Multiple myeloma and Chagas disease: qPCR as a marker for preemptive antiparasitic therapy: a case reports series and review

ABSTRACT Multiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56±32.10 months (mean±SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR.

Human papillomavirus DNA testing with the urine sample is not yet available: the accuracy of two distinct kits

SUMMARY OBJECTIVE: The aim of this study was to assess the results and efficiency of two real-time polymerase chain reaction procedures for detecting human papillomavirus utilizing urine samples. METHODS: This study comprised 151 patients who had previously tested positive for human papillomavirus in their cervical samples. Two different commercial real-time polymerase chain reaction techniques were used for identification and genotyping human papillomavirus in urine specimens. The urine samples of 151 patients were evaluated via the Roche Cobas test, and the urine samples of 91 patients were also evaluated via the Qiagen test. RESULTS: The overall consistency of urine and cervical swab specimens for the identification of human papillomavirus in Roche Cobas and Qiagen tests were 44.8 and 44%, respectively. The rates of positive human papillomavirus results from urine samples were 57 and 70.3%, respectively. The overall concordance among Roche Cobas and Qiagen tests utilizing urine samples for human papillomavirus type 16/18 was 84.3% with a kappa value of 0.675, and for other high-risk-human papillomavirus, it was 75.60% with a kappa value of 0.535. Roche Cobas showed high concordance with Qiagen test. CONCLUSION: human papillomavirus positivity was not detected in all urine samples. It is still inappropriate to recommend the use of urine liquid biopsy for the accurate and reliable detection of human papillomavirus. Due to the lack of a standardized tool, the utilization of urine samples as a screening human papillomavirus test remains a challenge.

Anti HCV activity and expression inhibition of HCC markers by protein extract from Iberis gibraltarica / Atividade anti-HCV e inibição da expressão de marcadores HCC por extrato de proteína de Iberis gibraltarica

Hepatitis C virus infection (HCV) is the foremost reason of progressive hepatic fibrosis and cirrhosis, with an elevated risk of hepatocellular carcinoma (HCC) development. Medicinal plants have been used for human health benefits for several years, but their therapeutic potential needs to be explored. The main objective of this study was to figure out the in vitro antiviral and anticancer characteristics of total crude protein of Iberis gibraltarica against HCV and HCC. Total crude protein of Iberis gibraltarica was isolated and quantified. The level of cytotoxicity was measured against the HepG2 cell line and it shows no significant cytotoxicity at the concentration of 504µg/ml. The anti-HCV effect was determined by absolute quantification via real time RT-PCR method and viral titer was reduced up to 66% in a dose dependent manner against the total protein of Iberis gibraltarica. The anticancer potential of Iberis gibraltarica was also examined through mRNA expression studies of AFP and GPC3 genes against the total protein of Iberis gibraltarica-treated HepG2 cells. The results show up to 90% of the down-regulation expression of AFP and GPC3. The obtained results indicate the therapeutic potential of total protein of Iberis gibraltarica against HCV and hepatocellular carcinoma in vitro.

A infecção pelo vírus da hepatite C (HCV) é a principal causa de fibrose hepática progressiva e cirrose, com risco elevado de desenvolvimento de carcinoma hepatocelular (HCC). As plantas medicinais vêm sendo utilizadas para benefícios à saúde humana há vários anos, mas seu potencial terapêutico precisa ser explorado. O principal objetivo deste estudo foi descobrir as características antivirais e anticancerígenas in vitro da proteína bruta total de Iberis gibraltarica contra HCV e HCC. A proteína bruta total de Iberis gibraltarica foi isolada e quantificada. O nível de citotoxicidade foi medido contra a linha celular HepG2 e não apresenta citotoxicidade significativa na concentração de 504µg/ml. O efeito anti-HCV foi determinado por quantificação absoluta através do método RT-PCR em tempo real e o título viral foi reduzido em até 66% de forma dose-dependente contra a proteína total de Iberis gibraltarica. O potencial anticancerígeno de Iberis gibraltarica também foi examinado através de estudos de expressão de mRNA dos genes AFP e GPC3 contra a proteína total de células HepG2 tratadas com Iberis gibraltarica. Os resultados mostram até 90% da expressão de regulação negativa de AFP e GPC3. Os resultados obtidos indicam o potencial terapêutico da proteína total de Iberis gibraltarica contra HCV e carcinoma hepatocelular in vitro.

Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells / 中国生物制品学杂志

@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines

Identification of Scolopendra Dispensing Granules by Allele-specific PCR / 中国实验方剂学杂志

ObjectiveTo establish an allele-specific polymerase chain reaction （PCR） method for identifying Scolopendra dispensing granules， so as to ensure the quality and therapeutic effects of Scolopendra and its preparations. MethodThe primer interval suitable for the PCR was selected based on the cytochrome c oxidase subunit 3（COX-3） gene sequence of Scolopendra， and the single nucleotide polymorphism （SNP） loci of Scolopendra and its adulterants were mined from the interval for the design of specific primers. The samples of Scolopendra and its adulterants were collected. The PCR system was established and optimized regarding the annealing temperature， cycles， Taq enzymes， DNA template amount， PCR instruments， and primer concentrations， and the specificity and applicability of this method were evaluated. ResultThe PCR system was composed of 12.5 μL 2×M5 PCR Mix， 0.4 μL forward primer （10 μmol·L-1）， 0.4 μL reverse primer （10 μmol·L-1）， 2.5 μL DNA template， and 9.2 μL sterile double distilled water. PCR parameters： Pre-denaturation at 94 ℃ for 3 min， 30 cycles （94 ℃ for 20 s， 62 ℃ for 20 s， 72 ℃ for 45 s）， and extension at 72 ℃ for 5 min. After PCR amplification with the system and parameters above， the electrophoresis revealed a bright band at about 135 bp for Scolopendra and no band for the adulterants. ConclusionThe established allele-specific PCR method can accurately identify the medicinal materials， decoction pieces， and standard decoction freeze-dried powder of Scolopendra， as well as the intermediates and final products of Scolopendra dispensing granules， which is of great significance for ensuring the quality and clinical efficacy of Scolopendra and its preparations.

PCR-RFLP for Distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method for rapid distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex， so as to avoid the influence of genetic confusion on drug safety. MethodThe DSS-tagged sequences of Periplocae Cortex were obtained from the Chloroplast Genome Information Resource （CGIR） and analyzed to find the enzymatic cleavage sites that were different from those of Acanthopanacis Cortex and Lycii Cortex. The specific enzymatic cleavage site， Cla I， of Periplocae Cortex was selected， on the basis of which the primers for PCR-RFLP were designed. Furthermore， the factors such as annealing temperature， number of cycles， Taq enzyme， PCR instruments， and enzymatic treatment time that may influence PCR-RFLP were studied. The established PCR-RFLP method was applied to the identification of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex samples produced in different regions. ResultThe PCR-RFLP at the annealing temperature of 59 ℃ and with 40 cycles showed clear bands of the samples. When the enzyme digestion time was 30 min. The reaction produced the target bands at about 140 bp and 290 bp for both Periplocae Cortex and its original plant and only a band at about 430 bp for Acanthopanacis Cortex， Lycii Cortex， and their original plants. The method can accurately distinguish Periplocae Cortex from its confounders Acanthopanacis Cortex and Lycii Cortex. ConclusionThe PCR-RFLP method for distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex was established. It has high stability， sensitivity， and applicability， providing a reference for the quality control of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex.

A Rapid PCR-RFLP Method for Assessing Heterozygosity of Murraya paniculata Germplasm / 中国实验方剂学杂志

ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.

Transcriptomic Analysis of Wuzi Yanzongwan on Testicular Spermatogenic Function in Semi-castrated Male Mice / 中国实验方剂学杂志

ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg-1·d-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg-1·d-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep / Mutações no gene da proteína morfogenética óssea 15 causadoras de defeitos na secreção proteica em ovelhas inférteis Cholistani

Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep

Abstract Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Resumo Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.

