Molecular Diagnostic Assays Research Articles

Multi-repeat sequences identification using genome mining techniques for developing highly sensitive molecular diagnostic assay for the detection of Chlamydia trachomatis

Chlamydia trachomatis (C. trachomatis) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed C. trachomatis infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the C. trachomatis genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/mL to 1×10-3pg/mL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, C. trachomatis IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting C. trachomatis DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.

Rapid Laboratory Diagnosis of Epizootic Hemorrhagic Disease Virus Using a Loop-Mediated Isothermal Amplification Assay.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a molecular diagnostic assay that is particularly useful for the detection of viral diseases of livestock. A major advantage of RT-LAMP is that it can be used either as a rapid field test or as a high-throughput screening tool in veterinary laboratories, with sensitivity comparable to the real-time RT-PCR assay. Unlike conventional or qPCR, RT-LAMP uses a strand displacement polymerase and a set of four to six primers that bind to several regions of the target nucleic acid. Amplification occurs without thermal cycling, and coupled with the numerous primers, RT-LAMP offers a rapid and highly specific molecular assay. In this chapter, we describe the RT-LAMP protocol for the detection of epizootic hemorrhagic disease virus (EHDV) as a low-cost, specific, and sensitive screening tool in veterinary diagnostic laboratories. We also provide guidance on how to adapt the RT-LAMP assay for rapid field testing.

Diagnostic Performance of STANDARD™ M10 Multidrug-resistant Tuberculosis Assay for Detection of Mycobacterium tuberculosis and Rifampicin and Isoniazid Resistance in Zimbabwe.

Although Zimbabwe has transitioned out of the 30 high-burden countries, it still remained in the 30 high multidrug-resistant (MDR)/rifampicin-resistant tuberculosis (TB) burden. Rapid detection of rifampicin (RIF) and isoniazid (INH) is essential for the diagnosis of MDR-TB. The World Health Organization has recommended the use of molecular WHO-recommended rapid diagnostic (mWRD) for TB and DR-TB. STANDARD™ M10 MDR-TB assay is a new molecular rapid diagnostic assay developed by SD Biosensor for the detection of Mycobacterium tuberculosis (MTB) and RIF and INF resistance. This study aims to determine the diagnostic accuracy of STANDARD™ M10 MDR-TB assay. The study was conducted on 214 samples with different MTB and RIF and INH resistance status. The STANDARD™ M10 MDR-TB assay was performed according to the manufacturer's instructions. Xpert MTB/RIF Ultra, MGIT culture, and phenotypic drug susceptibility testing are used as comparative methods. The sensitivity and specificity of STANDARD™ M10 MDR-TB assay for the detection of MTB are 99% and 97.9%, respectively. The sensitivity and specificity of the assay for detection of MDR-TB were 97.8% and 100%, respectively. The STANDARD™ M10 MDR-TB assay demonstrated high diagnostic accuracy in the detection of MTB and RIF and INH resistance. This molecular assay can also be used as an alternative to other mWRD assays.

