Fecal carriage of extended-spectrum beta-lactamase-producing Enterobacterales in healthy Spanish schoolchildren

Background: Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) are a serious threat among emerging antibiotic resistant bacteria. Particularly, the number of cases of ESBL-E infections reported in children has been increasing in recent years, and approved antibiotic treatments for this age group are limited. However, information regarding the prevalence of colonization in European children, risk factors associated with colonization, and the characteristics of the colonizing strains is scarce. The aims of this study were to determine the prevalence of ESBL-E colonization in fecal samples of apparently healthy schoolchildren, to identify lifestyle routines associated with colonization, and to characterize clonal relationships and mechanisms of resistance in ESBL-E isolates. Methods: A cohort of 887 healthy children (3-13 years old) from seven primary and secondary schools in the Madrid metropolitan area was recruited between April-June 2018, and sociodemographic information and daily habits were collected. Fecal samples were screened for ESBL-E carriage in selective medium. ESBL-E isolates were further characterized by assessing molecular epidemiology (PFGE and MLST), ESBL gene carriage, and antibiotic resistance profile. This information was analyzed in conjunction with the metadata of the participants in order to identify external factors associated with ESBL-E carriage. Results: Twenty four ESBL-E, all but one Escherichia coli, were detected in 23 children (prevalence: 2.6%; 95% CI: 1.6-3.6%). Of these, seven contained the blaCTX-M-14 allele, five the blaCTX-M-15, five the blaSHV-12, three the blaCTX-M-27, three the blaCTX-M-32, and one the blaCTX-M-9. Significant clonal diversity was observed among the isolates that grouped into 22 distinct clusters (at <85% similarity of PFGE profile). ESBL-producing E. coli isolates belonged to 12 different STs, with ST10 (25%) and ST131 (17%) being the most frequent. Apart from ß-lactams, resistance to trimethoprim/sulfamethoxazole (46%), ciprofloxacin (33%), levofloxacin (33%), tobramycin (21%), and gentamicin (8%) were the most frequently detected. Conclusion: The prevalence of ESBL-E in the studied cohort of children was lower than the average colonization rate previously detected in Europe for both children and adults. E. coli was the main ESBL-producing species detected and CTX-M were the most frequently identified ESBLs. High ST diversity suggests polyclonal dissemination. Compared to other STs, ST131 isolates were associated with resistance to various antimicrobials.ML-S was supported by the Sara Borrell Program of the Instituto de Salud Carlos III (ISCIII) (CD17CIII/00017). ZM was supported by the Río Hortega Program of the ISCIII. AÁ was supported by the Garantía Juvenil Program of the Comunidad Autónoma de Madrid. SS was supported by the Miguel Servet program of ISCIII (CPII18CIII/00005). This study was funded by the ISCIII, Ministry of Economy and Competitiveness (Spain), under projects PI16CIII/00024, PI18CIII/00030, MPY380/18, and MPY516/19.S

Parasitemia Levels in Trypanosoma cruzi Infection in Spain, an Area Where the Disease Is Not Endemic: Trends by Different Molecular Approaches

