Ha-eun Cho, M.D., Yujin Han, M.D., Ph.D., Jae Joon Lee, M.D., Ph.D., Kyung Sun Park, M.D., Ph.D., Young Jin Kim, M.D., Ph.D., Sun Young Cho, M.D., Ph.D., Hoi Soo Yoon, M.D., Ph.D., Woo-In Lee, M.D., Ph.D., and Tae Sung Park, M.D., Ph.D.. Ann Lab Med 2022;42:485-7. https://doi.org/10.3343/alm.2022.42.4.485

Conventional diagnostic methods (CDMs) for lower respiratory infections (LRIs) have limitations in detecting causative pathogens. This study evaluates the utility of shotgun metagenomic sequencing (SMS) as a complementary diagnostic tool using bronchoalveolar lavage (BAL) fluid. Sixteen BAL fluid samples from pneumonia patients with positive CDM results—including bacterial/fungal cultures; PCR for Mycobacterium tuberculosis or cytomegalovirus; and the BioFire^® FilmArray^® Pneumonia Panel (BioFire Diagnostics LLC, Salt Lake City, UT, USA)—underwent 10 Gb SMS on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Reads were aligned to the NCBI RefSeq database; with fungal identification further supported by internal transcribed spacer (ITS) analysis. Antibiotic resistance genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database. Microbial reads accounted for 0.00002–0.04971% per sample. SMS detected corresponding bacteria in 63% of cases, increasing to 69% when subdominant taxa were included. Fungal reads were low; however, Candida species were identified in four samples via ITS. No viral reads were detected. ARGs meeting perfect match criteria were found in two cases. This is the first real-world study comparing SMS with CDMs, including semiquantitative PCR, in BAL fluid for LRI. SMS shows promise as a supplementary diagnostic method, with further research needed to optimize its performance and cost-effectiveness.

Latent tuberculosis (TB) refers to a state in which an individual is infected with Mycobacterium tuberculosis but shows no clinical symptoms. The World Health Organization estimates that 23% of the global population has latent TB, which poses a significant public health challenge owing to the risk of progression to active TB. Diagnosis of latent TB involves tests, such as the tuberculin skin test (TST) and interferon-gamma release assays (IGRAs). The TST can yield false positives due to prior Bacillus Calmette-Guérin (BCG) vaccination, whereas IGRAs offer higher specificity and are unaffected by BCG vaccination. Factors, such as age and recent vaccinations, can affect test performance. Treatment with isoniazid and rifampicin is recommended for those diagnosed, as it has been shown to prevent 80%-90% of active TB cases, although more extended follow-up studies are needed to confirm its long-term efficacy. Indeterminate IGRA results, especially in immunocompromised individuals, add complexity to the diagnosis and treatment decisions, highlighting the need for careful interpretation. Further research is vital to improve the diagnostic accuracy, interpretation, and treatment effectiveness.

Background and Clinical Significance: Isolated prolongation of activated partial thromboplastin time (aPTT) without a bleeding tendency presents a frequent diagnostic challenge and often leads to prolonged, inconclusive evaluations. Case Presentation: We report the case of a pregnant woman with long-standing isolated aPTT prolongation in whom clinical exome sequencing enabled a definitive diagnosis. Two compound heterozygous variants in F12 were identified: NM_000505.4:c.1561G>A, p.(Glu521Lys), previously reported in Factor XII deficiency, and a novel in-frame insertion, NM_000505.4:c.1423_1425dup, p.(Cys475dup), absent from population databases and the prior literature. Familial genetic testing confirmed a trans configuration. Factor XII activity was markedly reduced to 1%, and mixing studies showed complete correction, consistent with coagulation factor deficiency without inhibitors. Variant interpretation using ClinGen specifications within a Bayesian framework classified both variants as likely pathogenic. Despite significant laboratory abnormalities, the patient experienced no bleeding or thrombotic complications and underwent cesarean delivery without adverse events. Conclusions: This case highlights that early integration of next-generation sequencing and quantitative variant interpretation frameworks can facilitate timely diagnosis, clarify clinical significance, and support appropriate management in patients with unexplained isolated aPTT prolongation.

Introduction: Diagnosing lower respiratory infections (LRIs) using conventional diagnostic methods (CDMs) presents limitations in detecting suspected pathogens. This study compares the latest CDMs with shotgun metagenomic sequencing (SMS) for bronchoalveolar lavage (BAL) fluid. The primary objective is to enhance pathogen detection using SMS. Materials and Methods: A total of 16 BAL fluid samples from patients with pneumonia with positive results in various CDMs—bacterial/fungal cultures, real-time PCR for Mycobacterium tuberculosis , cytomegalovirus, or the BioFire® FilmArray Pneumonia Panel—were included. Samples were subjected to 10 Gb SMS on the NovaSeq 6000 (Illumina) and were aligned against the NCBI RefSeq database. For eukaryotic reads, an additional matching process was performed using the internal transcribed spacer (ITS) region of fungi. Antibiotic resistance genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database model. To identify significant pathogens, thresholds for the relative abundance of SMS reads were applied to evaluate the concordance between CDM- and SMS-detected microbes. Results: The proportion of microbial reads ranged from 0.00002–0.04971% per sample. SMS detected corresponding bacterial reads (2–23,869) with relative abundance between 0.02% and 87.5%. Eukaryotic reads varied from 0 to 32, with no fungal alignment at the genus level. Candida species were identified in four samples using ITS. No viral reads were detected. In 10 out of 16 cases (63%), SMS detected pathogens above the threshold by SMS. When subdominant taxa were included, SMS detected pathogens in 11 out of 16 cases (69%). ARGs meeting perfect criteria via the Resistance Gene Identifier were observed in two cases. Conclusion: This study represents the first comparison of SMS and CDMs, including the FilmArray Pneumonia Panel, in the context of LRI diagnostics. SMS may serve as a valuable supplementary tool for LRI diagnosis. Further research is necessary to improve sensitivity and cost-effectiveness.