333 A molecular diagnostic assay for syphilis infection among a high-risk pregnant cohort.

333 A molecular diagnostic assay for syphilis infection among a high-risk pregnant cohort.

B-Lymphoblastic Leukemia Presenting with Eosinophilia: A Case Report and Review

B-lymphoblastic leukemia (B-ALL) presenting with eosinophilia represents a rare entity. In particular, the unique clinical manifestations as well as the genetic and molecular pathogenesis of the associated eosinophilia have been topics of debate and investigation. Herein, we present a case of B-ALL presenting with concomitant eosinophilia and a review of the published cases, including 120 additional cases. The patient, at presentation, was a 20-year-old female who presented with cardiac, neurologic, and cutaneous signs of eosinophilia and with leukocytosis (WBC 51, 40% eosinophils, 0% blasts), anemia, and thrombocytopenia. Imaging revealed multi-focal brain infarcts, pulmonary infiltrates, and myocarditis. Empiric steroids, colchicine, and intravenous immunoglobulin were initiated. One week after presentation, bone marrow biopsy revealed a hypercellular (>95%) marrow with 50% blasts and marked eosinophilia, which on later review revealed an immunophenotype most consistent with B-ALL (positive for Pax5, CD10, CD19, CD20, cCD22, CD34, CD45, CD79a, HLA-DR, and TdT and negative for MPO and CD117). Conventional cytogenetic analysis of the bone marrow was unable to be performed. Fluorescence in situ hybridization (FISH) revealed an IGH rearrangement in 10.5% of nuclei analyzed. After three weeks of treatment with steroids and resolution of symptomatic organ involvement, a repeat bone marrow biopsy did not show any evidence of malignancy or genetic abnormalities. The patient was continued on a steroid taper, did not initiate further antileukemic therapy, and was lost to follow up. Ten weeks after initial presentation and after completing a steroid taper, the patient presented with diffuse bone pain. Repeat bone marrow biopsy showed B-ALL with 64% blasts and increased eosinophils, normal conventional cytogenetics and hematologic malignancy sequencing panel, and FISH with gain of Xp in 20.5% of nuclei analyzed; a lumbar puncture did not detect blasts in the cerebrospinal fluid. The patient began induction therapy with aBFM, rituximab, PEG-asparaginase, and intrathecal cytarabine. A bone marrow biopsy performed four months after her initial presentation revealed a mildly hypocellular marrow with 3% B-lymphoblasts and no eosinophilia, suggesting residual disease, with no evidence of cytogenetic or molecular genetic abnormalities. As a result, therapy with blinatumomab was initiated. A follow up bone marrow biopsy performed six months after her initial presentation revealed a hypocellular marrow with multilineage maturation and no excess blasts with normal cytogenetic and molecular genetic studies. A literature review identified 120 additional cases of ALL with eosinophilia. In summary, it tends to present in adolescent males, often with mild anemia and thrombocytopenia and no peripheral blasts, which can lead to a delay in diagnosis of ALL. The eosinophilia is often symptomatic, with cardiopulmonary involvement being the most common, and can lead to rapid mortality in a substantial number of cases. A significant proportion of cases of ALL with eosinophilia involve t(5;14)(q31;q32), leading to overexpression of IL-3, which was not definitively found in this patient; however, there are numerous other recurrent abnormalities that may be contributing to the pathogenesis, both concurrently with and in the absence of this translocation, including other rearrangements of IGH as well as IKZF1 and CDKN2A abnormalities. Prompt diagnosis, now commonly using sensitive molecular diagnostic assays, and initiation of glucocorticoids for symptom control and subsequent antileukemic therapy, are essential to avoiding complications of eosinophilia in such patients.

Comparison of the MeltPro TB assay and whole-genome sequencing assay for rapid molecular diagnosis of drug resistant tuberculosis in guangdong province

BackgroundRifampicin (RIF) and multidrug-resistant tuberculosis (TB) are major public health threats. As conventional phenotypic drug susceptibility testing requires two–eight weeks, molecular diagnostic assays are widely used to determine drug resistance. MethodsClinical Mycobacterium tuberculosis isolates with consistent drug susceptibility results, tested using microbroth dilution and proportion methods in Löwenstein-Jensen medium from patients with TB in Guangdong province were utilized to evaluate MeltPro TB and whole-genome sequencing (WGS) assays in detecting resistance to RIF, isoniazid (INH), ethambutol (EMB), fluoroquinolones (FQ), and streptomycin (SM). Solid phenotypic drug susceptibility testing was used as the gold standard to evaluate the detection capacity of MeltPro TB on clinical sputum samples of patients with TB. ResultsSimilar to WGS, MeltPro TB successfully detected RIF, INH, and SM resistance with sensitivities of 86.3, 84.8, and 86.6 %, respectively. However, the resistant isolate detection rates were only 58.1 and 69.6 % for EMB and FQ-resistant strains. For clinical specimens, MeltPro TB still showed good detectable rates of RIF and INH resistance, with sensitivities of 82.4 % and 95.2 %, respectively. Detectable rates of FQ and EMB resistance were low: 77.8 % and 35.3 %, respectively. ConclusionsMeltPro TB can detect known DNA mutations associated with drug resistance in Mycobacterium tuberculosis strains with comparable efficacy to WGS. For FQ and EMB resistance testing, MeltPro TB requires optimization and is unsuitable for general use. MeltPro TB can be used for diagnosis of RIF and multidrug-resistant tuberculosis to rapidly initiate appropriate anti-TB drug therapy.

Molecular diagnostic assay for pre-harvest detection of Tilletia indica infection in wheat plants.

