Detección y cuantificación de VHB y VHC en muestras de plasma mediante la utilización de diferentes ensayos moleculares: estudio comparativo / Detection and quantification of HBV and HCV in plasma samples by means of different molecular assays: Comparative study

Introducción: La carga viral es un marcador muy útil para realizar el seguimiento de los pacientes infectados por VHB y VHC. Este trabajo compara ensayos basados en amplificación mediada por transcripción y en PCR a tiempo real con el objetivo de comprobar si pueden ser intercambiables. Material y métodos: Estudio bicéntrico en el que se analizó la carga viral de 147 muestras de plasma de pacientes infectados por VHB y 229 por VHC, mediante ensayos basados en amplificación mediada por transcripción (Aptima® HBV Quant y Aptima® HCV Quant Dx, que utilizan el sistema Panther (Hologic®)) y PCR a tiempo real (COBAS® AmpliPrep / COBAS® TaqMan® y COBAS® 6800), calculando el grado de concordancia entre ellos. Resultados: Se detectó carga viral en ambos equipos en 60 (40,82%) muestras de VHB (mediana del log de la carga viral: COBAS®: 2,51UI/mL (RIC 2,20-3,17), Panther: 2,71UI/mL (RIC 2,21-3,22)) y en 39 (16,96%) muestras de VHC (mediana del log de la carga viral: COBAS®: 3,93UI/mL (RIC 2,24-6,01), Panther: 3,80UI/mL (RIC 1,99-6,14)). La concordancia entre ambos equipos fue de κ=0,943 para VHB y κ=0,925 para VHC. La comparación de las muestras con carga viral detectada mediante los 2 ensayos mostró una correlación alta tanto para VHB (R2=0,86) como para VHC (R2=0,97). Conclusiones: Los ensayos basados tanto en amplificación mediada por transcripción como en PCR a tiempo real pueden ser intercambiables para el manejo de pacientes infectados con VHB y VHC.(AU)

Introduction: Viral load is a very useful marker for monitoring patients infected with HBV and HCV. This work compares assays based on transcription-mediated amplification and on real-time PCR to verify whether they can be interchangeable. Material and methods: a bicentric study, in which 147 plasma samples from patients infected with HBV and 229 with HCV were analyzed, was carried out. Transcription-mediated amplification-based assays (Aptima® HBV Quant and Aptima® HCV Quant Dx, employing Panther system (Hologic®)) and on real-time PCR (COBAS® AmpliPrep / COBAS® TaqMan® and COBAS® 6800) were used and the degree of concordance between them was calculated. Results: Viral load was detected in both systems in 60 (40.82%) HBV samples (median log viral load: COBAS®: 2.51IU/mL (IQR 2.20-3.17), Panther: 2.71IU/mL (IQR 2.21-3.22)) and in 39 (16.96%) HCV samples (median log viral load: COBAS®: 3.93IU/mL (IQR 2.24-6.01), Panther: 3.80IU/mL (IQR 1.99-6.14)). The agreement between both systems was κ=0.943 for HBV and κ=0.925 for HCV. Comparison of viral load samples detected by both assays showed a hight correlation for HBV (R2=0.86) and for HCV (R2=0.97). Conclusions: Both transcription-mediated amplification and on real-time PCR based assays may be interchangeable for the management of patients infected with HBV and HCV.(AU)

Detection and quantification of HBV and HCV in plasma samples by means of different molecular assays: Comparative study.

