Ribosomic DNA intergenic spacer 1 region is useful when identifying Candida parapsilosis spp. complex based on high-resolution melting analysis.

The epidemiology of Candida parapsilosis and the closely related species C. orthopsilosis and C. metapsilosis has changed in recent years, justify the need to identify this complex at the species level. In this study we investigate the intergenic spacer 1 (IGS1) of the ribosomal DNA (rDNA) to evaluate the utility of this gene region as a phylogenetic molecular marker and the suitability of a high-resolution melting (HRM) strategy based on this region for identification of members of the C. parapsilosis spp. complex. We sequenced the IGS1 and the internal transcribed spacer (ITS) regions of the rDNA from 33 C. parapsilosis sensu lato strains. Although both regions are useful in identifying species, comparative sequence analysis showed that the diversity in the IGS1 region was higher than in the ITS sequences. We also developed an HRM analysis that reliably identifies C. parapsilosis spp. complex based on the amplification of 70 bp in the IGS1 region. All isolates were correctly identified with a confidence interval >98%. Our results demonstrate that HRM analysis based on the IGS1 region is a powerful tool for distinguishing C. parapsilosis from cryptic species.

Cutaneous Alternaria infectoria infection diagnosed by molecular techniques in a renal transplant patient.

Organ transplant recipients under immunosuppressive therapy have a highly increased risk of opportunistic fungal infections. Cutaneous infection caused by Alternaria species are relatively rare in humans and most cases reported in the literature are in immunocompromised individuals. We report here on a 33-year old male renal transplant patient with diabetes mellitus who presented with cutaneous alternariosis caused by Alternaria infectoria, two years after the transplant. The diagnosis was performed by real-time polymerase chain reaction assay and histopathologic examination. The extension of the lesion under itraconazole treatment required treatment consisting of a combination of surgical excision and liposomal amphotericin B.

Digital Platform for Automatic Qualitative and Quantitative Reading of a Cryptococcal Antigen Point-of-Care Assay Leveraging Smartphones and Artificial Intelligence.

Cryptococcosis is a fungal infection that causes serious illness, particularly in immunocompromised individuals such as people living with HIV. Point of care tests (POCT) can help identify and diagnose patients with several advantages including rapid results and ease of use. The cryptococcal antigen (CrAg) lateral flow assay (LFA) has demonstrated excellent performance in diagnosing cryptococcosis, and it is particularly useful in resource-limited settings where laboratory-based tests may not be readily available. The use of artificial intelligence (AI) for the interpretation of rapid diagnostic tests can improve the accuracy and speed of test results, as well as reduce the cost and workload of healthcare professionals, reducing subjectivity associated with its interpretation. In this work, we analyze a smartphone-based digital system assisted by AI to automatically interpret CrAg LFA as well as to estimate the antigen concentration in the strip. The system showed excellent performance for predicting LFA qualitative interpretation with an area under the receiver operating characteristic curve of 0.997. On the other hand, its potential to predict antigen concentration based solely on a photograph of the LFA has also been demonstrated, finding a strong correlation between band intensity and antigen concentration, with a Pearson correlation coefficient of 0.953. The system, which is connected to a cloud web platform, allows for case identification, quality control, and real-time monitoring.

HIV and fungal priority pathogens.

The burden of invasive fungal infections associated with opportunistic fungal pathogens is a persistent challenge, particularly among people with advanced HIV disease. In October, 2022, WHO published the Fungal Priority Pathogens List (FPPL)-the first global effort to systematically prioritise fungal pathogens. Of the 19 pathogens in the WHO FPPL, four opportunistic pathogens in particular cause invasive diseases in people living with HIV: Cryptococcus neoformans, Histoplasma spp, Pneumocystis jirovecii, and Talaromyces marneffei. These four fungal pathogens are major causes of illness and death in people with advanced HIV and overwhelmingly affect those in low-income and middle-income countries. Access to diagnostics, improved surveillance, targeted support for innovation, and an enhanced public health focus on these diseases are needed in the effort to reduce HIV-associated deaths.

