Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization for detecting chromosome abnormalities in myelodysplastic syndromes: A retrospective study.

ABSTRACT: This study aimed to compare interphase fluorescence in situ hybridization (iFISH) and multiplex ligation dependent probe amplification (MLPA) for identifying genetic changes in myelodysplastic syndromes (MDS).The frequencies of cytogenetic changes in MDS patients treated at the Institute of Hematology and Blood Disease Hospital (China) in 2009 to 2018 were assessed by iFISH based on bone marrow samples. Then, the effectiveness of MLPA in detecting these anomalies was evaluated.Specimens from 287 MDS patients were assessed. A total of 36.9% (103/279) of MDS cases had chromosomal abnormalities detected by iFISH; meanwhile, 44.1% (123/279) harbored ≥1 copy-number variation (CNV) based on MLPA: +8 (n=46), -5 (n = 39), -7 (n = 27), del 20 (n = 32) and del 17 (n = 17). Overall, 0 to 4 aberrations/case were detected by MLPA, suggesting the heterogeneous and complex nature of MDS cytogenetics. There were 29 cases detected by MLPA, which were undetected by FISH or showed low signals. Sixteen of these cases had their risk classification changed due to MLPA detection, including 9 reassigned to the high-risk IPSS-R group. These findings demonstrated that MLPA is highly efficient in assessing cytogenetic anomalies, with data remarkably corroborating FISH findings (overall consistency of 97.1%). The sensitivities of MLPA in detecting +8, -5, -7, del 20 and del 17 were 92.3%, 97.1%, 100%, 100%, and 90%, respectively, with specificities of 95.8%, 97.6%, 97.7%, 97.6%, and 97%, respectively.MLPA represents a reliable approach, with greater efficiency, accuracy, and speed than iFISH in identifying cytogenetic aberrations in MDS.

Use of microarrays and MLPA for integrating diagnostics and personalizing treatment - Case report of a patient with Ph-like acute B-cell lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood cancer. A special subtype of high risk BCP-ALL is Philadelphia-like ALL (Ph-like ALL), in which the gene expression profile is similar to BCR-ABL1-positive leukemia; however, fusion of the mentioned genes does not occur. The unfavourable clinical course and incidence of 15% of cases means that the diagnosis and therapeutic strategy of Ph-like ALL must be carefully developed and implemented into clinical practice. The study presents the case of a patient with diagnosed Ph-like ALL. The use of molecular analytical techniques has made it possible to identify a patient who is likely to relapse and who may benefit from personalized therapy This study shows the advantages of using genomic analyses to identify therapeutic targets, which is especially important for patients with high-risk disease. This model of management could be extended to other cancer subtypes, allowing for tailored diagnosis.

Ultrasensitive digital quantification of cytokines and bacteria predicts septic shock outcomes.

Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. Here, we demonstrate sensitive and rapid quantification of bacterial load and cytokines from human biological samples to generate actionable hypotheses. Our digital assay measures IL-6 and TNF-α proteins, gram-negative (GN) and gram-positive (GP) bacterial DNA, and the antibiotic-resistance gene blaTEM with femtomolar sensitivity. We use our method to characterize bronchoalveolar lavage fluid from patients with asthma, and find elevated GN bacteria and IL-6 levels compared to healthy subjects. We then analyze plasma from patients with septic shock and find that increasing levels of IL-6 and blaTEM are associated with mortality, while decreasing IL-6 levels are associated with recovery. Surprisingly, lower GN bacteria levels are associated with higher probability of death. Applying decision-tree analysis to our measurements, we are able to predict mortality and rate of recovery from septic shock with over 90% accuracy.

A Retrospective Study about the Impact of Switching from Nested PCR to Multiplex Real-Time PCR on the Distribution of the Human Papillomavirus (HPV) Genotypes.

