Small airway brush gene expression predicts chronic lung allograft dysfunction and mortality.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) limits survival following lung transplant, but substantial lung damage occurs before diagnosis by traditional methods. We hypothesized that small airway gene expression patterns could identify CLAD risk before spirometric diagnosis and predict subsequent graft failure. METHODS: Candidate genes from 4 rejection-associated transcript sets were assessed for associations with CLAD or graft failure in a derivation cohort of 156 small airway brushes from 45 CLAD cases and 37 time-matched controls with >1-year stable lung function. Candidate genes not associated with CLAD and time to graft failure were excluded, yielding the Airway Inflammation 2 (AI2) gene set. Area under the receiver operating curve (AUC) for CLAD and competing risks of death or graft failure were assessed in an independent validation cohort of 37 CLAD cases and 37 controls. RESULTS: Thirty-two candidate genes were associated with CLAD and graft failure, comprising the AI2 score, which clustered into 3 subcomponents. The AI2 score identified CLAD before its onset, in early and late post-CLAD brushes, as well as in the validation cohort (AUC 0.69-0.88). The AI2 score association with CLAD was independent of positive microbiology, CLAD stage, or CLAD subtype. However, transcripts most associated with CLAD evolved over time from CLAD onset. The AI2 score predicted time to graft failure and retransplant-free survival in both cohorts (p ≤ 0.03). CONCLUSIONS: This airway inflammation gene score is associated with CLAD development, graft failure, and death. Future studies defining the molecular heterogeneity of airway inflammation could lead to endotype-targeted therapies.

The importance of geographic and sociodemographic aspects in the characterization of mucopolysaccharidoses: a case series from Ceará state (Northeast Brazil).

Geographic and sociodemographic aspects may influence the natural history and epidemiology of mucopolysaccharidoses (MPS). The main objective in this work was to evaluate the clinical, molecular, and geographic profile of MPS in a population from Ceará (Northeast Brazil). For this, we have performed a descriptive cross-sectional study based on clinical evaluation, interviews with patients and/or family members, and review of medical records of 76 MPS patients. MPS II was the most common type, with the most affected individuals presenting missense pathogenic variants. Patients with MPS I proved to be the most severe clinical phenotype, presenting the first symptoms (mean: 7.1 months; SD = 4.5) and being diagnosed earlier (2.2 years; SD = 2.1) in comparison with the other types. In addition, we have shown that 13 individuals with MPS VI were born of consanguineous marriages in small, nearby cities, in a place where geographical isolation, consanguinity, and clusters of genetic diseases were previously reported. Ten of these individuals (at least, seven different families) presented a rare pathogenic variant in the ARSB gene, c.1143-8T > G in homozygosity, previously reported only among Iberian and South American patients. The results presented here provide a comprehensive picture of MPS in an important state of the Brazilian Northeast, a region that concentrates many risk factors for rare genetic diseases, such as endogamy, inbreeding, and reproductive isolation. We discuss the possible evolutionary processes and biosocial dynamics that can help to explain this finding in terms of population medical genetics and public health.

Mesothelioma: molecular pathology and biomarkers.

Diffuse mesotheliomas are characterized by recurrent genomic alterations involving tumor suppressors and epigenetic regulators such as BAP1, CDKN2A, MTAP, and NF2. Depending on the differential diagnosis as informed by histologic assessment, one can apply the appropriate immunohistochemical and/or molecular panels to reach the correct pathologic diagnosis, sometimes even in cases with limited tissues. Biomarkers aid in the diagnosis of mesothelioma in the following scenarios: 1) For a tumor that is overtly malignant, how can one distinguish mesothelioma from other tumors? 2) For a mesothelial proliferation, how can one distinguish mesothelioma from a reactive process? To distinguish mesotheliomas from carcinomas, at least two positive and two negative markers are currently recommended. To distinguish sarcomatoid mesothelioma from pleomorphic carcinoma, even more markers-and sometimes molecular testing-are needed. To distinguish mesothelioma from reactive mesothelial conditions, useful immunohistochemical biomarkers include BAP1, MTAP, and merlin, which serve as surrogates for the corresponding gene mutation status. In patients with unusual clinical history, for tumors with a peculiar microscopic appearance, and/or in cases with an equivocal immunophenotypic profile, molecular testing can help to exclude mimics and to confirm the pathologic diagnosis.

Comprehensive assessment of vaginal infections using a single swab.

