Estandarización de PCR en tiempo real para detección y cuantificación de los oncogenes E7 de seis Virus del Papiloma Humano de Alto Riesgo

El cáncer de cuello uterino es causado por la infección persistente del epitelio cervical con los genotipos de alto riesgo del Virus del Papiloma Humano. Para su detección se realizan pruebas moleculares que detectan el gen L1 del VPH. Este gen puede perderse hasta en el 11 % de los casos durante la integración del ADN viral en el genoma del hospedero originando falsos negativos. Por otra parte, el oncogén E7 se expresa durante todas las etapas de progresión de la enfermedad. El objetivo de este trabajo fue estandarizar una PCR en tiempo real del oncogén E7 (E7-qPCR) para genotipificación y cuantificación de 6 VPH-AR. Los resultados muestran que la E7- qPCR amplificó VPH-16, -18, -31, -33, -35 y -45 con una alta sensibilidad con límites de detección desde 102 copias, eficiencias entre 90 y 110 %, valores R2 > 0,97 y análisis de curva de fusión que revelan productos específicos.

Cervical cancer is caused by persistent infection of the cervical epithelium with the high-risk genotypes of the Human Papilloma Virus (HR-HPV). For its detection, molecular tests are carried out that detect the L1 gene of HPV. This gene can be lost in up to 11 % of cases during the integration of viral DNA into the host genome, causing false negatives. On the other hand, the E7 oncogene is expressed during all stages of disease progression. The aim of this work was to standardize a real-time PCR of the E7 oncogene (E7-qPCR) for genotyping and quantification of 6 HR-HPV. The results show that the E7-qPCR amplified HPV-16, -18, -31, -33, -35 and -45 with high sensitivity with detection limits from 102 copies, efficiencies between 90 and 110 %, R2 values >0,97 and melting curve analysis revealing specific products.

Validación de la reacción de polimerasa en cadena en tiempo real como herramienta diagnóstica en la aspergilosis asociada a COVID-19 en pacientes hospitalizados en Clínica Dávila de Santiago, Chile, durante los años 2020 y 2021

Introducción: Desde el inicio de la pandemia por COVID-19 se han registrado casos de infecciones de aspergilosis pulmonar asociada a esta infección, la cual tiene características diferentes a la aspergilosis pulmonar clásica y, por lo tanto, han significado un desafío diagnóstico. Objetivo: Validar una reacción de polimerasa en cadena (RPC) en tiempo real (sigla en inglés RT-PCR) comercial, como herramienta diagnóstica alternativa a la técnica de galactomanano (GM) en el diagnóstico de aspergilosis pulmonar asociada a COVID-19 (sigla en inglés CAPA). Pacientes y Método: Se analizaron resultados de RT-PCR de Aspergillus spp y GM en lavado bronco-alveolar (LBA) de 72 pacientes, hospitalizados por COVID-19 de Clínica Dávila entre los años 2020 y 2021. De estos pacientes, 33 presentaron CAPA. Resultados: La RT-PCR de Aspergillus y GM presentaron una correlación positiva (r = 0,6351, valor p < 0,0001). La técnica de RT-PCR presentó una sensibilidad (S), especificidad (E), valor predictor positivo (VPP) y valor predictor negativo (VPN) de 100, 44, 66 y 100%, respectivamente, mientras que en GM fueron de 64, 89, 84 y 73%, respectivamente para LBA. Al utilizar ambas técnicas en combinación se obtuvo una S, E, VPP y VPN de 100, 82, 88 y 100%, respectivamente. Conclusión: Este estudio concluyó que usar una técnica de RT-PCR de Aspergillus y GM en conjunto en LBA mejoraron los parámetros de desempeño de ambas técnicas usadas de manera individual para diagnosticar CAPA. Se requieren más estudios para evaluar el desempeño de técnicas combinadas en otros tipos de aspergilosis.

Background: Since the beginning of the COVID-19 pandemic, there have been cases of pulmonary aspergillosis infections associated with this infection, which has different characteristics from classical pulmonary aspergillosis and therefore, have been diagnostic challenges. Aim: To validate a commercial real-time PCR (RT-PCR) method as an alternative diagnostic tool to the galactomannan (GM) technique in the diagnosis of COVID-19-associated pulmonary aspergillosis (CAPA). Methods: Results of RT-PCR of Aspergillus spp and GM in broncho-alveolar lavage (BAL) of 72 patients hospitalized for COVID-19 at Clínica Dávila between 2020 and 2021 were analyzed. Of these patients, 33 presented CAPA. Results: The RT-PCR for Aspergillus and GM showed a positive correlation (r = 0.6351, p-value < 0.0001). The RT-PCR for Aspergillus technique presented a sensitivity (S), specificity (S), positive predictive value (PPV) and negative predictive value (NPV) of 100, 44, 66 and 100% respectively, while the GM technique presented 64, 89, 84 and 73%, respectively for BAL. Using both techniques in combination a S, E, PPV and NPV of 100, 82, 88 and 100% were obtained respectively. Conclusion: This study concluded that using RT-PCR and GM techniques in combination in BAL improved the performance parameters of both techniques from those used individually to diagnose CAPA. Further studies are required to evaluate the performance of combined techniques in other aspergillosis focus.

The conventional diagnosis challenge: Real-time PCR and nested PCR correlation with the scoring system for individuals at high-risk of Pneumocystis jirovecii pneumonia

Introduction. Pneumocystis jirovecii is an opportunistic fungus that affects mainly people living with HIV (CD4 cell count lower than 200 cells/ml) and other immunosuppressed patients. Since P. jirovecii does not grow on routine mycological media, diagnosis of P. jirovecii pneumonia relies on indirect evidence of its presence in respiratory samples. Objectives. To associate the results of direct immunofluorescence and two molecular methods with a score to predict P. jirovecii pneumonia in patients with AIDS. Materials and methods. A prospective study was conducted with 40 patients. A respiratory sample collected before treatment was subjected to direct immunofluorescence using the Merifluor kit, to nested PCR targeting the mitochondrial large subunit ribosomal RNA, and to the VIASURE real-time PCR kit. Results. These three techniques revealed P. jirovecii in 6, 12, and 15 samples, respectively. All positive samples by direct immunofluorescence were positive by nested PCR, and all positive samples by nested PCR amplified by real-time PCR. There was a statistically significant association between the P. jirovecii pneumonia score and the molecular methods. Two patients were early diagnosed and responded well to treatment. Conclusion. Molecular methods, especially real-time PCR, are recommended for early diagnosis of P. jirovecii pneumonia in AIDS patients.