BACKGROUND AND OBJECTIVES: Human papillomavirus (HPV) is the most prevalent etiological agent of viral sexually-transmitted infection. This study retrospectively evaluated the impact of a switch to a real-time PCR assay in the HPV prevalence and genotypes distribution by a quasi-experimental before-and-after approach. MATERIALS AND METHODS: In total, 1742 samples collected from 1433 patients were analyzed at the UOC Microbiology and Virology of Policlinico of Bari, Italy. HPV DNA detection was performed using initially nested PCR and subsequently multiplex real-time PCR assay. RESULTS: Statistically significant difference in HPV overall prevalence after the introduction of the real-time assay was not detected (48.97% vs. 50.62%). According to different extraction-DNA amplification methods, differences were observed in the prevalence rates of HPV-45, 68, 40, 42, and 43. The lowest prevalence for HPV-45 was observed in the Magna Pure-Real Time PCR group, while HPV-68, 40, 42, and 43 were less observed in the Qiagen-Real Time PCR group. After, a multivariate logistic regression, an increase in the prevalence of HPV-42 (aOR: 4.08, 95% CI: 1.71-9.73) was associated with the multiplex real-time PCR assay. CONCLUSIONS: Although this study is a not a direct comparison between two diagnostic methods because it has a sequential structure, it serves to verify the impact of a new molecular assay on HPV distribution. Moreover, the stability of HPV prevalence over time suggests that the population composition and the behavioral variables did not likely change during the observation period. Our study proposes that the introduction of a molecular test for HPV detection may be related to changes of HPV genotypes distribution.

Padronização de multiplex PCR para detecção de dermatófitos em pelos e crostas de cães e gatos / Standardization of a PCR multiplex for the detection of dermatophytes in dogs and cats fur and crusts

Objetivou-se padronizar uma reação do tipo multiplex PCR (mPCR) para detectar Microsporum canis, Microsporum gypseum e o complexo Trichophyton mentagrophytes em amostras de pelos e/ou crostas de cães e gatos. 250 amostras de pelos e/ou crostas de cães e gatos foram analisadas por meio de exame direto e cultura, o DNA das mesmas foi extraído para mPCR. Primers foram desenhados e como controle positivo da reação utilizou-se o DNA extraído de colônias de M. canis (URM 6273), M. gypseum (URM 6921) e T. mentagrophytes (URM 6211), provenientes da Coleção de Culturas (Micoteca URM), Departamento de Micologia, Centro de Ciências Biológicas da Universidade Federal de Pernambuco (CCB/UFPE). Como controles negativos de reação, utilizou-se água destilada esterilizada e DNA extraído de Alternaria sp. para verificar a especificidade dos primers. Do total de amostras analisadas, 15 (6%) foram identificadas, em cultura, como dermatófitos, e destas, 10 foram M. canis, três M. gypseum e dois T. mentagrophytes (complexo). Destas 15 amostras positivas, 11 (73,3%) foram detectadas por meio da mPCR. Além destas, seis outras, negativas em cultura, foram identificadas como M. gypseum. Verificou-se uma boa concordância entre os resultados da cultura e mPCR (Kappa: 0,66). O protocolo padronizado neste estudo pode ser utilizado como um método de triagem, por apresentar uma sensibilidade maior que a da cultura, usado paralelamente aos exames de rotina, permitindo um diagnóstico em menor tempo.(AU)

The aim of this study was to standardize a multiplex PCR (mPCR) reaction to detect Microsporum canis, Microsporum gypseum and the Trichophyton mentagrophytes complex in dog and cat fur and/or crusts. 250 fur and/or crusts samples from dogs and cats were analyzed by direct examination and culture, DNA from them was extracted for mPCR. Primers were designed and the DNA extracted from colonies of M. canis (URM 6273), M. gypseum (URM 6921) and T. mentagrophytes (URM 6211) from the Collection of Cultures - URM Micoteca - Department of Mycology, Biological Sciences Center of the Federal University of Pernambuco (CCB / UFPE). As negative controls, sterile distilled water and DNA extracted from Alternaria sp., were used to verify the specificity of the primers. Of the total samples analyzed, 15 (6%) were identified in culture as dermatophytes, and of these, 10 were M. canis, three M. gypseum and two T. mentagrophytes (complex). Of these 15 positive samples, 11 (73.3%) were detected by mPCR. Besides these, six others, negative in culture, were identified as M. gypseum. There was good agreement between culture results and mPCR (Kappa: 0.66). The protocol standardized in this study can be used as a screening method, because it has a sensitivity greater than that of the culture, used in parallel to the routine exams, allowing a diagnosis in a shorter time.(AU)

Detecção de Aeromonas spp. e do gene de virulência aerolisina em tilápias do Nilo (Oreochromis niloticus) com a técnica de mPCR / Detection of Aeromonas spp. and virulence gene aerolysin in Nile tilapia (Oreochromis niloticus) using PCR technique