Direct analysis and identification of pathogenic Lichtheimia species by matrix-assisted laser desorption ionization-time of flight analyzer-mediated mass spectrometry.

Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor, Rhizopus, and Lichtheimia. Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant (Lichtheimia corymbifera, L. ramosa, and L. ornata) and irrelevant (L. hyalospora and L. sphaerocystis) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time.

Fungal cell gigantism during mammalian infection.

The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

Diagnostic Mycology Laboratories Should Have a Central Role for the Management of Fungal Disease.

The absence of awareness of fungal diseases as part of the differential diagnosis in at-risk populations has severe consequences. Here, we show how the active role of laboratories can improve patients' survival. Recently, major advances have been made in non-culture-based assays for fungal diseases, improving accuracy and turnaround time. Furthermore, with the introduction of proficiency control systems, laboratories are an easily monitored environment with good analytical accuracy. Diagnostic packages for opportunistic infections can overcome many deficiencies caused by the absence of awareness. In Guatemala, to make diagnosis accessible, we set up a diagnostic laboratory hub (DLH) providing screening for cryptococcosis, histoplasmosis and tuberculosis to a network of 13 healthcare facilities attending people living with HIV (PLWHIV). In two years, we screened 2127 newly HIV-diagnosed patients. The frequency of opportunistic infections was 21%, rising to 30.3% in patients with advanced HIV disease (<200 CD4); 8.1% of these patients had more than one infection. With the implementation of this diagnostic package, mortality decreased by 7%, a key goal of many public health interventions. Screening for serious infection in high-risk populations can partially overcome training or experiential deficiencies among clinicians for life-threatening fungal diseases.

Epidemiology and Mortality of Cryptococcal Disease in Guatemala: Two-Year Results of a Cryptococcal Antigen Screening Program.

Cryptococcal disease is an important opportunistic infection among people living with HIV. The cryptococcal antigen (CrAg) can be detected before the clinical onset of meningitis and its screening is recommended. Here, we evaluated CrAg frequency, and describe the epidemiological characteristics and mortality at 180 days in a cohort of HIV patients from Guatemala. A total of 3457 patients were screened with a CrAg lateral flow assay in serum between January 2017 and December 2018. CrAg positivity was 11.5% in patients with ≤100 CD4/mm3, 8.7% in patients with <200 CD4/mm3, and 6.3% in patients with <350 CD4/mm3. In Latin America, we estimated 9.2% CrAg positivity (IC95% 7.9−10.7%) in patients with ≤100 CD4/mm3. Among patients newly diagnosed with HIV, we estimated 4416 incident cases per year in Latin America in those with <200 CD4/mm3 and 5289 in those with <350 CD4/mm3. In addition, we calculated the burden in people not on ARV or without viral suppression and found 28,672 cases. CrAg screening should be considered in patients who have a CD4 cell count < 350 cells/mm3. Cryptococcal meningitis was associated with 30.8% mortality in Guatemala. Global access to diagnosis as well as to liposomal amphotericin B and flucytosine is a priority.

The interaction between Candida krusei and murine macrophages results in multiple outcomes, including intracellular survival and escape from killing.

