Association between index finger and ring finger length ratios and polymorphism of homeobox A11 gene locus among Ningxia college students / 解剖学报

Objective To investigate the association between the index finger and ring finger length ratio (2D ∶ 4D) and of four loci (rs6461992‚ rs6968828‚ rs7801581‚ rs17427875) polymorphism of homeobox (HOX) A11 gene among Ningxia college students. Methods Digit camera was used to collect frontal hand photos of 667 Han college students (348 males and 319 females) from Ningxia province; Image analysis software was used to mark the anatomical points and measure finger lengths of the index and ring fingers of both hands; multiplex PCR was used to detect each locus polymorphisms of HOXA11 gene; statistical software was used to compare and analyze the differences and associations of 2D ∶4D and gene polymorphisms between different genders. Results Among Ningxia Han college students‚ both left hand and right hand 2D ∶ 4D were significantly higher in females than those of in males (all P< 0. 05)‚ and there were no significant sex differences in right-left hand 2D ∶4D; the genotypes and allele frequencies of rs7801581 locus of HOXA11 gene differed significantly between genders (all P < 0. 05)‚ and none of the other locus polymorphisms showed any significant sex differences; only female left hand 2D ∶4D was significantly associated with rs6461992 locus genotype in the relationship between 2D ∶4D and HOXA11 polymorphisms (P<0. 05). Conclusion There were significant sex differences in 2D ∶ 4D among Han college students in Ningxia‚ and the rs6461992 locus polymorphism of HOXA11 gene may be associated with the formation of 2D ∶4D in females.

Development and verification of multiplex fluorescent quantitative PCR for detection of bacteria and fungi in cell culture medium / 中国生物制品学杂志

@#Objective To develop and verify a multiplex fluorescent quantitative PCR method for detection of common bacterial and fungal contaminants in cell culture medium.Methods According to NUC gene of Staphylococcus aureus,COLA gene of Clostridium spore,ITS-2 segment sequence of Candida albicans,a set of primers and probes were designed for each respectively,and using UBI3 gene of capsicum introduced as external standard gene,a triple reaction system of Staphylococcus aureus,Clostridium spore and external standard gene and a double reaction system of Candida albicans and external standard gene were established.The primer specificity,linear range,limit of detection,specificity,anti-interference performance and precision of the method were verified.Finally,100 samples of 293T cell culture medium were detected by using the developed method,which was compared with the common PCR method.Results Three pairs of primers all amplified about 100 bp specific gene bands corresponding to the three strains at different annealing temperatures(56,57,58 and59 ℃),and the size was consistent with the expected.In the range of 5.80 × 10~6 — 5.80 × 10~2 copies/μL,the standard plasmids of the three strains showed a good linear relationship with the Ct values.The standard curve equations were:Y=-3.373 X+37.48,Y=-3.557X+36.59 and Y=-3.536 X+39.78,each R~2> 0.99,respectively,and the amplification efficiency was in the range of 90%—110%.All the limits of detection of the three strains were 10~1 CFU/mL.The primers and probes of the three strains showed no specific amplification on the genomic DNA of six kinds of cells that were prone to cross-reaction.The genomic DNA of 293T cells,Yeast,Escherichia coli and Mycoplasma sp.had no effect on the detection.The CVs of repeatability and intermediate precision verification were both less than 15%.Among 100 cell culture medium samples,14 positive and 86 negative samples were detected,and the results of common PCR method for three positive and two negative samples randomly selected were consistent with the developed method.Conclusion The multiplex fluorescent quantitative PCR method developed in this study for the detection of bacteria and fungi in cell culture medium has good specificity,anti-interference performance and precision,and is simple to operate with low cost and high sensitivity,which can quickly detect the contaminants during cell culture.

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.

