Molecular Laboratory Research Articles

Bacteriological Assessment of Stethoscope Used by Health Care Personnel in Attat Hospital, Snnp, Gurage Zone, Ethiopia

The stethoscope has always been an important element of a physician's toolkit when it comes to examining patients. The widespread use of stethoscopes by health-care workers for patient examinations makes them a potential source of nosocomial infection transmission. The goal of this study was to see if stethoscopes used by different health-care professionals in Attat Hospital may transmit bacteria. From April to June 2018, a cross-sectional study was done in the molecular laboratory of Wolkite University's department of biotechnology and biology. A total of 26 stethoscopes from health workers who had direct contact with patients were gathered during the study period. The sample was obtained using a sterile cotton-tipped applicator saturated in a sterile solution of physiologic saline (0.85% sodium chloride) to swab the whole surface of the stethoscope's diaphragm and then inoculated into macconkey agar, tryptone soya agar, and blood agar medium. 18 (69.2%) stethoscopes out of total collected stethoscopes had bacterial growth, and 12 bacterial isolates were selected and characterized to genus level. Isolates include staphylococcus aureus (37.5%), coagulase negative staphylococci (28.12%), Streptococcus sp. (21.88%), and Bacillus sp. (12.5%). All isolates were susceptible to the co-trimoxazole and ciprofloxacine, while resistant to cifoxitine. They showed intermediate growth against vancomycine. All except streptococcus were found resistant against penicillin. Both S. aureus and CoNS were sensitive to the chloramphenicol; Streptococcus was intermediate while bacillus was resistant to the chloramphenicol. All stethoscopes (42.2%) that had never been cleaned and were last cleaned a week ago were severely contaminated, while those washed multiple times a day and cleansed between each patient before the examination of the patient had lower levels of contamination (27%).

Lower Genital Tract Microbiome—A Work in Progress

The lower genital tract microbiome is composed of millions upon millions of microbes colonizing this unique anatomical niche reflecting the influence of genetic, biologic, and behavioral factors. Over the last decade, as a result of ever advancing technology and research investment, our knowledge of this complex microbiome ecosystem has grown enormously. All too often such progress remains invisible to practitioners. In this special issue, clinical scientists, in a unique contribution, have bridged the ever-increasing distance between vaginal microbiota science and clinical disease. Approaching a myriad of pathologic entities, authors describe the clinical application of recent molecular laboratory findings encouraging diagnostic, prognostic, and therapeutic advances. Nevertheless, challenges are recognized and the authors outline vulvovaginal disease management opportunities. Above all, exposing the dynamism of vaginal microbiota affords a better understanding of pathogenesis of lower genital tract disease.

Time to Negative Test Result among Patients with COVID-19: A Retrospective Study

Background: Coronavirus disease 2019 (COVID-19) is a public health emergency, however, there is still limited data on the length of infectiveness of the disease especially in Nigeria. The aim of this study was to ascertain the duration of viral shedding in patients testing positive for COVID-19 to deduce the appropriate time to discharge SAR-CoV2 Positive patients from follow-up by the healthcare team in our setting.
 Methods: In this retrospective study, time to a negative test result in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was evaluated in a cohort of 490 patients who tested positive at the Satellite Molecular Laboratory of the Rivers State University Teaching Hospital, Port Harcourt.
 Results: The mean age of the 490 patients was 39.8 ± 13.9. There were 379 (77.3%) males and 111 (22.7%) females. The mean duration of SARS-CoV-2 RNA detection was approximately 15±2 days. When comparing patients who were tested twice with those who were tested more than two times, a significant difference between mean time to a negative result and the number of tests done was identified (t= P-Value = <0.001).
 Conclusions: Findings from this study show that the average time of transition from a positive to negative PCR test result was approximately 15 days, regardless of the gender. This can provide an estimated duration of hospital stay among COVID-19 patients to prevent unnecessary healthcare costs and minimize re-infection.