The current study describes a new diagnostic method for the rapid and accurate detection of Tilletia indica, the pathogen accountable for causing Karnal bunt (KB) disease in wheat. This method uses quantitative real-time polymerase chain reaction (qPCR) and a primer set derived from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of T. indica to identify the presence of the pathogen. The qPCR assay using this primer set was found highly sensitive, with a limit of detection (LOD) value of 4 pg of T. indica DNA. This level of sensitivity allows for the detection of the pathogen even in cases of different growth stages of wheat, where no visible symptoms of infection on the wheat plants can be seen by naked eyes. The study also validated the qPCR assay on ten different wheat cultivars. Overall, this study presents a valuable molecular tool for rapid, specific and sensitive detection of KB fungus in wheat host. This method has practical applications in disease management, screening of wheat genotypes against KB and can aid in the development of strategies to mitigate the impact of Karnal bunt disease on wheat production.

Converging Epidemics: A Narrative Review of Tuberculosis (TB) and Human Immunodeficiency Virus (HIV) Coinfection.

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis (MTB), remains a leading cause of mortality in individuals living with human immunodeficiency virus (HIV) infection, posing a significant strain on healthcare systems. Coinfection of HIV and TB results in a mutually advantageous relationship that accelerates the progression of both diseases. TB is a major contributor to mortality in individuals with HIV. However, diagnosing coinfected individuals is challenging due to the prevalence of extrapulmonary TB and smear-negative disease. Overthe past decade, significant progress has been made in the fight against TB, thanks to advances in molecular techniques. Yet, these molecular diagnostic assays remain inaccessible to many individuals coinfected with HIV and TB due to their high cost. To expedite treatment and reduce transmission, it is crucial to integrate HIV and TB control programs more closely, thereby minimizing diagnostic delays and enhancing early case detection. This review aims to provide a comprehensive overview of the current state of knowledge regarding the interplay between HIV and TB. It highlights recent developments in sensitive and rapid TB diagnostic tests, cutting-edge preventive strategies, and the screening of individuals coinfected with both HIV and TB. The objectives of this review are to shed light on the complex relationship between these two diseases and to emphasize the importance of integrated efforts in combating their impact on public health.

A rapid glove-based inoculum sampling technique to monitor Erysiphe necator in commercial vineyards.

Information on the presence and severity of grape powdery mildew (GPM), caused by Erysiphe necator, has long been used to guide management decisions. While recent advances in the available molecular diagnostic assays and particle samplers have made monitoring easier, there is still need for more efficient field collection of E. necator. The use of vineyard worker gloves worn during canopy manipulation as a sampler (glove swab) of E. necator was compared with samples identified by visual assessment with subsequent molecular confirmation (leaf swabs) and airborne spore samples collected by rotating-arm impaction traps (impaction traps). Samples from U.S. commercial vineyards in Oregon, Washington, and California were analyzed using two TaqMan qPCR assays targeting the internal transcribed spacer regions or cytochrome b gene of E. necator. Based on qPCR assays, visual disease assessments misidentified GPM up to 59% of the time with a higher frequency of misidentification occurring earlier in the growing season. Comparison of the aggregated leaf swab results for a row (n=915) to the row's corresponding glove swab had 60% agreement. The latent class analysis (LCA) indicated that glove swabs were more sensitive than leaf swabs in detecting E. necator presence. The impaction trap results had 77% agreement to glove swabs (n=206) taken from the same blocks. The LCAs estimated that the glove swabs and impaction trap samplers varied each year in which was more sensitive for detection. This likely indicates that these methods have similar levels of uncertainty and provide equivalent information. Additionally, all samplers, once E. necator was detected, were similarly sensitive and specific for detection of the A-143 resistance allele. Together, these results suggest that glove swabs are a viable sampling method for monitoring the presence of E. necator and, subsequently, the G143A amino acid substitution associated with resistance to quinone outside inhibitor fungicides in vineyards. Glove swabs could significantly reduce sampling costs due to the lack of need for specialized equipment, and time required for swab collection and processing.

CovidShiny: An Integrated Web Tool for SARS-CoV-2 Mutation Profiling and Molecular Diagnosis Assay Evaluation In Silico.

