A strategy based on Amplified Fragment Length Polymorphism (AFLP) for routine genotyping of nontuberculous mycobacteria at the clinical laboratory.

The increasing worldwide incidence of mycobacteriosis and the need to achieve improved clinical management makes nontuberculous mycobacteria (NTM) genotyping a useful tool. However, because of technical difficulties, medium size microbiology laboratories do not attempt to compare the genetic patterns that each of their isolates present. We have aimed to optimize a genotyping method with a reduced hands-on experimental time and that requires few technical resources. A strategy based on the Amplified Fragment Length Polymorphism (AFLP) methodology was developed using two rare-cutters enzymes (SacI and BglII). One out of seven primers was sequentially used in each amplification reaction that was analyzed by agarose gel electrophoresis. This approach makes it possible the timely genotyping of a moderate number of strains and its characterization without the need of image analysis software. We have genotyped 28 Mycobacterium intracellulare and 4 M. abscessus. Clinical researchers are encouraged to routinely genotype their NTM isolates.

Sinus fungal balls caused by Syncephalastrum spp.: 2 clinical cases and literature review.

INTRODUCTION: Syncephalastrum spp. is a mucoral that rarely produces pathology. Fungal balls caused by this genus are infrequent. It requires early diagnosis and treatment to avoid associated morbidity and mortality. METHODS: We describe two clinical cases of sinus fungus balls caused by Syncephalastrum spp. and review the literature. RESULTS: Two patients were treated for sinus fungus balls. When their samples were analysed, the aetiology was determined to be Syncephalastrum spp. A case of pulmonary fungus ball due to Syncephalastrum spp. is described in the literature. All cases, including these described in the present study, had a good evolution after treatment. CONCLUSIONS: Syncephalastrum spp. is a filamentous fungus that should be considered as an aetiology of sinus fungus ball.

Bacterial/fungal infection in hospitalized patients with COVID-19 in a tertiary hospital in the Community of Castilla y León, Spain.

INTRODUCTION: Bacterial/fungal coinfection and superinfections contribute to the increased morbi-mortality of viral respiratory infections (RIs). The main objective of this study was to determine the incidence of these infections in hospitalized patients with COVID-19. METHOD: Retrospective observational study of all patients admitted for COVID-19 and bacterial/fungal infections at the Hospital Clínico Universitario of Valladolid, Spain (March 1-May 31, 2020). Demographic, clinical and microbiological data were compared based on Intensive Care Unit (ICU) admission and predictors of mortality by were identified using multivariate logistic regression analyses. RESULTS: Of the 712 COVID-19 patients, 113 (16%) presented bacterial/fungal coinfections or superinfections. Their median age was 73 years (IQR 57-89) and 59% were men. The profiles of ICU patients (44%) included male, SARS-CoV-2 pneumonia, leukocytosis, elevated inteleukin-6, with interferon ß-1b and tocilizumab and superinfection (p < 0.05). Coinfections were diagnosed in 5% (39/712) patients. Most common pathogens of respiratory coinfection (18) were Streptococcus pneumoniae (6) and Staphylococcus aureus (6). Superinfections were detected in 11% (80/712) patients. Urinary (53) and RI (39) constituted the majority of superinfections Acinetobacter baumannii multidrug-resistant was the main agent of IR and bacteremia. An outbreak of A. baumannii contributed to this result. Three patients were considered to have probable pulmonary aspergillosis. Mortality was higher in UCI patients (50% vs. 29%, p = 0.028). The predictive factors of mortality included being a male with various comorbidities, SARS-CoV-2 pneumonia, bacteremia and superinfections from A. baumannii. CONCLUSION: The outbreak of A. baumannii was a determining factor in the increases of the incidence of infection and the morbi-mortality of ICU patients.

Global Emergence of Resistance to Fluconazole and Voriconazole in Candida parapsilosis in Tertiary Hospitals in Spain During the COVID-19 Pandemic.

