Evaluation of Fourier transform-infrared spectroscopy (FT-IR) as a control measure for nosocomial outbreak investigations.

Whole-genome sequencing (WGS) provides greater resolution than other molecular epidemiology strategies and is emerging as a new gold standard approach for microbial strain typing. The Bruker IR Biotyper is designed as a screening tool to identify bacterial isolates that require WGS to establish accurate relationships, but its performance and utility in nosocomial outbreak investigations have not been thoroughly investigated. Here, we evaluated the IR Biotyper by retrospectively examining isolates tested by WGS during investigations of potential nosocomial transmission events or outbreaks. Ninety-eight clinical isolates from 14 different outbreak investigations were examined: three collections of Acinetobacter baumannii (n = 2, n = 9, n = 5 isolates in each collection), one of Escherichia coli (n = 16), two of Pseudomonas aeruginosa (n = 2 and n = 5), two of Serratia marcescens (n = 9 and n = 7), five of Staphylococcus aureus (n = 8, n = 4, n = 3, n = 3, n = 17), and one of Stenotrophomonas maltophilia (n = 8). Linear regression demonstrated a weak, positive correlation between the number of pairwise genome-wide single-nucleotide polymorphisms (SNPs) and IR Biotyper spectral distance values for Gram-positive (r = 0.43, P ≤ 0.0001), Gram-negative (r = 0.1554, P = 0.0639), and all organisms combined (r = 0.342, P ≤ 0.0001). Overall, the IR Biotyper had a positive predictive value (PPV) of 55.81% for identifying strains that were closely related by genomic identity, but a negative predictive value (NPV) of 86.79% for identifying unrelated isolates. When experimentally adjusted cut-offs were applied to A. baumannii, P. aeruginosa, and E. coli, the PPV was 62% for identifying strains that were closely related and the NPV was 100% for identifying unrelated isolates. Implementation of the IR Biotyper as a screening tool in this cohort would have reduced the number of Gram-negative isolates requiring further WGS analysis by 50% and would reduce the number of S. aureus isolates needing WGS resolution by 48%.

High Clinical Impact of Broad-Range Fungal PCR in Suspected Fungal Sinusitis.

Broad-range fungal PCR is a powerful tool for identifying pathogens directly from patient specimens; however, reported estimates of clinical utility vary and costs discourage universal testing. We investigated the diagnostic and clinical utility of broad-range fungal PCR by examining 9 years of results from sinonasal specimens, hypothesizing that this anatomic location would identify immunocompromised patients at high risk for invasive fungal disease. We retrospectively identified 644 PCRs and 1,446 fungal cultures from sinus sites. To determine the relative performance of each testing modality, we performed chart review on 52 patients having specimens submitted for culture and PCR on the same day. Positivity rates were significantly higher for PCR (37.1%) than culture (13.7%) but similar for formalin-fixed and fresh tissues (42.3% versus 34.6%). Relative to culture, PCR had significantly faster turnaround time to both preliminary (94.5 versus 108.8 h) and final positive (137.9 versus 278.5 h) results. Among chart-reviewed patients, 88% were immunocompromised, 65% had proven or probable fungal disease, and testing sensitivities for culture and PCR (67.5% and 85.0%) were not statistically different. Nevertheless, PCR identified pathogens not recovered by culture in 14.9% of cases and informed clinical decision-making in 16.7% of all reviewed cases, and sensitivity of PCR combined with culture (90.0%) was higher than that of culture alone. We conclude that broad-range fungal PCR is frequently informative for patients at risk of serious fungal disease and is complementary to and has faster turnaround time than culture. Formalin-fixed tissue does not adversely affect diagnostic yield, but anatomic site may impact assay positivity rates.

Identification of Leptotrichia goodfellowii infective endocarditis by next-generation sequencing of 16S rDNA amplicons.

The oral aerotolerant anaerobe Leptotrichia goodfellowii is an unusual cause of endocarditis and is amenable to treatment with ß-lactam antibiotics. Because this organism is difficult to identify by conventional methods, molecular detection is a key diagnostic modality. Broad-range 16S rDNA PCR followed by Sanger sequencing constitute the first-line molecular approach, yet poor DNA quality, contaminating DNA, or low template quantity make identification challenging. Here we report a case of culture-negative, aortic and mitral valve endocarditis in a 66-yr-old woman with a history of cardiomyopathy, atrial fibrillation with intracardiac pacer, poor dentition, and recent tooth infection. In this case, 16S rDNA amplicon Sanger sequencing was not sufficient for pathogen identification because of interfering DNA, but deconvolution of the clinical sample using reflexive next-generation amplicon sequencing enabled confident identification of a single pathogenic organism, L. goodfellowii The patient developed a sigmoid colon perforation and died despite additional surgical treatment. Most Leptotrichia endocarditis cases have been subacute and have been successfully treated with antibiotics, with or without valve replacement. This case highlights both an unusual etiologic agent of endocarditis, as well as the rational utilization of advanced molecular diagnostics tools for characterizing serious infections.

