Longitudinal evaluation of hepatitis C viral persistence in HIV-infected patients with spontaneous hepatitis C clearance.

Hepatitis C virus (HCV) viral persistence in patients with spontaneous viral clearance is controversial. Several studies have shown HCV-RNA in peripheral blood mononuclear cells (PBMCs) and/or liver tissue among patients who have cleared the virus spontaneously, suggesting that viral persistence is a common situation that could involve the entire population studied. Thus, our aim was to evaluate HCV-RNA persistence in PBMCs and hepatocytes in subjects infected with the human immunodeficiency virus (HIV). A total of 1508 patients were prospectively followed and tested for anti-HCV antibodies and HCV-RNA to identify the patients who achieved spontaneous viral clearance. In all of the patients, the persistence of HCV-RNA in PBMCs was evaluated longitudinally during 2 years of follow-up. Fifty-nine patients fulfilled the inclusion/exclusion criteria and were included in the study. HCV-RNA was not detected in the PBMCs at baseline [59 PBMCs samples tested; 0 %; 95 % confidence interval (CI): 0-3.3 %] or during the follow-up (147 PBMCs samples tested; 0 %; 95 % CI: 0-2.02 %). Our study shows that HCV viral persistence is not a frequent occurrence in HIV-infected patients who have spontaneously resolved an HCV infection. Thus, the lack of serum HCV-RNA should continue to be addressed as the standard of healing.

Association of complement receptor 2 polymorphisms with innate resistance to HIV-1 infection.

HIV-1 induces activation of complement through the classical and lectin pathways. However, the virus incorporates several membrane-bound or soluble regulators of complement activation (RCA) that inactivate complement. HIV-1 can also use the complement receptors (CRs) for complement-mediated antibody-dependent enhancement of infection (C-ADE). We hypothesize that hypofunctional polymorphisms in RCA or CRs may protect from HIV-1 infection. For this purpose, 139 SNPs located in 19 RCA and CRs genes were genotyped in a population of 201 Spanish HIV-1-exposed seronegative individuals (HESN) and 250 HIV-1-infected patients. Two SNPs were associated with infection susceptibility, rs1567190 in CR2 (odds ratio (OR) = 2.27, P = 1 × 10(-4)) and rs2842704 in C4BPA (OR = 2.11, P = 2 × 10(-4)). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Although not significant (P = 0.25, OR = 1.57), similar genotypic proportions were obtained for the CR2 marker rs1567190. The results of the two association analyses were combined through a random effect meta-analysis, with a significant P-value of 2.6 x 10(-5) (OR = 2.07). Furthermore, we found that the protective CR2 genotype is correlated with lower levels CR2 mRNA as well as differences in the ratio of the long and short CR2 isoforms.

Detection and identification of Bartonella sp. in fleas from carnivorous mammals in Andalusia, Spain.

A total of 559 fleas representing four species (Pulex irritans, Ctenocephalides felis, Ctenocephalides canis and Spilopsyllus cuniculi) collected on carnivores (five Iberian lynx Lynx pardinus, six European wildcat Felis silvestris, 10 common genet Genetta genetta, three Eurasian badger Meles meles, 22 red fox Vulpes vulpes, 87 dogs and 23 cats) in Andalusia, southern Spain, were distributed in 156 pools of monospecific flea from each carnivore, and tested for Bartonella infection in an assay based on polymerase chain reaction (PCR) amplification of the 16 S-23 S rRNA intergenic spacer region. Twenty-one samples (13.5%) were positive and the sequence data showed the presence of four different Bartonella species. Bartonella henselae was detected in nine pools of Ctenocephalides felis from cats and dogs and in three pools of Ctenocephalides canis from cats; Bartonella clarridgeiae in Ctenocephalides felis from a cat, and Bartonella alsatica in Spilopsyllus cuniculi from a wildcat. DNA of Bartonella sp., closely related to Bartonella rochalimae, was found in seven pools of Pulex irritans from foxes. This is the first detection of B. alsatica and Bartonella sp. in the Iberian Peninsula. All of these Bartonella species have been implicated as agents of human diseases. The present survey confirms that carnivores are major reservoirs for Bartonella spp.

High rate of long-term survival for high-risk lymphoma patients treated with hematopoietic stem cell transplantation as consolidation or salvage therapy.

