Clinical validation of an in-house quantitative real time PCR assay for cytomegalovirus infection using the 1st WHO International Standard in kidney transplant patients / Validação clínica de um ensaio de PCR in house quantitativo em tempo real para infecção por citomegalovírus usando o 1º Padrão Internacional da OMS em pacientes com transplantados renais

Abstract Introduction: Cytomegalovirus (CMV) is one of the most common agents of infection in solid organ transplant patients, with significant morbidity and mortality. Objective: This study aimed to establish a threshold for initiation of preemptive treatment. In addition, the study compared the performance of antigenemia with qPCR results. Study design: This was a prospective cohort study conducted in 2017 in a single kidney transplant center in Brazil. Clinical validation was performed by comparing in-house qPCR results, against standard of care at that time (Pp65 CMV Antigenemia). ROC curve analysis was performed to determine the ideal threshold for initiation of preemptive therapy based on the qPCR test results. Results: Two hundred and thirty two samples from 30 patients were tested with both antigenemia and qPCR, from which 163 (70.26%) were concordant (Kappa coefficient: 0.435, p<0.001; Spearman correlation: 0.663). PCR allowed for early diagnoses. The median number of days for the first positive result was 50 (range, 24-105) for antigenemia and 42 (range, 24-74) for qPCR (p<0.001). ROC curve analysis revealed that at a threshold of 3,430 IU/mL (Log 3.54), qPCR had a sensitivity of 97.06% and a specificity of 74.24% (AUC 0.92617 ± 0.0185, p<0.001), in the prediction of 10 cells/105 leukocytes by antigenemia and physician's decision to treat. Conclusions: CMV Pp65 antigenemia and CMV qPCR showed fair agreement and a moderate correlation in this study. The in-house qPCR was revealed to be an accurate method to determine CMV DNAemia in kidney transplant patients, resulting in positive results weeks before antigenemia.

Resumo Introdução: Citomegalovírus (CMV) é um dos agentes infecciosos mais comuns em pacientes com transplante de órgãos sólidos, com morbidade e mortalidade significativas. Objetivo: Este estudo visou estabelecer um limite para o início do tratamento preemptivo. Além disso, comparou o desempenho da antigenemia com os resultados da qPCR in house. Desenho do estudo: Este foi um estudo de coorte prospectivo realizado em 2017 em um centro único de transplante renal no Brasil. A validação clínica foi realizada comparando resultados de qPCR in house, com o padrão de atendimento na época (Antigenemia para CMV Pp65). A análise da curva ROC foi realizada para determinar o limite ideal para o início da terapia preemptiva baseado nos resultados do teste qPCR in house. Resultados: 232 amostras de 30 pacientes foram testadas com antigenemia e qPCR, das quais 163 (70,26%) foram concordantes (Coeficiente Kappa: 0,435, p<0,001; Correlação Spearman: 0,663). PCR permitiu diagnósticos precoces. O número médio de dias para o primeiro resultado positivo foi 50 (intervalo, 24-105) para antigenemia e 42 (intervalo, 24-74) para qPCR (p<0,001). A análise da curva ROC revelou que em um limite de 3.430 UI/mL (Log 3,54), qPCR teve sensibilidade de 97,06% e especificidade de 74,24% (AUC 0,92617 ± 0,0185, p<0,001), na previsão de 10 células/10(5) leucócitos por antigenemia e na decisão do médico de tratar. Conclusões: Antigenemia para CMV Pp65 e qPCR para CMV mostraram uma concordância aceitável e uma correlação moderada neste estudo. qPCR in house revelou-se um método preciso para determinar DNAemia do CMV em pacientes transplantados renais, obtendo resultados positivos semanas antes da antigenemia.

Clinical validation of an in-house quantitative real time PCR assay for cytomegalovirus infection using the 1st WHO International Standard in kidney transplant patients.

INTRODUCTION: Cytomegalovirus (CMV) is one of the most common agents of infection in solid organ transplant patients, with significant morbidity and mortality. OBJECTIVE: This study aimed to establish a threshold for initiation of preemptive treatment. In addition, the study compared the performance of antigenemia with qPCR results. STUDY DESIGN: This was a prospective cohort study conducted in 2017 in a single kidney transplant center in Brazil. Clinical validation was performed by comparing in-house qPCR results, against standard of care at that time (Pp65 CMV Antigenemia). ROC curve analysis was performed to determine the ideal threshold for initiation of preemptive therapy based on the qPCR test results. RESULTS: Two hundred and thirty two samples from 30 patients were tested with both antigenemia and qPCR, from which 163 (70.26%) were concordant (Kappa coefficient: 0.435, p<0.001; Spearman correlation: 0.663). PCR allowed for early diagnoses. The median number of days for the first positive result was 50 (range, 24-105) for antigenemia and 42 (range, 24-74) for qPCR (p<0.001). ROC curve analysis revealed that at a threshold of 3,430 IU/mL (Log 3.54), qPCR had a sensitivity of 97.06% and a specificity of 74.24% (AUC 0.92617 ± 0.0185, p<0.001), in the prediction of 10 cells/105 leukocytes by antigenemia and physician's decision to treat. CONCLUSIONS: CMV Pp65 antigenemia and CMV qPCR showed fair agreement and a moderate correlation in this study. The in-house qPCR was revealed to be an accurate method to determine CMV DNAemia in kidney transplant patients, resulting in positive results weeks before antigenemia.