A localized small-scale external quality assessment (EQA) for PCR testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the molecular laboratories

The aim of this study was to estimate the PCR results for SARS-CoV-2 testing in 32 participating laboratories in a localized small-scale external quality assessment (EQA) scheme. EQA samples were distributed to the participants and detected immediately on the day of delivery. Qualitative results were submitted to the EQA provider, including negative or positive results along with cycle threshold (Ct) values for different target genes. Although the variability of Ct values differed among the laboratories in the EQA, a total of 32 (100 %) participants reported correct qualitative results. The study showed that the mean loads of N or E gene were higher than those of ORF1ab in SARS-CoV-2 RNA samples. Regardless of the analyzed gene target, the mean Ct values for weak positive and positive samples varied by fewer than 1.74 and 1.91 cycles, respectively. Less than 12 % of reported Ct values for ORF1ab and N genes deviated by more than ±4 cycles (maximum: −9.92 cycles), while none deviated by more than ±4 cycles for the E gene. The current EQA program can provide a robust practical basis for follow-up planning to conduct evaluations for SARS-CoV-2 PCR testing and other novel emerging pathogens in the future.

Molecular characterization of xerosis cutis: A systematic review.

Xerosis cutis or dry skin is a highly prevalent dermatological disorder especially in the elderly and in patients with underlying health conditions. In the past decades, numerous molecular markers have been investigated for their association with the occurrence or severity of skin dryness. The aim of this review was to summarize the molecular markers used in xerosis cutis research and to describe possible associations with different dry skin etiologies. We conducted a systematic review of molecular markers of xerosis cutis caused by internal or systemic changes. References published between 1990 and September 2020 were searched using 'MEDLINE', 'EMBASE' and 'Biological abstracts' databases. Study results were summarized and analyzed descriptively. The review protocol was registered in PROSPERO database (CRD42020214173). A total of 21 study reports describing 72 molecules were identified including lipids, natural moisturizing factors (NMFs), proteins including cytokines and metabolites or metabolic products. Most frequently reported markers were ceramides, total free fatty acids, triglycerides and selected components of NMFs. Thirty-one markers were reported only once. Although, associations of these molecular markers with skin dryness were described, reports of unclear and/or no association were also frequent for nearly every marker. An unexpectedly high number of various molecules to quantify xerosis cutis was found. There is substantial heterogeneity regarding molecular marker selection, tissue sampling and laboratory analyses. Empirical evidence is also heterogeneous regarding possible associations with dry skin. Total free fatty acids, total ceramide, ceramide (NP), ceramide (NS), triglyceride, total free amino acids and serine seem to be relevant, but the association with dry skin is inconsistent. Although the quantification of molecular markers plays an important role in characterizing biological processes, pathogenic processes or pharmacologic responses, it is currently unclear which molecules work best in xerosis cutis.

Study of Parental Polymorphism and Allelic Variation for Grain Quality and Yield Traits in Rice (Oryza sativa L.) Using SSR Markers

Aim: Identification of polymorphic markers is prerequisite for conducting any QTL mapping experiment because if the parents are polymorphic for the traits of interest, then further selection of plants in the progenies becomes easy. Hence, the objective of the present study was to identify polymorphic markers for grain quality and yield traits among the parental lines Improved Samba Mahsuri and Badshabhog.
 Place and Duration of Study: It was carried out at Molecular Breeding Lab, Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi - 221 005, India, during 2019.
 Methodology: Two parents Improved Samba Mahsuri and Badshabhog were used for the present study. The DNA extraction was done as per the CTAB method suggested by Murray and Thompson. Standard PCR protocol was followed.
 Results: For parental polymorphism survey, a total of 576 randomly selected SSR markers including 26 gene specific markers related to aroma, cooking and eating quality, grain dimension and yield related traits distributed across the 12 chromosomes of rice were used. Overall, 96 markers including 4 gene specific markers were found to be polymorphic between the two genotypes indicating a total polymorphism percentage of 16.67%. The highest polymorphism percentage was recorded on chromosome 6 (26.67%) followed by chromosome 4 (21.43%) and the lowest polymorphism percentage was observed on chromosome 10 (8.93%). The gene specific markers nksbad2, ARO7, BADEX7_5 and SSI were found to be polymorphic.
 Conclusion: Based on the present study it may be concluded that the polymorphic markers identified will further be utilized in genotyping of F2:3 population, linkage analysis and mapping QTL’s for grain quality and yield traits.

