Use Of Molecular Diagnostic Tests Research Articles

Antibiotic resistance genes in the subgingival microbiome and implications for periodontitis therapy.

Antibiotic resistance is emerging as a global public threat. However, it remains poorly investigated in the context of periodontal therapy. The aim of the study was to investigate the complete diversity of antibiotic resistance genes in a German population. Thirty-nine volunteers with periodontitis contributed to the present study with one to four periodontal pockets for a total of 124 subgingival samples. Samples were analyzed using shotgun metagenomics. A total of 19 antibiotic resistance genes from six antibiotic classes were detected in subgingival biofilm. Two thirds of the volunteers (n=26/39) showed antibiotic resistance genes for at least one of the antibiotic classes used for periodontal treatment in dental practice or research: beta-lactam, lincosamide, macrolide, nitroimidazole, and tetracycline. Macrolide was the most abundant class detected (21/39 patients). Findings from our study suggest a high prevalence of antibiotic resistance genes in periodontal pockets from German volunteers. We recommend the development and broader use of molecular diagnostic tests for antibiotic resistance in dental practice to ensure treatment success and to minimize antibiotic resistance.

Variation of use of targeted therapies and molecular diagnostic testing by practice type for non-small cell lung cancer and colorectal cancer.

6551 Background: Targeted therapies are important first-line treatments for many patients with non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). All patients with newly-diagnosed metastatic NSCLC and CRC should undergo molecular diagnostic testing to guide treatment selection. Methods: We used 100% Medicare fee-for-service data from 2015 through 2019 to identify beneficiaries with incident metastatic NSCLC or CRC receiving systemic therapy and to assign beneficiaries to oncology practices. We then assessed for use of molecular diagnostic testing and targeted therapies in these cohorts. We used linear mixed effects models to assess patient and practice characteristics associated with molecular diagnostic testing and targeted therapy use. Results: Rates of molecular diagnostic testing increased between 2015 and 2019 for NSCLC and CRC. In 2019, rates of molecular diagnostic testing were 85% and 65% for NSCLC and CRC, respectively. Rates of targeted therapy use did not increase over time for NSCLC or CRC, and were 8% and 5%, respectively, in 2019. Compared to National Cancer Institute (NCI)-designated cancer centers, rates of molecular diagnostic testing for CRC were 3.7 percentage points lower at practices associated with non-academic hospitals and 10.6 percentage points lower at small independent practices. Rates of targeted therapy use for NSCLC were 4.8, 5.9 and 5.5 percentage points lower at academic medical centers, large independent practices and small independent practices, respectively, compared to NCI centers. Conclusions: Rates of molecular diagnostic testing for NSCLC and CRC increased in recent years, but testing rates remain below recommended levels, and targeted therapy use remains low. Substantial variation in testing and targeted therapy use by practice type suggest that the practice where a patient is treated may impact access to recommended testing and efficacious treatments. [Table: see text]

Clinical Profile and Molecular Genetic Analysis of Prader - Willi Syndrome: A Single Center Experience.

Aim:The prevalence of childhood and adolescent obesity is increasing worldwide as well as in India. Prader–-Willi syndrome (PWS) is one of the most common causes of syndromic obesity with varied clinical manifestations across different lifespan. Herewith, we describe clinical and molecular characteristics of eight PWS who were diagnosed in an obesity clinic of tertiary care hospital.Materials and Methods:Clinically suspected cases of PWS were screened between January 2014 and January 2022. Detailed history and clinical examination were done to look for typical features of PWS like characteristic facial appearance, short stature, obesity, hyperphagia, delayed puberty or hypogonadism, diabetes mellitus, developmental delay, cognitive dysfunction, learning disabilities or abnormal behavior. All were evaluated, with 75 g oral glucose tolerance tests (GTT), HbA1c, Free T4, TSH, LH, FSH, testosterone, and growth hormone level. Intelligent quotient (IQ) of each patient was assessed by a psychiatrist using Binet–Kamat test. Molecular confirmation of clinically suspected PWS was done by either Methylation-specific polymerase chain reaction (MS-PCR) or Fluorescence in situ Hybridization (FISH) methods.Results:Based on clinical and molecular characteristics, eight were diagnosed as PWS. Except one, all were male with characteristic facies, mean age of study cohort was 12 years and mean BMI of 44.58. Obesity, short stature, hyperphagia, hypotonia, and mild to moderate mental retardation were noted in entire (100%) PWS study population. All male PWS patients had cryptorchidism, which was bilateral in six patients and unilateral (right undescended testes) in one. Apart from obesity, short stature, other endocrine associations noted were diabetes mellitus in 50% and subclinical hypothyroidism in 37% of PWS. Molecular characteristics of PWS were confirmed by Methylation-specific PCR in seven and by FISH method in one.Conclusion:Prader–Willi syndrome should be kept in mind in case of childhood or adolescent obesity with short stature, hypotonia, cryptorchidism, and developmental delay or cognitive dysfunction. Judicious use of molecular diagnostic testing should be made in all clinically suspected cases. Early diagnosis and appropriate management of this complex disorder by a multidisciplinary team will improve the quality of life and treatment outcome.

