Footprints of BK and JC polyomaviruses in specimens from females affected by spontaneous abortion.

STUDY QUESTION: Are JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) infections associated with spontaneous abortion (SA)? SUMMARY ANSWER: There is no association of JCPyV or BKPyV with SA. WHAT IS KNOWN ALREADY: A large number of risk factors have been associated with SA. The role of polyomaviruses, including JCPyV and BKPyV, in SA remains to be clarified. STUDY DESIGN, SIZE, DURATION: This is a case-control study including women affected by spontaneous abortion (SA, n = 100, the cases) and women who underwent voluntary interruption of pregnancy (VI, n = 100, the controls). PARTICIPANTS/MATERIALS, SETTING, METHODS: Viral DNAs were investigated by qualitative PCR and quantitative droplet-digital PCR (ddPCR) in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from SA (n = 100) and VI (n = 100). Indirect ELISAs with mimotopes/synthetic peptides corresponding to JCPyV and BKPyV viral capsid protein 1 epitopes were then employed to investigate specific IgG antibodies against JCPyV and BKPyV in human sera from SA (n = 80) and VI (n = 80) cohorts. MAIN RESULTS AND THE ROLE OF CHANCE: JCPyV DNA was detected in 51% and 61% of SA and VI samples, respectively, with a mean viral DNA load of 7.92 copy/104 cells in SA and 5.91 copy/104 cells in VI (P > 0.05); BKPyV DNA was detected in 11% and 12% of SA and VI specimens, respectively, with a mean viral DNA load of 2.7 copy/104 cells in SA and 3.08 copy/104 cells in VI (P > 0.05). JCPyV was more prevalent than BKPyV in both SA and VI specimens (P < 0.0001). In PBMCs from the SA and VI cohorts, JCPyV DNA was detected with a prevalence of 8% and 12%, respectively, with a mean viral DNA load of 2.29 copy/104 cells in SA and 1.88 copy/104 cells in VI (P > 0.05). The overall prevalence of serum IgG antibodies against JCPyV detected by indirect ELISAs was 52.5% and 48.7% in SA and VI groups, respectively, whereas BKPyV-positive sera were found in 80% SA and 78.7% VI samples. LIMITATIONS, REASONS FOR CAUTION: This study did not investigate the presence of viral mRNA and/or proteins, which are indicative of an active viral infection, and these might be taken into consideration in future studies. WIDER IMPLICATIONS OF THE FINDINGS: JCPyV and BKPyV DNA sequences were detected and quantitatively analyzed for the first time by PCR/ddPCR in chorionic villi tissues and PBMCs from SA and VI specimens. Moreover specific immunological approaches detected serum IgG against JCPyV/BKPyV. Statistical analyses, however, do not indicate an association between these polyomaviruses and SA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University of Ferrara, FAR research grants and the University Hospital of Ferrara/University of Ferrara joint grant. No potential conflicts of interest were disclosed.

Rates of antibiotic resistance/sensitivity in bacterial cultures of hidradenitis suppurativa patients.

BACKGROUND: Antibiotic (AB) treatment is one of the first steps in the management of hidradenitis suppurativa (HS). Bacteria, in HS patients, may play a double role, as triggering factors of inflammatory reactions and/or agents of infection. OBJECTIVES: The aims of this study are as follows: (i) to assess prevalence and AB resistance of bacterial growths in HS patients (ii) assessment of the clinical relevance of obtained data in guiding the selection of the most effective AB therapy. METHODS: Purulent material from 137 skin lesions of HS patients was collected with swabs. Bacterial flora and AB sensitivity were determined using microbiological cultures for aerobic and anaerobic bacteria. RESULTS: A total of 114 samples resulted positive for bacteria. Sample was collected from the axillae, groin and perianal areas. A total of 163 single bacterial growths were observed; 55% were Gram-positive and 44% were Gram-negative. Among them, 18.4% were anaerobic. The most frequent bacterial families included enterobacteriaceae (30.7%), Staphylococcus (25.2%) and Streptococcus (14.1%). The most frequent genus or species were proteus spp. (13.5%) and Escherichia coli (9.8%). The prevalence of AB resistance observed was clindamycin 65.7%, rifampicin 69.3%, penicillin 70.0%, ciprofloxacin 74%, tetracycline 84.7% and erythromycin 89.0%. A limitation of the study is represented the short culture period adopted which may have impaired the isolation of anaerobes. CONCLUSIONS: Bacterial growth in HS patients has shown a high level of resistance to ABs, including rifampicin, clindamycin and tetracyclines, cited as an empiric choice in HS therapeutic guidelines. A targeted and specific AB therapy, driven by microbiological evaluations with prolonged culture periods, seems more appropriate than empiric, generic, non-specific, therapeutic approaches. Current knowledge regarding HS bacterial AB resistance should be considered in the update of current therapeutic guidelines for HS.

