Epidemiology and susceptibility of Nakaseomyces (formerly Candida) glabrata bloodstream isolates from hospitalised adults in South Africa.

During 2016-2017, Nakaseomyces glabrata (formerly Candida glabrata) caused 14% of cases of candidaemia in South Africa. We aimed to describe the clinical characteristics of adults with N. glabrata candidaemia at 20 sentinel hospitals (accounting for 20% (172/917) of cases) and the antifungal susceptibility of the corresponding isolates. A higher proportion of patients with N. glabrata candidaemia were older (median age: 55 years [interquartile range (IQR): 41-65 years] vs. 49 years [IQR: 35-63 years]; p = 0.04), female (87/164, 53% vs. 283/671, 42%; p = 0.01), admitted to a public-sector hospital (152/172, 88% vs. 470/745, 63%; p < 0.001), treated with fluconazole only (most with suboptimal doses) (51/95, 54% vs. 139/361, 39%; p < 0.001), and had surgery (47/172, 27% vs. 123/745, 17%; p = 0.001) and a shorter hospital stay (median 7 days [IQR: 2-20 days] vs. 13 days [IQR: 4-27 days]; p < 0.001) compared to patients with other causes of candidaemia. Eight N. glabrata isolates (6%, 8/131) had minimum inhibitory concentrations in the intermediate or resistant range for ≥ 1 echinocandin and a R1377K amino acid substitution encoded by the hotspot 2 region of the FKS2 gene. Only 11 isolates (8%, 11/131) were resistant to fluconazole. Patients with confirmed N. glabrata candidaemia are recommended to be treated with an echinocandin (or polyene), thus further guideline training is required.

Nakaseomyces (formerly Candida) glabrata is a yeast-like fungus that forms part of the commensal gut flora and among people with certain risk factors, can invade into the bloodstream. Nakaseomyces glabrata is a relatively more common cause of candidaemia in high-income vs. low- and middle-income countries. There are no N. glabrata clinical isolates that are considered susceptible to fluconazole, and thus echinocandins are recommended for treatment. However, echinocandin resistance is emerging. We described the characteristics of South African patients with N. glabrata bloodstream infections and the antifungal susceptibility of corresponding isolates. We found that patients infected with N. glabrata were more likely to be older, female, admitted to public hospitals and to be post-surgery and these patients were also more likely to be treated with fluconazole monotherapy and to have stayed a shorter time in hospital compared to patients infected with other Candida species. Only 6% of N. glabrata isolates were echinocandin-resistant with mutations in specific resistance genes that we have found in South African N. glabrata isolates previously. Eight percent of N. glabrata isolates were resistant to fluconazole and the remainder were in the susceptible dose dependent category, requiring higher fluconazole treatment doses. Patients with confirmed N. glabrata bloodstream infection should ideally be treated with an echinocandin or polyene rather than fluconazole and training is required for doctors treating these patients.

Epidemiology of Culture-confirmed Candidemia Among Hospitalized Children in South Africa, 2012-2017.

BACKGROUND: We aimed to describe the epidemiology of candidemia among children in South Africa. METHODS: We conducted laboratory-based surveillance among neonates (≤28 days), infants (29 days to <1 year), children (1-11 years) and adolescents (12-17 years) with Candida species cultured from blood during 2012-2017. Identification and antifungal susceptibility of viable isolates were performed at a reference laboratory. We used multivariable logistic regression to determine the association between Candida parapsilosis candidemia and 30-day mortality among neonates. RESULTS: Of 2996 cases, neonates accounted for 49% (n = 1478), infants for 27% (n = 806), children for 20% (n = 589) and adolescents for 4% (n = 123). The incidence risk at tertiary public sector hospitals was 5.3 cases per 1000 pediatric admissions (range 0.39-119.1). Among 2943 cases with single-species infections, C. parapsilosis (42%) and Candida albicans (36%) were most common. Candida auris was among the 5 common species with an overall prevalence of 3% (n = 47). Fluconazole resistance was more common among C. parapsilosis (55% [724/1324]) versus other species (19% [334/1737]) (P < 0.001). Of those with known treatment (n = 1666), 35% received amphotericin B deoxycholate alone, 32% fluconazole alone and 30% amphotericin B deoxycholate with fluconazole. The overall 30-day in-hospital mortality was 38% (n = 586) and was highest among neonates (43% [323/752]) and adolescents (43% [28/65]). Compared with infection with other species, C. parapsilosis infection was associated with a reduced mortality among neonates (adjusted odds ratio 0.41, 95% confidence interval: 0.22-0.75, P = 0.004). CONCLUSIONS: Candidemia in this setting mainly affected neonates and infants and was characterized by fluconazole-resistant C. parapsilosis with no increased risk of death.