Candida krusei is a fungal pathogen of interest for the scientific community for its intrinsic resistance to fluconazole. Little is known about the interaction of this yeast with host immune cells. In this work, we have characterized the outcome of the interaction between C. krusei and murine macrophages. Once C. krusei was internalized, we observed different phenomena. In a macrophage-like cell line, C. krusei survived in a significant number of macrophages and induced filamentation and macrophage explosion. Phagocytosis of C. krusei led to actin polymerization around the yeast cells at the site of entry. Fluorescent specific staining with anti-Lamp1 and LysoTracker indicated that after fungal internalization, there was a phagolysosome maturation defect, a phenomenon that was more efficient when the macrophages phagocytosed killed yeast cells. Using cell line macrophages, we also observed macrophage fusion after cell division. When we used primary resident peritoneal macrophages in addition to macrophage explosion, we also observed a strong chemotaxis of uninfected macrophages to regions where C. krusei-infected macrophages were present. We also noticed yeast transfer phenomena between infected macrophages. Primary macrophages inhibited pseudohypha elongation more efficiently than the macrophage-like cell line, suggesting that C. krusei infection was better controlled by the former macrophages. Primary macrophages induced more tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) than the macrophage-like cell line. Our results demonstrate that C. krusei can exploit the macrophages for replication, although other different outcomes are also possible, indicating that the interaction of this pathogen with phagocytic cells is very complex and regulated by multiple factors.

Process analysis of variables for standardization of antifungal susceptibility testing of nonfermentative yeasts.

Nonfermentative yeasts, such as Cryptococcus spp., have emerged as fungal pathogens during the last few years. However, standard methods to measure their antifungal susceptibility (antifungal susceptibility testing [AST]) are not completely reliable due to the impaired growth of these yeasts in standard media. In this work, we have compared the growth kinetics and the antifungal susceptibilities of representative species of nonfermentative yeasts such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus albidus, Rhodotorula spp., Yarrowia lipolytica, Geotrichum spp., and Trichosporon spp. The effect of the growth medium (RPMI medium versus yeast nitrogen base [YNB]), glucose concentration (0.2% versus 2%), nitrogen source (ammonium sulfate), temperature (30°C versus 35°C), shaking, and inoculum size (10(3), 10(4), and 10(5) cells) were analyzed. The growth rate, lag phase, and maximum optical density were obtained from each growth experiment, and after multivariate analysis, YNB-based media demonstrated a significant improvement in the growth of yeasts. Shaking, an inoculum size of 10(5) CFU/ml, and incubation at 30°C also improved the growth kinetics of organisms. Supplementation with ammonium sulfate and with 2% glucose did not have any effect on growth. We also tested the antifungal susceptibilities of all the isolates by the reference methods of the CLSI and EUCAST, the EUCAST method with shaking, YNB under static conditions, and YNB with shaking. MIC values obtained under different conditions showed high percentages of agreement and significant correlation coefficient values between them. MIC value determinations according to CLSI and EUCAST standards were rather complicated, since more than half of isolates tested showed a limited growth index, hampering endpoint determinations. We conclude that AST conditions including YNB as an assay medium, agitation of the plates, reading after 48 h of incubation, an inoculum size of 10(5) CFU/ml, and incubation at 30°C made MIC determinations easier without an overestimation of MIC values.

Echinocandin susceptibility testing of Candida spp. Using EUCAST EDef 7.1 and CLSI M27-A3 standard procedures: analysis of the influence of bovine serum albumin supplementation, storage time, and drug lots.

The MICs of echinocandins against Candida isolates with fks mutations are higher than those for wild-type (WT) isolates. However, the MIC ranges for susceptible and mutant populations overlap or are poorly separated. It was recently reported that a greater separation could be achieved in the presence of serum. To more fully explore this possibility, we compared the performances of the reference microdilution methods by using standard and bovine serum albumin (BSA)-supplemented growth medium. Anidulafungin, caspofungin, and micafungin MICs were determined according to EUCAST and CLSI methods and with 50% BSA in the medium for 93 clinical isolates, including Candida albicans (20/10 [number of isolates/number of mutants]), C. glabrata (19/10), C. dubliniensis (2/1), C. krusei (16/3), C. parapsilosis (19), and C. tropicalis (19/4) isolates. Stability of the plates was tested after storage at -80°C for 2 and 6 months, and the performance of two different lots of caspofungin was investigated. The addition of BSA to the medium resulted in higher MICs (1 to 9 2-fold dilution steps) for all isolates and compounds. The increases were greatest for anidulafungin and micafungin and, among WT isolates, for C. parapsilosis. The number of very major errors (VMEs) was reduced (24% [20/84 isolates] versus ≤ 7% [6/84 isolates]) using BSA-supplemented EUCAST medium but not using BSA-supplemented CLSI medium (6% versus 9%). MIC results were unchanged after 6 months of storage of test plates. The two lots of caspofungin yielded identical results. Addition of BSA to the EUCAST medium increases the ability to differentiate between WT isolates and isolates harboring resistance mutations.

