Analysis of Clostridioides difficile Infection in Children with Diarrhea in Two Hospitals in Southern Brazil.

Clostridioides difficile infection (CDI) has been increasingly observed in children, but there is a lack of epidemiological and molecular data on CDI in Latin America. This prospective cohort study aimed to investigate the role of CDI in children with diarrhea. It included 105 children with antimicrobial-associated diarrhea (AAD) and analyzed the molecular characteristics of strains isolated from two hospitals in southern Brazil between 2017 and 2020. Fecal samples from the participants were tested for glutamate dehydrogenase (GDH) and A/B toxins using a rapid enzyme immunoassay. GDH-positive samples underwent automated real-time polymerase chain reaction and toxigenic culture. Toxigenic C. difficile isolates were selected for whole genome sequencing. Out of the 105 patients, 14 (13.3%) met the criteria for CDI. Children with a history of previous CDI and the presence of mucus in their stool were more likely to have CDI. Metronidazole was the most used treatment (71.4%), and three patients (23.1%) experienced CDI recurrence (rCDI). Although the number of sequenced isolates was limited, a wide diversity of sequence types (ST) was observed. In addition to toxin genes (tcdA, tcdB, cdtA, and cdtB), the isolates also exhibited virulence factors involved in adhesion (cwp66, groEL, slpA, fbpA/fbp68) and immune evasion (rmlA, rmlB, rmlC, gnd, rfbA-1), along with multiple resistance factors (gyrA mutation, norA, ermB, dfrF, and vanG). These findings highlight the prevalence and recurrence of CDI among hospitalized children. Longitudinal studies are needed to better understand the characteristics of CDI-associated diarrhea and its impact on the healthcare system in this population.

Laboratory diagnosis of Clostridioides difficile infection in symptomatic patients: what can we do better?

The laboratory diagnosis of Clostridioides difficile infection (CDI) is challenging since this bacteria may be detected in healthy people and toxin production detection is not sensitive enough to be used alone. Thus, there is no single test with adequate sensitivity and specificity to be used in laboratory diagnosis. We evaluated the performance of tests used in the diagnosis of CDI in symptomatic patients with risk factors in hospitals in southern Brazil. Enzyme immunoassays (EIA) for glutamate dehydrogenase antigen (GDH) and toxins A/B, real-time polymerase chain reaction (qPCR), GeneXpert system, and a two-step algorithm comprising GDH/TOXIN EIA performed simultaneously followed by GeneXpert for outliers were evaluated. Toxigenic strain in stool culture was considered CDI positive (gold standard). Among 400 samples tested, 54 (13.5%) were positive for CDI and 346 (86.5%) were negative. The diagnosis of the two-step algorithm and qPCR had an excellent performance with an accuracy of 94.5% and 94.2%, respectively. The Youden index showed that GeneXpert as a single test (83.5%) and the two-step algorithm (82.8%) were the most effective assays. Diagnosing CDI and non-CDI diarrhea could be successfully attained by the combination of clinical data with accuracy of laboratory tests.

Prevalence and Seasonality of Clostridiodes difficile over 12 Years at a Tertiary Hospital in Brazil.

Clostridioides difficile is the main pathogen responsible for antibiotic-associated diarrhea in adults. Besides its challenging diagnosis, C. difficile infection (CDI) causes substantial morbidity and mortality. Commercially, there are assays with different targets and performances in sensitivity and specificity. The objectives of this study were to: (1) evaluate the prevalence and seasonal variability of CDI rates at a tertiary hospital in southern Brazil over 12 years and (2) determine the impact of using a two-step algorithm test in the laboratory diagnosis. Between January 2007 and May 2019, fecal samples from 2275 patients were analyzed in a cross-sectional study. Four commercial tests were adopted for the diagnosis of CDI, the immunochromatographic test for toxin A from 2007 to 2010; the enzyme-linked immunosorbent assay method for toxins A and B from 2011 to March 2017; and the rapid enzyme immunoassay (EIA) for GDH and toxins A and B, associated with a Polymerase Chain Reaction (PCR) for the toxin B gene from June 2017 to 2019. The annual prevalence was 8.7% from 2007 to March 2017, increasing between June 2017 and 2019 to 14.7% when the C. diff Quik Chek Complete + GeneXpert C. difficile (two-step algorithm) test was adopted. The number of samples (691) and percentage of CDI cases (10.5%) were higher in winter, but the difference has no statistical significance (P > 0.05). An accurate diagnosis and adequate knowledge of the local seasonality of CDI allow the effective implementation of prevention and control strategies for nosocomial CDI, in addition to effective treatment for patients.