Trypanosoma cruzi infection has expanded globally through human migration. In Spain, the mother-to-child route is the mode of transmission contributing to autochthonous Chagas disease (CD); however, most people acquired the infection in their country of origin and were diagnosed in the chronic phase (imported chronic CD). In this context, we assessed the quantitative potential of the Loopamp Trypanosoma cruzi detection kit (Sat-TcLAMP) based on satellite DNA (Sat-DNA) to determine parasitemia levels compared to those detected by real-time quantitative PCRs (qPCRs) targeting Sat-DNA (Sat-qPCR) and kinetoplast DNA minicircles (kDNA-qPCR). This study included 173 specimens from 39 autochthonous congenital and 116 imported chronic CD cases diagnosed in Spain. kDNA-qPCR showed higher sensitivity than Sat-qPCR and Sat-TcLAMP. According to all quantitative approaches, parasitemia levels were significantly higher in congenital infection than in chronic CD (1 × 10-1 to 5 × 105 versus >1 × 10-1 to 6 × 103 parasite equivalents/mL, respectively [P < 0.001]). Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR results were equivalent at high levels of parasitemia (P = 0.381). Discrepancies were significant for low levels of parasitemia and older individuals. Differences between Sat-TcLAMP and Sat-qPCR were not qualitatively significant, but estimations of parasitemia using Sat-TcLAMP were closer to those by kDNA-qPCR. Parasitemia changes were assessed in 6 individual cases in follow-up, in which trends showed similar patterns by all quantitative approaches. At high levels of parasitemia, Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR worked similarly, but significant differences were found for the low levels characteristic of late chronic CD. A suitable harmonization strategy needs to be developed for low-level parasitemia detection using Sat-DNA- and kDNA-based tests. IMPORTANCE: Currently, molecular equipment has been introduced into many health care centers, even in low-income countries. PCR, qPCR, and loop-mediated isothermal amplification (LAMP) are becoming more accessible for the diagnosis of neglected infectious diseases. Chagas disease (CD) is spreading worldwide, and in countries where the disease is not endemic, such as Spain, the parasite Trypanosoma cruzi is transmitted from mother to child (congenital CD). Here, we explore why LAMP, aimed at detecting T. cruzi parasite DNA, is a reliable option for the diagnosis of congenital CD and the early detection of reactivation in chronic infection. When the parasite load is high, LAMP is equivalent to any qPCR. In addition, the estimations of T. cruzi parasitemia in patients living in Spain, a country where the disease is not endemic, resemble natural evolution in areas of endemicity. If molecular tests are introduced into the diagnostic algorithm for congenital infection, early diagnosis and timely treatment would be accomplished, so the interruption of vertical transmission can be an achievable goal.This research was supported by the Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland (WO klob-0003), and the Surveillance Program of Chagas Disease of the National Centre for Microbiology (CNM), Instituto de Salud Carlos III (ISCIII). CNM-ISCIII research team is supported by Fundación Mundo Sano, Spain (MVP 237/19). The ISGlobal research team is supported by the Agència de Gestió d’Ajuts Universitaris i de Recerca AGAUR) (2017 SGR 00924). ISGlobal is a member of the Centres de Recerca de Catalunya (CERCA) Programme, Government of Catalonia (Spain).S

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3.

CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain. Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were bla OXA-48 (263/377), bla KPC-3 (62/377), bla VIM-1 (28/377), and bla NDM-1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Table_4_CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3.pdf

[Objectives] CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.[Methods] In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.[Results] In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).[Conclusion] This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3.Peer reviewe

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC &gt; 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Serodiscordancia en el diagnóstico de la infección por Trypanosoma cruzi en área no endémica

La enfermedad de Chagas es endémica en Latinoamérica, pero debido a los flujos migratorios de las últimas décadas se ha convertido en un problema de salud global. Según la Organización Mundial de la Salud (OMS) el diagnóstico serológico de la infección por Trypanosoma cruzi se basa en la concordancia de dos técnicas serológicas de principios diferentes usadas en paralelo. Cuando los resultados son discrepantes se recomienda el uso de una tercera técnica confirmatoria no siempre disponible. El objetivo general de esta tesis es conocer la prevalencia de la serodiscordancia en pacientes latinoamericanos fuera de la zona endémica, y analizar el rendimiento de dos técnicas confirmatorias basadas en la metodología Western-blot (WB) para facilitar el diagnóstico de la infección por T. cruzi en este grupo de población serodiscordante. Como resultado del presente trabajo se ha podido concluir que con las técnicas de cribado actuales y siguiendo el algoritmo actual de la OMS, se detecta un porcentaje nada despreciable de pacientes sin un diagnóstico claro. Con el uso de una tercera técnica se confirma la infección en aproximadamente la mitad de los individuos. Estos hallazgos ponen de manifiesto la necesidad de desarrollo de nuevas técnicas diagnósticas lo suficientemente sensibles y específicas para poder ser utilizadas en solitario, así como el establecimiento de una técnica confirmatoria al alcance de los centros de referencia fuera del área endémica.Chagas disease is a parasitic zoonosis endemic in Latin America. However due to the migratory flows in the last decades, it has become a global concern. The serological diagnosis of Trypanosoma cruzi infection is based on the agreement of two different techniques used in parallel. This fact can lead, sometimes, to discordant results and when these results are persistent, the use of a third confirmatory technique is recommended. The main objective of this Thesis is to know the prevalence of discrepant results in Latin American patients outside the endemic area, and to evaluate the performance of two different confirmatory techniques in this population group. As a result of the present study it was possible to conclude that with the current screening techniques and following the diagnosis algorithm of the World Health Organization (WHO), a non-negligible percentage of patients without a clear diagnosis was found. According to our results, the use of a third technique confirms the infection in approximately half of the individuals with the implications that this entails. These findings highlight the need for the development of new diagnostic techniques sensitive and specific enough to be used alone, as well as the establishment of a confirmatory technique available in reference units inside and outside the endemic area