Patients with high-relapse-risk lymphomas or those relapsing after initial therapy have a limited probability of cure with conventional treatment. There is recent inconclusive evidence that, in such cases, intensification or salvage treatment with high-dose chemotherapy followed by hematopoietic stem cell transplantation (HSCT) increases the response rate and may improve survival. Nevertheless, published data on long-term follow-up of high-risk lymphoma patients treated with HSCT are scarce. We analyzed 101 consecutive patients receiving high-dose chemotherapy followed by HSCT after induction with standard chemotherapy. The median age was 38 years (range, 12-63 years). The diagnoses were Hodgkin's disease (n = 32), follicular lymphoma (n = 33), diffuse large B-cell lymphoma (n = 12), mantle cell lymphoma (n = 7), T-cell lymphoma (n = 14), and others (n = 3). Patients received either an autologous graft (n = 72) in first complete remission (1CR; n = 23) or in advanced stages (AS; n = 49), or an allogeneic graft (n = 29) in 1CR (n = 7) or in AS (n = 22). We concluded that transplant-related mortality was 2.7% for patients receiving an autologous HSCT and 27% for patients receiving an allogeneic HSCT. The main etiologies were graft-versus-host disease and infection in the allogeneic setting, and infection in the autologous setting. The probability of long-term (12-year) overall survival was 71%, higher than that described for high-relapse-risk lymphoma patients treated without HSCT and significantly better (P < .05) for patients who received the transplant in 1CR (89%) than in AS (65%). Finally, the probability of long-term survival was significantly better for patients treated with HSCT during the period from 2000-2007 (85%) compared with the period from 1989-1999 (72%).

Spotted fever group Rickettsia in brown dog ticks Rhipicephalus sanguineus in southwestern Spain.

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.

Ectoparasites of the endangered Iberian lynx Lynx pardinus and sympatric wild and domestic carnivores in Spain.

Ectoparasites can cause important skin disorders in animals and can also transmit pathogens. The Iberian lynx Lynx pardinus has been stated to be the most endangered felid in the world and such vector-borne pathogens may threaten its survival. We surveyed 98 wild carnivores (26 Iberian lynxes, 34 red foxes Vulpes vulpes, 24 Egyptian mongooses Herpestes ichneumon, 11 common genets Genetta genetta, two Eurasian badgers Meles meles, one polecat Mustela putorius) and 75 domestic but free-ranging carnivores (46 cats Felis catus, 29 dogs Canis familiaris) from June 2004 to June 2006 in the two areas where the last lynx metapopulations survive: Sierra Morena and Doñana (Andalusia, southern Spain). A total of 65% of lynxes were parasitized (50% by ticks, 19% by fleas, 4% by lice, 31% by hippoboscid flies), as were 75% of foxes (58%, 60%, 0%, 19%), 71% of mongooses (50%, 4%, 46%, 0%), 54% of genets (18%, 36%, 0%, 0%), 30% of cats (22%, 14%, 0%, 2%), and 7% of dogs (surveyed only for ticks). Both badgers presented ticks, fleas and lice. Five species of ixodid ticks (Rhipicephalus pusillus Gil Collado, Rhipicephalus turanicus Pomerantzev and Matikashvili, Ixodes ricinus (Linnaeus), Ixodes hexagonus Leach and Ixodes ventalloi Gil Collado; and Hyalomma sp.), four species of fleas (Ctenocephalides canis Curtis, Pulex irritans Linnaeus, Spilopsyllus cuniculi (Dale), Xenopsylla cunicularis Smit), three species of chewing lice (Felicola (Felicola) inequalis (Piaget), Trichodectes (Trichodectes) melis (Fabricius), and Felicola (Lorisicola) isidoroi Pérez and Palma), and one species of hippoboscid fly (Hippobosca longipennis (Fabricius)) were found. We did not detect any cases of mange. Hippobosca longipennis is a new record for Spanish wildlife, and all the flea species are new records for the Iberian lynx. Fleas were more frequent on lynxes and foxes in winter than in spring. Rhipicephalus spp. were more frequent on cats in spring than in any other season. These and other epidemiological findings are discussed with respect to the conservation of the Iberian lynx.

Prevalence data of Rickettsia slovaca and other SFG Rickettsiae species in Dermacentor marginatus in the southeastern Iberian peninsula.

In southern Spain, Dermacentor marginatus ticks can be infected with several genospecies of spotted fever Group (SFG) Rickettsia. We developed a nested polymerase chain reaction assay by using a species-specific probe targeting the ompA gene to detect and differentiate between the two groups of rickettsiae previously described in D. marginatus. SFG rickettsia has been detected in 85.15% of ticks studied (26.7% of positives have been to R. slovaca, the causative agent of TIBOLA-DEBONEL, and 73.3% to SFG rickettsia closely related to strains RpA4-JL-02-DnS14-DnS28).