BACKGROUND: The decision to use a particular test to diagnose patients presenting with symptoms of vaginitis and/or STI is based primarily on the prevailing standards of care in the clinic at which the patient evaluation takes place. As a result, laboratory testing of vaginal samples for these patients often involves either an STI or a vaginitis test, but rarely both options simultaneously, which complicates the diagnosis and management of concurrent infections. METHODS: Using de-identified remnant vaginal specimens from symptomatic patients previously tested for STI (Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC) and Trichomonas vaginalis (TV)) with the Becton Dickinson (BD) CTGCTV2 assay for BD MAX System, positivity for bacterial vaginosis (BV) and Candida spp (associated with vulvovaginal candidiasis (VVC)) were evaluated using the molecular-based BD MAX Vaginal Panel. FINDINGS: The rate of STI/BV co-infection was 79.4% (227/286) in this symptomatic population, while that of STI/VVC was 27.0% (77/285). Women diagnosed with any one of the three STIs tested had an OR 2.86 (95% CI, 1.99, 4.11; p<0.0001) for a concurrent BV infection and OR 0.96 (95% CI, 0.67, 1.37; p=0.8085) for infection with Candida species. CONCLUSION: Our results suggest that women being tested for STI have a high prevalence of co-infection with BV and a lower, although appreciable, prevalence of co-infection with VVC. The detection of co-occurring vaginal infections can be facilitated by molecular testing using a single sample.

Pathogen-Agnostic Advanced Molecular Diagnostic Testing for Difficult-to-Diagnose Clinical Syndromes-Results of an Emerging Infections Network Survey of Frontline US Infectious Disease Clinicians, May 2023.

During routine clinical practice, infectious disease physicians encounter patients with difficult-to-diagnose clinical syndromes and may order advanced molecular testing to detect pathogens. These tests may identify potential infectious causes for illness and allow clinicians to adapt treatments or stop unnecessary antimicrobials. Cases of pathogen-agnostic disease testing also provide an important window into known, emerging, and reemerging pathogens and may be leveraged as part of national sentinel surveillance. A survey of Emerging Infections Network members, a group of infectious disease providers in North America, was conducted in May 2023. The objective of the survey was to gain insight into how and when infectious disease physicians use advanced molecular testing for patients with difficult-to-diagnose infectious diseases, as well as to explore the usefulness of advanced molecular testing and barriers to use. Overall, 643 providers answered at least some of the survey questions; 478 (74%) of those who completed the survey had ordered advanced molecular testing in the last two years, and formed the basis for this study. Respondents indicated that they most often ordered broad-range 16S rRNA gene sequencing, followed by metagenomic next-generation sequencing and whole genome sequencing; and commented that in clinical practice, some, but not all tests were useful. Many physicians also noted several barriers to use, including a lack of national guidelines and cost, while others commented that whole genome sequencing had potential for use in outbreak surveillance. Improving frontline physician access, availability, affordability, and developing clear national guidelines for interpretation and use of advanced molecular testing could potentially support clinical practice and public health surveillance.

Epidemiology and laboratory detection of non-tuberculous mycobacteria.

The global incidence of non-tuberculous mycobacteria (NTM) infections is on the rise. This study systematically searched several databases, including PubMed, Web of Science, Google Scholar, and two Chinese libraries (Chinese National Knowledge Infrastructure and Wanfang) to identify relevant published between 2013 and 2023 related to the isolation of NTM in clinical specimens from various countries and provinces of China. Furthermore, a comprehensive literature review was conducted in PubMed and Google Scholar to identify randomized clinical trials, meta-analyses, systematic reviews, and observational studies that evaluated the diagnostic accuracy and impact of laboratory detection methods on clinical outcomes. This review presented the most recent epidemiological data and species distributions of NTM isolates in several countries and provinces of China. Moreover, it provided insights into laboratory bacteriological detection, including the identified strains, advantages and disadvantages, recent advancements, and the commercial Mycobacterium identification kits available for clinical use. This review aimed to aid healthcare workers in understanding this aspect, enhance the standards of clinical diagnosis and treatment, and enlighten them on the existing gaps and future research priorities.

The establishment of a molecular diagnostic platform for mitochondrial diseases: from conventional to next-generation sequencing.