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 Plasmid-Mediated Quinolone Resistance Genes (qnrA and aac(6?)-Ib-cr) in Drug Resistant Escherichia coli, Sudan

Background: The quinolone group, a synthetic antimicrobial, is widely used worldwide to treat many diseases, including those caused by Gram-negative bacteria. Escherichia coli and others are among the bacteria that produce quinolone resistance genes (qnr) such as qnrA and aac(6?)-Ib-cr. Objective: The present study aimed to the isolate Escherichia coli from patients attending some Hospitals in Wad Medani city, identification of drug resistance patterns and detection of the frequency of quinolones resistance genes; qnrA and aac(6?)-Ib-cr among isolated strains. Methods: A cross-sectional descriptive, hospital-based study included 119 Escherichia coli strains was conducted. A designed questionnaire used for demographic data collection and the attitude toward antimicrobials usage. Clinical specimens were processed for aerobic bacterial isolation and identification. Antimicrobial sensitivity performed by Kirby Bauer disc diffusion technique according to the CLSI guidelines. Presence of qnrA and aac(6?)-Ib-cr genes was assessed by multiplex PCR. Results: Most strains of Escherichia coli originated from urine 53.8% (64/119) and wounds 42.9% (51/119) specimens. Meropenem had the best effect against tested strains with susceptibility of 85% (101/119). Multiplex PCR assay, using specific primers, demonstrated that 41.2% (49/119) and 37.8% (45/119) of isolated Escherichia coli possessed qnrA and aac(6?)-Ib-cr genes respectively. Conclusion: The high rate of qnrA and aac (6)-Ib-cr genes among Escherichia coli necessitate the usage of molecular tools in detecting the genetic determinants of drug resistance microorganisms in countries such as Sudan.

Simultaneous detection of thirteen exons of dystrophin gene by optimized multiplex PCR assay to screen Duchenne/Becker muscular dystrophy

Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA)signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 µL PCR reaction includes primer(s) (0.3–0.5 µM each), dNTP mixture (160 µM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 µL), DNA template (250 ng/50 µL), BSA (0.4 µg/µL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics.

Investigation of etiology of community-acquired pneumonia in hospitalized patients in a tertiary hospital of São Paulo City, Brazil

Abstract Background Community-Acquired Pneumonia (CAP) is the primary cause of hospitalization in the United States and the third leading cause of death in Brazil. The gold standard for diagnosing the etiology of CAP includes blood culture, Gram-stained sputum, and sputum culture. However, these methods have low sensitivity. No studies investigating the etiology of CAP have been conducted in Brazil in the last 20-years, and the empirical choice of antimicrobials is mainly based on the IDSA guidelines. This is the first national study with this aim, and as a result, there's potential for the Brazilian consensus to be impacted and possibly modify its guidelines rather than adhering strictly to the IDSA's recommendations. Methods The aim of this study is to identify the main microorganisms implicated in CAP by employing a multiplex Polymerase Chain Reaction (mPCR) at the foremost public hospital in Brazil. All patients who were admitted to the emergency department and diagnosed with severe CAP underwent an mPCR panel using nasopharyngeal and oropharyngeal swabs, with the aim of detecting 13 bacterial and 21 viral pathogens. Results A total of 169 patients were enrolled in the study. The mPCR panel identified an etiological agent in 61.5% of patients, with viruses being the most common (42.01%), led by Rhinovirus, followed by Influenza and Coronavirus (non-SARS-CoV-2). Bacterial agents were identified in 34.91% of patients, with S. pneumoniae being the most common, followed by H. influenzae, M. catarrhalis, and S. aureus. Additionally, we found that the prescription for 92.3% of patients could be modified, with most changes involving de-escalation of antibiotics and antiviral therapy. Conclusion Our study revealed different etiological causes of CAP than those suggested by the Brazilian guidelines. Using molecular diagnostic tests, we were able to optimize treatment by using fewer antibiotics.