Detection of SARS-CoV-2 RNA in Upper Respiratory Swap Samples by Pooling Method

Background:Widespread and effective use of molecular diagnostic tests is indispensable for protecting public health and containing the severe respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. More than 1 year into the pandemic, as resources have reached a point of depletion, grouping samples in pools of certain sizes appears to be a reasonable method to reduce both the costs and the processing time without necessitating additional training, equipment, or materials.Aims:To assess whether the pooling strategy that was used in past outbreaks and is used in blood tests prior to transfusion for screening large populations can also be used in SARS CoV-2 tests.Study Design:Diagnostic accuracy study.Methods:This prospective study was conducted with 2815 samples, sent to the coronavirus disease 2019 (COVID-19) Laboratory of our hospital between February 12 and 21, 2021, to be tested for the presence of SARS-CoV-2. The samples were examined individually and in pools of five 100 μl taken from each sequential sample, using 3 different SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) kits, the Allplex™ 2019-nCoV Assay kit (Seegene, Republic of Korea), the GeneMAP™ 2019-nCoV detection V.3 kit (GenMark, Türkiye), and the Bio-Speedy™ SARS-CoV-2 Double Gene™ RT-qPCR kit (Bioeksen, Türkiye) on the BioRAD CFX96™ Touch (Bio-Rad Laboratories Inc., Hercules, CA, USA) platform available in our laboratory.Results:Following the extraction of serial dilutions prepared from the SARS-CoV-2 RNA positive (cycle of threshold: 20) sample, the standard curves of RT-PCR were analyzed. By evaluating the efficiency (E) values, all 3 kits showed high sensitivity and similar results; while the highest level was detected with the Allplex™ 2019-nCoV Assay kit in the nucleocapsid (N) gene (E: 124%), the lowest was detected with the Double Gene™ RT-qPCR kit in the N and ORF 1ab genes (E: 90%). Of the samples included in the study, only 1 positive sample with low viral load was found to be negative when studied by pooling. The total number of kits to be used in pooled tests and then to individually retest the 5 samples in positive pools was calculated as 827 and the savings rate as 69.91% (1968/2815).Conclusion:The pooling strategy is an effective approach to extend the impact of limited testing resources and reagents available in certain periods of the COVID-19 pandemic. Testing by pooling samples requires improvement of RNA extraction methods and careful monitoring of RT-PCR test sensitivity to avoid missing low-positive entities. Therefore, based on the prevalence of COVID-19 in their regions, laboratories should conduct their own validation of pooling studies for RNA extraction and amplification methods they use.

Considerations related to the use of molecular diagnostic tests in veterinary clinical and regulatory practice.

The article focuses on the Considerations related to the use of molecular diagnostic tests in veterinary clinical and regulatory practice . Topics discussed include technological advances are creating an ever widening array of options for detecting pathogens and diagnosing diseases of concern in clinical and regulatory veterinary medicine;and new technology can facilitate timelier responses and, in some instances, move assays from laboratory-based to patient-side.

The lag of genetic counseling in Mexico

Genetic counseling is the process of advising indivduals, partners and families affected by or at risk of genetic disorders to help them understand and adapt to the medical, psychological, social, family and reproductive implications of a genetic condition. Given the expansion of precision medicine and the recent increase in the use of molecular diagnostic tests, it is necessary to have duly certified personnel in our environment to perform it. In Mexico, unlike other countries, the figure of the genetic counselor is not formally recognized, which is why geneticists doctors give this advice within the healthcare context (as part of the outpatient consultation). The number of these health professionals is below the internationally proposed requirement standard of one medical geneticist per 100,000 inhabitants, which is only met in Mexico City, making it of utmost importance to increase the training of medical geneticists as well such as the inclusion of these in the different health institutions of the country.

Improving Antimicrobial Stewardship in Patients With Cancer With the Use of Molecular Diagnostic Tests

Improving Antimicrobial Stewardship in Patients With Cancer With the Use of Molecular Diagnostic Tests

Eye-Tracking Study to Enhance Usability of Molecular Diagnostics Reports in Cancer Precision Medicine.