Background: Candida parapsilosis is a frequent cause of candidemia worldwide. Its incidence is associated with the use of medical implants, such as central venous catheters or parenteral nutrition. This species has reduced susceptibility to echinocandins, and it is susceptible to polyenes and azoles. Multiple outbreaks caused by fluconazole-nonsusceptible strains have been reported recently. A similar trend has been observed among the C. parapsilosis isolates received in the last 2 years at the Spanish Mycology Reference Laboratory. Methods: Yeast were identified by molecular biology, and antifungal susceptibility testing was performed using the European Committee on Antimicrobial Susceptibility Testing protocol. The ERG11 gene was sequenced to identify resistance mechanisms, and strain typing was carried out by microsatellite analysis. Results: We examined the susceptibility profile of 1315 C. parapsilosis isolates available at our reference laboratory between 2000 and 2021, noticing an increase in the number of isolates with acquired resistance to fluconazole, and voriconazole has increased in at least 8 different Spanish hospitals in 2020-2021. From 121 recorded clones, 3 were identified as the most prevalent in Spain (clone 10 in Catalonia and clone 96 in Castilla-Leon and Madrid, whereas clone 67 was found in 2 geographically unrelated regions, Cantabria and the Balearic Islands). Conclusions: Our data suggest that concurrently with the coronavirus disease 2019 pandemic, a selection of fluconazole-resistant C. parapsilosis isolates has occurred in Spain, and the expansion of specific clones has been noted across centers. Further research is needed to determine the factors that underlie the successful expansion of these clones and their potential genetic relatedness.

Bacterial/fungal infection in hospitalized patients with COVID-19 in a tertiary hospital in the Community of Castilla y León, Spain. / Infección bacteriana/fúngica en pacientes con COVID-19 ingresados en un hospital de tercer nivel de Castilla y León, España.

INTRODUCTION: Bacterial/fungal coinfection and superinfections contribute to the increased morbi-mortality of viral respiratory infections (RIs). The main objective of this study was to determine the incidence of these infections in hospitalized patients with COVID-19. METHOD: Retrospective observational study of all patients admitted for COVID-19 and bacterial/fungal infections at the Hospital Clínico Universitario of Valladolid, Spain (March 1-May 31, 2020). Demographic, clinical and microbiological data were compared based on Intensive Care Unit (ICU) admission and predictors of mortality by were identified using multivariate logistic regression analyses. RESULTS: Of the 712 COVID-19 patients, 113 (16%) presented bacterial/fungal coinfections or superinfections. Their median age was 73 years (IQR 57-89) and 59% were men. The profiles of ICU patients (44%) included male, SARS-CoV-2 pneumonia, leukocytosis, elevated inteleukin-6, with interferon ß-1b and tocilizumab and superinfection (p < 0.05). Coinfections were diagnosed in 5% (39/712) patients. Most common pathogens of respiratory coinfection (18) were Streptococcus pneumoniae (6) and Staphylococcus aureus (6). Superinfections were detected in 11% (80/712) patients. Urinary (53) and RIs (39) constituted the majority of superinfections Acinetobacter baumannii multidrug-resistant was the main agent of IR and bacteremia. An outbreak of A. baumannii contributed to this result. Three patients were considered to have probable pulmonary aspergillosis. Mortality was higher in UCI patients (50 vs. 29%; p = 0.028). The predictive factors of mortality included being a male with various comorbidities, SARS-CoV-2 pneumonia, bacteremia and superinfections from A. baumannii. CONCLUSION: The outbreak of A. baumannii was a determining factor in the increases of the incidence of infection and the morbi-mortality of ICU patients.

Human Mycobacterium bovis infection in Castile and León (Spain), 2006-2015. / Tuberculosis por Mycobacterium bovis en la población de Castilla y León (España), 2006-2015.