Introducción. Pneumocystis jirovecii es un hongo oportunista que afecta principalmente a personas con HIV (recuento de CD4 menor de 200 células/ml) y a otros pacientes inmunosuprimidos. Como P. jirovecii no crece en los medios micológicos de rutina, el diagnóstico de neumonía por P. jirovecii se basa en la evidencia presente en muestras respiratorias. Objetivos. Asociar los resultados de la inmunofluorescencia directa y los de dos métodos moleculares con un puntaje para predecir la neumonía causada por P. jirovecii en pacientes con sida. Materiales y métodos. Se realizó un estudio prospectivo de 40 pacientes. Se recolectó una muestra respiratoria antes del inicio de tratamiento y se sometió a una prueba de inmunofluorescencia directa con el kit Merifluor, una PCR anidada para la amplificación de la subunidad larga del ribosoma mitocondrial y una PCR en tiempo real usando el kit VIASURE. Resultados. Estas tres técnicas evidenciaron la presencia de P. jirovecii en 6, 12 y 15 muestras, respectivamente. Todas las muestras positivas por inmunofluorescencia directa fueron positivas en la PCR anidada y todas las muestras positivas en la PCR anidada amplificaron por PCR en tiempo real. Se encontró una asociación estadística entre los valores de la neumonía causada por P. jirovecii y los métodos moleculares. Dos pacientes con diagnóstico temprano respondieron satisfactoriamente al tratamiento. Conclusión. Se recomiendan los métodos moleculares, especialmente la PCR en tiempo real, para el diagnóstico temprano de neumonía causada por P. jirovecii en pacientes con sida.

Hiperplasia pseudoepiteliomatosa: carcinoma escamocelular versus paracoccidioidomicosis oral, un caso con mirada dermatológica / Pseudoepitheliomatous hyperplasia: Squamous cell carcinoma versus oral paracoccidioidomycosis, a case from a dermatological perspective

La paracoccidioidomicosis es una micosis sistémica endémica en Latinoamérica. La presentación más frecuente compromete crónicamente los pulmones, la piel y las mucosas. Al inicio, este paciente presentó, por varios años, una lesión única en la mucosa oral que, en ausencia de otros síntomas, se relacionó con una neoplasia maligna, específicamente con un carcinoma escamocelular. La diferenciación entre los dos diagnósticos se hace mediante un examen directo, un estudio histopatológico y cultivos iniciales y subsecuentes. Sin embargo, tales estudios no fueron concluyentes. Después de varias consultas y pruebas, con los resultados del examen directo, la inmunodifusión y la PCR en tiempo real se confirmó el diagnóstico de paracoccidioidomicosis crónica multifocal. Este caso alerta sobre la ausencia de sospecha clínica de micosis endémicas, dada la presencia de lesiones mucocutaneas que pueden ser producidas por hongos como Paracoccidioides spp, y la importancia de considerarlas entre los diagnósticos diferenciales.

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America. The most frequent form involves a chronic compromise of the lungs, skin, and mucosa. The patient started with a single oral lesion that lasted for several years. The absence of other symptoms pointed out a possible malignant neoplasm, specifically a squamous cell carcinoma. Differentiation between both diagnoses-fungal infection and carcinoma-depends on the results of the direct examination, the histopathological study, and the initial and subsequent cultures. However, in this case, those findings were not conclusive. The coexistence of both diagnoses is frequent and increases the diagnostic challenge. After several consultations and tests, direct examination, immunodiffusion and real-time PCR findings the multifocal chronic paracoccidioidomycosis diagnosis was confirmed. This case warns about a systematical absence of clinical suspicion of endemic mycoses before the appereance of mucocutaneous lesions, which can be produced by fungi like Paracoccidioides spp, and the importance of considering those mycoses among the differential diagnoses.

Comparative evaluation of rapid diagnostic test and real-time PCR in clinically suspected malaria

Background: India represents 3% related to the global malaria problem. Early diagnosis and treatment that are complete alongside preventive measures are modalities essential to managing the situation. Rapid diagnostic tests (RDTs) and polymerase chain reaction (PCR) that are malaria that is real-time be used to obtain an exceedingly really very early diagnosis in acutely febrile customers. Aims and Objectives: This study aims to gauge the effectiveness of RDT bloodstream that is utilized entire from clients clinically suspected of malaria and compare it with real-time PCR. Materials and Methods: The cross-sectional study is observationally done and made up of 158 patients admitted to Index Hospital, Indore, having a serious illness that is febrile and medical suspicion of malaria. RDT for malaria antigen and PCR that are real-time done in the bloodstream that is whole examples depending on kit guidelines. Results: There exists a difference that is significant the nice and examples which are negative by both techniques. RT-PCR is diagnostic PCR that is real-time RDT has been good in 62 (44%) clients, whereas, real-time PCR detected the parasite in 136 (91%) customers. RDT was in reality negative for malaria antigen in 16 (12.8%) consumers, in whom RT-PCR was good. RDT failed to identify Plasmodium falciparum antigen in RT-PCR samples that can be good. RT-PCR indicates basic greater sensitiveness (82.4–95% CI 79.2–84.5%) in diagnosing malaria set alongside the quick test is an antigen. The sensitiveness of RT-PCR in detecting P. falciparum had been also high (74.2%, 95% CI 71.4–77.2). This has greater specificity than RDT in detecting P. falciparum disease (91.3%, 95% CI 89.4–95.4) in detecting P. falciparum than RDT. Conclusion: RT-PCR has better efficacy to look for the presence of malaria parasites in acutely clients being febrile remain undiscovered by RDT. Therefore, it might be helpful for the verification of diagnoses and studies which are epidemiological.

