Multicenter Evaluation of the Simplexa VZV Direct Assay for Detection of Varicella-Zoster Virus in Cerebrospinal Fluid and Lesion-Swab Specimens.

Varicella-zoster virus (VZV) is the etiologic agent of varicella (chickenpox) and herpes zoster (shingles) infections commonly involving skin, mucous membranes, and less frequently the central nervous system. Traditional methods for the laboratory diagnosis of these infections are time-consuming, labor-intensive, and often insensitive. As such, these tests are being replaced by more sensitive and rapid molecular methods. This study evaluated the performance of two different molecular assays, the Simplexa VZV Direct and Simplexa VZV Swab Direct, to detect VZV DNA in cerebrospinal fluid (CSF) and lesion-swab specimens, respectively. The Simplexa VZV Direct and Simplexa VZV Swab Direct assays were compared against individual composite reference methods that varied depending on the sample cohort examined. A total of 883 CSF and 452 cutaneous and mucocutaneous prospective, retrospective, and contrived specimens were evaluated in this multicenter study. The results of this study showed that the Simplexa assays demonstrated near perfect agreement (k = 0.98) compared to the composite reference methods for the detection of VZV in CSF and lesion swab specimens. A further comparison between the standard of care molecular assays employed at the site of specimen collection and the Simplexa assays demonstrated excellent agreement (k = 1.0). The Simplexa assays offer rapid and reliable alternatives for the detection of VZV in certain clinical specimens without the need for nucleic acid extraction.

Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica.

Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays.

Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens.

Two case reports of Clostridium difficile bacteremia, one with the epidemic NAP-1 strain.

Clostridium difficile bacteremia is rare. Here, we report two cases of C. difficile bacteremia in patients with significant underlying gastrointestinal pathology. In one case, the bacteremia was caused by the North American pulsed-field gel electrophoresis (PFGE) type 1 (NAP-1) strain, which is responsible for recent outbreaks of C. difficile infections of increased severity.

Whole-genome sequencing of clinical Clostridioides difficile isolates reveals molecular epidemiology and discrepancies with conventional laboratory diagnostic testing.

BACKGROUND: The high clinical burden of Clostridioides difficile infections merits rapid and sensitive identification of affected individuals. However, effective diagnosis remains challenging. Current best practice guidelines recommend molecular and/or direct toxin detection-based screening for symptomatic individuals, but previous work has called into question the concordance and performance of extant clinical assays. AIM: To better correlate the genomic and phenotypic properties of clinical C. difficile isolates with laboratory testing outcomes in both C. difficile-infected patients and asymptomatic carriers. METHODS: Whole-genome sequencing of clinical C. difficile isolates collected from an inpatient population at a single healthcare institution was performed, enabling examination of their molecular epidemiology and toxigenic gene content. Genomic findings were compared with clinical testing outcomes, identifying multiple diagnostic discrepancies. FINDINGS: Toxigenic culture, considered a 'reference standard', provided perfect sensitivity and specificity in predicting toxigenic gene content, whereas reduced performance was observed for Simplexa C. difficile Direct Assay (100% specificity, 88% sensitivity), Gene Xpert CD/Epi Assay (86% specificity, 83% sensitivity), and Quick Check Complete Tox A/B (100% specificity, 30% sensitivity). Genomic analysis additionally revealed variability in toxin gene sequences among C. difficile strains, phylogenomic equivalency between isolates from affected patients and carriers, and patient carriage with uncommon environmentally derived C. difficile lineages, as well as presenting opportunities for tracing pathogen transmission events. CONCLUSION: These results highlight the variable performance of clinical stool-based testing approaches as well as the potential diagnostic utility of whole-genome sequencing as an alternative to conventional testing algorithms.

Defining the landscape of ATP-competitive inhibitor resistance residues in protein kinases.

Kinases are involved in disease development and modulation of their activity can be therapeutically beneficial. Drug-resistant mutant kinases are valuable tools in drug discovery efforts, but the prediction of mutants across the kinome is challenging. Here, we generate deep mutational scanning data to identify mutant mammalian kinases that drive resistance to clinically relevant inhibitors. We aggregate these data with subsaturation mutagenesis data and use it to develop, test and validate a framework to prospectively identify residues that mediate kinase activity and drug resistance across the kinome. We validate predicted resistance mutations in CDK4, CDK6, ERK2, EGFR and HER2. Capitalizing on a highly predictable residue, we generate resistance mutations in TBK1, CSNK2A1 and BRAF. Unexpectedly, we uncover a potentially generalizable activation site that mediates drug resistance and confirm its impact in BRAF, EGFR, HER2 and MEK1. We anticipate that the identification of these residues will enable the broad interrogation of the kinome and its inhibitors.