Frecuencia y detección molecular de Gardnerella vaginalis en una institución de tercer nivel / Frequency and molecular detection of Gardnerella vaginalis in a third level institute

Resumen Antecedentes: Los padecimientos vaginales son la razón más común para que las mujeres busquen atención médica, con una prevalencia global que oscila entre el 23 y el 29% en mujeres en edad reproductiva. La vaginosis bacteriana es una de las principales causas de estos padecimientos, y el agente etiológico más frecuentemente identificado es Gardnerella vaginalis, sin embargo su diagnóstico es difícil, ya que requiere de medios artificiales selectivos enriquecidos y diferenciales. Objetivo: Determinar la frecuencia de G. vaginalis mediante la amplificación de ácidos nucleicos empleando la reacción en cadena de la polimerasa (PCR) en muestras cervicovaginales de pacientes que asisten a un instituto de tercer nivel. Método: Se analizaron 121 muestras cervicovaginales para la detección molecular del ARN ribosomal 16S de G. vaginalis. Resultados: G. vaginalis. se detectó en 34 muestras, de estas, 23 fueron de mujeres embarazadas y 11 de no embarazadas. Conclusión: La PCR de punto final detectó tres veces más la presencia de G. vaginalis que el medio de cultivo artificial.

Abstract Background: Vaginal conditions are the most common reason for women to seek medical care, with an overall prevalence ranging from 23 to 29% in women of reproductive age. Bacterial vaginosis is one of the main causes of these conditions, and the most frequently identified etiological agent is Gardnerella vaginalis, however, its diagnosis is difficult since it requires enriched and artificial selective culture media. Objective: To determine the frequency of G. vaginalis by nucleic acid amplification using polymerase chain reaction (PCR) in cervicovaginal samples from patients attending a third level institute. Method: One hundred twenty-one cervicovaginal samples were analyzed for molecular detection of 16S ribosomal RNA from G. vaginalis. Results: G. vaginalis was detected in 34 samples, of these, 23 were from pregnant women and 11 from non-pregnant women. Conclusion: Endpoint PCR detected three times more the presence of G. vaginalis than artificial culture medium.

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.

Neumonía en el paciente crítico: diagnóstico sindrómico rápido de PCR múltiple en un hospital general de agudos / Pneumonia in the critically ill patient: rapid syndromic diagnosis by multiplex PCR in a general hospital for acute care

Está demostrado en diversos estudios que los avances en el diagnóstico microbiológico reducen el tiempo de entrega de resultados y poseen un impacto clínico eviden-te. Hoy en día, las técnicas basadas en amplificación de ácidos nucleicos nos permiten hacer diagnóstico direc-tamente de la muestra y sumar la posibilidad de detectar más de un agente. Esto impacta tanto en el control de la multiresistencia (MR) como en el inicio de una terapéuti-ca apropiada. La implementación de un sistema de PCR múltiple rápido para neumonía puede ser útil en áreas crí-ticas, donde son frecuentes las infecciones respiratorias agudas (IRA) y el tiempo es un condicionante del éxito terapéutico. El objetivo de nuestro proyecto fue evaluar la implementación del diagnóstico sindrómico rápido por PCR múltiple para neumonía en el manejo del tratamiento de IRA en una unidad de cuidados intensivos. La con-ducta terapéutica fue la variable relevante. Este nuevo diagnóstico nos proporcionó una herramienta ágil, con un tiempo de respuesta de tres a cuatro horas. La ausencia o presencia de genes de resistencia y el microorganismo identificado fueron lo que condujo a la conducta terapéuti-ca acertada en el 75% de los casos. Constituyó una herra-mienta importante para el control de la multirresistencia bacteriana y aumentó la oportunidad de éxito terapéutico

It has been shown in various studies that advances in microbiological diagnosis reduce the delivery time of results and have an evident clinical impact. Today, techniques based on nucleic acid amplification allow us to diagnose directly from the sample and add the possibility of detecting more than one agent. This impacts both the control of MR and the initiation of appropriate therapy. The implementation of a rapid multiplex PCR system for pneumonia can be useful in critical areas where acute respiratory infections (ARI) are frequent and time is a determining factor for therapeutic success. The objective of our project was to evaluate the implementation of rapid syndromic diagnosis by multiple PCR for pneumonia in the management of ARI treatment in an Intensive Care Unit. The therapeutic behavior was the relevant variable. This new diagnosis provided us with an agile tool, with a response time of 3 to 4 hours. The absence or presence of resistance genes and the identified microorganism was what led to the correct therapeutic approach in 75% of the cases. It constituted an important tool for the control of bacterial multiresistance and increased the opportunity for therapeutic success.

