PCR múltiplex para el diagnóstico de microorganismos atípicos transmitidos sexualmente / Multiplex PCR testing for the atypical microorganisms diagnosis sexually transmitted

Resumen Antecedentes: Las infecciones de transmisión sexual son un problema de salud pública mundial. El análisis rutinario incluye solo pruebas microbiológicas y serológicas para el diagnóstico de patógenos. Los microorganismos atípicos como Chlamydia trachomatis y micoplasmas no son identificados debido a los requerimientos. Además, no es incluida Gardnerella vaginalis, aunque se asocia a la vaginosis bacteriana. Objetivo: Desarrollar una PCR múltiplex para el diagnóstico de C. trachomatis, micoplasmas y G. vaginalis. Método: Se estandarizó la PCR múltiplex utilizando oligonucleótidos para C. trachomatis (gen ompA, orf6 plasmídico), Mycoplasma/Ureaplasma y G. vaginalis (genes rRNA16s). Resultados: Se estandarizaron pruebas de PCR múltiplex para los microorganismos estudiados, optimizándose las concentraciones y condiciones de las reacciones múltiplex. Se obtuvieron PCR dúplex para C. trachomatis (ompA, orf6), Chlamydia/Gardnerella y Chlamydia/micoplasmas y tríplex para Chlamydia/Mycoplasma/Ureaplasma. También un cuádruplex para Chlamydia/Mycoplasma/Ureaplasma/Gardnerella. Los resultados fueron verificados por PCR e hibridación automática (HybriSpot 12) y análisis in silico. Conclusión: Se desarrollaron pruebas de PCR múltiplex con una alta sensibilidad y especificidad para la identificación de C. trachomatis, micoplasmas y G. vaginalis.

Abstract Background: Sexually transmitted infections are a global public health problem. Routine analysis includes microbiological and serological tests for the diagnosis of pathogens. Atypical microorganisms such as Chlamydia trachomatis and mycoplasmas are not determined due to the requirements for their identification. Furthermore, Gardnerella vaginalis is not included despite being associated with bacterial vaginosis. Objective: To develop a multiplex PCR to diagnose Chlamydia, mycoplasmas, and Gardnerella. Method: Standardization of multiplex PCR tests was carried out using oligonucleotides for the identification of Chlamydia (ompA gene, plasmid orf6), Mycoplasma/Ureaplasma and Gardnerella (rRNA16s genes). Results: Multiplex PCR tests were standardized for the microorganisms studied, optimizing the concentrations and conditions of the multiplex reactions. Duplex PCR was obtained for Chlamydia (ompA, orf6), Chlamydia/Gardnerella, and Chlamydia/mycoplasmas, and triplex PCR for Chlamydia/mycoplasmas. Also, a quadruplex for Chlamydia, Mycoplasma/Ureaplasma and Gardnerella. PCR and automatic hybridization verified the results obtained (HybriSpot 12) and in silico analysis. Conclusion: Multiplex PCR tests with high sensitivity and specificity were developed to identify C. trachomatis, mycoplasmas, and G. vaginalis.

Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus.

African swine fever virus (ASFV) was first identified in 1921 and is extensively prevalent around the world nowadays, which has a significant negative impact on the swine industry. In China, genotype II ASFV was first discovered in 2018, and has spread quickly to different provinces in a very short time; genotype I ASFV was first found in 2020, and has been reported in several provinces since then. To establish an accurate method for detection and differentiation of genotypes I and II ASFV, three primers and probes were designed targeting the ASFV B646L gene for different genotypes, the F1055L gene for genotype I, and the E183L gene for genotype II, and a triplex real-time quantitative PCR (qPCR) for differential detection of genotypes I and II ASFV was developed after optimizing the reaction conditions. The assay showed high sensitivity, and the limits of detection (LOD) of the B646L, F1055L, and E183L genes were 399.647 copies/reaction, 374.409 copies/reaction, and 355.083 copies/reaction, respectively; the coefficients of variation (CVs) of the intra-assay and the inter-assay were 0.22-1.88% and 0.16-1.68%, respectively, showing that this method had good repeatability; the assay could detect only ASFV, without cross-reactivity with other swine viruses including PRRSV, PEDV, PDCoV, CSFV, PRV, and PCV2, showing excellent specificity of this method. A total of 3,519 clinical samples from Guangxi province, southern China, were tested by the developed assay, and 8.16% (287/3,519) samples were found to be positive for ASFV, of which 0.17% (6/3,519) samples were positive for genotype I, 7.19% (253/3,519) samples for genotype II, and 0.80% (28/3,519) samples for genotypes I and II. At the same time, these clinical samples were also tested by a previously reported multiplex qPCR, and the agreement between these two methods was more than 99.94%. In summary, the developed triplex qPCR provided a fast, specific and accurate method for detection and differentiation of genotypes I and II ASFV.

