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Abstract Background: Arboviral diseases are a group of infectious diseases caused by viruses transmitted by arthropods, mainly mosquitoes. These diseases, such as those caused by the dengue (DENV), Zika (ZIKV), chikungunya (CHIKV), and yellow fever (YFV) viruses, have a significant impact worldwide. In this context, entomological surveillance plays a crucial role in the control and prevention of arboviruses by providing essential information on the presence, distribution, and activity of vector mosquitoes. Based on entomological surveillance, transovarian transmission provides information regarding the maintenance and dissemination of arboviruses. The objective of this study was to detect these arboviruses in Goiânia, Goiás, and analyze the occurrence of transovarian transmission. Methods: Aedes aegypti eggs were collected from different regions of Goiânia and cultivated under controlled laboratory conditions until the emergence of adult mosquitoes. Adult females were grouped into pools containing their heads and thoraxes. These pools were subsequently evaluated using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay. Results: A total of 157 pools (N=1570) were analyzed, with two pools testing positive for CHIKV and one pool testing positive for ZIKV, indicating that the offspring resulting from transovarian transmission are potentially infectious. Conclusions: In summary, the demonstration of the vertical transmission mechanisms of CHIKV and ZIKV in A. aegypti serves as an alert to health authorities, as these diseases are still underreported, and their primary urban vector has likely acquired this capacity, contributing to the dissemination of these infections.
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El ciclo del nitrógeno representa uno de los procesos biogeoquímicos más importantes para los ecosistemas terrestres y acuáticos. Las comunidades microbianas desempeñan un papel crucial en los procesos de transformación del nitrógeno en el suelo, ya que participan en diversas etapas como la nitrificación, de gran importancia para la producción agrícola. Dentro de los marcadores moleculares más utilizados para evaluar la actividad de poblaciones microbianas oxidantes de amonio se han considerado ampliamente los genes que codifican enzimas claves como la subunidad A de la actividad amonio monooxigenasa (AMO). Sin embargo, no se comprende completamente si la expresión de esta enzima tiene relación directa con el rendimiento de los cultivos. En este contexto, se evaluó la expresión del gen amo-A de comunidades bacterianas y archaeales presentes en un lote arrocero previamente caracterizado por ambientes. Para cuantificar la abundancia de arqueas y bacterias oxidantes de amonio, (AOA y AOB, respectivamente) se emplearon las técnicas de PCR en tiempo real (RT-qPCR) y PCR digital (RT-dPCR). En este trabajo se encontró a través del análisis de datos metagenómicos que hubo una mayor presencia de AOB en las muestras de suelo rizosférico mientras que las AOA fueron predominantes en las muestras de suelo de soporte "bulk", sin embargo, no se detectó la expresión del gen amo-A asociada a la comunidad de bacterias en las muestras de suelo analizadas. Por otra parte, no se presentaron diferencias entre los transcritos del gen amo-A asociados a la comunidad de AOA de los ambientes caracterizados. Además, la expresión de transcritos no estuvo relacionada con alguna de las propiedades químicas evaluadas. Finalmente, las estrategias de cuantificación para RT-qPCR (plásmido y templete) resultaron ser homólogas y funcionales para identificar la expresión del gen amo-A de AOA, mientras que la técnica de RT-dPCR fue más precisa para el análisis de la comunidad de AOB y AOA.
The nitrogen cycle represents one the most important biogeochemical process for terrestrial and aquatic ecosystems. Microbial communities play a crucial role in the processes of transformation of soil nitrogen in the, since they participate in various stages such as nitrification, which is of great importance for agricultural production. Among the most used molecular markers to assess ammonium oxidizing microbial populations activity have been considered widely the genes encoding key enzymes such as ammonium monooxygenase (AMO) subunit A. However, it is not fully understood whether the expression of this enzyme is directly related to the crop yield. In this context, this research work evaluated the expression of the amo-A gene of bacterial and archaeal communities present in a rice field previously characterized by environments. Real-time PCR (RT-qPCR) and digital PCR (RT-dPCR) techniques were used to quantify the abundance of archaea and ammonium-oxidizing bacteria (AOA and AOB, respectively). In this work it was found that in the analysis of metagenomic data there was a greater presence of AOB in rhizospheric soil samples while AOA were predominant in bulk soil samples, however, the expression of the amo-A gene was not detected. associated with the community of bacteria in the soil samples analyzed. On the other hand, it was found that the transcripts of the amo-A gene of the AOA community did not present differences between the characterized environments. Furthermore, the expression of transcripts is not related to any of the chemical properties evaluated. Finally, the quantification strategies for RT-qPCR (plasmid and quenching) turned out to be homologous and functional to identify the expression of the AOA amo-A gene, while the RT-dPCR technique was more precise for the analysis of the community of AOB and AOA.
