Novel Human Astroviruses: Prevalence and Association with Common Enteric Viruses in Undiagnosed Gastroenteritis Cases in Spain

Adenovirus; Children; Classic astrovirusAdenovirus; Nens; Astrovirus clàssicAdenovirus; Niños; Astrovirus clásicoA remarkable percentage of acute gastroenteritis cases remain etiologically undiagnosed. The aim of the study was to determine the prevalence of common and emerging enteric viruses, such as novel human astroviruses, among undiagnosed samples from children with acute gastroenteritis. Epidemiological studies for novel human astroviruses are still scarce. Stool samples collected over two consecutive winter seasons (2016-2017) from children with gastroenteritis in Spain, which were negative for bacteria, rotavirus, and adenovirus by routine diagnostics were screened by real-time RT-PCR assays for the presence of classical and novel astrovirus, rotavirus, norovirus GI and GII, sapovirus, and adenovirus. Overall, 220/384 stool samples (57.3%) were positive for at least one virus. Co-infections were identified in 21% of cases. Among a total of 315 viruses identified, adenovirus was the most prevalent (n = 103), followed by rotavirus (n = 51), sapovirus (n = 50), classical astrovirus (n = 43), novel astroviruses (n = 42), and norovirus (n = 26). Novel astroviruses were present in 13.3% of virus-positive cases. Most novel astroviruses were found in children <2-year-old (30/39 children, 77%, p = 0.01) and were found in co-infection (66%). Only classical astroviruses demonstrated significant differences in the Cq values during mono-infections compared to co-infections. In conclusion, common enteric viruses may be frequently found in children with undiagnosed gastroenteritis, indicating the need to implement more sensitive diagnostic methods. Novel astroviruses circulate in the community and could be the cause of gastroenteritis among young children.Supported in part by the Biotechnology Reference Network (XRB) program of the Generalitat de Catalunya. This work was also supported in part by the Certest Biotec Company. Diem-Lan Vu was recipient of a fellowship from the Geneva University Hospital

Blastocystis sp. Carriage and Irritable Bowel Syndrome: Is the Association Already Established?

Blastocystis sp.; Irritable bowel syndrome; PathogenesisBlastocystis sp.; Síndrome de l'intestí irritable; PatogènesiBlastocystis sp.; Síndrome del intestino irritable; PatogénesisBackground: The aim of the present study is to describe the occurrence of Blastocystis sp. detection among asymptomatic subjects and patients with irritable bowel syndrome in order to evaluate the potential association between irritable bowel syndrome and the parasitic infection. Methods: Cross-sectional study where adult patients with irritable bowel syndrome diagnosed according to Rome IV criteria were included. A control group was formed by asymptomatic subjects older than 18 years. Exclusion criteria were: immunosuppressive condition or having received any drug with demonstrated activity against Blastocystis sp. within the last 6 months before study inclusion. Epidemiological and clinical information was collected from all included participants. Two stool samples were obtained from all participants: one sample for microscopic examination and one sample for Blastocystis sp. PCR detection. Blastocystis sp. infection was defined by the positivity of any of the diagnostic techniques. Results: Seventy-two participants were included (36 asymptomatic subjects and 36 patients with irritable bowel syndrome). Thirty-five (48.6%) were men, and median age of participants was 34 (IQR 29–49) years. The overall rate of Blastocystis sp. carriage was 27.8% (20/72). The prevalence assessed through microscopic examination was 22.2% (16/72), while the prevalence measured by PCR was 15.3% (11/72). When comparing the presence of Blastocystis sp. between asymptomatic subjects and IBS patients, we did not find any statistically significant difference (36.1% vs. 19.4% respectively, p = 0.114). Conclusions: regarding the occurrence of Blastocystis sp., no differences were found between asymptomatic participants and patients with irritable bowel disease irrespective of the diagnostic technique performed.This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors

COVID-19: an opportunity of systematic integration for Chagas disease. Example of a community-based approach within the Bolivian population in Barcelona