Rapid detection of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in wound specimens and blood cultures: multicenter preclinical evaluation of the Cepheid Xpert MRSA/SA skin and soft tissue and blood culture assays.

A multicenter preclinical evaluation was conducted to evaluate the performance of two Cepheid Xpert assays for detection of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus. Sensitivity was 97.1% and 98.3% for MRSA in wound and blood culture specimens, respectively. Sensitivity was 100% for S. aureus from both specimen types.

Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares.

The first U.S. multicenter clinical trial to assess the performance of the Cepheid Xpert MRSA assay (Xpert MRSA) was conducted. The assay is a qualitative test designed for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from nares swabs. This novel test combines integrated nucleic acid extraction and automated real-time PCR for the detection of a MRSA-specific signature sequence. A total of 1,077 nares specimens were collected from seven geographically distinct health care sites across the United States with prevalence rates ranging from 5.2% to 44%. Nares specimens were tested by (i) the Xpert MRSA assay, (ii) direct culture on CHROMagar MRSA medium (direct CM culture), and (iii) broth-enriched culture (Trypticase soy broth with 6.5% sodium chloride) followed by plating onto CHROMagar MRSA medium (broth-enriched CM culture). When direct CM culture was designated the reference method, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MRSA assay were 94.3%, 93.2%, 73.0%, and 98.8%, respectively. When broth-enriched CM culture was used as the reference method, the clinical sensitivity, specificity, PPV, and NPV of the Xpert MRSA assay were 86.3%, 94.9%, 80.5%, and 96.6%, respectively. The BD GeneOhm MRSA (BDGO) assay was performed as a comparative molecular method. No statistical performance differences were observed between the Xpert MRSA and BDGO assays when they were compared to culture methods. From this large-scale, multicenter clinical comparison, we conclude that the Xpert MRSA assay is a simple, rapid, and accurate method for performing active surveillance for MRSA in a variety of health care populations.

Sequestration from immune CD4+ T cells of mycobacteria growing in human macrophages.

CD4+ helper T cells mediate resistance to tuberculosis, presumably by enhancing the antimicrobial activity of macrophages within which the Mycobacterium tuberculosis organism grows. A first step in resistance should be the presentation of mycobacterial antigens by macrophages to CD4+ T cells. However, when the antigenic stimulus is limited to organisms growing in human monocytes, the organisms become sequestered from immune CD4+ T cells. This block in presentation is selective for growing mycobacteria and not for other stimuli. Sequestration would allow replicating organisms to persist in infected individuals and may contribute to virulence.

Sexual transmission of hepatitis C: a review.

OBJECTIVES: To establish and understand the transmission of hepatitis C virus (HCV) and conclude on the possibility of sexual transmission as a route of transmission. DATA SOURCES: Review of Literature via medline, the internet and articles in indexed journals. DATA SELECTION: Published data from 1989 up to date found to have studied the sexual transmission of Hepatitis C Virus. DATA EXTRACTION: Abstracts of articles identified were assessed, read and analysed to determine possible relevance to the title. DATA SYNTHESIS: When relevance was established from the abstract the entire paper was then read and important points were included in the review. CONCLUSION: The data acquired so far cannot convincingly conclude that hepatitis C is not transmitted sexually, as the possibilities do exist. There are a lot of factors that suggest that HCV is sexually transmitted. Present data clearly indicates that more research needs to be carried out on different aspects of HCV that may have lead to the discrepancies mentioned herewith. There may be a role played by various factors such as the age, sex, race, immune status, presence of co-existing HIV infection, and any other concurrent illnesses upon the sexual transmission of HCV.

Extended-Spectrum ß-lactam Resistance in the Enteric Flora of Patients at a Tertiary Care Medical Centre.

