Architect i2000 SR has improved turnaround time for infectious disease serology testing over Vitros ECiQ.

BACKGROUND: Vitros ECiQ and Architect i2000 SR are two automated instruments used to detect serology biomarkers of hepatitis A, B and C viruses, and HIV infections. We compared performance of the Architect to the Vitro EciQ after implementation at our institution. METHODS: A retrospective review was performed to compare patient samples tested on the Vitros ECiQ or Architect for hepatitis and HIV serological assays. The positivity rate, frequency of equivocal results, turnaround times (TAT), and hands-on time (HOT) were analyzed. RESULTS: There was no statistical difference in the positivity rate between the two instruments, with the exception of two assays. An increase in equivocal results was observed for the Architect (0.2% vs 0.5%). Notably, the TAT for the Architect i2000 was shorter for all except one assay (31.6 vs 33.7 hours) and demonstrated improved workflow. CONCLUSIONS: Overall, both instruments performed comparably. Architect had shorter TAT over Vitros.

The Virulence of Escherichia coli O157:H7 Isolates in Mice Depends on Shiga Toxin Type 2a (Stx2a)-Induction and High Levels of Stx2a in Stool.

In this study we compared nine Shiga toxin (Stx)-producing Escherichia coli O157:H7 patient isolates for Stx levels, stx-phage insertion site(s), and pathogenicity in a streptomycin (Str)-treated mouse model. The strains encoded stx2a, stx1a and stx2a, or stx2a and stx2c. All of the strains elaborated 105-106 cytotoxic doses 50% (CD50) into the supernatant after growth in vitro as measured on Vero cells, and showed variable levels of increased toxin production after growth with sub-inhibitory levels of ciprofloxacin (Cip). The stx2a+stx2c+ isolates were 90-100% lethal for Str-treated BALB/c mice, though one isolate, JH2013, had a delayed time-to-death. The stx2a+ isolate was avirulent. Both an stx2a and a recA deletion mutant of one of the stx2a+stx2c+ strains, JH2010, exhibited at least a three-log decrease in cytotoxicity in vitro and both were avirulent in the mice. Stool from Str-treated mice infected with the highly virulent isolates were 10- to 100-fold more cytotoxic than feces from mice infected with the clinical isolate, JH2012, that made only Stx2a. Taken together these findings demonstrate that the stx2a-phage from JH2010 induces to higher levels in vivo than does the phage from JH2012. The stx1a+stx2a+ clinical isolates were avirulent and neutralization of Stx1 in stool from mice infected with those strains indicated that the toxin produced in vivo was primarily Stx1a. Treatment of mice infected with Stx1a+Stx2a+ isolates with Cip resulted in an increase in Stx2a production in vivo and lethality in the mice. Our data suggest that high levels of Stx2a in stool are predictive of virulence in mice.

False-Positive Results for Human Immunodeficiency Virus Type 1 Nucleic Acid Amplification Testing in Chimeric Antigen Receptor T Cell Therapy.

Chimeric antigen receptor (CAR) T cell immunotherapy has been a major advancement in cancer therapeutics. Reprogramming of T cells is achieved by using gammaretroviral or lentiviral vectors, which may interfere with human immunodeficiency virus type 1 (HIV-1) nucleic acid amplification testing (NAAT). Here, we describe three clinical scenarios in which CAR T cell immunotherapy interfered with HIV-1 testing, including (i) routine infectious disease screening prior to stem cell transplantation in a 16-year-old female with B cell acute lymphoblastic leukemia, post CAR T cell treatment; (ii) routine infectious disease screening prior to second CAR T cell collection in a 65-year-old male with diffuse large B cell lymphoma who failed initial CAR T cell treatment; and (iii) routine infectious risk assessment following an occupational health exposure from a 58-year-old male with multiple myeloma, who received CAR T cell treatment. In each case, patients initially tested negative by the "fourth-generation" HIV-1 screening enzyme immunoassay (targeting the p24 antigen and anti-HIV-1 antibodies), but positive by the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test v2.0 (targeting gag and the long terminal repeat [LTR]). These samples subsequently retested negative using the Abbott m2000 RealTime HIV-1 assay, which targets the integrase gene. These results indicated that cross-reactions between lentiviral vectors and LTR genomes targeted in the HIV-1 NAAT caused the HIV-1 NAAT false-positive results.

Streptococcus pneumoniae phosphotyrosine phosphatase CpsB and alterations in capsule production resulting from changes in oxygen availability.

Streptococcus pneumoniae produces a protective capsular polysaccharide whose production must be modulated for bacterial survival within various host niches. Capsule production is affected in part by a phosphoregulatory system comprised of CpsB, CpsC, and CpsD. Here, we found that growth of serotype 2 strain D39 under conditions of increased oxygen availability resulted in decreased capsule levels concurrent with an ∼5-fold increase in Cps2B-mediated phosphatase activity. The change in Cps2B phosphatase activity did not result from alterations in the levels of either the cps2B transcript or the Cps2B protein. Recombinant Cps2B expressed in Escherichia coli similarly exhibited increased phosphatase activity under conditions of high-oxygen growth. S. pneumoniae D39 derivatives with defined deletion or point mutations in cps2B demonstrated reduced phosphatase activity with corresponding increases in levels of Cps2D tyrosine phosphorylation. There was, however, no correlation between these phenotypes and the level of capsule production. During growth under reduced-oxygen conditions, the Cps2B protein was essential for parental levels of capsule, but phosphatase activity alone could be eliminated without an effect on capsule. Under increased-oxygen conditions, deletion of cps2B did not affect capsule levels. These results indicate that neither Cps2B phosphatase activity nor Cps2D phosphorylation levels per se are determinants of capsule levels, whereas the Cps2B protein is important for capsule production during growth under conditions of reduced but not enhanced oxygen availability. Roles for factors outside the capsule locus, possible interactions between capsule regulatory proteins, and links to other cellular processes are also suggested by the results described in this study.

Biochemical activities of Streptococcus pneumoniae serotype 2 capsular glycosyltransferases and significance of suppressor mutations affecting the initiating glycosyltransferase Cps2E.

The capsular polysaccharide (CPS) is essential for Streptococcus pneumoniae virulence. Its synthesis requires multiple enzymes, and defects that block completion of the pathway can be lethal in the absence of secondary suppressor mutations. In this study, we examined the functions of three capsular glycosyltransferases (Cps2F, Cps2G, and Cps2I) involved in serotype 2 CPS synthesis, whose deletions select for secondary mutations. We demonstrate that Cps2F is a rhamnosyltransferase that catalyzes addition of the third and fourth sugars in the capsule repeat unit, while Cps2G adds the fifth sugar (glucose). Addition of the terminal residue (glucuronic acid) could not be detected; however, activities of the other glycosyltransferases together with bioinformatic analyses suggest that this step is mediated by Cps2I. Most of the secondary suppressor mutations resulting from loss of these enzymes occur in cps2E, the gene encoding the initiating glycosyltransferase. Examination of the 69 S. pneumoniae serotypes containing Cps2E homologues yielded a consensus amino acid sequence for this protein and demonstrated that there is a highly significant association between the residues that are 100% conserved and those altered by suppressor mutations. Cps2E contains an extracytoplasmic loop whose function is unknown. Among our collection of mutants, six contained missense mutations affecting amino acids in the extracytoplasmic loop. These residues are highly conserved among S. pneumoniae Cps2E homologues, and mutations therein severely reduced CPS synthesis and Cps2E activity. The critical functions of these amino acids suggest a role for the Cps2E extracytoplasmic loop in initiation, and possibly regulation, of capsule synthesis.