INTRODUCTION: Viral load is a very useful marker for monitoring patients infected with HBV and HCV. This work compares assays based on transcription-mediated amplification (TMA) and on real-time PCR (RT-PCR) to verify whether they can be interchangeable. MATERIAL AND METHODS: A bicentric study, in which 147 plasma samples from patients infected with HBV and 229 with HCV were analyzed, was carried out. TMA-based assays (Aptima® HBV Quant and Aptima® HCV Quant Dx, employing Panther system (Hologic®)) and RT-PCR (COBAS® AmpliPrep/COBAS® TaqMan® and COBAS® 6800) were used and the degree of concordance between them was calculated. RESULTS: Viral load was detected in both systems in 60 (40.82%) HBV samples (median log viral load: COBAS: 2.51IU/mL (IQR 2.20-3.17), Panther: 2.71IU/mL (IQR 2.21-3.22)) and in 39 (16.96%) HCV samples (median log viral load: COBAS: 3.93IU/mL (IQR 2.24-6.01), Panther: 3.80IU/mL (IQR 1.99-6.14)). The agreement between both systems was κ=0.943 for HBV and κ=0.925 for HCV. Comparison of viral load samples detected by both assays showed a hight correlation for HBV (R2=0.86) and for HCV (R2=0.97). CONCLUSIONS: Both TMA and RT-PCR based assays may be interchangeable for the management of patients infected with HBV and HCV.

Commercial real time PCR implementation for rapid diagnosis of onychomycosis: A new workflow in a clinical laboratory.

INTRODUCTION: Onychomycosis is a frequent and underdiagnosed condition. Approximately 90% of toenail onychomycosis infections are caused by dermatophytes, but classical diagnosis based on culture and microscopy observation is slow and has low sensitivity. Both limitations can be solved incorporating molecular techniques to routine diagnosis of onychomycosis. OBJECTIVE: Prospective evaluation of the utility of incorporating in the clinical laboratory workflow a commercial real time PCR (qPCR) for dermatophytes detection in nails after potassium hydroxide direct observation screening. MATERIALS AND METHODS: 152 nail samples were included (34 KOH negative and 118 KOH positive) and processed by culture and qPCR. RESULTS: In the negative KOH group, only one dermatophyte grew in culture and three were detected by qPCR. In the group of positive KOH, 57 dermatophytes grew in culture and 81 were detected by qPCR. In this group, 25% of diagnosed dermatophytes were detected only by qPCR. The sensitivity of qPCR compared to culture is 92.8% and time of response decreases from days to hours. CONCLUSION: Based in our results, we propose a workflow algorithm for a clinical laboratory that eliminates culture for qPCR positive samples.

PCR a tiempo real comercial para diagnóstico rápido de las onicomicosis: un nuevo algoritmo de trabajo en el laboratorio clínico / Commercial real time PCR implementation for rapid diagnosis of onychomycosis: A new workflow in a clinical laboratory

Introduction: Onychomycosis is a frequent and underdiagnosed condition. Approximately 90% of toenail onychomycosis infections are caused by dermatophytes, but classical diagnosis based on culture and microscopy observation is slow and has low sensitivity. Both limitations can be solved incorporating molecular techniques to routine diagnosis of onychomycosis. Objective: Prospective evaluation of the utility of incorporating in the clinical laboratory workflow a commercial real time PCR (qPCR) for dermatophytes detection in nails after potassium hydroxide direct observation screening. Materials and methods: 152 nail samples were included (34 KOH negative and 118 KOH positive) and processed by culture and qPCR. Results: In the negative KOH group, only one dermatophyte grew in culture and three were detected by qPCR. In the group of positive KOH, 57 dermatophytes grew in culture and 81 were detected by qPCR. In this group, 25% of diagnosed dermatophytes were detected only by qPCR. The sensitivity of qPCR compared to culture is 92.8% and time of response decreases from days to hours. Conclusion: Based in our results, we propose a workflow algorithm for a clinical laboratory that eliminates culture for qPCR positive samples.(AU)