Etiología de las infecciones de transmisión sexual (ITS) diagnosticadas por la técnica de PCR múltiple-hibridación en población colombiana de la ciudad de Medellín atendida en el Laboratorio Clínico VID / Etiology of sexually transmitted infections (STIs) diagnosed by multiplex PCR-hibridization assay technique in a Colombian population attending the VID Clinical Laboratory in Medellin

Introducción. Las infecciones de transmisión sexual (ITS) son y seguirán siendo un serio problema de salud pública en todo el mundo según los datos de la OMS, con el agravante que la mayoría de los casos son asintomáticos y, además, no existe otro reservorio distinto al humano. El diagnóstico se puede realizar con pruebas tradicionales y moleculares, estas últimas incluyen la reacción en cadena de la polimerasa (PCR), de las cuales existen varios tipos, entre ellas, la PCR múltiple que tiene la capacidad de detectar ITS polimicrobianas a partir de una sola muestra. El objetivo de este estudio fue establecer cuáles fueron las infecciones de transmisión sexual más frecuentes en diferentes grupos de pacientes, así como determinar la utilidad del uso de la técnica de PCR múltiple en el diagnóstico de las ITS. Metodología. Se trata de un estudio observacional de corte transversal realizado entre los años 2021 y 2022 con pacientes que acudieron al servicio de diagnóstico del Laboratorio Clínico VID por sospecha de ITS. Las muestras recolectadas fueron evaluadas utilizando una prueba comercial basada en la técnica de PCR múltiple e hibridación. Las muestras procesadas fueron: orina e hisopados de endocérvix, uretra, recto, faringe y úlceras. Resultados. Se estudiaron 1.027 pacientes, de estos, 228 (22,2 %) fueron positivos para diferentes agentes de trasmisión sexual, distribuidos así: 50 (21,9 %) mujeres, 129 (56,6 %) hombres heterosexuales y 49 (21,5 %) hombres que tenían sexo con hombres (HSH). La edad promedio de las mujeres fue 30 años, y la de ambos grupos de hombres fue 36 años. Los microorganismos más frecuentemente identificados en mujeres fueron: C. trachomatis (A-K) en 28,6 %, seguido de virus herpes simplex tipo 2 (VHS-2) en 26,8 % y N. gonorrhoeae en 17,9 %. En hombres heterosexuales fueron C. trachomatis (A-K) en 37,5 %, N. gonorrhoeae en 21,5 % y VHS-2 en 18,7 %. En HSH fueron C. trachomatis (L1-L3) en 32,7 %, seguido de N. gonorrhoeae en 27,6 %, y de C. trachomatis (A-K) y VHS-2, ambos en 13,8 %. En 11 hombres heterosexuales, 8 HSH y en 6 mujeres, se identificó infección polimicrobiana. Conclusiones. C. trachomatis (A-K) fue el microorganismo más prevalente causante de ITS, seguido de N. gonorrhoeae en ambos grupos de hombres, y de VHS-2 en las mujeres, muy similar a lo reportado a nivel mundial. La prueba de PCR múltiple permite la detección de infecciones polimicrobianas comúnmente asociadas a ITS y el diagnóstico es preciso y confiable, incluso en pacientes asintomáticos

Sexually transmitted infections (STIs) are and will continue to be a serious public health problem throughout the world according to WHO data, with the aggravating factor that most cases are asymptomatic and, furthermore, there is no other reservoir other than humans. The diagnosis can be made with traditional and molecular tests, the latter include the polymerase chain reaction (PCR), of which there are several types, among them, multiplex PCR that has the capacity to detect polymicrobial STIs from a single sample. The objective of this study was to establish which were the most frequent sexually transmitted infections in different groups of patients, as well as to determine the usefulness of the multiplex PCR technique in the diagnosis of STIs. Methodology. This is an observational, cross-sectional study carried out between 2021 and 2022 with patients who attended the VID Clinical Laboratory for suspected STIs. The collected samples were evaluated using a commercial test based on the multiplex PCR technique and hybridization. The samples processed were: urine and swabs from endocervix, urethra, rectum, pharynx, and ulcers. Results. The study included 1,027 patients, of these, 228 (22.2%) were positive for different sexually transmitted agents, distributed as follows: 50 (21.9%) women, 129 (56.6%) heterosexual men and 49 (21.5%) men who had sex with men (MSM). The average age of the women was 30 years, and that of both groups of men was 36 years. The microorganisms most frequently identified in women were: C. trachomatis (A-K) in 28.6%, followed by herpes simplex virus type 2 (HSV-2) in 26.8% and N. gonorrhoeae in 17.9%. In heterosexual men they were C. trachomatis (A-K) in 37.5%, N. gonorrhoeae in 21.5% and HSV-2 in 18.7%. In MSM they were C. trachomatis (L1-L3) in 32.7%, followed by N. gonorrhoeae in 27.6%, and C. trachomatis (A-K) and HSV-2, both in 13.8%. Polymicrobial infection was identified in 11 heterosexual men, 8 MSM, and 6 women. Conclusions. C. trachomatis (A-K) was the most prevalent STI-causing microorganism, followed by N. gonorrhoeae in both groups of men, and HSV-2 in women, very similar to that reported worldwide. The multiplex PCR test allows the detection of polymicrobial infections commonly associated with STIs and the diagnosis is accurate and reliable, even in asymptomatic patients

