensitivity and 61% (38/62) specificity. Conclusion: BDG is detectable in the CSF of HIV-infected patients with Cryptococcus, and may provide useful diagnostic and prognostic information. Further research is needed to clarify the role of BDG in the immunology and management of cryptococcal disease.
Mortality due to AIDS-related Cryptococcal meningitis (CM) is often >50% in low-middle income ... more Mortality due to AIDS-related Cryptococcal meningitis (CM) is often >50% in low-middle income countries. Dissemination of CM can result in intracranial mass lesions known as cryptococcoma. Patients who develop cryptococcomas often have worse outcomes when compared to patients with cryptococcosis without cryptococcoma. We describe a cryptococcoma in the central nervous system (CNS) in a Ugandan patient with AIDS, and review the diagnosis and management with special focus on difficulties encountered in low or middle-income countries.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2015
Cryptococcus neoformans is the most common cause of adult meningitis in sub-Saharan Africa. The ... more Cryptococcus neoformans is the most common cause of adult meningitis in sub-Saharan Africa. The cryptococcal antigen lateral flow assay (CRAG 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 in Kampala, Uganda with suspected meningitis. Venous blood was also collected and centrifuged to obtain serum and/or plasma. CSF was tested after lumbar puncture. Of 207 participants, 149 (72%) were fingerstick CRAG-positive. 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%...
Earlier antiretroviral therapy (ART) initiation in cryptococcal meningitis resulted in higher mo... more Earlier antiretroviral therapy (ART) initiation in cryptococcal meningitis resulted in higher mortality compared with deferred ART initiation (1-2 weeks vs 5 weeks postmeningitis diagnosis). We hypothesized this was due to ART-associated immune pathology, without clinically recognized immune reconstitution inflammatory syndrome. Three macrophage activation markers and 19 cytokines/chemokines were measured from cryopreserved cerebrospinal fluid (CSF) and serum during the Cryptococcal Optimal ART Timing (COAT) trial. Comparisons were made between trial arms (early vs deferred) at 1, 8, 14, and 21 days following meningitis diagnosis. More participants with early ART initiation had CSF white cell count (WCC) ≥5/µL at day 14 (58% vs 40%; P = .047), after a median of 6-days ART. Differences were mainly driven by participants with CSF WCC <5/µL at meningitis diagnosis: 28% (10/36) of such persons in the early ART group had CSF WCC ≥5/µL by day 14, compared with 0% (0/27) in the defer...
Background: Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize ... more Background: Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. Delay in diagnosis leads to excess morbidity, mortality and healthcare costs related to unnecessary empiric treatment and isolation procedures. Methods: From January-April 2014, cerebrospinal fluid (CSF) from 39 HIV-infected persons with suspected meningitis in Kampala, Uganda was collected at time of diagnosis (n=18) and among persons with cryptococcal meningitis at therapeutic lumbar punctures (n=53). Standard bacterial, mycobacterial and fungal CSF diagnostics were performed on site. Cryopreserved CSF specimens (200 mcL) were then analyzed on the FilmArray™ System using a Meningitis/Encephalitis PCR panel (BioFire Diagnostics, Salt Lake City, UT; research use only). The panel targets 16 common pathogens: 6 bacterial, 8 viral, and Cryptococcus neoformans/gattii speciation. Operators were blinded to microbiology results. We assessed the diagnostic performance of the panel. Res...
Despite improvements in the antifungal regimens and the roll out of antiretroviral therapy (ART) ... more Despite improvements in the antifungal regimens and the roll out of antiretroviral therapy (ART) in sub-Saharan Africa, mortality due to cryptococcal meningitis remains high. Relapse of an initially successfully treated infection contributes to this mortality and is often a clinical dilemma in differentiating between paradoxical immune reconstitution inflammatory syndrome (IRIS) and culture-positive relapse or treatment failure. Herein, we present a clinical case scenario and review the case definitions, differential diagnosis, and management of relapse with an emphasis on the current diagnostic and management strategies. We also highlight the challenges of resistance testing and management of refractory relapse cases. The risk of relapse is influenced by: 1) the choice of induction therapy, with higher mortality risk with fluconazole monotherapy which can select for resistance; 2) non-adherence to or lack of secondary prophylaxis; 3) failure of linkage-to-care or retention-in-care of HIV ART programs.
Despite improvements in the antifungal regimens and the roll out of antiretroviral therapy (ART) ... more Despite improvements in the antifungal regimens and the roll out of antiretroviral therapy (ART) in sub-Saharan Africa, mortality due to cryptococcal meningitis remains high. Relapse of an initially successfully treated infection contributes to this mortality and is often a clinical dilemma in differentiating between paradoxical immune reconstitution inflammatory syndrome (IRIS) and culture-positive relapse or treatment failure. Herein, we present a clinical case scenario and review the case definitions, differential diagnosis, and management of relapse with an emphasis on the current diagnostic and management strategies. We also highlight the challenges of resistance testing and management of refractory relapse cases. The risk of relapse is influenced by: 1) the choice of induction therapy, with higher mortality risk with fluconazole monotherapy which can select for resistance; 2) non-adherence to or lack of secondary prophylaxis; 3) failure of linkage-to-care or retention-in-care of HIV ART programs.
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