Diagnosis of a neonatal ophthalmic discharge, Ophthalmia neonatorum, in the molecular age: investigation for a correct therapy.

An early double case of acute Ophthalmia neonatorum in 3-day-old twins is reported. Culture of eye swabs showed a wide bacterial polymorphism, in which common bacteria, such as Klebsiella pneumoniae, Streptococcus pneumoniae, Corynebacterium ulcerans and other Enterobacteriaceae, coexisted with atypical Mycoplasmataceae and Chlamydiaceae from resident cervical-vaginal maternal microbiota. The neonates were in an apparently healthy state, but showed red eyes with abundant greenish-yellow secretion, mild chemosis and lid edema. The maternal cervical-vaginal ecosystem resulted differently positive to the same common cultivable, atypical bacteria culturally and molecularly determined. This suggested a direct maternal-foetal transmission or a further foetal contamination before birth. An extended culture analysis for common bacteria to atypical ones was decisive to describe the involvement of Mycoplasmas (M. hominis and U. urealyticum) within the scenario of the Ophthalmia neonatorum in a Caucasian couple. The introduction of a routine PCR molecular analysis for Chlamydiaceae and N. gonorrhoeae allowed to establish which of these were present at birth, and contributed to determine the correct laboratory diagnosis and to define an adequate therapeutic protocol obtaining a complete resolution after one year for culture and atypical bacteria controls. This study suggests to improve the quality of laboratory diagnosis as unavoidable support to a correct clinical diagnosis and therapy, in a standardized modality both for swabbing and scraping, to check the new-born microbial programming starting in uterus, overtaking the cultural age to the molecular age, and to revise the WHO guidelines of SAFE Strategy for trachoma eye disease, transforming it into SAFES Strategy where the S letter is the acronym of Sexual ecosystem and behavioural valuation/education.

Severe pneumonia after intravesical BCG instillation in a patient with invasive bladder cancer: case report and literature review.

We present here the case of a 66 year old man with a severe bilateral community acquired pneumonia secondary to dissemination after an intravesical instillation of bacilllus Calmette-Guerin (BCG). Diagnosis was based on positive polymerase chain reaction (PCR) for mycobacterium tuberculosis complex in bronchoalveolar lavage and on the finding on transbronchial biopsy of non necrotising granulomas histopathologically similar to the granulomas found in bladder biopsies. These findings were confirmed using a validated real time PCR assay demonstrating the presence of the BCG genome in transbronchial and bladder biopsies.

Redescription of the male of Ixodes festai Rondelli, 1926 (Ixodida: Ixodidae) on specimens from Sardinia (Italy).

Ixodes festai Rondelli, 1926 is a poorly known bird parasite tick. Its immature forms have not been described yet, while the adult forms only insufficiently, especially the male. In this note the presence of the male of Ixodes festai for the first time in Sardinia (Italy) is reported and a detailed redescription is provided. Morphometric data as well as photographs performed both with optical and electron microscope (ESEM FEI Quanta 200) are also shown.

Thymosin ß4 and ß10 are highly expressed at the deep infiltrative margins of colorectal cancer - A mass spectrometry analysis.

