The aim of this study was to compare the clinical and laboratory characteristics of Clostridium difficile infection (CDI) in patients with discordant test results for the cytotoxin assay (CYT) and PCR assays. A retrospective study from May to August 2008 and March to May 2010 was performed. CDI was diagnosed in 128 patients. PCR increased the yield of C. difficile cases by 2-fold compared to that of the CYT assay. Fifty-six cases (44%) were detected by PCR only (CYT negative). Forty-nine percent of patients with non-NAP1 strains were detected by PCR only, compared to 28% of those infected with NAP1 strains (P < 0.05). No significant differences were found in the clinical severity of illness and outcome among patients that tested positive for CDI by both tests (CYT and PCR) compared to those that tested positive by PCR only. Clostridium difficile has been implicated in up to 25% of antibiotic-associated diarrhea and nearly all bacterial cases of hospital-acquired diarrhea (6, 10). The spectrum of disease can range from uncomplicated diarrhea to pseudomembranous colitis, toxic megacolon, sepsis with associated organ failure, and even death. North American hospitals began to experience an increased incidence of a particularly virulent strain of C. difficile in the early part of the decade, characterized by recurrent infection and increased mortality, especially in the elderly (3,8,11,14,20).The increase in the incidence of C. difficile infection (CDI) has been attributed in part to the emergence of a predominant strain, known as North American pulsed-field type 1 (NAP1), of ribotype 027 and group BI. The characteristics of the NAP1 strain include fluoroquinolone resistance, production of binary toxin in addition to toxins A and B, and various polymorphisms in the tcdC gene (13). Louie et al. were recently able to demonstrate both an increased severity of illness due to the NAP1 strain and lower incidence of relapse in those treated with fidaxomicin who were infected with non-NAP1 strains, suggesting that knowledge of the infecting strain may have a significant impact on treatment decisions (9). One of the FDA-cleared PCR diagnostic assays for the detection of C. difficile, the Xpert (Cepheid, Sunnyvale, CA), is able to identify NAP1 strains in a timely fashion; although only FDA cleared for infection control purposes, such results may potentially be used to make therapeutic decisions.Toxigenic culture has been considered the gold standard for the diagnosis of C. difficile, and its use is supported by the most recent practice guidelines issued by the Infectious Diseases Society of America (IDSA) and the Society for Healthcare Epidemiology of America (SHEA) (4). However, toxicogenic culture is time consuming and labor intensive. Alternative enzyme immunoassay (EIA)-based tests are more frequently used for routine diagnostic use, but sensitivities for EIAs have been reported to be as low as 30% (15). PCR-based detection of C. difficile is increasingly being used in many centers. PCR is rapid and highly sensitive but d...

Lyme disease is a chronic inflammatory disorder caused by the spirochetal bacterium, Borrelia burgdorferi. In vitro evidence suggests that binding of spirochetal lipoproteins to CD14, a pattern recognition receptor expressed on monocytes/macrophages and polymorphonuclear cells, is a critical requirement for cellular activation and the subsequent release of proinflammatory cytokines that most likely contribute to symptomatology and clinical manifestations. To test the validity of this notion, we assessed the impact of CD14 deficiency on Lyme disease in C3H/HeN mice. Contrary to an anticipated diminution in pathology, CD14−/− mice exhibited more severe and persistent inflammation than did CD14+/+ mice. This disparity reflects altered gene regulation within immune cells that may engender the higher bacterial burden and serum cytokine levels observed in CD14−/− mice. Comparing their in vitro stimulatory activity, live spirochetes, but not lysed organisms, were a potent CD14-independent stimulus of cytokine production, triggering an exaggerated response by CD14−/− macrophages. Collectively, our in vivo and in vitro findings support the provocative notion that: 1) pattern recognition by CD14 is entirely dispensable for elaboration of an inflammatory response to B. burgdorferi, and 2) CD14-independent signaling pathways are inherently more destructive than CD14-dependent pathways. Continued study of CD14-independent signaling pathways may provide mechanistic insight into the inflammatory processes that underlie development of chronic inflammation.

