1,721,036 research outputs found
Molecular markers to evaluate stage conversion of Toxoplasma gondii in recrudescence of Toxoplasmosis in immunocompetent host.
No full textMolecular epidemiology of HPV and Chlamydia trachomatis pathogenic agents in a large cohort of women from a North-East Italian area: prevalence and distribution of HPV genotypes in the setting of co-infection with Chlamydia trachomatis
Full text linkBackground: Human papillomavirus (HPV) and Chlamydia trachomatis (CT) are two of the most common and widespread sexually transmitted infections (STIs) in worldwide. Recently, a central role of high risk HPV (HR-HPV) genotypes associated to CT as a co-risk factor has been established in the development of cervical cancer (CC). Therefore, to investigate a pathogen as CT, potentially implicated as oncogenic factor for its tendency to cause chronic and persistent infections, together with HPV co-infection, could have a great importance for the public health. Epidemiological data on CT chronic infection and CT/HPV co-infection prevalence are not yet well defined in Italy. The aim of this study was to investigate CT/HPV co-infection in women from a Northern-East Italian area by analyzing the HPV prevalence and genotypes distribution in women at risk for CT chronic infection.
Methods: A retrospective study (2009-2014) was conducted on a large cohort of 7135 cervical swabs. 6214 at risk for CT and 921 from women at risk for HPV infection were tested by Real Time PCR. CT genotyping was realized by ompA gene sequencing. A quantitative Real time-PCR was then performed to assess the expression of the CT Hsp60–encoding gene (Ct604 portion), linked to a persistent status of infection. CT/HPV co-infection and prevalence of single or multiple genotypes was investigated using Luminex technology.
Results: the overall prevalence of the investigated pathogens resulted to be of 39% for HPV and 4% for CT, respectively. A CT/HPV co-infection was diagnosed in the 58% of the samples from women of which 68% with chronic infection. In women ≤ 25y, CT infection reached a peak of 14%(p< 0.0001), in co-infection with HPV, 68%, with a prevalence of chronic infection in this group of 72%. Of note, a CT infection was associated to HPV multiple genotypes in 78% (p< 0.0221), of these women. By analyzing the HPV genotypes distribution, a single infection was highly detected (60%) in women infected with HPV only, while multiple infections resulted prevalent (68%), in CT/HPV co-infected women. The CT serotype F was confirmed to be the most frequent serovar found in women samples, independently from HPV infection. Of interest, the levels of CT Hsp60 expression in HPV co-infected women were confirmed significantly lower compared to women infected with CT only.
Conclusions: Our large study, adds new findings regarding the epidemiology of HPV and CT distribution in women from this geographic area, confirming a high prevalence of multiple HPV infections associated with concomitant chronic disease by CT, especially in young women. Of remarkable interest, in these co-infection specific HPV genotypes were recovered, suggesting that the expression of CT Hsp60, interfering on immunologic pathways, might influence engraftment of HPV particulars genotypes. In comparison to the National data, our study highlights both the high
frequency of CT chronic infection and CT/HPV co-infection with multiple HPV infections in young women. This suggests that an early prophylactic HPV vaccination and a screening program for CT/HPV co-infection could play a significant role in STIs prevention and possible development of CC
Efficacy of a novel reverse transcriptase-polymerase chain reaction (RT-PCR) for detecting Toxoplasma gondii bradyzoite gene expression in human clinical specimens
No full textA reverse transcriptase-polymerase chain reaction (RT-PCR) assay, was performed to evaluate the transcription degree of bradyzoite- or tachyzoite-specific genes of Toxoplasma gondii on cerebrospinal fluid (CSF) specimens from AIDS patients with toxoplasmic encephalitis (TE), and to distinguish an asymptomatic latent infection from a reactivated disease. This method was compared with nested DNA amplification (n)-PCR. The mRNA expression of the representative T. gondii cystic matrix (MAG1) or bradyzoite-specific (SAG4) genes was investigated on CSF obtained from AIDS patients with first episode (no. 11) or relapse (no. 8) of TE. The mRNA expression of tachyzoite-specific (SAG1) gene was also studied. New designed oligonucleotide primers and probes, which identify a 212 bp fragment inside to the open reading MAG1 sequence, were employed in both RT-PCR and n-PCR assays. Oligo-dT primed cDNA synthesis appeared a suitable method for subsequent analysis by n-PCR. RT-PCR has been shown to be more sensitive and specific than n-PCR. MAG1 and SAG4 gene expression was detected in 8 (100%) and 6 (75%) patients with TE relapses, respectively, while SAG1 detected 7 (63%) patients with TE first episode. These findings suggest that RT-PCR method is able to identify the bradyzoite stage of T. gondii especially in patients who are at risk for TE relapse
Chlamydie ed Artropatie. Un possibile ruolo?