Development of a Quadruplex RT-qPCR for the Detection of Porcine Rotaviruses and the Phylogenetic Analysis of Porcine RVH in China.

Rotavirus A species (RVA), RVB, RVC, and RVH are four species of rotaviruses (RVs) that are prevalent in pig herds, and co-infections occur frequently. In this study, a quadruplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of four porcine RVs was developed by designing specific primers and probes based on the VP6 gene of RVA, RVB, RVC, and RVH, respectively. The method showed high specificity and could only detect RVA, RVB, RVC, and RVH, without cross-reaction with other porcine viruses; showed excellent sensitivity, with a limit of detection (LOD) of 1.5 copies/µL for each virus; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.15-1.14% and inter-assay CVs of 0.07-0.96%. A total of 1447 clinical fecal samples from Guangxi province in China were tested using the developed quadruplex RT-qPCR. The results showed that RVA (42.71%, 618/1447), RVB (26.95%, 390/1447), RVC (42.92%, 621/1447), and RVH (13.68%, 198/1447) were simultaneously circulating in the pig herds, and the co-infection rate of different species of rotaviruses was found to be up to 44.01% (579/1447). The clinical samples were also detected using one previously reported method, and the coincidence rate of the detection results using two methods was more than 99.65%. The phylogenetic tree based on the VP6 gene sequences of RVH revealed that the porcine RVH strains from Guangxi province belonged to the genotype I5, which was closely related to Japanese and Vietnamese strains. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of RVA, RVB, RVC, and RVH was developed and applied to investigate the prevalence of porcine RVs in Guangxi province, China. This study is the first to report the prevalence of porcine RVH in China.

Triplex Crystal Digital PCR for the Detection and Differentiation of the Wild-Type Strain and the MGF505-2R and I177L Gene-Deleted Strain of African Swine Fever Virus.

African swine fever (ASF) is a severe and highly contagious viral disease that affects domestic pigs and wild boars, characterized by a high fever and internal bleeding. The disease is caused by African swine fever virus (ASFV), which is prevalent worldwide and has led to significant economic losses in the global pig industry. In this study, three pairs of specific primers and TaqMan probes were designed for the ASFV B646L, MGF505-2R and I177L genes. After optimizing the reaction conditions of the annealing temperature, primer concentration and probe concentration, triplex crystal digital PCR (cdPCR) and triplex real-time quantitative PCR (qPCR) were developed for the detection and differentiation of the wild-type ASFV strain and the MGF505-2R and/or I177L gene-deleted ASFV strains. The results indicate that both triplex cdPCR and triplex qPCR were highly specific, sensitive and repeatable. The assays could detect only the B646L, MGF505-2R and I177L genes, without cross-reaction with other swine viruses (i.e., PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV). The limit of detection (LOD) of triplex cdPCR was 12 copies/reaction, and the LOD of triplex qPCR was 500 copies/reaction. The intra-assay and inter-assay coefficients of variation (CVs) for repeatability and reproducibility were less than 2.7% for triplex cdPCR and less than 1.8% for triplex qPCR. A total of 1510 clinical tissue samples were tested with both methods, and the positivity rates of ASFV were 14.17% (214/1510) with triplex cdPCR and 12.98% (196/1510) with triplex qPCR, with a coincidence rate of 98.81% between the two methods. The positivity rate for the MGF505-2R gene-deleted ASFV strains was 0.33% (5/1510), and no I177L gene-deleted ASFV strain was found. The results indicate that triplex cdPCR and triplex qPCR developed in this study can provide rapid, sensitive and accurate methods for the detection and differentiation of the ASFV B646L, MGF505-2R and I177L genes.