MiR-15b-5p Promotes Growth and Metastasis of Renal Clear Cell Carcinoma / MiR-15b-5p Promueve el Crecimiento y la Metástasis del Carcinoma Renal de Células Claras

SUMMARY: We investigated the expression and clinical significance of miR-15b-5p in clear cell renal cell carcinoma (RCC) through bioinformatics analysis and experimental verification. The differentially expressed miRNAs were screened in the GEO database. Venn diagram showed that there were 5 up-regulated miRNAs (has-miR-210, has-miR-142-3p, has-miR-142-5p, has-miR-15b-5p, and has-miR-193a-3p) and only 1 down-regulated miRNA (has-miR-532-3p) that were commonly expressed between GSE189331 and GSE16441 datasets. This was further confirmed in TCGA. Further analysis showed that the has-miR-193a-3p, has-miR-142-3p, has- miR-142-5p, and has-miR-15b-5p were closely related to tumor invasion, distant metastasis and survival probability. The expression of miR-15b-5p in ccRCC tissues was significantly higher than that in adjacent normal kidney tissues (P0.05). Following inhibition of miR-15b-5p expression, RCC cells had attenuated proliferation, increased apoptosis, and attenuated migration and invasion. has-miR-15b-5p-WEE1, has-miR-15b-5p-EIF4E, has-miR-15b-5p-PPP2R1B may be three potential regulatory pathways in ccRCC. miR-15b-5p is highly expressed in cancer tissues of ccRCC patients. It may promote proliferation, inhibit apoptosis and enhance cell migration and invasion of RCC cells. The has-miR-15b-5p-WEE1, has-miR-15b-5p-EIF4E, and has-miR-15b-5p-PPP2R1B may be three potential regulatory pathways in ccRCC.

Investigamos la expresión y la importancia clínica de miR-15b-5p en el carcinoma de células renales (CCR) de células claras mediante análisis bioinformático y verificación experimental. Los miARN expresados diferencialmente se examinaron en la base de datos GEO. El diagrama de Venn mostró que había 5 miARN regulados positivamente (has-miR-210, has-miR-142-3p, has-miR-142-5p, has-miR-15b-5p y has-miR-193a-3p). ) y solo 1 miARN regulado negativamente (has-miR-532-3p) que se expresaron comúnmente entre los conjuntos de datos GSE189331 y GSE16441. Esto fue confirmado aún más en TCGA. Un análisis más detallado mostró que has-miR-193a-3p, has-miR-142-3p, has- miR-142-5p y has-miR-15b-5p estaban estrechamente relacionados con la invasión tumoral, la metástasis a distancia y la probabilidad de supervivencia. La expresión de miR-15b-5p en tejidos ccRCC fue significativamente mayor que la de los tejidos renales normales adyacentes (P 0,05). Tras la inhibición de la expresión de miR-15b-5p, las células RCC tuvieron una proliferación atenuada, un aumento de la apoptosis y una migración e invasión atenuadas. has-miR-15b-5p-WEE1, has- miR-15b-5p-EIF4E, has-miR-15b-5p-PPP2R1B pueden ser tres posibles vías reguladoras en ccRCC. miR-15b-5p se expresa altamente en tejidos cancerosos de pacientes con ccRCC. Puede promover la proliferación, inhibir la apoptosis y mejorar la migración celular y la invasión de células RCC. has-miR-15b-5p-WEE1, has- miR-15b-5p-EIF4E y has-miR-15b-5p-PPP2R1B pueden ser tres posibles vías reguladoras en ccRCC.

