Nucleocapsid single point-mutation associated with drop-out on RT-PCR assay for SARS-CoV-2 detection.

BACKGROUND: Since its beginning, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been a challenge for clinical and molecular diagnostics, because it has been caused by a novel viral agent. Whole-genome sequencing assisted in the characterization and classification of SARS-CoV-2, and it is an essential tool to genomic surveillance aiming to identify potentials hot spots that could impact on vaccine immune response and on virus diagnosis. We describe two cases of failure at the N2 target of the RT-PCR test Xpert® Xpress SARS-CoV-2. METHODS: Total nucleic acid from the Nasopharyngeal (NP) and oropharyngeal (OP) swab samples and cell supernatant isolates were obtained. RNA samples were submitted to random amplification. Raw sequencing data were subjected to sequence quality controls, removal of human contaminants by aligning against the HG19 reference genome, taxonomic identification of other pathogens and genome recovery through assembly and manual curation. RT-PCR test Xpert® Xpress SARS-CoV-2 was used for molecular diagnosis of SARS-CoV-2 infection, samples were tested in duplicates. RESULTS: We identified 27 samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing of 2 of 27 samples revealed a single common mutation in the N gene C29197T, potentially involved in the failed detection of N target. CONCLUSIONS: This study highlights the importance of genomic data to update molecular tests and vaccines.

Cellular and Humoral Immune Responses to Vaccination for COVID-19 Are Negatively Impacted by Senescent T Cells: A Case Report.

BACKGROUND: Herein, we aimed to follow up on the cellular and humoral immune responses of a group of individuals who initially received the CoronaVac vaccine, followed by a booster with the Pfizer vaccine. METHODS: Blood samples were collected: before and 30 days after the first CoronaVac dose; 30, 90, and 180 days after the second CoronaVac dose, and also 20 days after the booster with the Pfizer vaccine. RESULTS: Whilst the positivity to gamma interferon-type cellular response increased after the first CoronaVac dose, neutralizing and IgG antibody levels only raised 30 days after the second dose, followed by a drop in these responses after 90 and 180 days. The booster with the Pfizer vaccine elicited a robust cellular and humoral response. A higher number of double-negative and senescent T cells, as well as increased pro-inflammatory cytokines levels were found in the participants with lower humoral immune responses. CONCLUSION: CoronaVac elicited an early cellular response, followed by a humoral response, which dropped 90 days after the second dose. The booster with the Pfizer vaccine significantly enhanced these responses. Furthermore, a pro-inflammatory systemic status was found in volunteers who presented senescent T cells, which could putatively impair the immune response to vaccination.

Study protocol: epidemiological and clinical characteristics of acute viral hepatitis in Brazilian health services.

INTRODUCTION: Acute viral hepatitis is a disease of great clinical importance. This study proposes actions to better characterise cases of acute hepatitis in Brazil and to provide relevant information to institutionalised health policies within the Unified Health System. Available data on acute hepatitis in Brazil need to be re-evaluated regarding the different hepatotropic agent (hepatitis A to E virus) frequencies, as well as other agents that can cause similar clinical conditions, such as Herpes Simplex Virus 1 and 2(HSV1, HSV2), Varicella Zoster Virus (VZV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Herpes Virus 6 and 7 (HHV6, HHV7), arbovirus (yellow fever, dengue, chikungunya, Zika), parvovirus B19, adenovirus, parechovirus, enterovirus, HIV, leptospirosis, toxoplasmosis and syphilis, in addition to autoimmune hepatitis. In this context, the primary aim of this study is the clinical-epidemiological and molecular characterisation of acute viral hepatitis in Brazilian health services from all geographical regions of the country. The present article describes the study protocol. METHODS AND ANALYSIS: This study will evaluate 2280 patients with symptoms and/or signs suggestive of acute liver disease in Brazilian health institutions in all five geographic Brazilian regions. Demographic, epidemiological and clinical data will be collected, as well as blood samples to be analysed at Hospital Israelita Albert Einstein Clinical Laboratory. ETHICS AND DISSEMINATION: Ethics approval was obtained at the national research ethics committee (Conselho Nacional de Ética em Pesquisa- CONEP-CAAE 00952818.4.1001.0071) and at all participating sites. Results will be published in journals and presented at scientific meetings.

