Melanie L. Yarbrough

Local Generation of Kynurenines Mediates Inhibition of Neutrophil Chemotaxis by Uropathogenic Escherichia coli

ABSTRACT During epithelial infections, pathogenic bacteria employ an array of strategies to attenuate and evade host immune responses, including the influx of polymorphonuclear leukocytes (PMN; neutrophils). Among the most common bacterial infections in humans are those of the urinary tract, caused chiefly by uropathogenic Escherichia coli (UPEC). During the establishment of bacterial cystitis, UPEC suppresses innate responses via multiple independent strategies. We recently described UPEC attenuation of PMN trafficking to the urinary bladder through pathogen-specific local induction of indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolic enzyme previously shown to have regulatory activity only in adaptive immunity. Here, we investigated the mechanism by which IDO induction attenuates PMN migration. Local tryptophan limitation, by which IDO is known to influence T cell longevity and proliferation, was not involved in its effect on PMN trafficking. Instead, metabolites in the IDO pathway, particularly l-kynurenine, directly suppressed PMN transepithelial migration and induced an attached, spread morphology in PMN both at rest and in the presence of chemotactic stimuli. Finally, kynurenines represent known ligands of the mammalian aryl hydrocarbon receptor (AHR), and UPEC infection of Ahr −/− mice recapitulated the derepressed PMN recruitment observed previously in Ido1 −/− mice. UPEC therefore suppresses neutrophil migration early in bacterial cystitis by eliciting an IDO-mediated increase in local production of kynurenines, which act through the AHR to impair neutrophil chemotaxis.

Analytical performance evaluation of the i-STAT Total β-human chorionic gonadotropin immunoassay

BACKGROUND The ability to perform quantitative hCG testing in whole blood at the point-of-care is desirable. The purpose of this study was to perform an analytical validation of the Abbott i-STAT Total β-hCG test. METHODS Whole blood, plasma, and serum samples were prepared by the addition of hCG and were used to evaluate precision, linearity, analytical sensitivity, accuracy, the high-dose hook effect, and dilution recovery. RESULTS Imprecision was highest with whole blood (CV = 16.0% and 6.7% at 10 and 1184 IU/l, respectively) and lowest in serum (CV = 8.1% and 4.3% at 11 and 1305 IU/l, respectively). The limits-of-quantitation were 8 and <5 IU/l for whole blood and both plasma and serum, respectively. The assay was linear between 5 and 2000 IU/l in all sample types (R(2) ≥ 0.998). i-STAT results agreed most closely with the Architect Total β-hCG assay and with greater differences observed with Beckman DxI Total βhCG and Roche Cobas e601 hCG+β assays (mean differences across all sample types were 9.3% and 12.3%, respectively). A high-dose hook effect was observed at concentrations > 400,000 IU/l. Accuracy was achieved in samples diluted with serum but not saline. CONCLUSIONS The i-STAT Total β-hCG test demonstrates acceptable performance for quantifying hCG in whole blood, plasma and serum.

Evaluation of Genotypic and Phenotypic Methods to Detect Carbapenemase Production in Gram-Negative Bacilli

BACKGROUND Carbapenemase-producing gram-negative bacteria (CP-GNB) are an urgent and expanding public health threat. Rapid and accurate identification of these organisms facilitates infection prevention efforts in healthcare facilities. The objective of our study was to evaluate methods to detect and identify CP-GNB. METHODS We examined 189 carbapenem-resistant GNB(CR-GNB), including Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex, using 3 different methods: 2 methods to screen isolates of GNB for carbapenemase production [the carbapenem inactivation method (CIM) and 2 chromogenic agars] and a molecular method (Cepheid GeneXpert Carba-R) to identify the mechanism of carbapenem resistance and the associated resistance genes (blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM). RESULTS The CIM was a simple and inexpensive phenotypic screen to differentiate between CR-GNB and CP-GNB, with improved analytical performance characteristics and inter-reader correlation compared to the modified Hodge test. Both chromogenic agars evaluated (HardyCHROM CRE and chromID CARBA) were able to support growth of most of the organisms tested, including isolates possessing the blaOXA-48-like gene. However, these media had a low analytical specificity for carbapenemase production, with breakthrough of CR-GNB that did not produce a carbapenemase. The Xpert Carba-R assay was rapid and easy to perform, and demonstrated 100% positive and negative agreement for characterization of genetic determinants of carbapenem resistance. CONCLUSIONS Screening by CIM followed by the Xpert Carba-R PCR is an accurate method for detecting and characterizing CP-GNB, including Enterobacteriaceae, P. aeruginosa, and A. baumannii complex.

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

ABSTRACT 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.

Identification of Nocardia, Streptomyces, and Tsukamurella using MALDI-TOF MS with the Bruker Biotyper

Nocardia species are the most commonly isolated aerobic actinomycetes from human clinical specimens. Our objective was to assess the identification of clinically relevant actinomycetes using the Bruker Biotyper MALDI-TOF system, including comparison of extraction methods, Biotyper library versions, score cutoffs, and media. Banked Streptomyces (n=10), Tsukamurella (n=2), and Nocardia isolates (n=60) were cultured and extracted using three methods: mycobacterial extraction, ethanol formic acid extraction, or direct on-target extraction. Following MALDI-TOF analysis, spectra were analyzed using versions 5 and 6 of the BDAL Biotyper library. Optimal species-level identifications for Nocardia were achieved using BDAL v6 at a score cutoff of ≥1.8 after direct extraction (49/60, 82%). Overall, the Biotyper platform with BDAL v6 accurately identified 12/16 species of Nocardia, demonstrating the utility of MALDI-TOF for identification of clinically relevant actinomycetes without the need for supplementation of the database.

