Etiología de las infecciones de transmisión sexual (ITS) diagnosticadas por la técnica de PCR múltiple-hibridación en población colombiana de la ciudad de Medellín atendida en el Laboratorio Clínico VID / Etiology of sexually transmitted infections (STIs) diagnosed by multiplex PCR-hibridization assay technique in a Colombian population attending the VID Clinical Laboratory in Medellin

Introducción. Las infecciones de transmisión sexual (ITS) son y seguirán siendo un serio problema de salud pública en todo el mundo según los datos de la OMS, con el agravante que la mayoría de los casos son asintomáticos y, además, no existe otro reservorio distinto al humano. El diagnóstico se puede realizar con pruebas tradicionales y moleculares, estas últimas incluyen la reacción en cadena de la polimerasa (PCR), de las cuales existen varios tipos, entre ellas, la PCR múltiple que tiene la capacidad de detectar ITS polimicrobianas a partir de una sola muestra. El objetivo de este estudio fue establecer cuáles fueron las infecciones de transmisión sexual más frecuentes en diferentes grupos de pacientes, así como determinar la utilidad del uso de la técnica de PCR múltiple en el diagnóstico de las ITS. Metodología. Se trata de un estudio observacional de corte transversal realizado entre los años 2021 y 2022 con pacientes que acudieron al servicio de diagnóstico del Laboratorio Clínico VID por sospecha de ITS. Las muestras recolectadas fueron evaluadas utilizando una prueba comercial basada en la técnica de PCR múltiple e hibridación. Las muestras procesadas fueron: orina e hisopados de endocérvix, uretra, recto, faringe y úlceras. Resultados. Se estudiaron 1.027 pacientes, de estos, 228 (22,2 %) fueron positivos para diferentes agentes de trasmisión sexual, distribuidos así: 50 (21,9 %) mujeres, 129 (56,6 %) hombres heterosexuales y 49 (21,5 %) hombres que tenían sexo con hombres (HSH). La edad promedio de las mujeres fue 30 años, y la de ambos grupos de hombres fue 36 años. Los microorganismos más frecuentemente identificados en mujeres fueron: C. trachomatis (A-K) en 28,6 %, seguido de virus herpes simplex tipo 2 (VHS-2) en 26,8 % y N. gonorrhoeae en 17,9 %. En hombres heterosexuales fueron C. trachomatis (A-K) en 37,5 %, N. gonorrhoeae en 21,5 % y VHS-2 en 18,7 %. En HSH fueron C. trachomatis (L1-L3) en 32,7 %, seguido de N. gonorrhoeae en 27,6 %, y de C. trachomatis (A-K) y VHS-2, ambos en 13,8 %. En 11 hombres heterosexuales, 8 HSH y en 6 mujeres, se identificó infección polimicrobiana. Conclusiones. C. trachomatis (A-K) fue el microorganismo más prevalente causante de ITS, seguido de N. gonorrhoeae en ambos grupos de hombres, y de VHS-2 en las mujeres, muy similar a lo reportado a nivel mundial. La prueba de PCR múltiple permite la detección de infecciones polimicrobianas comúnmente asociadas a ITS y el diagnóstico es preciso y confiable, incluso en pacientes asintomáticos