We conducted usability studies on commercially available molecular diagnostic (MDX) test reports to identify strengths and weaknesses in content and form that drive clinical decision making. Given routine genomic testing in cancer medicine, oncologists must interpret MDX reports as well as evidence concerning clinical utility of biomarkers accurately for treatment or trial selection. This work aims to evaluate effectiveness of MDX reports in facilitating cancer treatment planning. Fourteen clinicians at an academic tertiary care medical facility, with a wide range of experience in oncology and in the use of molecular testing, participated in this study. Three commercially available, widely used, Clinical Laboratory Improvement Amendments (CLIA)-certified, College of American Pathologists (CAP)-accredited test reports (labeled Laboratories A, B, and C) were used. Eye tracking, surveys, and think-aloud protocols were used to collect usability data for these MDX reports focusing on ease of comprehension and actionability. Clinicians found two primary areas in molecular diagnostic reports most useful for patient care: therapy options with benefit or lack of benefit to patients, including enrolling clinical trials; and pathogenic tumor molecular anomalies detected. Therapeutic implications and therapy classes such as US Food and Drug Administration-approved off-label, on-label, clinical trials were critical for decision making. However, all reports had usability and comprehension issues in these areas and could be improved. Focused usability studies can help drive our understanding of the clinical workflow for use of molecular diagnostic tests in cancer care. This in turn can have major effects on quality of care, outcomes, costs, and patient satisfaction. This study demonstrates the use of specific usability techniques (eye tracking and think-aloud protocols) to help clinical laboratories improve MDX report design in a precision oncology treatment setting.

Developments in molecular diagnostics

The present article provides an overview on the rapidly evolving scenario in clinical molecular diagnostics. The concept of clinical validity and utility underpins the use of molecular diagnostic tests in the clinical laboratory, and continuous sharing of expertise between clinical and laboratory partners will enhance appropriateness of molecular testing and interpretation, thus improving personalized medicine. Thanks to the technological advances made in molecular diagnostics, above all in next generation sequencing (NGS) and digital polymerase chain reaction (dPCR) platforms, clinical laboratories benefit from rapid translation of established and emerging molecular tests. The examples of their application which will be discussed encompass cystic fibrosis (CF) (qualitative molecular) and BCR/ABL1 (quantitative molecular) tests. A final consideration on total quality assurance in molecular testing is also made and the role of accreditation is underlined as one of the main factors in guaranteeing the overall quality of molecular test results.

Tuberculosis as a force health protection threat to the United States military.

Tuberculosis (TB) is a communicable disease that poses a threat to force health protection to the U.S. military. The rate of TB disease in the military is low; however, there are unique challenges for its control in this setting. As a low-risk population, TB testing in the U.S. military can be scaled back from the universal testing approach used previously. Reactivation of latent TB infection (LTBI) present at accession into service is the most important factor leading to TB disease; therefore, its diagnosis and treatment among recruits should be given a high priority. Deployment and overseas military service is an uncommon but important source of TB infection, and rigorous surveillance should be ensured. Case management of TB disease and LTBI can be improved by the use of cohort reviews at the service and installation levels and case finding and delays in the diagnosis of TB disease can be improved by education of providers, as well as increased use of molecular diagnostic tests. Program outcomes can be improved by making LTBI treatment compulsory, offering shorter treatment regimens, and increasing accountability through oversight and evaluation. The diagnosis of LTBI can be improved by implementing targeted testing in all settings and reducing confirmatory interferon-gamma release assay testing.

Novel approaches are needed to develop tomorrow's antibacterial therapies.

Society faces a crisis of rising antibiotic resistance even as the pipeline of new antibiotics has been drying up. Antibiotics are a public trust; every individual's use of antibiotics affects their efficacy for everyone else. As such, responses to the antibiotic crisis must take a societal perspective. The market failure of antibiotics is due to a combination of scientific challenges to discovering and developing new antibiotics, unfavorable economics, and a hostile regulatory environment. Scientific solutions include changing the way we screen for new antibiotics. More transformationally, developing new treatments that seek to disarm pathogens without killing them, or that modulate the host inflammatory response to infection, will reduce selective pressure and hence minimize resistance emergence. Economic transformation will require new business models to support antibiotic development. Finally, regulatory reform is needed so that clinical development programs are feasible, rigorous, and clinically relevant. Pulmonary and critical care specialists can have tremendous impact on the continued availability of effective antibiotics. Encouraging use of molecular diagnostic tests to allow pathogen-targeted, narrow-spectrum antibiotic therapy, using short rather than unnecessarily long course therapy, reducing inappropriate antibiotic use for probable viral infections, and reducing infection rates will help preserve the antibiotics we have for future generations.