Incidental identification of Strongyloides stercoralis infection by broad-range 28S rDNA gene sequencing in a patient with a hematolymphoid malignancy.

Strongyloides stercoralis is an important human parasite, especially in rural areas and developing countries. Infected immunosuppressed patients are at risk for hyperinfection, with severe clinical consequences. Here we describe the incidental detection and diagnosis of an unexpected S. stercoralis infection by methods designed to detect fungal 28S ribosomal DNA.

Clinical Next Generation Sequencing Outperforms Standard Microbiological Culture for Characterizing Polymicrobial Samples.

BACKGROUND: Humans suffer from infections caused by single species or more complex polymicrobial communities. Identification of infectious bacteria commonly employs microbiological culture, which depends upon the in vitro propagation and isolation of viable organisms. In contrast, detection of bacterial DNA using next generation sequencing (NGS) allows culture-independent microbial profiling, potentially providing important new insights into the microbiota in clinical specimens. METHODS: NGS 16S rRNA gene sequencing (NGS16S) was compared with culture using (a) synthetic polymicrobial samples for which the identity and abundance of organisms present were precisely defined and (b) primary clinical specimens. RESULTS: Complex mixtures of at least 20 organisms were well resolved by NGS16S with excellent reproducibility. In mixed bacterial suspensions (107 total genomes), we observed linear detection of a target organism over a 4-log concentration range (500-3 × 106 genomes). NGS16S analysis more accurately recapitulated the known composition of synthetic samples than standard microbiological culture using nonselective media, which distorted the relative abundance of organisms and frequently failed to identify low-abundance pathogens. However, extended quantitative culture using selective media for each of the component species recovered the expected organisms at the proper abundance, validating NGS16S results. In an analysis of sputa from cystic fibrosis patients, NGS16S identified more clinically relevant pathogens than standard culture. CONCLUSIONS: Biases in standard, nonselective microbiological culture lead to a distorted characterization of polymicrobial mixtures. NGS16S demonstrates enhanced reproducibility, quantification, and classification accuracy compared with standard culture, providing a more comprehensive, accurate, and culture-free analysis of clinical specimens.

Whole genome sequencing indicates Corynebacterium jeikeium comprises 4 separate genomospecies and identifies a dominant genomospecies among clinical isolates.

Corynebacterium jeikeium is an opportunistic pathogen which has been noted for significant genomic diversity. The population structure within this species remains poorly understood. Here, we explore the relationships among 15 clinical isolates of C. jeikeium (reference strains K411 and ATCC 43734, and 13 primary isolates collected over a period of 7 years) through genetic, genomic, and phenotypic studies. We report a high degree of divergence among strains based on 16S ribosomal RNA (rRNA) gene and rpoB gene sequence analysis, supporting the presence of genetically distinct subgroups. Whole genome sequencing indicates genomic-level dissimilarity among subgroups, which qualify as four separate and distinct Corynebacterium species based on an average nucleotide identity (ANIb) threshold of <95%. Functional distinctions in antibiotic susceptibilities and metabolic profiles characterize two of these genomospecies, allowing their differentiation from others through routine laboratory testing. The remaining genomospecies can be classified through a biphasic approach integrating phenotypic testing and rpoB gene sequencing. The genomospecies predominantly recovered from patient specimens does not include either of the existing C. jeikeium reference strains, implying that studies of this pathogen would benefit from examination of representatives from the primary disease-causing group. The clinically dominant genomospecies also has the smallest genome size and gene repertoire, suggesting the possibility of increased virulence relative to the other genomospecies. The ability to classify isolates to one of the four C. jeikeium genomospecies in a clinical context provides diagnostic information for tailoring antimicrobial therapy and may aid in identification of species-specific disease associations.

Repeat-region polymorphisms in the gene for the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-related molecule: effects on HIV-1 susceptibility.

In 1716 individuals--801 human immunodeficiency virus (HIV)-1-seropositive individuals, 217 high-risk HIV-1-seronegative individuals, and 698 general HIV-1-seronegative individuals--from a Seattle cohort and a Multicenter AIDS Cohort Study cohort, the association between HIV-1 susceptibility and repeat-region polymorphisms in the gene for the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-related molecule (DC-SIGNR) was investigated; 16 genotypes were found in the DC-SIGNR repeat region. The DC-SIGNR homozygous 7/7 repeat was found to be associated with an increased risk of HIV-1 infection (17.5% in high-risk HIV-1-seronegative individuals vs. 28.5% in HIV-1-seropositive individuals; P=.0015), whereas the DC-SIGNR heterozygous 7/5 repeat tended to be correlated with resistance to HIV-1 infection (35.5% in high-risk HIV-1-seronegative individuals vs. 27.6% in HIV-1-seropositive individuals; P=.0291). These findings suggest that DC-SIGNR polymorphisms may influence susceptibility to HIV-1.