Sexually transmitted infections (STIs) are and will continue to be a serious public health problem throughout the world according to WHO data, with the aggravating factor that most cases are asymptomatic and, furthermore, there is no other reservoir other than humans. The diagnosis can be made with traditional and molecular tests, the latter include the polymerase chain reaction (PCR), of which there are several types, among them, multiplex PCR that has the capacity to detect polymicrobial STIs from a single sample. The objective of this study was to establish which were the most frequent sexually transmitted infections in different groups of patients, as well as to determine the usefulness of the multiplex PCR technique in the diagnosis of STIs. Methodology. This is an observational, cross-sectional study carried out between 2021 and 2022 with patients who attended the VID Clinical Laboratory for suspected STIs. The collected samples were evaluated using a commercial test based on the multiplex PCR technique and hybridization. The samples processed were: urine and swabs from endocervix, urethra, rectum, pharynx, and ulcers. Results. The study included 1,027 patients, of these, 228 (22.2%) were positive for different sexually transmitted agents, distributed as follows: 50 (21.9%) women, 129 (56.6%) heterosexual men and 49 (21.5%) men who had sex with men (MSM). The average age of the women was 30 years, and that of both groups of men was 36 years. The microorganisms most frequently identified in women were: C. trachomatis (A-K) in 28.6%, followed by herpes simplex virus type 2 (HSV-2) in 26.8% and N. gonorrhoeae in 17.9%. In heterosexual men they were C. trachomatis (A-K) in 37.5%, N. gonorrhoeae in 21.5% and HSV-2 in 18.7%. In MSM they were C. trachomatis (L1-L3) in 32.7%, followed by N. gonorrhoeae in 27.6%, and C. trachomatis (A-K) and HSV-2, both in 13.8%. Polymicrobial infection was identified in 11 heterosexual men, 8 MSM, and 6 women. Conclusions. C. trachomatis (A-K) was the most prevalent STI-causing microorganism, followed by N. gonorrhoeae in both groups of men, and HSV-2 in women, very similar to that reported worldwide. The multiplex PCR test allows the detection of polymicrobial infections commonly associated with STIs and the diagnosis is accurate and reliable, even in asymptomatic patients

Current Status and Progress of Diagnostic Techniques for Cancer of Unknown Primary / 肿瘤防治研究

Cancer of unknown primary (CUP) is a heterogeneous tumor type that has been diagnosed as a metastatic tumor by pathological examination, but the primary tumor cannot be identified through comprehensive clinical examination. The incidence of CUP accounts for approximately 1%–2% of all tumors. CUP progresses rapidly and has a short course. The treatment and prognosis of patients with CUP are closely linked to the primary site. In clinical settings, identifying the primary tumor remains challenging. Scholars have focused on improving the detection rate. Novel technologies, such as gene expression profiling, high-throughput sequencing, epigenetics, and liquid biopsy, have been successively applied to identify the primary tumor of CUP accurately, sensitively and specifically. With the guidance of molecular diagnosis, targeted therapy, immunotherapy, and combination therapy will usher in the era of precision treatment for CUP, which may become a typical example for individualized therapy.

Practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a South African setting

Background: Formalin-fixed paraffin-embedded (FFPE) tissue archives in hospitals, biobanks, and others offer a vast collection of extensive, readily available specimens for molecular testing. Unfortunately, the use of tissue samples for molecular diagnostic applications is challenging; thus, the forensic pathology FFPE tissue archives in Africa have been a largely unexploited genetic resource, with the usability of DNA obtainable from these samples being unknown.Intervention: The study, conducted from January 2015 to August 2016, determined the usefulness of FFPE tissue as a reliable source of genetic material for successful post-mortem molecular applications and diagnostics. Formalin-fixed paraffin-embedded tissue samples were collected and archived from autopsies conducted over 13 years in the forensic medicine department of the University of Pretoria (Pretoria, South Africa). Deoxyribonucleic acid from FFPE tissue samples and control blood samples was amplified by high-resolution melt real-time polymerase chain reaction before sequencing. The procurement parameters and fixation times were compared with the quantity and quality of the extracted DNA and the efficiency of its subsequent molecular applications.Lessons learnt: This study has shown that FFPE samples are still usable in molecular forensics, despite inadequate sample preparation, and offer immense value to forensic molecular diagnostics.Recommendations: FFPE samples fixed in formalin for more than 24 h should still be used in molecular diagnostics or research, as long as the primer design targets amplicons not exceeding 300 base pairs.

