Can current molecular tests help in the diagnosis of indeterminate thyroid nodule FNAB?

ABSTRACT Approximately 15-30% of all thyroid nodules evaluated with fine-needle aspiration biopsy (FNAB) are classified as cytologically indeterminate. The stepwise unraveling of the molecular etiology of thyroid nodules has provided the basis for a better understanding of indeterminate samples and an opportunity to decrease diagnostic surgery in this group of patients. Over the last 15 years, several studies have tested different methodologies to detect somatic mutations (by polymerase chain reaction and next-generation sequencing, for example), and to identify differentially expressed genes or microRNA, aiming at developing molecular tests to improve the presurgical diagnosis of cytologically indeterminate nodules. In this review, we will provide an overview of the currently available molecular tests and the impact of mutation testing on the diagnosis of thyroid cancer. We will also review current published data and future perspectives in molecular testing of thyroid nodule FNAB and describe the current Brazilian experience with this diagnostic approach. Based on currently available data, especially for countries outside the US-Europe axis, a rational use of these tests must be made to avoid errors with regard to test indication and interpretation of test outcomes. In addition to clinical, radiological, and cytological features, we still need to determine local malignancy rates and conduct more independent validation and comparative performance studies of these tests before including them into our routine approach to indeterminate FNAB.

External Quality Assessment of MERS-CoV Molecular Diagnostics During the 2015 Korean Outbreak

BACKGROUND: The largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection outside Middle East Asia in 2015 has necessitated the rapid expansion of laboratories that conduct MERS-CoV molecular testing in Korea, together with external quality assessment (EQA) to evaluate the assays used. METHODS: The EQA program consisted of two phases; self-validation and blind assessment. For the first EQA phase, in vitro transcribed upstream region of the envelope gene (upE) and the open reading frame (ORF)1a RNAs were used at a concentration of 1,000 copies/microL. The test panel for the second EQA phase consisted of RNA extracts from three samples, which were obtained from two MERS-CoV positive patients and one MERS-CoV negative patient. RESULTS: The first EQA phase results for 46 participants showed a linear relationship between the threshold cycle (CT) values of RNA materials and the logarithmic concentrations for both upE and ORF1a gene targets (R2=0.73 and 0.75, respectively). The mean CT value for each concentration was different depending on which commercial kit was used for the assay. Among the three commonly used kits, PowerChek MERS Real-Time PCR kit (KogeneBiotech, Korea) showed the lowest CT values at all concentrations of upE and most concentrations of ORF1a. The second EQA phase results for 47 participants were 100% correct for all tested samples. CONCLUSIONS: This EQA survey demonstrates that the MERS-CoV molecular testing performed in Korea during the 2015 outbreak is of robust capability. However, careful establishment and validation of a cut-off value are recommended to ensure good analytical sensitivity.

Optimization of a molecular method for the diagnosis of canine babesiosis / Otimização de método molecular para diagnóstico da babesiose canina

Babesiosis is a hemolytic disease caused by protozoans of the genus Babesia (Apicomplexa). This disease occurs worldwide and is transmitted by ticks to a variety of mammals, including humans. The objective of the present study was to optimize a molecular approach for the detection of a fragment of 18S rDNA of Babesia canis, Babesia vogeli, Babesia rossi or Babesia gibsoni based on a single semi-nested Polymerase Chain Reaction (PCR), and compare the efficiency of this approach with that of a simple PCR protocol. To this end, 100 blood samples collected from dogs with suspected hemoparasite infections were analyzed. A comparison of the results of simple PCR and semi-nested PCR indicated a highly significant difference (p value = 0.0000). While only five (5%) of the samples tested positive using the simple protocol, 22 (22%) were positive using the snPCR technique. The results of this study reinforce the findings of previous studies, which have demonstrated the greater sensitivity of tests based on nested or semi-nested PCR. Therefore, to avoid false-negative results due to low levels of parasitemia, we suggest the preferential use of this protocol in epidemiological studies of canine babesiosis, particularly those that require reliable estimates of the prevalence of infection.

A babesiose é uma doença hemolítica de ocorrência mundial, causada por protozoários do gênero Babesia (Apicomplexa), que são transmitidos por carrapatos a diversos mamíferos, incluindo o homem. O objetivo deste estudo foi otimizar um método molecular para a detecção de fragmento do 18S rDNA de Babesia canis, Babesia vogeli, Babesia rossi ou Babesia gibsoni com base em uma única semi-nested (snPCR), comparando sua eficiência com um protocolo de PCR simples. Para isso, 100 amostras de sangue de cães com suspeita de hemoparasitoses foram analisadas e, enquanto o protocolo de PCR simples indicou somente 5% (5/100) de amostras positivas, o protocolo de snPCR, com 22% (22/100) de amostras positivas, apresentou maior sensibilidade (p valor = 0,0000). Este resultado está de acordo com outros estudos que mostram a maior sensibilidade de detecção dos testes baseado em nested ou snPCR. Assim, como uma forma de prevenir resultados falso-negativos devido à baixa parasitemia, sugere-se que este protocolo seja preferencialmente usado nos estudos epidemiológicos de babesiose canina, em especial naqueles que tratam da sua prevalência.