Cross-reactivity of a Histoplasma capsulatum antigen enzyme immunoassay in urine specimens from persons with emergomycosis in South Africa.

Histoplasma antigen detection in urine is a rapid diagnostic method for disseminated histoplasmosis, although cross-reactivity has been reported in specimens from patients with other thermally dimorphic fungal infections. We tested urine specimens, from persons with suspected invasive fungal infections, using a commercial monoclonal antibody Histoplasma enzyme immunoassay (EIA) at a South African national mycology reference laboratory from August 2014 through December 2018. Corresponding fungal culture and histopathology results were obtained from an electronic laboratory information system. In some cases, cultured fungal isolates were sent with the urine specimen for species-level identification by phenotypic and molecular methods. Cross-reactivity was confirmed using culture filtrates of several fungal pathogens. Of 212 referred cases, 41 (19%) were excluded since they had no recorded clinical history (n = 1), alternative diagnoses were confirmed (n = 2), or no fungal culture or histopathology results (n = 38). Eighty-seven of 212 (41%) had laboratory evidence of an invasive fungal disease, while 84 (40%) did not. Of the 87 cases, 37 (43%) were culture-confirmed mycoses: emergomycosis (n = 18), histoplasmosis (n = 8), sporotrichosis (n = 6), cryptococcosis (n = 2), talaromycosis (n = 1), and other fungi isolated (n = 2). The sensitivity and specificity of the EIA were calculated for two groups: culture-confirmed (n = 37) and histology-confirmed invasive fungal disease (n = 50). The sensitivity and specificity of the EIA for diagnosis of histoplasmosis compared to culture were 88% (7/8, 95%CI 47-100%) and 72% (21/29, 95%CI 53-87%), respectively, and for diagnosis of emergomycosis/histoplasmosis compared to histology was 83% (29/35, 95%CI 66-93%) and 93% (14/15, 95%CI 68-100%), respectively. Cross-reactions occurred in urine specimens of patients with Emergomyces africanus infection and in culture filtrates of E. africanus, T. marneffei and Blastomyces species. A commercial Histoplasma EIA had satisfactory accuracy for diagnosis of culture-confirmed histoplasmosis, but cross-reacted in urine specimens from patients with invasive disease caused by the closely-related pathogen, E. africanus and in culture filtrates of E. africanus and other related fungi. LAY SUMMARY: Emergomyces africanus and Histoplasma capsulatum are fungi that cause a multi-system disease among HIV-seropositive persons with a low CD4 cell count. Handling live cultures of these fungi to confirm a diagnosis requires specialized laboratory equipment and infrastructure which is infrequently accessible in low-resource settings. The features of the two diseases (i.e., disseminated histoplasmosis and emergomycosis) may be indistinguishable when infected tissue is prepared, stained, and examined under a microscope. Enzyme immunoassays (EIA) have been developed as rapid diagnostic tools for the detection of a cell wall component of H. capsulatum in urine specimens, although cross-reactions have been reported in specimens from patients with other fungal infections. We evaluated the accuracy of a commercial Histoplasma EIA to diagnose histoplasmosis and to assess cross-reactions in urine specimens from persons with emergomycosis and in cultures of E. africanus and related fungi. We report a sensitivity and specificity of 88% (95%CI 47-100%) and 72% (95%CI 53-87%) for diagnosis of histoplasmosis compared to culture and 83% (95%CI 66-93%) and 93% (95%CI 68-100%) for diagnosis of either histoplasmosis/emergomycosis compared to a diagnosis made by microscopic examination of infected tissue. The assay cross-reacted in urine specimens from patients with emergomycosis and in culture filtrates of related fungi. Although the EIA cross-reacted with other related fungi, this test can decrease the time to diagnosis and facilitate early treatment of emergomycosis and histoplasmosis in South Africa.

