Robert Schlaberg

Nocardia cyriacigeorgica, an Emerging Pathogen in the United States

ABSTRACT Nocardia cyriacigeorgica is recognized as an emerging pathogen in many parts of the world. We present the first case description of invasive N. cyriacigeorgica pulmonary infection in the United States identified to the species level by 16S rRNA and hsp65 sequence analysis. A subsequent retrospective molecular screening of recent Nocardia clinical isolates at our New York City medical center yielded an additional six N. cyriacigeorgica isolates. Because routine laboratory algorithms for the phenotypic identification of Nocardia species are limited in practice, the true prevalence of N. cyriacigeorgica infections may be greater than currently appreciated. Indeed, we present evidence confirming that N. cyriacigeorgica is coincident with the unofficial species designation Nocardia asteroides complex antimicrobial susceptibility pattern type VI and distinct from the N. asteroides sensu stricto strain ATCC 19247T. As nocardial species identity can predict antimicrobial susceptibility and guide clinical management, we offer simplified phenotypic and molecular protocols to assist the identification of N. cyriacigeorgica.

Absence of XMRV Retrovirus and Other Murine Leukemia Virus-Related Viruses in Patients with Chronic Fatigue Syndrome

ABSTRACT Chronic fatigue syndrome (CFS) is a multisystem disorder characterized by prolonged and severe fatigue that is not relieved by rest. Attempts to treat CFS have been largely ineffective primarily because the etiology of the disorder is unknown. Recently, CFS has been associated with xenotropic murine leukemia virus-related virus (XMRV) as well as other murine leukemia virus (MLV)-related viruses, though not all studies have found these associations. We collected blood samples from 100 CFS patients and 200 self-reported healthy volunteers from the same geographical area. We analyzed these in a blind manner using molecular, serological, and viral replication assays. We also analyzed samples from patients in the original study that reported XMRV in CFS patients. We did not find XMRV or related MLVs either as viral sequences or infectious viruses, nor did we find antibodies to these viruses in any of the patient samples, including those from the original study. We show that at least some of the discrepancy with previous studies is due to the presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. Our findings do not support an association between CFS and MLV-related viruses, including XMRV, and the off-label use of antiretrovirals for the treatment of CFS does not seem justified at present.

Susceptibility Profiles of Nocardia Isolates Based on Current Taxonomy

ABSTRACT The genus Nocardia has undergone rapid taxonomic expansion in recent years, and an increasing number of species are recognized as human pathogens. Many established species have predictable antimicrobial susceptibility profiles, but sufficient information is often not available for recently described organisms. Additionally, the effectiveness of sulfonamides as first-line drugs for Nocardia has recently been questioned. This led us to review antimicrobial susceptibility patterns for a large number of molecularly identified clinical isolates. Susceptibility results were available for 1,299 isolates representing 39 different species or complexes, including 11 that were newly described, during a 6-year study period. All tested isolates were susceptible to linezolid. Resistance to trimethoprim-sulfamethoxazole (TMP-SMX) was rare (2%) except among Nocardia pseudobrasiliensis (31%) strains and strains of the N. transvalensis complex (19%). Imipenem susceptibility varied for N. cyriacigeorgica and N. farcinica, as did ceftriaxone susceptibility of the N. nova complex. Resistance to more than one of the most commonly used drugs (amikacin, ceftriaxone, TMP-SMX, and imipenem) was highest for N. pseudobrasiliensis (100%), N. transvalensis complex (83%), N. farcinica (68%), N. puris (57%), N. brasiliensis (51%), N. aobensis (50%), and N. amikacinitolerans (43%). Thus, while antimicrobial resistance can often be predicted, susceptibility testing should still be considered when combination therapy is warranted, for less well characterized species or those with variable susceptibility profiles, and for patients with TMP-SMX intolerance.

Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel

ABSTRACT Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patients symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis. We compared detection of known respiratory virus-positive (n = 42) and unselected (n = 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness. Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information.

A Systematic Approach for Discovering Novel, Clinically Relevant Bacteria

We identified 95 isolates from novel taxa that may have clinical relevance.

Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection.