As infecções causadas por bactérias do gênero Aeromonas estão entre as doenças mais comuns em peixes cultivados em todo o mundo, com ocorrência de aeromoniose em todos os países que possuem cultivo de tilápia do Nilo (Oreochromis niloticus). O presente trabalho descreve o desenvolvimento de uma nova multiplex PCR (mPCR) para diagnóstico de Aeromonas spp. e identificação do gene aerolisina (aerA). Para padronização da mPCR foram utilizadas cepas de referência de várias espécies do gênero Aeromonas e de outros gêneros. Também foram usadas cepas de campo de A. hydrophila oriundas de cultivos de peixes pacamãs (Lophiosilurus alexandri) e Aeromonas spp. de tilápias do Nilo. Os primers foram desenhados com base na região 16S rRNA e aerA. Para verificar a melhor temperatura de anelamento foram utilizados gradientes entre 59°C a 61°C com 40ng de DNA molde. Os produtos da amplificação da região 16S rRNA e do gene aerA apresentaram 786 e 550pb, respectivamente. A mPCR apresentou melhor temperatura de anelamento a 57,6°C com limite de detecção das concentrações de DNA em ambos genes (16S rRNA and aerA) de 10-10g/μL. A mPCR padronizada é rápida, sensível e específica no diagnóstico de Aeromonas spp. e identificação do gene aerolisina. Esta metodologia apresenta vantagens quando comparada aos métodos de diagnóstico convencionais, podendo ser utilizada em cultivos comerciais de tilápias do Nilo ou outros peixes. A identificação do gene aerolisina é uma importante ferramenta na determinação do potencial patogênico dos isolados de Aeromonas spp. estudados.(AU)

Infections caused by bacteria of the genus Aeromonas are among the most common diseases in fish farming systems worldwide, and this disease occurs in all countries which have Nile tilapia (Oreochromis niloticus) farmed. The present work describes the development of a new multiplex PCR (mPCR) technique that diagnosis the genus Aeromonas and detects aerolysin gene (aerA). Reference strains of several Aeromonas species and other genera were used for standardization of mPCR. Strains of A. hydrophila from "pacaman" fish (Lophiosilurus alexandri) and Aeromonas spp. from Nile tilapia from farming systems were used too. Primers were designed based on the 16S rRNA region and aerA (aerolysin toxin). To verify a better annealing temperature were used gradients between 59°C and 61°C with 40ng of the DNA template. The 16S rRNA gene and the aerA gene amplification products showed 786 and 550 bp, respectively. The mPCR showed better annealing temperature at 57.6°C, and the detection limit for both genes (16S rRNA and aerA) was 10-10g/μL of the DNA. The standardized mPCR is quick, sensitive, and specific for Aeromonas spp. diagnosis and to detect aerolysin gene. This method showed advantages when compared to the conventional diagnostic methods and can be used in Nile tilapia or other fish farming systems. The detection of aerolysin gene is an important tool to determine the potential pathogenicity of Aeromonas spp. isolates.(AU)

Quantitative Thresholds Enable Accurate Identification of Clostridium difficile Infection by the Luminex xTAG Gastrointestinal Pathogen Panel.

Clostridium difficile colonizes the gastrointestinal (GI) tract, resulting in either asymptomatic carriage or a spectrum of diarrheal illness. If clinical suspicion for C. difficile is low, stool samples are often submitted for analysis by multiplex molecular assays capable of detecting multiple GI pathogens, and some institutions do not report this organism due to concerns for high false-positive rates. Since clinical disease correlates with organism burden and molecular assays yield quantitative data, we hypothesized that numerical cutoffs could be utilized to improve the specificity of the Luminex xTAG GI pathogen panel (GPP) for C. difficile infection. Analysis of cotested liquid stool samples (n = 1,105) identified a GPP median fluorescence intensity (MFI) value cutoff of ≥1,200 to be predictive of two-step algorithm (2-SA; 96.4% concordance) and toxin enzyme immunoassay (EIA) positivity. Application of this cutoff to a second cotested data set (n = 1,428) yielded 96.5% concordance. To determine test performance characteristics, concordant results were deemed positive or negative, and discordant results were adjudicated via chart review. Test performance characteristics for the MFI cutoff of ≥150 (standard), MFI cutoff of ≥1,200, and 2-SA were as follows (respectively): concordance, 95, 96, and 97%; sensitivity, 93, 78, and 90%; specificity, 95, 98, and 98%; positive predictive value, 67, 82, and 81%;, and negative predictive value, 99, 98, and 99%. To capture the high sensitivity for organism detection (MFI of ≥150) and high specificity for active infection (MFI of ≥1,200), we developed and applied a reporting algorithm to interpret GPP data from patients (n = 563) with clinician orders only for syndromic panel testing, thus enabling accurate reporting of C. difficile for 95% of samples (514 negative and 5 true positives) irrespective of initial clinical suspicion and without the need for additional testing.