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ABSTRACT The oral cavity constitutes a unique ecosystem with highly variable ecological niches that harbor a great variety of microorganisms, including yeasts. Molecular methods are currently considered the gold standard for identifying species, although they involve limitations associated with the disruption of yeast cell walls to release the genomic DNA (gDNA) for amplification. Aim: The aim of this study was to compare the performance of different methods for extracting gDNA from Candida albicans and Candida dubliniensis, subsequently amplifying DNA by PCR. Materials and Method: Fifty-two isolates (16 C. albicans and 36 C. dubliniensis) were obtained from subgingival biofilm of HIV+ patients with clinical signs of periodontal disease. The study evaluated 6 gDNA extraction methods and two PCR amplification methods. Furthermore, the presence of alleles of HWP1 gene was determined in C. albicans. Results: Comparisons of six methods show statistically significant differences (p<0.001) except for C. albicans in two of them. For C. dubliniensis, statistical differences were observed in all comparisons. Commercial methods were more efficient for concentrating gDNA than in-house methods, and both PCRs were effective. Ten heterozygous C. albicans isolates for this allele were positive for the HWP1-1 / HWP1-2 allele, one was homozygous for Wild Type HWP1-1 allele, and 5 were homozygous for novel/rare HWP1-2 allele. Conclusions: This study aims to provide simple, inexpensive strategies for phenotypic identification and molecular confirmation of Candida albicans and Candida dubliniensis for non-reference laboratories with low complexity and/or low budgets.
RESUMEN La cavidad oral constituye un ecosistema único con nichos ecológicos muy variables, capaz de albergar una gran variedad de microorganismos, incluidas las levaduras. Los métodos moleculares son considerados actualmente los métodos de identificación definitivos ya que a diferencia de los anteriores, nos brindan una correcta sensibilidad y especificidad. Sin embargo, existen limitaciones asociadas con la ruptura de las paredes celulares de estas levaduras para liberar el ADN genómico (gADN) necesario para la amplificación. Objetivo: El objetivo de este estudio fue comparar el rendimiento de diferentes métodos de extracción de gADN de Candida albicans y Candida dubliniensis, amplificando posteriormente por PCR. Materiales y Método: Se estudiaron 52 aislamientos, 16/52 de Candida albicans y 36/52 de Candida dubliniensis obtenidos de biofilm subgingival de pacientes VIH+ con signos clínicos de enfermedad periodontal. Se evaluaron seis métodos de extracción de gADN y la posterior amplificación se realizó por dos técnicas de PCR. Además en C. albicans se determinó la presencia de alelos para el gen HWP1. Resultados: Las comparaciones de seis métodos son estadísticamente significativas (p<0,001) excepto para C. albicans en dos de ellos. Para C. dubliniensis se observaron diferencias estadísticas en todas las comparaciones. Los métodos comerciales mostraron una mayor eficiencia en la concentración de gADN que los métodos caseros y ambos fueron efectivos en las dos PCR. 10 aislados de C. albicans resultaron positivos para el alelo HWP1-1/HWP1-2, siendo heterocigotos para este alelo. Solo un aislamiento fue homocigoto para el alelo HWP1-1 de tipo salvaje y 5 eran homocigotos para el alelo HWP1-2 nuevo/raro. Conclusiones: Este estudio tiene como objetivo proporcionar estrategias simples y económicas para la identificación fenotípica y confirmación molecular de Candida albicans y Candida dubliniensis para laboratorios de no referencia con baja complejidad y/o bajo presupuesto económico.
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Abstract Bovine pestiviruses are the causative agents of bovine viral diarrhea, a disease thatcauses severe economic losses in cattle. The aim of this study was to improve their diagnosisby developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set up a protocolfor collecting, shipping and preserving bovine pestiviral RNA on filter papers. The developedRT-qPCR showed high sensitivity in detecting these viruses in different matrices: viral stocks,semen and serum samples. With regard to the possibility of using the technique to test serumpools, it was possible to identify a positive serum sample within a pool containing 30 sera.In addition to evaluating the qPCR from fresh samples, the use of filter papers to sow bovinesamples was analyzed. The sampling method on two different filter papers using bovine blooddrops was a useful alternative for diagnostic purposes and allowed to preserve pestiviral RNAfor up to 12 months under refrigeration.