Comparación entre Xpert® y COBAS®-TaqMan® para la medición de carga viral de virus hepatitis B y C / Comparison between Xpert® and COBAS® TaqMan® for the measuring of viral load of hepatitis B and C viruses

INTRODUCCIÓN: La medición de carga viral (CV) de virus hepatitis B (VHB) y C (VHC) es fundamental en el seguimiento de pacientes con terapia antiviral. La metodología más utilizada para su determinación es COBAS ®-TaqMan ®. Recientemente, se desarrolló la tecnología Xpert ®, que se debe evaluar. OBJETIVO: Comparar la medición de la CV de VHB y VHC por metodología Xpert® con COBAS®-TaqMan® como método de referencia. MATERIAL Y MÉTODOS: 39 muestras de suero de pacientes con VHB y 39 con VHC, previamente cuantificadas por COBAS ®-TaqMan ®, fueron analizadas mediante Xpert® y los resultados se compararon utilizando la regresión de Deming y gráfico Bland-Altman. RESULTADOS: Hubo una alta correlación entre Xpert® y COBAS®-TaqMan®. Para VHB, la ecuación de Deming fue XpertHBV = 0,44 + 0,99xCOBASTaqManHBV, con coeficiente de correlación de 0,94 y diferencia entre medias de -0,401 log10 (IC95%: -1,985 a 1,183). Para VHC, la ecuación de Deming fue XpertHCV = 0,36 + 0,87x COBASTaqManHCV, con coeficiente de correlación de 0,98 y diferencia entre medias de 0,328 log (IC95%: -0,449 a 1,105). CONCLUSIÓN: El nuevo sistema Xpert® muestra una buena correlación con COBAS ®-TaqMan ® para la medición de la CV de VHB y VHC, siendo una buena alternativa para el seguimiento de pacientes en tratamiento.

BACKGROUND: The measurement of viral load (VL) of hepatitis B (HBV) and C (HCV) viruses is essential in the follow-up of patients with antiviral therapy. The most widely used methodology for this determination is COBAS®-TaqMan®. Recently, the Xpert® technology was developed and needs to be evaluated. AIM: To compare the measurement of the VL of HBV and HCV by Xpert® methodology with COBAS®-TaqMan® as a reference method. MATERIAL AND METHODS: 39 serum samples from patients with HBV and 39 with HCV, previously quantified by COBAS®-TaqMan®, were analyzed using Xpert® and the results were compared using Deming regression and Bland-Altman plot. RESULTS: There was a high correlation between Xpert® and COBAS®-TaqMan®. For HBV, the Deming equation was XpertHBV = 0.44 + 0.99xCOBASTaqManHBV, with a correlation coefficient of 0.94 and a difference between means of -0.401 log (95% CI: -1.985 to 1.183). For HCV, the Deming equation was XpertHCV = 0.36 + 0.87x COBASTaqManHCV, with a correlation coefficient of 0.98 and a difference between means of0.328 log10 (95% CI: -0.449 to 1.105). CONCLUSION: The new Xpert® system shows a good correlation with COBAS®-TaqMan® for the measurement of the VL of HBV and HCV, being a good alternative for the follow-up of patients under treatment.

Predictores de tuberculosis pulmonar y experiencia con su diagnóstico molecular rápido / Pulmonary tuberculosis predictors and rapid molecular diagnosis / Preditores de tuberculose pulmonar e experiência com o diagnóstico molecular rápido

Resumen: A nivel mundial se estima que en 2018 hubo alrededor de 10 millones de nuevos casos de tuberculosis (TBC). La detección molecular es una herramienta diagnóstica crecientemente utilizada para el diagnóstico de TBC. Los predictores de riesgo para TBC pulmonar son variados y varían de acuerdo a la población estudiada. Los objetivos del presente trabajo fueron: evaluar la performance de la detección de M. tuberculosis por la técnica Xpert® MTB/RIF para el diagnóstico de TBC pulmonar y determinar los factores predictores de presencia de esta enfermedad en pacientes asistidos en el Hospital Pasteur de Montevideo. Se realizó un estudio descriptivo, observacional y transversal. Se incluyeron 254 pacientes, 68 con TBC pulmonar. La sensibilidad de la prueba Xpert® MTB/RIF para detectar M. tuberculosis fue 100% (IC 95%: 91,2-100) y la especificidad 95,1% (IC 95%: 83,9-98,7). En el análisis multivariado se evidenció que los predictores independientes para presencia de TBC pulmonar fueron: contacto cercano con otro caso de TBC (p<0,001), consumo de pasta base de cocaína (p=0,006) y presentarse con adelgazamiento (p<0,001). En suma, la prueba Xpert® MTB/RIF se comportó como una excelente herramienta diagnóstica en nuestra población con elevada prevalencia de TBC pulmonar. Los predictores independientes para esta enfermedad indican que en la población analizada las estrategias de control de esta enfermedad requieren un abordaje multidisciplinario.

