Real-time polymerase chain reaction and laser capture microdissection: an efficient combination tool for Chlamydophila pneumoniae DNA quantification and localization of infection in atherosclerotic lesions.

Chlamydophila pneumoniae has been implicated in atherosclerosis, but the role of this obligate intracellular pathogen in the development of the above pathology is still unclear. In particular, its presence and quantitative distribution within lesional areas has not yet been defined. We studied 18 carotid biopsies obtained from patients undergoing endoartherectomy. By laser microdissection (LCM), two different sites (intra-plaque and plaque-adjacent areas) were taken from each lesion, and the presence and quantity of the pathogen DNA were determined by real-time polymerase chain reaction (Real-time PCR). A total of 8 plaques, exclusively, from patients with unstable angina, were positive in real-time PCR. The bacterial DNA was detected in both lesional areas of 3 plaques which contained the highest number of DNA copies (1,900 to 2,200 copy numbers), while C. pneumoniae DNA was detected only in the intra-plaque area of the other 5 positive (500 to 1,600 copy numbers). No C. pneumoniae DNA was found in the other 10 plaques of which 6 were from patients with unstable angina and 4 from stable angina patients. No DNA from Helicobacter pylori or Cytomegalovirus was found in any plaque. This is the first report where both the target lesion and an adjacent reference site were evaluated for the presence of C. pneumoniae DNA by the combination of LCM and Real-time PCR assays. The integration of these two methodologies offer an excellent tool for in situ studies and may help to elucidate the putative role of C. pneumoniae in atherosclerosis

Risk factors for Haemophilus influenzae and pneumococcal respiratory tract colonization in CVID

To the Editor: Disease-specific studies focused on infection risk in common variable immune deficiencies (CVIDs) are needed to define strategies for controlling respiratory infections predominantly due to bacteria such as Streptococcus pneumoniae and Haemophilus influenzae.1 Little information is available on the rate of airway bacterial carriage and its consequence in hypogammaglobulinemias. Despite IgG replacement, recurrent respiratory infections are common in CVID, possibly leading to chronic lung damage2 and poor quality of life.3 Thus, patients are often prescribed antibiotics and/or long-term antimicrobial prophylactic regimens. Several regimens are used including rotation or periodically changing antibiotics.4 However, antibiotics influence antimicrobial resistance among airway microbiota. In a recent meta-analysis on patients with chronic lung diseases, 30% of S pneumoniae showed resistance to macrolides.

Chlamydophila pneumoniae infection in patients undergoing carotid artery stent.

Although several reports have correlated Chlamydophila pneumoniae (CP) infection with carotid endarterectomy and coronary stent, no data have been reported on the potential relationship between this pathogen and carotid artery stenting (CAS). Hence, we evaluated 47 subjects, 27 symptomatic and 20 asymptomatic, before CAS intervention and during the follow up, for the presence of CP DNA and anti-CP antibodies, including chlamydial HSP60 (Cp-HSP60). Before stent placement, CP DNA was detected exclusively in symptomatic patients, all of whom were also positive for CP IgG and IgA and 85.7% of them also had CP-HSP60 antibodies. At the follow-up, all CP DNA positive and 11 out of the 13 symptomatic patients with Cp-HSP60 antibodies became negatives. In contrast, no change was observed for CP- IgA antibodies. Despite the small number of patients, the present study advocates an important role of CP infection in symptomatic patients with carotid artery disease. Our findings also suggest that stent placement and/or therapy might have a role in favouring resolution of inflammation, though not affecting persistence of CP infection

Erratum to: Respiratory diphtheria due to Corynebacterium ulcerans transmitted by a companion dog, Italy 2014

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Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

BACKGROUND: The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. RESULTS: Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. CONCLUSION: In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology

Meningitis with cranial polyneuritis and cavernous sinus thrombosis by Borrelia crocidurae: First autochthonous case in Europe