DeepCOVIDNet: Deep Convolutional Neural Network for COVID-19 Detection from Chest Radiographic Images.

The novel Coronavirus Disease 2019 (COVID-19) is a global pandemic that has infected millions of people causing millions of deaths around the world. Reverse Transcription Polymerase Chain Reaction (RT-PCR) is the standard screening method for COVID-19 detection but it requires specific molecular-biology training. Moreover, the general workflow is difficult e.g. sample collection, processing time, and analysis expertise, etc. Chest radiographic image analysis can be a good alternative screening method that is faster, more efficient, and requires minimal clinical or molecular biology trained laboratory personnel. Early studies have shown that abnormalities on the chest radiographic images are likely to be the consequence of COVID-19 infection. In this study, we propose DeepCOVIDNet, a deep learning based COVID-19 detection model. Our proposed deep-learning model is a multiclass classifier that can distinguish COVID-19, viral pneumonia, bacterial pneumonia, and healthy chest X-ray images. Our proposed model classifies radiographic images into four distinct classes and achieves the accuracy of 89.47% along with a high degree of precision, recall and F1 score. On a different dataset setting (COVID-19, bacterial pneumonia, viral pneumonia) our model achieves the maximum accuracy of 98.25%. We demonstrate generalizability of our proposed method using 5-fold cross validation for COVID-19 vs pneumonia and COVID-19 vs healthy classification that also manifests promising results.

French-Canadian families from Saguenay-Lac-Saint-Jean: a new founder population for APECED.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is more prevalent in some founder populations, but relatively unexplored in Canada. This study aimed at investigating the French-Canadian patients through phenotypic and genotypic characterization. Phenotype and demographic characterization were done for 12 affected individuals belonging to eight unrelated families. Samples from 11 cases were analyzed in a molecular clinical laboratory, and muscle biopsies were reviewed for two individuals with a limb-girdle muscle dystrophy. The clinical phenotype was similar to that observed in European Caucasian populations but differed in the non-endocrine spectrum from the American-reported series of cases. Two cases exhibited a limb-girdle muscle dystrophy, and we found preliminary evidence of a mitochondrial dysfunction, since all three biopsies examined showed COX-deficient fibers in excess of what would be expected for age. Electron microscopy showed mitochondrial accumulation without abnormal cristea or inclusions. The c.1616C > T variant in the AIRE gene was responsible for 100% of APECED cases in the French-Canadian population of Saguenay-Lac-Saint-Jean in Quebec, Canada. We report the first series of French-Canadian cases affected with APECED. The Saguenay-Lac-Saint-Jean region was uncovered as a new founder population for this condition. Muscle biopsy findings expanded the range of previously described APECED-related myopathology. Long term follow-up of our genetically homogeneous French-Canadian cases may help determine if the c.1616C > T variant increases the risk of muscle involvement. A neonatal screening program is under consideration to prevent undesired life-threatening endocrine manifestations.