Summary: According to global estimations, there were approximately 10 million new cases of tuberculosis in 2018. Molecular diagnosis constitutes a rapidly growing diagnostic tool for tuberculosis. Risk predictors for pulmonary tuberculosis are varied and they depend on the population studied. The study aimed to assess the performance of M. tuberculosis detection by use of Xpert® MTB/RIF diagnostic test to diagnose pulmonary tuberculosis and to identify predictive factors for this disease in patients assisted at Pasteur Hospital in Montevideo. A descriptive, observational and transversal study was conducted, which included 254 patients, 68 of which had pulmonary tuberculosis. Sensitivity of the Xpert MTB/RIF assay to detect M. tuberculosis was 100% (CI 95%: 91.2-100) and specificity 95.1% (CI 95%: 83.9-98.7). Multivariate analysis evidenced the following to be the independent predictors that detect pulmonary tuberculosis: close contact with other cases of tuberculosis (p<0.001), coca-paste consumption (p=0.006) and evidence of loss of weight (p<0,001). To sum up, the Xpert® MTB/RIF assay proved to be an excellent diagnostic tool in our population with a high prevalence of pulmonary tuberculosis. Independent predictors for this disease show that, in the population studied, control strategies require a multidisciplinary approach.

Resumo: Globalmente, estima-se que em 2018 ocorreram cerca de 10 milhões de novos casos de tuberculose (TB). A detecção molecular é uma ferramenta diagnóstica cada vez mais usada para seu diagnóstico. Os preditores de risco para TB pulmonar são diversos e variam de acordo com a população estudada. Os objetivos deste estudo foram: avaliar o desempenho da detecção do M. tuberculosis pela técnica Xpert MTB/RIF para o diagnóstico da TB pulmonar e determinar os fatores preditivos da presença desta doença em pacientes atendidos no Hospital Pasteur de Montevidéu. Foi realizado um estudo descritivo, observacional e transversal. 254 pacientes foram incluídos, 68 com TB pulmonar. A sensibilidade do teste Xpert® MTB/RIF para detectar M. tuberculosis foi de 100% (IC 95%: 91,2-100) e a especificidade de 95,1% (IC 95%: 83,9- 98,7). A análise multivariada mostrou que os preditores independentes para a presença de tuberculose pulmonar foram: contato próximo com outro caso de tuberculose (p <0,001), consumo de pasta base de cocaína (p = 0,006) e apresentar perda de peso (p <0,001). Em suma, o teste Xpert® MTB/RIF se comportou como uma excelente ferramenta diagnóstica em nossa população com alta prevalência de TB pulmonar. Os preditores independentes para essa doença indicam que, na população analisada, as estratégias de controle da doença requerem uma abordagem multidisciplinar.

Application of high-throughput sequencing in the diagnosis of inherited bleeding and thrombotic disorders / 中华检验医学杂志

Inherited bleeding and thrombotic disorders (BTD) are a group of heterogeneous diseases related to the coagulation system, platelet function and fibrinolytic system. In addition to the common BTD, it is difficult to diagnose such patients by routine laboratory tests, and special laboratory tests are often required to confirm the diagnosis. Therefore, the diagnosis and treatment of patients may be delayed, or even life-threatening. With the increasing use of high-throughput sequencing (HTS) technology in clinical practice, the diagnosis and differential diagnosis of BTD have made great progress.

