Tolerance to NADH/NAD+ imbalance anticipates aging and anti-aging interventions.

Redox couples coordinate cellular function, but the consequences of their imbalances are unclear. This is somewhat associated with the limitations of their experimental quantification. Here we circumvent these difficulties by presenting an approach that characterizes fitness-based tolerance profiles to redox couple imbalances using an in silico representation of metabolism. Focusing on the NADH/NAD+ redox couple in yeast, we demonstrate that reductive disequilibria generate metabolic syndromes comparable to those observed in cancer cells. The tolerance of yeast mutants to redox disequilibrium can also explain 30% of the variability in their experimentally measured chronological lifespan. Moreover, by predicting the significance of some metabolites to help stand imbalances, we correctly identify nutrients underlying mechanisms of pathology, lifespan-protecting molecules, or caloric restriction mimetics. Tolerance to redox imbalances becomes, in this way, a sound framework to recognize properties of the aging phenotype while providing a consistent biological rationale to assess anti-aging interventions.

Comparison of conventional and lipid emulsion formulations of amphotericin B: Pharmacokinetics and toxicokinetics in dogs.

The major limiting factor in the use of amphotericin B (AmB) is cumulative nephrotoxicity. In previous studies, AmB mixed with Intralipid® 20% (AmB-IL), a parenteral fat emulsion, reduces its toxicity, increases its efficacy and is less expensive than other commercial amphotericin B lipid formulations. The pharmacokinetics and toxicity of the conventional deoxycholate AmB formulation (Fungizone®) and AmB-IL were compared in dogs. The pharmacokinetic of AmB was significantly modified and renal toxicity and infusion-related side effects were reduced when the drug was prepared in fat emulsion. In addition, pharmacokinetics and toxicity were evaluated after the administration of multiple doses of AmB-IL with the purpose of determining an optimal treatment protocol in dogs. When using a consecutive day administration regime, there was a significant drug accumulation together with an increase in creatinine values after each dose. However, when using three doses per week administration regime, similar maximum and minimum plasma concentrations were maintained. During the four weeks of treatment a moderate increase in the creatinine values was observed but none of the treatments were ended prematurely. All these data suggest that Intralipid®, similar to that seen previously in humans, favors AmB distribution to the organs, decreasing drug toxicity and increasing its therapeutic index in the dogs. The dose protocol evaluated (25mg/m2/48h/three times per week) produces maintenance of AmB plasma levels that were close to that obtained by others authors after administration of liposomal formulations of AmB and that have been demonstrated to be clinically effective.

Cytokines and chemokines measured in dried SLA-stimulated whole blood spots for asymptomatic Leishmania infantum and Leishmania donovani infection.

Whole blood stimulation with soluble Leishmania antigen (SLA), followed by plasma cytokine and chemokine determination, provides means of detecting subjects with asymptomatic Leishmania infection. This work examines the potential of Protein Saver 903 cards for the storage and transport of SLA-stimulated dried plasma spot samples. Blood was collected from asymptomatic and negative control subjects living in a Leishmania infantum- (Spain) and Leishmania donovani-endemic area (Bangladesh). After SLA-stimulation, three types of sample were prepared: frozen liquid plasma (-20 °C), and plasma dropped onto Protein Saver cards kept at -20 °C (DPS-FZ), and at ambient temperature (DPS-AT). The concentrations of IFN-γ, IL-2, CXCL10, CXCL9, CCL2 and CXCL8 in the thawed liquid plasma (TLP), DPS-FZ and DPS-AT samples were then determined. Strong correlations were seen between the TLP and DPS-FZ/AT samples for all the studied cytokines/chemokines in both the L. infantum and L. donovani areas. Protein Saver 903 cards would therefore appear to allow for the transport of SLA-stimulated plasma samples by courier at ambient temperature. The CXCL10 and CXCL9 detectable in these plasma spots provided robust markers for identifying asymptomatic subjects from both endemic areas. This easy procedure opens up new possibilities for field studies in resource-limited settings, which could help in Leishmania control.

Interleukin-2 as a marker for detecting asymptomatic individuals in areas where Leishmania infantum is endemic.