Use of Direct PCR Technique Without DNA Extraction in Confirmation Test for Salmonella typhimurium Bacteria on Meatball Samples

The use of direct PCR technique without DNA extraction in the confirmation test for Salmonella typhimurium ATCC 14028 bacteria on meatball samples was carried out in the Food and Drug molecular biology testing laboratory Administration in Gorontalo. The basis of this research is to have an impact on economic value in carrying out the confirmation test for S. typhimurium ATCC 14028, where testing is carried out conventionally, namely DNA extraction, which requires a large amount of money. Hence, it is necessary to innovate to modify the testing phase so that it is more effective and efficient. The purpose of this study was to see whether the direct PCR technique without DNA extraction can be done for the confirmation test of S. typhimurium ATCC 14028 on meatball samples. This study's sample consisted of 20 types of meatball samples spiked with S. typhimurium ATCC 14028 cultures. The method used in this study was qPCR analysis using the SYBR Green method. Data analysis was carried out based on 2 main criteria: (1) Ct analysis and (2) Tm analysis. Real-time PCR analysis results obtained Ct values ​​in the range 14.14 - 15.20 with an average of 14.82 and Tm values ​​85.20 - 86.30 with an average of 85.79. Based on these data, it can be concluded that using direct PCR can be used for testing confirmation of S. typhimurium ATCC 14028 on meatball samples.

Keragaman Genetik Klon Lokal Kopi Robusta Asal Temanggung Berdasarkan Marka SSR

<em>Temanggung is one of the centers of Robusta coffee production in Central Java, with a variety of potential local coffee clones. The exploration found that several numbers of local Robusta coffee clones had the potential to have high productivity and were resistant to pests/diseases. However, their level of genetic similarity to the superior clones that have been released has not been clearly identified. This study aimed to investigate the genetic variability of Temanggung local Robusta coffee clones based on SSR markers. The study was conducted at the Molecular Laboratory of the Integrated Laboratory, Indonesian Industrial and Beverage Crops Research Institute from February to December 2018. A total of 29 local coffee clones derived from Temanggung along with two control coffee clones (BP 42 and BP 358) were used. All the 14 SSR markers used in the present study were polymorphic and could cluster those local coffee clones into 5 major groups at a genetic similarity coefficient of 0.57. Four local coffee clones (Putih Daun Lebar, Lokal, Tugusari Hijau and Tugusari Kuning) were in the same group with control clones in group I. One local clone (Tugusari Hijau) had a genetic similarity with the control clone BP 358 at 0.91. Meanwhile, the other 25 local clones were in different groups from the control clones. These local clones, which showed genetic distance far from the control clones, can be selected as candidates for local superior clones in coffee breeding program.</em>

Leading in a Clinical Molecular Laboratory: It Is Not Just About the Science

Although most molecular pathology professionals have a deep foundation in the science and clinical practice of pathology, many feel ill prepared for the soft skills necessary to lead a laboratory. These nonmedical skills, such as understanding the financials, productively communicating with stakeholders, and writing effective reports, are essential aspects of a laboratory leader's job. Although some of these skills are taught in trainee programs, laboratory directors and other molecular professionals frequently must learn on the job, impacting the smooth functioning of a laboratory and professional job satisfaction, which ultimately impacts patient care.

Characterizing the Diffusion and Rheological Properties of Aged Asphalt Binder Rejuvenated with Bio-Oil Based on Molecular Dynamic Simulations and Laboratory Experimentations.

Soybean-derived bio-oil is one of the vegetable-based oils that is gaining the most interest for potential use in the rejuvenation of aged asphalt binders. This laboratory study was conducted to characterize and quantify the diffusion and rheological properties of bio-oil-rejuvenated aged asphalt binder (BRAA) using soybean oil. In the study, the chemical structure of the soybean oil was comparatively characterized using an element analyzer (EA), gel permeation chromatography (GPC), and a Fourier infrared (FTIR) spectrometer, respectively. Based on the chemical structure of the bio-oil, BRAA molecular models were built for computing the diffusion parameters using molecular dynamic simulations. Likewise, a dynamic shear rheometer (DSR) test device was used for measuring and quantifying the rheological properties of the aged asphalt binder rejuvenated with 0%, 1%, 2%, 3%, 4%, and 5% soybean oil, respectively. The laboratory test results indicate that bio-oil could potentially improve the diffusion coefficients and phase angle of the aged asphalt binder. Similarly, the corresponding decrease in the complex shear modulus has a positive effect on the low-temperature properties of BRAA. For a bio-oil dosage 4.0%, the diffusion coefficients of the BRAA components are 1.52 × 10−8, 1.33 × 10−8, 3.47 × 10−8, 4.82 × 10−8 and 3.92 × 10−8, respectively. Similarly, the corresponding reduction in the complex shear modulus from 1.27 × 107 Pa to 4.0 × 105 Pa suggests an improvement in the low-temperature properties of BRAA. Overall, the study contributes to the literature on the potential use of soybean-derived bio-oil as a rejuvenator of aged asphalt binders.