Resumen Los Pestivirus bovinos son los agentes causales de la diarrea viral bovina, una enfermedad que genera importantes pérdidas económicas en el ganado vacuno. El objetivo de este trabajo fue mejorar su diagnóstico mediante el desarrollo de una RT-qPCR para detectar los Pestivirus bovinos A, B y H y disenar un protocolo de recolección, envío y conservación de ARN viral en papeles de filtro. La RT-qPCR desarrollada demostró alta sensibilidad en la detección de estos virus en diferentes matrices: stock viral, suero y semen. Respecto de la posibilidad de usar la técnica para testear pools de suero, fue posible identificar un suero positivo dentro de un pool compuesto por 30 sueros. Además de evaluar la qPCR en muestras frescas, se analizó el uso de papeles de filtro para sembrar muestras de bovinos. La metodología de toma de muestras en dos tipos de papeles de filtro usando gotas de sangre fue una alternativa útil para el diagnóstico y permitió conservar ARN viral por hasta 12 meses a temperaturas de refrigeración.
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Aims: Lactate acid functions as not only an energy source but a signaling molecule through the lactate receptor GPR81 under physiological conditions. However, the pathological role of lactic acid in the tumor microenvironment remains unclear, particularly for immune cells. Methodology: NK-92 cells were treated with L-lactic acid solutions at final concentrations of 10, 20, 30, and 40 mM, and its cell viability and cytotoxicity on A549 cells and A375 cells were evaluated by CCK8 assay and crystal violet assay, respectively. Furthermore, qPCR was used to assess the expression of GPR81 and cytotoxicity-related genes in NK-92 cells treated with antagonist and agonist. And their relationship between lactate/GPR81 pathway and cytotoxicity-related genes were analyzed by Pearson’s correlation. Results: The viability of NK-92 cells was inhibited by L-lactic acid with increasing concentration. Additionally, the cytotoxic activity against tumor cells of NK-92 cells treated with L-lactic acid decreased with increasing concentration. Moreover, qPCR results demonstrated that GPR81 can be activated by lactic acid or agonist (3,5-DHBA) and downregulate the expression cytotoxicity-related genes which included FASLG gene(Fas Ligand),TNF-? gene(Tumor necrosis factor-?), INFG gene (Interferon-?), RPF1 gene (Perforin 1), GZMA gene (Granzyme A), GZMB gene (Granzyme B), GZMH gene (Granzyme H), GAMK gene (Granzyme K) and GZMM gene (Granzyme M). And the expression of GPR81 returned to near-control level when treated with L-lactic acid in the presence of antagonist (3-OBA), the expression of cytotoxicity-related genes did as well. Pearson’s correlation analysis of cytotoxicity-related genes with GPR81 revealed that their correlation coefficient seems negative. Conclusion: Lactic acid can activate the GPR81 to downregulate the expression of cytotoxicity-related genes, subsequently lower the cytotoxicity of NK-92 cells.
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Objective: To investigate the expression of CD24 gene in human malignant pleural mesothelioma (MPM) cells and tissues, and evaluate its relationship with clinicopathological characteristics and clinical prognosis of MPM patients. Methods: In February 2021, UALCAN database was used to analyze the correlation between CD24 gene expression and clinicopathological characteristics in 87 cases of MPM patients. The TIMER 2.0 platform was used to explore the relationship between the expression of CD24 in MPM and tumor immune infiltrating cells. cBioportal online tool was used to analyze the correlation between CD24 and MPM tumor marker gene expression. RT-qPCR was used to analyze the expressions of CD24 gene in human normal pleural mesothelial cell lines LP9 and MPM cell lines NCI-H28 (epithelial type), NCI-H2052 (sarcoma type), and NCI-H2452 (biphasic mixed type). RT-qPCR was performed to detect the expressions of CD24 gene in 18 cases of MPM tissues and matched normal pleural tissues. The expression difference of CD24 protein in normal mesothelial tissue and MPM tissue was analyzed by immunohistochemistry. A Kaplan-Meier model was constructed to explore the influence of CD24 gene expression on the prognosis of MPM patients, and Cox regression analysis of prognostic factors in MPM patients was performed. Results: The CD24 gene expression without TP53 mutation MPM patients was significantly higher than that of patients in TP53 mutation (P<0.05). The expression of CD24 gene in MPM was positively correlated with B cells (r(s)=0.37, P<0.001). The expression of CD24 gene had a positive correlation with the expressions of thrombospondin 2 (THBS2) (r(s)=0.26, P<0.05), and had a negative correlation with the expression of epidermal growth factor containing fibulin like extracellular matrix protein 1 (EFEMP1), mesothelin (MSLN) and calbindin 2 (CALB2) (r(s)=-0.31, -0.52, -0.43, P<0.05). RT-qPCR showed that the expression level of CD24 gene in MPM cells (NCI-H28, NCI-H2052 and NCI-H2452) was significantly higher than that in normal pleural mesothelial LP9 cells. The expression level of CD24 gene in MPM tissues was significantly higher than that in matched normal pleural tissues (P<0.05). Immunohistochemistry showed that the expressions of CD24 protein in epithelial and sarcoma MPM tissues were higher than those of matched normal pleural tissues. Compared with low expression of CD24 gene, MPM patients with high expression of CD24 gene had lower overall survival (HR=2.100, 95%CI: 1.336-3.424, P<0.05) and disease-free survival (HR=1.800, 95%CI: 1.026-2.625, P<0.05). Cox multivariate analysis showed that compared with the biphasic mixed type, the epithelial type was a protective factor for the prognosis of MPM patients (HR=0.321, 95%CI: 0.172-0.623, P<0.001). Compared with low expression of CD24 gene, high expression of CD24 gene was an independent risk factor for the prognosis of MPM patients (HR=2.412, 95%CI: 1.291-4.492, P=0.006) . Conclusion: CD24 gene and protein are highly expressed in MPM tissues, and the high expression of CD24 gene suggests poor prognosis in MPM patients.