Incidence and distribution pattern of Rickettsia felis in peridomestic fleas from Andalusia, Southeast Spain.

The presence of Rickettsia felis was investigated in three species of pulicid fleas (Ctenocephalides felis, Ctenocephalides canis and Pulex irritans) collected in 38 locales in Andalusia (Spain) over the period 1999-2004. Amplification of a fragment of OmpB gene was positive in 54.17 % of lots of Ct. felis. The identity of the PCR bands was confirmed as R. felis by sequence data obtained directly from the PCR amplicon. No rickettsia was found in Ct. canis nor P. irritans.

Seroepidemiological study of Rickettsia felis, Rickettsia typhi, and Rickettsia conorii infection among the population of southern Spain.

Rickettsia typhi and Rickettsia conorii, the etiologic agents of, respectively, murine typhus and Mediterranean spotted fever, are recognized as frequent causes of fever of intermediate duration in southern Spain; in addition, in recent years Rickettsia felis has been detected in potential vectors in this area. Nevertheless, limited data exist regarding the actual prevalence of past infection due to these three pathogens. In the present study, the prevalence of past infection due to R. felis, R. typhi, and R. conorii was determined in a representative population of southern Spain during 2002. In addition, the possible risk factors associated with exposure to these pathogens were investigated. An epidemiological survey was completed by all subjects included in the study. Serum samples were tested by indirect immunofluorescence assay. The prevalence of past infection due to R. felis, R. typhi, and R. conorii among the 504 total subjects was 6.5, 3.8 and 8.7%, respectively. In multivariate analysis, infection due to R. felis was independently associated with a high-risk occupation (one that required working outdoors in nature, close contact with domestic animals, or potential contact with rodents) (OR=5.8; 95%CI 2.1-15.6), while infection due to R. typhi was associated with older age (factor of 1.04 [95%CI 1.008-1.068]) and frequent insect bites (OR=10.3; 95%CI 2.3-45.5). Two factors were associated with infection due to R. conorii: a high-risk occupation (OR=9.3; 95%CI 3.7-23.2), and participation in outdoor activities (OR=7.2; 95%CI 1.4-38.5). The results confirm the widespread prevalence of past infection due to R. felis, R. typhi, and R. conorii in the population of southern Spain.

Dermacentor-borne necrosis erythema and lymphadenopathy: clinical and epidemiological features of a new tick-borne disease.

This paper describes the epidemiological and clinical features of a tick-borne disease differing somewhat from other tick-borne diseases found previously in Spain. All patients were bitten by Dermacentor marginatus or a large tick. The clinical features include a crustaceous or necrotic lesion at the site of the tick's attachment, surrounded by an erythema (erythema migrans-like) and painful regional lymphadenopathies. The probable aetiological agent is Rickettsia slovaca. Similar cases have been reported in other European countries.

Genotypic identification of an undescribed spotted fever group rickettsia in Ixodes ricinus from southwestern Spain.

An undescribed rickettsia was directly analyzed with specific rickettsial molecular biology tools on Ixodes ricinus L. collected in different localities of the province of Cadiz (southwestern Spain). On the basis of the results of the citrate synthase (glta) gene, 190 kD-outer membrane protein (rOmpA) gene, and 16S ribosomal RNA (16S rRNA) gene partial sequence data, it was found that this rickettsia is sufficiently genetically distinct from other Rickettsia to be considered a distinct taxonomic entity. The isolation and culture of this organism, as well as comparative antigenic analysis, are required to ensure its conclusive taxonomic placement among spotted fever rickettsiae. The epidemiologic role of this new rickettsial agent and its possible pathogenicity to wild and domestic animals or humans is still unknown and needs to be investigated.

[Population dynamics of Rhipicephalus pusillus Gil Collado, 1938 (Acarina, Ixodidae) in 1986-1987 in south-eastern Spain: modelling of the biological cycle]. / Dynamique de la population de Rhipicephalus pusillus Gil Collado, 1938 (Acarina, Ixodidae) en 1986-1987 dans le sud-est de l'Espagne: modélisation du cycle biologique.