Complete screening of exons 2, 3, and 4 of kras and nras genes reveals a higher number of clinically relevant mutations than food and drug administration quantitative polymerase chain reaction-based commercial kits

Abstract Background: The presence of clinically relevant mutations in KRAS and NRAS genes determines the response of anti-epidermal growth factor receptor antibody therapy for metastatic colorectal cancer (mCRC). The only quantitative polymerase chain reaction (qPCR)-based diagnostic tests approved by the Food and Drug Administration (FDA) screen merely for mutations in codons 12 and 13 of KRAS. Objective: The objective of the study was to study the frequency of clinically relevant mutations in KRAS and NRAS genes that are not included in FDA-approved qPCR tests. Methods: Formalin-fixed paraffin-embedded tumor specimens from 1113 mCRC Mexican patients from different health institutions across the country were analyzed by Sanger sequencing for KRAS mutations in exons 2, 3, and 4. Furthermore, 83 were analyzed in exons 2, 3, and 4 of NRAS. Results: From the specimens tested for KRAS, 33.69% harbored a mutation. From these, 71.77% were in codon 12 and 27.69% in codon 13 (both located in exon 2). Codons 59 (exon 3) and 146 (exon 4) accounted for the remaining 0.54%. From the 83 specimens, in which NRAS was analyzed, three mutations were found in codon 12 (3.61%). Approximately 6% of RAS mutated specimens would have been falsely reported as RAS wild type if an FDA-approved qPCR diagnostic test had been used. Conclusions: While these kits based on qPCR can be very practical and highly sensitive, their mutation coverage ignores mutations from poorly genetically characterized populations.

Actinobacillosis outbreak in cattle with clinical manifestation of hippopotamus-like face / Actinobacilose em bovinos com manifestação clínica de cara de hipopótamo

Actinobacillosis outbreak with clinical manifestation of hippopotamus-like face observed in a property located in the municipality of Capão do Leão, Southern Brazil, in September 2016, is described. The cattle herd remained for most of the year in rice stubble. When these areas were occupied with new crops, they were transferred to areas where there were small native forests. Three cattle were affected. They presented a volume increase in the nasolabial and maxillary region, and there was also regional lymph node swelling. The evolution of the disease occurred in approximately six months. In tissue fragments collected for culture, Actinobacillus lignieresii was isolated. The diagnosis was based on clinical findings, histopathological evaluation characterized by the presence of piogranulomas with Splendore Hoepli reaction in its center, bacterial isolation, and identification of A. lignieresii by polymerase chain reaction (PRC) and genetic sequencing.(AU)

Descreve-se um surto de actinobacilose com manifestação clínica de cara de hipopótamo diagnosticado em uma propriedade localizada no município do Capão do Leão, Rio Grande do Sul em setembro de 2016. Os bovinos permaneciam durante a maior parte do ano em restevas de arroz e quando as áreas eram ocupadas com novas lavouras eram transferidos para áreas onde havia pequenas matas nativas. Foram afetados três bovinos adultos que apresentavam aumento de volume na região nasolabial e maxilar e havia, também, tumefação dos linfonodos regionais. A evolução da enfermidade era de aproximadamente seis meses. Nos fragmentos coletados para cultura houve isolamento de Actinobacillus lignieresii. O diagnóstico foi baseado nos achados clínicos, na avaliação histopatológica caracterizada pela presença de piogranulomas com reação de Splendori Hoepli no centro, no isolamento bacteriano, identificação de Actinobacillus lignieresii por reação em cadeia da polimerase (PRC) e sequenciamento genético.(AU)

Optimization of ultra-fast convection polymerase chain reaction conditions for pathogen detection with nucleic acid lateral flow immunoassay

Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to 3 µM or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, 2 µM of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

Exploration of verification and validation of clinical molecular diagnostics method inspection procedure / 中华检验医学杂志

Molecular testing are more and more widely used in clinical practice. The premise of clinical application is the verification or validation of the molecular testing procedure. Establishing a verification and validation model suitable for the testing procedures of clinical molecular testing is helpful for the practitioners of clinical molecular testing to carry out the verification and validation work more conveniently and efficiently. It is also helpful for the standardization of testing and quality control significantly. This article will elaborate on the status of verification and validation of molecular testing projects, the requirements of the ISO15189 accreditation system, the establishment of verification validation models and the prospects in the field.