Association between hydroxysteroid dehydrogenases gene family single-nucleotide polymorphisms and digit ratio / 解剖学报

Objective To investigate the association between 8 single-nucleotide polymorphisms (SNPs) of hydroxysteroid dehydrogenase (HSD) gene family and human digit ratio (2D ∶ 4D). Methods Randomly selected 808 college students (400 males and 408 females) as subjects, the digit ratio of left and right fingers were measured and calculated using computer image software. Eight SNPs (rs1000283, rs2236903, rs5479, rs56303414, rs676387, rs4445895, rs2066474, rs8190478) in HSD11B and HSD17B gene families were genotyped by multiplex PCR. The association between 2D ∶4D and different genotypes was analyzed by One-Way ANOVA. Results Female left hand(L)2D ∶ 3D, L2D ∶4D, L3D ∶4D, right hand(R)2D ∶4D, R2D ∶5D were significantly higher than male (P0. 05). The genotypes frequency of the 8 SNPs were not significantly associated with digit ratio (2D ∶4D) in both males and females (P>0. 05). Conclusion There are significant gender differences in digit ratio in Ningxia Han college students, but there is no correlation between digit ratio and 8 SNPs in HSD11B and HSD17B gene families, suggesting that HSD11B and HSD17B gene families may have nothing to do with the formation of human digit ratio.

Cerebrospinal fluid inflammatory cytokine profiles of patients with neurotropic parasitic infections

@#The pathogenesis of chronic parasitic central nervous system (CNS) infections, including granulomatous amoebic meningoencephalitis (GAE), cerebral toxoplasmosis (CT), and neurocysticercosis (NCC), is primarily due to an inflammatory host reaction to the parasite. Inflammatory cytokines produced by invading T cells, monocytes, and CNS resident cells lead to neuroinflammation which underlie the immunopathology of these infections. Immune molecules, especially cytokines, can therefore emerge as potential biomarker(s) of CNS parasitic infections. In this study, cerebral spinal fluid (CSF) samples from suspected patients with parasitic infections were screened for pathogenic free-living amoebae by culture (n=2506) and PCR (n=275). Six proinflammatory cytokines in smear and culture-negative CSF samples from patients with GAE (n = 2), NCC (n = 7), and CT (n = 23) as well as control (n = 7) patients were measured using the Multiplex Suspension assay. None of the CSF samples tested was positive for neurotropic free-living amoebae by culture and only two samples showed Acanthamoeba 18S rRNA by PCR. Of the six cytokines measured, only IL-6 and IL-8 were significantly increased in all three infection groups compared to the control group. In addition, TNFa levels were higher in the GAE and NCC groups and IL-17 in the GAE group compared to controls. The levels of IL-1b and IFNg were very low in all the infection groups and the control group. There was a correlation between CSF cellularity and increased levels of IL-6, IL-8, and TNFa in 11 patients. Thus, quantifying inflammatory cytokine levels in CSF might help with understanding the level of neuroinflammation in patients with neurotropic parasitic diseases. Further studies with clinico-microbiological correlation in the form of reduction of cytokine levels with treatment and the correlation with neurological deficits are needed.