Standardisation and validation of an in-house quantitative real-time polymerase chain reaction (qPCR) assay for the diagnosis of Clostridioides difficile infection.

OBJECTIVES: Clostridioides difficile is an emerging enteric pathogen that causes nosocomial diarrhoea in adults. The excessive cost of commercial molecular tests restricts the access of developing countries to its diagnosis. This study aimed to develop and validate in-house quantitative polymerase chain reaction (qPCR) targeting the C. difficile toxin B gene (tcdB) using two detection methodologies-SYBR Green and hydrolysis probes-for the diagnosis of C. difficile infection (CDI). METHODS: Glutamate dehydrogenase (GDH) plus toxigenic culture was the standard reference diagnostic method. The SYBR Green method and hydrolysis probes were used to study 392 samples simultaneously to assess the diagnostic value of these real-time PCR assays in detecting CDI from clinical samples. RESULTS: The SYBR Green and hydrolysis probe assays showed 97.9% and 87.5% sensitivity; 99.1% and 100.0% specificity; 94.0% and 100.0% positive predictive value; 99.7% and 98.3% negative predictive value; and 99.0% and 98.5% accuracy, respectively. CONCLUSIONS: The two qPCR methodologies evaluated could offer an adequate tool as part of an algorithm in the laboratory diagnosis of CDI.

Cost-Effectiveness of Molecular Method Diagnostic for Rapid Detection of Antibiotic-Resistant Bacteria.

OBJECTIVES: This study aimed to perform a cost-effectiveness analysis (CEA) of the molecular diagnostic method (MM) associated with conventional diagnostic method (CM) compared with the CM alone, for the detection of resistant profile in bacteremia, from the perspective of the Brazilian Public Health System, in intensive care units setting. METHODS: The clinical parameters regarding methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Gram-negative bacteria (CRGNB), and vancomycin-resistant Enterococcus spp. (VRE) infections were collected from searches on PubMed, Scopus, and SciELO, using specific keywords. Data on direct medical costs to treat these infections were collected according to Brazilian Public Health System perspective from Brazilian databases, in tables of 2018 to 2019. CEA was performed after building a dynamic model, which was calibrated and validated according to international recommendations. The incremental cost-effectiveness ratio of the MM + CM compared with the CM was calculated using the outcomes "avoided death" and "avoided resistant infections." One-way sensitivity analyses were performed. RESULTS: This CEA demonstrated that the MM + CM was dominant in all scenarios. Estimates showed that for MRSA, CRGNB, and VRE infections, every avoided death would lead to savings of Brazilian real (R$) 4.9 million ($937 301), R$2.2 million ($419 899), and R$1.3 million ($248 919), respectively. The same infections assessed by avoided resistant infections savings were projected to be R$24 964 ($4686), R$40 260 ($7558), and R$23 867 ($4480). CONCLUSIONS: MM leads to cost reduction and increased benefits, optimizing the use of financial resources on the health system in the intensive care unit setting, in bacteremia caused by MRSA, CRGNB, and VRE.