OBJECTIVE: Colorectal cancer (CRC) is a complicated tumor, involving several oncogenic signaling pathways, and with a molecular mechanism not fully understood yet. The implication of thymosin ß4 (Tß4) with tumor insurgence and in migration of CRC cells was evidenced in the past with different methodologies, while Tß10 connection with CRC has been sporadically investigated. This study focused on the implication of both types of thymosin in CRC progression and invasion by analyzing the changes in their levels according to different zones of the tumor, and to Dukes stage and budding index. PATIENTS AND METHODS: Tß4 and Tß10 were analyzed in deep and superficial tumor samples, and normal mucosa from 18 patients. Concentrations of Tß4 and Tß10 have been measured by high-pressure liquid chromatography (HPLC) coupled to electrospray-ion trap mass spectrometry (ESI-IT-MS). MS data were compared by t-test and ANOVA statistical analysis. Identification of thymosin and their proteoforms has been performed by HPLC-high resolution-ESI-IT-MSMS. RESULTS: Both Tß4 and Tß10, exhibited intra-tumoral quantitative differences, being upregulated in the deep part of the CRC. They exhibited, moreover, strong association with the Dukes stage and the budding grade, being more concentrated in patients at Dukes stage B and with budding index "2". CONCLUSIONS: The results obtained in the present investigation encouraged the hypothesis that the two thymosin are involved in colorectal cancer progression, and in promoting cancer invasion. Thus, they are good candidates to be diagnostic/prognostic biomarkers and therapy targets.

Argas (Persicargas) persicus (Oken, 1818) (Ixodida: Argasidae) in Sicily with considerations about its Italian and West-Mediterranean distribution.

Recently, in the province of Trapani (Western Sicily), some overwintering specimens of the argasid tick Argos (Persicargas) persicus (Oken, 1818) were observed and collected. Morphological and genetic analysis were utilized in order to reach a definitive identification. The species was found in two semi-natural sites where, having been found repeatedly, its presence does not appear accidental. Moreover the characteristics of the Sicilian findings seem to exclude a human-induced spread. This record, the first regarding Sicily and South Italy, is discussed together with the previous doubtful citations for Italy. These findings revalue not only all the old citations for Italy but also the hypothesis that the Mediterranean distribution of this argasid is of a natural origin.

Detection of parvovirus B19 and Chlamydophila pneumoniae in a patient with atypical sarcoidosis.

We present an elderly female patient with fever, aplastic anemia, arthralgic symptoms and atypical pneumonia. Serological and clinical findings suggested Parvovirus B19 and Chlamydophila pneumoniae infection. These supposed infections delayed the recognition of underlying sarcoidosis which definitive diagnosis was reached through a lung biopsy and histological demonstration of nonnecrotizing granulomas containing giant cells and noncaseating epithelioid cells. The present case highlights the potential difficulty to diagnose sarcoidosis in the presence of unusual infections which may complicate the course of this disease.

Clinical and diagnostic management of toxoplasmosis in the immunocompromised patient.

With the advent of the highly active antiretroviral therapy (HAART), the natural course of HIV infection has markedly changed and opportunistic infections including toxoplasmosis have declined and modified in presentation, outcome and incidence. However, TE is a major cause of morbidity and mortality especially in resource-poor settings but also a common neurological complication in some countries despite the availability of HAART and effective prophylaxis. In most cases toxoplasmosis occurs in brain and toxoplasmic encephalitis (TE) is the most common presentation of toxoplasmosis in immunocompromised patients with or without AIDS. The need of a definitive diagnosis is substantial because other brain diseases could share similar findings. Rapid and specific diagnosis is thus crucial as early treatment may improve the clinical outcome. Classical serological diagnosis is often inconclusive as immunodeficient individuals fail to produce significant titres of specific antibodies. Polymerase chain reaction (PCR) has a high diagnostic value in the acute disease, but like many 'in-house' PCR assays, suffers from lack of standardization and variable performance according to the laboratory. Molecular diagnosis of toxoplasmosis can be improved by performing real-time PCR protocols. This article summarises the clinical manifestations, diagnostic procedures and management strategies for this condition.