BACKGROUND: The aim of this study was to create a molecular diagnostic platform and establish a diagnostic pipeline for patients highly suspected of mitochondrial disorders. The effectiveness of three methods, namely, traditional restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), Sanger sequencing for hotspot detection and whole mitochondrial DNA (mtDNA), and third-generation (Nanopore) whole mtDNA sequencing, will be compared in diagnosing patients with suspected primary mitochondrial diseases (PMDs). The strengths and limitations of different methods are also discussed. MATERIAL AND METHODS: A single-center prospective cohort study was conducted to validate the diagnostic pipeline for suspected mitochondrial diseases. In the first stage, a PCR-based method with five sets of primers was used to screen for eight hotspots (m.3243A>G, m.3460G>A, m.8344A>G, m.8993T>G, m.9185T>C, m.11778G>A, m.13513G>A, and m.4977deletion) using either RFLP or direct Sanger sequencing. Sanger sequencing was also used to confirm the RFLP-positive samples. In the second stage, for samples with negative screening results for the eight hotspots, mitochondrial whole-genome sequencing was performed using Sanger sequencing or third-generation nanopore sequencing. RESULTS: Between June 2020 and May 2023, 30 patients from ages 0 to 63 with clinically suspected mitochondrial disease were enrolled. The positive yield for the diagnosis of PMDs was 8/30=26.7%, and the sensitivity of the heteroplasmy level for the RFLP-based method was approximately 5%. The remaining 22 patients who tested negative at the first stage were tested using Sanger sequencing or the third-generation sequencing Nanopore, and all tested negative for pathological mtDNA mutations. Compared to the Sanger sequencing method, the results of RFLP-PCR were compromised by the limitations of incomplete RFLP enzyme digestion. For whole-genome sequencing of mtDNA, Sanger sequencing, instead of nanopore sequencing, is preferred at our institution because of its cost-effectiveness. CONCLUSIONS: In our highly selective cohort, most tested positive in the first stage of the 8 hot spots screen. Sanger sequencing is a conventional and accurate method for mitochondrial disease screening, at least for the most common hot spots in the region. The results revealed that Sanger sequencing is an accurate method with the benefit of being more cost-effective. This integral platform of molecular diagnosis bears the advantages of being relatively low cost and having a shorter reporting time, facilitating crucial identification of patients with clinical evidence of such disorders. This diagnostic flowchart has also been translated into routine clinical use in the tertiary hospital.

Current laboratory testing practices for mismatch repair deficiency and microsatellite instability testing: A survey-based review of current laboratory practices.

OBJECTIVES: To describe mismatch repair (MMR) and microsatellite instability (MSI) testing practices in laboratories using the College of American Pathologists (CAP) MSI/MMR proficiency testing programs prior to the 2022 publication of the MSI/MMR practice guidelines copublished by CAP and the Association of Molecular Pathology (AMP). METHODS: Data from supplemental questionnaires provided with the 2020-B MSI/MMR programs to 542 laboratories across different practice settings were reviewed. Questionnaires contained 21 questions regarding the type of testing performed, specimen/tumor types used for testing, and clinical practices for checkpoint blockade therapy. RESULTS: Domestic laboratories test for MSI/MMR more often than international laboratories (P = .04) and academic hospitals/medical centers test more frequently than nonhospital sites/clinics (P = .03). The most commonly used testing modality is immunohistochemistry, followed by polymerase chain reaction, then next-generation sequencing. Most laboratories (72.6%; 347/478) reported awareness of the use of immune checkpoint inhibitor therapy for patients with high MSI or MMR-deficient results. CONCLUSIONS: The results demonstrate the state of MMR and MSI testing in laboratories prior to the publication of the CAP/AMP best practice guidelines, highlighting differences between various laboratory types. The findings indicate the importance of consensus guidelines and provide a baseline for comparison after their implementation.

The negative outlook: Long-term follow up of ThyroSeq negative and low-risk nodules.

BACKGROUND: Molecular testing of thyroid nodules is an essential tool to help risk stratify nodules with indeterminate cytology. Although ThyroSeq testing has been around for over a decade, there is a paucity of long-term follow-up data on cytologically indeterminate nodules that are determined to be molecularly negative or low-risk. The objective of this study is to assess the outcomes of nodules with indeterminate cytology (Bethesda III or IV) and negative or low-risk ThyroSeq results. METHODS: This is a single academic institution retrospective cohort study. Patients with at least one thyroid nodule sampled with fine-needle aspiration who underwent ThyroSeq testing from 2012 to 2018 and had negative or low-risk ThyroSeq results on a cytologically indeterminate sample (n = 159 patients, 167 nodules) were included in the study. Outcomes include the false-negative rate and negative predictive value of each test version, as well as follow-up length for each nodule. RESULTS: There were 159 patients with a mean age of 58 years (7-84 years) included in this study; the majority were female (81.8%). The mean follow-up was 4.0 years. Of 167 nodules, three were found to be malignant on resection (1.8%). The negative predictive value for the entire cohort was 98.2% and it was 89.3% for the surgical series. CONCLUSION: ThyroSeq testing has good negative predictive value and can help risk stratify cytologically indeterminate nodules. Routine follow-up allows for safe monitoring of nodules for features suggestive of malignancy.