Considerations on the identification and management of metastatic prostate cancer patients with DNA repair gene alterations in the Canadian context.

Olaparib is the first Health Canada-approved agent in metastatic prostate cancer to use a companion diagnostic to identify alterations in BRCA1, BRCA2, or ATM. As olaparib is introduced, clinicians must learn to access and interpret germline and somatic next-generation sequencing (NGS) results, and how to manage affected patients who appear to have distinct clinical features. The traditional model of referring patients to a hereditary cancer clinic (HCC) for germline testing is likely impractical in this disease, as the metastatic prostate cancer patient population would be overwhelming. Alternate approaches to this are clinician-ordered genetic testing (so-called “mainstreaming”), out-of-pocket payment for third-party private company genetic testing, or germline testing done in conjunction with somatic testing, particularly cell free circulating tumor DNA (ctDNA). Germline testing alone is not sufficient for identifying Olaparib-eligible patients, as less than half of BRCA1, BRCA2, or ATM alterations are germline in origin, but it is critically important to identify family members who are carriers so that risk-reduction measures can be undertaken. Somatic testing is not widely available in Canada, but some patients can access it through research protocols or by paying out-of-pocket. Somatic testing can be performed on archival or fresh solid tissue biopsy samples, or through whole blood samples to access plasma-derived circulating tumor DNA (ctDNA). Both testing approaches have relative advantages and disadvantages, but neither may be informative in all patients and, therefore, ideal somatic NGS pathways should provide options for both tissue and ctDNA testing. We advocate that clinicians begin discussions with their provincial lab formularies, HCC, and molecular pathology labs to highlight the importance of germline and somatic testing in this population and identify pathways for patient access. While olaparib has approval for use in BRCA1, BRCA2, and ATM-altered mCRPC, emerging evidence suggests that PARP inhibitors have variable activity in these three genes, with BRCA2 alterations appearing to be the most responsive. Retrospective and prospective series have reported varying outcomes to standard of care therapies, such as ARATs and taxane-based chemotherapy, in metastatic castration-resistant prostate cancer (mCRPC) patients with DNA damage repair (DDR) gene alterations, such as BRCA2. In the absence of high-level evidence showing a lack of benefit, we believe this patient population should still be considered for these treatments. In addition, platinum-based chemotherapy appears to have activity in DDR gene-altered mCRPC and should be considered another option when access to olaparib is not possible. At present, there is no evidence to support an optimal treatment sequence in this patient population, therefore, physician and patient preferences will need to be taken into consideration when selecting therapies. As olaparib and other PARP inhibitors are tested in different disease states and in combination with other therapies, we will likely see a more refined approach to use of these agents and management of this new biomarker-defined patient population.

Wetting and non-wetting liquid flow in nanopores: Wettability, non-Poiseuille profile and intermolecular fundamentals