Point-of-care multiplex molecular diagnosis coupled with procalcitonin-guided algorithm for antibiotic stewardship in lower respiratory tract infection: a randomized controlled trial.

OBJECTIVE: We aimed to show that coupling molecular syndromic respiratory panel (RP) testing with procalcitonin (PCT) measurement in the emergency department improves antibiotic (ATB) stewardship in lower respiratory tract infection. METHODS: Open-label, prospective, randomized interventional trial, conducted from 2019 to 2022 in an adult emergency department. Patients with a suspicion of lower respiratory tract infection were randomized into an intervention arm (PCT measurement and point-of-care BIOFIRE RP2.1 plus testing, accompanied by a recommended ATB algorithm) or a standard of care (SOC) arm (PCT allowed as current practice). The primary endpoint was the duration of antibiotic exposure. RESULTS: Four hundred fifty-one patients were randomized, median age 65 years (Q1-Q3: 49-77), the hospitalization rate was 59.9% (270/451), the median length of stay 5 days (Q1-Q3: 3 - 12), and the 28-day mortality rate 5.3% (23/451). The median duration of ATB exposure was 6 days (Q1-Q3: 0-9) and 5 days (Q1-Q3: 0-9) in the SOC and interventional arm respectively (p = 0.71). ATB was started in 29.6 % (67/226) and 33.8% (76/225) respectively (p = 0.54). The BIOFIRE RP2.1 plus identified at least one viral species in 112/225 patients (49.8%) of intervention arm. Two hundred twelve out of two hundred twenty-six (93.8%) SOC patients had PCT measurement. The adherence rate to algorithm in the intervention arm was 93.3 % (210/225). CONCLUSION: Displaying PCT and real-time RP results to emergency physicians failed to significantly reduce ATB exposure in lower respiratory tract infection suspicions. However, the median ATB duration and rate of initiation were already low in the SOC arm using PCT measurement routinely.

EOSAL-CNV for Easy and Rapid Detection of CNVs by Fragment Analysis : EOSAL: A Fast and Reliable New Method for CNV Detection.

Copy number variations (CNVs) are a type of genetic variation involving from 50 base pairs (bps) to millions of bps and, in a general point of view, can include alterations of complete chromosomes. As CNVs mean the gain or loss of DNA sequences, their detection requires specific techniques and analysis. We have developed Easy One-Step Amplification and Labeling for CNV Detection (EOSAL-CNV) by fragment analysis in a DNA sequencer. The procedure is based on a single PCR reaction for amplification and labeling of all fragments included. The protocol includes specific primers for the amplification of the regions of interest with a tail in each of the primers (one for forward and another for the reverse primers) together with primers for tail amplification. One of the primers for tail amplification is labeled with a fluorophore, allowing the amplification and labeling in the same reaction. Combination of several tail pairs and labels allows the detection of DNA fragment by different fluorophores and increases the number of fragments that can be analyzed in one reaction. PCR products can be analyzed without any purification on a DNA sequencer for fragment detection and quantification. Finally, simple and easy calculations allow the detection of fragments with deletions or extra copies. The use of EOSAL-CNV allows simplifying and reducing costs in sample analysis for CNV detection.

Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European genotype) and PRRSV type 2 (North American genotype) are prevalent all over the world. Nowadays, the North American genotype PRRSV (NA-PRRSV) has been widely circulating in China and has caused huge economic losses to the pig industry. In recent years, classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) have been the most common circulating strains in China. In order to accurately differentiate the circulating strains of NA-PRRSV, three pairs of specific primers and corresponding probes were designed for the Nsp2 region of C-PRRSV, HP-PRRSV, and NL-PRRSV. After optimizing the annealing temperature, primer concentration, and probe concentration, a multiplex real-time quantitative RT-PCR (qRT-PCR) and a multiplex Crystal digital RT-PCR (cdRT-PCR) for the differential detection of C-PRRSV, HP-PRRSV, and NL-PRRSV were developed. The results showed that the two assays illustrated high sensitivity, with a limit of detection (LOD) of 3.20 × 100 copies/µL for the multiplex qRT-PCR and 3.20 × 10-1 copies/µL for the multiplex cdRT-PCR. Both assays specifically detected the targeted viruses, without cross-reaction with other swine viruses, and indicated excellent repeatability, with coefficients of variation (CVs) of less than 1.26% for the multiplex qRT-PCR and 2.68% for the multiplex cdRT-PCR. Then, a total of 320 clinical samples were used to evaluate the application of these assays, and the positive rates of C-PRRSV, HP-PRRSV, and NL-PRRSV by the multiplex qRT-PCR were 1.88%, 21.56%, and 9.69%, respectively, while the positive rates by the multiplex cdRT-PCR were 2.19%, 25.31%, and 11.56%, respectively. The high sensitivity, strong specificity, excellent repeatability, and reliability of these assays indicate that they could provide useful tools for the simultaneous and differential detection of the circulating strains of C-PRRSV, HP-PRRSV, and NL-PRRSV in the field.

A Rare Case of a Prostatic Abscess Secondary to a Mycoplasma hominis Infection.

Mycoplasma hominis is one of the pathogenic organisms that may cause prostatitis with the development of a prostatic abscess in very rare cases. A 57-year-old man presented with lower urinary tract symptoms and low-grade fever. The transabdominal ultrasonography showed prostate enlargement suggesting acute prostatitis. The patient was started on empiric antibacterial therapy with fluoroquinolones. The urine and semen cultures showed no bacterial growth. A few days later, the patient presented again with symptoms progression and acute urinary retention. The transrectal ultrasound revealed diffuse calcifications and intraprostatic fluids. The computed tomography of the abdomen and pelvis showed a large abscess in the prostate with a periprostatic inflammatory reaction. While all bacterial cultures were negative, the multiplex polymerase chain reaction (PCR) test revealed a Mycoplasma hominis infection. The patient was managed with transurethral drainage. After six months of follow-up, the patient was free of symptoms and the repeat PCR study confirmed clearance of the Mycoplasma infection.

A Case of Frosted Branch Angiitis in the Age of Polymerase Chain Reaction: Diagnostic and Therapeutic Considerations.

To describe a unique case highlighting the limitations and caveats of multiplex polymerase chain reaction (mPCR) in the diagnosis of posterior infectious uveitis, specifically frosted branch angiitis (FBA), we present a case of FBA in which multiple diagnostic modalities, including mPCR, are inconclusive. A thorough literature review was carried out to discuss the validity of mPCR in the setting of posterior infectious uveitis, the theoretical effect of sample dilution, and to explore a management strategy in these difficult cases. It is known that mPCR has high sensitivity and specificity, with a low false negative rate. However, the rate of false negatives appears to increase in cases of FBA, and when samples are diluted. In such cases we suggest empiric treatment be initiated and targeted towards microorganisms most likely to be implicated based on exam and history.

Molecular techniques for the taxonomy of Aedes Meigen, 1818 (Culicidae: Aedini): A review of studies from 2010 to 2021.

The original description of Aedes Meigen in 1818, written in Latin, was very brief and included a single species, Aedes cinereus. In the last two decades the genus Aedes (Meigen, 1818) has undergone several revisions and reclassifications, with the current proposal being described by Wilkerson in 2015. However, the available keys for morphological identification are still not sufficient to differentiate cryptic species, damaged species, or those with confusing taxonomy. The current study aims to identify and describe the main taxonomic proposals and molecular methodologies available for the identification of the genus Aedes published between the years 2010 and 2021. The main molecular techniques used to identify the genus in the last 10 years, are: Multiplex PCR, DNA barcoding, nuclear and mitochondrial markers, environmental DNA, and bacterial microbiome analysis. This review highlights that there are catalogued data for only a few species of the genus Aedes, being restricted to medically important taxa such as Aedes albopictus and Aedes aegypti. The integrative taxonomy approach is a possibility to reconcile morphological and molecular data to improve species delimitation, contributing to future revisions of the genus.

