Regulatory Approach to Point-of-Care/At-Home Testing in the United States.

The Clinical Laboratory Improvement Amendments (CLIA) classifications were activated in the 1990s in partnership with the Centers for Medicare and Medicaid Services and Food and Drug Administration and included waived, moderate, and high complexity testing. The waived section of CLIA certificates allows laboratories to perform testing of analytes and methods of samples by the Food and Drug Administration. During the COVID-19 pandemic, many molecular or antigen laboratory testing methods for COVID-19 virus were quickly approved by emergency use authorization. Waived testing is now done in highly complex, moderately complex, and waived testing laboratories, and some at-home testing.

A Literature Review on How We Can Address Medical Laboratory Scientist Staffing Shortages.

OBJECTIVE: Laboratories are facing a critical shortage of medical laboratory scientists (MLS) and medical laboratory technicians (MLT) to address an increasing demand for laboratory testing. Training program closures, fewer student applicants, and financial decisions have contributed to staffing shortages. Lack of visibility, low wages, and perceived lack of opportunities for upward career mobility contribute to challenges in recruiting and retaining qualified individuals and students who are unaware of laboratory medicine careers. Our goal was to review the literature to determine the current state and consequences of staffing shortages, and potential solutions to address these shortages. METHODS: Medline/PubMed, PubMed Central, MeSH, Google Scholar, and Marshall Digital Scholar were used as resources. DISCUSSION/CONCLUSIONS: A collaboration of stakeholders is needed to identify staffing challenges, barriers, and solutions and to increase visibility of laboratory professionals. Early recruitment is best started in the middle and high school educational process.

Reducing Blood Culture Contamination Rates: Experiences of Four Hospital Systems.

INTRODUCTION: Blood cultures (BCs) frequently become contaminated during the pre-analytic phase of collection leading to downstream ramifications. We present a summary of performance improvement (PI) interventions provided by four hospital systems and common factors that contributed to decreased blood culture contamination (BCC) rates. METHODS: Each hospital independently formed a multidisciplinary team and action plan for implementation of their intervention, focusing on the use of educational and training tools. Their goal was to significantly decrease their BCC rates. Pre- and post-intervention data were compared during the sustainment period to determine their success. RESULTS: All hospitals met their goals of post-intervention BCC rates and with most achieving and sustaining BCC rates ≤ 1.0-2.0%. CONCLUSION: Our report highlights how four hospitals independently achieved their objective to decrease their BCC rate with the support of a multidisciplinary team. We propose a benchmark for BCC rates of 1.5 to < 2.0% as achievable and sustainable.

A Laboratory Medicine Best Practices Systematic Review and Meta-analysis of Nucleic Acid Amplification Tests (NAATs) and Algorithms Including NAATs for the Diagnosis of Clostridioides (Clostridium) difficile in Adults.

The evidence base for the optimal laboratory diagnosis of Clostridioides (Clostridium) difficile in adults is currently unresolved due to the uncertain performance characteristics and various combinations of tests. This systematic review evaluates the diagnostic accuracy of laboratory testing algorithms that include nucleic acid amplification tests (NAATs) to detect the presence of C. difficile The systematic review and meta-analysis included eligible studies (those that had PICO [population, intervention, comparison, outcome] elements) that assessed the diagnostic accuracy of NAAT alone or following glutamate dehydrogenase (GDH) enzyme immunoassays (EIAs) or GDH EIAs plus C. difficile toxin EIAs (toxin). The diagnostic yield of NAAT for repeat testing after an initial negative result was also assessed. Two hundred thirty-eight studies met inclusion criteria. Seventy-two of these studies had sufficient data for meta-analysis. The strength of evidence ranged from high to insufficient. The uses of NAAT only, GDH-positive EIA followed by NAAT, and GDH-positive/toxin-negative EIA followed by NAAT are all recommended as American Society for Microbiology (ASM) best practices for the detection of the C. difficile toxin gene or organism. Meta-analysis of published evidence supports the use of testing algorithms that use NAAT alone or in combination with GDH or GDH plus toxin EIA to detect the presence of C. difficile in adults. There is insufficient evidence to recommend against repeat testing of the sample using NAAT after an initial negative result due to a lack of evidence of harm (i.e., financial, length of stay, or delay of treatment) as specified by the Laboratory Medicine Best Practices (LMBP) systematic review method in making such an assessment. Findings from this systematic review provide clarity to diagnostic testing strategies and highlight gaps, such as low numbers of GDH/toxin/PCR studies, in existing evidence on diagnostic performance, which can be used to guide future clinical research studies.

Whole Genome Sequencing detects Inter-Facility Transmission of Carbapenem-resistant Klebsiella pneumoniae.