Antifungal susceptibility profile of clinical Alternaria spp. identified by molecular methods.

OBJECTIVES: To analyse the susceptibility pattern of a collection of Alternaria spp. clinical isolates. METHODS: The antifungal susceptibilities of 35 isolates identified by means of sequencing the internal transcribed spacer region of rDNA were analysed by European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology. RESULTS AND CONCLUSIONS: No clear differences among the activity of antifungals against Alternaria alternata and Alternaria infectoria were detected, except for echinocandins.

Impact of the COVID-19 pandemic on HIV care in Guatemala.

OBJECTIVES: To describe the impact of the coronavirus disease 2019 (COVID-19) pandemic on the diagnosis of human immunodeficiency virus (HIV) and deaths from opportunistic infections in Guatemala. METHODS: A retrospective study was conducted to investigate the impact of the COVID-19 pandemic on people with HIV at a referral clinic (Clinica Familiar Luis Angel García, CFLAG), as well as the disruption of services at a diagnostic laboratory hub (DLH) which provides diagnosis for opportunistic infections to a network of 13 HIV healthcare facilities. Comparative analysis was undertaken using the months March-August from two different time periods: (i) pre-COVID-19 (2017-2019); and (ii) during the COVID-19 period (2020). RESULTS: During the COVID-19 period, 7360 HIV tests were performed at Clinica Familiar Luis Angel García, compared with an average of 16,218 tests in the pre-COVID-19 period; a reduction of 54.7% [95% confidence interval (CI) 53.8-55.4%],Deaths from opportunistic infections at 90 days were 10.7% higher in 2020 compared with 2019 (27.3% vs 16.6%; P = 0.05). Clinical samples sent to the DLH for diagnosis of opportunistic infections decreased by 43.7% in 2020 (95% CI 41.0-46.2%). CONCLUSION: The COVID-19 pandemic is having a substantial impact on HIV care in Guatemala. Diagnostic services for HIV have been severely affected and deaths from opportunistic infections have increased. The lessons learnt must guide the introduction of strategies to reduce the impact of the pandemic.

Incidence of Histoplasmosis in a Cohort of People with HIV: From Estimations to Reality.

Among people with HIV, histoplasmosis represents an important cause of mortality. Previous studies provided estimates of the disease incidence. Here, we compared those estimates with the results obtained from a screening program implemented in Guatemala, which included histoplasmosis detection for people with HIV. To compare the results of this program with previous estimations, a literature search was performed and reports concerning histoplasmosis incidence were analyzed. The screening program enrolled 6366 patients. The overall histoplasmosis incidence in the screening program was 7.4%, which was almost double that estimated in previous studies. From 2017 to 2019, the screening program showed an upward trend in histoplasmosis cases from 6.5% to 8.8%. Histoplasmosis overall mortality among those who were newly HIV diagnosed showed a decrease at 180 days from 32.8% in 2017 to 21.2% in 2019. The screening approach using rapid diagnostic assays detects histoplasmosis cases more quickly, allowing a specific treatment to be administered, which decreases the mortality of the disease. Therefore, the use of these new techniques, especially in endemic areas of histoplasmosis, must be implemented.

A Rapid Screening Program for Histoplasmosis, Tuberculosis, and Cryptococcosis Reduces Mortality in HIV Patients from Guatemala.