Treatment of very preterm preeclampsia via heparin-mediated extracorporeal LDL-precipitation (H.E.L.P.) apheresis: The Freiburg preeclampsia H.E.L.P.-Apheresis study.

OBJECTIVE: Soluble Fms-like tyrosine kinase-1 (sFlt-1) is thought to be causative in the pathogenesis of preeclampsia (PE) and specific removal of sFlt-1 via dextran sulfate cellulose (DSC)-apheresis was suggested as cure to allow prolongation of pregnancy in preterm PE. However, in addition a deranged lipoprotein metabolism may impact endothelial and placental function in PE. Lipoprotein-apheresis by heparin-mediated extracorporeal LDL-precipitation (H.E.L.P.) was previously applied and has been shown to alleviate symptoms in PE. This clinical trial reevaluates the clinical efficacy of H.E.L.P.-apheresis in PE considering sFlt-1. STUDY DESIGN: Open pilot study assessing the prolongation by H.E.L.P.-apheresis in 6 women (30-41 years) with very preterm PE (24+4 to 27+0 gestational weeks (GW)) (NCT01967355) compared to a historic control-group matched for GW at admission (<28 GW; n = 6). Clinical outcome of mothers and babies, and pre- and post H.E.L.P.-apheresis levels of sFlt-1 and PlGF were monitored. MAIN OUTCOME MEASURES: In apheresis patients (2-6 treatments), average time from admission to birth was 15.0 days (6.3 days in controls; p = 0.027). Lung maturation was induced in all treated cases, and all children were released in healthy condition. Apheresis reduced triglycerides and LDL-cholesterol by more than 40%. Although H.E.L.P.-apheresis induced a transient peak baseline levels did not change and rather stabilized sFlt-1 levels at pre-apheresis levels throughout treatments, with sFlt-1/PLGF ratio remaining unaffected. CONCLUSIONS: H.E.L.P.-apheresis proved again to be safe and prolongs pregnancies in PE. However, without changing sFlt-1 levels below baseline lowering lipids or other yet undefined factors appear to be of more relevance than reducing sFlt-1.

Aedes albopictus in Sardinia: reappearance or widespread colonization?

The Asian tiger mosquito Aedes albopictus (Skuse, 1894) was first discovered in the South of Sardinia in October 1994, in a tyre depot not far from Cagliari-Elmas airport. Insecticide treatment was thought to have successfully eradicated the mosquito, but in 1996 and 1997 new breeding sites were discovered, a few at some distance from the first. More recently two sites have been reported in the heart of the city of Cagliari. It is not known whether the mosquito has spread from the first breeding place discovered, where treatment may not have been definitive, or whether they have been newly introduced. The recent sighting of Ae. albopictus in Olbia in the Northeast of the island tends to suggest the latter. Cagliari and Olbia are actually Sardinia's two largest sea ports of entry.

Production of plasminogen activator and plasminogen activator inhibitors by alveolar macrophages in control subjects and AIDS patients.

OBJECTIVE: To reveal a possible impairment of the plasminogen activator system in the pulmonary infections of AIDS patients. DESIGN: To test the plasminogen activator system functionality in alveolar macrophages and bronchoalveolar lavage fluid (BALF) in control subjects and AIDS patients. Procedures were designed to detect the presence of imbalance in plasminogen activator activity and to ascertain if this imbalance is due to a direct effect of the HIV virus on macrophages or to superimposed opportunistic infection. METHODS: Alveolar macrophages obtained by bronchoalveolar lavage (BAL) were either lysed with Triton X-100 or cultured for 24 h. Plasminogen activators and plasminogen activator inhibitors (PAI) were measured by chromogenic substrate assay and binding to 125I-urokinase followed by 10% sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. RESULTS: Plasminogen activator activity in BALF and in alveolar macrophages from AIDS patients was decreased. This reduction was independent of the presence of an infectious pulmonary process. In contrast, free PAI was increased in AIDS patients with Pneumocystis carinii infection. This increase is possibly caused by a different glycosylated form of PAI-2. CONCLUSIONS: Our data support the view that the pulmonary fibrogenic response is in part secondary to an imbalance within the plasminogen activator system and provide the basis for clarifying the role of these alterations in the pathophysiology of AIDS-related pulmonary infections.