The dissemination of Enterobacteriaceae expressing resistance to extended-spectrum cephalosporins, which are therapeutically used in both human and veterinary medicine, is of critical concern. The normal commensal flora of food animals may serve as an important reservoir for the zoonotic food-borne transmission of Enterobacteriaceae harbouring ß-lactam resistance. We hypothesized that the predominant AmpC and ESBL genes reported in US livestock and fresh retail meat products, blaCMY-2 and blaCTX-M , would also be predominant in human enteric flora. We recovered enteric flora from a convenience sample of patients included in a large tertiary medical centre's Clostridium difficile surveillance programme to screen for and estimate the frequency of carriage of AmpC and ESBL resistance genes. In- and outpatient diarrhoeic submissions (n = 692) received for C. difficile testing at the medical centre's clinical diagnostic laboratory from July to December, 2013, were included. Aliquoted to a transport swab, each submission was inoculated to MacConkey broth with cefotaxime, incubated at 37°C and then inoculated to MacConkey agars supplemented with cefoxitin and cefepime to select for the AmpC and ESBL phenotypes, with blaCMY and blaCTX-M genotypes confirmed by PCR and sequencing. From the 692 diarrhoeic submissions, our selective culture yielded 184 isolates (26.6%) with reduced susceptibility to cefotaxime. Of these, 46 (6.7%) samples harboured commensal isolates carrying the AmpC blaCMY . Another 21 (3.0%) samples produced isolates harbouring the ESBL blaCTX-M : 19 carrying CTX-M-15 and 2 with CTX-M-27. Our results indicate that ß-lactam resistance genes likely acquired through zoonotic food-borne transmission are present in the enteric flora of this hospital-associated population at lower levels than reported in livestock and fresh food products.

Immunogenicity of ribonucleic acid-protein fraction of Mycobacterium tuberculosis encapsulated in liposomes.

Immunologically potent RNA-protein extracted from Mycobacterium tuberculosis strain H37Ra, when entrapped in phosphatidylcholine multilamellar liposomes and injected into mice, induced both cellular and humoral immune responses. Significant protection against infection with M. tuberculosis H37Rv was also induced in the immunised mice, as monitored by (i) higher survival rates, (ii) decreased viable counts of M. tuberculosis H37Rv in lungs, livers and spleens, (iii) lower lung density, and (iv) lower root specific lung weight, in comparison with a control group of unimmunized mice.

Diagnostic Detection of Herpes Simplex and Hepatitis C Viral Amplicons by Capillary Electrophoresis: Comparison With Southern Blot Detection.

Background: As a result of the large number of DNA-based clinical assays, there is intense interest in making polymerase chain reaction (PCR)-amplified DNA product analysis faster, more cost-effective, and more automated. Methods and Results: In this study, an evaluation of the use of capillary gel electrophoresis with laser-induced fluorescence detection is described as a means of analyzing postamplification PCR products from clinical specimens. Sixty-six herpes simplex virus and 152 hepatitis C virus amplicons were analyzed after PCR and reverse transcription PCR, respectively. It is shown that the use of a physical gel buffer system in a short capillary in conjunction with laser-induced fluorescence detection allows for sensitive detection of herpes simplex virus- and hepatitis C virus-specific DNA fragments in an expedient manner. Interinstrument and intercapillary reproducibility of the migration time was evaluated and found to be excellent. The advantages and disadvantages over agarose gel electorphoresis-Southern blot analysis are summarized. Conclusions: The advantages offered by capillary gel electrophoresis with laser-induced fluorescent detection including rapid and sensitive analysis, ease of setup, reduced cost, and possibility for automation, make this procedure a viable alternative to more labor-intensive agarose gel electrophoresis-Southern blot analysis as molecular diagnostic methodology.

Immunobiological studies with mycobacterial ribonucleic acid-protein complex: Part I--Immune responses and protective role against experimental tuberculosis.

Ribonucleic acid (RNA) isolated from M. tuberculosis H37Ra was found to be native in nature as determined by hyperchromicity studies using ribonuclease. Mycobacterial RNA-protein (Myc. RNA-P) when injected as RNA-P-FIA complexes induced weak humoral immune responses and strong cell-mediated immune (CMI) responses which were directed against Myc. RNA. Protection comparable to BCG was induced in mice immunized with RNA-FIA complexes against LD50 dose of M. tuberculosis as monitored by increased survival rates, decreased lung density, root specific lung weight (RSLW) and by decreased viable counts of M. tuberculosis in lung, liver and spleen of immunized mice. Enzymatic degradation studies revealed Myc. RNA component to specifically mediate protection while the protein component was found ineffective.

Immunobiological studies with mycobacterial ribonucleic acid-protein complex: Part II--Mechanism of protective immunity against experimental tuberculosis.