Chagas disease; COVID-19; BoliviaEnfermedad de Chagas; COVID-19; BoliviaMalaltia de Chagas; COVID-19; BolíviaBackground As a Neglected Tropical Disease associated with Latin America, Chagas Disease (CD) is little known in non-endemic territories of the Americas, Europe and Western Pacific, making its control challenging, with limited detection rates, healthcare access and consequent epidemiological silence. This is reinforced by its biomedical characteristics—it is usually asymptomatic—and the fact that it mostly affects people with low social and financial resources. Because CD is mainly a chronic infection, which principally causes a cardiomyopathy and can also cause a prothrombotic status, it increases the risk of contracting severe COVID-19. Methods In order to get an accurate picture of CD and COVID-19 overlapping and co-infection, this operational research draws on community-based experience and participative-action-research components. It was conducted during the Bolivian elections in Barcelona on a representative sample of that community. Results The results show that 55% of the people interviewed had already undergone a previous T. cruzi infection screening—among which 81% were diagnosed in Catalonia and 19% in Bolivia. The prevalence of T. cruzi infection was 18.3% (with 3.3% of discordant results), the SARS-CoV-2 22.3% and the coinfection rate, 6%. The benefits of an integrated approach for COVID-19 and CD were shown, since it only took an average of 25% of additional time per patient and undoubtedly empowered the patients about the co-infection, its detection and care. Finally, the rapid diagnostic test used for COVID-19 showed a sensitivity of 89.5%. Conclusions This research addresses CD and its co-infection, through an innovative way, an opportunity of systematic integration, during the COVID-19 pandemic.This intervention was partially funded by the NGO Fundación Mundo Sano-España, financing part of the promotional material of the community intervention. The design of the study and the collection, the data analysis and its interpretation has not been funded

Analytical Evaluation of Dried Blood Spot and Rapid Diagnostic Test as a New Strategy for Serological Community Screening for Chronic Chagas Disease

Trypanosoma cruzi; Mancha de sangre seca (DBS); Cribado serológicoTrypanosoma cruzi; Taca de sang seca (DBS); Cribratge serològicTrypanosoma cruzi; Dried blood spot (DBS); Serological screeningBackground: Chagas disease is a public health problem not only in Latin America, but also in other regions, including Spain, due to migration movements. Conventional serological diagnosis requires an invasive sample (plasma or serum) and a well-equipped laboratory. To circumvent those limitations, blood samples dried on filter paper (DBS) or Rapid Diagnostic Test (RDT) could be a practical alternative to reference protocol for serological screening in epidemiological studies. We evaluated the usefulness of dried blood sampling and a rapid diagnostic test (Trypanosoma Detect™) for the detection of antibodies against T. cruzi for their use in community-based screening. Methodology/Principal Findings: A total of 162 stored paired whole-blood and serum samples from Latin American migrants and 25 negative-control blood samples were included. Diagnosis of chronic Chagas disease was performed in serum according to WHO algorithms. Blood samples were retrospectively collected as dried spots and then analyzed using two different serological techniques, enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence immunoassay (E-CLIA). Whole-blood samples were also used to evaluate a rapid diagnostic test based on immunochromatography. A better correlation with conventional serum was observed in dried blood elutes using E-CLIA than ELISA (97% vs. 77% sensitivity, respectively). Both assays reported 100% specificity. The median cut-off index values of E-CLIA for dried blood were significantly lower than those for serum (138.1 vs. 243.3, P<0.05). The Trypanosoma Detect™ test presented a sensitivity and specificity of 89.6% and 100%, respectively. Conclusions: The detection of antibodies against T. cruzi in dried blood samples shows a higher sensitivity when using E-CLIA compared with ELISA. Trypanosoma Detect™ is easier to use but has a lower sensitivity. Hence, we propose a sequential strategy based on performing the rapid test first, and a negative result will be confirmed by DBS-ECLIA for use in community Chagas disease screening programs.This work has been supported by the Fundació la Marató TV3 (project number 20182610)

Increasing trend of antimicrobial resistance in Shigella associated with MSM transmission in Barcelona, 2020–21: outbreak of XRD Shigella sonnei and dissemination of ESBL-producing Shigella flexneri