Introducción: La onicomicosis es una patología frecuentemente infradiagnosticada. Aproximadamente el 90% de las infecciones en las uñas del pie están causadas por dermatofitos, pero el diagnóstico microbiológico clásico basado en cultivo y microscopia es lento y tiene una baja sensibilidad. Ambas limitaciones pueden resolverse incorporando técnicas moleculares al diagnóstico de la onicomicosis. Objetivo: Evaluación prospectiva de la utilidad de incorporar en un laboratorio clínico una PCR a tiempo real (qPCR) comercial para detección de dermatofitos en uñas tras cribado por examen directo con hidróxido de potasio (KOH). Materiales y métodos: Se incluyeron 152 muestras de uñas (34 KOH negativas y 118 KOH positivas) y se procesaron mediante cultivo y qPCR. Resultados: En el grupo KOH negativo, solo un dermatofito creció en cultivo y 3 se detectaron mediante qPCR. En el grupo KOH positivo, 57 dermatofitos crecieron en cultivo y 81 se detectaron por qPCR. En este grupo, el 25% de los dermatofitos diagnosticados se detectaron únicamente mediante qPCR. La sensibilidad de la qPCR comparada con el cultivo es del 92,8% y el tiempo de respuesta disminuye de días a horas. Conclusión: En base a nuestros resultados, proponemos un algoritmo de flujo de trabajo para los laboratorios de microbiología clínica, que elimina el cultivo para aquellas muestras con qPCR positiva.(AU)

Evaluation of VIASURE Sexually Transmitted Diseases Real Time PCR Detection kit (CerTest Biotec) for the diagnostic of sexually transmitted infections / Evaluación de VIASURE® Sexually Transmitted Diseases Real-Time PCR Detection kit (CerTest Biotec) para el diagnóstico de las infecciones de transmisión sexual

Introduction: Sexually transmitted infections (STIs) are common in our environment, and trends have been increasing in the last few years. Different methods for STIs diagnosis have been applied by microbiology laboratories over years, but real-time PCR has improved this process. Our objective was to evaluate VIASURE Sexually Transmitted Diseases Real-Time PCR Detection kit (CerTestBiotec, S.L.) comparing with the real-time PCR technique used in our laboratory (Allplex™ STI7 Essential Assay, Seegene) which was considered as reference assay. Methods: A total of 948 samples from different sites (vaginal, endocervical, urethral, rectal, pharyngeal swabs and urine samples) were analyzed from July to September 2018. Results: A discordant result was obtained in 4.5% (43 samples). These discrepancies were mainly observed in threshold cycle (Ct) value next to the limit of detection. The k coefficient obtained shows a very high agreement between both methods with k values from 0.92 to 0.99. Conclusions: VIASURE Sexually Transmitted Diseases Real-Time PCR Detection kit provides a very good correlation with Allplex STI7 and therefore, it's a good tool for the diagnostic of STIs. Positive results with Ct value obtained from 35 and low amplification signal should be applied with caution and should be interpreted based on the patient's clinical data.(AU)

Introducción: Las infecciones de transmisión sexual (ITS) son frecuentes en nuestro entorno y con tendencia a aumentar. Las técnicas moleculares han mejorado su diagnóstico aportando sensibilidad y rapidez de resultados. Nuestro objetivo ha sido evaluar el nuevo kit de PCR a tiempo real VIASURE® Sexually Transmitted Diseases (CerTest Biotec, SL) comparándolo con la técnica de PCR a tiempo real empleada en nuestro laboratorio (AllplexTM STI7 Essential Assay, Seegene). Métodos: Se analizaron prospectivamente un total de 948 muestras de diferente localización (exudado vaginal, endocervical, uretral, rectal, faríngeo y orina) recibidas en nuestro laboratorio desde julio hasta septiembre de 2018. Resultados: En el 4,5% (43 muestras) se obtuvo un resultado discordante entre ambas técnicas. Estas discrepancias se observaron principalmente en ciclos próximos al límite de detección. El valor del coeficiente k osciló entre 0,92 a 0,99, mostrando una muy buena correlación entre técnicas. Conclusiones: VIASURE® Sexually Transmitted Diseases Real-Time PCR Detection kit es una buena herramienta para el diagnóstico de las ITS. Muestras con señal de amplificación en ciclos a partir de 35 y baja señal de fluorescencia, deben ser tratados con precaución e interpretarse en función de los datos clínicos del paciente.(AU)

Evaluation of VIASURE Sexually Transmitted Diseases Real Time PCR Detection kit (CerTest Biotec) for the diagnostic of sexually transmitted infections.