Opportunistic infections (OIs) and advanced HIV disease (AHD) contribute to HIV-related mortality. Here, we analyzed the situation of AHD and OIs in a cohort of newly diagnosed HIV patients from Guatemala. We included 2127 adult patients from 13 facilities across the country during 2017 to 2018. Patients were screened for tuberculosis (TB), nontuberculous mycobacteria (NTM), histoplasmosis, and cryptococcal disease, independently of their CD4 cell count. Of the 2127 enrolled patients, 1682 (79.1%) had a CD4 cell count available; of which 52% presented with AHD. Of the Mayan population, 65% had AHD. The overall OI incidence was 21%. Histoplasmosis was the most frequent OI (7.9%), followed by TB (7.1%); 94.4% of these infections occurred in patients with a CD4 < 350 cells/mm3. Mortality at 180 days was significantly higher in those with OIs than without OIs (29.7% vs. 5.9%, p < 0.0001). In one year, this program decreased the OI mortality by 7% and increased the OI treatment by 5.1%. Early OI diagnosis and appropriate therapy reduced OI mortality among newly diagnosed HIV patients in Guatemala. Screening for OIs should be considered in all newly diagnosed HIV patients who have a CD4 cell count < 350 cells/mm3 or those without a CD4 cell count available. To improve results, interventions such as early HIV detection and access to flucytosine and liposomal amphotericin B are required.

Antifungal susceptibility profile of human-pathogenic species of Lichtheimia.

Forty-four isolates belonging to human pathogenic species of Lichtheimia were tested against nine antifungal agents by using the EUCAST methodology. No remarkable differences were found between the clinical species, although L. ramosa showed slightly higher MICs for all drugs. Amphotericin B was the most active drug. Among azole drugs, posaconazole had the best activity in vitro and voriconazole was inactive. Echinocandins showed activity for some isolates, suggesting a potential role in combination therapy.

Environmental study of azole-resistant Aspergillus fumigatus and other aspergilli in Austria, Denmark, and Spain.

A single mechanism of azole resistance was shown to predominate in clinical and environmental Aspergillus fumigatus isolates from the Netherlands, and a link to the use of azoles in the environment was suggested. To explore the prevalence of azole-resistant A. fumigatus and other aspergilli in the environment in other European countries, we collected samples from the surroundings of hospitals in Copenhagen, Innsbruck, and Madrid, flowerbeds in an amusement park in Copenhagen, and compost bags purchased in Austria, Denmark, and Spain and screened for azole resistance using multidish agars with itraconazole, voriconazole, and posaconazole. EUCAST method E.DEF 9.1 was used to confirm azole resistance. The promoter and entire coding sequence of the cyp51A gene were sequenced to identify azole-resistant A. fumigatus isolates. A. fumigatus was recovered in 144 out of 185 samples (77.8%). Four A. fumigatus isolates from four Danish soil samples displayed elevated azole MICs (8%), and all harbored the same TR/L98H mutation of cyp51A. One A. lentulus isolate with voriconazole MIC of 4 mg/liter was detected in Spain. No azole-resistant aspergilli were detected in compost. Finally, A. terreus was present in seven samples from Austria. Multi-azole-resistant A. fumigatus is present in the environment in Denmark. The resistance mechanism is identical to that of environmental isolates in the Netherlands. No link to commercial compost could be detected. In Spain and Austria, only Aspergillus species with intrinsic resistance to either azoles or amphotericin B were found.

Echinocandin susceptibility testing of Candida species: comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest, disk diffusion, and agar dilution methods with RPMI and isosensitest media.

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC(50)) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S

Comparison of caspofungin MICs by means of EUCAST method EDef 7.1 using two different concentrations of glucose.

According to the product insert for Cancidas (caspofungin acetate), the drug must not be diluted in solutions containing glucose as this decreases caspofungin stability. The aim of this study was to compare caspofungin MICs for a collection of yeasts by means of EUCAST method EDef7.1 but using two different concentrations of glucose: 2% versus 0.2%. MICs were identical or within one 2-fold dilution for 93 out of 95 strains (97.9%), showing that glucose does not interfere with susceptibility.