Evaluation of two commercial methods for the susceptibility testing of Candida species: Vitek 2(R) and Sensititre YeastOne(R) / Evaluación de dos métodos comerciales para el estudio de la sensibilidad a los antifúngicos de especies de Candida: Vitek 2(R) y Sensititre YeastOne(R)

Background: An increased incidence of fungal infections caused by Candida species, especially Candida glabrata and Candida krusei, which are less susceptible to azoles, has been observed. Standardized susceptibility testing is essential for clinical management and for monitoring the epidemiology of resistance. Aims: We evaluated the performance of two different susceptibility testing commercial methods, Vitek 2(R) and Sensititre YeastOne(R), and compared them with the standard broth microdilution method (CLSI). Methods: A total of 80 isolates of several Candida species (Candida albicans, Candida parapsilosis complex, Candida tropicalis, C. glabrata and C. krusei) were selected for this study. Results: We analyzed the categorical agreement (CA) between the methods, stratifying the disagreements. The average CA between the methods was 96.3% for Vitek 2(R) and 84% for Sensititre YeastOne(R). No very major errors were observed. Major errors and minor errors were found for all the isolates tested. With the azoles, both Vitek 2(R) and Sensititre YeastOne(R) had good and similar performance levels, except for C. tropicalis and C. krusei (Sensititre YeastOne(R) showed low CA, 56.2%). With the echinocandins, both methods showed good performance for C. albicans, C. parapsilosis and C. tropicalis. However, we observed important discrepancies for C. krusei with caspofungin: Vitek 2(R) had 100% CA while Sensititre YeastOne(R) had only 25%. With amphotericin B, both Vitek 2(R) and Sensititre YeastOne(R) had good performance with high CA. Conclusions: Despite the limited isolates tested, we concluded that both methods have good performance and are reliable for antifungal susceptibility testing. However, caspofungin activity against C. krusei and C. glabrata should be interpreted carefully when using Sensititre YeastOne(R) because we observed a low CA

Antecedentes: La incidencia de infecciones fúngicas provocadas por especies de Candida, especialmente por Candida glabrata y Candida krusei, menos sensibles a los azoles, ha ido en aumento. Los métodos estandarizados de estudio de la sensibilidad a los antifúngicos son fundamentales para el manejo clínico y para un mejor seguimiento de la epidemiología de la resistencia. Objetivos: Se evaluó la actividad de dos métodos comerciales diferentes para el estudio de la sensibilidad in vitro a los antifúngicos, Vitek 2(R) y Sensititre YeastOne(R), y se compararon con la técnica estándar de microdilución en caldo del CLSI. Métodos: Para este estudio se seleccionó un total de 80 cepas aisladas de varias especies de Candida (Candida albicans, Candida parapsilosis, Candida tropicalis, C. glabrata y C. krusei). Resultados: Se analizó la concordancia categórica (CC) entre los métodos y se estratificaron los desacuerdos. La CC media entre los métodos fue del 96,3% para Vitek 2(R) y del 84% para Sensititre YeastOne(R). No se observaron errores muy altos. Se encontraron errores mayores y menores en todos los aislamientos probados. Con los azoles, tanto Vitek 2(R) como YeastOne(R) presentaron rendimientos buenos y similares, excepto para C. tropicalis y C. krusei (Sensititre YeastOne(R) mostró baja CC, el 56,2%). Con las equinocandinas, los dos métodos mostraron buen rendimiento para C. albicans, C. parapsilosis y C. tropicalis. Sin embargo, se observaron discrepancias importantes para C. krusei con la caspofungina: Vitek 2(R) presentó el 100% de CC, mientras que Sensititre YeastOne(R) solo el 25%. Para la anfotericina B, Vitek 2(R) y Sensititre YeastOne(R) presentaron un buen rendimiento con una CC alta. Conclusiones: Aunque el número de cepas aisladas probadas fue limitado, concluimos que los dos métodos tienen un buen rendimiento y son fiables para la prueba de sensibilidad antifúngica. Sin embargo, la actividad de la caspofungina frente a C. krusei y C. glabrata mediante el método Sensititre YeastOne(R) debe interpretarse cuidadosamente, ya que se observa un valor bajo de CC

Evaluation of two commercial methods for the susceptibility testing of Candida species: Vitek 2® and Sensititre YeastOne®.