No full textNegli ultimi anni la relazione tra sinoviti croniche e agenti infettivi “difficili”, tra cui Chlamydophila pneumoniae, ha suscitato l’interesse di numerosi studiosi. L’ipotesi patogenetica del “peptide artritogenico”, che prevede l’induzione di cellule T citotossiche in seguito ad una cross-reazione tra autoantigeni e antigeni batterici, appare particolarmente appropriata nel caso delle Artriti Reattive (ReA), ove tra i criteri maggiori di diagnosi rientra l’anamnesi positiva per una infezione enterica o uretrale precedente l’esordio delle manifestazioni articolari.
Tra i numerosi microorganismi chiamati in causa nelle ReA Chlamydophila appare essere il più comune; inoltre, nei monociti/macrofagi sinoviali di pazienti affetti da ReA Chlamydophila-associate sono state riscontrate forme di C. trachomatis in stato di persistenza.
Peraltro, recenti indagini molecolari sembrano confermare un ruolo di C. pneumoniae, non solo in alcuni casi di ReA, ma anche in altre forme di artropatie croniche; tuttavia il significato di tali riscontro presenta ancora numerosi aspetti controversi.
Non esistono pareri uniformi sui tests laboratoristici da utilizzare per l’identificazione di Chlamydophila anche se la PCR viene ritenuta una delle metodiche più efficaci per l’individuazione del microrganismo nel liquido sinoviale. Recentemente, lo sviluppo di un nuovo modello colturale ci ha consentito di dimostrare l’abilità di C. pneumoniae di sopravvivere all’interno di cellule mononucleate in una forma vitale e infettante.
Gli obiettivi di questo lavoro sono stati: a) valutare la presenza e la vitalità di C. pneumoniae nel sangue e nel fluido sinoviale di pazienti affetti da sinovite cronica; b) studiare l’implicazione di tale microorganismo nella patogenesi di artropatie croniche non ReA
Expression of Toxoplasmic 65 kDa Cystic mRNA by RT-PCR in Patients with Toxoplasma gondii Relapses
No full textThe 65 kDa MAG-1 antigen is abundantly located in the bradyzoite’s cyst matrix and it is poorly expressed in tachyzoites. To date no definitive systems are available to detect bradyzoites, responsible of T. gondii reactivation. Home made oligonucleotides deduced from MAG-1 gene and from 35 fold repetitive B1 gene were employed in RT-PCR and Southern blot in detecting relapses of Toxoplasma gondii encephalitis (TE), from nine AIDS patients under anti-TE prophylaxis or treatment. Cerebrospinal fluid (CSF) specimens were checked with both primers and a comparative evaluation was made. The expression of mRNA detected by MAG-1 gene was significantly higher (no. 9/9, 100%) compared to that observed for B1 gene (no. 6/9, 66.6%; p < 0.01), and a specific fragment (212 bp) was confirmed by Southern blot analysis and alignment by GenBank data base (dbEST). The MAG-1 amplified fragment, was internal to 1.2 kb exon, which codifies an antigenic fraction expressed in cystic stage only. Since MAG-1 transcription has been experimentally determined during bradyzoite conversion, our system might be useful in processing clinical specimens especially from patients with TE reactivation
Cultural and molecular isolation of Chlamydophila pneumoniae from patients with joint disease. Pathogenic and diagnostic perspectives.