Automated immature granulocyte count in patients of the intensive care unit with suspected infection / Contagem automatizada de granulócitos imaturos em pacientes de uma unidade de terapia intensiva com suspeita de infecção

ABSTRACT Introduction: Diagnosing infections in intensive care unit (ICU) patients is vital to provide appropriate therapies. Hematological analyzers perform automated immature granulocyte counts (IG) quickly and with no additional cost when compared to traditional microbiological cultures. Elevated IG is directly associated with infections and inflammation. Objectives: Evaluate IG as infection marker in adult inpatients at the ICU-Complexo Hospital de Clínicas da Universidade Federal do Paraná (CHC-UFPR), compared to cultures of biological materials (gold standard). Material And Methods: Samples of 200 adult inpatients at CHC-UFPR ICU with suspected infection were used. Absolute (IG#) and relative (IG%) counts were performed on the Sysmex XN-3000. Cultures and blood cultures were performed either manually or on Bactec FX. Diagnostic accuracy and agreement for IG# and IG% were evaluated. Results: The reference intervals (RI) obtained for IG# and IG% were 0.06 × 103/µl and 0.6%, respectively, with sensitivity for both of 74.4% and specificity of 25.3% for IG#, and 26.6% for IG%. The receiver operating characteristic (ROC) curve showed cut-off value of 0.33 × 103/µl for IG#, sensitivity of 28%, specificity of 82.3%, and area under the curve (AUC) of 0.521. For IG%, cut-off value was 1.35%, sensitivity 44.6%, specificity 64.6%, and AUC 0.532. CV < 3% increased specificity to 88%. Conclusion: RI of IG% and IG# showed high sensitivity and are useful in screening for infection in ICU patients. The CVs demonstrated by the ROC curves showed high specificity and are helpful on the exclusion of sepsis diagnosis in ICU patients. IG was shown to be useful for screening and confirmation of infection in ICU patients.

RESUMEN Introducción: Diagnosticar infecciones en pacientes de la unidad de cuidados intensivos (UCI) es de suma importancia para proporcionar el tratamiento adecuado. El contaje automatizado de granulocitos inmaduros (GI) en analizadores hematológicos es rápido y sin costes adicionales. La elevada tasa de GI está asociada a infecciones. Objetivos: Evaluar GI como indicador de infección en pacientes adultos de la UCI del Complexo Hospital de Clínicas da Universidade Federal do Paraná (CHC-UFPR) en comparación a culturas de materiales biológicos (estándar de oro). Material Y Métodos: Se analizaron muestras de 200 pacientes adultos con sospecha de infección de la UCI del CHC-UFPR. Los conteos automatizados de granulocitos inmaduros absolutos (GI#) e relativos (GI%) se realizaron en el Sysmex-XN-3000, y los cultivos y hemocultivos, manualmente o en el Baetec-FX. Se han evaluado precisión diagnóstica y concordancia para GI# y GI%. Resultados: Los rangos de referencia obtenidos para GI# y GI% fueron 0,06 × 103/µl y 0,6%, respectivamente, con sensibilidad para ambos de 74,4% y especificidad de 25,3% para IG# y 26,6% para IG%. La curva receiver operating characteristic (ROC) ha mostrado valor de corte de 0,33 × 103/µl para IG#, sensibilidad de 28%, especificidad de 82,3% y área bajo la curva (AUC) de 0,521. Para GI%, el valor de corte ha sido 135%, sensibilidad de 44,6%, especificidad de 64,6% y AUC de 0,532. Valores de corte de GI% < 3% aumentaron la especificidad para 88%. Conclusión: Rangos de referencia de GI% y GI# presentaron sensibilidad elevada y son útiles en el triaje de infecciones en pacientes de UCI. Los valores de corte enseñados por las curvas ROC presentaron alta especificidad, permitiendo la identificación adecuada de los pacientes sanos. GI se ha mostrado útil para triaje y confirmación de infección en pacientes de UCI.

