Valoración de un método de dilución para la resolución de resultados no valorables en la detección de Chlamydia trachomatis y Neisseria gonorrhoeae con la plataforma cobas 4800 / Evaluation of a dilution method for non-evaluable results in the detection of Chlamydia trachomatis and Neisseria gonorrhoeae with the Cobas 4800 platform

Introducción: Un porcentaje variable de muestras analizadas por el equipo cobas 4800 pueden dar un resultado invalidado por inhibición de la PCR o erróneo al no extraerse el ADN correctamente con el test cobas 4800 CT/NG. Método: Valoración de un protocolo de agitación y dilución de la muestra original (exudado u orina) en un total de 116 muestras. Para analizar la sensibilidad de este método, 100 muestras (exudados y orinas) con resultado conocido fueron retestadas. Resultados: Un 98,3% (114/116) de las muestras se resolvieron con este protocolo con un 100% de concordancia al consultar con datos clínicos, tinción de Gram y otras muestras analizadas en paralelo del mismo paciente. Discusión: Los datos indican que no hay pérdida de sensibilidad con este protocolo, por lo que los usuarios de esta plataforma podrían usarlo sin necesidad de métodos alternativos (AU)

Introduction: A variable percentage of samples analysed using the Cobas 4800 assay can give an invalid result by PCR inhibition or erroneous due to incorrect DNA extraction with the Cobas 4800 CT/NG test. Method: An analysis was performed using the vortex agitation and dilution protocol on the original sample (swab or urine) for a total of 116 samples. In order to analyse the sensitivity of this method, 100 samples (swabs and urine) with known results were retested. Results: A total of 98.3% (114/116) of the samples analysed were resolved with this protocol with 100% agreement after reviewing clinical data, Gram stain, and other samples analysed in parallel from the same patient. Discussion: The data indicate no loss of sensitivity with this protocol; thus Cobas 4800 users could use this method without the need for alternative methods (AU)

Evaluation of a dilution method for non-evaluable results in the detection of Chlamydia trachomatis and Neisseria gonorrhoeae with the Cobas 4800 platform. / Valoración de un método de dilución para la resolución de resultados no valorables en la detección de Chlamydia trachomatis y Neisseria gonorrhoeae con la plataforma cobas 4800.

INTRODUCTION: A variable percentage of samples analysed using the Cobas 4800 assay can give an invalid result by PCR inhibition or erroneous due to incorrect DNA extraction with the Cobas 4800 CT/NG test. METHOD: An analysis was performed using the vortex agitation and dilution protocol on the original sample (swab or urine) for a total of 116 samples. In order to analyse the sensitivity of this method, 100 samples (swabs and urine) with known results were retested. RESULTS: A total of 98.3% (114/116) of the samples analysed were resolved with this protocol with 100% agreement after reviewing clinical data, Gram stain, and other samples analysed in parallel from the same patient. DISCUSSION: The data indicate no loss of sensitivity with this protocol; thus Cobas 4800 users could use this method without the need for alternative methods.

Clinical and epidemiological characterisation of lymphogranuloma venereum in southwest Spain, 2013-2015.

OBJECTIVES: Lymphogranuloma venereum (LGV) infections caused by Chlamydia trachomatis L serovars have emerged in 2003 in Europe among HIV-positive men having sex with men (MSM). Our aim was to evaluate LGV prevalence and predictors in a high-risk population attending to two STI clinics in the southwest of Spain between December 2013 and April 2015. METHODS: Screening of C. trachomatis using commercial kits was carried out, followed by real-time pmpH-PCR discriminating LGV strains, and finally ompA gene was sequenced for phylogenetic reconstruction. RESULTS: A total of 6398 samples were tested, of which, 594 (9.3%) were C. trachomatis-positive specimens and successfully typed by pmpH PCR. Five hundred and eighty-one samples contained non-LGV and 13 (2.2%; 95% CI 1.3% to 3.7%) samples had LGV. One hundred and sixty-six (27.9%; 95% CI 24.5% to 31.7%) CT-positive results were found in MSM. All C. trachomatis LGV types were found in rectal samples from MSM (13/166, 7.8%; 95% CI 4.5% to 13.0%). Of these, five (38.5%; 95% CI 17.7% to 64.5%) patients were asymptomatic and 11 (84.6%; 95% CI 57.8% to 95.7%; p<0.001) were also HIV positive. Successful treatment of LGV was achieved in all patients including 11/13 (84.6%) who received single-dose azithromycin. All of the L types were confirmed to be genotype L2b with ompA PCR and sequencing. CONCLUSIONS: This analysis shows that LGV infections are occurring in MSM in southwest Spain, where no data about LGV have been described before, reinforcing the need for screening and genotyping for LGV. LGV should be taken into account when considering treatment and management of rectal C. trachomatis infections, including in asymptomatic HIV-positive MSM. Larger studies on appropriate treatment for asymptomatic LGV infection are needed.