Relevance of the correlation between tomography findings and laboratory test results in the accuracy of the diagnosis of pulmonary tuberculosis.

Objective: To evaluate the correlation between multidetector computed tomography (MDCT) findings and laboratory test results in patients with pulmonary tuberculosis (PTB). Materials and Methods: A total of 57 patients were evaluated. Patients with suspected PTB were divided into groups according to the final diagnosis (confirmed or excluded), and the groups were compared in terms of sociodemographic variables, clinical symptoms, tomography findings, and laboratory test results. Results: Among the patients with a confirmed diagnosis of PTB, small pulmonary nodules with a peribronchovascular distribution were significantly more common in the patients with a positive sputum smear microscopy result (47.4% vs. 8.3%; p = 0.046), as were a miliary pattern (36.8% vs. 0.0%; p = 0.026), septal thickening (84.2% vs. 41.7%; p = 0.021), and lymph node enlargement (52.6% vs. 8.3%; p = 0.020). Small pulmonary nodules with a centrilobular distribution were significantly more common among the culture-positive patients (75.0% vs. 35.7%; p = 0.045), as was a tree-in-bud pattern (91.7% vs. 42.9%; p = 0.014). A tree-in-bud pattern, one of the main tomography findings characteristic of PTB, had a sensitivity, specificity, positive predictive value, and negative predictive value of 71.0%, 73.1%, 75.9%, and 67.9%, respectively. Conclusion: MDCT presented reliable predictive values for the main tomography findings in the diagnosis of PTB, being a safe tool for the diagnosis of PTB in patients with clinical suspicion of the disease. It also appears to be a suitable tool for the selection of patients who are candidates for more complex, invasive examinations from among those with high clinical suspicion of PTB and a negative sputum smear microscopy result.

Objetivo: Avaliar a correlação entre os achados na tomografia computadorizada multidetectores (TCMD) comparativamente aos resultados laboratoriais em pacientes com tuberculose pulmonar (TBP). Materiais e Métodos: Amostra de 57 pacientes foi avaliada. Pacientes com suspeita clínica de TBP foram divididos de acordo com a positividade do diagnóstico, e as variáveis sociodemográficas, sintomas clínicos e achados tomográficos e laboratoriais foram comparados. Resultados: Nos pacientes com TBP e baciloscopia positiva, foram verificadas frequências significativas para pequenos nódulos pulmonares com distribuição peribroncovascular (47,4% vs. 8,3%; p = 0,046) e miliar (36,8% vs. 0,0%; p = 0,026), espessamento septal (84,2% vs. 41,7%; p = 0,021) e linfonodomegalias (52,6% vs. 8,3%; p = 0,020). Em relação à cultura, os pequenos nódulos pulmonares com distribuição centrolobular (75,0% vs. 35,7%; p = 0,045) e opacidades em árvore em brotamento (91,7% vs. 42,9%; p = 0,014) apresentaram frequências significativamente superiores. Medidas de sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo para árvore em brotamento, um dos principais achados tomográficos característicos da TBP, foram, respectivamente, 71.0%, 73,1%, 75,9% e 67,9%. Conclusão: A TCMD apresentou medidas preditivas confiáveis para os principais achados tomográficos no diagnóstico de TBP, sendo uma ferramenta segura para o diagnóstico da doença em pacientes com suspeita clínica. Também se mostrou adequada para selecionar os pacientes para exames mais complexos e invasivos entre os com alta suspeita clínica de TBP e baciloscopia negativa.

First Molecular Detection and Epidemiological Analysis of Equine Influenza Virus in Two Regions of Colombia, 2020-2023.