No field method exists for identifying asymptomatic individuals in areas where Leishmania infantum is endemic. This work reports that, 24 h after stimulating whole blood with soluble Leishmania antigen (SLA), plasma interferon-γ (IFN-γ) and interleukin-2 (IL-2) become significantly elevated in samples from asymptomatic individuals (n=47) compared with those from negative controls (n=50), all of them recruited from a blood bank. When compared with the reference test SLA-lymphoproliferative assay, IL-2 appears as a new, 100% sensitive and specific marker for asymptomatic individuals with a positive cellular response (compared with 100% and 84.78%, respectively, for IFN-γ). Further studies in other transmission areas and in other cohorts of exposed people need to be performed to confirm these results. Once validated, IFN-γ and IL-2 levels in SLA-stimulated whole blood could be reliably used in the field to estimate the prevalence of those asymptomatic individuals with Leishmania-specific cellular immune responses.

Leishmania infantum is clonal in AIDS patients too: epidemiological implications.

OBJECTIVE: To test, in AIDS patients, a previously proposed hypothesis of clonal population structure in Leishmania infantum, the agent of visceral leishmaniasis. DESIGN: Forty-three stocks of L. infantum isolated from AIDS patients in Spain were analysed by multilocus enzyme electrophoresis. METHODS: The results were analysed in terms of population genetics according to previously described statistical methods. Departures from panmixia were examined by linkage disequilibrium analysis. RESULTS: As previously shown in HIV-negative patients, classical manifestations of clonality were shown, namely strong linkage disequilibrium, over-representation of genotypes and overall lack of genotype diversity. The same dominant clonal genotype (MON1) was recorded in both HIV-positive and HIV-negative patients. Frequency of this dominant genotype was not statistically different in HIV-positive and HIV-negative patients. CONCLUSIONS: The parasite population under survey appears to be clonal; parasite genotypes can therefore be equated to natural clones, stable in space and time, which can be used as multilocus epidemiological markers. Nevertheless, additional studies are required to better estimate the long-term stability of these clonal genotypes and the possible interference of gene exchange at an evolutionary scale.

Treatment of visceral leishmaniasis in HIV-infected patients: a randomized trial comparing meglumine antimoniate with amphotericin B. Spanish HIV-Leishmania Study Group.

BACKGROUND: Visceral leishmaniasis is common in patients with HIV infection living in endemic areas, but the most effective and safe treatment remains unknown. OBJECTIVE: To compare the efficacy and safety of meglumine antimoniate versus amphotericin B in HIV-infected patients with first episodes of visceral leishmaniasis (VL). DESIGN: An open, multicentre, prospective and randomized trial. SETTING: Twelve tertiary hospitals. PATIENTS: Eighty-nine consecutive HIV-infected patients diagnosed with VL. Patients were randomly assigned to treatment with either meglumine antimoniate (20 mg pentavalent antimony per kilogram of body weight per day) or amphotericin B (0.7 mg/kg per day) both for 28 days. Treatment was considered successful if a bone marrow aspirate performed 1 month after the end of therapy did not detect parasites. Relapse was defined as the reappearance of parasites after an initial cure. RESULTS: An initial cure was attained in 29 of 44 patients (65.9%) randomly assigned to treatment with meglumine antimoniate and 28 of 45 (62.2%) randomly assigned to treatment with amphotericin B. The incidence of moderate to severe adverse events was similar in both groups. The patients treated with meglumine antimoniate had higher incidences of cardiotoxicity (14 versus 0%, P = 0.02) and chemical pancreatitis (30 versus 0%, P < 0.01). However, in the amphotericin B group, nephrotoxicity was more frequent (36 versus 5%, P < 0.01). There was no difference in survival or relapse-free interval according to the allocated group of therapy. CONCLUSION: Treatment of VL with meglumine antimoniate or amphotericin B was shown to have similar efficacy and toxicity rates in Spanish HIV-infected patients. The differences in the toxicity patterns could be useful in choosing one of these agents as first-line treatment.

Identification of 'Old World' Leishmania by DNA recombinant probes.

Leishmania are usually identified by iso-enzyme analysis. This method works well, but there is a need for an additional, more simple, method of identification. Here we present data that show that in a Southern blot analysis, recombinant DNA probes in combination with certain restriction enzymes can differentiate between taxa of Leishmania. Probes based on clones selected from a L. infantum cDNA library gave characteristic patterns on Southern blots for reference strains of the different types of Leishmania found in Europe, Africa and Asia. Within the different taxa little or no variation was observed. Although the L. infantum derived probes showed a somewhat stronger hybridization for strains of the L. donovani complex, the signal obtained with most probes was satisfactory for L. major, L. aethiopica and L. tropica. Within the L. donovani complex none of the selected probes differentiated between isolates belonging to L. infantum, L. chagasi or L. donovani. Probes containing kinetoplast DNA showed considerable variation in hybridization within a taxon.