BACKGROUND: An increased incidence of fungal infections caused by Candida species, especially Candida glabrata and Candida krusei, which are less susceptible to azoles, has been observed. Standardized susceptibility testing is essential for clinical management and for monitoring the epidemiology of resistance. AIMS: We evaluated the performance of two different susceptibility testing commercial methods, Vitek 2® and Sensititre YeastOne®, and compared them with the standard broth microdilution method (CLSI). METHODS: A total of 80 isolates of several Candida species (Candida albicans, Candida parapsilosis complex, Candida tropicalis, C. glabrata and C. krusei) were selected for this study. RESULTS: We analyzed the categorical agreement (CA) between the methods, stratifying the disagreements. The average CA between the methods was 96.3% for Vitek 2® and 84% for Sensititre YeastOne®. No very major errors were observed. Major errors and minor errors were found for all the isolates tested. With the azoles, both Vitek 2® and Sensititre YeastOne® had good and similar performance levels, except for C. tropicalis and C. krusei (Sensititre YeastOne® showed low CA, 56.2%). With the echinocandins, both methods showed good performance for C. albicans, C. parapsilosis and C. tropicalis. However, we observed important discrepancies for C. krusei with caspofungin: Vitek 2® had 100% CA while Sensititre YeastOne® had only 25%. With amphotericin B, both Vitek 2® and Sensititre YeastOne® had good performance with high CA. CONCLUSIONS: Despite the limited isolates tested, we concluded that both methods have good performance and are reliable for antifungal susceptibility testing. However, caspofungin activity against C. krusei and C. glabrata should be interpreted carefully when using Sensititre YeastOne® because we observed a low CA.

Clinical impact of Candida spp. biofilm production in a cohort of patients with candidemia.

Persistent candidemia refers to the continued isolation of the same Candida species in the blood of a candidemic patient despite appropriate therapy. Despite the clinical importance of persistent candidemia, studies have superficially addressed the biological conditions behind this phenomenon. The aim of this study was to evaluate the correlation between the biofilm-forming ability by Candida bloodstream isolates and the persistence of infection. A total of 55 isolates of Candida were tested and characterized in two groups: (i) group I, which included seven patients with persistent candidemia, and (ii) group II, which included 18 patients with nonpersistent candidemia. Microorganisms were identified at the species level by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Biofilm quantification was evaluated by the crystal violet staining method and confocal scanning laser microscopy (CSLM). Molecular tests confirmed the identification of Candida albicans (92% group I and 94% group II) and Candida dubliniensis isolates (8% group I and 6% group II). All 55 isolates were able to form biofilms, but a higher biofilm mass was produced by C. albicans/C. dubliniensis strains cultured from the persistent group (P < .05). Our data suggest that Candida sp. biofilm production should be considered a relevant biologic variable in explaining patients who fail to clear a bloodstream infection despite adequate antifungal treatment with triazoles.

Outbreak of candidemia caused by fluconazole resistant Candida parapsilosis strains in an intensive care unit.

BACKGROUND: Candidemia is an increasing problem in tertiary care hospitals worldwide. Here, we report the first outbreak of candidemia caused by fluconazole-resistant C. parapsilosis (FRCP) strains in Brazil. METHODS: This was a cross-sectional study of clinical and microbiological data of all candidemic episodes diagnosed from July 2011 to February 2012 in a 200-bed tertiary care hospital. Initial yeast identification and susceptibility testing were performed using the VITEK 2 - System. Isolates of Candida spp. resistant to fluconazole were sent to a reference laboratory (LEMI-UNIFESP) for further molecular identification and confirmation of resistance by CLSI microdilution test. A multivariate analysis was conducted to identify factors associated with FRCP infection. RESULTS: We identified a total of 40 critically ill patients with candidemia (15 women) with a median age of 70 years. The incidence of candidemia was 6 cases/1,000 patients admissions, including 28 cases (70 %) of infection with C. parapsilosis, 21 of which (75 %) were resistant to fluconazole. In only 19 % of FRCP candidemia cases had fluconazole been used previously. The results of our study indicated that diabetes is a risk factor for FRCP candidemia (p = 0.002). Overall, mortality from candidemia was 45 %, and mortality from episodes of FRCP infections was 42.9 %. CONCLUSIONS: The clustering of incident cases in the ICU and molecular typing of strains suggest horizontal transmission of FRCP. Accurate vigilant monitoring for new nosocomial strains of FRCP is required.

High rate of Candida deep-seated infection in patients under chronic hemodialysis with extended central venous catheter use / Altas tasas de infección profunda por Candida en pacientes en hemodiálisis crónica con catéter venoso central de uso prolongado

Background. Hemodialysis has been described as an important risk factor for the development of candidemia in patients suffering from chronic renal failure. Aims. The aim of this study was to evaluate the epidemiology of candidemia in outpatients with renal replacement therapy (RRT) by hemodialysis where the fungemia clearly represents a healthcare-associated infection. Methods. We retrospectively collected clinical and laboratory data from patients undergoing at least 3 months of RRT by hemodialysis who developed candidemia within 48 h of hospital admission. Results. We identified 14 patients with candidemia with central venous catheters (CVC) in place for 11-277 days before developing fungemia. Deep-seated infection was documented in 6 out of 14 candidiasis cases (43%), including 5 cases of endocarditis (36%). Conclusions. CVC in patients under RRT should be promptly replaced by fistulas and grafts to avoid bloodstream infections. Facing a case of candidemia, adequate source control and prompt initiation of antifungal therapy are mandatory to avoid morbidity and mortality (AU)

