Manuel Rodríguez-Iglesias

Comparison of human papillomavirus genotyping using commercial assays based on PCR and reverse hybridization methods

Different tests for human papillomavirus (HPV) screening are commercially available, detecting high‐risk oncogenic HPV types with a pool of genotype‐specific probes. However, it is necessary to establish reliable methods for the identification of individual genotypes. The purpose of this study was to compare three different commercial methods for HPV genotyping: INNO‐LiPA HPV Genotyping v2 (LiPA), Linear Arrays HPV Genotyping Test (LA) and Clinical Arrays Human Papillomavirus (CA). A total of 83 HPV DNA‐positive samples by hybrid capture method were genotyped (82, 78 and 81 by LiPA, LA and CA, respectively). Comparison analysis was limited to the HPV genotypes common to the three assays. There were concordant results (absolute agreement between assays) in 31 samples (39.7%) and compatible results (correspondence for some but not all genotypes) were found in 44 samples (56.4%). Only three samples (3.8%) were considered as discordant (did not show any similarity between the tests). Analyzing kappa values we have a very good agreement (>0.8) for HPV16 and HPV31 and good agreement (0.6–0.8) for HPV types 6, 18, 53 and 66 when all methods are compared. We conclude that all genotyping methods tested are highly comparable and suitable for clinical and epidemiological studies.

Hepatitis C virus core protein up-regulates anergy-related genes and a new set of genes, which affects T cell homeostasis

Hepatitis C virus (HCV) infection is the main cause for chronic hepatitis, leading to cirrhosis and hepatic carcinoma. Virally induced immune dysfunction has been called as the cause for viral persistence. Previous results demonstrate that CD4 Jurkat cells stably expressing the HCV core protein show an increased activation of NFAT transcription factor and an impaired IL‐2 promoter activity, affecting intracellular signaling pathways in a manner that mimics clonal anergy. We had shown previously that NFAT activates a transcriptional program, ensuing in immunological tolerance. In the present work, we have engineered lentiviral vectors expressing the HCV core to analyze the events, which unfold in the initial phase of HCV core‐induced anergy. We show that genes initially described to be up‐regulated by ionomycin‐induced anergy in mice are also up‐regulated in humans, not only by ionomycin but also by HCV core expression. We also show that HCV core is sufficient to cause NFAT nuclear translocation and a slow‐down in cell‐cycle progression, and using whole genome microarrays, we identify novel genes up‐regulated in Jurkat cells expressing HCV core. The relevance of our results is highlighted by the presence of HCV in CD4 T cells from HCV chronically infected patients.

Performance of the New INNO-LiPA HPV Extra to Genotype Human Papillomavirus in Cervical Cell Specimens

Objective: It was our aim to compare the INNO-LiPA HPV Genotyping Extra (LiPA; Innogenetics) versus the Linear Array test (LA; Roche) in cervical samples. Study Design: One hundred cervical samples were selected, obtained from a cancer prevention center. All samples were human papillomavirus (HPV) DNA positive by Hybrid Capture 2 HPV test and genotyped by LA HPV genotyping test. All data were analyzed using Cohen’s ĸ tests. ĸ values ranged from poor, fair, moderate and good to very good agreement strength. Results: Detection of multiple infection and HPV genotypes per sample was higher by LA than by LiPA (61.5 vs. 52.1 and 2.2 vs. 1.7%, respectively). There were concordant results in 65 samples and compatible results in 33 samples. Only 2 samples were considered as discordant. In 21 samples, additional types were detected by LA, and in 13 samples, additional types were detected by LiPA. Analyzing the ĸ values, we have found very good agreement for 14 genotypes (6, 16, 26, 31, 33, 35, 45, 51, 52, 53, 58, 66, 68 and 70). Conclusions: We considered that the new LiPA is highly comparable with other methods and suitable for clinical and epidemiological studies.