During the summer of 2015, New York, New York, USA, had one of the largest and deadliest outbreaks of Legionnaires’ disease in the history of the United States. A total of 138 cases and 16 deaths were linked to a single cooling tower in the South Bronx. Analysis of environmental samples and clinical isolates showed that sporadic cases of legionellosis before, during, and after the outbreak could be traced to a slowly evolving, single-ancestor strain. Detection of an ostensibly virulent Legionella strain endemic to the Bronx community suggests potential risk for future cases of legionellosis in the area. The genetic homogeneity of the Legionella population in this area might complicate investigations and interpretations of future outbreaks of Legionnaires’ disease.

Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n ‫؍‬ 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n ‫؍‬ 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n ‫؍‬ 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.Clostridium difficile is one of the leading causes of infectious antibiotic-associated diarrhea and pseudomembranous colitis worldwide (2, 16). This is illustrated by the increased incidence and severity of C. difficile infection, suggesting the emergence of a new hypervirulent strain (5, 13-15, 17, 25, 32).While TcdB, a cytotoxin, is the known established virulence factor of C. difficile, toxin A (TcdA), a cytotoxic enterotoxin, works synergistically with TcdB, causing damage to the intestinal mucosa in cases of C. difficile infection (17). The genes that encode these toxins are located on the pathogenicity locus of C. difficile (4,10,24). Additionally, several deletions in the tcdC gene, a putative negative regulator of the expression of the toxin A (tcdA) and the toxin B (tcdB) genes, have been identified, and these deletions result in higher levels of cytotoxin expression (11). Furthermore, research has shown that some C. difficile strains produce another toxin, known as the binary toxin (19,22,28). The genes that encode this toxin, cdtA and cdtB, together produce an actin-specific ADP-ribosyltransferase that induces damage to the actin skeleton, leading to cytopathic effects in cell lines (1). It has been suggested that the binary toxin genes and deletions in the tcdC gene are potential virulence factors in the recent emerging hypervirulent strain (22, 29).The "gold standard" for the detection of C. difficile toxin production is a cytotoxin assay with stool specimens or isolates from anaerobic culture. The cytotoxin assay is difficult to perform and time-consuming, and it is often less sensitive than molecular assays ...

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.

bClostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

No abstract

Primary cutaneous marginal zone lymphoma (PCMZL) has rarely been reported in teenagers and is occasionally associated with Borrelia burgdorferi infection. Juxta-articular fibrotic nodules represent a unique, localized fibrosing response to spirochete infections, namely Borreliosis. Herein, we report a 15-year-old healthy boy who presented with a 4-year history of progressive acquisition of asymptomatic, erythematous nodules, ≤ 3 cm, beginning with his right forearm (3), then right arm (1) and lastly his right inner thigh (1). Biopsy showed PCMZL in three of five samples, and inflamed, fibrotic nodules, near the elbow in two. The bottom heavy lymphomatous nodules consisted of mostly small CD20+ CD43+ lymphocytes, some with plasmacytoid features. Mature plasma cells were lambda light chain restricted by in situ hybridization. The juxta-articular fibrotic nodules were located in the deep dermis and subcutis, had peripheral plasma cell-rich infiltrates, and showed nodular sclerosis (morphea profunda-like) in one, and lamellar and angiocentric sclerosis in the other reminiscent of quiescent lesions of chronic localized fibrosing leukocytoclastic vasculitis. Immunohistochemistry for B. burgdorferi revealed rare positive organisms; however, polymerase chain reaction (PCR) and serology were negative for B. burgdorferi as were serologic and/or PCR assays for Bartonella henselae, Ba. quintana, Ehrlichia chaffeensis, Treponema pallidum, Helicobacter pylori and Babesia microti. No evidence of extracutaneous disease was found by the review of systems and imaging studies. A 4-week trial of doxycycline therapy failed, whereas intralesional (IL) corticosteroid therapy induced rapid regression of his nodules. After two local recurrences, also treated with IL corticosteroids, he is well, without cutaneous disease, 20 months later. A literature review of 19 pediatric cases PCMZL reveals a similar natural history as adult PCMZL. Despite negative serology and PCR for B. burgdorferi, the occurrence of ipsilateral juxta-articular fibrotic nodules, positive B. burgdorferi immunohistochemistry and rapid response to IL corticosteroids implicate the presence of a replicative or non-replicative infectious (spirochetal) antigen in the initiation and promotion of this teenager's PCMZL.