Antimicrobial resistance; Shigella sonnei; Sexually transmitted infectionResistencia antimicrobiana; Shigella sonnei; Infección de transmisión sexualResistència antimicrobiana; Shigella sonnei; Infecció de transmissió sexualBackground Several countries have recently reported the detection of ESBL-producing Shigella sonnei associated with transmission among MSM. In a previous study by our group, 2.8% of Shigella spp. obtained from MSM in Barcelona between 2015 and 2019 were ESBL producers. Objectives To describe and characterize the emerging ESBL-producing Shigella spp. associated with sexual transmission among MSM detected from 2020 to 2021 in Barcelona, elucidating their connectivity with contemporaneous ESBL-producing Shigella spp. from other countries. Results From 2020 to 2021, we identified that among MSM, 68% of S. sonnei were XDR harbouring blaCTX-M-27 and 14% of Shigella flexneri were MDR harbouring blaCTX-M-27. WGS analysis showed that the ESBL-producing S. sonnei were part of a monophyletic cluster, which included isolates responsible for the prolonged outbreak occurring in the UK. Our data also reveal the first emergence and clonal dissemination of ESBL-producing and fluoroquinolone-resistant S. flexneri 2a among MSM. Conclusions We report an increasing trend of antimicrobial resistance in Shigella spp. among MSM in Barcelona since 2021, mainly as a consequence of the dissemination of XDR ESBL-producing S. sonnei, previously reported in the UK. These results highlight the importance of international collaborative surveillance of MDR/XDR S. sonnei and S. flexneri for rapid identification of their emergence and the prevention of the transmission of these pathogens.This work was partially supported by the ‘Ministerio de Economía y Competitividad’, ‘Instituto de Salud Carlos III’, and co-financed by the European Regional Development Fund (ERDF) ‘A Way to Achieve Europe’ (Spanish Network for Research in Infectious Diseases, grant number RD16/0016/0003) and by the Centro de Investigación Biomédica en Red (CIBER de Enfermedades Infecciosas, grant no. CB21/13/00054). A.M.M. is supported by a grant from the ‘Fondo de Investigación Sanitaria’ (Contratos Predoctorales de Formación en Investigación, grant number FI19/00315)

Unexpected Loa loa Finding in an Asymptomatic Patient From The Gambia: A Case Report

Gambia; Epidemiology; LoiasisGambia; Epidemiología; LoiasisGàmbia; Epidemiologia; LoiasiA 17-year-old asymptomatic male from The Gambia presented for a routine health examination after migration to Spain. Laboratory diagnosis confirmed the presence of Loa loa microfilariae. This unusual finding emphasizes the importance of screening in newly arrived migrants and the need of an extended anamnesis including migratory route and previous travels

Evaluation of Two Different Strategies for Schistosomiasis Screening in High-Risk Groups in a Non-Endemic Setting

Diagnosis; Non-endemic; SchistosomiasisDiagnóstico; No endémico; EsquistosomiasisDiagnòstic; No endèmic; EsquistosomiasiA consensus on the recommended screening algorithms for schistosomiasis in asymptomatic high-risk subjects in non-endemic areas is lacking. The objective of this study was to evaluate the real-life performance of direct microscopy and ELISA serology for schistosomiasis screening in a high-risk population in a non-endemic setting. A retrospective cohort study was conducted in two out-patient Tropical Medicine units in Barcelona (Spain) from 2014 to 2017. Asymptomatic adults arriving from the Sub-Saharan region were included. Schistosomiasis screening was conducted according to clinical practice following a different strategy in each setting: (A) feces and urine direct examination plus S. mansoni serology if non-explained eosinophilia was present and (B) S. mansoni serology plus uroparasitological examination as the second step in case of a positive serology. Demographic, clinical and laboratory features were collected. Schistosomiasis cases, clinical management and a 24 month follow-up were recorded for each group. Four-hundred forty individuals were included. The patients were mainly from West African countries. Fifty schistosomiasis cases were detected (11.5% group A vs. 4 % group B, p = 0.733). When both microscopic and serological techniques were performed, discordant results were recorded in 18.4% (16/88). Schistosomiasis cases were younger (p < 0.001) and presented eosinophilia and elevated IgE (p < 0.001) more frequently. Schistosomiasis is a frequent diagnosis among high-risk populations. Serology achieves a similar performance to direct diagnosis for the screening of schistosomiasis in a high-risk population