Antecedentes. La hemodiálisis se ha descrito como un importante factor de riesgo para el desarrollo de candidemia en pacientes con insuficiencia renal crónica. Objetivos. El objetivo de este estudio fue evaluar la epidemiología de la candidemia en pacientes en hemodiálisis con terapia renal sustitutiva (TRS), en la que la fungemia representa claramente una infección asociada a los cuidados hospitalarios. Métodos. Se recogieron retrospectivamente datos clínicos y microbiológicos de pacientes con, al menos, 3 meses de hemodiálisis con TRS que desarrollaron candidemia dentro de las primeras 48 horas tras la admisión hospitalaria. Resultados. Identificamos a 14 pacientes con candidemia asociada con el uso de catéter venoso central (CVC) durante períodos de 11 a 277 días previos al desarrollo de la fungemia. En 6 de los 14 casos de candidemia, el diagnóstico fue de candidiasis invasiva (43%), incluidos 5 casos de endocarditis (36%). Conclusiones. Los CVC en pacientes con TRS deberían ser sustituidos inmediatamente por fístulas o injertos arteriovenosos para evitar infecciones del torrente sanguíneo. Ante los casos de candidemia, un control adecuado de las posibles fuentes de infección y el comienzo inmediato de la terapia antifúngica deberían ser imperativos para reducir tanto la morbilidad como la mortalidad (AU)

High rate of Candida deep-seated infection in patients under chronic hemodialysis with extended central venous catheter use.

BACKGROUND: Hemodialysis has been described as an important risk factor for the development of candidemia in patients suffering from chronic renal failure. AIMS: The aim of this study was to evaluate the epidemiology of candidemia in outpatients with renal replacement therapy (RRT) by hemodialysis where the fungemia clearly represents a healthcare-associated infection. METHODS: We retrospectively collected clinical and laboratory data from patients undergoing at least 3 months of RRT by hemodialysis who developed candidemia within 48h of hospital admission. RESULTS: We identified 14 patients with candidemia with central venous catheters (CVC) in place for 11-277 days before developing fungemia. Deep-seated infection was documented in 6 out of 14 candidiasis cases (43%), including 5 cases of endocarditis (36%). CONCLUSIONS: CVC in patients under RRT should be promptly replaced by fistulas and grafts to avoid bloodstream infections. Facing a case of candidemia, adequate source control and prompt initiation of antifungal therapy are mandatory to avoid morbidity and mortality.

Epidemiology and Microbiologic Characterization of Nosocomial Candidemia from a Brazilian National Surveillance Program.

Candidemia is a growing problem in hospitals all over the world. Despite advances in the medical support of critically ill patients, candidiasis leads to prolonged hospitalization, and has a crude mortality rate around 50%. We conducted a multicenter surveillance study in 16 hospitals distributed across five regions of Brazil to assess the incidence, species distribution, antifungal susceptibility, and risk factors for bloodstream infections due to Candida species. From June 2007 to March 2010, we studied a total of 2,563 nosocomial bloodstream infection (nBSI) episodes. Candida spp. was the 7th most prevalent agent. Most of the patients were male, with a median age of 56 years. A total of 64 patients (46.7%) were in the ICU when candidemia occurred. Malignancies were the most common underlying condition (32%). The crude mortality rate of candidemia during the hospital admission was 72.2%. Non-albicans species of Candida accounted for 65.7% of the 137 yeast isolates. C. albicans (34.3%), Candida parapsilosis (24.1%), Candida tropicalis (15.3%) and Candida glabrata (10.2%) were the most prevalent species. Only 47 out of 137 Candida isolates were sent to the reference laboratory for antifungal susceptibility testing. All C. albicans, C. tropicalis and C. parapsilosis isolates were susceptible to the 5 antifungal drugs tested. Among 11 C. glabrata isolates, 36% were resistant to fluconazole, and 64% SDD. All of them were susceptible to anidulafungin and amphotericin B. We observed that C. glabrata is emerging as a major player among non-albicans Candida spp. and fluconazole resistance was primarily confined to C. glabrata and C. krusei strains. Candida resistance to echinocandins and amphotericin B remains rare in Brazil. Mortality rates remain increasingly higher than that observed in the Northern Hemisphere countries, emphasizing the need for improving local practices of clinical management of candidemia, including early diagnosis, source control and precise antifungal therapy.