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing, with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuing to evolve and accumulate mutations. While various bioinformatics tools have been developed for SARS-CoV-2, a well-curated mutation-tracking database integrated with in silico evaluation for molecular diagnostic assays is currently unavailable. To address this, we introduce CovidShiny, a web tool that integrates mutation profiling, in silico evaluation, and data download capabilities for genomic sequence-based SARS-CoV-2 assays and data download. It offers a feasible framework for surveilling the mutation of SARS-CoV-2 and evaluating the coverage of the molecular diagnostic assay for SARS-CoV-2. With CovidShiny, we examined the dynamic mutation pattern of SARS-CoV-2 and evaluated the coverage of commonly used assays on a large scale. Based on our in silico analysis, we stress the importance of using multiple target molecular diagnostic assays for SARS-CoV-2 to avoid potential false-negative results caused by viral mutations. Overall, CovidShiny is a valuable tool for SARS-CoV-2 mutation surveillance and in silico assay design and evaluation.

A-252 Differentiation of SARS-CoV-2, Influenza A and B, and Respiratory Syncytial Virus Using a Single Swab: A Clinical Performance Evaluation of the BD MAX System Multiplex PCR Assay

Abstract Background Some shared clinical features of SARS-CoV-2, influenza (flu), and respiratory syncytial virus (RSV) infection often pose a diagnostic challenge for clinicians. In that context, the use of molecular diagnostic systems multiplex assays, using a nasopharyngeal or nasal swab, facilitates the detection of the causing virus by differentiating between the RNA of each disease. Our study evaluated the clinical accuracy of one such multiplex PCR assay, the BD Respiratory Viral Panel for BD MAX™ System (BD MAX RVP). Methods Symptomatic patients that had a nasopharyngeal and nasal specimen prospectively collected from 6 geographical locations in the US were enrolled in the study between January and April 2022 along with nasopharyngeal swabs retrospectively collected between December 2019 and January 2022. All specimen testing was performed using the BD MAX System. Hologic® Flu A/B/RSV assay on Panther Fusion™ System was the comparator for flu A, flu B, and RSV while a composite of two out of three assays authorized under FDA Emergency Use Authorization (Roche cobas® SARS-CoV-2 on cobas® 6800 System, Hologic® Aptima® SARS-CoV-2 on Panther® System, and Quidel Lyra® SARS-CoV-2 on Applied Biosystems® 7500 Fast Dx) was used as comparator for SARS-CoV-2. Positive and negative percentage agreements (PPA and NPA, respectively) were calculated to determine performance. Reproducibility and precision assessments were also performed as part of the study. Results Tested specimens included 252 nasopharyngeal and 254 nasal swabs, prospectively collected, and 240 retrospectively acquired nasopharyngeal specimens. For prospectively collected SARS-CoV-2 nasopharyngeal specimens, PPA of 98.8% (95% CI 93.6, 99.8) and NPA of 98.2% (95% CI 94.9, 99.4) were observed while for flu A, PPA was 100% (95% CI 61.0, 100) and NPA 99.6% (95% CI 97.7, 99.9). PPA and NPA for prospectively collected SARS-CoV-2 anterior nasal specimens were 98.8% (95% CI 93.3, 99.8) and 98.3 (95% CI 95.1, 99.4), respectively. For flu A, PPA of 100% (95% CI 61.0, 100) and NPA of 99.6% (95% CI 97.8, 99.9) were observed. Lack of enrolled positive patients with flu B or RSV precluded the PPA calculation for those viruses. However, NPA for both flu B and RSV was 100% for the two specimen types. In retrospectively obtained nasopharyngeal specimens, PPA was 100% (95% CI 93.7, 100) for flu A, 100% (95% CI 93.8, 100) for flu B, and 98.4% (95% CI 91.5, 99.7) for RSV while NPA was 98.9% (95% CI 96.1, 99.7) for flu A, 98.9% (95% CI 96.1, 99.7) for flu B, and 100% (95% CI 97.9, 100) for RSV. Conclusion The BD MAX RVP assay showed a strong clinical performance in detecting and differentiating between the RNA of SARS-CoV-2, flu A and B, and RSV.

Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay.

