Discrepancy between antihypertensive effect and angiotensin converting enzyme inhibition by captopril

Captopril, an inhibitor of angiotensin converting enzyme, was administered twice daily to 13 hypertensive patients for a mean period of 9 weeks. Continuous blood pressure control in the ambulatory patients was established with a portable blood pressure recorder. Notwithstanding, in eight patients with normal renal function, plasma converting enzyme was found to resume normal activity before administration of the morning dose of captopril. Only in 5 patients with impaired renal function did some blockade of plasma converting enzyme persist for more than 12 hours. Measured plasma converting enzyme activity seemed to reflect total conversion of angiotensin I, including conversion in the pulmonary vascular bed, since changes in its activity were closely paralled by changes in plasma aldosterone levels. Bradykinin accumulation seems unlikely when converting enzyme and thus, presumably, kininase II has resumed normal activity. Captopril administration does not seem to alter plasma epinephrine or norepinephrine levels. Blood pressure reduction in the face of normal angiotensin converting enzyme activity is probably due to hyporesponsiveness of the arterioles to pressor hormones, which may be due to specific renin-related and/or nonspecific effects of captopril.

Gender difference in the response to an angiotensin-converting enzyme inhibitor and a diurectic in hypertensive patients of African descent

Résumé de la thèse en français Titre : Différence entre hommes et femmes dans la réponse à un inhibiteur de l'enzyme de conversion de l'angiotensine et un diurétique chez des patients hypertendus d'origine Africaine. Introduction: L'efficacité des inhibiteurs de l'enzyme de conversion de l'angiotensine (ACEI) dans le traitement de l'hypertension artérielle chez les patients africains est controversée. Objectif: Nous avons examiné la baisse de la tension artérielle ambulatoire (ABP) en réponse à un diurétique et un ACEI chez des patients hypertendus d'origine africaine et nous avons évalué les différentes caractéristiques déterminant l'efficacité du traitement. Méthodes: Etude en simple-aveugle randomisée, en crossover AB/BA. Arrangement Familles hypertendues d'origine africaine de la population générale des Seychelles. Participants : 52 patients (29 hommes et 23 femmes) sur 62 patients hypertendus éligibles ont été inclus. Le principal résultat était la mesure de la réponse de l'ABP à 20 mg de lisinopril (LIS) ou 25 mg d'hydrochlorothiazide (HCT) quotidiennement pendant quatre semaines. Résultats: Le jour, la réponse systolique/diastolique de l'ABP sous HCT était de 4.9 (95% intervalle de confiance (IC) 1.2-8.6)/3.6 (1.0-6.2) mm Hg pour les hommes et 12.9 (9.216.6)/6.3 (3.7-8.8) mm Hg pour les femmes. Sous LIS, la réponse était de 18.8 (15.022.5)/14.6 (12.0-17.1) mm Hg pour les hommes et de 12.4 (8.7-16.2)/7.7 (5.1-10.2) mm Hg pour les femmes. La nuit, la réponse systolique/diastolique sous HCT était de 5.0 (0.6-9.4)/2.7 ((-0.4)-5.7) mm Hg pour les hommes et de 11.5 (7.1-16.0)/5.7 (2.6-8.8) mm Hg pour les femmes, et sous LIS était de 18.7 (14.2-22.1)/15.4 (12.4-18.5) mm Hg pour les hommes et de 3.5 ((-1.0)-7.9)/2.3 ((-0.8)-5.4) mm Hg pour les femmes. L'analyse de régression linéaire multiple a montré que le sexe est un prédicteur indépendant de la réponse tensionnelle à l'HCT et au LIS. Conclusions : Les patients hypertendus d'origine africaine ont présenté une baisse tensionnelle plus grande en réponse au LIS qu'à l'HCT. Les hommes ont mieux répondu au LIS qu'à l'HCT alors que les femmes ont répondu de manière similaire aux deux traitements.

Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by enzyme-linked immunosorbent assay

The present study was conducted to detected IgG antibodies using Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by the enzyme-linked immunosorbent assay (ELISA). Sera from 90 subjects were analyzed (30 with strongyloidiasis, 30 with other parasites and 30 healthy individuals). Results were expressed in antibody titers, which were considered as positive when titer was > 80. Sensibility and specificity of the assay were 100% and 96.7%, respectively. It can be concluded that the heterologous alkaline extract could be employed in ELISA as a diagnostic aid in human strongyloidiasis, due to its advantages as easiness of obtaining, practicability in preparing, and high indexes of sensitivity and specificity.