Performance of the HSV OligoGen kit for the diagnosis of herpes simplex virus type 1 and 2.

PCR methods are nowadays between the most rapid and sensitive methods for screening and diagnosing herpes simplex virus (HSV) type 1 and 2. The aim of this study was to analyze the reliability, accuracy, and usefulness of the new assay HSV OligoGen kit in comparison with the Roche LightCycler HSV ½ Qual Kit assay for the detection of HSV in clinical samples. For this analysis, a prospective study was designed for detection of HSV-1 and HSV-2 including 110 ulcer specimens, 48 urine, 48 endocervical, 43 cerebral spinal fluids, 4 urethral and 3 pharyngeal swabs that were sent from a regional STI clinic or an Intensive Clinical Unit, both in Seville, Spain. In comparison to the Roche LightCycler HSV ½ Qual Kit assay, sensitivity, specificity, positive and negative predicative values, and kappa value for HSV detection using the HSV OligoGen kit were 96.2%, 100%, 100%, 98.3%, and 0.97 for HSV-1, respectively. For HSV-2, the corresponding values were 98.3%, 100%, 100%, 99.5%, and 0.98, respectively. Statistical data obtained in this study confirms the usefulness and reliable results of this new assay.

Comparison of the CT OligoGen kit with cobas 4800 assay for detection of Chlamydia trachomatis / Comparación del kit CT OligoGen con el ensayo Cobas 4800 para la detección de Chlamydia trachomatis

INTRODUCTION: A prospective study was designed to assess the performance of the new CT OligoGen kit and the cobas 4800 assay for detection of Chlamydia trachomatis. METHODS: A set of samples that included urine samples (n = 212), endocervical (n = 167), rectal (n = 53), pharyngeal (n = 7) and urethral swabs (n = 3). The samples were sent from a regional sexually transmitted diseases (STD) clinic in Seville, Spain, and were collected from 261 men and 181 women. Discordant results were re-analyzed and clinical data and other tests were reviewed in order to resolve them. RESULTS: Sensitivity, specificity, positive predicative value (PPV), negative predictive value (NPV) and kappa value for C. trachomatis detection using the CT OligoGen kit were 98.5%, 100%, 100%, 95.4% and 0.97, respectively. Conclusions This new kit had a high sensitivity, specificity, PPV and NPV for C. trachomatis, therefore the performance profile confirms the usefulness and reliable results of this new assay

INTRODUCCIÓN: Se diseñó un estudio prospectivo para evaluar las características del nuevo kit CT OligoGen en comparación con el test cobas 4800 para la detección de Chlamydia trachomatis. MÉTODOS: Se analizaron una serie de muestras que incluían orinas (n = 212), exudados endocervicales (n = 167), rectales (n = 53), faríngeos (n = 7) y uretrales (n = 3). Estas muestras provenían de un centro de infecciones de transmisión sexual (Sevilla) y pertenecían a 261 hombres y 181 mujeres. Los resultados discordantes se reanalizaron y revisaron historias clínicas y otras pruebas para resolverlas. RESULTADOS: Los valores de sensibilidad, especificidad, valor predictivo positivo (VPP) y negativo (VPN) y valor kappa para el kit CT OligoGen fue 98,5%, 100%, 100%, 95,4% and 0,97, respectivamente. CONCLUSIONES: Este nuevo kit tuvo una alta sensibilidad, especificidad, VPP y VPN para la detección de C. trachomatis, por lo que esta evaluación confirma su utilidad y fiabilidad

Comparison of the CT OligoGen kit with cobas 4800 assay for detection of Chlamydia trachomatis.

INTRODUCTION: A prospective study was designed to assess the performance of the new CT OligoGen kit and the cobas 4800 assay for detection of Chlamydia trachomatis. METHODS: A set of samples that included urine samples (n=212), endocervical (n=167), rectal (n=53), pharyngeal (n=7) and urethral swabs (n=3). The samples were sent from a regional sexually transmitted diseases (STD) clinic in Seville, Spain, and were collected from 261 men and 181 women. Discordant results were re-analyzed and clinical data and other tests were reviewed in order to resolve them. RESULTS: Sensitivity, specificity, positive predicative value (PPV), negative predictive value (NPV) and kappa value for C. trachomatis detection using the CT OligoGen kit were 98.5%, 100%, 100%, 95.4% and 0.97, respectively. CONCLUSIONS: This new kit had a high sensitivity, specificity, PPV and NPV for C. trachomatis, therefore the performance profile confirms the usefulness and reliable results of this new assay.