Wetting and non-wetting liquid flow in nanoscale pores or channels is of great interest in many science and technology fields, such as biotechnology, energy storage/conversion and micro-devices. It is also of considerable significance in geosciences and geoengineering. For instance, for the geological disposal of contaminated waste understanding flow and transport in nanoporous rocks is critical for accurately evaluating potential environmental hazards. Similarly, for oil recovery from tight reservoirs, understanding flow mechanisms in nanopores may improve production forecasts and enable the development of strategies to enhance production.This paper presents a comprehensive study on wetting and non-wetting liquid flow in nanopores, including the development of a new nanoflow model and its demonstration using published findings from molecular dynamics simulations and laboratory tests. The new nanoflow model is formulated based on the general two-dimensional Navier-Stokes equations. The novel aspects include: 1) the model incorporates the parameters relating to the intermolecular interactions between the fluid and the solid wall in a nanopore, which are lacking in traditional continuum models; 2) the model contains the effects of wettability and the nanochannel’s dimension on flowrate, which are unique to nanoflows and not considered in traditional continuum models; and 3) the model is based on a two-dimensional non-Poiseuille flow model described by a set of coupled ordinary differential equations. Therefore, the numerical solutions are much easier to obtain than the partial differential equations of non-Poiseuille flow models. The classic Poiseuille flow is its limiting case.Three examples are presented to demonstrate the model’s capability. The first is the determination of the contact angle of water in hydrophobic carbon nanochannels or nanotubes using published results from MD simulations and laboratory tests. The second example uses the new nanoflow model to simulate non-wetting water flow in hydrophobic nanochannels or nanotubes, including flow in a nanoscale graphene slit representative of those present in graphene batteries for energy storage. The results obtained are compared with those obtained from MD simulations. The last example discusses the wetting or partially wetting liquid flow profiles in nanoscale pores. The three examples provide new insights into wetting and non-wetting liquid flow behaviour in nanopores.

Positivity Rate as a Performance Indicator for COVID-19 Response in Edo State, Nigeria

Background: Positivity rate is a very reliable indicator in determining the effectiveness of public health interventions of infectious disease magnitude.
 Aim: To use positivity rate as performance indicator of effectiveness of COVID-19 Control measures in Edo State, Southern Nigeria.
 Materials and Methods: Laboratory based record review of 20,220 Polymerase Chain Reaction (PCR) based laboratory results from COVID-19 line-list analysed from the four (4) NCDC accredited molecular laboratory within Edo State. The weekly COVID-19 test positivity was computed in relation to the total samples tested between 23rd March to 30th November, 2020. Data collected were analysed using IBM SPSS version 20.0 software with statistical significance set at p˂0.050 and 95% Confidence Interval.
 Results: A total of 20,220 PCR results reviewed with 2696 (13.3%) COVID-19 cases identified while 17,524 (86.7%) were negative. The average weekly sample collection and testing was 552.28 ± 369.98, with average weekly COVID-19 case yield and positivity rate (%) of 77.89 ±106.23 and 12.20±13.75% respectively. A statistically significant positive correlation was identified between sample tested and COVID-19 case yield (R = 0.569; < 0.01). The first wave of the COVID-19 in Edo State witnessed a consistent and progressive decline in test positivity rate till it deepened at 0.6 % by the end of November, 2020.
 Conclusion: Increasing sample testing has significant positive correlation with COVID-19 case detection in Edo State. Positivity rate was a good indicator to monitor COVID-19 outbreak response in Edo State.
 Targeted surveillance and sample collection for testing can significantly help improve the quality and case yield during epidemic outbreak response. Bridging the gap in sample collection for testing can greatly influence how quickly an epidemic outbreak response and control is achieved.

Stakeholder attitudes towards establishing a national genomics registry of inherited cancer predisposition: a qualitative study.

This study aimed to describe the acceptability and perceived barriers and enablers to establish a national registry targeting carriers of pathogenic variants in cancer susceptibility genes from stakeholders' perspectives. Such a registry may effectively target carriers to translate existing research findings into optimised clinical care and provide a population-level resource for further clinical research and new gene and therapy discovery. In-depth interviews were conducted with individuals from four stakeholder groups: carriers of pathogenic variants, healthcare professionals, data custodians from the field of familial cancer, and heads of molecular pathology laboratories. Interview data were subjected to a qualitative analysis guided by a thematic analysis framework using NVivo software. A total of 28 individuals were interviewed: 11 carriers, 8 healthcare professionals, 5 laboratory heads, and 4 data custodians. All carriers and healthcare professionals were enthusiastic about the potential research applications of the registry. Carriers described that altruistic motivations provided the foundation of their support of the planned registry. Some carriers felt comfortable with a broad consent (consenting once, prospectively), while others preferred a narrow consent approach (consenting each time data is accessed). Some carriers and data custodians and registry developers also expressed a reluctance to link family member data without appropriate consent. Participants' enthusiasm and support for a national registry herald a productive and responsive research partnership once the registry has been established. Participants' views can be used to inform the approaches to be taken to develop and manage such a registry as an implicit codesign approach.