Subject(s)
Humans , Mesothelioma, Malignant , Mesothelioma/diagnosis , Lung Neoplasms/genetics , Pleural Neoplasms/diagnosis , Prognosis , Biomarkers, Tumor/analysis , Extracellular Matrix Proteins , CD24 Antigen/geneticsABSTRACT
Several agents can cause hemoparasitic diseases in dogs, and blood-sucking arthropods transmit these diseases. These agents can cause several clinical manifestations and, in some cases, can kill the host. Because these agents are essential in animal health, this study aims to detect the frequency of Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, and Rangelia vitalii by real-time PCR and Babesia vogeli in dogs in the southern region of the city of São Paulo, São Paulo. Of the 98 dog samples, 18 (18.4%) tested positive with real-time polymerase chain reaction for at least one studied agent. Of these 18 samples, 17 tested positive for a single agent (11.2% for B. canis vogeli, 1.02% for R. vitalii, and 5.1% for E. canis), and one showed co-infection with B. canis vogeli and R. vitalii. The results demonstrate the presence of hemoparasites in the studied animals, which can influence the quality and life expectancy of these animals. The Rangeliadetection warns small animal clinicians to include it as a differential diagnosis for hemoparasitosis.(AU)
As hemoparasitoses em cães podem ser causadas por diversos agentes, sendo essas doenças transmitidas por artrópodes hematófagos. Esses agentes podem causar diversas manifestações clínicas e, em alguns casos, podem matar o hospedeiro. Este estudo teve como objetivo detectar por PCR em tempo real a frequência de Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, Rangelia vitalii e Babesia canis vogeli em amostras de cães da zona sul da cidade de São Paulo, Brasil. Das 98 amostras de cães, 18 (18,4%) testaram positivo com reação em cadeia da polimerase em tempo real para pelo menos um agente estudado. Destas 18 amostras, 17 testaram positivo para um único agente (11,2% para B. canis vogeli, 1,02% para R. vitalii e 5,1% para E. canis), e uma apresentou coinfecção com B. canis vogeli e R. vitalii. Os resultados demonstram a presença de hemoparasitas nos animais estudados, o que pode influenciar a qualidade e a expectativa de vida desses animais. Além disso, é o primeiro relato da detecção de R. vitalli na zona sul de São Paulo e serve de alerta para os clínicos de pequenos animais incluírem esse agente como diagnóstico diferencial para as hemoparasitoses.(AU)
Subject(s)
Animals , Protozoan Infections/diagnosis , Babesiosis/diagnosis , Ehrlichiosis/diagnosis , Dogs/microbiology , Brazil , Polymerase Chain Reaction/veterinary , Piroplasmida , Molecular Diagnostic Techniques/veterinary , Ehrlichia canisABSTRACT
ABSTRACT Background: Leptospirosis represents a One Health issue, affecting humans and animals. This study investigated pathogenic leptospires in small wild rodents in São Paulo, Brazil. Methods: Kidney samples from 164 rodents underwent qPCR testing, targeting pathogenic Leptospira spp. Results: Thirty-five animals (21.34%) tested positive, including five rodent species: Akodon montensis (2/21; 9.5%), Necromys lasiurus (1/4; 25%), Oligoryzomys nigripes (24/92; 26.1%), Oligoryzomys flavescens (5/26; 19.2%), and Sooretamys angouya (3/14; 21.4%). Botucatu municipality exhibited the highest prevalence, with 42.5% (20/47) of the animals testing positive. Conclusions: The presence of Leptospira spp. in wild rodents suggests they may be chronic carriers, contaminating the environment.
