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.The program implemented in Guatemala was supported by Global Action for Fungal Infections and JYLAG, a charity foundation based in Switzerland (E.A. received this funding under the proposal: “Minimizing HIV deaths through rapid fungal diagnosis and better care in Guatemala”).S

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.This work was supported by Global Action Fund for Fungal Infections and JYLAG, a charity Foundation based in Switzerland (E.A. received this funding under the proposal: “Minimising HIV deaths through rapid fungal diagnosis and better care in Guatemala”). Other contributions came from Intrahealth International and the Ministry of health in Guatemala (MSPAS).S

Método para la detección simultánea de histoplasma capsulatum y paracoccidioides brasiliensis

La presente invención se refiere a un método de detección simultánea de ADN de Histoplasma capsulatum y Paracoccidioides brasiliensis mediante la técnica de PCR multiplex cuantitativa en tiempo real, basada en sondas específicas marcadas con dos fluoróforos diferentes. Esta técnica es aplicable tanto en muestras biológicas como en cultivos microbiológicos y muestras ambientales. Además, otro aspecto de la invención es un kit que permite detectar los dos microorganismos de forma simultánea.REIVINDICACIONES: 1. Método para la detección simultánea de Histoplasma capsulatum y Paracoccidioides brasiliensis que comprende: a. obtener una muestra y aislar su ADN, b. amplificar un fragmento de la región ITS del ADN ribosómico de Histoplasma capsulatum y/o Paracoccidioides brasiliensis contenido en el ADN aislado en el paso (a) , c. determinar la desviación del paso (b) con respecto a los controles y d. analizar la desviación del paso (c) y atribuir la misma a la presencia de los citados microorganismos. 2. Método según la reivindicación 1 donde la concentración del ADN aislado para llevar a cabo la amplificación es de al menos 10 fg/μl. 3. Método según la reivindicación 1 donde la muestra es: a. una muestra biológica, b. un cultivo microbiológico o c. una muestra ambiental. 4. Método según la reivindicación 3 donde la muestra biológica está relacionada con el aparato respiratorio. 5. Método según cualquiera de las reivindicaciones 1 a 4 donde la amplificación de la región ITS2 del ADN ribosómico de Histoplasma capsulatum se realiza por medio de los cebadores SEQ ID NO: 1 y SEQ ID NO: 2 y la amplificación de la región ITS1 del ADN ribosómico de Paracoccidioides brasiliensis se realiza por medio de los cebadores SEQ ID NO: 4 y SEQ ID NO: 5. 6. Método según cualquiera de las reivindicaciones 1 a 5 donde la amplificación se realiza por medio de PCR multiplex en tiempo real caracterizada porque se emplean sondas específicas marcadas con dos fluoróforos diferentes. 7. Método según la reivindicación 6 donde las sondas son: a. SEQ ID NO: 3 para el fragmento amplificado de Histoplasma capsulatum y se marca en el extremo 5' con un fluoróforo y en el extremo 3' con un quencher y b. SEQ ID NO: 6 para el fragmento amplificado de Paracoccidioides brasiliensis y se marca en el extremo 5' con un fluoróforo distinto del usado en el apartado (a) y en el extremo 3' con un quencher. 8. Método según la reivindicación 7 donde el fluoróforo del apartado (a) es FAM, el fluoróforo del apartado (b) es HEX y el quencher es BHQ1. 9. Método según cualquiera de las reivindicaciones 1 a 8, para la monitorización de la respuesta a un tratamiento de histoplasmosis y/o paracoccidioidomicosis. 10. Kit para la detección simultánea de Histoplasma capsulatum y Paracoccidioides brasiliensis que comprende pares de cebadores que pueden amplificar, mediante la técnica de la PCR, un fragmento de la región ITS del ADN ribosómico de Histoplasma capsulatum y/o Paracoccidioides brasiliensis. 11. Kit según la reivindicación 10 que comprende los cebadores SEQ ID NO: 1 y SEQ ID NO: 2 para amplificar un fragmento de la región ITS2 del ADN ribosómico de Histoplasma capsulatum y los cebadores SEQ ID NO: 4 y SEQ ID NO: 5 para amplificar un fragmento de la región ITS1 del ADN ribosómico de Paracoccidioides brasiliensis. 12. Kit según la cualquiera de las reivindicaciones 10 u 11 donde la amplificación se realiza por medio de PCR multiplex en tiempo real y se emplean sondas específicas marcadas con dos fluoróforos diferentes. 13. Kit según la reivindicación 12 donde las sondas son: a. SEQ ID NO: 3 para el fragmento amplificado de Histoplasma capsulatum y se marca en el extremo 5'con un fluoróforo y en el extremo 3' con un quencher y b. SEQ ID NO: 6 para el fragmento amplificado de Paracoccidioides brasiliensis y se marca en el extremo 5' con un fluoróforo distinto del usado en el apartado (a) y en el extremo 3' con un quencher. 14. Kit según la reivindicación 13 donde el fluoróforo del apartado (a) es FAM, el fluoróforo del apartado (b) es HEX y el quencher es BHQ1. 15. Kit según cualquiera de las reivindicaciones 10 a 14, para el diagnóstico de histoplasmosis y/o paracoccidioidomicosis en una muestra biológica, ambiental o en un cultivo microbiológico. 16. Kit según cualquiera de las reivindicaciones 10 a 14, para la monitorización de la respuesta a un tratamiento de histoplasmosis y/o paracoccidioidomicosis.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Instituto de Salud Carlos IIISolicitud de patent