Detección molecular de patógenos en deposiciones en niños consultantes por diarrea aguda en Servicios de Urgencia

Introducción: La diarrea aguda continúa siendo una de las principales causas de morbilidad en niños; sin embargo, el diagnóstico etiológico presenta limitaciones dada la baja sensibilidad de los métodos tradicionales. Objetivo: Describir los microorganismos identificados en niños que acudieron al Servicio de Urgencia (SU) de un hospital universitario en Santiago, Chile, por diarrea aguda y a los que se le solicitó panel molecular gastrointestinal. Métodos: Se revisaron fichas clínicas y resultados de panel gastrointestinal realizados entre junio de 2017 y marzo de 2020. Resultados: Se incluyeron 198 pacientes, edad promedio de 54,5 meses y 60,6% (120/198) de sexo masculino. La positividad del panel fue de 78,8% (156/198) con 35,3% (55/156) de las muestras polimicrobianas. Se identificaron 229 microorganismos, de los cuales 72,9% (167/229) corresponden a bacterias, 25,8% (59/229) a virus y 1,3% (3/229) a parásitos. Destacaron Campylobacter spp. y Escherichia coli enteropatógena (ECEP) como las bacterias más frecuentemente identificadas. Los pacientes con detección de Campylobacter spp. presentaron con mayor frecuencia fiebre (p = 0,00). ECEP se aisló principalmente (82,5%) en muestras polimicrobianas. Discusión: Los resultados enfatizan el potencial que poseen los estudios moleculares para mejorar el diagnóstico etiológico de la diarrea, pero a la vez llevan a cuestionar el rol patogénico de algunos microorganismos identificados.

Background: Acute diarrhea continues to be one of the main causes of morbidity in children, however the etiologica diagnosis presents limitations given the low sensitivity of traditional methods. Aim: To describe the microorganisms identified in children who attended the emergency department (ED) in Santiago, Chile, due to acute diarrhea and to whom a gastrointestinal panel was requested as part of their study. Material and Methods: Clinical records and results of the gastrointestinal panel carried out between June 2017 and March 2020 were reviewed. Results: 198 patients were included, the average age was 54.5 months and 60.6% (120/198) were males. Positivity was 78.8% (156/198) with 35.3% (55/156) of the samples being polymicrobial. 229 microorganisms were identified, of which 72.9% (167/229) corresponded to bacteria, 25.8% (59/229) to viruses, and 1.3% (3/229) to parasites. Campylobacter spp. and enteropathogenic Escherichia coli (EPEC) were the most frequently identified bacteria. Patients with detection of Campylobacter spp. presented a higher frequency of fever (p = 0.00). EPEC was isolated in 82.5% of the cases in polymicrobial samples. Discussion: The results emphasize the potential of molecular studies to improve the etiological diagnosis of diarrhea and at the same time lead to question the pathogenic role of some microorganisms.

Molecular detection of SARS-CoV-2 in Argentina: Evaluation of alternative diagnostic tools for the decentralization of the diagnosis

Abstract The rocketing number of COVID-19 cases highlighted the critical role that diagnostic tests play in medical and public health decision-making to contain and mitigate the SARS-CoV-2 pandemic. This study reports the evaluation and implementation of different tests for the molecular detection of SARS-CoV-2 in the central region of Argentina. We evaluated 3 real time RT-PCR kits (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit and WGene SARS-CoV-2 RT Detection), 2 nucleic acid extraction methods [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-min vs. 9-min, a pre-analytical reagent (FlashPrep®) and 2 isothermal amplification tests (Neokit Plus and ELA CHEMSTRIP®). The order according to the best performance of the 3 real-time RT-PCR kits evaluated was: DisCoVery > GeneFinderTM> WGene. The 2 RNA extraction methods showed similar good results: MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min was selected due to its faster performance. FlashPrep® reagent showed excellent results to perform direct RNA detection. Isothermal amplification assays showed acceptable sensitivity and specificity values (>80%), except in samples with Ct> 30. Our data show optimal real time RT-PCR kits and alternative molecular methods for SARS-CoV-2 diagnostic. These alternative assays proved to be aceptable.