A retrospective study examining the clinical significance of testing respiratory panels in children who presented to a tertiary hospital in 2019.

Background. Respiratory tract infections are a leading cause of hospital visits in the paediatric population and carry significant associated morbidity and mortality in this population. The introduction of respiratory panel testing has been said to guide clinicians in the overall management of patients. Methods. We conducted a retrospective study examining all respiratory panels carried out in our hospital during 2019 on paediatric patients. Patients included were those who had symptoms indicative of respiratory infections who presented acutely, including those with chronic respiratory conditions. A total of 188 respiratory panel results were obtained along with collected patient data. These were analysed using SPSS V. 25.0 to get the below mentioned results. Results. The majority (76.6 %) of patients were less than 3 years with 59 % of total population being males. The majority (80.9 %) had mild clinical severity score. The most common pathogen that was detected on the respiratory panel was Enterovirus Human Rhinovirus spp, followed by the influenza viruses. Only four cases were positive for bacterial pathogens (two Mycoplasma pneumoniae , one Bordetella pertussis and one Chlamydia pneumoniae ), which accounts for 2.1 % of all panels analysed. The significance of respiratory panels in influencing treatment were analysed in the forms of change of management plans before and after results of respiratory panels. This was observed in 14.4 % of patients who were not on any empiric medication and then based on panel results were started on medications, as well as 11.7 % who were on medications already, and the medications were altered based on the result of the panel (Chi square P=0.057). This was mainly seen with cases of influenza A H1N1 patients and to a lesser extent, Mycoplasma pneumonia. Conclusion. The use of respiratory panels in our hospital had little impact on patient care and management. The main organisms that influenced clinician decision in treatment were influenza A viruses and bacterial organisms ( Mycoplasma pneumoniae , Chlamydia pneumoniae and Bordetella pertussis ). Other than that, the use of clinical judgement proved more beneficial. We recommend use of specific testing for these organisms rather than the whole panel as case to case bases, which would be more cost-effective and consistent with patient management.

Utilidad del panel de PCR multiplex en el diagnóstico microbiológico temprano y adecuación antimicrobiana en pacientes críticos con neumonía / Usefulness of the multiplex PCR panel in the early microbiological diagnosis and antimicrobial suitability in critical patients with pneumonia / Utilidade do painel de PCR multiplex no diagnóstico microbiológico precoce e adequação antimicrobiana em pacientes críticos com pneumonia

Resumen: Introducción: el inicio temprano de la antibioticoterapia adecuada en infecciones graves se asocia con reducción de la mortalidad. La identificación precoz del microorganismo es fundamental para realizar un tratamiento dirigido y disminuir la terapéutica inicial inapropiada. Objetivo: valorar la utilidad de una técnica de biología molecular por amplificación de ácidos nucleicos mediante reacción en cadena de polimerasa en tiempo real para diagnóstico microbiológico temprano y adecuación de la antibioticoterapia en pacientes con neumonías graves. Metodología: estudio retrospectivo observacional llevado a cabo en la unidad de cuidados intensivos del Hospital Maciel. Se analizaron muestras respiratorias de pacientes con diagnóstico o sospecha de neumonía. Se compararon los resultados microbiológicos obtenidos por técnicas convencionales y por biología molecular multiplex (panel neumonía). Resultados: se incluyeron 53 muestras obtenidas de 51 pacientes. El multiplex detectó al menos un microorganismo en 38 (71,7%) muestras frente a 30 (56.6%) desarrollos en cultivos tradicionales. La mayoría de las muestras se obtuvieron bajo antibioticoterapia previa (86.8%). El panel neumonía mostró un porcentaje de concordancia positiva combinado de 100% y un porcentaje de concordancia negativa del 94% para la identificación bacteriana en comparación con los métodos microbiológicos tradicionales. En 27 (51%) casos el resultado del panel de neumonía determinó un cambio en la conducta terapéutica. Conclusiones: la técnica de PCR permite la identificación temprana de microorganismos causantes de neumonía optimizando la terapéutica empírica inicial y racionalizando el uso de antimicrobianos. Un panel negativo aleja el planteo de infección respiratoria a gérmenes habituales y permite considerar diagnósticos diferenciales en cuanto a foco y/o etiología.