Fetal RHD screening from circulating cell-free DNA in RhD-negative pregnant women / 中华围产医学杂志

Objective:To investigate the RHD genotypes of RhD-negative pregnant women and explore the optimum strategy for fetal RHD screening among this population in the region. Methods:This prospective study recruited 33 cases of RhD-negative singleton pregnancies at ≥12 weeks of gestation in Nanjing Drum Tower Hospital from March to November 2021. On the basis of RHD genotyping, quantitative real-time polymerase chain reaction (PCR) was used to amplify the exons 5 and 10 of RHD gene in the circulating cell-free DNA of RhD-negative pregnant women harboring whole RHD gene deletion and RHD-CE(2-9)- D. High-throughput sequencing was performed to detect chr1:25648453 locus from circulating cell-free DNA in plasma of RhD-negative pregnant women harboring RHD 1227A mutation to screen the fetal RhD blood group. Neonatal umbilical cord blood samples were collected for verifying fetal RHD genotyping. Descriptive statistical analysis was used. Results:Whole RHD gene deletion homozygous genotype ( n=20, 60.6%), RHD-CE(2-9) -D/whole RHD gene deletion heterozygous genotype ( n=5, 21.2%), RHD 1227A/whole RHD gene deletion heterozygous genotype ( n=7, 15.2%) and RHD 711delC/whole RHD gene deletion heterozygous genotype ( n=1) were identified in the 33 RhD-negative pregnant women. In the 25 cases with whole RHD gene deletion homozygous genotype or RHD-CE(2-9)- D/whole RHD gene deletion heterozygous genotype, 22 fetuses were RhD-positive and three were RhD-negative based on prenatal screening, which were confirmed by the neonatal serological test results after birth. In the seven cases carrying RHD 1227A/whole RHD gene deletion heterozygous genotype, all fetuses were RhD-positive, which were consistent with the results of serological detection after delivery. The case harboring RHD 711delC/whole RHD gene deletion heterozygous genotype did not receive fetal RHD screening. Conclusions:This study suggests that whole RHD gene deletion homozygous genotype is the most common allele in RhD-negative population in this area, followed by RHD 1227A/whole RHD gene deletion heterozygous genotype and RHD- CE(2-9)- D/whole RHD gene deletion heterozygous genotype. For women with whole RHD gene deletion homozygous genotype, RHD- CE(2-9)- D, or RHD 1227A mutation, fetal RHD screening with quantitative real-time PCR and high-throughput sequencing are important for the management of RhD-negative pregnant women.

Mycoplasma pneumoniae infection and macrolide resistance of children hospitalized in Ningbo Area， Zhejiang Province in 2019‒2021 / 上海预防医学

ObjectiveTo investigate Mycoplasma pneumoniae （MP） infection and macrolide resistance of hospitalized children in Ningbo Area in 2019‒2021. MethodsA total of 6 782 respiratory throat swab specimens were collected from hospitalized pediatric patients with pneumoniae， admitted in Ningbo Women and Children's Hospital from January， 2019 to December 2021. MP and its mutations in 23S rRNA were detected by real-time polymerase chain reaction. ResultsAmong 6 782 respiratory throat swab specimens from 2019‒2021， 1 290 cases （19.02%） were MP positive， and the positive rate decreased year by year （P<0.05）. The positive rate in 2019 was 28.12%， higher than that in 2020 （7.16%） and 2021 （5.16%） （all P<0.017）. The mutation of 23S rRNA occurred in 947 cases， with a mutation rate of 73.41%. The mutation rate in 2020 was 84.04%， higher than that in 2019 （73.01%） and 2021 （66.23%）. The differences of positive rate and mutation rate in different seasons were significant （P<0.05） （all P<0.008）. The positive rate was the highest in summer （25.00%）， and the mutation rate was the highest in winter （78.89%）. The positive rate of female children was 20.52%， higher than that of male children （17.82%） （P<0.05）， and the mutation rates of female and male children were 74.93% and 71.77% （P>0.05）， respectively. The difference of positive rate among the different age was significant （P<0.05）. The positive rates in the 5‒ and 8‒ years groups were 27.24% and 26.38%， higher than those in the 1‒ and 2‒ years groups， respectively. The difference of mutation rate among the four groups in age was not significant （P>0.05）. ConclusionThe infection rate of MP in children decreases in Ningbo Area from 2019 to 2021. MP infection may be related with gender， seasonal distribution， age， and the resistance rate of MP to macrolide is high.

Factors associated with postoperative signs and symptoms in teeth with periapical lesion: a longitudinal study

Abstract This study aimed to evaluate the association of the variables age, gender, arch position, tooth length, root canal amplitude, and periapical lesion size with the occurrence of postoperative signs and symptoms (pain, tenderness, and edema) and the use of postoperative analgesics following root canal treatment with foraminal enlargement in single-rooted teeth with apical periodontitis. This prospective longitudinal study included 105 patients requiring root canal treatment of maxillary or mandibular single-rooted teeth with periapical lesion. After root canal treatment in a single session, pain intensity and tenderness were recorded daily for 7 days and on days 14 and 30. Edema was evaluated by two independent evaluators within 48 h, 72 h, and 7 days after treatment. Ordinal and logistic regressions were performed (p < 0.05). Female gender (beta = 1.02; p < 0.01), mandibular teeth (beta = 25.50; p < 0.01), medium root canal amplitude (beta = 0.93; p = 0.03), and edema (beta = 1.88; p < 0.01) were associated with increased postoperative pain and tenderness, while the use of analgesics (beta = -1.82; p < 0.01) and time in days (beta = -0.23; p < 0.01) were associated with a decrease in these signs and symptoms. Edema was considered a risk factor for analgesic requirement (Odds Ratio [OR] = 61.46; p < 0.01). Factors such as gender, arch position, and root canal amplitude were associated with postoperative signs and symptoms. The use of analgesics was more required in edema and was associated with decreased pain.

Resumo Este estudo teve como objetivo avaliar a associação das variáveis idade, sexo, posição no arco, comprimento do dente, amplitude do canal radicular e tamanho da lesão periapical com a ocorrência de sinais e sintomas pós-operatórios (dor, dor ao toque e edema) e o uso de analgésicos após o tratamento endodôntico com alargamento foraminal em dentes uniradiculares com lesão periapical. Este estudo longitudinal prospectivo incluiu 105 pacientes que necessitavam de tratamento endodôntico em dentes uniradiculares superiores ou inferiores com lesão periapical. Após o tratamento do canal radicular em uma sessão, a intensidade da dor e a dor ao toque foram registradas diariamente por 7 dias e nos dias 14 e 30. O edema foi avaliado por dois avaliadores independentes dentro de 48 h, 72 h e 7 dias após o tratamento. Foram realizadas regressões ordinal e logística, e a significância estatística foi fixada em um valor de p < 0,05. Gênero feminino (beta = 1,02; p < 0.01), dentes inferiores (beta = 25,50; p < 0.01), amplitude média do canal radicular (beta = 0,93; p = 0,03) e edema (beta = 1,88; p < 0.01) foram associados ao aumento da dor e dor ao toque pós-operatória, enquanto o uso de analgésicos (beta = -1,82; p < 0.01) e o tempo em dias (beta = -0,23; p < 0.01) foram associados à diminuição desses sinais e sintomas. O edema foi considerado fator de risco para necessidade de analgésico (Odds Ratio [OR] = 61,46; p < 0.01). Fatores como sexo, posição do arco e amplitude do canal radicular foram associados aos sinais e sintomas pós-operatórios. O uso de analgésicos foi mais necessário no edema e foi associado à diminuição da dor.