Detección de virus papiloma humano en la prevención del cáncer cérvico-uterino / Human papilomavirus detection in cervical cancer prevention

El cáncer cérvico-uterino (CCU), que está fuertemente asociado a la infección por virus papiloma humano de alto riesgo (VPH-AR), sigue siendo un problema de salud pública en Latinoamérica. El uso de la citología para la detección de lesiones pre-cancerosas no ha tenido mayor impacto en las tasas de incidencia y mortalidad del CCU, que aún se mantienen altas en la región. La disponibilidad de nuevas técnicas de tamizaje para la detección de lesiones pre-cancerosas y de vacunas altamente eficaces que previenen casi todas las lesiones relacionadas con los VPH-AR de alto potencial oncogénico VPH 16 y 18, en mujeres no expuestas previamente al virus brindan una gran oportunidad para la prevención del CCU. La detección de VPH-AR representa actualmente un valioso componente de las guías clínicas para el tamizaje, manejo y tratamiento del CCU y sus lesiones precursoras. Se han desarrollado estrategias metodológicas que detectan un amplio espectro de tipos de VPH-AR; sin embargo, solo un pequeño subgrupo de ellas ha documentado la validación clínica para cualquiera de las indicaciones habituales de la detección de estos virus. Las pruebas de VPH que no estén validadas y que no hayan demostrado confiabilidad, reproducibilidad y exactitud no deben ser usadas en el manejo clínico. Una vez incorporada una prueba de VPH en el laboratorio, es esencial que el procedimiento completo sea sometido a un continuo y riguroso control de calidad para evitar prácticas subóptimas, potencialmente dañinas. Este artículo discute los recientes progresos y el estado actual de estos métodos.

Cervical cancer (CC), which is strongly associated to high-risk human papillomavirus (hr-HPV) infection, continues being a significant health problem in Latin America. The use of conventional cytology to detect precancerous cervical lesions has had no major impact on reducing CC incidence and mortality rates, which are still high in the region. New screening tools to detect precancerous lesions became available, which provide great opportunities for CC prevention, as do highly efficacious HPV vaccines able to prevent nearly all lesions associated with HPV-16 and -18 when applied before viral exposure. Currently, hr-HPV testing represents an invaluable component of clinical guidelines for screening, management and treatment of CC and their precursor lesions. Many testing strategies have been developed that can detect a broad spectrum of hr-HPV types in a single assay; however, only a small subset of them has documented clinical performance for any of the standard HPV testing indications. HPV tests that have not been validated and lack proof of reliability, reproducibility and accuracy should not be used in clinical management. Once incorporated into the lab, it is essential to submit the whole procedure of HPV testing to continuous and rigorous quality assurance to avoid sub-optimal, potentially harmful practices. Recent progress and current status of these methods are discussed in this article.

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection.

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10 6 -10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.

Estandarización de un método de detección molecular del virus influenza (H5N1) de alta patogenicidad / Standardization of a molecular detection method of highly pathogenic avian influenza virus (H5N1)

El virus de la influenza aviar H5N1 de alta patogenicidad mantiene el alerta mundial debido a su potencial zoonótico y pandémico. Surge entonces la necesidad de contar con herramientas para la detección temprana y de esta forma reducir el impacto potencial a la salud humana y animal. En este estudio se estandarizó un método de detección molecular de los genes de la matriz (M), hemaglutinina (H5) y neuraminidasa (N1) del virus de la influenza aviar H5N1 de alta patogenicidad de linaje asiático, mediante transcripción-reversa y reacción en cadena de la polimerasa en tiempo real (RRT-PCR). A partir de un ARN viral de referencia cepa A/Vietnam/1203/2004 (H5N1) se construyeron controles positivos mediante clonación de productos de PCR. Los estándares de naturaleza plasmídica se emplearon en la obtención de curvas estándar para determinar los límites de detección de la técnica. La sensibilidad observada para todos los genes analizados fue de 10² copias de ADN/μL. Las curvas mostraron una eficiencia superior al 90%, y R²>0,99. Este método puede ser útil en las campañas de monitoreo del virus en aves migratorias, así como para el tamizaje de muestras clínicas de humanos, en una emergencia de salud.

Highly pathogenic avian influenza virus (HPAI) H5N1 is a global threat due to its zoonotic and pandemic potential. Then, concern arises and the need to have early detection tools to minimize the impact on human and animal health. In this work, a molecular detection method was implemented to detect matrix (M), hemagglutinin (H5) and neuraminidase (N1) genes of HPAI avian influenza virus H5N1, based on real time RT-PCR (RRT-PCR). Positive controls were constructed from reference RNA viral A/Vietnam/1203/2004 (H5N1), cloned into plasmidic vectors and sequenced. Assay detection sensitivity was assessed with standard curves for each gene. Assay sensitivity was 10² DNA copies/μl in all cases. Curves showed amplification efficiency higher than 90% and R²>0.99. This method could be useful for bird monitoring campaigns and as a screening procedure for clinical samples.