RESUMO Introdução: Diagnosticar infecções em pacientes da unidade de terapia intensiva (UTI) é vital para implementar terapias apropriadas. A contagem automatizada de granulócitos imaturos (IG) em analisadores hematológicos é rápida e sem custos adicionais. A taxa de IG elevada está associada a infecções. Objetivos: Avaliar IG como indicador de infecção em pacientes adultos da UTI do Complexo Hospital de Clínicas da Universidade Federal do Paraná (CHC-UFPR) em comparação com culturas de materiais biológicos (padrão-ouro). Material E Métodos: Foram analisadas amostras de 200 pacientes adultos com suspeita de infecção da UTI do CHC-UFPR. As contagens automatizadas de granulócitos imaturos absolutas (IG#) e relativas (IG%) foram realizadas no Sysmex-XN-3000, e as culturas e as hemoculturas, manualmente ou no Bactec-FX. As características de desempenho de teste diagnóstico para IG# e IG% foram avaliadas. Resultados: Os intervalos de referência (IR) obtidos para IG# e IG% foram 0,06 × 103/µl e 0,6%, respectivamente, com sensibilidade para ambos de 74,4% e especificidade de 25,3% para IG# e 26,6% para IG%. A curva receiver operating characteristic (ROC) mostrou valor de corte de 0,33 × 103/µl para IG#, sensibilidade de 28%, especificidade de 82,3% e área sob a curva (AUC) de 0,521. Para IG%, o valor de corte foi de 1,35%, sensibilidade de 44,6%, especificidade de 64,6% e AUC de 0,532. Valores de corte de IG% < 3% aumentaram a especificidade para 88%. Conclusão: IRs de IG% e IG# apresentaram sensibilidade elevada e são úteis na triagem de infecção nos pacientes da UTI. Os VCs demonstrados pelas curvas ROC para IG% e IG# apresentaram elevada especificidade, sendo, portanto, úteis para exclusão de diagnóstico de sepse nos pacientes da UTI. IG mostrou-se útil para triagem e confirmação de infecção em pacientes de UTI.

Colonização microbiana precoce de pacientes identificados por triagem neonatal para fibrose cística, com ênfase em Staphylococcus aureus / Early microbial colonization of cystic fibrosis patients identified by neonatal screening, with emphasis on Staphylococcus aureus

OBJETIVOS: Avaliar prospectivamente a colonização bacteriana de pacientes com fibrose cística identificados por triagem neonatal. Avaliar a suscetibilidade a antimicrobianos e caracterizar molecularmente as cepas de Staphylococcus aureus isoladas da orofaringe dos pacientes no período do estudo. MÉTODOS: Foram estudados 25 pacientes com fibrose cística, identificados por tripsina imunorreativa e com diagnóstico confirmado por duas ou mais provas de suor, atendidos regularmente no ambulatório de fibrose cística do Hospital de Clínicas da Universidade Federal do Paraná. Foram coletadas amostras de orofaringe com swab e cultivadas por métodos rotineiros; as colônias bacterianas foram identificadas fenotipicamente e testadas quanto à suscetibilidade a antimicrobianos. Os isolados de S. aureus foram submetidos a tipagem molecular por eletroforese em campo pulsado. RESULTADOS: De um total de 234 amostras de orofaringe, S. aureus foi isolado em maior número (76 por cento dos pacientes, 42 por cento das amostras), seguido de Pseudomonas aeruginosa (36 por cento dos pacientes, 16 por cento das amostras) e Haemophilus spp. (76 por cento dos pacientes; 19 por cento das amostras). Dos 19 pacientes colonizados com S. aureus, foram obtidos 73 isolados, 18 oxacilina-resistentes (24,6 por cento), isolados de dois pacientes, com perfis eletroforéticos idênticos ao do clone brasileiro. Os demais isolados oxacilina-sensíveis distribuíram-se entre 18 perfis eletroforéticos distintos. CONCLUSÃO: Observou-se uma maior prevalência de S. aureus, com isolamento mais precoce em relação aos outros patógenos pesquisados. Os isolados multissensíveis distribuíram-se em clones distintos, caracterizando a não transmissibilidade entre as cepas comunitárias. Os S. aureus resistentes a oxacilina isolados apresentaram perfis eletroforéticos idênticos, provavelmente adquiridos no ambiente hospitalar. P. aeruginosa foi pouco freqüente na população estudada.