Screening for invasive fungal disease using non-culture-based assays among inpatients with advanced HIV disease at a large academic hospital in South Africa.

INTRODUCTION: Despite widespread access to antiretroviral therapy (ART), the burden of advanced HIV disease in South Africa is high. This translates into an increased risk of AIDS-related opportunistic infections, including invasive mycoses. METHODS: Using a limited number of non-culture-based diagnostic assays, we aimed to determine the prevalence of invasive mycoses and tuberculosis among hospitalised adults with very advanced HIV (CD4 counts < 100 cells/µL) at a large academic hospital. We conducted interviews and prospective medical chart reviews. We performed point-of-care finger stick and serum cryptococcal antigen lateral flow assays; serum (1 → 3) ß-D-glucan assays; urine Histoplasma galactomannan antigen enzyme immunoassays and TB lipoarabinomannan assays. RESULTS: We enrolled 189 participants from 5280 screened inpatients. Fifty-eight per cent were female, with median age 37 years (IQR: 30-43) and median CD4 count 32 cells/µL (IQR: 13-63). At enrolment, 60% (109/181) were receiving ART. Twenty-one participants (11%) had a diagnosis of an invasive mycosis, of whom 53% (11/21) had cryptococcal disease. Thirteen participants (7%) had tuberculosis and a concurrent invasive mycosis. ART-experienced participants were 60% less likely to have an invasive mycosis than those ART-naïve (adjusted OR: 0.4; 95% CI 0.15-1.0; P = .03). Overall in-hospital mortality was 13% (invasive mycosis: 10% [95% CI 1.2-30.7] versus other diagnoses: 13% (95% CI 8.4-19.3)). CONCLUSIONS: One in ten participants had evidence of an invasive mycosis. Diagnosis of proven invasive fungal disease and differentiation from other opportunistic infections was challenging. More fungal-specific screening and diagnostic tests should be applied to inpatients with advanced HIV disease.

Multilocus sequence typing of azole-resistant Candida auris strains, South Africa.

BACKGROUND: Candida auris is an emerging multidrug-resistant fungal pathogen associated with high mortality. METHODS: We investigated the genetic relatedness of clinical C. auris isolates from patients admitted to either public- or private-sector hospitals, which were submitted to a reference laboratory from 2012 to 2015. Patient demographics and clinical details were recorded. We performed antifungal susceptibility testing, sequencing of the hotspot 1 and 2 regions of the FKS1 and FKS2 genes for all isolates with an echinocandin minimum inhibitory concentration (MIC) of ≥1 µg/mL and cluster analysis using multilocus sequence typing. RESULTS: Eighty-five isolates were confirmed as C. auris. The median patient age was 59 years [inter-quartile range (IQR): 48-68 years], with male patients accounting for 68% of cases. Specimen types included urine (29%), blood (27%), central venous catheter tips (25%), irrigation fluid (7%), tissue (5%), respiratory tract specimens (4%) and other (3%). Ninety-seven per cent of isolates were resistant to fluconazole, 7% were resistant to both fluconazole and voriconazole, 8% were resistant to both fluconazole and echinocandins (considered multidrug resistant) and all were susceptible to amphotericin B. Of the 15 randomly selected fluconazole-resistant isolates, 14 isolates had an isavuconazole MIC ≤ 1 µg/mL. No FKS mutations were detected. Multilocus sequence typing (MLST) analysis grouped isolates into two clusters: cluster 1 and cluster 2 comprising 83 and 2 isolates, respectively. CONCLUSIONS: Azole-resistant C. auris strains circulating in South African hospitals were related by MLST, but the possibility of nosocomial transmission should be explored using a more discriminatory technique, for example, whole genome sequencing.