OBJECTIVES: To identify transmission patterns of Carbapenem-resistant Klebsiella pneumoniae infection during an outbreak at a large, tertiary care hospital and to detect whether the outbreak organisms spread to other facilities in the integrated healthcare network. METHODS: We analyzed 71 K. pneumoniae whole genome sequences collected from clinical specimens before, during and after the outbreak and reviewed corresponding patient medical records. Sequence and patient data were used to model probable transmissions and assess factors associated with the outbreak. RESULTS: We identified close genetic relationships among carbapenem-resistant K. pneumoniae isolates sampled during the study period. Transmission tree analysis combined with patient records uncovered extended periods of silent colonization in many study patients and transmission routes that were likely the result of asymptomatic patients transitioning between facilities. CONCLUSIONS: Detecting how and where Carbapenem-resistant K. pneumoniae infections spread is challenging in an environment of rising prevalence, asymptomatic carriage and mobility of patients. Whole genome sequencing improved the precision of investigating inter-facility transmissions. Our results emphasize that containment of Carbapenem-resistant K. pneumoniae infections requires coordinated efforts between healthcare networks and settings of care that acknowledge and mitigate transmission risk conferred by undetected carriage and by patient transfers between facilities.

Pseudomonas aeruginosa Regulated Intramembrane Proteolysis: Protease MucP Can Overcome Mutations in the AlgO Periplasmic Protease To Restore Alginate Production in Nonmucoid Revertants.

The progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogen Pseudomonas aeruginosa from a nonmucoid form to a mucoid form in the lung. This conversion involves the constitutive synthesis of the exopolysaccharide alginate, whose production is under the control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in the algT (algU) gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the mucA22 allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes, algO, also known as prc or PA3257, and mucP Interestingly, our algO mutations were complemented by both mucP and algO, leading us to delete, clone, and overexpress mucP, algO, mucE, and mucD in both wild-type PAO1 and PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest that the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild-type strain but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants of Pseudomonas aeruginosaIMPORTANCE Infection by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality seen in CF patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated posttranslationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF.

Rapid Identification of Five Classes of Carbapenem Resistance Genes Directly from Rectal Swabs by Use of the Xpert Carba-R Assay.

Carbapenemase-producing organisms (CPO) have been identified by global health leaders as an urgent threat. Detection of patients with gastrointestinal carriage of CPO is necessary to interrupt their spread within health care facilities. In this multisite study, we assessed the performance of the Xpert Carba-R test, a rapid real-time quantitative PCR (qPCR) assay that detects five families of carbapenemase genes (blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM) directly from rectal swab specimens. Using dual swabs, specimens from 755 patients were collected and tested prospectively. An additional 432 contrived specimens were prepared by seeding well-characterized carbapenem-susceptible and -nonsusceptible strains into a rectal swab matrix and inoculating them onto swabs prior to testing. Antimicrobial susceptibility testing, broth enriched culture, and DNA sequencing were performed by a central laboratory blind to the Xpert Carba-R results. The Xpert Carba-R assay demonstrated a positive percentage of agreement (PPA) between 60 and 100% for four targets (blaKPC, blaNDM, blaVIM, and blaOXA-48) and a negative percentage of agreement (NPA) ranging between 98.9 and 99.9% relative to the reference method (culture and sequencing of any carbapenem-nonsusceptible isolate). There were no prospective blaIMP-positive samples. Contrived specimens demonstrated a PPA between 95 and 100% and an NPA of 100% for all targets. Testing of rectal swabs directly using the Xpert Carba-R assay is effective for rapid detection and identification of CPO from hospitalized patients.

Effectiveness of Preanalytic Practices on Contamination and Diagnostic Accuracy of Urine Cultures: a Laboratory Medicine Best Practices Systematic Review and Meta-analysis.