Cost minimization analysis of an in-house molecular test for cytomegalovirus in relation to a commercial molecular system

ABSTRACT Introduction: Cytomegalovirus may cause severe disease in immunocompromised patients. Nowadays, quantitative polymerase chain reaction is the gold-standard for both diagnosis and monitoring of cytomegalovirus infection. Most of these assays use cytomegalovirus automated molecular kits which are expensive and therefore not an option for small laboratories, particularly in the developing world. Objective: This study aimed to optimize and validate an in-house cytomegalovirus quantitative polymerase chain reaction test calibrated using the World Health Organization Standards, and to perform a cost-minimization analysis, in comparison to a commercial cytomegalovirus quantitative polymerase chain reaction test. Study design: The methodology consisted of determining: optimization, analytical sensitivity, analytical specificity, precision, curve variability analysis, and inter-laboratorial reproducibility. Patients (n = 30) with known results for cytomegalovirus tested with m2000 RealTime System (Abbott Laboratories, BR) were tested with the in-house assay, as well as patients infected with other human herpes virus, in addition to BK virus. A cost-minimization analysis was performed, from a perspective of the laboratory, assuming diagnostic equivalence of the methodologies applied in the study. Results: The in-house assay had a limit of detection and quantification of 60.3 IU/mL, with no cross-reactivity with the other viral agents tested. Moreover, the test was precise and had a R 2 of 0.954 when compared with the m2000 equipment. The cost analysis showed that the assay was economically advantageous costing a median value of 37.8% and 82.2% in comparison to the molecular test in use at the hospital and the m2000 equipment, respectively. Conclusions: These results demonstrated that in-house quantitative polymerase chain reaction testing is an attractive alternative in comparison to automated molecular platforms, being considerably less expensive and as efficacious as the commercial methods.

Cost minimization analysis of an in-house molecular test for cytomegalovirus in relation to a commercial molecular system.

INTRODUCTION: Cytomegalovirus may cause severe disease in immunocompromised patients. Nowadays, quantitative polymerase chain reaction is the gold-standard for both diagnosis and monitoring of cytomegalovirus infection. Most of these assays use cytomegalovirus automated molecular kits which are expensive and therefore not an option for small laboratories, particularly in the developing world. OBJECTIVE: This study aimed to optimize and validate an in-house cytomegalovirus quantitative polymerase chain reaction test calibrated using the World Health Organization Standards, and to perform a cost-minimization analysis, in comparison to a commercial cytomegalovirus quantitative polymerase chain reaction test. STUDY DESIGN: The methodology consisted of determining: optimization, analytical sensitivity, analytical specificity, precision, curve variability analysis, and inter-laboratorial reproducibility. Patients (n=30) with known results for cytomegalovirus tested with m2000 RealTime System (Abbott Laboratories, BR) were tested with the in-house assay, as well as patients infected with other human herpes virus, in addition to BK virus. A cost-minimization analysis was performed, from a perspective of the laboratory, assuming diagnostic equivalence of the methodologies applied in the study. RESULTS: The in-house assay had a limit of detection and quantification of 60.3IU/mL, with no cross-reactivity with the other viral agents tested. Moreover, the test was precise and had a R2 of 0.954 when compared with the m2000 equipment. The cost analysis showed that the assay was economically advantageous costing a median value of 37.8% and 82.2% in comparison to the molecular test in use at the hospital and the m2000 equipment, respectively. CONCLUSIONS: These results demonstrated that in-house quantitative polymerase chain reaction testing is an attractive alternative in comparison to automated molecular platforms, being considerably less expensive and as efficacious as the commercial methods.

Clostridium difficile contamination in retail meat products in Brazil.

INTRODUCTION: Clostridium difficile is an important cause of diarrhoea, particularly in patients receiving antibiotic therapy. Recent studies have shown that a substantial proportion of C. difficile infections are acquired in the community, as a zoonotic disease. Brazil is a large exporter of meat and so far no study has evaluated meat contamination with C. difficile spores. METHODS: Here we analysed 80 retail meat products purchased from local supermarkets in a Brazilian metropolis (Porto Alegre, Southern Brazil). Samples from these products were grown in anaerobic conditions, and tested with a real time polymerase chain reaction test. RESULTS: Contamination with C. difficile spores was not found in the study. Bacteria isolated from meat included Streptococcus gallolyticus, Lactobacillus plantarum, Enterococcus gallinarum and Pediococcus acidilactici. DISCUSSION: Close vigilance is required in order to guarantee the quality of Brazilian retail meat in the long term.

Clostridium difficile contamination in retail meat products in Brazil

ABSTRACT Introduction Clostridium difficile is an important cause of diarrhoea, particularly in patients receiving antibiotic therapy. Recent studies have shown that a substantial proportion of C. difficile infections are acquired in the community, as a zoonotic disease. Brazil is a large exporter of meat and so far no study has evaluated meat contamination with C. difficile spores. Methods Here we analysed 80 retail meat products purchased from local supermarkets in a Brazilian metropolis (Porto Alegre, Southern Brazil). Samples from these products were grown in anaerobic conditions, and tested with a real time polymerase chain reaction test. Results Contamination with C. difficile spores was not found in the study. Bacteria isolated from meat included Streptococcus gallolyticus, Lactobacillus plantarum, Enterococcus gallinarum and Pediococcus acidilactici. Discussion Close vigilance is required in order to guarantee the quality of Brazilian retail meat in the long term.