INTRODUCTION: The annual incidence of tuberculosis (TB) from Mycobacterium bovis in humans has considerably declined in industrialised countries since the early twentieth century. The objective of this study was to determine the epidemiological, clinical and microbiological characteristics of patients with this illness in Castile and León (CyL). METHODS: Retrospective study of all M. bovis TB cases in CyL over a 10-year period, comparing the risk factors, the epidemiology and the clinical course between pulmonary (PTB) and extrapulmonary TB (EPTB). RESULTS: 75 cases of TB were due to M. bovis: 45 PTB and 31 EPTB. The annual incidence of TB due to M. bovis was 0.3 cases per 100,000. It remained stable between the first and second five-year period (0.27 vs. 0.33, p=0.656). However, the overall incidence of TB fell in both five-year periods (13.58 vs. 10.71, p<0.0001). The mean age was 66.2+21.3 years, mainly men (63%) and Spanish patients (92%). PTB was significantly more frequent in men, aged over 66 years, with immunosuppressive conditions or who were smokers. Mortality was 9%, associated with higher age, immunosuppression or treatment different from that recommended by the WHO. CONCLUSIONS: The incidence of M. bovis TB in CyL was higher than that for Spain and for other European countries, and remained stable despite the decreased the TB due to MTC. It affected mostly Spanish-born patients who lived in rural areas and with a high mean age.

Buruli ulcers in a Spanish aid worker after a stay in Peru.

Buruli ulcer (BU) is a chronic and destructive infection of the skin and soft tissues caused by Mycobacterium ulcerans. Recently, population flows have triggered the appearance of several sporadic cases of BU in non-endemic countries. This represents a significant diagnostic challenge for clinicians and microbiologists. We describe the first case of BU imported to Spain. The patient was a Spanish woman who had stayed 5 months in the jungle of Peru.

Lady Windermere syndrome in Castile and León. / Síndrome de Lady Windermere en Castilla y León.

INTRODUCTION: Lady Windermere syndrome (LWS) is a pulmonary disease caused by Mycobacterium avium complex (MAC). The objective of this study is to ascertain its frequency and characteristics in the northern area of the autonomous community of Castile and León. METHODS: A retrospective study of patients with MAC isolates in respiratory samples from five public hospitals in the autonomous community over a six-year period, following the ATS/IDSA criteria. The MAC strains were identified by GenoType Mycobacterium reverse hybridisation probes or PCR-RFLP analysis of the hsp65 gene. RESULTS: Of 183 cases of MAC identified, only five women (2.7%) aged 68.8±10.7years met LWS criteria. In three cases, MAC was isolated jointly and intermittently with other pathogens. Only one patient was treated according to ATS/IDSA criteria. DISCUSSION: LWS remains underestimated, with affected patients representing a significant burden on healthcare resources over long periods of time. As a result, greater microbiological and therapeutic knowledge of the syndrome is needed.

Contribution of microbiology in the diagnosis of tuberculosis in Castile and León (Spain): Findings of the GRUMICALE 2013 study. / Contribución de la microbiología al diagnóstico de la tuberculosis en Castilla y León: conclusiones del estudio GRUMICALE 2013.

INTRODUCTION AND OBJECTIVES: A retrospective study was conducted by collecting microbiological tuberculosis (TB) data in Castile and León during the year 2013 in order to determine the incidence and distribution of TB, and resistance to the tuberculostatic drug, and compare them with the epidemiological data provided by the Department of Epidemiological Surveillance (SIVE). MATERIAL AND METHODS: Microbiologists of the 14 hospitals of the Castile and León public health network (GRUMICALE) collected epidemiological, microbiological, and management data from the Microbiology laboratories in the community during the year 2013. A single isolate of Mycobacterium tuberculosis complex (MTC) per patient was considered. RESULTS: The study included a total of 270 MTC isolates (an incidence rate of 11.63 cases/100,000 inhab./year). A total of 288 cases of TB (11.43 cases/100,000 inhab. year) were recovered using epidemiological data, which included 243 confirmed, 29 suspected, and 16 as probable cases. Pulmonary TB was predominant, followed a long way off by the pleural TB and the remaining locations. A total of 27,620 samples were processed for mycobacterial detection. Mycobacterial growth was observed in 3.46% of automated fluid cultures, and 50.37% were positive by direct staining of the smear. Resistance to one tuberculostatic drug, mostly to isoniazid, was observed in 16 (5.92%) isolates of Mycobacterium tuberculosis (MT). The province with greater incidence and number of isolates was León (24.23 cases/100,000 inhab./year), with the highest being observed in El Bierzo health area (30.46 cases/100,000 inhab./year). CONCLUSIONS: An adequate collection of microbiological information is essential to determine the epidemiology of TB in our region.