Advances in mass-encoded probes for multiplex mass spectrometric detection / 中国药科大学学报

@#Mass-encoded probe is a probing tool that specifically identifies target molecules and thus outputs their characteristic ion signals with mass tags.It plays an important role in multiplex assay of disease markers, drug target screening and other biomedical applications.Based on various mass spectrometric methods, researchers have developed an array of mass tag-encoded probes with different structures and functions, providing powerful technical tools for multiplex detection of biomolecules in physiological environments and for mass spectrometry imaging of tissue samples.This review introduces the latest research progress of mass tag-encoded probes in multiplex mass spectrometric detection from three aspects, i.e. structural composition of the probes, mass spectrometric methods and their application in biochemical analysis, with a prospect of the future development of mass tag-encoded probes.

Development and verification of multiplex fluorescence quantitative PCR (Taqman probe) for detection of telomerase activity / 中国生物制品学杂志

@#Objective To develop and verify a multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity.Methods Specific reverse transcription primers,two pairs of quantitative primers and probes were designed for the CDS sequence of telomerase catalytic subunit telomerase reverse transcriptase(TERT).After optimization of the reverse transcription primers(specific reverse transcription primers and random primers) and quantitative primers(two pairs of quantitative primer probes used alone or in combination) in the reaction system,with the primer probe of internal reference gene GAPDH,multiplex fluorescence quantitative PCR was performed in a single tube.In addition,telomerase positive standard and negative standard were prepared with 293T and MRC-5 cells respectively,and the stability and precision of the method were verified.The telomerase activity in 19 normal mesenchymal cell samples and 32 breast cancer cell samples were detected by the developed method.Results The optimum reaction system was as follows:using cDNA synthesized with specific reverse transcription primers as the template,2 pairs of quantitative primer probes of TERT gene were mixed with internal reference gene GAPDH primer probes for multiplex fluorescence quantitative PCR reaction in a single tube.After optimization,the sensitivity and TERT fluorescence signal quantity of the system were greatly improved,and the ΔRn was enhanced by 3 times.The amplification curve of positive standard TERT gene was normal,and the ΔCt between TERT gene and GAPDH gene remained stable.The amplification curve of GAPDH gene in negative standard was normal,while there was no amplification curve of TERT gene.There was a little difference in ΔCt between TERT and GAPDH genes in the positive standard frozen and thawed for 3 and 5 times repeatedly and the positive standard without freezing and thawing,and the CVs of precision in intra-and inter-groups were all less than 1%.Telomerase activity was negative in 19 normal mesenchymal cell samples and positive in 32 breast cancer cell samples,and significant difference in Ct value of TERT gene between them was observed(t=4.236,P <0.001).Conclusion The developed multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity has good stability and precision,which is expected to be used in early diagnosis and gene therapy of tumors.

Development and verification of multiplex real-time RT-PCR assay for classing and identification of HPIV1,HPIV2 and HPIV3 / 中国生物制品学杂志

@#ObjectiveTo develop and verify a multiplex real-time RT-PCR assay for simultaneous identification of human parainfluenza virus type 1(HPIV1),type 2(HPIV2)and type 3(HPIV3).MethodsThe whole genome sequences of HPIV1,HPIV2 and HPIV3 were downloaded from the database for alignment analysis,and the conserved regions were selected. Specific primers and probes were designed for the three viruses respectively to develop a multiplex real-time RTPCR assay with human ribonuclease P(RNase P)as theinternal quality control. The method was verified for the sensitivity,specificity and precision and used to detect 192 clinical samples.ResultsAfter optimization,the multiplex real-time RTPCR reaction system was determined to be 30 μL,including 10 × NeoscriptRTase/UNG Multi mix 3 μL,5 × Neoscript RT Premix Multi Buffer 6 μL,upstream and downstream primers of HPIV1,HPIV2 and HPIV3(100 μmol/L)0. 1 μL respectively,HPIV1,HPIV2,HPIV3 probes(100 μmol/L)0. 05 μL respectively,RNase P upstream and downstream primers(50 μmol/L)0. 06 μL respectively,RNase P probe(50 μmol/L)0. 03 μL respectively,template 15 μL,and ddH2O supplemented to 30 μL. The reaction conditions were 50 ℃ 20 min,95 ℃ 3 min and 45 cycles of 95 ℃ 15 s and54 ℃ 30 s. Fluorescence signals were collected during annealing in each cycle. The minimum detection limits of HPIV1,HPIV2 and HPIV3 were all 500 copies/mL by the multiplex real-time RT-PCR assay;The method showed no cross-reaction with influenza A virus,influenza B virus,respiratory syncytial virus andnovel coronaviruses. The coefficients of variation(CVs)in intra-and inter-groups of the recombinant plasmid standard mixture with three different concentrations were all less than 3%. HPIV1,HPIV2 and HPIV3 were detected in 192 clinical samples,and the positive rates were7. 81%,0. 05% and 3. 1%,respectively.ConclusionThe multiplex real-time RT-PCR assay for detection of HPIV1,HPIV2 and HPIV3 developed in this study has good sensitivity,specificity and precision,which has a high clinical application prospect in the field of rapid diagnosis and identification of HPIV.