Evidence of cerebrospinal fluid free kappa light chains in AIDS patients with Toxoplasma gondii encephalitis.

Cerebrospinal fluid (CSF) free light chains of kappa or lambda (FLC kappa/lambda) type were investigated by affinity mediated blotting technique (AMI) and ELISA in 28 patients of which nine with AIDS and Toxoplasma gondii encephalitis (AIDS, TE), 11 with AIDS with or without other CNS AIDS-related opportunistic infections (non-TE AIDS) and eight control patients with or without inflammatory neurological disorders (control group). CSF restricted oligoclonal FLC bands either of k or lambda isotype or both were found by AMI in 18 (90%) out of 20 AIDS patients, while a CSF pattern predominantly characterized by FkappaLC rather than FlambdaLC was observed in eight (88.8%) out of nine TE patients. No FLC components were detected in the matched sera of TE or non-TE AIDS patients or in the CSF and sera from control group. The anti-parasite-specific FkappaLC CSF/serum mean levels and the T. gondii-specific FkappaLC index values were found by ELISA to be significantly more elevated in TE patients when compared to non-TE AIDS or control group. These findings suggest that the increased production of T. gondii-specific FkappaLC could provide insights into pathogenesis of reactivated TE in immunocompromised patients and may have important diagnostic usefulness.

Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECL-western blotting.

The polymerase chain reaction (PCR) for detection of cerebral spinal fluid (CSF) Toxoplasma gondii DNA was combined with the study of intrathecal antibody synthesis by antibody specific index calculation (ASI) and the detection of specific oligoclonal IgG bands (OCB) by affinity mediated immunoblotting (AMI) in 11 AIDS patients with T. gondii encephalitis (TE) and in 20 control patients with or without neurological disorders. Enhanced chemiluminescence (ECL) western-blot technique was employed to evaluate the antigenic specificity of CSF-IgG towards individual T. gondii antigens. PCR was positive in all TE patients which displayed brain-derived or blood-derived specific OCB, even when comparative ASI failed. Four TE patients had a unique anti-T. gondii OCB restricted to the CSF and a strong antibody response toward the 29 kDa band by ECL western blot. This response could be an important marker to discriminate TE from other opportunistic central nervous system (CNS) infections in the course of AIDS.

Assessment of HIV-intrathecal humoral immune response in AIDS-related neurological disorders.

Intrathecal synthesis of IgG directed to HIV antigens was investigated by antibody specific index (ASI), affinity-mediated immunoblot (AMI) and Western blot (WB) assay in a group of 88 AIDS patients of which 28 with HIV-associated neurological disorders (HAND), 13 without associated neurological disorders (WAND) and 47 with non-HIV-associated neurological disorders (non-HAND). CD4+ count was above 50 cells/mm3 (CD4+>50) in 30 and below 50/mm3 (CD4+<50) in 58 patients, respectively. A significantly higher frequency for CSF complete anti-gag profile (p<0.001), and for HIV-specific oligoclonal patterns ("mixed" pattern=p<0.01) was observed in HAND as compared to patterns from the other clinical groups. A decrease in complete anti-env, anti-pol and anti-gag reactivity was present in CSF of patients with CD4+<50 as compared to those with CD4+>50. Our findings suggest that AIDS appears to be characterized by an anti-HIV intrathecal humoral immune response which is principally directed to env products with a prevalence of oligoclonal patterns and CSF complete anti-gag profile in HIV-associated neurological involvement.

Evidence of Pneumocystis carinii in cell line cultures infected with peripheral blood mononuclear cells isolated from AIDS patients with P. carinii pneumonia.