Resumen La explosión de casos de COVID-19 resaltó el papel fundamental que desempeñan las pruebas de diagnóstico en la toma de decisiones médicas y de salud pública para contener y mitigar la pandemia de SARS-CoV-2. Este estudio reporta la evaluación y la implementación de diferentes test para la detección molecular de SARS-CoV-2 en la región central de Argentina. Evaluamos tres kits de RT-PCR en tiempo real (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit y WGene SARS-CoV-2 RT Detection), dos métodos de extracción de ácidos nucleicos (MagaBio plus Virus DNA/RNA Purification Kit II [BioFlux, 35-min vs. 9-min), un reactivo pre-analítico (FlashPrep®) y dos test de amplificación isotérmica (Neokit Plus and ELA CHEMSTRIP®). El orden de rendimiento de los tres kits de RT-PCR en tiempo real evaluados fue el siguiente: DisCoVery GeneFinder™ WGene. Los dos métodos de extracción de RNA mostraron buenos y similares resultados; se seleccionó MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min debido a su rápido tiempo de procesamiento. El reactivo FlashPrep® mostró excelentes resultados para realizar detección directa de RNA. Los ensayos de amplificación isotérmica mostraron valores de sensibilidad y de especificidad aceptables (80%), excepto en muestras con Ct 30. Nuestros resultados muestran kits de RT-PCR en tiempo real óptimos, como así también métodos moleculares alternativos para el diagnóstico de SARS-CoV-2 que resultan aceptables para su uso en contextos adversos, de descentralización y en diferentes escenarios epidemiológicos, para la detección rápida y precisa del SARS-CoV-2.

Identificación y selección de Saccharomyces cerevisiae nativas para mejorar el proceso productivo del Pisco a partir de uva Quebranta

El Pisco es el destilado del Perú, elaborado a partir de mostos recientemente fermentados con uvas criollas denominadas "pisqueras". Las levaduras son los microorganismos clave en la fermentación y el uso de cepas nativas seleccionadas presenta ventajas competitivas para la tipicidad del producto, así como también para la estandarización del producto y el control microbiológico del proceso. El objetivo fue identificar y seleccionar las cepas de levaduras nativas para la producción del Pisco de uva Quebranta aisladas de procesos productivos de Pisco en el valle de Ica. Para ello, se emplearon técnicas microbiológicas y moleculares mediante el análisis de ITS1-5.8S-ITS2 PCR-RFLP para la identidad taxonómica. La evaluación de las cepas para producir Pisco consistió en el análisis fisicoquímico y organoléptico del destilado obtenido con las cepas seleccionadas. Se evaluaron 3 aislados para la producción de Pisco identificados como Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49 y UNA SC - 54, de los cuales la cepa UNA SC-49 destacó por mostrar aptitudes enológicas diferentes a las otras cepas. Este trabajo constituye el primer registro de levaduras nativas del Perú para mostos procedentes de uva Quebranta.

El Pisco stands as Peru's distilled spirit, crafted from recently fermented musts derived from native grape varieties known as "pisqueras". Yeasts serve as decisive microorganisms in the fermentation process, and the utilization of selected native strains confers competitive advantages for product typicity, standardization, and microbiological control within the production process. The aim was to identify and select native yeast strains for Quebranta grape Pisco production, isolated from Pisco production processes in the Ica Valley. Microbiological and molecular techniques were employed, utilizing ITS1-5.8S-ITS2 PCR-RFLP analysis for taxonomic identification. The assessment of yeast strains for Pisco production involved the physicochemical and organoleptic analysis of the distilled product obtained using the selected strains. Three isolates were evaluated for Pisco production, identified as Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49, and UNA SC - 54. Among these, the UNA SC-49 strain stood out due to displaying oenological characteristics distinct from the other strains. This work is the first documentation of native yeasts in Peru for musts derived from Quebranta grapes.

Material genético do SARS-CoV-2 em superfícies de equipamentos utilizados na prática de atividades físicas / Genetic material of SARS-CoV-2 on the surface of equipment used in the practice of physical activities

