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.

In Vitro Antifungal Resistance of Candida auris Isolates from Bloodstream Infections, South Africa.

Candida auris is a multidrug-resistant fungal pathogen that is endemic in South African hospitals. We tested bloodstream C. auris isolates that were submitted to a reference laboratory for national laboratory-based surveillance for candidemia in 2016 and 2017. We confirmed the species identification by phenotypic/molecular methods. We tested susceptibility to amphotericin B, anidulafungin, caspofungin, micafungin, itraconazole, posaconazole, voriconazole, fluconazole, and flucytosine using broth microdilution and Etest methods. We interpreted MICs using tentative breakpoints. We sequenced the genomes of a subset of isolates and compared them to the C. auris B8441 reference strain. Of 400 C. auris isolates, 361 (90%) were resistant to at least one antifungal agent, 339 (94%) to fluconazole alone (MICs of ≥32 µg/ml), 19 (6%) to fluconazole and amphotericin B (MICs of ≥2 µg/ml), and 1 (0.3%) to amphotericin B alone. Two (0.5%) isolates from a single patient were pan-resistant (resistant to fluconazole, amphotericin B, and echinocandins). Of 92 isolates selected for whole-genome sequencing, 77 clustered in clade III, including the pan-resistant isolates, 13 in clade I, and 2 in clade IV. Eighty-four of the isolates (91%) were resistant to at least one antifungal agent; both resistant and susceptible isolates had mutations. The common substitutions identified across the different clades were VF125AL, Y132F, K177R, N335S, and E343D in ERG11; N647T in MRR1; A651P, A657V, and S195G in TAC1b; S639P in FKS1HP1; and S58T in ERG3. Most South African C. auris isolates were resistant to azoles, although resistance to polyenes and echinocandins was less common. We observed mutations in resistance genes even in phenotypically susceptible isolates.

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.

Human Blastomycosis in South Africa Caused by Blastomyces percursus and Blastomyces emzantsi sp. nov., 1967 to 2014.

We reevaluated 20 cases of blastomycosis diagnosed in South Africa between 1967 and 2014, with Blastomyces dermatitidis considered to be the etiological agent, in light of newly described species and the use of more advanced technologies. In addition to histopathological and/or culture-based methods, all 20 isolates were phenotypically and genotypically characterized, including multilocus typing of five genes and whole-genome sequencing. Antifungal susceptibility testing was performed as outlined by Clinical and Laboratory Standards Institute documents M27-A3 and M38-A2. We merged laboratory and corresponding clinical case data, where available. Morphological characteristics and phylogenetic analyses of five-gene and whole-genome sequences revealed two groups, both of which were closely related to but distinct from B. dermatitidis, Blastomyces gilchristii, and Blastomyces parvus The first group (n = 12) corresponded to the recently described species Blastomyces percursus, and the other (n = 8) is described here as Blastomyces emzantsi sp. nov. Both species exhibited incomplete conversion to the yeast phase at 37°C and were heterothallic for mating types. All eight B. emzantsi isolates belonged to the α mating type. Whole-genome sequencing confirmed distinct species identities as well as the absence of a full orthologue of the BAD-1 gene. Extrapulmonary (skin or bone) disease, probably resulting from hematogenous spread from a primary lung infection, was more common than pulmonary disease alone. Voriconazole, posaconazole, itraconazole, amphotericin B, and micafungin had the most potent in vitro activity. Over the 5 decades, South African cases of blastomycosis were caused by species that are distinct from B. dermatitidis Increasing clinical awareness and access to simple rapid diagnostics may improve the diagnosis of blastomycosis in resource-limited countries.

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.

Epidemiologic Shift in Candidemia Driven by Candida auris, South Africa, 2016-20171.

Candida auris is an invasive healthcare-associated fungal pathogen. Cases of candidemia, defined as illness in patients with Candida cultured from blood, were detected through national laboratory-based surveillance in South Africa during 2016-2017. We identified viable isolates by using mass spectrometry and sequencing. Among 6,669 cases (5,876 with species identification) from 269 hospitals, 794 (14%) were caused by C. auris. The incidence risk for all candidemia at 133 hospitals was 83.8 (95% CI 81.2-86.4) cases/100,000 admissions. Prior systemic antifungal drug therapy was associated with a 40% increased adjusted odds of C. auris fungemia compared with bloodstream infection caused by other Candida species (adjusted odds ratio 1.4 [95% CI 0.8-2.3]). The crude in-hospital case-fatality ratio did not differ between Candida species and was 45% for C. auris candidemia, compared with 43% for non-C. auris candidemia. C. auris has caused a major epidemiologic shift in candidemia in South Africa.

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.

In Vitro Antifungal Susceptibility of Yeast and Mold Phases of Isolates of Dimorphic Fungal Pathogen Emergomyces africanus (Formerly Emmonsia sp.) from HIV-Infected South African Patients.