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction- restriction fragment length polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Sustained 24-hour blockade of the renin-angiotensin system: a high dose of a long-acting blocker is as effective as a lower dose combined with an angiotensin-converting enzyme inhibitor.

Whether a higher dose of a long-acting angiotensin II receptor blocker (ARB) can provide as much blockade of the renin-angiotensin system over a 24-hour period as the combination of an angiotensin-converting enzyme inhibitor and a lower dose of ARB has not been formally demonstrated so far. In this randomized double-blind study we investigated renin-angiotensin system blockade obtained with 3 doses of olmesartan medoxomil (20, 40, and 80 mg every day) in 30 normal subjects and compared it with that obtained with lisinopril alone (20 mg every day) or combined with olmesartan medoxomil (20 or 40 mg). Each subject received 2 dose regimens for 1 week according to a crossover design with a 1-week washout period between doses. The primary endpoint was the degree of blockade of the systolic blood pressure response to angiotensin I 24 hours after the last dose after 1 week of administration. At trough, the systolic blood pressure response to exogenous angiotensin I was 58% +/- 19% with 20 mg lisinopril (mean +/- SD), 58% +/- 11% with 20 mg olmesartan medoxomil, 62% +/- 16% with 40 mg olmesartan medoxomil, and 76% +/- 12% with the highest dose of olmesartan medoxomil (80 mg) (P = .016 versus 20 mg lisinopril and P = .0015 versus 20 mg olmesartan medoxomil). With the combinations, blockade was 80% +/- 22% with 20 mg lisinopril plus 20 mg olmesartan medoxomil and 83% +/- 9% with 20 mg lisinopril plus 40 mg olmesartan medoxomil (P = .3 versus 80 mg olmesartan medoxomil alone). These data demonstrate that a higher dose of the long-acting ARB olmesartan medoxomil can produce an almost complete 24-hour blockade of the blood pressure response to exogenous angiotensin in normal subjects. Hence, a higher dose of a long-acting ARB is as effective as a lower dose of the same compound combined with an angiotensin-converting enzyme inhibitor in terms of blockade of the vascular effects of angiotensin.

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.

Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis

Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species - M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti" - were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, São Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.

Detection of Entamoeba histolytica/Entamoeba dispar in stool specimens by using enzyme-linked immunosorbent assay

Entamoeba histolytica actually comprises two genetically distinct but morphologically indistinguishable species. E. histolytica can cause invasive intestinal and extra intestinal disease, while E. dispar cannot. Identification and differentiation of E. dispar and E. histolytica in stool sample by microscopy is imprecise. Several weeks of culture and isoenzyme analysis are required to differentiate E. histolytica from E. dispar. In this study, we have used an enzyme-linked immunosorbent assay (ELISA) for detection of E. histolytica/E.dispar and compared it with microscopy. Eighty-eight samples were evaluated, trichrome staining was positive in 20.4% (18) and ELISA was positive in 29.5% (26). Both tests were positive in 14 (15.9%) samples, 4 (4.5%) only with direct microscopy, and 12 (13.6%) only with ELISA. Both tests were negative in 58 (65.9%) samples. Microscopy has low sensitivity and high specificity, low negative predictive value and high positive predictive value in comparison with ELISA. E. histolytica/E. dispar antigen detection ELISA tests are inexpensive compared to the specific tests, yield objective results and do not require experienced microscopists and can therefore be recommended for screening of stools worldwide and the results can be taken for treatment that are fitting with its clinic.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

