Culture of urine specimens by use of chromID CPS Elite medium can expedite Escherichia coli identification and reduce hands-on time in the clinical laboratory

Urine is one of the most common specimen types submitted to the clinical microbiology laboratory; the use of chromogenic agar is one method by which the laboratory might expedite culture results and reduce hands-on time and materials required for urine culture analysis. The objective of our study was to compare chromID CPS Elite (bioMérieux), a chromogenic medium, to conventional primary culture medium for evaluation of urine specimens. Remnant urine specimens (n = 200) were inoculated into conventional media and into chromID CPS Elite agar (chromID). The time to identification and consumables used were documented for both methods. Clinically significant pathogen(s) were recovered from 51 cultures using conventional media, with Escherichia coli being the most frequently recovered organism (n = 22). The rate of exact uropathogen agreement between conventional and chromogenic media was 82%, while overall categorical agreement was 83.5% The time interval between plating and final organism identification was decreased with chromID agar versus conventional media for E. coli (mean of 24.4 h versus 27.1 h, P < 0.001). Using chromID, clinically significant cultures required less hands-on time per culture (mean of 1 min and 2 s [1:02 min]) compared to conventional media (mean of 1:31 min). In addition, fewer consumables (2.4 versus 3.3 sticks and swabs) and rapid biochemical tests (1.0 versus 1.9) were necessary using chromID versus conventional media. Notably, antimicrobial susceptibility testing demonstrated good overall agreement (97.4%) between the chromID and conventional media for all antibiotics tested. chromID CPS Elite is accurate for uropathogen identification, reduces consumable usage, and may expedite the identification of E. coli in clinical specimens

Unbiased Metagenomic Sequencing for Pediatric Meningitis in Bangladesh Reveals Neuroinvasive Chikungunya Virus Outbreak and Other Unrealized Pathogens.

The burden of meningitis in low-and-middle-income countries remains significant, but the infectious causes remain largely unknown, impeding institution of evidence-based treatment and prevention decisions. We conducted a validation and application study of unbiased metagenomic next-generation sequencing (mNGS) to elucidate etiologies of meningitis in Bangladesh. This RNA mNGS study was performed on cerebrospinal fluid (CSF) specimens from patients admitted in the largest pediatric hospital, a World Health Organization sentinel site, with known neurologic infections (n = 36), with idiopathic meningitis (n = 25), and with no infection (n = 30), and six environmental samples, collected between 2012 and 2018. We used the IDseq bioinformatics pipeline and machine learning to identify potentially pathogenic microbes, which we then confirmed orthogonally and followed up through phone/home visits. In samples with known etiology and without infections, there was 83% concordance between mNGS and conventional testing. In idiopathic cases, mNGS identified a potential bacterial or viral etiology in 40%. There were three instances of neuroinvasive Chikungunya virus (CHIKV), whose genomes were &gt;99% identical to each other and to a Bangladeshi strain only previously recognized to cause febrile illness in 2017. CHIKV-specific qPCR of all remaining stored CSF samples from children who presented with idiopathic meningitis in 2017 (n = 472) revealed 17 additional CHIKV meningitis cases, exposing an unrecognized meningitis outbreak. Orthogonal molecular confirmation, case-based clinical data, and patient follow-up substantiated the findings. Case-control CSF mNGS surveys can complement conventional diagnostic methods to identify etiologies of meningitis, conduct surveillance, and predict outbreaks. The improved patient- and population-level data can inform evidence-based policy decisions.IMPORTANCE Globally, there are an estimated 10.6 million cases of meningitis and 288,000 deaths every year, with the vast majority occurring in low- and middle-income countries. In addition, many survivors suffer from long-term neurological sequelae. Most laboratories assay only for common bacterial etiologies using culture and directed PCR, and the majority of meningitis cases lack microbiological diagnoses, impeding institution of evidence-based treatment and prevention strategies. We report here the results of a validation and application study of using unbiased metagenomic sequencing to determine etiologies of idiopathic (of unknown cause) cases. This included CSF from patients with known neurologic infections, with idiopathic meningitis, and without infection admitted in the largest children's hospital of Bangladesh and environmental samples. Using mNGS and machine learning, we identified and confirmed an etiology (viral or bacterial) in 40% of idiopathic cases. We detected three instances of Chikungunya virus (CHIKV) that were &gt;99% identical to each other and to a strain previously recognized to cause systemic illness only in 2017. CHIKV qPCR of all remaining stored 472 CSF samples from children who presented with idiopathic meningitis in 2017 at the same hospital uncovered an unrecognized CHIKV meningitis outbreak. CSF mNGS can complement conventional diagnostic methods to identify etiologies of meningitis, and the improved patient- and population-level data can inform better policy decisions

Implementation Plan to Integrate the Nanosphere Verigene Assay in a High-volume Reference Laboratory and the Anticipated Workflow Benefits to Adopting Rapid Blood Culture Testing

Laboratory processing pathways have a significant impact on the overall management of patients with sepsis. Retrieval and isolation of the suspected pathogen from a patient blood culture specimen is required for a definitive diagnosis of bacterial septicemia. Reference laboratories are high-volume facilities most often located some distance away from the collecting facility. Given the lengthy work up already required for blood culture pathogen analysis, reference laboratories must identify ways to optimize every step of the blood culture pathway in the effort to decrease turnaround time and mitigate lag time to final pathogen identification incurred by prolonged collection-to-incubation times. Rapid molecular diagnostic methods independent of culture results is an available potential solution. The focus of this paper is to consider published literature on the evaluation of rapid blood culture testing to identify its potential benefits and ultimately layout a properly developed implementation plan that integrates the Verigene microarray-based rapid blood culture testing system (Nanosphere, Northbrook, IL, USA) into the blood culture workflow at a high-volume reference laboratory