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Abstract Respiratory Syncytial Virus (RSV) poses a global health concern, particularly affecting young children, the elderly, and immunosuppressed individuals. RSV viral load is essential for understanding transmission, disease severity, prevention, and treatment. This retrospective study aimed to analyze the frequency rates and viral loads of RSV infections in different patient cohorts and age groups over an eight-year period in a university hospital in São Paulo, Brazil. This study analyzed 1380 Immunocompetent (IC) and Immunosuppressed (IS) patients with acute respiratory tract infections. IC included patients with chronic Heart Disease (HD), Primary Care service recipients (PC), and a subgroup suspected of having Severe Acute Respiratory Syndrome caused by Influenza A (H1N1)pdm09 virus (SARS H1N1). IS comprised transplant patients and those with HIV infection. Respiratory samples were collected between February 2005 and October 2013, with RSV detection and viral load quantification (Log10 copies of RNA/mL) using RT-qPCR. Overall RSV infection rate was 17.3 %, with higher rates in children (23.9 %) than in adults (12.9 %), particularly in children under two years of age (28.2 %). Children in the SARS H1N1 and PC subgroups had higher infection rates (16.4 % and 34.9 %, respectively), with the highest rate in PC children aged 1 to < 2 years (45.45 %). Adults with HD had a significantly higher frequency rate (27.83 %) than those in the SARS H1N1 (2.65 %) and IS (15.16 %) subgroups and higher hospitalization rate among adults under 65 years. RSV viral load ranged from 2.43 to 10.15 Log10 RNA copies/mL (mean ± SD 5.82 ± 2.19), with hospitalized patients exhibiting significantly higher viral loads (7.34 ± 1.9) than outpatients (4.38 ± 1.89). Elderly bone marrow transplant patients also had significantly higher viral loads (7.57 ± 2.41) than younger adults (5.12 ± 1.87). This study provides insights into the RSV infection patterns in different patient cohorts in Brazil. Further investigations are needed to understand susceptibility and risk factors associated with RSV infection. In conclusion, high RSV viral load among hospitalized patients could serve as a surrogate marker of disease severity. Additionally, patients with chronic heart disease deserve greater attention regarding complications associated with RSV infection.
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ABSTRACT Background: This study aimed to describe the kinetics of Leishmania parasite load determined using kinetoplast DNA (kDNA)-based quantitative polymerase chain reaction (qPCR) in visceral leishmaniasis (VL) patients. Methods: Parasite load in blood was assessed by qPCR at five time points, up to 12 months post-diagnosis. Sixteen patients were followed up. Results: A significant reduction in the parasite load was observed after treatment (P < 0.0001). One patient had an increased parasite load 3 months post-treatment and relapsed clinically at month six. Conclusions: We have described the use of kDNA-based qPCR in the post-treatment follow-up of VL cases.
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@#Objective To develop and verify a qPCR method for the qualitative and quantitative analysis of residual host DNA in human rabies vaccine(Vero cells) stock solution.Methods The qPCR standard curve was established by using the Vero cell DNA quantitative national standard,and the residual host DNA was extracted using magnetic beads.The specificity,repeatability,intermediate precision,accuracy and durability of the method were verified,and the linear range and limit of quantification were determined.The residual DNA of three batches of human rabies vaccine(Vero cells) stock solution was quantitatively analyzed and the fragment size was qualitatively analyzed by using this method.Results The correlation coefficients(R~2) of Vero cell DNA quantitative national standard amplification standard curve were all more than 0.99 by qPCR,and the quantitative range was 0.3 pg/mL～30 ng/mL.The method showed good specificity and repeatability.In the verification of intermediate precision,accuracy and durability,the relative standard deviations(RSD)of detection results of the samples were all less than 10%.The residual DNA content of Vero cells in three batches of stock solution was 0.20～0.77 ng/dose,which met the relevant standard of Chinese Pharmacopoeia(Volume Ⅲ,2020edition).The residual DNA fragments greater than 154 bp accounted for 52%～63%.Conclusion The developed qPCR method for the detection of residual DNA in human rabies vaccine(Vero cells) stock solution had good specificity,repeatability,intermediate precision and durability,and qualitatively and quantitatively analyzed the residual DNA rapidly and accurately,which was of great significance for improving the detection and control of residual DNA content in the production process and final product of human rabies vaccine(Vero cells).