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

This work was presented in part at 31st European Congress of Clinical Microbiology & Infectious Diseases (ECCMID), which will take place online from 9 – 12 July 2021. Abstract number 03467.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.CrAg LFA tests were provided by IMMY at no cost. This research was funded by Global Action For Fungal Infections (www.GAFFI.org), JYLAG, a charity Foundation based in Geneva, Switzerland, and Fondo de Investigación Sanitaria from Instituto de Salud Carlos III (PI20CIII/00043). D.B.-P. was supported by grant PTQ2020-011340/AEI/10.13039/501100011033 funded by the Spanish State Investigation Agency. J.C.S.-D. was supported by a fellowship from the Fondo de Investigación Sanitaria (grant FI17CIII/00027).S

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.S

Echinocandin susceptibility testing of Candida spp. using the 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.Fil: Arendrup, Maiken Cavling. Statens Serum Institut. Unit of Mycology and Parasitology; DinamarcaFil: Rodriguez Tudela, Juan Luis. Instituto de Salud Carlos III. Centro Nacional de Microbiología. Servicio de Micología; EspañaFil: Park, Steven. UMDNJ-New Jersey Medical School. Public Health Research Institute; Estados UnidosFil: Garcia, Guillermo Manuel. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. UMDNJ-New Jersey Medical School. Public Health Research Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe; ArgentinaFil: Delmas, Guillaume. UMDNJ-New Jersey Medical School. Public Health Research Institute; Estados UnidosFil: Cuenca Estrella, Manuel. Instituto de Salud Carlos III. Centro Nacional de Microbiología. Servicio de Micología; EspañaFil: Gómez López, Alicia. Instituto de Salud Carlos III. Centro Nacional de Microbiología. Servicio de Micología; EspañaFil: Perlin, David Scott. UMDNJ-New Jersey Medical School. Public Health Research Institute; Estados Unido

The Diagnostic Laboratory Hub: A New Health Care System Reveals the Incidence and Mortality of Tuberculosis, Histoplasmosis, and Cryptococcosis of PWH in Guatemala.

A Diagnostic Laboratory Hub (DLH) was set up in Guatemala to provide opportunistic infection (OI) diagnosis for people with HIV (PWH). Patients newly presenting for HIV, PWH not receiving antiretrovirals (ARVs) for >90 days but returned to care (Return/Restart), and PWH on ARVs with symptoms of OIs (ARV treatment) were prospectively included. Screening for tuberculosis, nontuberculous mycobacteria (NTM), histoplasmosis, and cryptococcosis was done. Samples were couriered to the DLH, and results were transmitted electronically. Demographic, diagnostic results, disease burden, treatment, and follow-up to 180 days were analyzed. In 2017, 1953 patients were included, 923 new HIV infections (an estimated 44% of all new HIV infections in Guatemala), 701 on ARV treatment, and 315 Return/Restart. Three hundred seventeen (16.2%) had an OI: 35.9% tuberculosis, 31.2% histoplasmosis, 18.6% cryptococcosis, 4.4% NTM, and 9.8% coinfections. Histoplasmosis was the most frequent AIDS-defining illness; 51.2% of new patients had <200 CD4 cells/mm3 with a 29.4% OI incidence; 14.3% of OIs in new HIV infections occurred with CD4 counts of 200-350 cells/mm3. OIs were the main risk factor for premature death for new HIV infections. At 180 days, patients with OIs and advanced HIV had 73-fold greater risk of death than those without advanced disease who were OI-free. The DLH OI screening approach provides adequate diagnostic services and obtains relevant data. We propose a CD4 screening threshold of <350 cells/mm3. Mortality remains high, and improved interventions are required, including expansion of the DLH and access to antifungal drugs, especially liposomal amphotericin B and flucytosine.Financial support. This work was supported by Global Action Fund for Fungal Infections and JYLAG, a charity Foundation based in Switzerland (E.A. received this funding under the proposal: “Minimising HIV deaths through rapid fungal diagnosis and better care in Guatemala”). Other contributions came from AIDS Health Foundation (AHF) Guatemala, Intrahealth International and Ministry of health in Guatemala (MSPAS).S

Cistopatia experimental inducida por corynebacterium grupo D2

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai