Difficulties in the Mutation Analysis of Plasminogen Gene: A Study in Two Patients with Ligneous Conjunctivitis

The absence or very low levels of plasminogen cause a rare disabling disease called ligneous conjunctivitis, characterized by the growth of fibrin-rich pseudomembranes in the conjunctiva and on other mucosal surfaces. Several mutations have been detected in the plasminogen gene of patients affected with ligneous conjunctivitis. The human plasminogen gene, located on chromosome 6, has a marked homology with the genes belonging to the plasminogenapo(a) family, and with a number of pseudogenes and plasminogen-like genes located on chromosome 2. This work describes a series of nucleotide variations related to genes other than the plasminogen one, found during the genetic characterization of plasminogen defect in two unrelated patients with ligneous conjunctivitis. The results of automated sequences of each exon and intron-exon boundaries were compared with those of the human plasminogen gene from the NCBI gene bank. In particular, a co-amplified gene on chromosome 2 mimicking a 14 bp deletion in exon 5 of the plasminogen gene was identified by sequencing two different bands obtained from a long run of the PCR exon 5 product in NuSieve agarose gel, and by PstI restriction enzyme analysis of the same amplicons. Moreover, 21 single nucleotide exchanges due to plasminogen-like genes co-amplification were observed, namely one in exon 1, two in exon 4, three in exons 3, 5 and 16, four in exon 13, and five in exon 17. In conclusion, these data confirm the difficulty of plasminogen genetic analysis and may help researchers to better identify the true plasminogen gene mutations causing molecular defects

Viral infections of the central nervous system in elderly patients: a retrospective study

Summary Objectives Very few data exist on viral meningitis and encephalitis in elderly patients (>65 years old). Methods This study investigated the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, cytomegalovirus (CMV), Epstein–Barr virus (EBV), enterovirus (EV), human adenovirus (HAdV), human parechoviruses (HPeVs), and tick-borne encephalitis virus (TBEV) through real-time PCR (RT-PCR) in patients >65 years old who had cerebrospinal fluid (CSF) tested for a suspected central nervous system infection. Results A total of 2868 RT-PCRs were performed on 502 CSF samples. Overall, 65 positive RT-PCRs were found: 23 for HSV (35.4% of positives), 15 for EV (23.1% of positives), 14 for EBV (21.5% of positives), 12 for VZV (18.5% of positives), and one for CMV (1.5% of positives). A positive RT-PCR in CSF was detected in 24 (17.4%) patients aged ≥80 years and in 35 (9.6%) patients aged 65–79 years ( p =0.02). VZV was more frequently detected in the oldest subjects (5.9% vs. 1.6%, p =0.03). Conclusions HSV was the most common viral aetiology identified in the study, with VZV infection being recognized more frequently in those patients aged ≥80 years

Subclinical finding in the perception of tactile sensation involvement after SARS-CoV2 infection: comparison with healthy controls using Semmes–Weinstein monofilament testing

BackgroundPost-acute COVID-19 syndrome patients complain of sensory alterations, mainly positive symptoms such as paresthesia or neuropathic pain but also decreased tactile sensation. Using the Semmes–Weinstein monofilament test (SWMT), our study aims to confront recently infected SARS-CoV2 subjects with a control group.MethodsThis is a cross-sectional, single-centric study. We performed the SWMT (North Coast Medical Inc.) on 30 patients with previous SARS-CoV2 infection (COVID group) and 46 controls (control group). These patients did not present comorbidities or sensory impairment and did not take any medications. The control group tested negative for SARS-CoV2 infection since the COVID-19 pandemic; the COVID group was examined for this study after the resolution of the infection. We tested the threshold of tactile sensation of the tips of the thumb, index, and little finger of each hand, one hand at a time; the dorsum and the hypothenar regions were also tested.ResultsBoth groups presented the perception of tactile sensation within the reference value. Despite this result, subclinical changes suggestive of the involvement in peripheral sensory nerve function have been identified in the tested sites in the COVID group compared to the control group. The overall mean target force (grams) was higher in the COVID group than in the control group: 27 (7) vs. 19 (10) mg, p < 0.001.ConclusionControls and the COVID group infection had normal tactile sensation thresholds. However, the COVID group presented a higher threshold than the control group, suggesting a possible subclinical perception of tactile sensation involvement of A-beta nerve fibers

Daclatasvir plasma level and resistance selection in HIV patients with hepatitis C virus cirrhosis treated with daclatasvir, sofosbuvir, and ribavirin

ObjectivesEffective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV–HCV co-infected patients.MethodsThis study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV–HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing.ResultsDCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA <12 IU/ml at week 12 and undetectable at week 16.ConclusionsSub-optimal DCV drug levels allow the selection of resistance-associated variants and fail to contribute to antiviral activity. No definite reason for the low DCV level was found. Quantifying the drug is suggested in difficult-to-treat patients

Serum levels of VCAM-1 are associated with survival in patients treated with nivolumab for NSCLC

Background High circulating levels of cellular adhesion molecules (CAMs) in non-small cell lung cancer (NSCLC) have been supposed to act as a negative prognostic factor. Here, we explored the predictive role of pre-treatment levels of CAMs in previously treated patients receiving nivolumab for NSCLC. Materials and methods Seventy one patients with advanced NSCLC, treated with nivolumab at the dose of 3 mg/kg every 14 days, were enrolled. Maximum follow-up time was 3 years. Serum levels of Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intracellular Adhesion Molecule-1 (ICAM-1) were measured at baseline and before each nivolumab administration. Endpoints of the study were a composite outcome of survival &gt;= 2 years or absence of disease progression at the end of the follow-up, and the overall survival. Results Composite outcome and overall survival were positively associated with VCAM-1 baseline levels and with the reduction of VCAM-1 during the treatment. After adjustment for potential confounders, the change in VCAM-1 serum levels during the treatment was an independent predictor of overall survival. Conclusions High baseline serum levels of VCAM-1 are associated with a longer survival in patients treated with nivolumab as second line treatment for NSCLC. Surviving patients experience also a significant reduction in CAMs expression during the treatment. Hence, CAMs might be promising prognostic factors in patients with NSCLC underoing immunotherapy

Anal and oral human papillomavirus (HPV) infection in HIV-infected subjects in northern Italy: a longitudinal cohort study among men who have sex with men

<p>Abstract</p> <p>Background</p> <p>A study including 166 subjects was performed to investigate the frequency and persistence over a 6-month interval of concurrent oral and anal Human Papillomavirus (HPV) infections in Human Immunodeficiency Virus (HIV)-infected men who have sex with men (MSM).</p> <p>Methods</p> <p>Patients with no previously documented HPV-related anogenital lesion/disease were recruited to participate in a longitudinal study. Polymerase chain reaction (PCR) was performed to detect HPV from oral and anal swabs and to detect Human Herpes Virus 8 (HHV-8) DNA in saliva on 2 separate specimen series, one collected at baseline and the other collected 6 months later. A multivariate logistic analysis was performed using anal HPV infection as the dependent variable versus a set of covariates: age, HIV plasma viral load, CD4+ count, hepatitis B virus (HBV) serology, hepatitis C virus (HCV) serology, syphilis serology and HHV-8 viral shedding. A stepwise elimination of covariates with a p-value > 0.1 was performed.</p> <p>Results</p> <p>The overall prevalence of HPV did not vary significantly between the baseline and the follow-up, either in the oral (20.1 and 21.3%, respectively) or the anal specimens (88.6 and 86.3%). The prevalence of high-risk (HR) genotypes among the HPV-positive specimens was similar in the oral and anal infections (mean values 24.3% and 20.9%). Among 68 patients with either a HR, low-risk (LR) or undetermined genotype at baseline, 75% had persistent HPV and the persistence rates were 71.4% in HR infections and 76.7% in LR infections. There was a lack of genotype concordance between oral and anal HPV samples. The prevalence of HR HPV in anus appeared to be higher in the younger patients, peaking (> 25%) in the 43-50 years age group. A decrease of the high level of anal prevalence of all genotypes of HPV in the patients > 50 years was evident. HHV-8 oral shedding was positively related to HPV anal infection (p = 0.0046). A significant correlation was found between the persistence of HHV-8 shedding and HIV viral load by logistic bivariate analysis (Odds Ratio of HHV-8 persistence for 1-log increase of HIV viral load = 1.725 ± 0.397, p = 0.018).</p> <p>Conclusions</p> <p>A high prevalence of HPV infection was found in our cohort of HIV-infected MSM, with a negative correlation between anal HPV infection and CD4 cell count.</p

A dysfunctional factor X (factor X San Giovanni Rotondo) present at homozygous and double heterozygous level: Identification of a novel microdeletion (delC556) and missense mutation (Lys(408)-> Asn) in the factor X gene - A study of an Italian family

Abstract Low levels of factor X (F.X) were detected in a 4-year-old boy who experienced acute lymphoblastic leukemia and bleeding manifestations. Laboratory data suggested the presence of a dysfunctional F.X molecule. Two novel F.X gene mutations were identified in the proband that was double heterozygous for both: a microdeletion (delC556) in exon VI resulting in a frameshift leading to a termination codon at position 226. This deletion was found in six family members with reduced F.X antigen and activity levels. A second mutation characterised by a G(1344)-->C transversion in exon VIII was detected in the proband resulting in a Lys(408)-->Asn substitution. This latter mutation was present in several asymptomatic family members from the paternal and the maternal side. The proband's sister was homozygous for the Lys(408)-->Asn substitution and exhibited low F.X activity with a normal antigen level. The naturally occurring F.X Lys(408)-->Asn (F.X(K408N)) variant was isolated from plasma of either homozygous or double heterozygous individuals. NH(2)-terminal sequencing of the heavy chain of F.X(K408N) failed to show any sequence abnormality in patients who were also carriers of the delC556, suggesting that this latter lesion accounted for the lack of F.X synthesis. Purified F.X Lys(408)-->Asn had an identical behaviour to normal F.X as judged by SDS-PAGE and immunoblotting. Clotting assay using purified F.X(K408N) and F.X-deficient plasma resulted in a laboratory phenotype similar to that observed in a homozygous subject for F.X Lys(408)-->Asn substitution. This is the first characterisation of a naturally occurring F.X variant with a mutation at the COOH-terminal end of the molecule

Conformation sensitive gel electrophoresis for detection of factor X gene mutations

Fifteen patients from five families with laboratory data suggesting factor X (FX) deficiency were screened for causative mutations by conformation sensitive gel electrophoresis (CSGE) followed by sequencing. All exonic and flanking intronic regions of factor X gene were amplified using PCR. After heteroduplex formation, samples were analyzed onto a polyacrylamide gel for possible mismatch. An abnormal CSGE profile indicating an heteroduplex was identified in 10/15 cases. All the 10 patients with a patter of migration suggesting a mismatch had a laboratoristic pattern of FX deficiency whereas the five cases with a normal CSGE aspect referred to the normal components of the families who did not carry any FX defect. Sequencing demonstrated that the 10 exons, which showed a suspect CSGE pattern, harbored a mutation responsible for the factor X defect. Of the five mutation identified, two were recognized to be novel mutations (a 871C>T substitution and a 11696>T transversion in exon 8), both located in the catalytic portion of FX. CSGE may be an effective and simple procedure for screening factor X gene mutation

Distribution of human papillomavirus types in the anogenital tract of females and males.

Human papillomavirus (HPV) infection is the most common sexually transmitted infection in both men and women, but there are limited data comparing the prevalence of HPV infection between genders and in different anogenital sites. This cross-sectional analysis describes the distribution of HPV types in the genital tract of 3,410 consecutive females and 1,033 males undergoing voluntary screening for HPV and referred to a single institution. The relationship between specific HPV types and the presence of anogenital lesions was examined. In both females and males, the overall prevalence of HPV infection was about 40%. A wide variety of HPV types was identified, but the prevalence of different types was remarkably similar in the two genders, even when considering different anatomical sites. HPV-6 was the most frequent (prevalence 13%) type in all anogenital sites in men followed by HPV-16 (7%), while HPV-16 was the most common type in women (about 6%), either in the cervix, vagina, or vulva, followed by HPV-6. In addition to HPV-16, HPV-58, HPV-33, HPV-31, and HPV-56 were the carcinogenic types detected most commonly and were significantly associated with high-grade squamous intraepithelial cervical lesions, while HPV-53 and HPV-66 were the most common among possibly carcinogenic types. In both genders, anogenital warts were associated with HPV-6 and HPV-11 infection, and, less frequently, with other types, like HPV-54, HPV-62, and HPV-66. These results show that genital HPV infection involves numerous HPV types, which have similar distribution patterns in females and males and in different anogenital anatomical sites