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Objective:To develop a rapid, simple and cost-effective quantitative TaqMan RT-PCR (RT-qPCR) that could be used as an alternative to sequencing for the detection of Omicron variants and to evaluate its performance.Methods:Primers and TaqMan probes targeting the conserved domains of SARS-CoV-2 ORF1ab and the high-frequency mutation sites in the S gene of Omicron variants were designed. Then a RT-qPCR for the detection of Omicron variants was established. The consistency of the method was verified using samples identified by whole-genome sequencing. The specificity and sensitivity of the method were also evaluated.Results:The established RT-qPCR could distinguish Omicron variants from early epidemic A strains and Alpha and Delta variants of SARS-CoV-2, and the results were consistent with those of whole-genome sequencing with a coincidence rate of 100% (28/28). There was no cross-reactivity with other six respiratory viruses or coxsackievirus group A16. For RNA standards, this method showed good linearity in the range of 10 9-10 3 copies/μl with a correlation coefficient ( R2) greater than 0.99 and detection sensitivity of 10 3 copies/μl. Conclusions:The RT-qPCR designed in this study for Omicron variant detection had good sensitivity and specificity and could be easily performed in laboratories, which would greatly facilitate the monitoring of Omicron variants.
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La papa (Solanum tuberosum) Diacol Capiro es uno de los cultivares con mayor producción y consumo interno en Colombia, siendo los departamentos de Cundinamarca y Boyacá, los principales productores. Este cultivo, se ve afectado por un complejo de virus, que disminuye la calidad de los tubérculos y los rendimientos. En este trabajo, se evaluó la prevalencia de los virus de ARN: PLRV, PVY, PVX, PVS, PVV, PYVV, PMTV y PVB, en brotes de tubérculos-semilla certificados, provenientes de la sabana Cundiboyacense, mediante RT-qPCR. Los resultados revelan la ocurrencia de siete de los ochos virus en las muestras, con niveles de infección de 100 % (PVS, PVX y PYVV), 75 % (PLRV), 50 % (PVY), 37,5 % (PMTV) y 12,5 % (PVB). Adicionalmente, con el fin de obtener información de los genomas de los virus detectados, se utilizó secuenciación de alto rendimiento (HTS), de una muestra compuesta (bulk) de brotes, siendo posible obtener el genoma completo del PLRV y el genoma parcial del PVY. Los análisis filogenéticos realizados con dichas secuencias ubicaron a los virus PLRV y PVY en clados, conformados por aislamientos colombianos, con niveles de identidad superiores al 97 %. Estos hallazgos evidencian la necesidad de fortalecer los programas de certificación de tubérculos-semilla de papa en el país, mediante la utilización de pruebas moleculares de detección viral.
Diacol-Capiro is one of the most important potato (Solanum tuberosum) cultivars in Colombia with most production concentrated in the provinces of Cundinamarca and Boyacá. Unfortunately, this crop is seriously affected by several viruses that compromise the quality of tubers and yields. In this work, it was evaluated the prevalence of the RNA viruses: PLRV, PVY, PVX, PVS, PVV, PYVV, PMTV, and PVB in certified tuber-seed sprouts produced in the highlands of Cundinamarca and Boyacá by RT-qPCR. Results revealed a prevalence of 100 % for PVS, PVX, and PYVV; 75 % for PLRV, 50 % for PVY, 37.5 % for PMTV, and 12.5 % for PVB. Additionally, high-throughput sequencing from a sprout´s bulk sample was used to gather genomic information of infecting viruses, which resulted in a partial PVY sequence, and a complete PLRV genome. Phylogenetic analysis revealed that both assemblies cluster within clades comprising other Colombian isolates with more than 97 % nucleotide sequence identity. These findings highlight the need to update potato seed-tuber certification programs in Colombia with the implementation of more sensitive molecular tests.
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El objetivo de este estudio fue determinar la presencia del Virus Papiloma Humano (VPH) tipo 16 y 18 en biopsias de tejido mamario parafinado de pacientes con diagnóstico clínico de cáncer de mama. Se analizaron 32 biopsias de cáncer de mama embebidas en parafina para detectar el ADN de VPH mediante PCR en tiempo real, los iniciadores estuvieron dirigidos al gen E6. Se evaluaron el tipo histológico, grado histológico y la sobreexpresión de C-erB2 y Ki-67 mediante inmunohistoquímica. El 84,38% (27) fueron positivos para VPH, el 25% (8) fueron positivos para VPH-16 y el 59,38% (19) para VPH-18. El 15,63% (5) de las muestras presentaron infección mixta. Se evidenció la sobrexpresión de C-erbB2 y Ki-67 en 6,25% (2) de las muestras positivas para VPH-16 y 15,63% (5) de las muestras positivas para VPH-18. Se detectó ADN de VPH-16 y VPH-18 en las muestras de biopsias analizadas mediante PCR en tiempo real.