ASM LabCap’s contributions to disease surveillance and the International Health Regulations (2005)

The revised International Health Regulations [IHR(2005)], which requires the Member States of the World Health Organization (WHO) to develop core capacities to detect, assess, report, and respond to public health threats, is bringing new challenges for national and international surveillance systems. As more countries move toward implementation and/or strengthening of their infectious disease surveillance programs, the strengthening of clinical microbiology laboratories becomes increasingly important because they serve as the first line responders to detect new and emerging microbial threats, re-emerging infectious diseases, the spread of antibiotic resistance, and the possibility of bioterrorism. In fact, IHR(2005) Core Capacity #8, “Laboratory”, requires that laboratory services be a part of every phase of alert and response

Practice guidelines for clinical microbiology laboratories: Mycobacteria

Mycobacteria are the causative organisms for diseases such as tuberculosis (TB), leprosy, Buruli ulcer, and pulmonary nontuberculous mycobacterial disease, to name the most important ones. In 2015, globally, almost 10 million people developed TB, and almost half a million patients suffered from its multidrug-resistant form. In 2016, a total of 9,287 new TB cases were reported in the United States. In 2015, there were 174,608 new case of leprosy worldwide. India, Brazil, and Indonesia reported the most leprosy cases. In 2015, the World Health Organization reported 2,037 new cases of Buruli ulcer, with most cases being reported in Africa. Pulmonary nontuberculous mycobacterial disease is an emerging public health challenge. The U.S. National Institutes of Health reported an increase from 20 to 47 cases/100,000 persons (or 8.2% per year) of pulmonary nontuberculous mycobacterial disease among adults aged 65 years or older throughout the United States, with 181,037 national annual cases estimated in 2014. This review describes contemporary methods for the laboratory diagnosis of mycobacterial diseases. Furthermore, the review considers the ever-changing health care delivery system and stresses the laboratory’s need to adjust and embrace molecular technologies to provide shorter turnaround times and a higher quality of care for the patients who we serve

Committee II: Guidelines for cytologic sampling techniques of lung and mediastinal lymph nodes

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146505/1/dc23975.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146505/2/dc23975_am.pd

Optimizing molecular detection and sequencing of Porcine Reproductive and Respiratory Syndrome virus in clinical oral fluid specimens

This thesis begins with discussion of molecular diagnostics used in diagnosis of the Porcine Reproductive and Respiratory (PRRS) virus with specific emphasis on swine oral fluids are discussed through a review of literature. Risks contributing to missed detection are discussed including: virus mutation, viral degradation in clinical samples, and polymerase chain reaction (PCR) inhibition. Chapter two reports optimization of RNA extraction and PCR protocols for swine oral fluids. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids. Chapter three presents efforts to improve success rate of PRRS sequencing from PCR-positive oral fluids. Sequencing open reading frame 5 (ORF5) of the PRRS virus is a commonly used molecular diagnostic test for virus characterization and epidemiologic investigations in the United States. Attempts in recent years to obtain PRRS virus sequencing from swine oral fluids has been less successful than serum, limiting the cost effectiveness of using this specimen type in disease eradication efforts. In this study, viral RNA was extracted from swine oral fluid diagnostic submissions and tested with three different modifications of the PCR reaction; regular or standard method, diluted extract prior to PCR, and tough or inhibition-resistant PCR enzyme mix. Success rate of generating PCR product was evaluated along with concentration of amplified product. The diluted method yielded the highest cDNA concentration of the target band, resulting in a significantly higher success rate of amplifying the target than the `regular\u27 method, and had the highest proportion of samples successfully sequence

PLoS One

Identification of etiology remains a significant challenge in the diagnosis of infectious diseases, particularly in resource-poor settings. Viral, bacterial, and fungal pathogens, as well as parasites, play a role for many syndromes, and optimizing a single diagnostic system to detect a range of pathogens is challenging. The TaqMan Array Card (TAC) is a multiple-pathogen detection method that has previously been identified as a valuable technique for determining etiology of infections and holds promise for expanded use in clinical microbiology laboratories and surveillance studies. We selected TAC for use in the Aetiology of Neonatal Infection in South Asia (ANISA) study for identifying etiologies of severe disease in neonates in Bangladesh, India, and Pakistan. Here we report optimization of TAC to improve pathogen detection and overcome technical challenges associated with use of this technology in a large-scale surveillance study. Specifically, we increased the number of assay replicates, implemented a more robust RT-qPCR enzyme formulation, and adopted a more efficient method for extraction of total nucleic acid from blood specimens. We also report the development and analytical validation of ten new assays for use in the ANISA study. Based on these data, we revised the study-specific TACs for detection of 22 pathogens in NP/OP swabs and 12 pathogens in blood specimens as well as two control reactions (internal positive control and human nucleic acid control) for each specimen type. The cumulative improvements realized through these optimization studies will benefit ANISA and perhaps other studies utilizing multiple-pathogen detection approaches. These lessons may also contribute to the expansion of TAC technology to the clinical setting