Visceral leishmaniasis in Angola due to Leishmania (Leishmania) infantum.

A 26-year-old man from Angola with no history of travel outside the country presented with typical symptoms of visceral leishmaniasis. The parasite was isolated and biochemically characterized using both kinetoplast DNA and nuclear DNA probes and showed a strong homology with Leishmania (Leishmania) donovani sensu lato (s.l.). When the nuclear DNA of the isolate was hybridized with a specific Leishmania (L.) infantum probe, the pattern obtained showed a clear signal with this species. To establish its identity more specifically, this isolate was typed using a 15-system isoenzyme panel and thick-starch gel electrophoresis, and was identified as L. (L.) infantum zymodeme 1 (MAD-1), the most widespread zymodeme in Mediterranean countries. One case of visceral leishmaniasis has been reported in Angola, but this case is the first report of L. (L.) infantum in Africa south of the equator.

In vitro susceptibility of Plasmodium falciparum to chloroquine, amodiaquine, quinine, mefloquine, and sulfadoxine/pyrimethamine in Equatorial Guinea.

Between March 1990 and June 1992, a study was carried out in Equatorial Guinea on the in vitro response of Plasmodium falciparum to different antimalarial drugs. Field work for the study was conducted both in the country's island region as well as on the mainland, and resistant isolates were found to exhibit interregional differences. On the island of Bioko, 204 tests were performed with 16% (11 of 69) resistant to chloroquine, 9% (4 of 46) resistant to quinine, 14% (6 of 43) resistant to a combination of sulfadoxine/pyrimethamine, and 6.5% (3 of 46) resistant to amodiaquine. In the mainland area of Bata, the same antimalarial drugs and mefloquine were tested with the following results: 9% (5 of 58) resistant to chloroquine; 2% (1 of 58) resistant to amodiaquine, and 3% (2 of 58) resistant to a combination of sulfadoxine/pyrimethamine. No isolates resistant to quinine or mefloquine were found. Effective concentrations (EC50, EC90, and EC99) and regression lines (log dose/response) for each antimalarial drug were calculated to establish a surveillance system for antimalarial drug chemosensitivity in Equatorial Guinea. Finally, 12 isolates from 12 patients previously treated with chloroquine were studied to compare both tests (in vivo-in vitro) and obtain a correlation between the RII and RIII types of in vivo and in vitro resistances. No correlation for the RI type was found between the two methods.

Human subcutaneous dirofilariasis caused by Dirofilaria repens in Ibiza, Spain.

A case of human subcutaneous dirofilariasis contracted in Ibiza, Spain is reported. An incomplete nematode extracted from the eyelid of a woman patient was identified on the basis of its anatomic and histologic characteristics as a nongravid adult female of the species Dirofilaria repens. The subcutaneous location of the worm, together with the epidemiologic data, support this finding.

Infection of sand flies by humans coinfected with Leishmania infantum and human immunodeficiency virus.

To determine the role that Leishmania infantum/human immunodeficiency virus (HIV) coinfected patients could play in the epidemiology of visceral leishmaniasis (VL), we applied direct xenodiagnosis of VL in this study to test the infectivity of six coinfected patients to colonized Phlebotomus perniciosus. All patients proved to be infective for the sand flies. The infectivity of patients who had still not received specific treatment for VL was inversely proportional to their absolute CD4+ T lymphocyte cell count. It has been proven that P. perniciosus can acquire and allow the development of L. infantum by feeding on L. infantum/HIV coinfected patients. Since this sand fly is an important vector of VL in southern Europe, a new natural anthroponotic cycle could be considered in the epidemiology of L. infantum/HIV coinfection. The design of leishmaniasis control programs and the management of coinfected individuals should take these findings into account.

The role of serology in the diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus type-1.