Bovine mastitis, a common and costly disease in dairy cattle, is primarily caused by Staphylococcus aureus. Timely and accurate detection of this pathogen is crucial for effective disease management. In this study, we developed and validated a novel molecular diagnostic assay based on the CRISPR/Cas12a system coupled with Recombinase Polymerase Amplification (RPA) and Loop-Mediated Isothermal Amplification (LAMP). We utilized specific primers targeting the nucleotide sequences of the S.aureus genes of interest, such as nuc and sea. RPA/LAMP reactions were performed under optimized conditions, and the resulting products were subsequently subjected to CRISPR/Cas12a detection. The CRISPR/Cas12a assay successfully detected the target nuc and sea genes, with a limit of detection of 104 and 102 gene copies per reaction, respectively. All 13 S.aureus clinical isolates were identified by RPA-CRISPR/Cas12a assay. The total reaction time is approximately 1h. The assay demonstrated high sensitivity for the detection of S.aureus in both laboratory and clinical samples.

Loop mediated isothermal amplification as a molecular diagnostic assay: Application and evaluation for detection of Enterohaemorrhagic Escherichia coli (O157:H7)

PurposeThis study aimed at evaluating the performance of the Loop Mediated Isothermal Amplification (LAMP) diagnostic test, which targets the putative Fimbria protein-encoding gene (Z3276) for rapid and specific detection of locally isolated enterohemorrhagic Escherichia coli (EHEC) O157:H7. ResultsA total number of 40 locally available bacteria isolates and standard strains, among them 6 entrohemorrhagic (O157:H7) and 10 entropathogenic E. coli, 7 non diarrheic E. coli strains and 13 non entrohemorrhagic shiga toxic (stx) E. coli isolates as well as 4 pathogenic non E. coli species were used to optimize and evaluate the LAMP assay. The LAMP amplified DNA samples were visualized as turbid DNA both by naked eye and gel electrophoresis followed by staining. The assay had a sensitivity of 100% (6/6), a specificity of 97.05% (33/34), and an efficiency of 97.5% (39/40). The assay was also exhibited with 100% negative predicted value and 85.7% positive predicted value. The LAMP assay was also 10-fold more sensitive than the conventional PCR assay; sensitivity was determined by serial dilution. The results of LAMP and the PCR tests showed very high agreement (k = 0.97) in the detection of the bacteria studied. ConclusionCompared with the performance of PCR and SMAC, LAMP assay was better in terms of efficiency, rapidity and cost-effectiveness, which can be used as a point-care diagnostic test in resource-limited laboratories.

Diagnostic validity of molecular diagnostic assays for white spot syndrome virus at different severity grades

The high virulence of the white spot syndrome virus (WSSV) in acute infections and the lack of effective treatments underscore the necessity for a rapid, accurate, and efficient diagnostic process to control white spot disease. The analytical sensitivity of diagnostic assays (polymerase chain reactions; PCRs and rapid diagnostic kit) at different severity grades of WSSV infection were determined using the 95% limit of detection (LOD95%). The LOD95% of nested, real-time, and one-step PCRs and rapid diagnostic kit were 0.70, 4.67, 1.19, and 79,434.65 viral genome copies/reactions, respectively. From the intramuscular challenge tests conducted under different dose and temperature conditions, the WSSV severity grades of time-course collected whiteleg shrimp (Litopenaeus vannamei) and dead or live shrimps were determined based on the viral loads of the pleopod. By applying the WSSV severity grades, a total of 92 shrimps were classified as G0. Furthermore, 92, 66, 199, and 79 shrimps were classified as G1, G2, G3, and G4, respectively. Additionally, 222 shrimps were classified as negative as WSSV was not confirmed in the nested PCR assay. Diagnostic sensitivity (DSe) and specificity (DSp) values of molecular diagnostic assays were compared with those of nested PCR in artificial WSSV-infected shrimp to evaluate diagnostic performance. The real-time and one-step PCRs exhibited a DSe >92.4% for G0 grade (approximately 101–102 copies/mg), indicating WSSV detection at low copy numbers. The rapid diagnostic kit presented a DSe >92.4% for G2 grade (approximately 104–105 copies/mg), suggesting the detection of WSSV-infected shrimp with clinical signs during the endemic period. These results suggest that the strategy of presumptive diagnosis using one-step PCR and rapid diagnostic kit at different seasonal periods followed by confirmatory diagnosis using real-time PCR assay could aid in controlling WSD with rapidity, accuracy, and cost-effectiveness.