Disseminated emmonsiosis is an important AIDS-related mycosis in South Africa that is caused by Emergomycesafricanus, a newly described and renamed dimorphic fungal pathogen. In vitro antifungal susceptibility data can guide management. Identification of invasive clinical isolates was confirmed phenotypically and by sequencing of the internal transcribed spacer region. Yeast and mold phase MICs of fluconazole, voriconazole, itraconazole, posaconazole, caspofungin, anidulafungin, micafungin, and flucytosine were determined with custom-made frozen broth microdilution (BMD) panels in accordance with Clinical and Laboratory Standards Institute recommendations. MICs of amphotericin B, itraconazole, posaconazole, and voriconazole were determined by Etest. Fifty unique E. africanus isolates were tested. The yeast and mold phase geometric mean (GM) BMD and Etest MICs of itraconazole were 0.01 mg/liter. The voriconazole and posaconazole GM BMD MICs were 0.01 mg/liter for both phases, while the GM Etest MICs were 0.001 and 0.002 mg/liter, respectively. The fluconazole GM BMD MICs were 0.18 mg/liter for both phases. The GM Etest MICs of amphotericin B, for the yeast and mold phases were 0.03 and 0.01 mg/liter. The echinocandins and flucytosine had very limited in vitro activity. Treatment and outcome data were available for 37 patients; in a multivariable model including MIC data, only isolation from blood (odds ratio [OR], 8.6; 95% confidence interval [CI], 1.3 to 54.4; P = 0.02) or bone marrow (OR, 12.1; 95% CI, 1.2 to 120.2; P = 0.03) (versus skin biopsy) was associated with death. In vitro susceptibility data support the management of disseminated emmonsiosis with amphotericin B, followed by itraconazole, voriconazole, or posaconazole. Fluconazole was a relatively less potent agent.

In Vitro Antifungal Activity of Manogepix and Other Antifungal Agents against South African Candida auris Isolates from Bloodstream Infections.

We determined the susceptibility of South African Candida auris bloodstream surveillance isolates to manogepix, a novel antifungal, and several registered antifungal agents. C. auris isolates were submitted to a reference laboratory between 2016 and 2017. Species identification was confirmed by phenotypic methods. We determined MICs for amphotericin B, anidulafungin, caspofungin, micafungin, itraconazole, posaconazole, voriconazole, fluconazole, and flucytosine using Sensititre YeastOne and manogepix using a modified Clinical and Laboratory Standards Institute broth microdilution method. Clade distribution was determined for a subset of isolates using whole-genome sequencing. Of 394 tested isolates, 357 were resistant to at least 1 antifungal class. The manogepix MIC range was 0.002 to 0.06 µg/mL for 335 isolates with fluconazole monoresistance. Nineteen isolates were resistant to both fluconazole and amphotericin B yet still had low manogepix MICs (range, 0.004 to 0.03 µg/mL). Two isolates from the same patient were panresistant but had manogepix MICs of 0.004 µg/mL and 0.008 µg/mL. Comparing MIC50 values, manogepix was >3-fold more potent than azoles, 4-fold more potent than echinocandins, and 9-fold more potent than amphotericin B. Of 84 sequenced isolates, the manogepix MIC range for 70 clade III isolates was 0.002 to 0.031 µg/mL, for 13 clade I isolates was 0.008 to 0.031 µg/mL, and for one clade IV isolate, 0.016 µg/mL. Manogepix exhibited potent activity against all isolates, including those resistant to more than one antifungal agent and in three different clades. These data support manogepix as a promising candidate for treatment of C. auris infections. IMPORTANCE Since C. auris was first detected in South Africa in 2012, health care-associated transmission events and large outbreaks have led to this pathogen accounting for more than 1 in 10 cases of candidemia. A large proportion of South African C. auris isolates are highly resistant to fluconazole but variably resistant to amphotericin B and echinocandins. There is also an emergence of pandrug-resistant C. auris isolates, limiting treatment options. Therefore, the development of new antifungal agents such as fosmanogepix or the use of new combinations of antifungal agents is imperative to the continued effective treatment of C. auris infections. Manogepix, the active moiety of fosmanogepix, has shown excellent activity against C. auris isolates. With the emergence of C. auris isolates that are pandrug-resistant in South Africa, our in vitro susceptibility data support manogepix as a promising new drug candidate for treatment of C. auris and difficult-to-treat C. auris infections.

Decreasing fluconazole susceptibility of clinical South African Cryptococcus neoformans isolates over a decade.

BACKGROUND: Fluconazole is used in combination with amphotericin B for induction treatment of cryptococcal meningitis and as monotherapy for consolidation and maintenance treatment. More than 90% of isolates from first episodes of cryptococcal disease had a fluconazole minimum inhibitory concentration (MIC) ≤4 µg/ml in a Gauteng population-based surveillance study of Cryptococcus neoformans in 2007-2008. We assessed whether fluconazole resistance had emerged in clinical cryptococcal isolates over a decade. METHODOLOGY AND PRINCIPAL FINDINGS: We prospectively collected C. neoformans isolates from 1 January through 31 March 2017 from persons with a first episode of culture-confirmed cryptococcal disease at 37 South African hospitals. Isolates were phenotypically confirmed to C. neoformans species-complex level. We determined fluconazole MICs (range: 0.125 µg/ml to 64 µg/ml) of 229 C. neoformans isolates using custom-made broth microdilution panels prepared, inoculated and read according to Clinical and Laboratory Standards Institute M27-A3 and M60 recommendations. These MIC values were compared to MICs of 249 isolates from earlier surveillance (2007-2008). Clinical data were collected from patients during both surveillance periods. There were more males (61% vs 39%) and more participants on combination induction antifungal treatment (92% vs 32%) in 2017 compared to 2007-2008. The fluconazole MIC50, MIC90 and geometric mean MIC was 4 µg/ml, 8 µg/ml and 4.11 µg/ml in 2017 (n = 229) compared to 1 µg/ml, 2 µg/ml and 2.08 µg/ml in 2007-2008 (n = 249) respectively. Voriconazole, itraconazole and posaconazole Etests were performed on 16 of 229 (7%) C. neoformans isolates with a fluconazole MIC value of ≥16 µg/ml; only one had MIC values of >32 µg/ml for these three antifungal agents. CONCLUSIONS AND SIGNIFICANCE: Fluconazole MIC50 and MIC90 values were two-fold higher in 2017 compared to 2007-2008. Although there are no breakpoints, higher fluconazole doses may be required to maintain efficacy of standard treatment regimens for cryptococcal meningitis.