Equine influenza is a viral disease caused by the equine influenza virus (EIV), and according to the WOAH, it is mandatory to report these infections. In Latin America and Colombia, EIV risk factors have not been analyzed. The objective of this research is to perform an epidemiological and molecular analysis of the EIV in horses with respiratory symptoms from 2020 to 2023 in Colombia. Molecular EIV detection was performed using RT-qPCR and nanopore sequencing. A risk analysis was also performed via the GEE method. A total of 188 equines with EIV respiratory symptoms were recruited. The positivity rate was 33.5%. The descriptive analysis showed that only 12.8% of the horses were vaccinated, and measures such as the quarantine and isolation of symptomatic animals accounted for 91.5% and 88.8%, respectively. The variables associated with the EIV were the non-isolation of positive individuals (OR = 8.16, 95% CI (1.52-43.67), p = 0.014) and sharing space with poultry (OR = 2.16, 95% CI (1.09-4.26), p = 0.027). In conclusion, this is the first EIV investigation in symptomatic horses in Colombia, highlighting the presence of the virus in the country and the need to improve preventive and control measures.

Molecular detection of ceftriaxone resistance in Neisseria gonorrhoeae clinical specimens: a tool for public health control.

OBJECTIVES: This study aimed to validate and implement a rapid screening assay for molecular detection of the penA-60 allele that is associated with ceftriaxone resistance in Neisseria gonorrhoeae for use on both isolate lysates and clinical specimen DNA extracts. METHODS: A N. gonorrhoeae penA real-time (RT)-PCR was adapted to include a species-specific pap confirmation target and a commercially available internal control to monitor for PCR inhibition.The modified assay was validated using N. gonorrhoeae-positive (n=24) and N. gonorrhoeae-negative (n=42) clinical specimens and isolate lysates. The panel included seven samples with resistance conferred by penA alleles targeted by the assay and four samples with different penA alleles. The feasibility of using the penA RT-PCR for molecular surveillance was assessed using clinical specimens from 54 individuals attending a London sexual health clinic who also had a N. gonorrhoeae isolate included in the 2020 Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP). RESULTS: The assay correctly identified N. gonorrhoeae specimens (n=7) with penA-60/64 alleles targeted by the assay. No penA false negatives/positives were detected, giving the penA target of the assay a sensitivity, specificity, positive and negative predicted values (PPV, NPV) of 100% (95% CIs; sensitivity; 56.1-100%, specificity; 93.6-100%, PPV; 56.1-100%, NPV; 93.6-100%).No cross-reactivity with other Neisseria species or other urogenital pathogens was detected. The N. gonorrhoeae target (pap) was detected in 73 out of 78 of the N. gonorrhoeae-positive specimens, resulting in 92.6% sensitivity (95% CI 83.0% to 97.3%), 100% specificity (95% CI 75.9% to 100%) and PPV, and a NPV of 89.4% (95% CI 52.5% to 90.9%). No penA-59/60/64 alleles were detected within the clinical specimens from the GRASP 2020 feasibility molecular surveillance study (n=54 individuals). CONCLUSION: The implementation of this PCR assay for patient management, public health and surveillance purposes enables the rapid detection of gonococcal ceftriaxone resistance conferred by the most widely circulating penA alleles.

BD Vaginal Panel assay results on the high-throughput BD COR system compared to the BD MAX system.

Molecular-based assays demonstrate excellent sensitivity for the detection of vaginitis causes. Here, the high-throughput BD Vaginal Panel for BD COR System (VP-COR) performance was compared to that of the predicate, BD MAX Vaginal Panel for BD MAX System (VP-MAX). Clinical or contrived samples were used to determine the agreement between VP-COR and VP-MAX. Acceptance criteria for VP-COR agreement were as follows: bacterial vaginosis (BV) required a positive percent agreement (PPA) point estimate of ≥95% and a negative percent agreement (NPA) point estimate of ≥98%; Candida group, Candida glabrata, Candida krusei, and Trichomonas vaginalis (TV) required a PPA and NPA point estimate of ≥95% [with lower bound of 95% confidence interval (95% CI) ≥90%]. PPA was 99.5% (95% CI: 97.5-100) and 97.9% (95% CI: 96.5-98.8) for BV contrived (n = 516) and BV clinical (n = 1,050) specimens, respectively. For the Candida group (clinical; n = 724), C. glabrata (contrived; n = 544), C. krusei (contrived; n = 522), and TV (clinical; n = 702), PPA was 99.4% (95% CI: 98.0-99.9), 100% (95% CI: 97.9-100), 100% (95% CI: 97.6-100), and 99.7% (95% CI: 98.3-100), respectively; the lowest lower bound CI value was 97.6%. NPA was >95% for BV contrived and BV clinical specimens. For the Candida group, C. glabrata, C. krusei, and TV, NPA was ≥98.9%; the lowest lower bound CI value was 97.3%. These results demonstrate the equivalent performance of the VP-COR assay when compared to VP-MAX.IMPORTANCEVaginitis is common among women of reproductive age, resulting in around 10 million office visits a year. Diagnosis is often difficult due to its multiple causes-including bacterial vaginosis, vulvovaginal candidiasis, and trichomoniasis-as well as variation in symptom presentation. Typically, cases are identified with a combination of symptomology, medical history, physical examination, and office- or laboratory-based testing. These traditional techniques involve subjective elements and demonstrate varying sensitivity and specificity. Inaccurate or delayed diagnosis leads to continued symptoms, repeat visits, inappropriate treatment, and unnecessary costs. Alternatively, the use of molecular-based assays increases sensitivity for the detection of vaginitis causes. With the validation of the vaginal panel molecular assay on COR (a high-throughput platform), a workflow can be streamlined in high-demand laboratories while providing high sensitivity for vaginitis detection.

