Using next-generation sequencing (NGS), we developed and validated a whole-genome sequencing (WGS)-based clinical test for fungal species identification on clinical isolates. The identification is mainly based on the fungal ribosomal internal transcribed spacer (ITS) region as the primary marker, and additional marker and genomic analysis applied for species within the Mucorales family (using the 28S rRNA gene) and Aspergillus genus (using the beta-tubulin gene and k-mer tree-based phylogenetic clustering). The validation study involving 74 unique fungal isolates (22 yeasts, 51 molds, and 1 mushroom-forming fungus) showed high accuracy, with 100% (74/74) concordance on the genus-level identifications and 89.2% (66/74) concordance on the species level. The 8 discrepant results were due to either the limitation of conventional morphology-based methodology or taxonomic changes. After one year of implementation in our clinical laboratory, this fungal NGS test was utilized in 29 cases; the majority of them were transplant and cancer patients. We demonstrated the utility of this test by detailing five case studies, in which accurate fungal species identification led to correct diagnosis, treatment adjustment or was ruled out for hospital acquired infection. This study provides a model for validation and implementation of WGS for fungal identification in a complex health system that serves a large immunocompromised patient population.

The global emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a critical public healthcare concern due to treatment challenges and high mortality. In recent years, there has been an increase in cases of CRKP co-producing New Delhi metallo-β-lactamases (NDM) and oxacillinase 48 (OXA-48)-like carbapenemases in the US. The aim of this study was to correlate the clinical and genomic characteristics of CRKP co-producing NDM and OXA-48-like carbapenemases isolated from patients in Southern California since 2016. Whole-genome sequencing was performed on clinical isolates obtained from various sources, including blood, abdominal fluid, wounds, and urine. Genetic diversity was observed in these CRKP, including ST-14, ST-16, ST-167, ST-437, ST-2096, and ST-2497 lineages. Phylogenetic analysis revealed two closely related clusters (ST-14 and ST-2497), with single nucleotide polymorphism (SNP) differences ranging from 0 to 36, suggesting a possible local spread of these CRKP. Significant antimicrobial resistance (AMR) genes were identified in these CRKP, including bla_NDM-1, bla_NDM-5, bla_OXA-232, bla_OXA-181, bla_CTX-M-15, armA, tet(A), and tet(D). Moreover, pColKP3-type and Inc-type plasmids known to harbor AMR genes were also detected in these isolates. Most of the patients infected with this rare type of CRKP died, although their severe comorbidities also played important roles in their demise. Our study highlighted the extremely limited treatment options and poor clinical outcomes associated with these dual-carbapenemase-producing CRKP. Real-time genomic surveillance of these unusual and deadly CRKP can provide critical information for infection prevention and treatment guidance.

Invasive fungal infections are an increasing global health concern, particularly in low-resource settings where diagnostic capacity is limited. In The Gambia, where tuberculosis is highly prevalent, fungal infections may be misdiagnosed as mycobacterial disease due to overlapping clinical symptoms and limited access to fungal testing. This study aimed to determine the presence of three priority fungal pathogens by the World Health Organization (WHO) classification system— Aspergillus species ( spp .), Histoplasma spp. , and Pneumocystis jirovecii —in patients with symptoms suggestive of tuberculosis, and to develop a molecular tool to support future surveillance. A multiplex quantitative polymerase chain reaction assay was developed and validated for simultaneous detection of Aspergillus spp. , Histoplasma spp. , and Pneumocystis jirovecii DNA in human sputum. The assay was applied to 273 stored sputum samples collected from adult patients presenting with respiratory symptoms concerning for tuberculosis in The Gambia. The multiplex assay demonstrated high sensitivity and specificity, detecting as few as ten DNA copies per reaction for each target. Among the 273 sputum samples analyzed, Aspergillus DNA was identified in five samples (1.8%), Pneumocystis jirovecii DNA in three samples (1.1%), and no samples were positive for Histoplasma . Cough and weight loss were the most frequently reported symptoms among participants with positive results. This study represents the first molecular detection of Aspergillus spp. and Pneumocystis jirovecii in adults in The Gambia. These findings suggest that fungal colonization or infection may occur in a small proportion of patients presenting with tuberculosis-like symptoms. The multiplex molecular platform developed here provides an accessible approach for fungal surveillance and may improve diagnosis and management of fungal infections in resource-limited settings.