Ultra-deep sequencing of 45S rDNA to discern intragenomic diversity in three Chrysodeixis species for molecular identification

Species identification is necessary to prevent introductions of exotic plant pests through global trade. Many of these pests are understudied and lack publicly available DNA sequence data on which rapid molecular identification methods can be based. One such lineage is the genus Chrysodeixis, which includes three species of potential concern for United States trade initiatives: C. includens, C. chalcites, and C. eriosoma. Here we describe a method to generate robust 45S rDNA profiles using long read sequencing in order to clarify evolutionary relationships and develop a real-time PCR identification technique. Such an identification tool will be useful in rapidly differentiating between Chrysodeixis species of quarantine concern where traditional morphological identification methods are insufficient. Molecular methods such as this greatly reduce the time spent identifying each specimen, allow for detection of eDNA, vastly increase throughput, and increase the probability of detection. The methods presented here will be generally adaptable to any understudied lepidopteran taxa that necessitates a molecular diagnostic assay and, with adjustment or testing of the primers, could be applied to any group for which development of rDNA profiles in a benchtop setting would prove useful.

Rapid and visual identification of HIV-1 using reverse transcription loop-mediated isothermal amplification integrated with a gold nanoparticle-based lateral flow assay platform.

Human immunodeficiency virus type one (HIV-1) infection remains a major public health problem worldwide. Early diagnosis of HIV-1 is crucial to treat and control this infection effectively. Here, for the first time, we reported a novel molecular diagnostic assay called reverse transcription loop-mediated isothermal amplification combined with a visual gold nanoparticle-based lateral flow assay (RT-LAMP-AuNPs-LFA), which we devised for rapid, specific, sensitive, and visual identification of HIV-1. The unique LAMP primers were successfully designed based on the pol gene from the major HIV-1 genotypes CRF01_AE, CRF07_BC, CRF08_BC, and subtype B, which are prevalent in China. The optimal HIV-1-RT-LAMP-AuNPs-LFA reaction conditions were determined to be 68°C for 35 min. The detection procedure, including crude genomic RNA isolation (approximately 5 min), RT-LAMP amplification (35 min), and visual result readout (<2 min), can be completed within 45 min. Our assay has a detection limit of 20 copies per test, and we did not observe any cross-reactivity with any other pathogen in our testing. Hence, our preliminary results indicated that the HIV-1-RT-LAMP-AuNPs-LFA assay can potentially serve as a useful point-of-care diagnostic tool for HIV-1 detection in a clinical setting.

Development of the NOGGO GIS v1 Assay, a Comprehensive Hybrid-Capture-Based NGS Assay for Therapeutic Stratification of Homologous Repair Deficiency Driven Tumors and Clinical Validation.

The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution's specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25-85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which BRCA1 and BRCA2 mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The "NOGGO GIS v1 assay" performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.

State of the Art for Diagnosis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge among reproductive-age women. It is associated with multiple adverse health outcomes, including increased risk of acquisition of HIV and other sexually transmitted infections (STIs), in addition to adverse birth outcomes. While it is known that BV is a vaginal dysbiosis characterized by a shift in the vaginal microbiota from protective Lactobacillus species to an increase in facultative and strict anaerobic bacteria, its exact etiology remains unknown. The purpose of this minireview is to provide an updated overview of the range of tests currently used for the diagnosis of BV in both clinical and research settings. This article is divided into two primary sections: traditional BV diagnostics and molecular diagnostics. Molecular diagnostic assays, particularly 16S rRNA gene sequencing, shotgun metagenomic sequencing, and fluorescence in situ hybridization (FISH), are specifically highlighted, in addition to multiplex nucleic acid amplification tests (NAATs), given their increasing use in clinical practice (NAATs) and research studies (16S rRNA gene sequencing, shotgun metagenomic sequencing, and FISH) regarding the vaginal microbiota and BV pathogenesis. We also provide a discussion of the strengths and weaknesses of current BV diagnostic tests and discuss future challenges in this field of research.

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis.

In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available. To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories. Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing. qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA. All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.

TaqMan qPCR assays improve Pseudomonas syringae pv. actinidiae biovar 3 and P. viridiflava (PG07) detection within the Pseudomonas sp. community of kiwifruit

TaqMan qPCR assays improve Pseudomonas syringae pv. actinidiae biovar 3 and P. viridiflava (PG07) detection within the Pseudomonas sp. community of kiwifruit