A humanidade foi impactada por uma Pandemia que expôs a população ao contato com um vírus de elevado contágio e com o índice de letalidade alarmante. Este estudo objetivou avaliar a possibilidade da persistência de material genético do SARS-CoV-2 na superfície dos equipamentos de estabelecimentos de prática de atividades físicas indoor e outdoor. Foram coleta das amostras de equipamentos utilizados para a prática de exercícios físicos em cinco academias, cinco praças e entre os frequentadores desses ambientes. Aplicou-se a técnica RT-PCR para a detecção doRNA do SARS-CoV-2 e posterior análise dos resultadose foi detectada a existência de partículas de RNA viral do SARS-CoV-2 em 48,57% das amostras coletadas dos equipamentos das academias e 12,85% das amostras coletadas nas praças, evidenciando uma incidência menor em equipamentos utilizados em locais abertos em todas as áreas comparadas.Além disso, constatou-se que 35,7% dos participantes do estudo testaram positivo para COVID-19.Os casos positivos para COVID-19 detectados apresentaram sintomas classificados como levesa moderados e uma recuperação rápida.A presença de material genético nos equipamentos,por sua vez, leva-nos a perceber a importância da higienização adequada das superfícies, como forma de prevenção.

Humanity was impacted by a Pandemic that exposed the population to contact with a highly contagious virus with an alarming lethality rate. The present study aimed to evaluate the possibility of the persistence of genetic material from SARS-CoV-2 on the surface of equipment used to practice indoor and outdoor physical activities. A sample of equipment used for the practice of physical activity was collected in five gyms and five squares and among the regulars of these environments. The RT-PCR technique was applied to detect the RNA of SARS-CoV-2 and subsequent analysis of the results. The existence of SARS-CoV-2 viral RNA particles was detected in 48.57% of the samples collected from gym equipment and 12.85% of the samples collected in squares, evidencing a lower incidence in equipment used in open spaces in all areas compared and it was found that 35.7% of the study participants tested positive for COVID-19. The positive cases for COVID-19 detected, had symptoms classified as mild to moderate and a quick recovery. The presence of genetic material in the equipment, in turn, leads us to realize the importance of proper cleaning of surfaces, as a form of prevention.

Estudio comparativo entre un test de antígeno y la prueba de la reacción en cadena de la polimerasa en tiempo real (RT-PCR) para el diagnóstico de infección por COVID-19 en pediatría / Comparative study between an antigen test and the reverse transcription-polymerase chain reaction (RT-PCR) test for the diagnosis of COVID-19 in pediatrics

Introducción. La pandemia por COVID-19 ha puesto de manifiesto la necesidad de pruebas diagnósticas rápidas. La prueba de referencia es la reacción en cadena de la polimerasa en tiempo real (RT-PCR). Requiere un equipo y personal capacitado, y su resultado puede llevar un tiempo de espera prolongado. El sistema BD Veritor® es el método rápido cromatográfico utilizado para la detección del antígeno del coronavirus de tipo 2 del síndrome respiratorio agudo grave, en individuos sintomáticos. El objetivo primario del siguiente trabajo es evaluar sensibilidad y especificidad del test de antígeno (TA) comparadas con la RT-PCR en población pediátrica. Población y métodos. Estudio prospectivo, de prueba diagnóstica. Se incluyó a todo menor de 17 años en los primeros 5 días de inicio de síntomas, que consultó desde julio de 2021 hasta febrero de 2022. Se calculó un mínimo de 300 muestras para lograr una precisión de ± 8,76 % y de ± 3,68 % para sensibilidad y especificidad respectivamente. Se analizaron en paralelo las muestras por ambas metodologías. Resultados. De 316 muestras pareadas, 33 fueron positivas por ambos métodos; 6 fueron positivas solo por RT-PCR. La especificidad del TA fue del 100 %; la sensibilidad, del 84,6 %, con un valor predictivo positivo y negativo del 100 % y del 98 % respectivamente. Conclusiones. El TA demostró ser útil en el diagnóstico de pacientes pediátricos con COVID-19 en los primeros 5 días de inicio de síntomas, aunque aquellos con TA negativo y alta sospecha clínica deberían confirmar su resultado con la RT-PCR.