Higher frequency of specific periodontopathogens in hypertensive patients. A pilot study

Abstract Periodontitis and arterial hypertension are two of the pathologies with the highest global prevalence; evidence reported so far has been favorable to an association between them. This cross-sectional study aimed to evaluate and compare the microbiological counts of hypertensive and normotensive patients with periodontitis. Sociodemographic, behavioral, systemic health data and periodontal clinical parameters were assessed. Counts of A. actinomycetemcomitans, P. intermedia, P. gingivalis and F. nucleatum were performed by real-time polymerase chain reaction using subgingival biofilm samples. Thirty-eight patients were included in this preliminary analysis, divided into two groups: Normotensive Group (NG) (n = 14) and Hypertensive Group (HG) (n = 24). Patients diagnosed with periodontitis composed both groups. Data analysis was performed with significance level of 5%. There was no significant difference between groups for clinical periodontitis diagnosis. In addition, hypertensive individuals had higher P. intermedia, P. gingivalis, and F. nucleatum counts when compared to normotensive individuals. The parameters probing pocket depth, bleeding on probing, and A. actinomycetemcomitans count did not presented statistical differences between groups. With these preliminary results, it can be concluded that the presence of arterial hypertension may be associated with a greater quantity of periodontopathogenic bacterial of some species in individuals with periodontitis.

Resumo A periodontite e a hipertensão arterial são duas das patologias com maior prevalência global, as evidências relatadas até o momento têm sido favoráveis ​​a uma associação entre elas. Este estudo transversal teve como objetivo avaliar e comparar contagem microbiológicas de pacientes hipertensos e normotensos com periodontite. Dados sociodemográficos, comportamentais, de saúde sistêmica e parâmetros clínicos periodontais foram avaliados. Contagens de A. actinomycetemcomitans, P. intermedia, P. gingivalis e F. nucleatum foram realizadas por reação em cadeia da polimerase em tempo real utilizando amostras de biofilme subgengival. Trinta e oito pacientes foram incluídos nesta análise preliminar, divididos em dois grupos: Grupo Normotenso (GN) (n = 14) e Grupo Hipertenso (GH) (n = 24). Pacientes diagnosticados com periodontite compuseram os dois grupos. A análise dos dados foi realizada com nível de significância de 5%. Não houve diferença significativa entre os grupos para o diagnóstico clínico de periodontite. Além disso, os hipertensos apresentaram maior contagem de P. intermedia, P. gingivalis e F. nucleatum quando comparados aos normotensos. Os parâmetros profundidade de sondagem, sangramento à sondagem e contagem de A. actinomycetemcomitans não apresentaram diferenças estatísticas entre os grupos. Com esses resultados preliminares, pode-se concluir que a presença de hipertensão arterial pode estar associada a uma maior quantidade de bactérias periodontopatogênicas de algumas espécies em indivíduos com periodontite.

Zika virus RNA detection in blood donors in São Paulo, Brazil

ABSTRACT Introduction: The Zika Virus (ZIKV) is a single-stranded RNA genome virus, belonging to the family Flaviviridae, genus Flavivirus. Outbreaks around the world have demonstrated that the presence of asymptomatic viremic blood donors provides an increase in the risk of transfusion transmission (TT) and nucleic acid test (NAT) screening has been proposed to ensure the blood safety. This study implemented an "in-house" method to detect ZIKV RNA in blood sample donations. Methods: Primary plasma tubes are submitted to nucleic acid extraction on an automated platform. After extraction, the NAT set-up is performed in the robotic pipettor, in which an amplification mixture containing primers and probes for ZIKV and Polio vaccine virus (PV) are added in duplex as an internal control. The real-time polymerase chain reaction is then performed in a thermocycler, using the protocol established by the supplier. Results: From May 2016 to May 2018, 3,369 samples were collected from 3,221 blood donors (confidence coefficient 95%), of which 31 were considered false positive (0.92%), as they did not confirm initial reactivity when repeated in duplicates and 14 (0.42%) had their results invalid due to repeat failure in the internal control, 4 (0.12%), due to insufficient sample volume and 2 (0.05%), due to automatic pipettor failures. No Zika RNA reactive sample was identified. Conclusion: The test showed feasible to be incorporated into the blood screening routine. Our data do not indicate the need to screen for ZIKV RNA in São Paulo during the evaluated period. However, a generic NAT system covering a group of flaviviruses which are circulating in the region, such as DENV and YFV, among others, could be a useful tool.

Respiratory pathogens in children with acute lower respiratory infection detected through multiplex real-time PCR / Patógenos respiratorios en niños con infección respiratoria aguda baja detectados mediante PCR multiplex en tiempo real

Abstract Introduction: Acute respiratory infection in children has a high burden of disease. Detection of multiple micro -organisms through molecular testing of nasopharyngeal swab samples could change the paradigm of a single pathogen being the cause of respiratory disease in children and prove its usefulness in clinical practice. Objective: To characterize the pathogens identified in nasopharyngeal swab samples by means of multiplex realtime polymerase chain reaction (RT-PCR), as well as clinical variables and laboratory findings in children <5 years diagnosed with acute lower respiratory tract infection (ALRTI) and hospitalized in Bogotá D.C., Colombia. Materials and methods: Cross-sectional study conducted in 81 children hospitalized between September 2019 and March 2020 at the Clínica Cafam and in whom nasopharyngeal swab samples were collected for microbiological identification using the Allplex™ multiplex RT-PCR assay. Correlations between the number of pathogens and blood cells and C-reactive protein levels were determined by Spearman's rank correlation coefficient. Results: Patients' mean age was 17.23 months (±14.44), 54.32% were males, and 51.85% were young infants. A total of 149 microorganisms (60.40% viruses) were identified in 63 children (77.78%). Mixed infection and coinfection were reported in 48.15% and 11.11% of children, respectively. Regarding clinical findings, shortness of breath, upper airway obstruction, cough, fever and pharyngitis were the most common clinical signs and/or symptoms in patients with mixed infection (32.97%), coinfection (64.40%), mixed infection (29.78%), and absence of microorganism (22.00%), respectively. A negative correlation was observed between the number of leukocytes and the number of neutrophils and the number of microorganisms detected in the preschoolers group (r=-0.46; p =0.058 and r=-0.51; p =0.033, respectively). Furthermore, a positive correlation was found between monocyte count and the number of microorganisms detected (r=0.53; p =0.0096). Conclusion: Multiplex RT-PCR assay allowed the identification of microorganisms in most children, as well as cases of mixed infection and coinfection in more than half of the sample. In addition, clinical findings in these children were highly heterogeneous as per the assay result..