Serodiscordancia en el diagnóstico de la infección por Trypanosoma cruzi en área no endémica /

La enfermedad de Chagas es endémica en Latinoamérica, pero debido a los flujos migratorios de las últimas décadas se ha convertido en un problema de salud global. Según la Organización Mundial de la Salud (OMS) el diagnóstico serológico de la infección por Trypanosoma cruzi se basa en la concordancia de dos técnicas serológicas de principios diferentes usadas en paralelo. Cuando los resultados son discrepantes se recomienda el uso de una tercera técnica confirmatoria no siempre disponible. El objetivo general de esta tesis es conocer la prevalencia de la serodiscordancia en pacientes latinoamericanos fuera de la zona endémica, y analizar el rendimiento de dos técnicas confirmatorias basadas en la metodología Western-blot (WB) para facilitar el diagnóstico de la infección por T. cruzi en este grupo de población serodiscordante. Como resultado del presente trabajo se ha podido concluir que con las técnicas de cribado actuales y siguiendo el algoritmo actual de la OMS, se detecta un porcentaje nada despreciable de pacientes sin un diagnóstico claro. Con el uso de una tercera técnica se confirma la infección en aproximadamente la mitad de los individuos. Estos hallazgos ponen de manifiesto la necesidad de desarrollo de nuevas técnicas diagnósticas lo suficientemente sensibles y específicas para poder ser utilizadas en solitario, así como el establecimiento de una técnica confirmatoria al alcance de los centros de referencia fuera del área endémica.Chagas disease is a parasitic zoonosis endemic in Latin America. However due to the migratory flows in the last decades, it has become a global concern. The serological diagnosis of Trypanosoma cruzi infection is based on the agreement of two different techniques used in parallel. This fact can lead, sometimes, to discordant results and when these results are persistent, the use of a third confirmatory technique is recommended. The main objective of this Thesis is to know the prevalence of discrepant results in Latin American patients outside the endemic area, and to evaluate the performance of two different confirmatory techniques in this population group. As a result of the present study it was possible to conclude that with the current screening techniques and following the diagnosis algorithm of the World Health Organization (WHO), a non-negligible percentage of patients without a clear diagnosis was found. According to our results, the use of a third technique confirms the infection in approximately half of the individuals with the implications that this entails. These findings highlight the need for the development of new diagnostic techniques sensitive and specific enough to be used alone, as well as the establishment of a confirmatory technique available in reference units inside and outside the endemic area

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

Objectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain. Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA−48 (263/377), blaKPC−3 (62/377), blaVIM−1 (28/377), and blaNDM−1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Evaluation of the Performance of the Loopamp Detection Kit for the Diagnosis of Chagas Disease in an Area Where It Is Not Endemic, Spain

In Spain, PCR is the tool of choice for the diagnosis of congenital Chagas disease (CD) and serology for diagnosing chronic CD. A loop-mediated isothermal amplification test for DNA detection showed good analytical performance and ease of use. In Spain, PCR is the tool of choice for the diagnosis of congenital Chagas disease (CD) and serology for diagnosing chronic CD. A loop-mediated isothermal amplification test for DNA detection showed good analytical performance and ease of use. We aimed to evaluate the performance of the Loopamp detection kit (Eiken Chemical Co. Ltd., Japan) (Tcruzi -LAMP) for congenital and chronic CD diagnosis using well-characterized samples. We included samples from 39 congenital and 174 chronic CD cases and from 48 uninfected children born to infected mothers and 34 nonchagasic individuals. The sensitivity, specificity, and accuracy of Tcruzi -LAMP were estimated using standard case definitions for congenital CD (positive result by parasitological or PCR tests or serology after 9 months of age) and chronic CD (positive serology by at least two tests). The Tcruzi -LAMP results were read by visual examination and a real-time fluorimeter. For congenital CD, Tcruzi -LAMP sensitivity was 97% for both types of reading; specificity was 92% by visual examination and 94% by fluorimeter. For chronic CD, sensitivity was 47% and specificity 100%. The accuracy in congenital CD was >94% versus 56% in chronic CD. The agreement of Tcruzi -LAMP with PCR tests was better in congenital CD (kappa, 0.86 to 0.91) than in chronic CD (kappa, 0.67 to 0.83). The Loopamp detection kit showed good performance for the diagnosis of congenital CD. Tcruzi -LAMP, like PCR, can be useful for the screening and early diagnosis of congenital infection