High frequency of HPV16 European variant E350G among Mexican women from Sinaloa

Background & objectives: Human papillomavirus (HPV) infections play a crucial role in the aetiology of cervical cancer (CC), and HPV16 is the primary viral genotype associated with CC. A number of variants of the HPV16 E6 gene are involved in the progression of CC, differing in their prevalence and biological and biochemical properties. This study was designed to determine the frequency of HPV types 16/18 and to identify the presence of HPV16 E6-variants in asymptomatic Mexican women. Methods: A total of 189 cervical Pap smears were collected from women attending public health services in three different cities in Sinaloa, Mexico. Viral DNA was identified by amplification of E6 viral gene fragments using polymerase chain reaction (PCR). Identification of variants was done by sequencing a DNA fragment (321bp) of the HPV16 E6 gene. Results: More than half of the women tested were HPV-positive (52.38%), with HPV16 being the most frequent genotype (21.16%), followed by HPV18 (8.99%). Sequence analysis of the E6-HPV16 PCR products showed that in all cases, the viruses corresponded to European variants. It was further observed that the E350G intra-variant was the most common (>76%). Interpretation & conclusions: This study showed a predominance of European lineage variants of HPV16 among asymptomatic women from Sinaloa, Mexico, predominantly with of the E350G variant. This variant has been shown to be associated with an increased risk of early development of CC. The use of molecular identification of carcinogenic HPV and Pap test screening may be a good strategy for monitoring women to prevent CC.

Recent Advancement of the Molecular Diagnosis in Pediatric Brain Tumor

Recent discoveries of brain tumor-related genes and fast advances in genomic testing technologies have led to the era of molecular diagnosis of brain tumor. Molecular profiling of brain tumor became the significant step in the diagnosis, the prediction of prognosis and the treatment of brain tumor. Because traditional molecular testing methods have limitations in time and cost for multiple gene tests, next-generation sequencing technologies are rapidly introduced into clinical practice. Targeted sequencing panels using these technologies have been developed for brain tumors. In this article, focused on pediatric brain tumor, key discoveries of brain tumor-related genes are reviewed and cancer panels used in the molecular profiling of brain tumor are discussed.

Evaluation of clinical application of two molecular assays for detection of toxigenic Clostridium difficile / 中华检验医学杂志

Objective In comparison with Xpert C.difficile/Epi through detection of Clostridium difficile toxin genes from clinical stool , the performance of a laboratory-developed ( LD) assay was evaluated in detail.Methods A total of 176 stool specimens collected from patients with diarrhea in the First People′s Hospital of Yuhang District and the People′s Hospital of Yingzhou , Ningbo from August 1 to December 30 were detected by the two assays in parallel , and meanwhile the C.difficile strains will be isolated and identified for C.difficile toxin genes by a conventional PCR assay .The Cross-tabs Analysis was used for the results by using SPSS20.0 software.Results In comparison with the results of Xpert C.difficile/Epi as the standard, the LD assay had a sensitivity of 91.7％(22/24), a specificity of 100％(152/152), a positive predictive value (PPV) of 100％(22/22), and negative predictive value (NPV) 98.7％(152/154).The results of two assays were statistically coherent (Kappa=0.950, P<0.001).In comparison with culture and detection of toxin genes results , the LD assay had a sensitivity of 90.0％ ( 18/20 ) , a specificity of 97.0％(152/156), a PPV of 81.8％ (18/22), and NPV of 98.7％ (152/154)(Kappa=0.838, P<0.001), and the Xpert C.difficile/Epi assay had a sensitivity of 90.0％ (18/20), a specificity of 96.0％(150/156), a PPV of 75.0％(18/24), and NPV of 98.7％ (150/152)(Kappa=0.792, P<0.001). Conclusions The performance of the LD assay was similar to that of the Xpert C .difficile/Epi kit in detection of toxigenic C.difficile.The LD assay could be directly applied to detection of toxigenic C.difficile from clinical stool samples .The clinical application of this LD assay will also provide a domestic and promising diagnostic assay for diagnosis of C.difficile infection in China.