BACKGROUND: Urinary tract infection (UTI) in the United States is the most common bacterial infection, and urine cultures often make up the largest portion of workload for a hospital-based microbiology laboratory. Appropriately managing the factors affecting the preanalytic phase of urine culture contributes significantly to the generation of meaningful culture results that ultimately affect patient diagnosis and management. Urine culture contamination can be reduced with proper techniques for urine collection, preservation, storage, and transport, the major factors affecting the preanalytic phase of urine culture. OBJECTIVES: The purposes of this review were to identify and evaluate preanalytic practices associated with urine specimens and to assess their impact on the accuracy of urine culture microbiology. Specific practices included collection methods for men, women, and children; preservation of urine samples in boric acid solutions; and the effect of refrigeration on stored urine. Practice efficacy and effectiveness were measured by two parameters: reduction of urine culture contamination and increased accuracy of patient diagnosis. The CDC Laboratory Medicine Best Practices (LMBP) initiative's systematic review method for assessment of quality improvement (QI) practices was employed. Results were then translated into evidence-based practice guidelines. SEARCH STRATEGY: A search of three electronic bibliographic databases (PubMed, SCOPUS, and CINAHL), as well as hand searching of bibliographies from relevant information sources, for English-language articles published between 1965 and 2014 was conducted. SELECTION CRITERIA: The search contained the following medical subject headings and key text words: urinary tract infections, UTI, urine/analysis, urine/microbiology, urinalysis, specimen handling, preservation, biological, preservation, boric acid, boric acid/borate, refrigeration, storage, time factors, transportation, transport time, time delay, time factor, timing, urine specimen collection, catheters, indwelling, urinary reservoirs, continent, urinary catheterization, intermittent urethral catheterization, clean voided, midstream, Foley, suprapubic, bacteriological techniques, and microbiological techniques. MAIN RESULTS: Both boric acid and refrigeration adequately preserved urine specimens prior to their processing for up to 24 h. Urine held at room temperature for more than 4 h showed overgrowth of both clinically significant and contaminating microorganisms. The overall strength of this body of evidence, however, was rated as low. For urine specimens collected from women, there was no difference in rates of contamination for midstream urine specimens collected with or without cleansing. The overall strength of this evidence was rated as high. The levels of diagnostic accuracy of midstream urine collection with or without cleansing were similar, although the overall strength of this evidence was rated as low. For urine specimens collected from men, there was a reduction in contamination in favor of midstream clean-catch over first-void specimen collection. The strength of this evidence was rated as high. Only one study compared midstream collection with cleansing to midstream collection without cleansing. Results showed no difference in contamination between the two methods of collection. However, imprecision was due largely to the small event size. The diagnostic accuracy of midstream urine collection from men compared to straight catheterization or suprapubic aspiration was high. However, the overall strength of this body of evidence was rated as low. For urine specimens collected from children and infants, the evidence comparing contamination rates for midstream urine collection with cleansing, midstream collection without cleansing, sterile urine bag collection, and diaper collection pointed to larger reductions in the odds of contamination in favor of midstream collection with cleansing over the other methods of collection. This body of evidence was rated as high. The accuracy of diagnosis of urinary tract infection from midstream clean-catch urine specimens, sterile urine bag specimens, or diaper specimens compared to straight catheterization or suprapubic aspiration was varied. AUTHORS' CONCLUSIONS: No recommendation for or against is made for delayed processing of urine stored at room temperature, refrigerated, or preserved in boric acid. This does not preclude the use of refrigeration or chemical preservatives in clinical practice. It does indicate, however, that more systematic studies evaluating the utility of these measures are needed. If noninvasive collection is being considered for women, midstream collection with cleansing is recommended, but no recommendation for or against is made for midstream collection without cleansing. If noninvasive collection is being considered for men, midstream collection with cleansing is recommended and collection of first-void urine is not recommended. No recommendation for or against is made for collection of midstream urine without cleansing. If noninvasive collection is being considered for children, midstream collection with cleansing is recommended and collection in sterile urine bags, from diapers, or midstream without cleansing is not recommended. Whether midstream collection with cleansing can be routinely used in place of catheterization or suprapubic aspiration is unclear. The data suggest that midstream collection with cleansing is accurate for the diagnosis of urinary tract infections in infants and children and has higher average accuracy than sterile urine bag collection (data for diaper collection were lacking); however, the overall strength of evidence was low, as multivariate modeling could not be performed, and thus no recommendation for or against can be made.

Consolidated clinical microbiology laboratories.

The manner in which medical care is reimbursed in the United States has resulted in significant consolidation in the U.S. health care system. One of the consequences of this has been the development of centralized clinical microbiology laboratories that provide services to patients receiving care in multiple off-site, often remote, locations. Microbiology specimens are unique among clinical specimens in that optimal analysis may require the maintenance of viable organisms. Centralized laboratories may be located hours from patient care settings, and transport conditions need to be such that organism viability can be maintained under a variety of transport conditions. Further, since the provision of rapid results has been shown to enhance patient care, effective and timely means for generating and then reporting the results of clinical microbiology analyses must be in place. In addition, today, increasing numbers of patients are found to have infection caused by pathogens that were either very uncommon in the past or even completely unrecognized. As a result, infectious disease specialists, in particular, are more dependent than ever on access to high-quality diagnostic information from clinical microbiology laboratories. In this point-counterpoint discussion, Robert Sautter, who directs a Charlotte, NC, clinical microbiology laboratory that provides services for a 40-hospital system spread over 3 states in the southeastern United States explains how an integrated clinical microbiology laboratory service has been established in a multihospital system. Richard (Tom) Thomson of the NorthShore University HealthSystem in Evanston, IL, discusses some of the problems and pitfalls associated with large-scale laboratory consolidation.

Development of PCR assays for detection of Trichomonas vaginalis in urine specimens.