A comprehensive review of iPS cell line-based disease modelling of the polyglutamine spinocerebellar ataxias 2 and 3: a focus on the research outcomes.

Spinocerebellar ataxias (SCAs) are a rare autosomal dominant neurodegenerative disorder. To date, approximately 50 different subtypes of SCAs have been characterized. The prevalent types of SCAs are usually of PolyQ origin, wherein the disease pathology is a consequence of multiple glutamine residues being encoded onto the disease proteins, causing expansions. SCAs 2 and 3 are the most frequently diagnosed subtypes, wherein affected patients exhibit certain characteristic physiological manifestations, such as gait ataxia and dysarthria. Nevertheless, other clinical signs were exclusive to these subtypes. Recently, multiple molecular diagnostic methods have been developed to identify and characterize these subtypes. Despite these advancements, the molecular pathology of SCAs remains unknown. To further understand the mechanisms involved in neurodegenerative SCAs 2 and 3, patient-derived induced pluripotent stem cell (iPSC)-based modelling is a compelling avenue to pursue. We cover the present state of iPSC-based in-vitro illness modelling of SCA subtypes 2 and 3 below, along with a list of cell lines created, and the relevance of research outcomes to personalized autologous therapy.

Nanopore sequencing for smear-negative pulmonary tuberculosis-a multicentre prospective study in China.

PURPOSE: In this prospective study, the diagnosis accuracy of nanopore sequencing-based Mycobacterium tuberculosis (MTB) detection was determined through examining bronchoalveolar lavage fluid (BALF) samples from pulmonary tuberculosis (PTB) -suspected patients. Compared the diagnostic performance of nanopore sequencing, mycobacterial growth indicator tube (MGIT) culture and Xpert MTB/rifampin resistance (MTB/RIF) assays. METHODS: Specimens collected from suspected PTB cases across China from September 2021 to April 2022 were tested then assay diagnostic accuracy rates were compared. RESULTS: Among the 111 suspected PTB cases that were ultimately diagnosed as PTB, the diagnostic rate of nanopore sequencing was statistically significant different from other assays (P < 0.05). Fleiss' kappa values of 0.219 and 0.303 indicated fair consistency levels between MTB detection results obtained using nanopore sequencing versus other assays, respectively. Respective PTB diagnostic sensitivity rates of MGIT culture, Xpert MTB/RIF and nanopore sequencing of 36.11%, 40.28% and 83.33% indicated superior sensitivity of nanopore sequencing. Analysis of area under the curve (AUC), Youden's index and accuracy values and the negative predictive value (NPV) indicated superior MTB detection performance for nanopore sequencing (with Xpert MTB/RIF ranking second), while the PTB diagnostic accuracy rate of nanopore sequencing exceeded corresponding rates of the other methods. CONCLUSIONS: In comparison with MGIT culture and Xpert MTB/RIF assays, BALF's nanopore sequencing provided superior MTB detection sensitivity and thus is suitable for testing of sputum-scarce suspected PTB cases. However, negative results obtained using these assays should be confirmed based on additional evidence before ruling out a PTB diagnosis.