To define the possible role of serology in the diagnosis and prognosis of visceral leishmaniasis (VL) in patients with human immunodeficiency virus type-1 (HIV-1) infection, the dynamics of humoral immune responses was investigated in 20 coinfected patients. Sequential sera obtained before, during, and after VL diagnosis were analyzed by an indirect immunofluorescent antibody test (IFAT), a recombinant ELISA (using the rK39 protein), and immunoblotting. During the active course of the disease, positive results were found by IFAT or ELISA in 22% of the cases and by immunoblotting in 78% of the cases. A great variability in the response was observed during the follow-up with a trend to more positive results near the time of VL diagnosis. Forty-six percent of the patients were positive by IFAT or ELISA on at least one time point before VL and 37.5% were positive during the period following treatment. These results confirm the limited usefulness of the IFAT and ELISA in the diagnosis of VL in coinfected patients and demonstrate their low ability to predict the development or the outcome of disease. In these patients, immunoblotting could be a useful tool for studying the natural course of leishmaniasis, although it has limited value for diagnosis or treatment control.

Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea.

A semi-nested, multiplex polymerase chain reaction (PCR) based on the amplification of the sequences of the 18S small subunit ribosomal RNA (ssrRNA) gene was tested in a field trial in Equatorial Guinea (a hyperendemic focus of malaria in west central Africa). The method uses a primary PCR amplification reaction with a universal reverse primer and two forward primers specific for the genus Plasmodium and to mammals (the mammalian-specific primer was included as a positive control to distinguish uninfected cases from inhibition of the PCR). The second amplification is carried out with the same Plasmodium genus-specific forward primer and four specific reverse primers for each human Plasmodium species. The PCR amplified products are differentiated by fragment size after electrophoresis on a 2% agarose gel. Four villages from three regions of the island of Bioko (Equatorial Guinea) and two suspected Plasmodium vivax-P. ovale infections from the hospital of Malabo were tested by microscopy and PCR. The PCR method showed greater sensitivity and specificity than microscopic examination and confirmed the existence of a focus of P. vivax infections in Equatorial Guinea suspected by microscopic examination. It also provided evidence of several mixed infections, mainly P. falciparum and P. malariae, the two predominant species causing malaria in Equatorial Guinea.

Clinicoepidemiologic characteristics, prognostic factors, and survival analysis of patients coinfected with human immunodeficiency virus and Leishmania in an area of Madrid, Spain.

From 1987 to 1995, a retrospective case study was conducted at the Ramon y Cajal Hospital in Madrid, Spain, a public teaching hospital with 1,100 beds, to determine the clinicoepidemiologic characteristics, survival, and prognostic factors of patients with visceral leishmaniasis (VL) and human immunodeficiency virus (HIV) infection. The prevalence of VL in HIV+ patients compared with HIV- patients was studied. Epidemiologic, clinical, and parasitologic characteristics, as well as the effects of treatment, prognosis, and survival in 54 HIV+ patients (90 episodes) with VL were defined. Comparative survival studies among patients with and without acquired immunodeficiency syndrome (AIDS)-defining criteria and multivariate analysis of survival risk factors were performed. The prevalence of VL in patients with AIDS was much higher than in immunocompetent individuals. In spite of a good initial response to treatment for VL, 60.6% of the patients had relapsed by the end of one year. Mortality from the first episode was 18.5%, and 24% died in the first month after diagnosis of any VL episode. The mean survival of the 29 patients who died was 10.27 months. Survival in patients with and without AIDS at the time of the first episode of VL was compared at 30 months: 53.7% versus 20.5% (P = 0.00149). We found no significant difference (P = 0.24) in the survival of HIV+ patients who had died of VL without AIDS at the time of the first episode of VL compared with those of a control group of 413 dead patients with AIDS without VL. A diagnosis of AIDS at the time of the first episode of VL and thrombocytopenia were the only risk factors found related to survival. We conclude that in AIDS patients, VL is a recurrent disease that is highly prevalent and whose clinical course is modified by HIV.

Mucocutaneous leishmaniasis due to Leishmania (Leishmania) infantum: biochemical characterization.

A vegetative mass in the right nasal cavity of a 62-year-old man from Palma de Mallorca, Spain, was found to be due to Leishmania. The organism was isolated in culture and characterized by in situ hybridization, Southern blot hybridization, and isoenzyme analysis; it was thus demonstrated to be the most common enzyme variant 1 (MON 1) of Leishmania (Leishmania) infantum.

In vitro chloroquine-resistant falciparum malaria in Malabo, Equatorial Guinea: case reports.