The aim of this study was to determine the presence of Human Papillomavirus (HPV) type 16 and 18 in biopsies of paraffin-embedded breast tissue from patients with clinically diagnosed breast cancer. 32 paraffin-embedded breast cancer biopsies were analyzed in order to detect HPV DNA by real-time PCR, the primers were directed at the E6 gene. The histological type, histological grade and overexpression of C-erB2 and Ki-67 were evaluated by immunohistochemistry. 84.38% (27) of the samples were positive for HPV, 25% (8) were positive for HPV-16 and 59.38% (19) were positive for HPV-18. Mixed infection was found in 15.63% (5) of the samples. Overexpression of C-erbB2 and Ki-67 was seen in 6.25% (2) of the samples positive for HPV-16 and in 15.63% (5) samples positive for HPV-18. HPV-16 and HPV-18 DNA was detected in the biopsy samples analyzed by real-time PCR.
Subject(s)
Humans , Female , Breast Neoplasms , Human papillomavirus 16 , Human papillomavirus 18 , Papillomaviridae , Tissues , Biopsy , Immunohistochemistry , Clinical Diagnosis , Polymerase Chain ReactionABSTRACT
Abstract Objective To evaluate the sensitivity and specificity of the quantitative real-time polymerase chain reaction (qPCR) for 16S rDNA gene screening using sonicated fluid from orthopedic implants. Methods A retrospective study was conducted on 73 sonicated fluids obtained from patients with infection associated with orthopedic implants. The samples were subjected to conventional culture and molecular testing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and qPCR for 16S rDNA. The cycle threshold values were used to define a cut-off of the qPCR of the 16S rDNA for negative and positive cultures. Results No statistical differences were observed between the positive and negative culture groups based on the time from the first surgery to infection (p= 0.958), age (p =0.269), or general comorbidities. Nevertheless, a statistical difference was found between the mean duration of antibiotic use before device removal (3.41 versus 0.94; p =0.016). Bacterial DNA was identified in every sample from the sonicated fluids. The median cycle thresholds of the positive and negative cultures were of 25.6 and 27.3 respectively (p< 0.001). As a diagnostic tool, a cycle threshold cut-off of 26.89 demonstrated an area under the curve of the receiver operating characteristic of 0.877 (p≤ 0.001). Conclusion The presence of antimicrobial agents for more than 72 hours decreased culture positivity, but did not influence the qPCR results. Despite this, amplification of the 16S rDNA may overestimate infection diagnosis.
Resumo Objetivo Avaliar a sensibilidade e a especificidade da reação em cadeia de polimerase em tempo real quantitativa (quantitative real-time polymerase chain reaction, qPCR, em inglês) para a triagem do gene rDNA 16S, com a utilização do fluido sonicado de implantes ortopédicos. Métodos Um estudo retrospectivo foi realizado em 73 fluidos sonicados obtidos de pacientes com infecção associada aos implantes ortopédicos. As amostras foram submetidas a cultura convencional e a teste molecular utilizando ionização e dessorção a laser assistida por matriz com espectrometria de massa por tempo de voo (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS, em inglês) e qPCR para o gene rDNA 16S. Os valores limiares do ciclo foram usados para definir um ponto de corte para a qPCR do gene rDNA 16S para culturas negativas e positivas. Resultados Não foram observadas diferenças estatísticas entre os grupos de cultura positiva e negativa com base no tempo desde a primeira cirurgia até a infecção (p= 0,958), na idade (p= 0,269), ou nas comorbidades em geral. No entanto, uma diferença estatística foi encontrada entre a duração média do uso de antibióticos antes da remoção do dispositivo (3,41 versus 0,94; p= 0,016). O DNA bacteriano foi identificado em todas as amostras dos fluidos sonicados. Os limiares do ciclo médio de culturas positivas e negativas foram de 25,6 e 27,3, respectivamente (p< 0,001). Como uma ferramenta de diagnóstico, um corte do limite do ciclo de 26,89 demonstrou uma área sob a curva da característica de operação do receptor de 0,877 (p ≤ 0,001). Conclusão A presença de agentes antimicrobianos por mais de 72 horas diminuiu a positividade da cultura, mas não influenciou os resultados da qPCR. Apesar disso, a amplificação do rDNA 16S pode sobrestimar o diagnóstico de infecção.
Subject(s)
Humans , Prostheses and Implants/microbiology , Sonication , Polymerase Chain Reaction , Retrospective Studies , Infection Control , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Anti-Infective AgentsABSTRACT
Background & objectives: Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations. Methods: Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes. Results: In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x10-1 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV. When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV. All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100 . In full combination assays, CHIKV was detected to 1.32x10-1, DENV1-4 to 3.79x101 and ZIKV to 1.07x100 . Interpretation & conclusion: This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.