INTRODUCTION: Sexually transmitted infections (STIs) are common in our environment, and trends have been increasing in the last few years. Different methods for STIs diagnosis have been applied by microbiology laboratories over years, but real-time PCR has improved this process. Our objective was to evaluate VIASURE Sexually Transmitted Diseases Real-Time PCR Detection kit (CerTestBiotec, S.L.) comparing with the real-time PCR technique used in our laboratory (Allplex™ STI7 Essential Assay, Seegene) which was considered as reference assay. METHODS: A total of 948 samples from different sites (vaginal, endocervical, urethral, rectal, pharyngeal swabs and urine samples) were analyzed from July to September 2018. RESULTS: A discordant result was obtained in 4.5% (43 samples). These discrepancies were mainly observed in threshold cycle (Ct) value next to the limit of detection. The k coefficient obtained shows a very high agreement between both methods with k values from 0.92 to 0.99. CONCLUSIONS: VIASURE Sexually Transmitted Diseases Real-Time PCR Detection kit provides a very good correlation with Allplex STI7 and therefore, it's a good tool for the diagnostic of STIs. Positive results with Ct value obtained from 35 and low amplification signal should be applied with caution and should be interpreted based on the patient's clinical data.

The diagnostic accuracy of three rapid diagnostic tests for typhoid fever at Chittagong Medical College Hospital, Chittagong, Bangladesh.

OBJECTIVE: To determine the diagnostic accuracy of three rapid diagnostic tests (RDTs) for typhoid fever in febrile hospitalised patients in Bangladesh. METHODS: Febrile adults and children admitted to Chittagong Medical College Hospital, Bangladesh, were investigated with Bact/Alert(®) blood cultures and real-time PCR to detect Salmonella enterica Typhi and Paratyphi A and assays for Rickettsia, leptospirosis and dengue fever. Acute serum samples were examined with the LifeAssay (LA) Test-it™ Typhoid IgM lateral flow assay detecting IgM antibodies against S. Typhi O antigen, CTKBiotech Onsite Typhoid IgG/IgM Combo Rapid-test cassette lateral flow assay detecting IgG and IgM antibodies against S. Typhi O and H antigens and SD Bioline line assay for IgG and IgM antibodies against S. Typhi proteins. RESULTS: In 300 malaria smear-negative febrile patients [median (IQR) age of 13.5 (5-31) years], 34 (11.3%) had confirmed typhoid fever: 19 positive by blood culture for S. Typhi (three blood PCR positive) and 15 blood culture negative but PCR positive for S. Typhi in blood. The respective sensitivity and specificity of the three RDTs in patients using a composite reference standard of blood culture and/or PCR-confirmed typhoid fever were 59% and 61% for LifeAssay, 59% and 74% for the CTK IgM and/or IgG, and 24% and 96% for the SD Bioline RDT IgM and/or IgG. The LifeAssay RDT had a sensitivity of 63% and a specificity of 91% when modified with a positive cut-off of ≥2+ and analysed using a Bayesian latent class model. CONCLUSIONS: These typhoid RDTs demonstrated moderate diagnostic accuracies, and better tests are needed.

Detection of low-intensity Schistosoma mansoni infection by Percoll sedimentation and real-time PCR techniques in a low-endemicity Egyptian setting.