Blood antimicrobial activity varies against different Mycobacterium spp.

In vitro analysis of mycobacterial pathogenicity or host susceptibility has traditionally relied on the infection of macrophages, the target cell of mycobacteria, despite difficulties reproducing their antimycobacterial activity. We have employed alternative models, namely whole blood and leukocytes in plasma, from QuantiFERON negative individuals, and performed infections with the pathogenic M. tuberculosis, the less pathogenic M. avium, M. kansasii and M. chelonae and the occasionally pathogenic M. gordonae and M. bovis. The anticoagulant used in blood extraction, heparin or EDTA, had a major influence in the outcome of the infection. Thus, while in the heparinized models a similar number of bacteria were enumerated in the inoculum and after seven days, in the presence of EDTA a killing effect was observed, despite the inhibitory effect of EDTA on cellular functions like the production of cytokines or reactive oxygen species (ROS). A special case was the rapidly growing mycobacteria M. chelonae, that multiplied in heparinized models but was eliminated in models with EDTA. We verified that EDTA is not responsible for the bactericidal effect, but acts as a bacteriostatic agent. Further work will determine whether blood derived models are a better alternative to the classical macrophage.

Immunological response to Mycobacterium tuberculosis infection in blood from type 2 diabetes patients.

The convergence of tuberculosis and diabetes represents a co-epidemic that threatens progress against tuberculosis. We have investigated type 2 diabetes as a risk factor for tuberculosis susceptibility, and have used as experimental model whole blood infected in vitro with Mycobacterium tuberculosis. Blood samples from diabetic patients were found to have a higher absolute neutrophil count that non-diabetic controls, but their immune functionality seemed impaired because they displayed a lower capacity to phagocytose M. tuberculosis, a finding that had been previously reported only for monocytes. In contrast, an increased production of TNFα was detected in infected blood from diabetic patients. Despite the altered phagocytic capacity showed by cells from these patients, the antimicrobial activity measured in both whole blood and monocyte derived macrophages was similar to that of controls. This unexpected result prompts further improvements in the whole blood model to analyze the immune response of diabetes patients to tuberculosis.

In vitro infection with Mycobacterium tuberculosis induces a distinct immunological pattern in blood from healthy relatives of tuberculosis patients.

Part of the susceptibility to tuberculosis has a genetic basis, which is clear in primary immunodeficiencies, but is less evident in apparently immunocompetent subjects. Immune responses were analysed in blood samples from tuberculosis patients and their healthy first-degree relatives who were infected in vitro with mycobacteria (either Mycobacterium tuberculosis or M. bovis BCG). The antimicrobial activity against M. tuberculosis in blood from relatives was significantly lower than that observed in healthy controls. Tuberculosis patients exhibited a higher number of neutrophils, and monocyte phagocytosis was inhibited in both relatives and tuberculosis patients. A remarkable finding was that the production of reactive oxygen species by infected neutrophils was higher in relatives than in healthy controls. A higher production of TNFα in infected blood from relatives was also observed. These results may indicate that relatives display a stronger inflammatory response and that their immune response to M. tuberculosis is different from those of unrelated controls. First-degree relatives may represent a highly informative group for the analysis of tuberculosis susceptibility.