Summary: Introduction: the early initiation of the adequate antibiotic therapy in severe infections is associated to a reduction in mortality. Early identification of the microorganism is essential to define directed therapy and decrease the initial inadequate treatment. Objective: to assess usefulness of a molecular biology technique by nucleic acid amplification through a polymerase chain reaction in real time for an early microbiological diagnosis and correction of the antibiotic therapy in patients with severe pneumonias. Method: retrospective, observational study conducted in the intensive care unit of Maciel Hospital. The respiratory samples of patients with a diagnosis of pneumonia or suspicious to have pneumonia were analyzed. The microbiological results obtained were compared using conventional techniques and multiplex molecular biology (pneumonia panel). Results: 53 samples obtained from 51 patients were included in the study. Multiplex detected at least one microorganism in 38 (71.7%) samples compared to 30 (56.6%) in traditional cultures. Most samples were obtained under the previous antibiotic therapy (86.8%). The pneumonia panel showed a combined positive agreement percentage of 100% and a negative agreement of 94% for the identification of bacteria when compared to the traditional microbiological methods. In 27 cases (51%) the pneumonia panel results determined changing the therapeutic behavior. Conclusions: the PCR technique allows for the early identification of microorganisms causing pneumonia, thus optimizing initial empirical therapy and rationalizing the use of antibiotics. A negative panel reduces the suspicion of a respiratory infection caused by the usual germs and enables considering differential diagnosis in terms of etiology or cause.

Resumo: Introdução: o início precoce da antibioticoterapia adequada em infecções graves está associado à redução da mortalidade. A identificação precoce do microrganismo é essencial para realizar o tratamento dirigido e reduzir o uso inicial inadequado de antimicrobianos. Objetivo: avaliar a utilidade de uma técnica de biologia molecular para amplificação de ácidos nucleicos por reação em cadeia da polimerase em tempo real para diagnóstico microbiológico precoce e adequação da antibioticoterapia em pacientes com pneumonia grave. Metodologia: estudo observacional retrospectivo realizado na unidade de terapia intensiva do Hospital Maciel. Amostras respiratórias de pacientes com diagnóstico ou suspeita de pneumonia foram analisadas. Os resultados microbiológicos obtidos por técnicas convencionais e por biologia molecular multiplex (painel de pneumonia) foram comparados. Resultados: foram incluídas 53 amostras obtidas de 51 pacientes. O multiplex detectou pelo menos um microrganismo em 38 (71,7%) amostras em comparação com 30 (56,6%) usando culturas tradicionais. A maioria das amostras foi obtida com antibioticoterapia prévia (86,8%). O painel de pneumonia mostrou uma concordância percentual positiva combinada de 100% e uma concordância percentual negativa de 94% para identificação bacteriana em comparação com métodos microbiológicos tradicionais. Em 27 (51%) casos, o resultado do painel de pneumonia determinou mudança no comportamento terapêutico. Conclusões: a técnica de PCR permite a identificação precoce de microrganismos causadores de pneumonia, otimizando a terapia empírica inicial e racionalizando o uso de antimicrobianos. Um painel negativo afasta a suspeita de infecção respiratória pelos germes usuais e permite considerar diagnósticos diferenciais em termos de foco e/ou etiologia.

SARS-CoV-2 RNA Detection on Environmental Surfaces in a University Setting of Central Italy.