Pneumocystis jirovecii en el siglo XXI

La pneumònia per Pneumocystis jirovecii (PJP) ha experimentat canvis en la seva epidemiologia en les últimes dècades, sobretot en països amb accés als tractaments antiretrovirals d'alta eficàcia. La prevalença de la PJP en pacients immunocompromesos VIH negatius (oncològics, hematològics, trasplantats, malalties autoimmunes o aquells que reben teràpies immunodepressores) està en augment. En ells, en general, la infecció és més aguda i greu, presentant major requeriment d'ingrés a UCI i mortalitat més elevada, comparat amb els VIH positius. El seu diagnòstic es veu dificultat perquè característicament cursa amb menor càrrega fúngica i, sovint, no es poden sotmetre a una fibrobroncoscòpia, posant en evidència la necessitat de millorar el rendiment les eines convencionals per al diagnòstic de laboratori. D'altra banda, conèixer la diversitat genètica de Pneumocystis pot facilitar la comprensió de la seva ecologia. Aquest coneixement s'ha vist limitat fins a la data per l'absència d'un mètode de tipat estandarditzat amb rendiment acceptable. Els objectius principals d'aquesta tesi són: (i) avaluar el rendiment de dues PCR a temps real sobre mostres obtingudes de manera invasiva i no invasiva per a optimització de la diagnosi de la PJP, especialment en població no VIH, així com la influència de la diana utilitzada, i generant els corresponents criteris d'interpretació; (ii) comparar el rendiment de dos esquemes de MLST (β-TUB, DHPS, mt26S, ITS versus CYB, mt26S, SOD) proposats prèviament i possibles combinacions alternatives de les seves loci per identificar l'estratègia MLST més adequada per al genotipat de Pneumocystis, prestant atenció a la influència de la càrrega fúngica sobre la taxa d'amplificació i (iii) utilitzar la combinació de loci amb millor rendiment descriure l'epidemiologia molecular de Pneumocystis en una cohort retrospectiva en un hospital terciari de Barcelona, incloent pacients colonitzats i infectats. Independentment de la diana de la PCR utilitzada, les mostres de rentat oral van resultar útils per al diagnòstic ràpid i precís de la PJP, mitjançant una interpretació qualitativa dels resultats. També van resultar útils en la seva aplicació a mostres invasives, encara que es requereix interpretació quantitativa dels resultats. La càrrega fúngica va tenir impacte en les taxes d'èxit de seqüenciació, principalment en els loci nuclears. Una selecció acurada de dianes i l'optimització de protocols tècnics va permetre identificar la combinació de CYB, mt26S, β-TUB i SOD com la més adequada i discriminatòria per al genotipat de Pneumocystis, millorant el rendiment pel que fa als dos esquemes MLST prèviament descrits. La nova combinació de loci va mostrar una elevada diversitat genètica present en la cohort, sense predomini de seqüenciotips. No es va identificar associació entre variants al·lèliques o seqüenciotips i trets clínics. L'esquema MLST seleccionat va permetre descartar la transmissió creuada del fong entre pacients.La neumonía por Pneumocystis jirovecii (PJP) ha experimentado cambios en su epidemiología en las últimas décadas, sobre todo en países con acceso a los tratamientos antirretrovirales de alta eficacia. La prevalencia de la PJP en pacientes inmunocomprometidos VIH negativos (oncológicos, hematológicos, trasplantados, enfermedades autoinmunes o aquellos que reciben terapias inmunodepresoras) está en aumento. En ellos, por lo general, la infección es más aguda y grave, presentando mayor requerimiento en UCI y mortalidad más elevada, comparado con los VIH positivos. Su diagnóstico se ve dificultado porque característicamente cursa con menor carga fúngica y, con frecuencia, no pueden someterse a una fibrobroncoscopia, poniendo en evidencia la necesidad de mejorar el rendimiento las herramientas convencionales para el diagnóstico de laboratorio. Por otra parte, conocer la diversidad genética de Pneumocystis puede facilitar la comprensión de su ecología. Este conocimiento se ha visto limitado hasta la fecha por la ausencia de un método de tipado estandarizado con rendimiento aceptable. Los objetivos principales de esta tesis son: (i) evaluar el rendimiento de dos PCR a tiempo real sobre muestras obtenidas de forma invasiva y no invasiva para optimización del diagnóstico de la PJP, especialmente en población no VIH, así como la influencia de la diana utilizada, y generando los correspondientes criterios de interpretación; (ii) comparar el rendimiento de dos