For many years the chloroquine-resistant problem in Africa was circumscribed to East Africa, but in the last two years it has been spreading progressively to the West. We report here the two first cases of chloroquine-resistant falciparum malaria imported into Spain from Equatorial Guinea. Both cases show a parasitological grade III resistance in a W.H.O. in vitro macrotest. The clinical recovery with the alternative treatment (Fansidar) was satisfactory.

HIV--Leishmania infantum co-infection: humoral and cellular immune responses to the parasite after chemotherapy.

Specific serum antibodies, peripheral blood T-cell subsets, cellular response in vitro to soluble Leishmania antigens, phenotype of stimulated cells, and serum levels of tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta 1 were studied in Spain in 17 patients co-infected with HIV and Leishmania infantum who had been previously treated with pentavalent antimony. Both humoral and cellular responses to Leishmania sp. appeared diminished, 8 out of 17 patients were positive by indirect immunofluorescence, and immunoblotting detected heterogeneous antibody-binding pattern in 11 out of 13 subjects. A blastogenesis test was positive in 4 cases; 2 of them presented proliferation of CD4+ cells while CD8+ cells proliferated in the other 2 patients. Serum levels of TNF-alpha were similar to those observed in patients infected with HIV only, while serum levels of TGF-beta 1 were significantly lower in the co-infected patients. The inability of antibody response to control the parasite and the absence of specific T-cell immunity to Leishmania sp. would explain the high frequency of relapses reported in these patients. The decreased levels of TGF-beta 1 could have an important role in the interaction between the 2 pathogens.

Molecular epidemiology of Leishmania infantum on the island of Majorca: a comparison of phenotypic and genotypic tools.

In the Mediterranean basin, Leishmania infantum is the causative agent of both visceral and cutaneous leishmaniasis, and is an important opportunistic parasite in patients infected with human immunodeficiency virus (HIV). The commonest method used to study the variability of Leishmania spp. is isoenzyme analysis. In addition to this, we employed 3 assays based on the polymerase chain reaction (PCR): random amplified polymorphic deoxyribonucleic acid (RAPD), intergenic region typing (IRT), based on the amplification of ribosomal ribonucleic acid internal transcribed spacers and restriction fragment length polymorphism (PCR-RFLP). We used 54 L. infantum stocks isolated from HIV co-infected patients, 38 isolated from dogs, 3 isolated from immunocompetent patients and 3 isolated from 1826 sand files in the island of Majorca (Spain), a closed ecological niche. Zymodemes MON-1 (70%), MON-24 (11%) and MON-34 (18%) were found among the human isolates, and MON-1 (95%) and MON-108 (5%) among those from dogs. RAPD and IRT could not discriminate among the strains as they all gave the same pattern, even when different zymodemes were examined. In contrast, PCR-RFLP was able to distinguish the strains and, furthermore, a dendrogram (unweighted pair group method with arithmetic average [UPGMA]) was constructed from the genetic distances derived from RFLP data. The Leishmania isolates from HIV-infected subjects formed a single cluster, supporting the existence of an artificial anthroponotic cycle previously proposed by our group, in which syringes have been substituted for sand flies, and in which certain clones have been spread among intravenous drug users. This contrasts with the clusters representing a zoonotic cycle, involving dogs, sand flies and both immunocompetent and immunocompromised humans.

Molecular tracking of infections by Leishmania infantum.

Leishmania infantum is a major opportunistic parasite in patients with acquired immune deficiency syndrome and is very variable in these subjects. Isoenzyme characterization is not able to explain this variability, since half of the stocks isolated from patients co-infected with human immunodeficiency virus and Leishmania belong to zymodeme MON-1. Amplification of L. infantum minicircles by the polymerase chain reaction (PCR) and digestion of the amplified product to reveal restriction fragment length polymorphisms (RFLP) has proved very useful in distinguishing between relapses and reinfections in co-infected, treated patients. We have confirmed the existence of a leishmaniasis outbreak among intravenous drug users in north-east Spain, previously detected by isoenzymatic analysis. We have documented persistence of the same strain of Leishmania in 2 treated co-infected patients throughout several years, regardless of the theoretical rapid evolution ascribed to kinetoplast deoxyribonucleic acid minicircle sequences. We suggest using this PCR-RFLP technique to detect reinfections in treated co-infected subjects.