High-risk human papillomavirus in Turkish patients with clinically suspicious cervical lesions analyzed by multiplex-PCR

Background & objectives: Human papillomavirus (HPV) infection is known to be the main cause of cervical cancer. This study aimed to determine the prevalence of high-risk HPV genotypes in smear specimens taken from women who had normal or abnormal cytology using a multiplex PCR method. Methods: The study included 270 women aged between 19 and 69 yr with or without suspicious cervical abnormalities. A Pap smear sample from each patient was cytologically examined, and HPV typing was performed using a multiplex fluorescent PCR method. Those who were high-risk HPV positive and had a normal or abnormal cytology were further evaluated by colposcopy and biopsy. Results: The total HPV positivity was 43 per cent (116/270). HPV positivity in the patients with an abnormal cytology was 77 per cent (33/43), whereas it was only 37 per cent (83/227) in women with normal cytology, which showed a significant difference (P<0.05). HPV positivity was also related to the age group when all the subjects were considered (P<0.05), and the highest prevalence of HPV infection was in the 30-39 yr age group. High-risk HPV types 16, 18, 31, 35, 51 and 56 were more common in the normal cytology patients, whereas high-risk HPV types 16, 31, 35, 45, 58 and 68 were commonly found in the abnormal cytology patients. Interpretation & conclusions: The determination of high-risk HPV genotypes in women with clinically suspicious cervical lesions should be conducted during an annual follow-up, irrespective of a normal or abnormal cytology by the age of 30 years or above.

Dengue Serotype Prevalence and Laboratory Profile Correlation in a Tertiary Care Hospital in Gandhinagar

Background: Dengue fever and dengue haemorrhagic fever currently rank highly among the newly emerging infectious diseases and are the most important arboviral disease worldwide. Dengue virus can be distinguished by both serological and molecular methods. This study was aimed at analysing the prevalence and laboratory dynamics of the four dengue serotypes in tertiary care patients attending GMERS Medical College Gandhinagar. Material & methods: This study was an observational retrospective study. A total 105 samples were tested for Dengue serotyping by RT-PCR. Results: Among positive patients Dengue virus serotype-2 was the most common serotype 94 (89%) followed by DENV3 7(6%) and DENV4 2(2%). Co-infection with DENV 2/4 was 2(2%). A higher prevalence of dengue haemorrhagic fever was noted in serotype 2 compared to serotypes 3, 4, and coinfection. Thrombocytopenia was present in all serotypes of infection. There was a significant difference in the disturbance of liver function in DENV2, as compared to others serotype. Dengue serotype 2 was very common in rural areas, while dengue serotype 3 was seen in the urban Gandhinagar zone. Discussion: Dengue is the most extensively spread mosquito-borne disease. As per previous studies most common prevalent and severity of serotype wasDENV2, however in our study we were able to identify DENV3, DENV4 and confection with serotypes (DENV2 & DENV4).

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.