esquemas de MLST (β-TUB, DHPS, mt26S, ITS versus CYB, mt26S, SOD) propuestos previamente y posibles combinaciones alternativas de sus loci para identificar la estrategia MLST más adecuada para el genotipado de Pneumocystis, prestando atención a la influencia de la carga fúngica sobre la tasa de amplificación y (iii) utilizar la combinación de loci con mejor rendimiento describir la epidemiología molecular de Pneumocystis en una cohorte retrospectiva en un hospital terciario de Barcelona, incluyendo pacientes colonizados e infectados. Independientemente de la diana de la PCR utilizada, las muestras de lavado oral resultaron útiles para el diagnóstico rápido y certero de la PJP, mediante una interpretación cualitativa de los resultados. También resultaron útiles en su aplicación a muestras invasivas, aunque se requiere interpretación cuantitativa de los resultados. La carga fúngica tuvo impacto en las tasas de éxito de secuenciación, principalmente en los loci nucleares. Una selección cuidadosa de dianas y la optimización de protocolos técnicos permitió identificar la combinación de CYB, mt26S, β-TUB y SOD como la más adecuada y discriminatoria para el genotipado de Pneumocystis, mejorando el rendimiento con respecto a los dos esquemas MLST previamente descritos. La nueva combinación de loci revelo una elevada diversidad genética presente en la cohorte, sin predominancia de secuenciotipos. No se identificó asociación entre variantes alélicas o secuenciotipos y rasgos clínicos. El esquema MLST seleccionado permitió descartar la transmisión cruzada del hongo entre pacientes.In recent decades the epidemiology of Pneumocystis jirovecii pneumonia (PJP) has experienced changes, particularly in countries with access to highly effective antiretroviral treatment. The prevalence of PJP in immunocompromised HIV-negative patients (oncological, hematological, transplanted, autoimmune diseases or those receiving immunosuppressive therapies) is increasing. They experience an infection that, in general, is more acute, more severe, with a greater requirement of advanced vital support and a higher mortality as compared to HIV-positive patients. The diagnosis of PJP in the former is hampered by their low fungal loads and a critical condition that impede the performance of a bronchoscopy, highlighting the need to improve the performance of conventional laboratory diagnostic tools. On the other hand, a deeper knowledge of the genetic diversity of Pneumocystis could facilitate the understanding of its ecology. To date, this knowledge has been limited by the absence of a standardized typing method with acceptable performance. The main objectives of this thesis are: (i) to optimize the diagnosis of PJP, especially in non-HIV population, by evaluating the performance of two real-time PCRs on invasively and non-invasively obtained samples, the assessment of the influence of the genetic target and the generation of interpretation criteria; (ii) to compare the performance of two previously proposed MLST schemes (β-TUB, DHPS, mt26S, ITS versus CYB, mt26S, SOD) as well as possible alternative combinations of loci to identify the most suitable MLST strategy for Pneumocystis genotyping, paying attention to the influence of fungal load on the rate of amplification and (iii) using the best performing combination of loci, to describe the molecular epidemiology of Pneumocystis in a retrospective cohort in a tertiary hospital in Barcelona, including colonized and infected patients. Regardless of the PCR target used, the oral lavage samples proved to be useful for the rapid and accurate diagnosis of PJP, through a qualitative interpretation of the results. Both PCR were also useful in their application to invasive samples, although quantitative interpretation of the results is required. The fungal load had an impact on sequencing success rates, mainly in nuclear loci. A careful selection of targets and the optimization of technical protocols allowed the identification of CYB-mt26S-β-TUB-SOD as the most suitable and discriminatory combination for Pneumocystis genotyping, improving the performance with respect to the two previously described MLST schemes. The new combination of loci revealed a high genetic diversity present in the cohort, without predominance of sequence types. No association was identified between allelic variants or sequence types and clinical features. The selected MLST scheme allowed to rule out cross transmission of the fungus between patients