Candida auris in South Africa, 2012-2016.

To determine the epidemiology of Candida auris in South Africa, we reviewed data from public- and private-sector diagnostic laboratories that reported confirmed and probable cases of invasive disease and colonization for October 2012-November 2016. We defined a case as a first isolation of C. auris from any specimen from a person of any age admitted to any healthcare facility in South Africa. We defined probable cases as cases where the diagnostic laboratory had used a nonconfirmatory biochemical identification method and C. haemulonii was cultured. We analyzed 1,692 cases; 93% were from private-sector healthcare facilities, and 92% of cases from known locations were from Gauteng Province. Of cases with available data, 29% were invasive infections. The number of cases increased from 18 (October 2012-November 2013) to 861 (October 2015-November 2016). Our results show a large increase in C. auris cases during the study period, centered on private hospitals in Gauteng Province.

Large Outbreaks of Fungal and Bacterial Bloodstream Infections in a Neonatal Unit, South Africa, 2012-2016.

Candidemia is a major cause of healthcare-associated infections. We describe a large outbreak of Candida krusei bloodstream infections among infants in Gauteng Province, South Africa, during a 4-month period; a series of candidemia and bacteremia outbreaks in the neonatal unit followed. We detected cases by using enhanced laboratory surveillance and audited hospital wards by environmental sampling and epidemiologic studies. During July-October 2014, among 589 patients, 48 unique cases of C. krusei candidemia occurred (8.2% incidence). Risk factors for candidemia on multivariable analyses were necrotizing enterocolitis, birthweight <1,500 g, receipt of parenteral nutrition, and receipt of blood transfusion. Despite initial interventions, outbreaks of bloodstream infection caused by C. krusei, rarer fungal species, and bacterial pathogens continued in the neonatal unit through July 29, 2016. Multiple factors contributed to these outbreaks; the most functional response is to fortify infection prevention and control.

Cryptococcal antigen positivity combined with the percentage of HIV-seropositive samples with CD4 counts <100 cells/µl identifies districts in South Africa with advanced burden of disease.

INTRODUCTION: Cryptococcal meningitis (CM) is an opportunistic fungal disease with a high mortality among HIV-positive patients with severe immunosuppression (CD4 count <100 cells/µl). Reflexed screening for cryptococcal antigen (CrAg) in remnant blood samples was initially piloted at selected CD4 testing laboratories of the National Health Laboratory Service (NHLS) prior to the implementation of a national screening programme using a lateral flow assay (LFA) (IMMY, Norman, OK, USA). The aim of this study was to assess CrAg positivity nationally, per province and district in combination with the percentage of CD4 samples tested with a CD4 count <100 cells/µl to identify areas with advanced HIV/CrAg disease burden. METHODS: CrAg and CD4 laboratory result data were extracted from the NHLS corporate data warehouse. Monthly test volumes were used to assess CrAg test volumes and coverage, while bubble charts were used to display the relationship between CD4 <100 cells/µl, CrAg positivity and number of positive CrAg samples by district. ArcGIS software was used to spatially report CrAg positivity. RESULTS: CrAg screening coverage was stable at around 96% after November 2016. Samples with a CD4 <100 cell/µl and CrAg positivity were also stable over the study period at 10% and ~5% respectively. The highest CrAg positivity was reported for the Kwa-Zulu Natal province (7.3%), which also had the lowest percentage of samples with a CD4 <100 cells/µl (7.2%). Uthungulu and Umkhanyakude districts had the highest CrAg positivity (9.3% and 8.9% respectively). Ethekwini and Johannesburg Metro districts contributed to 22% of the total number of CrAg-positive samples tested across South Africa for the period reported. CONCLUSION: Existing CD4 testing services were used to rapidly scale up CrAg reflex testing in South Africa. Districts with advanced HIV and CrAg disease burden were identified that need further investigation of patient management interventions.