Trichomonas vaginalis infections are usually asymptomatic or can result in nonspecific clinical symptoms, which makes laboratory-based detection of this protozoan parasite essential for diagnosis and treatment. We report the development of a battery of highly sensitive and specific PCR assays for detection of T. vaginalis in urine, a noninvasive specimen, and development of a protocol for differentiating among Trichomonas species that commonly infect humans.

A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and algT/U activity results in the loss of alginate production.

Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg(+)) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg(+) due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.

Anogenital human papillomavirus in sexually abused and nonabused children: a multicenter study.

OBJECTIVES: To characterize the epidemiology of genital human papillomavirus (HPV) infection in children without previous consensual sexual activity, comparing HPV prevalence by certainty of child sexual abuse (CSA). PATIENTS AND METHODS: Patients presenting for evaluation of CSA in 8 sites in Atlanta, Houston, Harrisburg, and New York City were recruited along with patients presenting for unrelated health visits. CSA certainty was classified as definite, probable, possible, or no evidence following published guidelines and the results of history, physical examination, and laboratory tests. Urine and swabs of external genitalia were tested for HPV using L1 consensus polymerase chain reaction. RESULTS: The study included 576 participants (89.9% female) aged 6 months to 13 years (mean: 7.9); 534 of whom were evaluated for CSA and 42 for unrelated reasons. Of those evaluated for CSA, 14 had genital warts. One or more HPV types were detected in 11.8% (61 of 517) of participants with adequate samples. HPV detection was more likely among abused participants (definite, probable, or possible) than among participants without evidence of CSA (13.7% and 1.3%, respectively; P < .0001) and increased with certainty of abuse (8.4%, 15.6%, and 14.5% in participants with possible, probable, and definite CSA, respectively; P < .0001). Participants aged 10 years or older had a higher prevalence of HPV (20.6%) than others (5.6%) (P < .0001). CSA, anogenital warts, and age were independently associated with HPV detection. CONCLUSIONS: HPV detection was associated with CSA and increased with CSA certainty. In this population, genital HPV seemed to behave as a sexually transmitted infection.

A two-site analytical evaluation of the BD Viper System with XTR Technology in Nonextracted Mode and Extracted Mode with seeded simulated specimens.

The BD ProbeTec CT Q(x) Amplified DNA Assay and the BD ProbeTec GC Q(x) Amplified DNA Assay (both BD Diagnostics, Sparks, MD) represent two new assays developed for use with the BD Viper System with XTR Technology (BD Diagnostics). These assays were built on the foundation of the former BD ProbeTec ET assays (BD Diagnostics) and its accompanying instrumentation. The study described below compared the new assay format, Extracted Mode, to the former assay format, Nonextracted Mode, with the primary objective of examining and measuring overall time expenditures and efficiency of operation. An 80-142-min reduction in "hands-on" total processing time was observed for the Extracted Mode whether testing urines or swabs. The second objective was to assess the accuracy of performance at low simulated analytical loads for the pathogens Chlamydia trachomatis and Neisseria gonorrhoeae. Performance accuracies for simulated positive and negative urine or swab specimens were calculated to be 99.97% for Extracted Mode and 99.76% for Nonextracted Mode.

Co-regulation of {beta}-lactam resistance, alginate production and quorum sensing in Pseudomonas aeruginosa.

Development of ß-lactam resistance, production of alginate and modulation of virulence factor expression that alters host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. In this study, we propose that a co-regulatory network exists between these mechanisms. We compared the promoter activities of ampR, algT/U, lasR, lasI, rhlR, rhlI and lasA genes, representing the ß-lactam antibiotic resistance master regulatory gene, the alginate switch operon, the las and rhl quorum-sensing (QS) genes, and the LasA staphylolytic protease, respectively. Four isogenic P. aeruginosa strains, the prototypic Alg(-) PAO1, Alg(-) PAOampR, the mucoid Alg(+) PAOmucA22 (Alg(+) PDO300) and Alg(+) PAOmucA22ampR (Alg(+) PDOampR) were used. We found that in the presence of AmpR regulator and ß-lactam antibiotic, the extracytoplasmic function sigma factor AlgT/U positively regulated P(ampR), whereas AmpR negatively regulated P(algT/U). On the basis of this finding we suggest the presence of a negative feedback loop to limit algT/U expression. In addition, the functional AlgT/U caused a significant decrease in the expression of QS genes, whereas loss of ampR only resulted in increased P(lasI) and P(lasR) transcription. The upregulation of the las QS system is likely to be responsible for the increased lasA promoter and the LasA protease activities in Alg(-) PAOampR and Alg(+) PDOampR. The enhanced expression of virulence factors in the ampR strains correlated with a higher rate of Caenorhabditis elegans paralysis. Hence, this study shows that the loss of ampR results in increased virulence, and is indicative of the existence of a co-regulatory network between ß-lactam resistance, alginate production, QS and virulence factor production, with AmpR playing a central role.