Recent Advancement of the Molecular Diagnosis in Pediatric Brain Tumor

Recent discoveries of brain tumor-related genes and fast advances in genomic testing technologies have led to the era of molecular diagnosis of brain tumor. Molecular profiling of brain tumor became the significant step in the diagnosis, the prediction of prognosis and the treatment of brain tumor. Because traditional molecular testing methods have limitations in time and cost for multiple gene tests, next-generation sequencing technologies are rapidly introduced into clinical practice. Targeted sequencing panels using these technologies have been developed for brain tumors. In this article, focused on pediatric brain tumor, key discoveries of brain tumor-related genes are reviewed and cancer panels used in the molecular profiling of brain tumor are discussed.

Applicability of PCR-based clonality assay in dogs with multicentric lymphoma / Aplicabilidade do ensaio de clonalidade por PCR em cães com linfoma multicêntrico

Linfoma multicêntrico apresenta alta prevalência dentre as neoplasias em cães, e o diagnóstico rotineiro não é eficaz para avaliação de prognóstico. A PCR para rearranjos de receptores de antígeno (PRRA) apresenta potencial para classificação e estadiamento de linfomas. Este trabalho objetiva relatar o desenvolvimento de um protocolo de PRRA para aplicação em cães, baseando-se em condições e primers descritos na literatura. Foram coletados aspirados de linfonodo de 10 cães com linfoma multicêntrico e 15 lâminas de linfonodo positivas para linfoma já secas ao ar, fixadas e coradas. O protocolo utilizado demonstrou-se eficaz na amplificação de DNA das amostras frescas e das lâminas, com sensibilidade de 75%, similar à de estudos anteriores. Resultados parciais sugerem prevalência de linfomas de células B (60%) sobre células T (40%). O presente estudo abre precedentes para uma série de novos estudos com diagnóstico molecular de linfomas.(AU)

Origins and History of Laboratory Medicine

Medical diagnostics plays a significant role in clinical decisions. The first medical laboratory test to be developed was urine analysis, in which urine properties were analyzed for diagnosis. Urine analysis has been long used as a routine laboratory test that was improved with the development of sampling and test methods. As the field of hematology progressed with the invention of the microscope, blood tests were developed. Demands for tests based on clinical chemistry have existed since the 17th century, and research using patient blood began in the 18th century. In the 20th century, with the development of the spectrophotometer, chemical analyses were performed for diagnostic purposes. With the appearance of cholera outbreaks, the identification of microorganisms was necessary for patient diagnosis, and the development of specific test methods contributed to microorganism detection in the laboratory. Blood transfusion, which started with blood collection in the 15th century, is currently used as a therapeutic method in medicine. Moreover, once the hypothesis of acquired immunity was proven in the 18th century, various methods for measuring immunity were developed. Molecular diagnosis, which was established during the 20th century after the presentation of Mendel's Genetic Laws in the 19th century, developed rapidly and became the predominant field in medical laboratory diagnostics. Thus, medical laboratory technology became an academic field, with foundations based on basic sciences. Modern medicine will further progress thanks to medical advancements, leading to an extension of average human lifespan up to 100 years. Laboratory medicine will provide significant support for this development.

Molecular Testing of Lymphoproliferative Disorders: Current Status and Perspectives

Molecular pathologic testing plays an important role for the diagnosis, prognostication and decision of treatment strategy in lymphoproliferative disease. Here, we briefly review the molecular tests currently used for lymphoproliferative disease and those which will be implicated in clinical practice in the near future. Specifically, this guideline addresses the clonality test for B- and T-cell proliferative lesions, molecular cytogenetic tests for malignant lymphoma, determination of cell-of-origin in diffuse large B-cell lymphoma, and molecular genetic alterations incorporated in the 2016 revision of the World Health Organization classification of lymphoid neoplasms. Finally, a new perspective on the next-generation sequencing for diagnostic, prognostic, and therapeutic purpose in malignant lymphoma will be summarized.