[Detection of methicillin resistance and identification of Staphylococcus spp. from positive blood culture bottles using the mecA and nucA genes with the LightCycler System]. / Detección de la resistencia a meticilina e identificación de Staphylococcus spp. en hemocultivos positivos amplificando los genes mecA y nucA con el sistema LightCycler.

INTRODUCTION: The aim of this study was to evaluate the feasibility of detecting Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and of identifying methicillin resistance directly in positive BACTEC blood culture bottles using the LightCycler system. METHODS: One hundred thirty-one positive blood culture bottles in which Gram-positive cocci in cluster were observed after Gram staining and 40 positive bottles with microorganisms other than staphylococci were studied. A molecular assay based on an automated DNA extraction protocol with a MagNA Pure LC instrument was used. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both a 408-pb fragment within the mecA gene and a 279-pb fragment within the S. aureus-specific nucA gene. RESULTS: All the bottles that yielded methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA) or methicillin-resistant CoNS (MRCoNS) strains were correctly identified by the nucA and mecA PCR assays. One bottle that yielded a mixed culture of MSSA and MRCoNS gave positive results for both genes. In the 21 bottles with methicillin-susceptible CoNS (MSCoNS), nucA PCR were negative, but two of these bottles gave positive results for the mecA gene. The sensitivity and specificity of the nucA gene assay were 100%. The sensitivity and specificity of the PCR assay for detection of methicillin resistance with the mecA gene were 100% and 97.5%, respectively. CONCLUSION: This is a sensitive and highly specific method for identifying staphylococci in positive blood cultures, allowing discrimination between methicillin-susceptible and -resistant strains in less than 3 hours after Gram stain.

Detección de la resistencia a meticilina e identificación de Staphylococcus spp. en hemocultivos positivos amplificando los genes mecA y nucA con el sistema Light Cycler / Detection of methicillin resistance and identification of Staphylococcus spp. from positive blood culture bottles using the mecA and nucA genes with the LightCycler System

INTRODUCCIÓN. El objetivo de este trabajo fue evaluarla eficacia del sistema LightCycler para la detección de Staphylococcus aureus y estafilococos coagulasa negativos(ECN), y la identificación de resistencia a meticilina directamente en botellas Bactec de hemocultivos positivos. MÉTODOS. Se procesaron 131 botellas de hemocultivos positivos en los que se observaron cocos grampositivos en racimos tras la tinción de Gram y 40 botellas con microorganismos diferentes a estafilococos. Para la extracción del ADN se utilizó el sistema automático MagNAPure LC (Roche Diagnostic). Los cebadores y las sondas dehibridación se diseñaron para la amplificación y detección de las secuencias específicas de un fragmento de 408 pb del gen mecA y de un fragmento de 279 pb del gen nucA. RESULTADOS. Todas las botellas con cepas de S. aureusresistente a meticilina (SARM), sensible a meticilina(SASM) o ECN resistentes a meticilina (ECNRM) fueron correctamente identificados mediante reacción en cadena de la polimerasa (PCR) de los genes nucA y mecA. Una botella con un cultivo mixto de SASM y ECNRM dio resultados positivos para ambos genes. Las 21 botellas con cepas de ECN sensible a meticilina (ECNSM) fueron correctamente identificadas con el gen nucA, pero dos de ellas fueron positivas para el gen mecA. La sensibilidad y la especificidad fueron del 100% para el gen nucA, y del 100 y 97,5%, respectivamente, para el gen mecA. CONCLUSIÓN. Este es un método sensible y específico para la identificación de estafilococos en hemocultivos, y permite también la identificación de cepas resistentes a meticilina en un tiempo máximo de 3 h a partir de la visualización de la tinción de Gram (AU)