Diagnostic accuracy of Afirma gene expression classifier, Afirma gene sequencing classifier, ThyroSeq v2 and ThyroSeq v3 for indeterminate (Bethesda III and IV) thyroid nodules: a meta-analysis.

Objective: The management of thyroid nodules with indeterminate cytology (ITN) is still a challenge. To evaluate the performance of commercial molecular tests for ITN, we performed this comprehensive meta-analysis. Methods: We performed an electronic search using PubMed/Medline, Embase, and the Cochrane Library. Studies assessing the diagnostic accuracy of Afirma gene expression classifier (GEC), Afirma gene sequencing classifier (GSC), ThyroSeq v2 (TSv2), or ThyroSeq v3 (TSv3) in patients with ITN (only Bethesda category III or IV) were selected; Statistical analyses were performed by using Stata. Results: Seventy-one samples (GEC, n = 38; GSC, n = 16; TSv2, n = 9; TSv3, n = 8) in 53 studies, involving 6490 fine needle aspirations (FNAs) with ITN cytology with molecular diagnostics (GEC, GSC, TSv2, or TSv3), were included in the study. The meta-analysis showed the following pooled estimates: sensitivity 0.95 (95% CI: 0.94-0.97), specificity 0.35 (0.28-0.43), positive likelihood ratio (LR+) 1.5 (1.3-1.6), and negative likelihood ratio (LR-) 0.13 (0.09-0.19), with the best performance for TSv3 (area under the ROC curve 0.95 (0.93-0.96), followed by TSv2 (0.90 (0.87-0.92)), GSC (0.86 (0.82-0.88)), and GEC (0.82 (0.78-0.85)); the best rule-out property was observed for GSC (LR-, 0.07 (0.02-0.19)), followed by TSv3 (0.11 (0.05-0.24)) and GEC (0.16 (0.10-0.28), and the best rule-in was observed for TSv2 (LR+, 2,9 (1.4-4.6)), followed by GSC (1.9 (1.6-2.4)). A meta-regression analysis revealed that study design, Bethesda category, and type of molecular test were independent factors. Conclusion: We showed that in patients with ITN, TSv3 has the best molecular diagnostic performance, followed by TSv2, GSC, and GEC. As regards rule-out malignancy, GSC, and rule-in, TSV2 is superior to other tests.

Unravelling the diagnostic methodologies for SARS-CoV-2; the Indispensable need for developing point-of-care testing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-caused COVID-19 pandemic that continues to be a global menace and since its emergence in the late 2019, SARS-CoV-2 has been vigorously spreading throughout the globe putting the whole world into a multidimensional calamity. The suitable diagnosis strategies are on the front line of the battle against preventing the spread of infections. Since the clinical manifestation of COVID-19 is shared between various diseases, detection of the unique impacts of the pathogen on the host along with the diagnosis of the virus itself should be addressed. Employing the most suitable approaches to specifically, sensitively and effectively recognize the infected cases may be a real game changer in controlling the outbreak and the crisis management. In that matter, point-of-care assays (POC) appears to be the potential option, due to sensitivity, specificity, affordable, and availability. Here we brief the most recent findings about the virus, its variants, and the conventional methods that have been used for its detection, along with the POC strategies that have been applied to the virus diagnosis and the developing technologies which can accelerate the diagnosis procedure yet maintain its efficiency.

Impact of molecular ciprofloxacin resistance testing in management of gonorrhoea in a large urban clinic.

OBJECTIVES: Antibiotic resistance in gonorrhoea is of significant public health concern with the emergence of resistance to last-line therapies such as ceftriaxone. Despite around half of Neisseria gonorrhoeae isolates tested in the UK being susceptible to ciprofloxacin, very little ciprofloxacin is used in clinical practice. Testing for the S91F mutation associated with ciprofloxacin resistance is now available in CE-marked assays and may reduce the requirement for ceftriaxone, but many patients are treated empirically, or as sexual contacts, which may limit any benefit. We describe the real-world impact of such testing on antimicrobial use and clinical outcomes in people found to have gonorrhoea in a large urban UK sexual health clinic. METHODS: Molecular ciprofloxacin resistance testing (ResistancePlus GC assay (SpeeDx)) was undertaken as an additional test after initial diagnosis (m2000 Realtime CT/NG assay (Abbott Molecular)) in those not already known to have had antimicrobial treatment. Data from a 6-month period (from March to September 2022) were analysed to determine treatment choice and treatment outcome. RESULTS: A total of 998 clinical samples tested positive for N. gonorrhoeae in 682 episodes of infection. Of the 560 (56%) samples eligible for resistance testing, 269 (48.0%) were reported as wild-type, 180 (32.1%) were predicted to be resistant, 63 (11.3%) had an indeterminate resistance profile, and in 48 (8.6%) samples, N. gonorrhoeae was not detected. Ciprofloxacin was prescribed in 172 (75%) of 228 episodes in which the wild-type strain was detected. Four (2%) of those treated with ciprofloxacin had a positive test-of-cure sample by NAAT, with no reinfection risk. All four had ciprofloxacin-susceptible infection by phenotypic antimicrobial susceptibility testing. CONCLUSIONS: In routine practice in a large UK clinic, molecular ciprofloxacin resistance testing led to a significant shift in antibiotic use, reducing use of ceftriaxone. Testing can be targeted to reduce unnecessary additional testing. Longer term impact on antimicrobial resistance requires ongoing surveillance.

