No abstract

In a large prospective comparison, the illumigene test detected Clostridium difficile in 98% of toxin-positive and 58% of toxinnegative samples confirmed positive by other methods. The Xpert was uniformly sensitive. Most samples with discrepant results had C. difficile concentrations below the illumigene limit of detection. The significance of low-level C. difficile detection needs investigation.T he incidence and severity of Clostridium difficile infection (CDI) have increased dramatically in North America and Europe over the last decade (1). In the United States, rates of CDI are now at an all-time high (2). With this change in incidence, the performance of C. difficile testing has been reexamined and there has been a movement toward tests that detect C. difficile antigens or DNA directly versus tests that detect C. difficile toxins (3). As a group, C. difficile detection tests (e.g., PCR, loop-mediated amplification [LAMP], glutamate dehydrogenase immunoassay) are more sensitive than toxin tests, but the reported sensitivities still vary for unclear reasons. For example, the published sensitivity of the illumigene C. difficile assay ranges from 81 to 98% (4-11), leaving laboratories with uncertainty about which test to use and what performance to expect at their own institution.To address this issue, we evaluated the fecal C. difficile DNA load of positive samples as part of a large, prospective comparison of two FDA-approved nucleic acid amplification tests (NAATs) for C. difficile, the illumigene C. difficile test and the Xpert C. difficile/Epi test, with toxigenic culture. When the clinical results and fecal C. difficile load comparisons suggested a genuine difference in the sensitivity of these two NAATs, we evaluated the inoculum size and analytical limit of detection (LOD) of both assays to further investigate the causes and significance of discrepant results.Study population and test methods. Consecutive diarrheal stool samples submitted for C. difficile testing from adult inpatients Ն72 h after admission between January and October 2011 were included in the study; nonconforming stool samples were rejected. Each sample was tested for toxigenic C. difficile by three tests, (i) an illumigene C. difficile LAMP assay (Meridian Bioscience), (ii) an Xpert C. difficile/Epi real-time PCR assay (Cepheid), and (iii) a toxigenic culture. In addition, the fecal toxin status of each sample was determined by a toxin immunoassay (Premier C. difficile Toxins A & B; Meridian Bioscience) performed on all samples and a cytotoxicity assay (Wampole C. difficile Tox-B [TechLab]; MHRF cells [Diagnostic Hybrids]) performed on immunoassay-negative, C. difficile-positive samples from frozen aliquots when available (55/61; 90.2%) (12). All tests except cytotoxicity were performed daily on fresh stool by following the manufacturer's instructions. For toxigenic culture, 0.5 ml stool was mixed 1:1 with 95% EtOH for 10 min and a swab was used to inoculate a prereduced agar medium (cycloserine cefoxitin fructose agar supplemented wit...

Chlamydia trachomatis infection of the female genital tract can lead to irreversible fallopian tube scarring. In the mouse model of genital infection using Chlamydia muridarum, IL-1R signaling plays a critical role in oviduct tissue damage. In this study, we investigated the pathologic role of IL-1α, one of the two proinflammatory cytokines that bind to IL-1R. Il1a−/− mice infected with C. muridarum cleared infection at their cervix at the same rate as wild-type (WT) mice, but were significantly protected from end point oviduct damage and fibrosis. The contribution of IL-1α to oviduct pathology was more dramatic than observed in mice deficient for IL-1β. Although chlamydial burden was similar in WT and Il1a−/− oviduct during peak days of infection, levels of IL-1β, IL-6, CSF3, and CXCL2 were reduced in Il1a−/− oviduct lysates. During infection, Il1a−/− oviducts and uterine horns exhibited reduced neutrophil infiltration, and this reduction persisted after the infection resolved. The absence of IL-1α did not compromise CD4 T cell recruitment or function during primary or secondary chlamydial infection. IL-1α is expressed predominantly by luminal cells of the genital tract in response to infection, and low levels of expression persisted after the infection cleared. Ab-mediated depletion of IL-1α in WT mice prevented infection-induced oviduct damage, further supporting a key role for IL-1α in oviduct pathology.

Genital infections with Chlamydia trachomatis can lead to uterine and oviduct tissue damage in the female reproductive tract. Neutrophils are strongly associated with tissue damage during chlamydial infection, while an adaptive CD4 T cell response is necessary to combat infection. Activation of triggering receptor expressed on myeloid cells-1 (TREM-1) on neutrophils has previously been shown to induce and/or enhance degranulation synergistically with TLR-signaling. Additionally, TREM-1 can promote neutrophil transepithelial migration. In this study, we sought to determine the contribution of TREM-1,3 in immunopathology in the female mouse genital tract during Chlamydia muridarum infection. Relative to control mice, trem1,3 -/- mice had no difference in chlamydial burden or duration of lower genital tract infection. We also observed a similar incidence of oviduct hydrosalpinx 45 days post-infection in trem1,3 -/- compared to WT mice. However, compared to WT, trem1,3 -/- mice developed significantly fewer uterine horn hydrometra. Early in infection, trem1,3 -/- mice displayed a notable decrease in the number of uterine glands containing polymorphonuclear cells and uterine horn lumens had fewer neutrophils, with increased G-CSF. Trem1,3 -/- mice also had reduced erosion of the luminal epithelium. These data indicate TREM-1,3 contributes to transepithelial neutrophil migration in the uterus and uterine glands, promoting the development of uterine hydrometra in infected mice.

It has been shown that caspase-1, but not its upstream activator, ASC, contributes to oviduct pathology during mouse genital Chlamydia muridarum infection. We hypothesized that this dichotomy is due to the inadvertent absence of caspase-11 in previously used caspase-1-deficient mice. To address this, we studied the independent contributions of caspase-1 and -11 during genital Chlamydia infection. Our results show that caspase-11 deficiency was sufficient to recapitulate the effect of the combined absence of both caspase-1 and caspase-11 on oviduct pathology. Further, mice that were deficient for both caspase-1 and -11 but that expressed caspase-11 as a transgene (essentially, caspase-1-deficient mice) had no significant difference in oviduct pathology from control mice. Caspase-11-deficient mice showed reduced dilation in both the oviducts and uterus. To determine the mechanism by which caspase-11-deficient mice developed reduced pathology, the chlamydial burden and immune cell infiltration were determined in the oviducts. In the caspase-11-deficient mice, we observed increased chlamydial burdens in the upper genital tract, which correlated with increased CD4 T cell recruitment, suggesting a contribution of caspase-11 in infection control. Additionally, there were significantly fewer neutrophils in the oviducts of caspase-11-deficient mice, supporting the observed decrease in the incidence of oviduct pathology. Therefore, caspase-11 activation contributes to pathogen control and oviduct disease independently of caspase-1 activation.

No abstract

Chlamydia trachomatis-genital infection in women can be modeled in mice using Chlamydia muridarum. Using this model, it has been shown that the cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1α lead to irreversible tissue damage in the oviducts. In this study, we investigated the contribution of TNFα on IL-1α synthesis in infected epithelial cells. We show that C muridarum infection enhanced TNFα-induced IL-1α expression and release in a mouse epithelial cell line. In addition to IL-1α, several TNFα-induced inflammatory genes were also highly induced, and infection enhanced TNF-induced cell death. In the mouse model of genital infection, oviducts from mice lacking the TNFα receptor displayed minimal staining for IL-1α compared with wild-type oviducts. Our results suggest TNFα and IL-1α enhance each other’s downstream effects resulting in a hyperinflammatory response to chlamydial infection. We propose that biologics targeting TNF-induced IL-1α synthesis could be used to mitigate tissue damage during chlamydial infection.

No abstract