Pneumocystis jirovecii en el siglo XXI

La pneumònia per Pneumocystis jirovecii (PJP) ha experimentat canvis en la seva epidemiologia en les últimes dècades, sobretot en països amb accés als tractaments antiretrovirals d’alta eficàcia. La prevalença de la PJP en pacients immunocompromesos VIH negatius (oncològics, hematològics, trasplantats, malalties autoimmunes o aquells que reben teràpies immunodepressores) està en augment. En ells, en general, la infecció és més aguda i greu, presentant major requeriment d’ingrés a UCI i mortalitat més elevada, comparat amb els VIH positius. El seu diagnòstic es veu dificultat perquè característicament cursa amb menor càrrega fúngica i, sovint, no es poden sotmetre a una fibrobroncoscòpia, posant en evidència la necessitat de millorar el rendiment les eines convencionals per al diagnòstic de laboratori. D’altra banda, conèixer la diversitat genètica de Pneumocystis pot facilitar la comprensió de la seva ecologia. Aquest coneixement s’ha vist limitat fins a la data per l’absència d’un mètode de tipat estandarditzat amb rendiment acceptable. Els objectius principals d’aquesta tesi són: (i) avaluar el rendiment de dues PCR a temps real sobre mostres obtingudes de manera invasiva i no invasiva per a optimització de la diagnosi de la PJP, especialment en població no VIH, així com la influència de la diana utilitzada, i generant els corresponents criteris d’interpretació; (ii) comparar el rendiment de dos esquemes de MLST (β-TUB, DHPS, mt26S, ITS versus CYB, mt26S, SOD) proposats prèviament i possibles combinacions alternatives de les seves loci per identificar l’estratègia MLST més adequada per al genotipat de Pneumocystis, prestant atenció a la influència de la càrrega fúngica sobre la taxa d’amplificació i (iii) utilitzar la combinació de loci amb millor rendiment descriure l’epidemiologia molecular de Pneumocystis en una cohort retrospectiva en un hospital terciari de Barcelona, incloent pacients colonitzats i infectats. Independentment de la diana de la PCR utilitzada, les mostres de rentat oral van resultar útils per al diagnòstic ràpid i precís de la PJP, mitjançant una interpretació qualitativa dels resultats. També van resultar útils en la seva aplicació a mostres invasives, encara que es requereix interpretació quantitativa dels resultats. La càrrega fúngica va tenir impacte en les taxes d’èxit de seqüenciació, principalment en els loci nuclears. Una selecció acurada de dianes i l’optimització de protocols tècnics va permetre identificar la combinació de CYB, mt26S, β-TUB i SOD com la més adequada i discriminatòria per al genotipat de Pneumocystis, millorant el rendiment pel que fa als dos esquemes MLST prèviament descrits. La nova combinació de loci va mostrar una elevada diversitat genètica present en la cohort, sense predomini de seqüenciotips. No es va identificar associació entre variants al·lèliques o seqüenciotips i trets clínics. L’esquema MLST seleccionat va permetre descartar la transmissió creuada del fong entre pacients.La neumonía por Pneumocystis jirovecii (PJP) ha experimentado cambios en su epidemiología en las últimas décadas, sobre todo en países con acceso a los tratamientos antirretrovirales de alta eficacia. La prevalencia de la PJP en pacientes inmunocomprometidos VIH negativos (oncológicos, hematológicos, trasplantados, enfermedades autoinmunes o aquellos que reciben terapias inmunodepresoras) está en aumento. En ellos, por lo general, la infección es más aguda y grave, presentando mayor requerimiento en UCI y mortalidad más elevada, comparado con los VIH positivos. Su diagnóstico se ve dificultado porque característicamente cursa con menor carga fúngica y, con frecuencia, no pueden someterse a una fibrobroncoscopia, poniendo en evidencia la necesidad de mejorar el rendimiento las herramientas convencionales para el diagnóstico de laboratorio. Por otra parte, conocer la diversidad genética de Pneumocystis puede facilitar la comprensión de su ecología. Este conocimiento se ha visto limitado hasta la fecha por la ausencia de un método de tipado estandarizado con rendimiento aceptable. Los objetivos principales de esta tesis son: (i) evaluar el rendimiento de dos PCR a tiempo real sobre muestras obtenidas de forma invasiva y no invasiva para optimización del diagnóstico de la PJP, especialmente en población no VIH, así como la influencia de la diana utilizada, y generando los correspondientes criterios de interpretación; (ii) comparar el rendimiento de dos esquemas de MLST (β-TUB, DHPS, mt26S, ITS versus CYB, mt26S, SOD) propuestos previamente y posibles combinaciones alternativas de sus loci para identificar la estrategia MLST más adecuada para el genotipado de Pneumocystis, prestando atención a la influencia de la carga fúngica sobre la tasa de