The evolution of the Rhipicephalus pusillus population, a parasite of the european wild rabbit, Oryctolagus cuniculus, is studied in a locality of the Province of Granada (Spain), in 1986-1987. The moving average technique was applied to the sampling data (358 hosts), for each stage of the postembryonic cycle. This technique permitted us, together with the habitual usage of the statistics, to understand in a more complete manner, the evolution of daily changes which affect the average number of ticks over the population of the host rabbits. Various factors allowed us to envisage a strong dependency of the parasitism of the females and the larvae in relation with the developing environmental conditions, which determine the apparition and concentration of the parasites on the hosts. A very strong correlation between the parasitism by male ticks and that of the female ticks was observed for the two year duration of our investigation. However, the moment at which the distribution of the ectoparasites is contagious, the male abundance is nearly twice that of the females. The larvae respond to a similar pattern of distribution both years, but it is necessary to note a delay, which we can attribute to climatic changes registered from one year to the next. The nymphs, on the other hand, present a different distribution curve for each year. Their presence depends on climatic conditions and especially that of the situation of the ticks and hosts population in the moments proceeding: these conditions determine a deplacement of their apparition in one sense or another.

Effect of palmitate, acetate and glucose on glutamine metabolism in Ehrlich ascites tumor cells.

Acetate and the long chain free fatty acid palmitate provoked a decrease in the rates of glutamine utilization and glutamate production in Ehrlich ascites tumor cells incubated with 0.5 mM glutamine. There was a cumulative effect with glucose on glutamine metabolism.

Inorganic phosphate and energy charge compartmentation in Ehrlich ascites tumour cells in the presence of glucose and/or glutamine.

Inorganic phosphate concentrations and the values of energy charge were determined in the cytosolic and mitochondrial compartments of Ehrlich ascites tumour cells incubated with 5 mM glucose and/or 0.5 mM glutamine. The initial value of inorganic phosphate concentration in the cytosolic compartment decreased in the three incubation conditions assayed; this decrease was greater when glucose was present in the incubation medium. Mitochondrial inorganic phosphate concentration diminished in incubations with only glucose and raised in incubations with only glutamine.

Glutamine and glucose as energy substrates for Ehrlich ascites tumour cells.

Energy metabolism of freshly harvested Ehrlich ascites tumour cells in the presence of 5 mM glucose and/or 0.5 mM glutamine was studied. The rate of oxygen utilization was not altered by the addition of 0.5 mM glutamine; 5 mM glucose induced an inhibition of respiration. In the presence of both glucose and glutamine, the Crabtree effect decreased. In these conditions, the rates of oxygen uptake, the CO2 evolution and the changes in the redox states of cytochromes indicate that glucose is preferred by Ehrlich ascites tumour cells as energy substrate. Glucose decreased the rate of glutamine utilization by 34%. On the other hand, glutaminolysis did not inhibit glycolysis.

Malate-citrate cycle during glycolysis and glutaminolysis in Ehrlich ascites tumor cells.

The malate-citrate cycle was studied during aerobic glycolysis and glutaminolysis in a strain of Ehrlich ascites tumor cells which showed a very low malate-aspartate shuttle system activity. The experimental approach includes: estimation of mitochondrial NAD[P]+-dependent malic enzyme activity; respiratory activity of freshly harvested or fasted cells, and of isolated mitochondria; and determination of the metabolites involved in the glycolytic and glutaminolytic pathways. The results suggest that in this strain, the malate-citrate shuttle is not an effective pathway for transferring glycolytic reducing equivalents from cytosol to mitochondria. Less than 15% of the glucose uptake was affected by the 1,2,3-benzenetricarboxylate inhibition of the malate-citrate shuttle. Moreover, in the presence of glucose, the malate-citrate cycle did not appear to play an important role in the glutaminolytic process. The present work supports and extends the finding of previous studies, since the results showed that the glucose metabolism depressed the oxidative processes in Ehrlich ascites tumor mitochondria, not only alone, but also in the presence of glutamine. Interestingly, the high glutamine uptake was maintained in the presence of glucose.

Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase high-performance liquid chromatography.

The concentrations of free amino acids in plasma and in ascitic liquid of mice with Ehrlich ascitic tumours were determined by reversed-phase high-performance liquid chromatography using pre-column derivatization with Dns chloride and UV detection at 254 nm. Sample preparation is simple, and the Dns derivatives are stable. Complete separation of 27 amino acids, including proline and cysteine, was achieved in 70 min with detection limits of less than 25 pmol. There was no interference from Dns-Cl, Dns-OH and Dns-NH2. Retention time reproducibility was better than 1%. The described method enables a rapid, economical and reproducible quantification of free amino acids in biological fluids.