Up-regulation of FOXP3 and induction of suppressive function in CD4 + Jurkat T-cells expressing hepatitis C virus core protein

HCV (hepatitis C virus) infection is a serious health care problem that affects more than 170 million people worldwide. Viral clearance depends on the development of a successful cellular immune response against the virus. Interestingly, such a response is altered in chronically infected patients, leading to chronic hepatitis that can result in liver fibrosis, cirrhosis and hepatocellular carcinoma. Among the mechanisms that have been described as being responsible for the immune suppression caused by the virus, Treg-cells (regulatory T-cells) are emerging as an essential component. In the present work we aim to study the effect of HCV-core protein in the development of T-cells with regulatory-like function. Using a third-generation lentiviral system to express HCV-core in CD4+ Jurkat T-cells, we describe that HCV-core-expressing Jurkat cells show an up-regulation of FOXP3 (forkhead box P3) and CTLA-4 (cytotoxic T-lymphocyte antigen-4). Moreover, we show that HCV-core-transduced Jurkat cells are able to suppress CD4+ and CD8+ T-cell responses to anti-CD3 plus anti-CD28 stimulation.

Evaluation of INNO-LiPA mycobacteria v2 assay for identification of rapidly growing mycobacteria

A total of 54 rapidly growing mycobacteria (RGM) isolated from patients attended in the two hospitals of Cadiz Bay (Spain) were selected during a seven-year-period (2000-2006) in order to evaluate the INNO-LiPA Mycobacteria v2 assay for mycobacterial identification, based on the reverse hybridization principle. The strains were cultured in Lowenstein-Jensen and Middlebrook 7H9 media and identified to the species level by sequencing of the 16S rRNA, PCR-restriction enzyme analysis of the hsp65 gene, conventional tests and INNO-LiPA Mycobacteria v2 assay. By the molecular methods we identified a total of 12 different species: 23 Mycobacterium fortuitum, 11 M. chelonae, 10 M. abscessus, 2 M. senegalense, 1 M. alvei, 1 M. brumae, 1 M. mageritense, 1 M. mucogenicum, 1 M. neoaurum, 1 M. peregrinum, 1 M. septicum and 1 M. smegmatis. Fifty two strains (96.3%) were correctly identified by conventional techniques and 47 strains (87.0%) by INNO-LiPA Mycobacteria v2 assay. We find INNO-LiPA Mycobacteria v2 assay simple to perform but it provides few advantages in comparison with conventional methods and sometimes needs complementary tests to identify Mycobacterium fortuitum complex, M. chelonae complex and specific species due to the great heterogeneity in the RGM group.

Aspectos actuales del virus de la hepatitis E

La hepatitis E es la causa principal de hepatitis no A no B de transmision enterica en areas con un clima tropical o subtropical y condiciones sanitarias pobres. Asimismo, es responsable de epidemias de magnitud variable vinculadas al consumo de aguas y de casos esporadicos de hepatitis aguda. El agente causal es el virus de la hepatitis E (VHE), no envuelto, con ARN monocatenario, de polaridad positiva y de aproximadamente 7,2 kb de longitud. Recientemente se han aislado en paises industrializados cepas del VHE procedentes de cerdos y ademas se ha informado en Europa, Japon y EE.UU. de casos de hepatitis aguda sin los factores de riesgo reconocidos para la hepatitis E. Algunas de las nuevas cadenas del VHE encontradas en personas de estos paises se han relacionado con aislamientos de VHE porcino de la misma area geografica, lo que indicaria que la hepatitis E es una enfermedad zoonotica. Asi, la hepatitis E ha empezado a aparecer con mayor prevalencia en paises donde, tradicionalmente, no era endemica. Esta revision resume el conocimiento actual en la biologia, la estructura y la transmision del virus, asi como el diagnostico de la infeccion, y describe el estado actual en areas con una incidencia baja de la hepatitis aguda E y el papel de los animales como vectores potenciales del virus.

Genetic analysis of human clinical isolates of Lactococcus garvieae: Relatedness with isolates from foods

Lactococcus garvieae is a Gram-positive bacterium well-known as an important pathogen in aquaculture, and it is also a human pathogen of increasing clinical significance. Forty-three human L. garvieae isolates from clinical specimens were characterized by Multilocus Sequence Typing (MLST). Twenty-six different sequence types (STs) were identified among the human isolates, of which 20 were novel STs. Most human isolates clustered into four clonal complexes, with a predominance of CC3. Within CC3, ST10 was the most common genotype, indicating the existence of a circulating genetic lineage among the human isolates analyzed. The four CCs also grouped L. garvieae strains isolated from meat, dairy and fish, indicating a genetic overlap between isolates from human and these foods. Genetic relatedness among human and food L. garvieae isolates was confirmed by phylogenetic analysis based on the concatenated sequences of the seven MLST genes. These results represent the first evidence of genetic relatedness between isolates of L. garvieae of human and those isolated meat, milk and dairy products and suggest that, in addition to fish and seafood, these foods might represent important sources of human L. garvieae infections.