INTRODUCTION. The aim of this study was to evaluate the feasibility of detecting Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and of identifying methicillin resistance directly in positive BACTEC blood culture bottles using the LightCycler system. METHODS. One hundred thirty-one positive blood culture bottles in which Gram-positive cocci in cluster were observed after Gram staining and 40 positive bottles with microorganisms other than staphylococci were studied. A molecular assay based on an automated DNA extraction protocol with a MagNA Pure LC instrument was used. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both a 408-pb fragment within the mecA gene and a 279-pb fragment within the S. aureus-specific nucA gene. RESULTS. All the bottles that yielded methicillin-resistant S.aureus (MRSA), methicillin-sensitive S. aureus (MSSA) ormethicillin-resistant CoNS (MRCoNS) strains were correctly identified by the nucA and mecA PCR assays. One bottle that yielded a mixed culture of MSSA and MRCoNSgave positive results for both genes. In the 21 bottles with methicillin-susceptible CoNS (MSCoNS), nucA PCR were negative, but two of these bottles gave positive results forthe mecA gene. The sensitivity and specificity of the nucA gene assay were 100%. The sensitivity and specificity of the PCR assay for detection of methicillin resistance with the mecA gene were 100% and 97.5%, respectively. CONCLUSION. This is a sensitive and highly specific method for identifying staphylococci in positive blood cultures, allowing discrimination between methicillin-susceptible and –resistant strains in less than 3 hours after Gramstain (AU)

[Genotypic resistance of human immunodeficiency virus type 1 in patients with therapeutic failure]. / Resistencia genotípica del virus de la inmunodeficiencia humana tipo 1 en pacientes con fracaso terapéutico.

BACKGROUND AND OBJECTIVE: The aim of the present study is to analyse both the frequency of resistances to the different antiretrovirals and the implication of the mayor associated resistance mutations in a group of patients with therapeutic failure. PATIENTS AND METHOD: He sequence of protease (pr) and retrotranscriptase (rt) of HIV-1 isolated from blood were analysed in 284 patients. The viral load level was between 500 and 106 copies/ml. The genotype was made with TRUGENETM HIV-1 kit, which is based in the synthesis of cDNA by RT-PCR and sequenciation of this cDNA by PCR. RESULTS: 39,78% of the patients have not associated resistance mutations in the antiretrovirales used in the treatment, including 73 patients who have not associated resistance mutations to antiretrovirales. 60,2% of the patients have associated resistance mutations in the antiretrovirales used in the treatment. In the patients multitreated, 88,2% presented resistance to some and 53% presented genotypic resistance or possible resistance to some of the TARGA treatment. CONCLUSIONS: The prevalence of resistance mutations in patients with therapeutic failure is bigger than 50% with both genes.

Resistencia genotípica del virus de la inmunodeficiencia humana tipo 1 en pacientes con fracaso terapéutico / Genotypic resistance of human immunodeficiency virus type 1 in patients with therapeutic failure

FUNDAMENTO Y OBJETIVO: El objetivo del presente estudio es analizar tanto la frecuencia y secuencia de aparición de resistencias a los distintos tipos de antirretrovirales como la implicación en éstas de las principales mutaciones asociadas a resistencia en un grupo de pacientes infectados por el virus de la inmunodeficiencia humana tipo 1 en los que el tratamiento antirretroviral parece haber fracasado. PACIENTES Y MÉTODO: Se examinaron las secuencias de la proteasa y de la retrotranscriptasa del virus de la inmunodeficiencia humana tipo 1 aislado de muestras de plasma sanguíneo en un total de 284 pacientes. La carga viral de las muestras debía estar comprendida entre 500 y 106 copias/ml. La genotipificación se realizó con el sistema TrugeneTM HIV-1, que se basa en la síntesis de ADNc mediante reacción en cadena de la polimerasa previa transcripción inversa y posterior secuenciación de este ADNc mediante reacción en cadena de la polimerasa. RESULTADOS: El 39,78 por ciento de los pacientes estaban siendo tratados eficazmente, es decir, no tenían mutaciones que confiriesen resistencia a ninguno de los antirretrovirales usados en el tratamiento, incluidos 73 pacientes que no presentaban ninguna mutación relacionada con resistencia genotípica a algún tipo de antirretroviral, mientras que el 60,2 por ciento de los pacientes presentaba mutaciones que conferían resistencia a alguno de los antirretrovirales del tratamiento. En los pacientes en seguimiento, el 88,2 por ciento presentó resistencia a algún antirretroviral y el 53 por ciento presentó resistencia genotípica o posible resistencia a algún fármaco del tratamiento antirretroviral de gran actividad que recibía. CONCLUSIONES: La prevalencia de mutaciones de resistencia en pacientes con fracaso terapéutico es mayor del 50 por ciento con ambos genes (AU)