Resumen Introducción. La infección respiratoria aguda en niños tiene una alta carga de enfermedad. La detección de múltiples microorganismos a través de pruebas moleculares en hisopados nasales podría cambiar el paradigma de patógeno único causal de enfermedad respiratoria en niños y ser de utilidad en la práctica clínica. Objetivo. Caracterizar los patógenos identificados mediante la técnica de reacción en cadena de polimerasa multiplex en tiempo real (RT-PCR) en hisopado nasal, así como las variables clínicas y los resultados de laboratorio en niños <5 años diagnosticados con infección respiratoria aguda baja (IRAB) y hospitalizados en Bogotá D.C., Colombia. Materiales y métodos. Estudio transversal realizado en 81 niños hospitalizados entre septiembre de 2019 y marzo de 2020 en la clínica Cafam y en quienes se hizo hisopado nasal para realizar la identificación microbiològica mediante la prueba RT-PCR multiplex Allplex. Las correlaciones entre el número de patógenos y los niveles de células del hemograma y el nivel de proteína C reactiva se determinaron mediante el coeficiente de correlación de Spearman. Resultados. La edad promedio fue 17.23 meses (±14.44), 54.32% fueron varones y 51.85%, lactantes menores. Se identificaron 149 microorganismos (60.40% virus) en 63 niños (77.78%). Hubo infección mixta en el 48.15% y coinfección en 11.11% de los niños. Respecto a los hallazgos clínicos, la dificultad respiratoria, la obstrucción de la vía respiratoria alta, la tos, la fiebre y la faringitis fueron más comunes en los casos de infección mixta (32.97%), ausencia de microorganismo (16.00%), coinfección (64.40%), infección mixta (29.78%) y ausencia de microorganismo (22.00%), respectivamente. Se observó una correlación negativa entre el número de leucocitos y neutrófilos y el número de microorganismos detectados en preescolares (r=-0.46; p=0.058 y r=-0.51; p=0.033) y una positiva entre el recuento de monocitos y el número de microorganismos detectados (r=0.53; p =0.0096). Conclusión. La prueba RT-PCR multiplex permitió identificar microorganismos en la mayoría de niños, así como casos de infección mixta y coinfección en más de la mitad de la muestra. Además, los hallazgos clínicos fueron altamente heterogéneos entre los niños según el resultado de la prueba.

Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos / Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method

RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

Surgical Approach and Management Strategies in a Pediatric Cardiovascular Surgery Clinic During the COVID-19 Outbreak

ABSTRACT Introduction: The coronavirus disease 2019 (COVID-19) pandemic has required changes in the management of pediatric cardiac surgery. We would like to share the patient treatment and surgical management strategies employed in our Pediatric Cardiovascular Surgery Clinic during the COVID-19 pandemic. Methods: A total of 112 patients were followed up in our clinic between 11.03.2020 and 02.07.2020. Their mean age was 1,118 (4-5,740) days. Management and treatment were performed by our pediatric heart team (pediatric cardiac anesthetists, general pediatricians, pediatric cardiologists, pediatric cardiac surgeons, and an infectious diseases specialist). We prepared new protocols and a surveillance system specific to the pandemic to prevent in-hospital transmission and reduce postoperative mortality and morbidity; our operations were performed according to these protocols. All decisions pertaining to the operation timing and treatment strategy of our COVID-19-positive patients were made by the same team. Results: During the study period, a total of 112 patients, 69 boys and 43 girls, were hospitalized in our clinic. A total of 333 COVID-19 real-time polymerase chain reaction tests were performed on patients and accompanying persons; positive results were found in three patients and two accompanying individuals. Conclusion: By employing new protocols and a surveillance system throughout the healthcare system, we think that early diagnosis and treatment of the pediatric congenital heart disease population, which is susceptible to infections, can continue unperturbed. This and similar approaches can increase postoperative success and prevent transmission in the pediatric population - which are frequently COVID-19 asymptomatic.

Expression Levels of the CA9, WT1, and PRAME genes and genotyping-associated antigens for the diagnosis and prognosis of colorectal cancer

Background: Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide, and is one of the major health problems in Asia, Africa, Europe, and America. The tumor antigens recently are of interesting indicators as diagnostic and prognostic tools, The aim of the present study is to detect the expression levels of carbonic anhydrase IX (CA9), the Wilms tumor gene (WT1), and the preferentially expressed antigen in melanoma (PRAME) in the peripheral blood of CRC patients in comparison with healthy controls. Methods: A prospective case-control study of CRC patients was conducted. We included 25 newly-diagnosed CRC eligible patients and obtained peripheral blood samples of them as well as 10 blood samples from the control group. All samples were then submitted to deoxyribonucleic acid (DNA) extraction and a molecular study through real-time polymerase chain reaction (PCR). Results: The CRC group consisted of 15 (60%) female and 10 (40%) male patients with a mean age of 50.52 ± 9.8 years, while the control group included 4 (40%) female and 6 (60%) male patients with a mean age of 47.7 ± 7.9 years. The CRC group, 24 (96%) of patient samples were CA9-positive with strong statistically significant differences (p < 0.00001; sensitivity: 96%; specificity: 90%). Regarding the WT1 gene, there were 11 (44%) positive samples in the CRC group, with no statistically significant differences (p = 0.055; sensitivity: 44%; specificity: 90%). The PRAME gene was positive in 9 (36%) samples in the CRC group, with no statistically significant differences (p = 0.357; sensitivity: 36%; specificity: 80%. Among CA9 (24 patients; 96%) of patients with CRC expressed positive results, in WT1 11(91.6%) CRC patients expressed gene, and in PRAME gene, 9 patients with CRC (81.8%) expressed positive results. Conclusion: Overexpression of the CA9 gene in CRC of high sensitivity and specificity to be used as a tool to discriminate CRC from benign associate with high accuracy compare to WT1 and PRAME genes. (AU)