No full textChlamydophila pneumoniae is an ubiquitous intracellular pathogen which causes acute respiratory diseases and may be associated with chronic inflammatory diseases including atherosclerosis, multiple sclerosis and arthritis. C. pneumoniae is rarely cultured from the synovial fluid or blood, and serology is seldom useful. So far most of the studies concerning the possible association between C. pneumoniae and arthritis have been made by molecular methods. Recent advances in the standardization of polymerase chain reaction techniques have shown to confirm a role of C. pneumoniae not only in reactive arthritis (ReA) but also in chronic arthritis. In this study, we investigated whether C. pneumoniae could be isolated in synovial fluid and PBMC specimens of patients with different forms of arthritis including ReA. Advanced PCR and Reverse transcriptase PCR techniques targeting different chlamydial genes associated to a novel culture method based on combination of additional centrifugation and extension of culture time, were applied to detect C. pneumoniae in 6 patients with chronic synovitis including one with Anchylosing Spondylitis and relapsing joint swelling. For this patient, serological, coltural as well as molecular assays did detect C. pneumoniae only. Particularly, a high expression of Heat shock protein 60 and 70 of C. pneumoniae was found in the PBMC and the synovial compartments, thus confirming the ability of C. pneumoniae to survive inside blood ad synovia in vital and metabolically active forms. By contrast, the selective decrease of MOMP and 16sRNA, leads to hypotesize a different expression of Chlamydophyla genes during the different phases of infection
Detection of clinical-stage specific molecular Toxoplasma gondii gene patterns in patients with toxoplasmic lymphadenitis
No full textThree cases of symptomatic toxoplasmic lymphadenitis, together with a serologic profile of recent infection, are described, for which quantitative real-time PCR (LightCycler PCR) targeting different parasite genes was designed, in order to quantify Toxoplasma gondii DNA in acute and follow-up blood specimens. Similar parasite gene kinetics and DNA concentrations were observed in the patients studied. However, the profile of each target gene investigated was different. While the level of B1 DNA remained elevated for the entire time of observation, irrespective of clinical and serologic resolution, the SAG-1 gene was detected at the end of acute symptomatic disease, overlapping with a strong anti-T. gondii IgA antibody response, and persisting for over 3 months after infection and clinical recovery. With respect to the two bradyzoite genes investigated (SAG-4 and MAG-1), levels peaked during the symptomatic phase, but did not fall until 2 or 3 months of follow up. The real-time PCR assay with new alternative targets to the B1 gene may have potential for monitoring the clinical outcome of disease and for providing molecular information regarding the actual state of infection
Unusual Mycoplasma detection in PBMCs of patients with underfined fever.
No full textBACKGROUND AND AIM
Infections by Mycoplasma and Chlamydia represent a challenge for the clinician, because of the changing clinical expression and the potential pathological sequelae. The routine microbiological investigations do not always provide elements for definitive diagnosis.
We retrospectively evaluated Mycoplasma and Chlamydia spp. in sera and in blood specimens from patients with fever as main symptom in the absence of other clinical or laboratory signs indicating the presence of direct or indirect causal organism.
METHODS
Serum and PBMC specimens were collected from 34 immunocompetent patients between the ages of 17 and 39 years (mean age, 28•1 years), who attended our outpatient clinic (years 2004-09) because of persistent fever, but pulmonary function test and radiography negative or not significant. Specific serum anti-Mycoplasma pneumoniae and Chlamydia Spp antibodies searched with commercial kits (M. pneumoniae IFA IgM/IgG, Delta Biologicals, Italy; Chlamydophila pneumoniae - Cp Quant IgA/G, Eurospital, Trieste, Italy), were based on M. pneumoniae FH strain (ATCC 15531) cultured in McCoy cells and on purified EB, for C. pneumoniae. For molecular techniques, PBMCs were preliminary screened by PCR for DNA Mycoplasma genus (MGSO/GPO1) and positive results were then analyzed with species-specific primers for human Mycoplasmas. C. pneumoniae and C. trachomatis, were investigated by n-PCR using primers able to amplify fragment 16s rRNA and MOMP genes. The specific products obtained, were confirmed by sequencing with ABI PRISM 3130 DNA Sequencer (Applied Biosystem,The Netherlands).
RESULTS
Mycoplasma or C. pneumoniae positive serology and or PCR were found in 4 (11.7%) and 9 (26.4%) patients respectively (Fig. 1). Among the patients with Mycoplasma infection, the most prominent symptom was low grade and persistent (for over 3 months) fever associated with fatigue (three cases) or fever with cough or sore throat (one case). These patients had a positive (IgM) serology (four, 11.7%) or PCR (three, 9%) with a positive concordance between both methods in three. M. pneumoniae DNA was found in two (5.8%), while M. hominis in one (2.9%). This last finding was also confirmed in follow-up specimens by two different species-specific primer copies. The sequence analysis also confirmed that the amplified products belonged to M. pneumoniae and M. hominis (Fig. 2). Of the 9 patients with C. pneumoniae positive finding, 6 displayed a positive IgG and IgA serology, while 7, DNA. Their symptoms were: persistent fever with chills and fatigue (two cases) and cough (one), fever alone (three) or associated with CMV infection (one), fever associated with myalgia and pneumonia (1 case, respectively).
CONCLUSIONS
In general, the distribution of Cp serology and Cp PCR was more abundant than Mp serology or Mp PCR results. These last however, were predominant in patients with fever and fatigue. This suggests that a low percentage of patients with unexplained fever and fatigue without respiratory symptoms may have systemic mycoplasmal infections. Fever is often problematical in absence of clinical or microbiological criteria. The finding of M. hominis DNA in sequential PBMCs should not be underestimated and the use of macrolide therapy may be appropriate
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