A Multiplex PCR assay for a differential diagnostic of rickettsiosis, Lyme disease and scrub typhus

Background & objectives: Coexistence of tick-borne diseases in some regions in Latin America makes the diagnosis difficult due to shared initial signs and symptoms. Rickettsiosis, Lyme disease and recently, scrub typhus are gaining more importance. The objective of this study is to develop a multiplex-PCR assay for a differential diagnosis of rickettsiosis, Lyme disease and scrub typhus. Methods: By using bibliographic and bioinformatic analysis, we identify candidate regions to perform the multiplexPCR assay for Rickettsia sp., Borrelia burgdorferi and Orientia tsutsugamushi as well as identify optimal melting temperature and sensibility analysis. Results: We identified specific primer pairs for Rickettsia sp, Borrelia burgdorferi and Orientia tsutsugamushi with different PCR fragment length but a common melting temperature, 58°C. Interpretation & conclusion: We successfully developed a Multiplex PCR assay for differential diagnosis of rickettsiosis, Lyme disease and scrub typhus that could be a rapid and easy option in clinical and epidemiological practice.Laboratorio de Enfermedades Infecciosas y Parasitarias 1. Unidad Interinstitucional de Investigación Clínica y Epidemiológica, Facultad de Medicina, Universidad Autónoma de Yucatán. Yucatán, Mexico

Laboratorio de Enfermedades Emergentes y Re-emergentes. Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”. Universidad Autonoma de Yucatan. Yucatán, Mexico

RADIOGRAPH BASED DIAGNOSIS OF ARTHROGRYPOSIS MULTIPLEX CONGENITA

Arthrogryposis multiplex congenita (AMC) is a rare disorder, presenting with multiple contractures and limb deformities.Various theories have been proposed for the development of this congenital anomaly. The final diagnosis is made based upon the clinical signs and imaging findings. This case report aims to outline the imaging features that can help radiologists to make the diagnosis of AMC through radiographs and skeletal survey. Since, very few cases have been reported in literature, no specific treatment protocols have been outlined. Therefore, antenatal diagnosis based on characteristic imaging findings is crucial for early termination and parental counselling.

Impact of quality of response on survival outcomes among multiple myeloma patients treated with novel agents - a retrospective analysis

Abstract BACKGROUND: In this era of target therapies, novel data on the correlation between response endpoints and survival outcomes in multiple myeloma have arisen. OBJECTIVE: To determine the impact of quality of response on clinical outcomes, using first-line treatment, and identify risk factors influencing progression-free survival (PFS) and overall survival (OS) among myeloma patients. DESIGN AND SETTING: Retrospective analysis on myeloma patients who were treated at the Clinic of Hematology and Clinical Immunology, University Clinical Centre, Niš, Serbia, over a four-year period. METHODS: A total of 108 newly diagnosed patients who received first-line therapy consisting of conventional chemotherapy or novel agent-based regimens were included in this analysis. RESULTS: The quality of response to first-line therapy for the whole cohort was classified as follows: complete response (CR) in 19%; very good partial response (VGPR) in 23%; partial response (PR) in 38%; and less than PR for the remaining patients. After a median follow-up of 25.4 months, the three-year PFS and OS for the entire study population were 47% and 70%, respectively. Achievement of CR was the main factor associated with significantly prolonged PFS and OS, in comparison with patients who reached VGPR and PR. Likewise, addition of the new drugs bortezomib and thalidomide to standard chemotherapy led to considerably extended PFS and OS, compared with conventional therapy alone. CONCLUSIONS: This analysis demonstrated that the quality of response after application of first-line treatment using novel agent-based regimens among multiple myeloma patients was a prognostic factor for PFS and OS, which are the most clinically relevant outcomes.

Multiplex loop mediated isothermal amplification (m-LAMP) as a point of care technique for diagnosis of malaria

Diagnosis of malaria is a prominent challenge due to the endemic nature of infection. Malaria poses a great threat to global public health. The disease can be diagnosed by several techniques out of which microscopy is a known gold standard. High sensitivity of molecular techniques is making them more reliable and popular as tools for diagnosis of malaria. However, new methods are required which can fulfill the criteria of being Point of Care Test (POCT) as defined by WHO. Loop-mediated isothermal amplification (LAMP) technique amplifies DNA in an isothermal condition, and surpasses the disadvantages of conventional molecular techniques such as polymerase chain reaction. Multiplex LAMP, a modification of LAMP may emerge as a new POC for malaria diagnosis. This review deals with the use of LAMP and multiplex LAMP in diagnosis of malaria and its prospective use as point of care techniques.