Estimating the hCGβcf in urine during pregnancy.

OBJECTIVE Elevated urine concentrations of hCG beta core fragment (hCGβcf) are known to cause false negative qualitative point-of-care hCG test results, but limited information is available regarding urine hCGβcf. In this study, we evaluate the relationship between serum and urine hCG concentrations and the frequency of elevated urine hCGβcf concentrations. DESIGN AND METHODS Paired serum and urine specimens were obtained from 60 women at various stages of pregnancy and hCG was measured using the Abbott Architect and Roche Cobas e602 assays. Urine specimens with the greatest difference in urine hCG concentrations between these two instruments were tested using a qualitative point-of-care device and hCGβcf was quantified using LC-MS/MS. RESULTS Urine hCG concentrations were lower than serum and the magnitude of the difference depended on whether the hCG assay detected hCGβcf. Elevated hCGβcf concentrations (>280,000pmol/L) were observed in 12% of specimens from an unselected patient population. There was a significant correlation (r=0.97; p<0.0001) between the difference (Roche hCG-Abbott hCG) and the hCGβcf concentration as measured by LC-MS/MS (Roche-Abbott difference IU/L=(hCGβcf (pmol/L)∗0.131+656)). CONCLUSIONS A correlation exists between serum and urine hCG concentrations but this correlation is variable. hCGβcf concentrations can be estimated using two automated assay reagent platforms that differ in their recognition of hCGβcf.

The Brief Case: A Reactive HIV Rapid Antibody Test in a Pregnant Woman

A 32-year-old pregnant woman presented to her obstetrician for routine prenatal care during her 3rd month of pregnancy. She reported no major health concerns, with the exception of mild morning sickness that had been gradually improving. Upon physical examination, she appeared healthy and her vitals

The ABCs of STIs: An Update on Sexually Transmitted Infections

BACKGROUND Sexually transmitted infections (STIs) are spread primarily through sexual contact and are a major cause of morbidity and mortality worldwide. Once identified, some STIs can be cured following appropriate therapy; for others, suppressive regimens and approaches to prevent ongoing transmission are important. The incidence of many common STIs is increasing in the US as well as worldwide, and hundreds of millions of people are currently infected. Laboratory testing plays a major role in the diagnosis and treatment of STIs, and clinical laboratorians should be familiar with the current guidelines and methods for testing. CONTENT Accurate and sensitive methods to diagnose STIs are essential to direct appropriate antimicrobial therapy and interrupt the cycle of disease transmission. This review summarizes laboratory testing for common bacterial, viral, and parasitic causes of STIs. Disease manifestations reviewed include cervicitis and urethritis, genital ulcerative disease, human immunodeficiency virus, viral hepatitis, human papilloma virus, and vaginitis. Recent advancements in the recognition and management of STIs, including updates to diagnostic algorithms, advances in testing methods, and emerging challenges with antimicrobial resistance, are summarized. SUMMARY Diagnostic methods and therapeutic guidelines for STIs are rapidly evolving. In combination with changing epidemiology, the development of novel therapeutics, and advancements in diagnostic methods, this has resulted in changing practices in laboratory testing and, subsequently, management of disease. Molecular methods have facilitated personalized therapy and follow-up regimens targeted for individual types or strains of some STIs.

Characterizing urinary hCGβcf patterns during pregnancy

OBJECTIVE Elevated concentrations of hCG beta core fragment (hCGβcf) are known to cause false-negative results in qualitative urine pregnancy test devices, but the pattern of urinary hCGβcf during normal pregnancy has not been well characterized. Here, we evaluate the relationship between urine hCG, hCGβcf, and hCG free β subunit (hCGβ) during pregnancy. DESIGN AND METHODS Banked second trimester urine specimens from 100 pregnant women were screened for high concentrations of hCGβcf using a qualitative point-of-care device known to demonstrate false-negative results in the presence of elevated hCGβcf concentrations. Additional first and third trimester specimens from the same pregnancy were obtained from 10 women who generated negative/faint positive results, 5 women who generated intermediate positive results, and 10 women who generated strong positive results on the point-of-care device. Intact hCG, hCGβcf, hCGβ, and specific gravity were quantified in these 75 specimens. RESULTS Urinary hCGβcf concentrations were greater than intact hCG concentrations at all times. A strong correlation (r(2)=0.70) was observed between urine intact hCG and hCGβcf concentrations. A poor correlation was observed between specific gravity and intact hCG (r(2)=0.32), hCGβ (r(2)=0.32), and hCGβcf (r(2)=0.32). The highest hCGβcf concentrations were observed between 10 and 16weeks gestation but individual women demonstrated very different patterns of hCGβcf excretion. CONCLUSIONS Urine specimens with elevated hCGβcf are frequently encountered during pregnancy but hCGβcf excretion patterns are unpredictable. Manufacturers and clinicians must appreciate that hCGβcf is the major immunoreactive component in urine during pregnancy and must design and interpret qualitative urine hCG test results accordingly.

Fetal lung maturity testing: the end of an era

Respiratory distress syndrome is a major cause of neonatal morbidity and mortality that is most commonly caused by a deficiency in lung surfactant in premature infants. Therefore, laboratory tests were developed to measure the presence and/or concentration of lung surfactant in amniotic fluid in order to estimate maturity of the fetal lung. Although these tests were once widely employed, their utilization by physicians has decreased in recent years. Several studies have shown that demonstration of a mature fetal lung index by antenatal testing does not improve neonatal outcomes. Instead, decreased respiratory and nonrespiratory morbidities are most highly correlated with gestational age of the fetus. Therefore, fetal lung maturity testing may have passed the point of being clinically useful.