Results of a sample-to-cutoff ratio using Abbott Architect rHTLV-I/II assay allow to predict detection of HTLV-1 and HTLV-2 proviral DNA by real-time PCR / Resultados da razão sinal da amostra sobre o cutoff da reação no ensaio Abbott Architect rHTLV-I/II permite prever a detecção de DNA proviral por PCR em tempo real

The present study aims to correlate the sample-to-cutoff ratios (S/CO) distributions of reactive results for HTLV-1/2 antibodies with the detection of proviral DNA in a population of blood donor candidates. It was carried out a retrospective data search of 632 HTLV-1/2 reactive samples, submitted to confirmatory testing from January 2015 to December 2019. Serological screening was performed by chemiluminescent microparticle immunoassay Architect rHTLV-I/II, whereas confirmatory testing was performed by in-house real-time polymerase chain reaction method. 496 out of 632 samples (78%) had undetectable HTLV-1/2 proviral DNA and 136 (22%) had detectable proviral DNA. HTLV infection was not confirmed in any individual for whom the S/CO ratio value was <4, and proviral DNA detection rates gradually escalated as S/CO ratio values increased. The sensitivity and predictive positive value found for the Architect rHTLV-I/II was 100% and 22%, respectively. The receiver operating characteristic (ROC) curve analysis showed that the optimal S/CO ratio value for predicting the presence of HTLV-1/2 was 18.11. High S/CO ratios were more associated with the detection of proviral DNA. The S/CO ratio value <4 suggests excluding true HTLV infection and the risk of blood transmission (AU).

O estudo tem como objetivo correlacionar às distribuições das razões sample-to-cutoff (S/CO) de resultados reagentes para anticorpos HTLV-1/2 com a detecção de DNA proviral em uma população de candidatos à doação de sangue. Realizou-se uma busca retrospectiva de dados de 632 amostras reagentes para HTLV-1/2 submetidas à testagem confirmatória entre janeiro de 2015 a dezembro de 2019. A triagem sorológica foi realizada pelo imunoensaio quimioluminescente de micropartículas Architect rHTLV-I/II, enquanto o teste confirmatório foi realizado pelo método de PCR em tempo real in-house. 496 de 632 amostras (78%) apresentaram DNA proviral indetectável e 136 (22%) apresentaram DNA proviral detectável. A infecção por HTLV não foi confirmada em nenhum indivíduo com valor de S/CO <4 e as taxas de detecção de DNA proviral escalonaram gradualmente à medida que as razões S/CO aumentaram. A sensibilidade e valor preditivo positivo encontrados para o Architect rHTLV-I/II foram 100% e 22%, respectivamente. Utilizando análise de curva ROC, o valor de razão S/CO ideal para predizer a presença de DNA proviral foi de 18,11. Razões S/CO elevadas foram mais associadas à detecção de DNA proviral. Em suma, o valor de S/CO <4 sugere a exclusão de infecção por HTLV e o risco de transmissão pelo sangue (AU).

Experiencia en el diagnóstico de tuberculosis meníngea en un hospital de alta complejidad en Colombia / The experience of a tertiary care hospital in Colombia in the diagnosis of meningeal tuberculosis

Resumen Introducción: la tuberculosis es una de las enfermedades infecciosas de mayor distribución mundial y la tuberculosis meníngea es una de sus manifestaciones más devastadoras. Su diagnóstico y confirmación microbiológica no siempre es fácil. Objetivo: describir la experiencia en el diagnóstico de tuberculosis meníngea por pruebas moleculares comparado con cultivo, caracterizando las principales manifestaciones clínicas y determinar los factores asociados a mortalidad. Métodos: identificamos retrospectivamente a los pacientes adultos con diagnóstico de tuberculosis meníngea, mediante técnicas de pruebas moleculares y/o cultivo para M. tuberculosis, que ingresaron en nuestra institución entre enero de 2018 y marzo de 2020, se realizó un análisis estadístico descriptivo. Se excluyeron mujeres gestantes, pacientes que no contaran con prueba molecular para M. tuberculosis. Resultados: se obtuvo una muestra de 33 pacientes, los hallazgos más relevantes en el citoquímico de líquido cefalorraquídeo (LCR) fue la presencia de hipoglucorraquia, con una mediana de 34.2 mg/dL (RIQ 2.0-95.0 mg/dL) y de hiperproteinorraquia, con mediana de 265 mg/dL (RIQ 24.0-600 mg/dL). El resultado más significativo fue la presencia de proteína C reactiva elevada en suero en todos los casos, con una mediana de 53.3 mg/L (RIQ 22.9-89.6 mg/L) y neutrofilia en 75.8% (25). La mortalidad fue de 54.5% (18), la sensibilidad de la prueba molecular en LCR fue del 38.46% y el valor predictivo positivo de 58.82%. Conclusiones: el diagnóstico de TB meníngea sigue siendo todo un reto, aunque las pruebas moleculares pueden ayudar en el diagnóstico temprano, su sensibilidad es baja en formas extrapul-monares. (Acta Med Colomb 2022; 47. DOI:https://doi.org/10.36104/amc.2022.2115).

Abstract Introduction: tuberculosis is one of the most widely disseminated infectious diseases worldwide, and meningeal tuberculosis is one of its most devastating manifestations. Its diagnosis and microbiological confirmation is not always easy. Objective: to describe the experience in diagnosing meningeal tuberculosis through molecular tests compared to a culture, characterize the main clinical manifestations, and determine factors associated with mortality. Methods: we retrospectively identified adult patients diagnosed with meningeal tuberculosis through molecular and/or culture tests for M. tuberculosis who were admitted to our institution between January 2018 and March 2020. A descriptive analysis was performed. Pregnant women and patients who did not have a molecular test for M. tuberculosis were excluded. Results: a sample of 33 patients was obtained. The most relevant cerebrospinal fluid (CSF) cytochemical analysis findings were low glucose, with a median of 34.2 mg/dL (IQR 2.0-95.0 mg/ dL) and high protein, with a median of 265 mg/dL (IQR 24.0-600 mg/dL). The most significant result was elevated serum C-reactive protein in all cases, with a median of 53.3 mg/L (IQR 22.9 -89.6 mg/L) and neutrophilia in 75.8% (25). Mortality was 54.5% (18), the sensitivity of the CSF molecular test was 38.46% and the positive predictive value was 58.82%. Conclusions: the diagnosis of meningeal TB continues to be a challenge. While molecular tests can help provide an early diagnosis, their sensitivity is low in extrapulmonary forms. (Acta Med Colomb 2022; 47. DOI:https://doi.org/10.36104/amc.2022.2115).