The detection of Pneumocystis carinii was investigated in an in-vitro system consisting of a human lung epithelial cell line (A-549) inoculated with infected peripheral blood mononuclear cells (PMBC) from HIV-infected patients with proven or suspected P. carinii pneumonia (PCP), and from HIV-negative patients with other lung infections. Supernates from cultures were sampled daily and evaluated for the presence of P. carinii by Giemsa and immunofluorescence staining. P. carinii was isolated from 98 (95.1%) of 103 culture supernate samples from patients with proven pneumocystosis and 45 (66.1%) of 68 from patients with suspected PCP 40 or 72 h after PBMC inoculation. This system has been shown to support the growth of P. carinii but did not seem to be adequate for the production of large numbers of organisms, although long-term survival in vitro for up to 3 weeks was observed. Recovery of P. carinii from infected PBMC strongly supports previous observations about its ability to disseminate haematogenously and could represent a further advance in understanding the pathogenesis and diagnosis of PCP.

Use of PCR to detect mycoplasma DNA in respiratory tract specimens from adult HIV-positive patients.

The polymerase chain reaction (PCR) was evaluated retrospectively for its ability to detect Ureaplasma urealyticum and Mycoplasma spp. in respiratory tract specimens obtained from adult patients with AIDS. Mycoplasma DNA was detected in specimens from 12 of 84 patients. Of the 107 specimens tested, 13 and seven positive PCR results were obtained with the genus- and species-specific oligonucleotide primers used, respectively, in two different steps. With the latter, one sample was positive for U. urealyticum plus M. hominis, another for M. fermentans plus M. salivarium, and five others were positive for M. salivarium. The unexpected detection of U. urealyticum DNA in respiratory secretions from an adult AIDS patient suggested that this urogenital mycoplasma could have a role in determining or exacerbating respiratory tract infections in the HIV-positive population, but that its low rate of isolation could be related to the frequent failure of methods used currently to detect mycoplasmas.

Investigation of the simian polyomavirus SV40 as a potential causative agent of human neurological disorders in AIDS patients.

Neurological diseases and a variety of neoplasms frequently occur in AIDS patients. Human JC and BK polyomaviruses have been associated with neurological disorders in such patients. SV40 polyomavirus sequences have been detected in human brain tumours, other neoplasms and normal tissues. JCV, BKV and SV40 DNA sequences were investigated in cerebrospinal fluid (CSF) samples from 12 AIDS patients affected by different neurological disorders, by PCR assay and filter hybridisation with specific internal oligoprobes, and DNA sequencing. Three of the 12 CSF samples were positive for JCV (one sample) or SV40 (one) DNA, or both (one). No sample was positive for BKV DNA. JCV- and SV40-specific genomic regions were confirmed by DNA sequencing. CSF samples from the two patients diagnosed clinically as having progressive multifocal leukoencephalopathy (PML) contained either JCV (one sample) or SV40 (one) DNA. The CSF found to contain both JCV and SV40 DNA originated from a patient with a cerebral mass lesion of unknown aetiology. These results suggest that SV40 may be involved in the aetiology of PML in AIDS patients, and raise the possibility that SV40 and JCV may act synergically in vivo to enhance their pathogenicity.

Cytomegalovirus in bronchoalveolar lavage specimens from patients with AIDS: comparison with antigenaemia and viraemia.

Pulmonary infection with cytomegalovirus (CMV) is a well recognised complication of AIDS. It is often possible to detect CMV-infected cells in bronchoalveolar lavage (BAL) specimens with monoclonal antibodies, but the clinical significance of their presence remains unclear. To investigate this, 24 AIDS patients were tested for CMV antigenaemia and viraemia, in addition to CMV detection in BAL. CMV was detected in the BAL of nine patients (38%), five with clinical and laboratory evidence of pulmonary infection and four without pulmonary involvement. Blood samples positive for CMV antigen were observed in two patients with CMV-positive BAL specimens and, in both cases, antigenaemia resolved without therapy. No case of viraemia was detected. Pneumocystis carinii was detected concomitantly with CMV in the BAL of four of the patients with pulmonary involvement and in one without signs of pulmonary infection. These data suggest that CMV-positive BAL results are of limited significance in the diagnosis of CMV pneumonia in AIDS patients, unless associated with high levels of antigenaemia or viraemia and compatible clinical symptoms.