The transmission of SARS-CoV-2 occurs through direct contact (person to person) and indirect contact by means of objects and surfaces contaminated by secretions from individuals with COVID-19 or asymptomatic carriers. In this study, we evaluated the presence of SARS-CoV-2 RNA on surfaces made of different materials located in university environments frequented by students and staff involved in academy activity during the fourth pandemic wave (December 2021). A total of 189 environmental samples were collected from classrooms, the library, computer room, gym and common areas and subjected to real-time PCR assay to evaluate the presence of SARS-CoV-2 RNA by amplification of the RNA-dependent RNA polymerase (RdRp) gene. All samples gave a valid result for Internal Process Control and nine (4.8%) tested very low positive for SARS-CoV-2 RNA amplification with a median Ct value of 39.44 [IQR: 37.31-42.66] (≤1 copy of viral genome). Our results show that, despite the prevention measures implemented, the presence of infected subjects cannot be excluded, as evidenced by the recovery of SARS-CoV-2 RNA from surfaces. The monitoring of environmental SARS-CoV-2 RNA could support public health prevention strategies in the academic and school world.

Tuberculosis in Cirrhosis - A Diagnostic and Management Conundrum.

Background: Diagnosis and management of tuberculosis (TB) in patients with cirrhosis remains challenging. We studied the clinical spectrum, diagnosis, and management of TB along with the assessment of the diagnostic utility of various laboratory investigations in this cohort. Methods: A retrospective review of records of patients with cirrhosis (July 2017 and December 2019) was done. Out of 30 patients with cirrhosis and TB, 20 patients with pleural/peritoneal TB (cases) were compared with 20 consecutively selected spontaneous bacterial peritonitis (SBP) controls. Composite of clinical, laboratory, radiologic features and response to antituberculosis therapy (ATT) was taken as the gold standard to diagnose TB. Results: Extrapulmonary TB (EPTB) (n = 23, 76.7%) was more common. Overall, 9 (30%) patients presented with ATT-induced hepatitis. Patients with pleural/peritoneal TB had less severe hepatic dysfunction as compared to SBP group with significantly lower CTP [8 ± 1.5 vs. 9 ± 1.7 (P = 0.01)], MELD [16.3 ± 5.8 vs. 20.2 ± 6.6 (P = 0.02)] and MELD-Na [18.8 ± 5.9 vs. 22.5 ± 7.1 (P = 0.03)] scores. Median ascitic/pleural fluid total protein [2.7 (2.4-3.1) vs. 1.1 (0.9-1.2); P < 0.0001] and adenosine deaminase (ADA) levels [34.5 (30.3-42.7) vs. 15 (13-16); P < 0.0001] were significantly higher in the TB group. Total protein levels had a sensitivity and specificity 81% and 93.3%, respectively, at cut off value of >2 g/dl with an AUROC of 0.89 [(0.79-0.96); P < 0.001] whereas ADA levels at cutoff >26 IU/L showed 80% sensitivity and 90% specificity to diagnose pleural/peritoneal TB with an AUROC of 0.93 [(0.82-0.97); P < 0.001]. Only 11 (36.7%), and 8 (26.6%) patients showed positivity on GeneXpert and mTB-PCR, respectively. Patients with Child-Turcotte-Pugh scores of ≤7 and 8-10 tolerated well two and one hepatotoxic drugs, respectively. Conclusions: EPTB is more frequent in patients with cirrhosis. Relatively lower cutoffs of ascitic/pleural fluid total protein and ADA may be useful to diagnose EPTB in patients with high pretest probability. Individualized ATT with close monitoring and dynamic modifications is effective and well-tolerated.

A Pipeline for the Development of Microsatellite Markers using Next Generation Sequencing Data.

Background: Also known as Simple Sequence Repetitions (SSRs), microsatellites are profoundly informative molecular markers and powerful tools in genetics and ecology studies on plants. Objective: This research presents a workflow for developing microsatellite markers using genome skimming. Methods: The pipeline was proposed in several stages that must be performed sequentially: obtaining DNA sequences, identifying microsatellite regions, designing primers, and selecting candidate microsatellite regions to develop the markers. Our pipeline efficiency was analyzed using Illumina sequencing data from the non-model tree species Pterodon emarginatus Vog. Results: The pipeline revealed 4,382 microsatellite regions and drew 7,411 pairs of primers for P. emarginatus. However, a much larger number of microsatellite regions with the potential to develop markers were discovered from our pipeline. We selected 50 microsatellite regions with high potential for developing markers and organized 29 microsatellite regions in sets for multiplex PCR. Conclusion: The proposed pipeline is a powerful tool for fast and efficient development of microsatellite markers on a large scale in several species, especially nonmodel plant species.