Occurrence of Mycoplasma bovigenitalium and Ureaplasma diversum in dairy cattle from to Pernambuco state, Brazil / Ocorrência de Mycoplasma bovigenitalium e Ureaplasma diversum em bovinos leiteiros do estado de Pernambuco, Brasil

The objective of this study was to conduct an investigation of Mycoplasma bovigenitalium and Ureaplasma diversum infections in cattle in the microregion of the Ipanema Valley, state of Pernambuco, Brazil. Vaginal swabs were collected from 355 breeding cows in reproductive age and were analyzed by multiplex PCR (mPCR) and culture. An epidemiological investigation of risk factors was performed for Mollicutes. mPCR analysis showed that, 9.29% (33/355) of the cows were positive for M. bovigenitalium and 21.69% (77/355) for U. diversum; coinfection was observed in 2.81% (10/355) of the cows. The microbiological isolation showed, 81.81% (27/33) of Mycoplasma spp. and 24.67% (19/77) of Ureaplasma spp.. The risk factors related to Mollicutes infection identified were semi-intensive breeding system (OR= 4.6), pasture rent (OR= 3.6), non-isolation of animals with reproductive disorders (OR= 3.2), and natural mounting and artificial insemination (OR= 3.5). There was a significant association between Mollicutes infection and abortions in the first gestational third (P= 0.001). This is the first record of M. bovigenitalium and U. diversum infection in cows in the semiarid region of the state of Pernambuco, Brazil. Preventive measures directed to the identified risk factors can decrease the occurrence of Mollicutes in these herds.(AU)

O objetivo deste estudo foi realizar uma investigação de Mycoplasma bovigenitalium e Ureaplasma diversum em bovinos leiteiros da microrregião do Vale do Ipanema, estado de Pernambuco, Brasil. Foram coletados suabes vaginais de 355 vacas em idade reprodutiva. As amostras foram analisadas por multiplex PCR (mPCR) e cultura. Foi realizada uma investigação dos fatores de risco para Mollicutes. Na mPCR, 9,29% (33/355) das vacas foram positivas para M. bovigenitalium e 21,69% (77/355) para U. diversum; coinfecção foi observada em 2,81% (10/355) das vacas. O isolamento microbiológico mostrou crescimento de Mycoplasma spp. em 81,81% (27/33) das amostras e em 24,67% (19/77) para Ureaplasma spp. Os fatores de risco relacionados à infecção por Mollicutes identificados foram sistema de produção semi-intensivo (OR= 4,6), aluguel de pastagem (OR= 3,6), não isolamento de animais com desordens reprodutivas (OR= 3,2) e monta natural e inseminação artificial (OR= 3,5). Houve uma associação significativa entre a infecção por Mollicutes e abortos no primeiro terço gestacional (P=0,001). Este é o primeiro relato da infecção por M. bovigenitalium e U. diversum em vacas na região semiárida do estado de Pernambuco, Brasil. As medidas preventivas direcionadas aos fatores de risco identificados podem diminuir a ocorrência de Mollicutes nesses rebanhos.(AU)

Screening for BRCA1 large genomic rearrangements in female Egyptian hereditary breast cancer patients.

Approximately 5%-10% of all breast cancers are inherited as the result of germline mutations in the BRCAl gene. Large genomic rearrangements (LGRs) in BRCA1 have not been well-researched in the Egyptian population. Using multiplex ligation-dependent probe amplification, we showed BRCA1 rearrangements in 4/22 cases (18.2%) of familial breast cancer. No influence of having multiple breast cancer cases within the family was observed in patients diagnosed at or=45 years and having BRCAl-positive LGRs. However, focusing on cases with first- and second-degree relatives affected, we observed a significant difference between the percentage of patients with BRCA1-positive versus BRCAl-negative LGRs. Our results provide the first evidence that LGRs in BRCA1 exist in the Egyptian population. Screening for these alterations may be desirable when breast cancer patients are diagnosed at an early age, especially if these cases have first- and second-degree of relatives with breast cancer.