CONTEXT - Metagenomic sequencing can be used for detection of any pathogens using unbiased, shotgun next-generation sequencing (NGS), without the need for sequence-specific amplification. Proof-of-concept has been demonstrated in infectious disease outbreaks of unknown causes and in patients with suspected infections but negative results for conventional tests. Metagenomic NGS tests hold great promise to improve infectious disease diagnostics, especially in immunocompromised and critically ill patients. OBJECTIVE - To discuss challenges and provide example solutions for validating metagenomic pathogen detection tests in clinical laboratories. A summary of current regulatory requirements, largely based on prior guidance for NGS testing in constitutional genetics and oncology, is provided. DATA SOURCES - Examples from 2 separate validation studies are provided for steps from assay design, and validation of wet bench and bioinformatics protocols, to quality control and assurance. CONCLUSIONS - Although laboratory and data analysis workflows are still complex, metagenomic NGS tests for infectious diseases are increasingly being validated in clinical laboratories. Many parallels exist to NGS tests in other fields. Nevertheless, specimen preparation, rapidly evolving data analysis algorithms, and incomplete reference sequence databases are idiosyncratic to the field of microbiology and often overlooked.

Neuroprotection and Reduced Proliferation of Microglia in Ribavirin-Treated Bornavirus-Infected Rats

ABSTRACT In a rat model of Borna disease, intracerebral ribavirin caused clinical improvement without changes in virus titer or nucleic acid. Levels of microglia and infiltrating CD4 and CD8 cells were decreased, despite increases in mRNAs encoding interleukin-1β (IL-1β), IL-10, and gamma interferon in the brain. Intracerebral ribavirin may reduce morbidity through effects on microglia cell proliferation.

A Method for Optimizing and Validating Institution-Specific Flagging Criteria for Automated Cell Counters

CONTEXT Automated cell counters use alerts (flags) to indicate which differential white blood cell counts can be released directly from the instrument and which samples require labor-intensive slide reviews. The thresholds at which many of these flags are triggered can be adjusted by individual laboratories. Many users, however, use factory-default settings or adjust the thresholds through a process of trial and error. OBJECTIVE To develop a systematic method, combining statistical analysis and clinical judgment, to optimize the flagging thresholds on automated cell counters. DESIGN Data from 502 samples flagged by Sysmex XE-2100/5000 (Sysmex, Kobe, Japan) instruments, with at least 1 of 5 user-adjustable, white blood cell count flags, were used to change the flagging thresholds for maximal diagnostic effectiveness by optimizing the Youden index for each flag (the optimization set). The optimized thresholds were then validated with a second set of 378 samples (the validation set). RESULTS Use of the new thresholds reduced the review rate caused by the 5 flags from 6.5% to 2.9% and improved the positive predictive value of the flagging system for any abnormality from 27% to 37%. CONCLUSIONS This method can be used to optimize thresholds for flag alerts on automated cell counters of any type and to improve the overall positive predictive value of the flagging system at the expense of a reduction in the negative predictive value. A reduced manual review rate helps to focus resources on differential white blood cell counts that are of clinical significance and may improve turnaround time.

Antepartum Screening for Group B Streptococcus by Three FDA-Cleared Molecular Tests and Effect of Shortened Enrichment Culture on Molecular Detection Rates

ABSTRACT Neonatal Streptococcus agalactiae infections cause significant morbidity and mortality, and antenatal screening is recommended. We compared three U.S. Food and Drug Administration (FDA)-cleared nucleic acid amplification tests (NAATs) to culture using 314 vaginal/rectal swabs after 18 to 24 h (recommended period) and 4 to 8 h (shortened period) of broth enrichment. Agreement of the NAATs with each other was high (97.1% to 98.4%), but culture was less sensitive than all NAATs (67% to 73%). A shortened period of broth culture enrichment resulted in 1 false-negative result in 68 (1.5%). The NAATs performed comparably and were more sensitive than culture.

Viral pathogen detection by metagenomics and pan-viral group polymerase chain reaction in children with pneumonia lacking identifiable etiology

Summary Two broad-spectrum pathogen detection methods, next-generation sequencing and pan-viral group polymerase chain reaction, detected previously missed, putative pathogens in 34% of children hospitalized with community-acquired pneumonia with no identified etiology.