Improved diagnosis of periprosthetic joint infection by multiplex PCR of sonication fluid from removed implants.

The microbiological diagnosis of periprosthetic joint infection (PJI) is crucial for successful antimicrobial treatment. Cultures have limited sensitivity, especially in patients receiving antibiotics. We evaluated the value of multiplex PCR for detection of microbial DNA in sonication fluid from removed orthopedic prostheses. Cases of PJI in which the prosthesis (or part of it) was removed were prospectively included. The removed implant was sonicated, and the resulting sonication fluid was cultured and subjected to multiplex PCR. Of 37 PJI cases (17 hip prostheses, 14 knee prostheses, 4 shoulder prostheses, 1 elbow prosthesis, and 1 ankle prosthesis), pathogens were identified in periprosthetic tissue in 24 (65%) cases, in sonication fluid in 23 (62%) cases, and by multiplex PCR in 29 (78%) cases. The pathogen was detected in 5 cases in sonication fluid only (Propionibacterium acnes in all cases; none of these patients had previously received antibiotics) and in 11 cases by multiplex PCR only (all of these patients had previously received antibiotics). After exclusion of 8 cases caused by P. acnes or Corynebacterium species, which cannot be detected due to the absence of specific primers in the PCR kit, sonication cultures were positive in 17 cases and multiplex PCR sonication cultures were positive in 29 cases (59% versus 100%, respectively; P < 0.01). Among 19 cases (51%) receiving antibiotics, multiplex PCR was positive in all 19 (100%), whereas sonication cultures grew the organism in 8 (42%) (P < 0.01). Multiplex PCR of sonication fluid is a promising test for diagnosis of PJI, particularly in patients who previously received antibiotics. With modified primer sets, multiplex PCR has the potential for further improvement of the diagnosis of PJI.

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples.

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression.

An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ.

Variations of plasmid content in Rickettsia felis.

BACKGROUND: Since its first detection, characterization of R. felis has been a matter of debate, mostly due to the contamination of an initial R. felis culture by R. typhi. However, the first stable culture of R. felis allowed its precise phenotypic and genotypic characterization, and demonstrated that this species belonged to the spotted fever group rickettsiae. Later, its genome sequence revealed the presence of two forms of the same plasmid, physically confirmed by biological data. In a recent article, Gillespie et al. (PLoS One. 2007;2(3):e266.) used a bioinformatic approach to refute the presence of the second plasmid form, and proposed the creation of a specific phylogenetic group for R. felis. METHODOLOGY/PRINCIPAL FINDINGS: In the present report, we, and five independent international laboratories confirmed unambiguously by PCR the presence of two plasmid forms in R. felis strain URRWXCal(2) (T), but observed that the plasmid content of this species, from none to 2 plasmid forms, may depend on the culture passage history of the studied strain. We also demonstrated that R. felis does not cultivate in Vero cells at 37 degrees C but generates plaques at 30 degrees C. Finally, using a phylogenetic study based on 667 concatenated core genes, we demonstrated the position of R. felis within the spotted fever group. SIGNIFICANCE: We demonstrated that R. felis, which unambiguously belongs to the spotted fever group rickettsiae, may contain up to two plasmid forms but this plasmid content is unstable.