Detection by multiplex PCR of Mycoplasma species associated with dairy cattle in Argentina.

There is scarce information about the frequency and epidemiological and clinical features associated with the presence of Mycoplasma spp. in Argentine dairy herds. The objectives of this study were to develop a multiplex PCR for identifying M.bovis and M.canadense and to describe the frequency of Mycoplasma spp. isolated from clinical samples submitted to a diagnostic laboratory. Of a total of 1548 samples from intramammary infections, bulk tank milk and biological fluids, 38 Mycoplasma isolates were obtained. M. bovis, M. canadense, M.californicum and M.leachii were detected by using two multiplex PCRs, confirming their presence in clinical conditions in dairy cattle. The techniques used in the present study can be useful to broaden the knowledge about Mycoplasma infections in cattle, since the search for these organisms is not usually included in routine diagnoses.

Development of a multiplex qRT-PCR assay for detection of African swine fever virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus.

BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. OBJECTIVES: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. METHODS: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. RESULTS: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 100 copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. CONCLUSIONS: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

Use of Molecular Genetic Methods to Reduce the Risk of Incorrect Identification of Fish Strains in Brazilian Aquaculture.

The identification of fish species using traditional methods is generally based only on morphological characteristics and these methods are currently under review. This kind of identification of hybrid fishes solely based on their morphologies may be unreliable, especially when the samples include juveniles and post-F1 lineage fishes. Therefore, in the present study, we used molecular markers to accurately identify the fish species of economic interest that are used as materials in the projects developed in research institutions. We evaluated six lots of fishes sampled from different research centers, containing a total of 84 specimens acquired from private fish farms that were considered to be the representatives of pure species. Genetic analyses of all the specimens revealed that, globally, 22 samples (26.2%) were interspecific hybrids, while 20 (90.9%) samples were surprisingly characterized as post-F1 hybrids. This result confirms that hybrids are sold in markets without adequate labeling and also indicates the lack of proper control of the commercialization and management of stocks and products in fish farms. In addition, we determined that molecular diagnosis was an extremely effective and necessary method to test the reliability of biological materials currently used in scientific research.

Monitoring Sexually Transmitted Infections in Cervicovaginal Exfoliative Samples in Mexican Women.

BACKGROUND: Globally, Sexually Transmitted Infections (STIs) are a major cause of morbidity in sexually active individuals, having complications in reproduction health and quality of life. In concordance with the Sustainable Development Goals (SDG), the study aimed to investigate the prevalence of Candida spp., Ureaplasma spp., Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, HSV, and Mycoplasma spp. from cervicovaginal samples and to correlate them with the gynecological history of the patients. METHODS: Our analytical, prospective, and cross-sectional study included 377 women who participated in a reproductive health campaign during 2015-2016. Anthropometric and gynecological variables were obtained. Cervicovaginal specimens were collected and analyzed with a multiplex in-house PCR to detect Candida spp., Ureaplasma spp., Trichomonas vaginalis, Neisseria gonorrhoeae, HSV, Mycoplasma spp., and Chlamydia trachomatis. RESULTS: The positive cases were 175/377 (46.4%) to at least one of the microorganisms. The most frequent pathogen detected in this population was Ureaplasma spp. (n = 111, 29.4%), followed by Mycoplasma spp. (n = 56, 14.9%) and Candida spp. (n = 47, 12.5%); 33.7% of the positive cases were single infections, whereas 12.7% had coinfection. The multiplex PCR assay was designed targeting nucleotide sequences. CONCLUSIONS: Our data demonstrated that monitoring STIs among asymptomatic patients will encourage target programs to be more precisely and effectively implemented, as well as make these programs more affordable, to benefit society by decreasing the prevalence of STIs.

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]