Passive transfer of protective antituberculous immunity against LD50 dose of M. tuberculosis H37Rv was found to be mainly mediated by immune T-cells harvested from spleens of donor mice immunized with Myc. RNA-P-FIA complexes as monitored by indices of percent survival, root specific lung weight, lung density and by bacterial enumeration from different organs. Treatment of immune T-cells with anti-Thy 1.2. monoclonal antibodies plus complement prior to passive transfer, completely abrogated its protective effect thereby confirming their protective nature. Passive transfer of immune sera as well as immune T + B cells did not induce any enhancement in protective immunity.

An approach to isolating T cell lines that react to antigens presented on the surface of dendritic cells.

We describe an approach that might be useful for identifying antigens on surfaces of antigen presenting cells. It is known that dendritic cells carry antigens in situ and are efficient at clustering antigen-specific T cells. Using the human mixed lymphocyte reaction (MLR) system, we have shown that alloreactive CD4+ T cells can be selected by their capacity to cluster with dendritic cells in the first 2 days of the MLR. Small numbers of clustered cells, 1-10/culture well, could then be expanded as antigen-specific lines in presence of either antigen or mitogen, sodium periodate. Few antigen-specific lines could be isolated from the nonclustered fraction. When T cell lines derived from the dendritic T cell clusters were maintained without antigen, i.e. using second party (syngeneic antigen-presenting cells (APC] or irrelevant antigen bearing APC, i.e. third-party (HLA-mismatched) stimulator cells plus mitogen, the T cells retained their specificity for the original stimulating alloantigen over the time course tested, several weeks to months. These findings show that by using dendritic cells as immunoadsorbents one can prepare antigen-specific cell lines and maintain the specificity of the lines without the need for adding exogeneous antigen during either immunoselection or cloning. We discuss the possible use of dendritic cells as a means for raising T cell lines and clones that recognize antigens being carried by APC and which might be pertinent to protective immunity and autoimmunity.

Dendritic cells efficiently immunoselect mycobacterial-reactive T cells in human blood, including clonable antigen-reactive precursors.

Given the persistence of tuberculosis throughout the world, the delineation of mechanisms that lead to protective immunity to Mycobacterium tuberculosis is important. We have evaluated the presenting function of human dendritic cells for mycobacterial antigens, since these antigen-presenting cells (APC) are particularly effective in initiating antigen-specific T-cell responses. Dendritic cells from blood prove to be active APC for mycobacteria-specific proliferative responses by CD4+ T cells from bacillus Calmette-Guérin (BCG)-vaccinated individuals. In the first 24-48 hr of the response, dendritic cells that have been pulsed with mycobacterial antigens, including live BCG, effectively bind T cells forming discrete cell clusters. The clusters represent about 1% of the applied T cells. Clusters are highly enriched in mycobacterial reactivity while the non-clusters are depleted. Clustered T cells can be used as a starting point to expand antigen-specific cell lines. Mitogen and allogeneic feeder cells were used as APC to expand the mycobacterial-reactive lines, because the antigen-specific T cells had been preselected by virtue of their binding to antigen-pulsed dendritic cells. We discuss the advantages of obtaining antigen-reactive T cells by using dendritic cells as immunoadsorbents. These lines should help delineate the range of mycobacterial antigens and T-cell responses that participate in host responses to mycobacteria.

DNA prime-canarypox boost with polycistronic hepatitis C virus (HCV) genes generates potent immune responses to HCV structural and nonstructural proteins.

DNA vaccination was employed to study immune responses to hepatitis C virus (HCV) proteins. As an immunizing strategy, we studied immune responses of BALB/c (H-2d) and C57BL/6 mice (H-2b) to HCV genes delivered intramuscularly as a polycistronic construct capsid/E1/E2/NS2/NS3 (pRC/C-NS3) encoding 5 structural and nonstructural proteins. We also evaluated canarypox virus containing the same HCV genes as a means for potentiating immune responses to naked DNA. Our results indicate that mice that received a polycistronic pRC/C-NS3 with canarypox booster had enhanced antibody and cellular responses to HCV proteins. Immunodominant CD8(+) T cell responses to several HCV structural and nonstructural proteins, characterized by cytotoxicity and interferon (IFN)-gamma production or IFN-gamma production without significant cytotoxicity, were observed in both strains of mice. The combination of naked DNA with a nonreplicating canarypox booster encoding HCV polycistronic pRC/C-NS3 genes appears to diversify and enhance T cell responses to HCV proteins.