Comparison of Luminex NxTAG Respiratory Pathogen Panel and xTAG Respiratory Viral Panel FAST Version 2 for the Detection of Respiratory Viruses

Owing to advancements in molecular diagnostics, recent years have seen an increasing number of laboratories adopting respiratory viral panels to detect respiratory pathogens. In December 2015, the NxTAG respiratory pathogen panel (NxTAG RPP) was approved by the United States Food and Drug Administration. We compared the clinical performance of this new assay with that of the xTAG respiratory viral panel (xTAG RVP) FAST v2 using 142 clinical samples and 12 external quality assessment samples. Discordant results were resolved by using a laboratory-developed respiratory viral panel. The NxTAG RPP achieved 100% concordant negative results and 86.6% concordant positive results. It detected one coronavirus 229E and eight influenza A/H3N2 viruses that were missed by the xTAG RVP FAST v2. On the other hand, the NxTAG RPP missed one enterovirus/rhinovirus and one metapneumovirus that were detected by FAST v2. Both panels correctly identified all the pathogens in the 12 external quality assessment samples. Overall, the NxTAG RPP demonstrated good diagnostic performance. Of note, it was better able to subtype the influenza A/H3N2 viruses compared with the xTAG RVP FAST v2.

Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting

Abstract Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting.

Infecção natural de pombos (Columba livia) por Cryptosporidium spp / Natural infection of pigeons (Columba livia) with Cryptosporidium spp

O estudo teve como objetivo diagnosticar microscopicamente e molecularmente a presença de oocistos de Cryptosporidium spp. provenientes de amostras fecais de pombos (Columba livia) no município do Rio de Janeiro. As amostras fecais foram coletadas frescas, logo após as aves defecarem e acondicionadas sob refrigeração e encaminhadas para o processamento e pesquisa de oocistos de Cryptosporidium spp. ao laboratório de Protozoologia do Departamento de Parasitologia, da Universidade Federal Rural do Rio de Janeiro. As amostras foram submetidas a técnica de centrifugação e flutuação com solução saturada de açúcar e as lâminas observadas com objetiva de 40X. Nas amostras positivas para a presença de oocistos de Cryptosporidium spp. foi realizada a extração de ácido desoxirribonucleico (DNA) para uso nas reações em cadeia pela polimerase (PCR) e Nested-PCR. Um total de 387 amostras fecais de pombos foram obtidas, no diagnóstico microscópico foi possível a observação de oocistos deste parasita em 81 amostras (20,93%). Destas, 53 amostras amplificaram DNA específico para Cryptosporidium spp. na reação da Nested-PCR. Mundialmente, Cryptosporidium spp. parasitando pombos é assinalado em apenas quatro países: Turquia, China, Irã e Tailândia. O resultado encontrado neste trabalho, em três bairros populosos do município do Rio de Janeiro, revelou ser preocupante, por serem áreas onde há uma quantidade acentuada da população animal e humana, facilitando a dispersão e contaminação ambiental dos oocistos. Este é o primeiro registro no Brasil de parasitismo de Cryptosporidium spp. tendo como hospedeiro o pombo e estes resultados, não devem ser negligenciados.

The aim of this study was to diagnose microscopically and molecularly the presence of Cryptosporidium spp. oocysts from pigeons (Columba livia) faecal samples in Rio de Janeiro city. The fecal samples were collected fresh, after the birds defecated and conditioned under refrigeration and sent to the processing and research of Cryptosporidium spp. oocysts to the protozoology laboratory of the Parasitology Department, Universidade Federal Rural do Rio de Janeiro. The samples were submitted to centrifugation and flotation with saturated sugar solution technique and the slides observed with a 40X objective. In the positive samples for the Cryptosporidium spp. oocysts the extraction of deoxyribonucleic acid (DNA) for polymerase chain reaction (PCR) and NestedPCR was performed. A total of 387 pigeons faecal samples were obtained, in the microscopic diagnosis it was possible to observe oocysts of this parasite in 81 samples (20.93%). Of these, 53 samples amplified DNA specific for Cryptosporidium spp. in the Nested-PCR reaction. Worldwide, Cryptosporidium spp. parasitizing pigeons is reported in only four countries: Turkey, China, Iran and Thailand. The results found in this study, in three populated neighborhoods of Rio de Janeiro city, were worrisome because they are areas where there is a marked quantity of animal and human population, facilitating the dispersion and environmental contamination of oocysts. This is the first record in Brazil of parasitism of Cryptosporidium spp. having as host the pigeon and these results, should not be neglected.