Dipylidium caninum infection in an infant: a rare case report and literature review

ABSTRACT Dipylidiasis is a zoonotic parasitic infection caused by Dipilydium caninum , a common intestinal tapeworm of dogs and cats. Humans may be accidental hosts when the cysticercoid larva is ingested, mainly infants and young children due to their playing habits and their proximity with dogs and cats. It is considered a rare infection in the world. In the past 20 years only 16 cases have been reported in Europe, China, Japan, India, Sudan, Latin America and the United States. We describe a case of dipylidiasis observed in a 9-month-old girl who likely acquired the infection through games with her pet dog. In a stool sample, we observed 6 proglottids of tapeworm. Each proglottid segment was about 8–9 mm long and 2–3 mm thick. A wet mount revealed proglottids with two genital pores, one on each side, and eggs were clustered in packets containing 8–12 and surrounded by a thin membrane. The patient was successfully treated with a single dose of praziquantel. The pet dog was seen by the veterinary and also showed parasitism by Dipylidium . To the best of our knowledge, this is the only human case reported in Spain according to the literature reviewed.

Evaluation of the BDProbeTec ET system as screening tool in the direct detection of mycobacterium tuberculosis complex in respiratory specimens

We evaluated the BDProbeTec ET System (Becton Dickinson) for the routine detection of Mycobacterium tuberculosis complex (MTC) in respiratory specimens and pleural fluids, comparing with microscopy (Ziehl Neelsen stain, ZN) and culture in liquid (BACTEC MGIT 960, MGIT) and solid (Löwenstein Jensen, LJ) media. Five hundred and two specimens, collected from 266 patients, of which 257 with suspected tuberculosis and 9 receiving anti-tuberculosis treatment, were investigated. Thirty-nine specimens were positive by any method, including false positives. Mycobacteria were isolated from 33 specimens (32 Mycobacterium tuberculosis and 1 Mycobacterium chelonae). Thirty-six specimens were BDProbeTec ET positive, 33 specimens were MGIT positive, 27 were LJ positive and 22 were ZN positive. With BDProbeTec ET, 2 specimens were false negative (culture positive), and 2 specimens from non-treated patients were false positive (culture negative). The overall sensitivity, specificity, and positive and negative predictive values for BDProbeTec ET compared to culture were 93.7, 98.7, 83.3, and 99.5%, respectively, while with smear-positive and smear-negative specimens the sensitivities were 100% and 81.5% respectively. In five treated patients the disappearance of MTC could be monitored using BDProbeTec ET in parallel with culture. The overall inhibition rate was 0.2%. BDProbeTec ET can be very useful for rapid detection of MTC, especially in smear-negative respiratory specimens.

The zoonotic potential of Lactococcus garvieae: An overview on microbiology, epidemiology, virulence factors and relationship with its presence in foods

Lactococcus garvieae is a relevant worldwide fish pathogen affecting various farmed and wild marine and freshwater species. It has also been isolated from other animals, such as ruminants with subclinical mastitis and pigs with pneumonia. From the early 90s, L. garvieae has been associated with different human infections, mainly endocarditis. During the last five years, human infections by this bacterium appear to be increasing, likely due to the improvement in microbiological methods for bacterial identification and the alertness of this bacterium by physicians. Human L. garvieae infections have been associated with the consumption or the handling of contaminated raw fish or seafood, and recently, a genetic study showed that meat, raw milk and dairy products may also be food sources of human L. garvieae infections. However, the status of L. garvieae as a potential zoonotic bacterium is still controversial to date. In this work, we describe four new human infections by L. garvieae in elderly and inmunocompromised patients, and we show an overview on L. garvieae microbiology, epidemiology, virulence factors and relationship with its presence in foods.