Ultrasensitive molecular tests for Plasmodium detection: applicability in control and elimination programs and reference laboratories / Pruebas moleculares ultrasensibles para la detección de Plasmodium: aplicabilidad en los programas de control y eliminación y en los laboratorios de referencia / Testes moleculares ultra-sensíveis para detecção de Plasmodium: aplicabilidade em programas de controlo e eliminação e laboratórios de referencia

ABSTRACT Objective. To evaluate molecular tools to detect low-level parasitemia and the five species of Plasmodium that infect humans for use in control and elimination programs, and in reference laboratories. Methods. We evaluated 145 blood samples from patients who tested positive by nested polymerase chain reaction (nPCR), from asymptomatic individuals and from the WHO Global Malaria Programme/United Kingdom National External Quality Assessment Service. Samples were assayed using the genus-specific RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) and the RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). The results from the molecular tests were compared with those from quantitative PCR (qPCR), nPCR and thick blood smear. Results. The levels of parasitemia ranged from 1 to 518 000 parasites/µL, depending on the species. Compared with nPCR, alt-S&T had a sensitivity of 100%, except for identifying P. falciparum, for which the sensitivity was 93.94%. All samples positive by alt-Gen were also positive by nPCR. When comparing alt-Gen to qPCR, the sensitivity was 100% for P. vivax, P. malariae and P. falciparum. For all Plasmodium species, the correlation between cycle threshold values of alt-S&T and alt-Gen compared with qPCR was significant (P < 0.0001, Spearman's test), with r = 0.8621 for alt-S&T and r = 0.9371 for alt-Gen. When all Plasmodium species were considered, there was a negative correlation between the level of parasitemia and real-time PCR cycle threshold values (P < 0.0001). In this study, only 2 of 28 samples from asymptomatic individuals were positive by thick blood smear; however, all 28 of these samples were positive by alt-S&T. Conclusions. The alt-Gen and alt-S&T assays are suitable for detecting submicroscopic infections for distinct epidemiological purposes, such as for use in surveys and reference laboratories, and screening in blood banks, which will contribute to global efforts to eliminate malaria.

RESUMEN Objetivo. Evaluar herramientas moleculares para detectar bajos niveles de parasitemia y las cinco especies de Plasmodium que infectan a los seres humanos, a fin de emplearlas en los programas de control y eliminación y en los laboratorios de referencia. Métodos. Se evaluaron 145 muestras de sangre de pacientes positivos por reacción en cadena de la polimerasa anidada (nPCR), de individuos asintomáticos y de muestras del Programa Mundial de Malaria de la Organización Mundial de la Salud/Servicio Nacional de Evaluación Externa de Calidad del Reino Unido. Las muestras se analizaron con el kit de PCR RealStar® Malaria 1.0 (alt-Gen; altona Diagnostics), específico para cada género, y con el kit de PCR RealStar® Malaria Screen & Type (alt-S&T; altona Diagnostics). Se compararon los resultados de las pruebas moleculares con los de la PCR cuantitativa (qPCR), la nPCR y el frotis de gota gruesa. Resultados. Los niveles de parasitemia oscilaron entre 1 y 518 000 parásitos/µl, según la especie. En comparación con la nPCR, la prueba alt-S&T tuvo una sensibilidad del 100%, excepto para la identificación de P. falciparum, para el cual la sensibilidad fue del 93,94%. Todas las muestras positivas por alt-Gen lo fueron también por nPCR. Al comparar alt-Gen con la qPCR, la sensibilidad fue del 100% para P. vivax, P. malariae y P. falciparum. Para todas las especies de Plasmodium, la correlación entre los valores del umbral de ciclo de alt-S&T y alt-Gen en comparación con la qPCR fue significativa (P < 0,0001, prueba de Spearman), con r = 0,8621 para alt-S&T y r = 0,9371 para alt-Gen. Cuando se consideraron todas las especies de Plasmodium hubo una correlación negativa entre el nivel de parasitemia y los valores de umbral de ciclo de PCR en tiempo real (P < 0,0001). En este estudio, solo 2 de las 28 muestras de individuos asintomáticos fueron positivas por frotis de gota gruesa; sin embargo, las 28 muestras fueron positivas por alt-S&T. Conclusiones. Los ensayos alt-Gen y alt-S&T son adecuados para detectar infecciones submicroscópicas con distintos fines epidemiológicos, como su uso en investigaciones y laboratorios de referencia y el cribado en bancos de sangre, lo que contribuirá a los esfuerzos mundiales para eliminar la malaria.

RESUMO Objectivo. Avaliar ferramentas moleculares para detectar parasitemia de baixo nível e as cinco espécies de Plasmodium que infectam humanos, para utilização em programas de controlo e eliminação e em laboratórios de referência. Métodos. Avaliámos 145 amostras de sangue de doentes que testaram positivo por reacção em cadeia da polimerase aninhada (nPCR), de indivíduos assintomáticos, e do Programa Global de Paludismo da Organização Mundial de Saúde/Serviço Nacional de Avaliação da Qualidade Externa do Reino Unido. As amostras foram ensaiadas utilizando o RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) e o RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). Os resultados dos testes moleculares foram comparados com os resultados da PCR quantitativa (qPCR), nPCR e exame da gota espessa. Resultados. Os níveis de parasitemia variaram de 1 a 518 000 parasitas/µL, dependendo da espécie. Em comparação com a nPCR, alt-S&T tinha uma sensibilidade de 100%, excepto na identificação de P. falciparum, para a qual a sensibilidade era de 93,94%. Todas as amostras positivas por alt-Gen foram também positivas por nPCR. Ao comparar alt-Gen com qPCR, a sensibilidade foi de 100% para P. vivax, P. malariae e P. falciparum. Para todas as espécies Plasmodium, a correlação entre os valores limiares de ciclo de alt-S&T e alt-Gen comparados com qPCR foi significativa (P < 0,0001, teste de Spearman), com r = 0,8621 para alt-S&T e r = 0,9371 para alt-Gen. Quando todas as espécies de Plasmodium foram consideradas, houve uma correlação negativa entre o nível de parasitemia e os valores limiares do ciclo de PCR em tempo real (P < 0,0001). Neste estudo, apenas 2 de 28 amostras de indivíduos assintomáticos foram positivas por exame da gota espessa; no entanto, todas estas 28 amostras foram positivas por alt-S&T. Conclusões. Os ensaios alt-Gen e alt-S&T são adequados para a detecção de infecções submicroscópicas para fins epidemiológicos distintos, tais como para utilização em inquéritos e laboratórios de referência e o rastreio em bancos de sangue, o que contribuirá para os esforços globais de eliminação da malária.