amplificación y (iii) utilizar la combinación de loci con mejor rendimiento describir la epidemiología molecular de Pneumocystis en una cohorte retrospectiva en un hospital terciario de Barcelona, incluyendo pacientes colonizados e infectados. Independientemente de la diana de la PCR utilizada, las muestras de lavado oral resultaron útiles para el diagnóstico rápido y certero de la PJP, mediante una interpretación cualitativa de los resultados. También resultaron útiles en su aplicación a muestras invasivas, aunque se requiere interpretación cuantitativa de los resultados. La carga fúngica tuvo impacto en las tasas de éxito de secuenciación, principalmente en los loci nucleares. Una selección cuidadosa de dianas y la optimización de protocolos técnicos permitió identificar la combinación de CYB, mt26S, β-TUB y SOD como la más adecuada y discriminatoria para el genotipado de Pneumocystis, mejorando el rendimiento con respecto a los dos esquemas MLST previamente descritos. La nueva combinación de loci revelo una elevada diversidad genética presente en la cohorte, sin predominancia de secuenciotipos. No se identificó asociación entre variantes alélicas o secuenciotipos y rasgos clínicos. El esquema MLST seleccionado permitió descartar la transmisión cruzada del hongo entre pacientes.In recent decades the epidemiology of Pneumocystis jirovecii pneumonia (PJP) has experienced changes, particularly in countries with access to highly effective antiretroviral treatment. The prevalence of PJP in immunocompromised HIV-negative patients (oncological, hematological, transplanted, autoimmune diseases or those receiving immunosuppressive therapies) is increasing. They experience an infection that, in general, is more acute, more severe, with a greater requirement of advanced vital support and a higher mortality as compared to HIV-positive patients. The diagnosis of PJP in the former is hampered by their low fungal loads and a critical condition that impede the performance of a bronchoscopy, highlighting the need to improve the performance of conventional laboratory diagnostic tools. On the other hand, a deeper knowledge of the genetic diversity of Pneumocystis could facilitate the understanding of its ecology. To date, this knowledge has been limited by the absence of a standardized typing method with acceptable performance. The main objectives of this thesis are: (i) to optimize the diagnosis of PJP, especially in non-HIV population, by evaluating the performance of two real-time PCRs on invasively and non-invasively obtained samples, the assessment of the influence of the genetic target and the generation of interpretation criteria; (ii) to compare the performance of two previously proposed MLST schemes (β-TUB, DHPS, mt26S, ITS versus CYB, mt26S, SOD) as well as possible alternative combinations of loci to identify the most suitable MLST strategy for Pneumocystis genotyping, paying attention to the influence of fungal load on the rate of amplification and (iii) using the best performing combination of loci, to describe the molecular epidemiology of Pneumocystis in a retrospective cohort in a tertiary hospital in Barcelona, including colonized and infected patients. Regardless of the PCR target used, the oral lavage samples proved to be useful for the rapid and accurate diagnosis of PJP, through a qualitative interpretation of the results. Both PCR were also useful in their application to invasive samples, although quantitative interpretation of the results is required. The fungal load had an impact on sequencing success rates, mainly in nuclear loci. A careful selection of targets and the optimization of technical protocols allowed the identification of CYB-mt26S-β-TUB-SOD as the most suitable and discriminatory combination for Pneumocystis genotyping, improving the performance with respect to the two previously described MLST schemes. The new combination of loci revealed a high genetic diversity present in the cohort, without predominance of sequence types. No association was identified between allelic variants or sequence types and clinical features. The selected MLST scheme allowed to rule out cross transmission of the fungus between patients.Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologi

Unexpected false-negative result in a traveller’s malaria diagnosis

A 4-year-old male presented at the emergency room for a 4-day-history of fever, diarrhoea and vomiting. The family had returned from Senegal 1 day before presenting to our clinic. None of the family had taken malaria prophylaxis. Because of a recent travel history, investigations to rule out malaria were initiated. These included a rapid antigen detection test (RDT; BinaxNow® Malaria, Abbott, USA) and microscopy examination.Postprint (published version