Activity of ancestral restriction factors against ancient retroviruses

Analysis of TRIM5α and APOBEC3G genes suggests that these two restriction factors underwent strong positive selection throughout primate evolution. This pressure was possibly imposed by ancient exogenous retroviruses, of which endogenous retroviruses are remnants. Our study aims to assess in vitro the activity of these factors against ancient retroviruses by reconstructing their ancestral gag sequences, as well as the ancestral TRIM5α and APOBEC3G for primates. Based on evolutionary genomics approach, we reconstructed ancestors of the two largest families of human endogenous retroviruses (HERV), namely HERV-K and HERV-H, as well as primate ancestral TRIM5α and APOBEC3G variants. The oldest TRIM5α sequence was the catarhinne TRIM5α, common ancestor of Old World monkeys and hominoids, dated from 25 million years ago (mya). From the oldest, to the youngest, ancestral TRIM5α variants showed less restriction of HIV-1 in vitro [1]. Likewise three ancestral APOBEC3Gs sequences common to hominoids (18 mya), Old World monkeys, and catarhinnes (25 mya) were reconstructed. All ancestral APOBEC3G variants inhibited efficiently HIV-1Δvif in vitro, compared to modern APOBEC3Gs. The ability of Vif proteins (HIV-1, HIV-2, SIVmac and SIVagm) to counteract their activity tallied with the residue 128 on ancestral APOBEC3Gs. Moreover we are attempting to reconstruct older ancestral sequences of both restriction factors by using prosimian orthologue sequences. An infectious onemillion- years-old HERV-KCON previously reconstituted was shown to be resistant to modern TRIM5α and APOBEC3G [2]. Our ancestral TRIM5α and APOBEC3G variants were inactive against HERV-KCON. Besides we reconstructed chimeric HERV-K bearing ancestral capsids (up to 7 mya) that resulted in infectious viruses resistant to modern and ancestral TRIM5α. Likewise HERV-K viruses bearing ancestral nucleocapsids will be tested for ancestral and modern APOBEC3G restriction. In silico reconstruction and structural modeling of ancestral HERV-H capsids resulted in structures homologous to that of the gammaretrovirus MLV. Thus we are attempting to construct chimeric MLV virus bearing HERV-H ancestral capsids. These chimeric ancestral HERVs will be tested for infectivity and restriction by ancestral TRIM5α. Similarly chimeric MLV viruses bearing ancestral HERV-H nucleocapsids will be reconstructed and tested for APOBEC3G restriction.

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

Molecular characterization of human T-cell lymphotropic virus coinfecting human immunodeficiency virus 1 infected patients in the Amazon region of Brazil

The present work evaluated the epidemiology of human immunodeficiency virus 1/human T-cell lymphotropic virus (HIV-1/HTLV) coinfection in patients living in Belém (state of Pará) and Macapá (state of Amapá), two cities located in the Amazon region of Brazil. A total of 169 blood samples were collected. The sera were tested by enzyme-linked immunosorbent assay to determine the presence of antibodies anti-HTLV-1/2. Confirmation of infection and discrimination of HTLV types and subtypes was performed using a nested polymerase chain reaction targeting the pX and 5' LTR regions, followed by restriction fragment length polymorphism and sequencing analysis. The presence of anti-HTLV1/2 was detected in six patients from Belém. The amplification of the pX region followed by RFLP analysis, demonstrated the presence of HTLV-1 and HTLV-2 infections among two and four patients, respectively. Sequencing HTLV-1 5' LTR indicated that the virus is a member of the Cosmopolitan Group, Transcontinental subgroup. HTLV-2 strains isolated revealed a molecular profile of subtype HTLV-2c. These results are a reflex of the epidemiological features of HIV-1/HTLV-1/2 coinfection in the North region of Brazil, which is distinct from other Brazilian regions, as reported by previous studies.

Efficacy of an enzyme-linked immunosorbent assay as a diagnostic tool for schistosomiasis mansoni in individuals with low worm burden

IgM-ELISA is an immunoenzymatic method useful for detection of IgM antibodies against a fraction of Schistosoma mansoni adult worm antigen (AWA) that is soluble in trichloroacetic acid (AWA-TCA). This method was applied to three groups of individuals with different clinical and epidemiological characteristics, and the results compared with those obtained by other diagnostic methods: immunofluorescence test for detection of IgM antibodies (IgM-IFT) or IgG antibodies (IgG-IFT), ELISA for detection of IgG antibodies (IgG-ELISA), and two parasitological methods, Kato-Katz and miracidium hatching. The IgM-ELISA presented a sensitivity of 98%, when the parasitologic fecal examination was defined as reference diagnostic method, and a specificity of 98 and 97.3%, respectively for the group of clinically healthy individuals and other helminth carriers. A comparative analysis between the results of IgM-ELISA and those obtained by other serologic tests showed a good degree of agreement, with Kappa indices ranging from 0.95 to 0.98. The diagnostic efficacy of 97.8%, as determined with schistosomiasis patients with low parasitic burden, suggests the excellent performance of the IgM-ELISA and its usefulness for the diagnosis of schistosomiasis when applied in low endemic areas.