Implementation of a molecular tumour board in LATAM: the impact on treatment decisions for patients evaluated at Instituto Alexander Fleming, Argentina.

BackgroundThe role of the molecular tumour board (MTB) is to recommend personalised therapy for patients with cancer beyond standard-of-care treatment. A comprehensive molecular analysis of the tumour in a molecular pathology laboratory is important for all targeted therapies approaches. Here we report the 1-year experience of the Instituto Alexander Fleming Molecular Tumour Board.Patients and methodsThe MTB of the Instituto Alexander Fleming was launched in December 2019 in a monthly meeting. In each interactive monthly session, five cases were presented and discussed by the members. These cases were referred by the treating oncologists. The MTB recommendations were sent to each physician individually, and to the rest of the meeting participants. This was discussed with the patients/families by the treating oncologist. The final decision to choose therapy was left to the treating physicians. Of the 32 patients presented at MTB, 28 (87.5%) had potentially actionable alterations and only 4 (12.5%) had no actionable mutation. Six (19%) patients received a local regulatory agency approved drug recommendation, nine (28%) patients received an off-label approval treatment recommendation and three (9%) patients did not receive the treatment due to access and reimbursement of the drug.ConclusionIn most of the cases evaluated, the MTB was able to provide treatment recommendations based on targetable genetic alterations. Molecular-guided extended personalised patient care is effective for a small but clinically significant proportion of patients in challenging clinical situations. We believe that the implementation of a MTB is feasible in the Latin America (LATAM) region.

Identification and molecular characterisation of cercospora leaf spot disease pathogen on cowpea (Vigna unguiculata L. Walp)

Cercospora leaf spot disease (CLSD) is a serious constraint to the production of cowpea worldwide, especially in the tropics. This study was conducted to identify the causative agent of CLSD on cowpea accessions. Infected cowpea leaf samples were collected from established cowpea experimental field at the Institute of Agricultural Research and Training (IAR&T), Ibadan. The leaves were placed on potato dextrose agar (PDA) and sub-cultured to obtain pure fungi isolates. Nucleic acid extraction from the pure culture was carried out at the molecular laboratory of National Horticultural Research Institute (NIHORT), Ibadan. Polymerase chain reaction (PCR) was carried out to amplify fungi nucleic acid using ITS1 and ITS4 universal primers. The amplicon obtained were subjected to Basic Alignment Search Tool (BLAST) to determine the sequence homology. These sequences were compared with known sequences on the National Centre for Biotechnology Information (NCBI) database. The sequences analysed had similarity with Cercospora canescens (MK029364.1 and MW756382.1) deposited on the NCBI database. This study confirms that C. canescens is the causal pathogen of CLSD found on the infected cowpea leaves.