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ABSTRACT Objective: Despite the treatment of anovulation, infertility is still one of the main complications in PCOS women during reproductive age, which appears to be mainly due to impaired uterine receptivity. This study investigated the transcriptome profiles of endometrium in PCOS patients and healthy fertile individuals as the control group. Material and methods: Total mRNA was extracted from endometrial tissues of PCOS patients (n = 12) and healthy fertile individuals (n = 10) during the luteal phase. After cDNA synthesis, PCR array was performed using Human Female Infertility RT² Profiler PCR Array kit (Qiagen, Cat. No: PAHS-164Z) for evaluating expression of 84 genes contributing to the female infertility. Results: PCR Array data analysis identified significantly greater expression of CSF, IL11, IL15, IL1r1, IL1b, TNF, LIF, TNFRSF10B, TGFβ, C3, ITGA4 (Cd49d), SPP1, and Calca in PCOS women than in controls (P < 0.05). However, the expression of LIFR, C2, CD55, CFD, CALCA, LAM1, LAMC2, MMP2, MMP7, MMP9, ESR, SELL, ITGB3, and VCAM1 was significantly lower in PCOS group than in controls (P < 0.05). The results revealed dysregulation of immune-inflammatory molecules, complement activation and downregulation of IGF-I as well as adhesion molecules in PCOS group. Conclusion: The findings of this study indicated some potential causes of reduced receptivity of endometrium thus compromising the fertility in PCOS patients.
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Objective:To investigate the expression of promyelocytic leukemia zinc finger (PLZF) in human peripheral blood with asthma and its clinical significance.Methods:Forty patients with stable asthma from May 2021 to October 2021 in the Department of Respiratory Medicine of the Shanghai Fifth People's Hospital were enrolled, and forty healthy controls were recruited in the study. The levels of cytokines in serum were measured by enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR (qPCR) was used to detect the expression of PLZF mRNA in plasma. The level and distribution of PLZF+ cells in PBMCs were detected by flow cytometry after isolating peripheral blood mononuclear cells (PBMCs). Independent sample t test, Mann-Whitney U test, χ 2 test, ROC curve and Logistic regression were used to analyze the results with SPSS 26.0 and Graphpad Prism 7.0. A P<0.05 was considered statistically significant. Results:The levels of cytokines IFN-γ, IL-2, IL-4, TNF-α and IL-17 in human peripheral blood from the asthma group were obviously higher than those in the control group ( P<0.05), whereas there was no significant difference in the level of cytokine IL-10 between the two groups. The level of PLZF mRNA in PBMCs from the asthma group was significantly up-regulated compared to that in the control group [(3.40%±2.52%) vs. (1.23%±0.78%), P<0.05]. CD8+PLZF+ and Vβ11+PLZF+T cells in the asthma group were significantly outnumbered than those in the control group ( P<0.05). Logistic regression and ROC curve analysis showed that PLZF expression in PBMC was a risk factor for the development of asthma ( OR =3.67, AUC=0.87, P<0.05). Conclusions:The high expression of PLZF in peripheral blood may play an important role in the development of asthma, which needs to be further confirmed by large sample studies.
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In forensic physical evidence identification, the accurate identification of the individual origin and their body fluid composition of the biological samples obtained from the crime scene play a critical role in determining the nature of a crime. In recent years, RNA profiling has become one of the fastest developing methods for body fluids identification. Due to the characteristics of tissue or body fluid specific expression, various types of RNA markers have been proven to be promising candidate markers for body fluids identification in previous studies. This review summarizes the research progress of RNA markers in body fluids identification, including the RNA markers that have been effectively verified in current research and their advantages and disadvantages. Meanwhile, this review prospects the application of RNA markers in forensic medicine.
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Forensic Medicine/methods , Body Fluids/chemistry , RNA/analysis , Feces , Forensic Genetics , Semen/chemistry , Saliva/chemistryABSTRACT
In criminal investigations, postmortem interval (PMI) is important information to be inferred in homicide investigations, as well as the focus and the difficulty in forensic pathology research. Because the DNA content in different tissues is relatively constant and shows changes regularly with the extension of PMI, it has become a research hotspot of PMI estimation. This paper reviews the recent progress of PMI estimation technologies including DNA-based single cell gel electrophoresis, image analysis, flow cytometry, real-time fluorescence quantitative PCR and high-throughput sequencing, hoping to provide references for forensic medicine practice and scientific research.