Tumor BRCA testing in ovarian cancer and EQA scheme: our experience of a critical evaluation

Next generation sequencing (NGS) is a widespread molecular biology method integrated into clinical practice to detect genetic variants, for diagnostic and prognostic purposes. The scheduled external quality assessments (EQA) is integral part of clinical molecular laboratory quality assurance. The EQA provides an efficient system to compare analytic test performances among different laboratories, which is essential to evaluate consistency of molecular test. EQA failures demands targeted corrective action plans. In this context, the complexity of the NGS techniques requires careful and continuous quality control procedures. We report a tumor BRCA1/2 (tBRCA) testing benchmark discrepancy provided by the European Molecular Genetics Quality Network in our laboratory during a round of EQA for somatic mutation testing of BRCA genes in relation to ovarian cancer. The critical analysis emerging from the tBRCA EQA is presented. We underline that harmonization processes are still required for the EQA in the molecular biology field, especially if applied to the evaluation of methods characterized by high complexity.

Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. Methods. The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). Results. The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. Conclusion. During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.

Screening and identification of pectinolytic bacteria for ramie degumming

To explore high-quality microbial resources with the capability of ramie degumming, we collected soil samples from rotten ramie and straw heaps. After enrichment culture by ramie raw materials, bacterial strains with the potential ramie-degumming function were screened using a pectin-hydrolysis plate. Dominant bacteria were identified by combining colonial morphological characteristics with the molecular biology method, and their ramie-degumming effects were verified through comprehensive biological degumming indices. Results demonstrated that Bacillus aryabhattai, Bacillus thuringiensis, Lysinibacillus fusiformis, and Acidovorax temperans were successfully obtained. The highest pectinase activity, 98.2 U/mg, was found by A. temperans. B. thuringiensis showed the best ramie-degumming effect. The residual gum content, single-fiber linear density, and bundle-breaking strength of the degummed ramie fiber treated with B. thuringiensis were 8.32%, 6.80 dtex, and 7.84 cN/dtex, respectively. The residual gum content of the ramie fiber treated with B. thuringiensis met the textile requirement (<10%), and the values of all other indicators were also satisfactory. Therefore, B. thuringiensis was an excellent strain for ramie degumming, indicating potential industrial applications.

Seven Years Leptospirosis Follow-Up in a Critical Care Unit of a French Metropolitan Hospital; Role of Real Time PCR for a Quick and Acute Diagnosis

(1) Background: Leptospirosis infection can lead to multiple organ failure, requiring hospitalization in an intensive care unit for supportive care, along with initiation of an adapted antibiotic therapy. Achieving a quick diagnosis is decisive in the management of these patients. (2) Methods: We present here a review of leptospirosis cases diagnosed in the intensive care unit of our hospital over seven years. Clinical and biological data were gathered, and we compared the differences in terms of diagnostic method. (3) Results: Molecular biology method by Polymerase Chain Reaction (PCR) allowed quick and reliable diagnosis when performed in the first days after the symptoms began. Moreover, we identified that sampling blood and urine for PCR was more efficient than performing PCR on only one type of biological sample. (4) Conclusions: Our results confirm the efficiency of PCR for the quick diagnosis of leptospirosis and suggest that testing both blood and urine early in the disease might improve diagnosis.

ISOLATE OF HETEROTROPHIC MICROALGAE AS A POTENTIAL SOURCE FOR DOCOHEXAENOIC ACID (DHA)

Docosahexaenoic acid (DHA) is one of essential fatty acids that are beneficial to health. Nowadays, the source of docosahexaenoic acid (DHA) is mainly obtained from fish which are extracted into fish oil products. However, the fish oil products still have some drawbacks in term of purity, acceptable flavor for costumers, and also their not environmental friendly production process. As an alternative solution, heterotrophic microalgae can be used as a potential source for DHA due to their excellence compared to fish oil products. The aim of this study is to isolate the heterotropic microalgae that can produce DHA. The heterotrophic microalgae were isolated from mangrove fallen leaves (Rhizophora apiculata) by using direct planting method. The morphology of pure microalgae colony were observed through light microscope and subsequently fermented for 14 days. Fatty acids were extracted and methylated through direct transesterification method. Identification and quantification of DHA were conducted by using gas chromatography. The results were four isolates of heterotropic microalgae, namely MTKC1, MTKC2, MTKC3, and MTKC4. The extract of MTKC2 that only showed the content of DHA with value of 9.2 % w/w. Therefore MTKC2 is a potential source for DHA. The MTKC2 was further identified by using molecular biology method and confirmed as Thraustochytrium aureum.