ROLE OF REAL TIME PCR (RT-PCR) IN CLINICAL DIAGNOSIS FOR COVID-19 REVIEW

The pandemic of Coronavirus Disease 2019 (COVID19) has compelled scientists to create highly reliable diagnostic tools quickly in order to successfully and properly diagnose this pathology and thereby prevent infection transmission. Even though structural and molecular properties of the severe acute respiratory syndrome coronavirus 2 (SARSCoV2) were previously unknown, private research institutes and biomedical firms quickly developed numerous diagnostic procedures beneficial for making a correct detection of COVID19. Rapid antigen or antibody testing, immunoenzymatic serological tests, and RT-PCR based molecular assays are the most frequently used and validated procedures now available. The PCR has grown in popularity in molecular diagnostics to the point where it is still considered the gold standard for finding nucleotides from a variety of sources becoming an indispensable tool in the research lab. Because of its improved speed, sensitivity, reproducibility, and lower likelihood of carry-over contamination, real-time PCR has gained greater popularity. Currently, five different chemistries are employed to detect PCR product during real-time PCR. The self- fluorescing amplicons, DNA binding fluorophores, 5? endonuclease, neighbouring linear and hairpin oligoprobes, and self-fluorescing amplicons are all detailed in depth. We also go through the problems that have hampered the development of multiplex real-time PCR and the importance of real-time PCR in nucleic acid quantification.

Evaluación del desempeño clínico del ensayo SUMASIGNAL VHC para la detección del ARN del virus de la hepatitis C

RESUMEN Introducción: La detección y cuantificación del genoma del virus de la hepatitis C (ARN-VHC), mediante la transcripción inversa-PCR en tiempo real (RT-qPCR), es vital para el diagnóstico y seguimiento del tratamiento antiviral de los pacientes con hepatitis C. Objetivo: Evaluar los indicadores de desempeño clínico como la especificidad, sensibilidad y reproducibilidad del ensayo SUMASIGNAL VHC, comercializado por el Centro de Inmunoensayo (Cuba), tomando como técnica de referencia la prueba Artus® HCV RG RT-qPCR. Métodos : Se empleó un panel conformado por 70 muestras de suero: 46 ARN-VHC positivas, 12 ARN-VHC negativas y 12 positivas a marcadores moleculares de otros virus. El coeficiente de correlación de Pearson (r) y la prueba de Bland-Altman, se utilizaron para comparar las cargas virales del VHC, obtenidas por las técnicas SUMASIGNAL VHC y la de referencia; las que fueron expresadas en logaritmo en base 10 (log10) UI/mL. Los valores de p< 0,05 se consideraron estadísticamente significativos. Resultados: La sensibilidad y especificidad clínica del SUMASIGNAL VHC fue 100 %; mientras que no se detectó reactividad cruzada con los otros virus evaluados. Se demostró que el ensayo en cuestión, tuvo una correlación buena (r= 0,89) y fuerte concordancia (media= -0,01 Log10 UI/mL) con la prueba de referencia (p< 0,0001). La reproducibilidad de la cuantificación del ARN-VHC en dos momentos diferentes, con la prueba SUMASIGNAL VHC mostró una correlación excelente (r= 0,97; p< 0,0001). Conclusiones: El ensayo SUMASIGNAL VHC proporciona datos válidos, reproducibles y técnicamente confiables para realizar el diagnóstico específico del VHC, incluso constituye una herramienta útil para monitorear la carga viral de este agente en el Sistema Nacional de Salud Cubano.

ABSTRACT Introduction: Detection and quantification of the hepatitis C virus genome (HCV RNA) by real time reverse transcription PCR (RT-qPCR) are vital for the diagnosis and antiviral treatment follow-up of hepatitis C patients. Objective: Evaluate the clinical performance of the SUMASIGNAL HCV assay for quantification of HCV viral load. Methods: A panel was formed with 70 serum samples: 46 HCV RNA positive, 12 HCV RNA negative and 12 positive for molecular markers of other viruses. Pearson's correlation coefficient (r) and the Bland-Altman plot were used to compare the HCV viral loads obtained by SUMASIGNAL HCV techniques with reference values, which were expressed as base 10 logarithm (log10) UI/ml. Values of p <0.05 were considered statistically significant. Results: The clinical sensitivity and specificity of SUMASIGNAL HVC were 100%, and no cross-reactivity was detected with the other viruses evaluated. The study assay exhibited a good correlation (r= 0.89) and strong concordance (mean= -0.01 Log10 UI/ml) with the reference test (p< 0.0001). Reproducibility of the HCV RNA quantification with the SUMASIGNAL HCV test at two different moments displayed an excellent correlation (r= 0.97; p< 0.0001). Conclusions: The SUMASIGNAL HVC assay provides valid, reproducible and technically reliable data for specific HCV diagnosis. It is also a useful tool to monitor the viral load of this agent in the Cuban National Health System.

Soluble ST2 as a Potential Biomarker in Pericardial Fluid of Coronary Artery Patients

Abstract Introduction: The growth Stimulation expressed gene 2 (ST2) (or interleukin 1 receptor-like 1, also known as IL1RL1) is considered a biomarker of poor prognosis in cardiovascular diseases. The aims of this study are to investigate ST2 in the pericardial fluid (PF) of coronary artery disease patients and to contribute to the understanding of the pathophysiology of coronary artery disease. Methods: 40 patients (blood plasma and PF) who underwent coronary artery bypass surgery and 40 controls (blood plasma only) were included in this study. Soluble ST2 (sST2) level was determined by enzyme-linked ımmunosorbent assay method in plasma and PF, and sST2 gene expression was determined by quantitative real-time polymerase chain reaction (QRT-PCR) method. Results: The sST2 level was found to be 44.89 ng/ml and 390.357 ng/ml in the control and patient groups' plasma, and 223.992 ng/ml in the PF of the patient group. An increase in sST2 level was detected in the patient group compared to the control group (P<0.001). The sST2 expression in plasma was higher in the patient group than in the control group. Additionally, sST2 was more expressed in the plasma of the patient group than PF (P<0.001). Conclusion: The fact that sST2 was detected for the first time in a high level in PF showed that this biomarker was closely related with the heart and strengthened its potential to be used as a biomarker. Therefore, sST2 can contribute to the understanding of the pathophysiology of coronary artery disease.