Multiplex Reverse Transcription Polymerase Chain Reaction Combined with a Microwell Hybridization Assay Screening for Arbovirus and Parasitic Infections in Febrile Patients Living in Endemic Regions of Colombia.

Acute febrile syndrome is a frequent reason for medical consultations in tropical and subtropical countries where the cause could have an infectious origin. Malaria and dengue are the primary etiologies in Colombia. As such, constant epidemiological surveillance and new diagnostic tools are required to identify the causative agents. A descriptive cross-sectional study was conducted to evaluate the circulation and differential diagnosis of six pathogens in two regions of Colombia. The results obtained via multiplex reverse transcription polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) were comparable to those obtained using rapid tests conducted at the time of patient enrollment. Of 155 patients evaluated, 25 (16.1%) and 16 (10.3%) were positive for malaria and dengue, respectively; no samples were positive for any of the other infectious agents tested. In most cases, m-RT-PCR-ELISA confirmed the results previously obtained through rapid testing.

Detección y genotipificación del virus del papiloma humano de alto riesgo mediante PCR multiplex en tiempo real (RT-PCR VPH AR) / High risk human papillomavirus detection and genotyping by multiplex real time PCR (RT-PCR HR HPV)

Utilidad clínica de la prueba La relación causal entre el desarrollo de cáncer de cérvix y la infección con genotipos de alto riesgo (AR) del virus del papiloma humano (VPH), ha llevado al desarrollo de estrategias para su detección y caracterización genotípica, como una medida de prevención de este tipo de cáncer. Dado que la presencia del VPH no puede ser determinada mediante los hallazgos clínicos de la paciente, como tampoco en los hallazgos morfológicos en la citología ni en la detección de anticuerpos específicos contra el VPH (pruebas serológicas), su detección y genotipificación recaen en el uso de pruebas moleculares, las cuales en su mayoría están dirigidas a la detección del ADN de los genotipos de alto riesgo, usando la técnica de reacción en cadena de la polimerasa (PCR) convencional y en tiempo real (RT-PCR) [1]. La técnica de PCR permite la amplificación de regiones específicas del ADN del VPH en los genes L1, E6 y E7, los cuales, por sus variaciones en la secuencia, permiten la genotipificación del virus [2,3]. Las pruebas de detección de ADN y/o genotipificación del VPH son consideradas herramientas de tamización en cáncer de cérvix, que detectan la infección causada por VPH. Su aplicación está enfocada en la clasificación de anormalidades citológicas, monitoreo de infecciones persistentes, seguimiento postratamiento de lesiones intraepiteliales de alto grado y vigilancia epidemiológica en salud pública [4-6]. La utilización de la citología y las pruebas de detección de ADN del VPH, aumenta la sensibilidad de la tamización para la detección de cáncer de cérvix y reduce de manera significativa el riesgo de sufrir lesiones cervicales premalignas por un periodo de 5 años [2,7]

Evaluation of two multiplex RT-PCR assays for detection and subtype differentiation of Brazilian swine influenza viruses.

Influenza A virus (IAV) subtypes H1N1, H1N2, and H3N2 are endemic in swine herds in most pork producing countries; however, the viruses circulating in different geographic regions are antigenically and genetically distinct. In this sense, the availability of a rapid diagnostic assay to detect locally adapted IAVs and discriminate the virus subtype in clinical samples from swine is extremely important for monitoring and control of the disease. This study describes the development and validation of a multiplex RT-PCR assay for detection and subtyping of IAV from pigs. The analytical and diagnostic specificity of the assays was 100% (94.3-100.0, CI 95%), and the limit of detection was 10-3 TCID50/mL. A total of 100 samples (IAV isolates and clinical specimens) were tested, and the virus subtype was determined for 80 samples (80%; 71.1-86.7, CI 95%). From these, 50% were H1N1, 22.5% were H1N2, and 7.5% were H3N2. Partial subtyping was determined for 8.75% samples (H1pdmNx and HxN2). Additionally, mixed infections with two virus subtypes (H1N2 + H3N2 and H1N1pdm + H1pdmN2; 2.5%) and reassortant viruses (H1pdmN2, 6.25%; and H1N1hu, 2.5%) were detected by the assay. A rapid detection of the most prevalent IAV subtypes and lineages in swine is provided by the assays developed here, improving the IAV diagnosis in Brazilian laboratories, and contributing to the IAV monitoring.

Simultaneous Detection of Both RNA and DNA Viruses Infecting Dry Bean and Occurrence of Mixed Infections by BGYMV, BCMV and BCMNV in the Central-West Region of Mexico.

A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed to simultaneously detect bean common mosaic virus (BCMV), bean common mosaic necrotic virus (BCMNV), and bean golden yellow mosaic virus (BGYMV) from common bean leaves dried with silica gel using a single total nucleic acid extraction cetyl trimethyl ammonium bromide (CTAB) method. A mixture of five specific primers was used to amplify three distinct fragments corresponding to 272 bp from the AC1 gene of BGYMV as well as 469 bp and 746 bp from the CP gene of BCMV and BCMNV, respectively. The three viruses were detected in a single plant or in a bulk of five plants. The multiplex RT-PCR was successfully applied to detect these three viruses from 187 field samples collected from 23 municipalities from the states of Guanajuato, Nayarit and Jalisco, Mexico. Rates of single infections were 14/187 (7.5%), 41/187 (21.9%), and 35/187 (18.7%), for BGYMV, BCMV, and BCMNV, respectively; 29/187 (15.5%) samples were co-infected with two of these viruses and 10/187 (5.3%) with the three viruses. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting these viruses in the common bean and can be used for routine molecular diagnosis and epidemiological studies.

Effect of several PEO-PPO amphiphiles on bax, bcl-2, and hTERT mRNAs: An insight into apoptosis and cell immortalization induced in hepatoma cells by these polymeric excipients.

UNLABELLED: Recent data have shown that synthetic polymers and nanomaterials display phenotypic effects in cells and signal transduction mechanisms involved in inflammation, differentiation, proliferation, and apoptosis. AIM: This article aims to investigate the effect of poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) block copolymers with a wide range of biomedical and pharmaceutical applications on apoptosis and/or cell immortalization, by flow cytometry and multiplex RT-PCR for bax, bcl-2, and human telomerase reverse transcriptase (hTERT). RESULTS: PEO-PPO amphiphiles upregulated bax and hTERT and induced apoptosis of two human hepatoma cell lines. CONCLUSIONS: PEO-PPO block copolymers-considered safe for human use-can drastically alter gene expression profiles of genes related to apoptosis/cell proliferation.

Simultaneous detection of bee viruses by multiplex PCR.

Honey bee mortality is a serious problem that beekeepers in Argentina have had to face during the last 3 years. It is known that the consequence of the complex interactions between environmental and beekeeping parameters added to the effect of different disease agents such as viruses, bacteria, fungi and parasitic mites may result in a sudden collapse of the colony. In addition, multiple viral infections are detected frequently concomitantly in bee colonies. The aim of this study was to establish a multiplex polymerase chain reaction method for rapid and simultaneous detection of the most prevalent bee viruses. This multiplex PCR assay will provide specific, rapid and reliable results and allow for the cost effective detection of a particular virus as well as multiple virus infections in a single reaction tube. This method could be a helpful tool in the surveillance of the most frequently found bee viruses and to study the dynamics and the interactions of the virus populations within colonies.