Isolation, characterization of Bacillus sp. producing heavy metal absorption γ-PGA

Poly-gamma-glutamic acid (γPGA), which is a biodegradable, non-immunogenic and unusual anionic amino-acid polymer consist of D- and L-glutamic acid units, was exploited for a wide array of useful applications. Bacillus are well known cellular system important for fermentation to synthesize γPGA, which is used as thickener, drugs carrier, cryoprotectant, humectant, biological adhesive, flocculants, or heavy metal absorbent. This study focused on the isolation of Bacillus spp. that is possible to produce γ-PGA from different soil samples from different places in Vietnam. Study the effect of precursors, temperature, carbon sources, times and pH on γ-PGA production. From 31 soil samples and 4 straws samples, strain 20.2 which produced the highest γ-PGA yields (riches 15.2 mg/ml), was identified as Bacillus sp. 20.2 by molecular biology method. The suitable conditions for growing of Bacillus sp. 20.2 strain to produce γ-PGA are at 37°C, pH 7 after 72 hours. Citric acid instead of glucose in a GSP medium is better for producing γ-PGA by strain Bacillus sp. 20.2. Poly-gamma-glutamic acid (γ-PGA) là một polymer amino-acid gồm D và L-glutamic acid, có khả năng phân hủy sinh học, không gây miễn dịch, đã được ứng dụng rộng rãi trong công nghiệp, y học. Bacillus subtilis được biết đến là hệ thống tế bào ý nghĩa quan trọng trong quá trình lên men để tổng hợp γ-PGA. γ-PGA hòa tan trong nước, phân hủy sinh học và không độc đối với con người và môi trường. γ-PGA ổn định với nhiều protease vì các protease thường không nhận acid γ-glutamic (Obst et al., 2004). γ-PGA có cấu trúc đồng phân đơn giản, không gây miễn dịch. Do đó, γ-PGA đã được quan tâm ứng dụng trong các lĩnh vực như y học, công nghiệp thực phẩm, mỹ phẩm và đặc biệt là xử lý nước nhiễm kim loại nặng. Trong nghiên cứu này chúng tôi tập trung phân lập, tuyển chọn các chủng Bacillus có khả năng sinh tổng hợp PGA cao. Sau đó định danh và đánh giá khả năng sinh tổng hợp PGA từ chủng đã phân lập được. Kết quả cho thấy từ 34 mẫu rơm và đất, chúng tôi đã phân lập được chủng với mã số 20.2 có khả năng sinh PGA cao nhất đạt 15.2 mg/ml. Chủng này đã được định danh bằng phân tích trình tự gene 16S rRNA và thuộc loài Bacillus sp. Môi trường thích hợp sinh tổng hợp PGA là GSP ở điều kiện 37oC pH7 sau 72 giờ nuôi cấy.

PCR-Based Detection and Genotyping ofHelicobacter pyloriin Endoscopic Biopsy Samples from Brazilian Patients

Helicobacter pylori(H. pylori) is considered the second most prevalent infection in man. A precise diagnosis is important for treating patients with the indicative gastrointestinal symptoms. The present study analyzes the effectiveness of a molecular biology method (PCR) comparing the results obtained with the histology and with the rapid urease tests. PCR was used in the detection and genotyping of theH. pyloriurease-C gene and the patterns which were obtained from the patients studied. 141 biopsy samples from 131 patients were evaluated. 59 paraffin biopsies samples were positive forH. pyloriaccording to the histological examination. Of those, 59/12 (20.3%) were amplified using PCR. Of the 82 samples from the fresh biopsies, 64 were positive forH. pyloriaccording to the rapid urease test (78%); there was an agreement of 100% with PCR. Sixty positiveH. pylorisamples were genotyped (58 samples of fresh biopsies and 2 samples of paraffin biopsies) using two restriction enzymes. The patterns observed were analyzed with the computational program BIO 1D; 11 patterns with the enzymeHhaIand 12 patterns with the enzymeMboIwere found. However, it was not possible to find a statistically significant correlation between the specific genotypes and digestive pathologies. Accordingly, future research should be performed to confirm a statistically significant relationship between genotyping and gastrointestinal symptoms.

Prevention of cervical cancer in women with ASCUS in the Brazilian Unified National Health System: cost-effectiveness of the molecular biology method for HPV detection

This study aimed to assess the performance of PCR as a means of detecting HPV 16/18 compared to the single probe-based PCR for detecting high-risk HPV, and evaluate these methods for detecting cervical intraepithelial neoplasia (CIN) in follow-ups for ASCUS testing. It also compares the costs of cytology, PCR methods, colposcopy and biopsy in the Brazilian Unified National Health System. Of the 81 patients with ASCUS, 41 (50.6%) tested positive for HPV 16/18 in PCR testing and 47 (58.02%) tested positive for high-risk HPV with single probe-based PCR testing. The negative predictive value was 93.75% for HPV 16/18 PCR and 100% for single probe-based PCR in cases that progressed to high-grade CIN. The annual costs of patient referral were the following: R$2,144.52 for referral of patients with ASCUS cytology for colposcopy; R$6,307.44 for referral of patients with ASCUS cytology and PCR positive for HPV 16/18 or colposcopy; R$3,691.80 for referral of patients with ASCUS cytology with single probe-based PCR positive for high-risk HPV. Therefore, cost per user can be reduced by performing single probe-based PCR for high-risk HPV on patients with ASCUS.