OBJECTIVE: To evaluate the performance of Percoll sedimentation and real-time polymerase chain reaction (PCR) for the detection of S. mansoni cases previously tested as negative by Kato-Katz technique in two low-endemic areas in Alexandria, Egypt, Abis 4 and 8 villages. METHODS: Stool samples of 824 primary schoolchildren were examined by Kato-Katz technique (three slides of 41.7 mg each). After obtaining the results of this survey, stool samples were recollected from a subset of 150 students, who gave negative results after Kato-Katz. These samples were microscopically examined after the concentration with Percoll technique. Part of the 150 negative stool samples and five positive samples (used as controls) were kept at -20 °C and further processed by SYBR Green PCR. RESULTS: Prevalence of S. mansoni infection as determined by three Kato-Katz thick smears was 1.82% (15 cases). Three more cases tested positive by Percoll sedimentation among the 150 samples that were negative by Kato-Katz. Specific amplification by SYBR Green PCR was noted in all positive controls and in three cases of Kato-Katz-negative samples, two of which were also positive by Percoll. CONCLUSION: Percoll sedimentation and SYBR Green PCR proved useful in detecting low-intensity S. mansoni infections in low-endemicity areas in Egypt.

Detection of carbapenemase-producing Enterobacteriaceae in various scenarios and health settings.

Detection of carbapenemase-producing Enterobacteriaceae (CPE) is an important task at microbiology laboratories in hospitals. As the prevalence of CPE is increasing worldwide, the implementation of phenotypically based screening as well as confirmatory procedures to detect CPE are important for microbiologists. In addition to detection of carbapenem hydrolysis, the inhibition of activity against a carbapenem in the presence of several inhibitor compounds specific to class A, B, or class C beta-lactamases is a useful method to confirm the presence of carbapenemases in bacterial isolates. There is also a proteomic approach that compares the MALDI-TOF spectrum generated by the intact carbapenem (non-hydrolyzed) with that obtained after hydrolysis of the beta-lactam ring by beta-lactamase to reveal the presence of carbapenemases in bacterial isolates. Proteomic methods will probably be more frequently implemented in laboratories in the near future. Finally, molecular methods to directly or indirectly detect the presence of a carbapenemase genes are increasingly being used in microbiology laboratories. One of the main advantages of these methods is their speed, and also that they can be used directly with the clinical sample without the need for an isolated bacterial colony. Multiplex PCR, real-time PCR, DNA microarrays and pyrosequencing are some examples of molecular-based tests. Their main disadvantage is their cost, although prices are going down as the range of services increases. Surveillance of carriers is also an important task for infection control purposes. In this case, commercially available chromogenic medium supplemented with low carbapenem concentrations has shown an excellent ability to detect CPE. Moreover, molecular methods to detect specific carbapenemase genes directly from rectal swabs, stools, or other colonization sources have had excellent results.

Schistosoma real-time PCR as diagnostic tool for international travellers and migrants.

OBJECTIVE: To evaluate the use of a genus-specific PCR that combines high sensitivity with the detection of different Schistosoma species for diagnosis in international travellers and migrants in comparison to standard microscopy. METHODS AND RESULTS: The genus-specific real-time PCR was developed to target the 28S ribosomal RNA gene of the major human Schistosoma species. It was validated for analytical specificity and reproducibility and demonstrated an analytical sensitivity of 0.2 eggs per gram of faeces. Its diagnostic performance was further evaluated on 152 faecal, 32 urine and 38 serum samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine in Antwerp (Belgium). We detected Schistosoma DNA in 76 faecal (50.0%) and five urine (15.6%) samples of which, respectively, nine and one were not detected by standard microscopy. Only two of the 38 serum samples of patients with confirmed schistosomiasis were positive with the presently developed PCR. Sequence analysis on positive faecal samples allowed identification of the Schistosoma species complex. CONCLUSION: The real-time PCR is highly sensitive and may offer added value in diagnosing imported schistosomiasis. The genus-specific PCR can detect all schistosome species that are infectious to humans and performs very well with faeces and urine, but not in serum.