End of an Era: Reduced Severity of Tricuspid Regurgitation in Cardiac Transplant Recipients Managed with Molecular Diagnostic Testing

Purpose A well-established complication of right ventricular endomyocardial biopsy (EMB) used for the diagnosis of acute cellular rejection is damage to the tricuspid valve and subsequent tricuspid regurgitant flow. Since the advent of Molecular Diagnostic Testing (MDT) as a screening test for determining which patients require EMB, the need for EMB at our center has decreased significantly. We hypothesized that the severity of tricuspid regurgitation (TR) should therefore be reduced with greater reliance on MDT and reduced utilization of EMB. Methods and Materials 58 echocardiograms from cardiac transplant patients who survived at least two years post-transplant were reviewed by observers blinded to post-transplant rejection surveillance strategies. 30 patients were in the pre-MDT group, from 2002-2004, when only EMB was used for post-transplant rejection surveillance. 28 patients were in the MDT group, from 2008-2010, when MDT was used as a screen to determine if EMB was required. TR was graded on a scale of zero to four, with zero representing no regurgitation present on echocardiography, one representing trace, two representing mild, three representing moderate and four representing severe TR. Results Of the 30 patients evaluated from the pre-MDT group, from 2002-2004, the average TR score was 1.87. In the 28 patients evaluated after routine MDT testing was implemented, from 2008-2010, the average score was 1.30, indicating a statistically significant reduction in tricuspid regurgitation severity(p = 0.0297). There was no evidence of severe TR in the MDT patients. Conclusions Our findings demonstrate that with greater use of MDT, the severity of tricuspid regurgitation has been reduced. MDT has become an established method for monitoring immune activation and as a screen for identifying the need for EMB in low risk cardiac transplant recipients. Our findings further support the continued use of MDT as a screening test before resorting to EMB.

Use of a Molecular Diagnostic Test in AFB Smear Positive Tuberculosis Suspects Greatly Reduces Time to Detection of Multidrug Resistant Tuberculosis

BackgroundThe WHO has recommended the implementation of rapid diagnostic tests to detect and help combat M/XDR tuberculosis (TB). There are limited data on the performance and impact of these tests in field settings.MethodsThe performance of the commercially available Genotype MTBDRplus molecular assay was compared to conventional methods including AFB smear, culture and drug susceptibility testing (DST) using both an absolute concentration method on Löwenstein-Jensen media and broth-based method using the MGIT 960 system. Sputum specimens were obtained from TB suspects in the country of Georgia who received care through the National TB Program.ResultsAmong 500 AFB smear-positive sputum specimens, 458 (91.6%) had both a positive sputum culture for Mycobacterium tuberculosis and a valid MTBDRplus assay result. The MTBDRplus assay detected isoniazid (INH) resistance directly from the sputum specimen in 159 (89.8%) of 177 specimens and MDR-TB in 109 (95.6%) of 114 specimens compared to conventional methods. There was high agreement between the MTBDRplus assay and conventional DST results in detecting MDR-TB (kappa = 0.95, p<0.01). The most prevalent INH resistance mutation was S315T (78%) in the katG codon and the most common rifampicin resistance mutation was S531L (68%) in the rpoB codon. Among 13 specimens from TB suspects with negative sputum cultures, 7 had a positive MTBDRplus assay (3 with MDR-TB). The time to detection of MDR-TB was significantly less using the MTBDRplus assay (4.2 days) compared to the use of standard phenotypic tests (67.3 days with solid media and 21.6 days with broth-based media).ConclusionsCompared to conventional methods, the MTBDRplus assay had high accuracy and significantly reduced time to detection of MDR-TB in an area with high MDR-TB prevalence. The use of rapid molecular diagnostic tests for TB and drug resistance should increase the proportion of patients promptly placed on appropriate therapy.

Practical Considerations for High-Throughput Influenza A Virus Surveillance Studies of Wild Birds by Use of Molecular Diagnostic Tests

Influenza A virus surveillance studies of wild bird populations are essential to improving our understanding of the role of wild birds in the ecology of low-pathogenic avian influenza viruses and their potential contribution to the spread of H5N1 highly pathogenic avian influenza viruses. Whereas the primary results of such surveillance programs have been communicated extensively, practical considerations and technical implementation options generally receive little attention. In the present study, the data obtained from 39,490 samples were used to compare the impacts of variables such as the sampling procedure, storage and transport conditions, and the choice of molecular and classical diagnostic tests on the outcome of the results. Molecular diagnostic tests allowed estimation of the virus load in samples, which has implications for the ability to isolate virus. Virus isolation in embryonated eggs was more sensitive than virus isolation in cell cultures. Storage and transport conditions had less of an impact on diagnostics by the use of molecular tests than by the use of classical approaches. These findings indicate that molecular diagnostic tests are more sensitive and more reliable than classical tests. In addition, molecular diagnostic tests facilitated analyses in real time and allowed the discrimination of H5 influenza viruses with low and high pathogenicities without the need for virus isolation. Critical assessment of the methods used in large surveillance studies like this will facilitate comparison of the results between studies. Moreover, the lessons learned from current large-scale influenza A virus surveillance activities could be valuable for other pathogen surveillance programs in the future.

Specimen Stability for DNA-based Diagnostic Testing

The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4 degrees C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4 degrees C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.