A novel test and treat program for hepatitis C virus infection utilizing HCV core antigen testing, among police and general population, Islamabad, Pakistan, 2022.

Hepatitis C virus core antigen (HCVcAg) testing can simplify and decrease costs of HCV infection confirmation compared to molecular testing (nucleic acid testing). We piloted HCVcAg testing for the confirmation of active infection. The study was conducted during June through December 2022 among the police and the general population of Islamabad, Pakistan age 18 years and older. Initial screening for HCV antibody was conducted using a rapid diagnostic test (RDT) for all consenting participants. Those who tested positive had venous blood samples tested for HCVcAg, platelets and aspartate aminotransferase (AST). Persons with HCVcAg values ≥3 fmol/L were defined as viremic, and they were offered treatment with direct acting antiviral (DAA) medications, sofosbuvir and daclatasvir. Aspartate aminotransferase to platelet ratio index (APRI) was calculated for each HCV infected person, and those with an APRI score <1.5 received treatment for 12 weeks, while those with APRI ≥ to 1.5 received 24 weeks of treatment. A total of 15,628 persons were screened for anti-HCV using RDT and 643 (4.1%) tested positive. HCVcAg values of ≥3 fmol/L was found in 399/643 (62.1%), and all were offered and accepted treatment. Of those treated, 273/399 (68.4%) returned for a follow-up SVR and HCVcAg was not detected in 261/273, a 95.6% cure rate. The pilot study demonstrated the effectiveness of reaching and treating an urban population using RDT for screening and HCVcAg for confirmation of infection and test of cure.

Whole Exome Sequencing as an Effective Molecular Diagnosis Tool for Craniofacial Fibrous Dysplasia with Ocular Complications.

PURPOSE: To summarize the clinical manifestations of craniofacial fibrous dysplasia (CFD) patients with ocular complications, and find effective methods to diagnose early. METHODS: Nine CFD patients with ocular complications, and their parents were recruited in this study. All patients underwent ocular and systemic examinations. Bone lesions from all patients and peripheral blood from patients and their parents were collected for whole exome sequencing (WES). According to the screening for low-frequency deleterious variants, and bioinformatics variants prediction software, possible disease-causing variants were found in multiple CFD patients. The variants were validated by Sanger sequencing. Trio analysis was performed to verify the genetic patterns of CFD. RESULTS: All patients were diagnosed with CFD, according to the clinical manifestations, classic radiographic appearance, and pathological biopsy. The main symptoms of the 9 CFD patients, included visual decline (9/9), craniofacial deformity (3/9) and strabismus (2/9), with few extraocular manifestations. The family backgrounds of all the CFD patients indicated that only the patient was affected, and their immediate family members were normal. GNAS variants were identified in all bone lesions from CFD patients, including two variant types: c.601C > T:p.R201C(6/9) and c.602G > A:p.R201H (3/9) in exon 8. The detection rate reached 100% by WES, but only 77.8% by Sanger sequencing. Interestingly, we found GNAS variants could not be detected in peripheral blood samples from CFD patients or their parents, and other potentially disease-causing gene variants related to CFD were not found. CONCLUSIONS: For CFD patients with bone lesions involving the optic canal or sphenoid sinus regions, ocular symptoms should also be considered. Furthermore, we confirmed that CFD is not inherited, somatic variants in the GNAS gene are the main pathogenic gene causing CFD. Compared to the traditional methods in molecular genetic diagnosis of CFD, WES is more feasible and effective but limited in the type of samples.