Borrelia crocidurae is endemic in West Africa, where it represents the leading cause of tick-borne relapsing fever (TBRF). TBRF typically presents with high fever and systemic symptoms, followed by recurrent episodes. Neurological complications may occur during febrile relapses. B. crocidurae is considered the most neurotropic agent of TBRF and is associated to severe neurological manifestations i.e. meningitis and encephalitis.To date, European cases of B. crocidurae infection have been reported in travelers returning from endemic areas. We report the first autochthonous case in Europe of B. crocidurae infection, presenting as meningitis with cranial polyneuritis and cavernous sinus thrombosis that were not preceded by classic febrile recurrences. Keywords: Borrelia crocidurae, Europe, Autochthonous, Meningitis, Cranial polyneuritis, Cavernous sinus thrombosi

The multidrug transporter P-glycoprotein: A mediator of melanoma invasion?

Malignant melanoma shows high levels of intrinsic drug resistance associated with a highly invasive phenotype. In this study, we investigated the role of the drug transporter P-glycoprotein (Pgp) in the invasion potential of drug-sensitive (M14 WT, Pgp-negative) and drug-resistant (M14 ADR, Pgp-positive) human melanoma cells. Coimmunoprecipitation experiments assessed the association of Pgp with the adhesion molecule CD44 in multidrug resistant (MDR) melanoma cells, compared with parental ones. In MDR cells, the two proteins colocalized in the plasma membrane as visualized by confocal microscopy and immunoelectron microscopy on ultrathin cryosections. MDR melanoma cells displayed a more invasive phenotype compared with parental cells, as demonstrated by quantitative transwell chamber invasion assay. This was accomplished by a different migration strategy adopted by resistant cells ("chain collective") previously described in tumor cells with high metastatic capacity. The Pgp molecule, after stimulation with specific antibodies, appeared to cooperate with CD44, through the activation of ERK1/2 and p38 mitogen-activated protein kinase (MAPK) proteins. This activation led to an increase of metalloproteinase (MMP-2, MMP-3, and MMP-9) mRNAs, and proteolytic activities, which are associated with an increased invasive behavior. RNA interference experiments further demonstrated Pgp involvement in migration and invasion of resistant melanoma cells. A link was identified between MDR transporter Pgp, and MAPK signaling and invasion. © 2007 The Society for Investigative Dermatology

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

<p>Abstract</p> <p>Background</p> <p>Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs.</p> <p>Methods</p> <p>Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR.</p> <p>Results</p> <p>The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC₅₀in the low nanomolar range. Inhibition of FIV integration <it>in situ </it>was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810.</p> <p>Conclusion</p> <p>We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication <it>in vitro</it>. The present study helped establish L-870,810, a compound successfully tested in human clinical trials, as one of the most potent anti-FIV agents ever tested <it>in vitro</it>. This finding may provide new avenues for treating FIV infection and contribute to the development of a small animal model mimicking the effects of ART in humans.</p

Characterization of spotted fever group Rickettsiae in ticks from a city park of Rome, Italy

 Background: Ticks are vectors and important reservoirs for microbial agents that cause disease in humans and animals. Among these pathogens, the members of Rickettsia species play an important role in public health.Aim and Methods: One hundred twenty-nine ticks belonging to four tick species (Ixodes ricinus, Rhipicephalus turanicus, Dermacentor marginatus, and Haemaphysalis punctata) were collected at different sites of the Insugherata Natural Reserve, localized in the urban area of Rome, Italy. Questing ticks were tested by PCR for Rickettsia spp., amplifying partial gene of ompA.Results: Forty-six ticks were found to be infected with Rickettsia species. Five SFG rickettsiae were identified: three human pathogens Rickettsia conorii, Rickettsia massiliae and Rickettsia aeschlimannii, and two putative new strains Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2. The phylogenetic analysis of partial gene sequences of ompA, gltA, and 17-kd antigen showed that they clustered with several rickettsiae with unidentified pathogenicity. However, Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2 clustered in a statistically supported clade with R. massiliae, and R. monacensis, respectively.Conclusion: Our findings suggest that Rickettsia species other than R. conorii are implicated in human disease in Italy