Introduction. The COVID-19 pandemic has brought to light the need for rapid diagnostic tests. The gold standard test is reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR requires equipment and trained personnel, and results may take a long waiting time. The BD Veritor® System is a rapid chromatographic method used for the detection of severe acute respiratory syndrome coronavirus 2 antigen in symptomatic individuals. The primary objective of this study is to assess the sensitivity and specificity of the antigen test (AT) compared to the RT-PCR in the pediatric population. Population and methods. Prospective study with a diagnostic test. All children younger than 17 years in the first 5 days of symptom onset, who consulted between July 2021 and February 2022, were included. A minimum of 300 specimens was estimated to achieve an accuracy of ±8.76% and ±3.68% for sensitivity and specificity, respectively. Specimens were analyzed in parallel using both methodologies. Results. Of 316 paired samples, 33 were positive by both methods; 6 were positive only by RT-PCR. The specificity of the AT was 100%; sensitivity was 84.6%, with a positive and negative predictive value of 100% and 98%, respectively. Conclusions. The AT proved to be useful in the diagnosis of pediatric patients with COVID-19 in the first 5 days of symptom onset, although those with a negative AT and high clinical suspicion should confirm their result with a RT-PCR.

Utilidad de la implementación de un método molecular automatizado en el diagnóstico de agentes causales de infecciones de transmisión sexual / Usefulness of the implementation of an automated molecular method in the diagnosis of causal agents of sexually transmitted infections / Utilidade da implementação de um método molecular automatizado no diagnóstico de agentes causais de infecções sexualmente transmissíveis

Resumen Se presentan los resultados obtenidos en los primeros meses desde la implementación del diagnóstico de agentes causales de infecciones de transmisión sexual (ITS) por PCR en tiempo real en un sistema automatizado. Se estudiaron 46 muestras endocervicales y 39 muestras de uretra masculina, por examen microscópico en fresco, coloración de Gram, cultivo en agar sangre, agar Thayer Martin, galerías miniaturizadas para investigar Ureaplasma spp. y Mycoplasma hominis (Mycoplasma IST3, bioMérieux, Francia) y PCR múltiple para Neisseria gonorrhoeae, Chlamydia trachomatis . Trichomonas vaginalis (BDMaxTM System, Becton Dickinson, EE.UU.). Mediante PCR múltiple se detectó un agente vinculado a ITS en el 48,7% de las muestras uretrales y en el 21,7% de las muestras endocervicales, además de 5 casos de coinfección. El 18,8% de las infecciones se diagnosticaron sólo por PCR. Estos datos demuestran que mediante PCR se optimizó el diagnóstico de ITS en personas de ambos sexos.

Abstract The results obtained in the first months after the implementation of the diagnosis of causative agents of sexually transmitted infections (STIs) by real- time PCR in an automated system are presented. Forty-six endocervical samples and 39 male urethral samples were studied by fresh microscopic examination, Gram staining, blood agar culture, Thayer Martin agar, miniaturised galleries to investigate Ureaplasma spp. and Mycoplasma hominis (Mycoplasma IST3, bioMérieux, France), and multiplex PCR for Neisseria gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis (BDMaxTM System, Becton Dickinson, USA). Using multiplex PCR, an agent linked to STIs was detected in 48.7% of the urethral samples and in 21.7% of the endocervical samples, in addition to 5 cases of coinfection. A total of 18.8% of the infections were diagnosed only by PCR. These data show that PCR optimised the diagnosis of STIs in people of both sexes.

Resumo São apresentados os resultados obtidos nos primeiros meses a partir da implementação do diagnóstico de agentes causadores de infecções sexualmente transmissíveis (ISTs) por PCR em tempo real em um sistema automatizado. Quarenta e seis amostras endocervicais e 39 amostras uretrais masculinas foram estudadas por exame microscópico fresco, coloração de Gram, cultura de ágar sangue, ágar Thayer Martin, galerias miniaturizadas para investigar Ureaplasma spp. e Mycoplasma hominis (Mycoplasma IST3, bioMérieux, França) e PCR múltiplo para Neisseria gonorrhoeae, Chlamydia trachomatis e Trichomonas vaginalis (BDMaxTM System, Becton Dickinson, EUA). Utilizando PCR múltiplo, um agente ligado a IST foi detectado em 48,7% das amostras uretrais e em 21,7% das amostras endocervicais, além de 5 casos de coinfecção; 18,8% das infecções foram diagnosticadas apenas por PCR. Esses dados mostram que através do PCR foi otimizado o diagnóstico de IST em pessoas de ambos os sexos.