OBJECTIVES: To assess bacterial colonization prospectively in patients with cystic fibrosis identified by neonatal screening. To assess susceptibility to antimicrobials and to perform the molecular typing of Staphylococcus aureus strains isolated from the oropharynx of patients during the study. METHODS: Twenty-five cystic fibrosis patients receiving regular treatment at the Cystic Fibrosis Outpatient Clinic of Hospital de Clínicas of Universidade Federal do Paraná Brazil, were included in the study. All patients were identified by trypsin-like immunoreactivity and their diagnosis was confirmed by two or more sweat tests. Oropharyngeal swabs were collected and cultured according to routine methods; bacterial colonies were phenotypically identified and their susceptibility to antimicrobials was tested. S. aureus isolates were submitted to molecular typing using pulsed-field gel electrophoresis. RESULTS: Out of 234 oropharyngeal swabs, S. aureus was the most frequently isolated strain (76 percent of patients, 42 percent of swabs), followed by Pseudomonas aeruginosa (36 percent of patients, 16 percent of swabs) and Haemophilus spp. (76 percent of patients; 19 percent of swabs). Seventy-three isolates were obtained from 19 patients colonized with S. aureus, of which 18 were oxacillin-resistant (24.6 percent), isolated from two patients, with the same electrophoretic profiles as that of the Brazilian clone. The remaining oxacillin-sensitive isolates were distributed into 18 electrophoretic profiles. CONCLUSION: There was higher prevalence of S. aureus, with earlier isolation than other pathogens. Multi-sensitive isolates were distributed into different clones, characterizing non-transmissibility among community-acquired strains. The isolated oxacillin-resistant S. aureus showed identical electrophoretic profiles, probably acquired in hospital. P. aeruginosa was not so frequent in the studied population.

Early microbial colonization of cystic fibrosis patients identified by neonatal screening, with emphasis on Staphylococcus aureus.

OBJECTIVES: To assess bacterial colonization prospectively in patients with cystic fibrosis identified by neonatal screening. To assess susceptibility to antimicrobials and to perform the molecular typing of Staphylococcus aureus strains isolated from the oropharynx of patients during the study. METHODS: Twenty-five cystic fibrosis patients receiving regular treatment at the Cystic Fibrosis Outpatient Clinic of Hospital de Clínicas of Universidade Federal do Paraná, Brazil, were included in the study. All patients were identified by trypsin-like immunoreactivity and their diagnosis was confirmed by two or more sweat tests. Oropharyngeal swabs were collected and cultured according to routine methods; bacterial colonies were phenotypically identified and their susceptibility to antimicrobials was tested. S. aureus isolates were submitted to molecular typing using pulsed-field gel electrophoresis. RESULTS: Out of 234 oropharyngeal swabs, S. aureus was the most frequently isolated strain (76% of patients, 42% of swabs), followed by Pseudomonas aeruginosa (36% of patients, 16% of swabs) and Haemophilus spp. (76% of patients; 19% of swabs). Seventy-three isolates were obtained from 19 patients colonized with S. aureus, of which 18 were oxacillin-resistant (24.6%), isolated from two patients, with the same electrophoretic profiles as that of the Brazilian clone. The remaining oxacillin-sensitive isolates were distributed into 18 electrophoretic profiles. CONCLUSION: There was higher prevalence of S. aureus, with earlier isolation than other pathogens. Multi-sensitive isolates were distributed into different clones, characterizing non-transmissibility among community-acquired strains. The isolated oxacillin-resistant S. aureus showed identical electrophoretic profiles, probably acquired in hospital. P. aeruginosa was not so frequent in the studied population.