Identifying unnecessary duplicate genetic testing in a large medical center

Abstract Introduction Genetic testing has become ubiquitous in clinical medicine and plays an important role in making diagnoses and guiding treatment plans. Indiscriminate use of these tests can cause duplicate testing, which is typically not indicated because results from repeated constitutional molecular genetic testing should not change over time. Thus, duplicate genetic testing often represents inappropriate test utilization that can contribute an unnecessary burden on the laboratory and health care system. Objective The purpose of our study is to determine the incidence of duplicate testing of in-house genetic testing offered at a large medical center, which includes cystic fibrosis, factor V Leiden, prothrombin G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, and MTHFR A1298C, and to develop a tool to identify and block duplicate testing. Methods We retrospectively analyzed internal laboratory databases of all cystic fibrosis (n = 36164), factor V Leiden (n = 3264), prothrombin G20210A (n = 2890), MTHFR C667T (n = 1451), and MTHFR A1293C (n = 1290) testing performed at the molecular pathology laboratory at a large medical center from either January 2010 or January 2014 to July 2019. We analyzed and cleaned the databases with the R programming language, and we developed a prototype web-based app to proactively identify duplicate test requests with the Shiny R package. Results From January 2010 to July 2019, 3535 (9.8%) of the 36164 cystic fibrosis tests performed were duplicate tests for 3257 unique patients. Of these duplicate cystic fibrosis tests, 2997 were repeated once in the same patient, 244 were repeated twice in the same patient, 14 were repeated three times in the same patient, and 2 were repeated four times in the same patient. From January 2014 to July 2019, 99 (3.0%) of the 3264 factor V Leiden tests, 86 (3.0%) of the 2890 prothrombin G20210A tests, 49 (3.4%) of the 1451 MTHFR C667T, and 46 (3.6%) of the 1290 MTHFR A1298C tests performed were duplicate tests. We developed a proof-of-concept Shiny web-browser app that provides a user-friendly interface to determine if a patient has been previously tested in the molecular pathology lab. This app operates locally on a laboratory computer and uses spreadsheets automatically exported from the electronic medical record. These features allow for the app to be deployed quickly without needing to be integrated into the electronic medical record. Conclusions The results of this study indicate that unnecessary duplicate testing represents a small but significant proportion of genetic testing performed by the molecular pathology laboratory. Duplicate testing occurred more frequently with cystic fibrosis testing, which reflects its high volume at the medical center. Deployment of web-based apps using Shiny can provide straightforward and efficient tools for reducing duplicate tests.

Multiplex Fragment analysis detects all COVID-19 variants of concern

Introduction/ObjectiveThe majority of tracking methods have employed whole genome sequencing, which can be very expensive and time consuming. An alternative method has been to use genotyping of specific mutations to identify variants. However, tracking SARS-CoV-2 variants by targeted methods has been a moving target. Most methods only multiplex four targets per reaction, but we have multiplexed 8 targets in a single tube using fragment analysis.Methods/Case ReportFluorescently labeled primers targeted a combination of insertion/ deletion mutations and single nucleotide mutations. The PCR amplified products, amplicons, were separated by capillary electrophoresis. Primers were designed to detect changes in size indicative of insertion or deletion mutations including: ORF1A:Del3675_3677, S:Del69_70, S:Del144, S:Del157_158, S:Del242_244, ORF8:Del119_120, and ORF8:ins28269-28273. Allele-specific primers were designed to detect both the wild-type and mutated versions of S:N501Y, S:E484K, and S:L452R.Residual nasopharyngeal and nasal specimens testing positive for SARS-CoV-2 by RT-PCR or isothermal amplification (IDnow) methods were selected from May 1- June 24, 2021. Variant analysis was performed by multiplex targeted PCR and whole genome sequencing in parallel on the same specimens to determine positive percent agreement.Results (if a Case Study enter NA)Variant analysis was performed on 250 specimens detecting each of the major variants of concern Alpha (B.1.1.7, U.K. origin, n= 108), Beta (B.1.351, South Africa origin, n=3), Gamma (P.1, Brazil origin, n=12), Delta (B.1.617.2, Indian origin, n=17), and Iota (B.1.526, New York, n=5). Some specimens with low viral load were detected by only PCR (n=18), only WGS (n=41), or neither (n=20). Overall positive percent agreement was 95% (163/171).ConclusionThis adjustable method robustly and accurately identifies COVID-19 VOCs utilizing a platform amenable to multiple targets (20-40 targets ranging from 100-500b.p. across four fluorescent channels) using equipment commonly found in routine molecular pathology laboratories. Future directions include adjusting targets to detect new variants.