Evaluation of the serologic response against two consensus V3 loop peptides from human immunodeficiency virus-1 in Cuban patients.

OBJECTIVES: A retrospective study was conducted to evaluate the antibody response of Cuban patients infected with human immunodeficiency virus (HIV)-1 against two consensus peptides from the third variable domain (V3) loop of glycoprotein gp120. METHODS: The study included sera from 10 individuals at different stages of disease. Two 15-meric synthetic peptides designed from a consensus sequence, belonging to group B or C of HIV-1, were used to determine antibody titers and avidity indexes in an indirect enzyme-linked immunoassay. RESULTS: A high reactivity against both peptides was detected, with 80% of the sera reacting with at least one of the peptides. The antibody titers and avidity indexes did not correlate with disease progression. Additionally, for one of the patients from whom the virus had been isolated, a higher avidity index was found against the homologous peptide. CONCLUSIONS: This study showed high reactivity against two consensus peptides from the V3 loop of gp120 among patients with HIV. Large scale studies are needed to determine whether the titers or avidity of anti-V3 antibodies, at the early stages of infection, are predictive of disease progression. Both peptides are candidates for inclusion in experimental vaccines.

Discrete Markov image modeling and inference on the quadtree.

Noncasual Markov (or energy-based) models are widely used in early vision applications for the representation of images in high-dimensional inverse problems. Due to their noncausal nature, these models generally lead to iterative inference algorithms that are computationally demanding. In this paper, we consider a special class of nonlinear Markov models which allow one to circumvent this drawback. These models are defined as discrete Markov random fields (MRF) attached to the nodes of a quadtree. The quadtree induces causality properties which enable the design of exact, noniterative inference algorithms, similar to those used in the context of Markov chain models. We first introduce an extension of the Viterbi algorithm which enables exact maximum a posteriori (MAP) estimation on the quadtree. Two other algorithms, related to the MPM criterion and to Bouman and Shapiro's (1994) sequential-MAP (SMAP) estimator are derived on the same hierarchical structure. The estimation of the model hyper parameters is also addressed. Two expectation-maximization (EM)-type algorithms, allowing unsupervised inference with these models are defined. The practical relevance of the different models and inference algorithms is investigated in the context of image classification problem, on both synthetic and natural images.

[Ultramicro-ELISA for the detection of IgM antibodies anti M. leprae]. / Ultramicroelisa para la detección de anticuerpos IgM anti M. leprae.

The availability of an ultramicroanalytic system (SUMA) and species-specific antigen of M. leprae obtained by chemical synthesis, have made possible the standardization and validation of an ultramicroELISA assay for detecting specific human IgM antibodies to this mycobacterium. The specificity of this test to demonstrate the infection with M. leprae was corroborated through a screening of 433 blood bank serum samples and other 265 from different groups (100, control group, 50 tuberculosis patients, 65 leprosy patients, 50 from household). The results obtained in the additional study of 140 household sero showed a high correlation (r = 0.98) with the conventional microELISA method. The use of SUMA allows saving reagents and time since sample handling, plate reading, print out and storing the data are computer assisted.

[Indirect ultramicroElisa for the detection of total antibodies against cytomegaloviruses in human blood]. / UltramicroELISA indirecto para la detección de anticuerpos totales a Citomegalovirus en suero humano.

We have standardized an indirect ultramicro ELISA assay for detecting antibodies to human Cytomegalovirus (CMV) using human serum samples (UMELISA CMV). The optimal concentration of coating antigen (30 ug/ml), serum dilution (1:40) and anti-human conjugate working dilution (1:1500), were determined by a check board titration method. The UMELISA CMV was compared with the latex agglutination test for antibodies to CMV (Dupont de Nemours) and with an indirect immunofluorescent method. The results have showed the high coincidence, sensitivity and especificity of the proposed assay regarding the two methods compared with, and supporting its use either for a blood donors screening or in the serological diagnosis of this infection by paired serum samples.

[Indirect ultra-micro-ELISA for detecting IgG antibodies in patients with fascioliasis]. / UltramicroELISA indirecto para la detección de anticuerpos IgG en pacientes con fascioliasis.

The standardization of an ultramicroELISA for the detection of IgG antibodies anti excretory-secretory antigens of Fasciola hepatica (UME-Fasciola) is described. It was studied a considerable group of sera of which 56 were from patients with fascioliasis, 168 from patients with other parasitic diseases, and 300 from sound persons that were used as negative controls. As regards the parasitology test considered as "Gold Standard", the UME-Fasciola showed a sensitivity of 100%, an specificity of 98%, and predictive values for positives and negatives of 90.3% and 100%, respectively. Cross-reaction was only observed with the sera from patients infected with Opistorchis felineus. On comparing the UME-Fasciola with the conventional ELISA, it was obtained a concordance index of 95.5%.

[Normalization and application of an ELISA ultramicro for the detection of herpes simplex virus antibodies]. / Normalización y aplicación de un ultramicro ELISA para la detección de anticuerpos al virus herpes simple.

An ELISA ultramicro was normalized for detecting herpes simplex virus antibodies. A study was made on 145 samples from the Pedro Kourí Tropical Medicine Institute and the Blood Bank, both by ELISA ultramicro and immunofluorescence and ELISA, and 98% and 99% of coincidence was reached, respectively. It indicates good correspondence between these techniques.

[Estimation of IgG Adenovirus antibody titers using a standard curve and an indirect ultramicroELISA essay]. / Estimación del título de anticuerpos IgG a Adenovirus utilizando una curva patrón y un ensayo de ultramicroELISA indirecto.

It was possible to standardize a procedure which combined an indirect microELISA assay with a standard curve and that allowed to estimate the titre of IgG antibodies to Adenovirus in samples of human serum, using only one dilution of these. Based on the end-point titre previously determined for a panel of 117 serum samples, we selected 90 of these samples (r2 = 0.98) to build 4 standard curves that related the natural logarithm of the fluorescence responses to the natural logarithm of the end-point titre for a wide range of serum dilutions (1:40 = 1:320). It was selected the curve corresponding to serum dilution 1:40 (r2 = 0.81), which made possible an optimum utilization of those accessories designed to handle the volumes in the ultramicro range and, therefore, the automation of the whole procedure. The results obtained as regards the complement fixation test (100% of sensitivity and 97.3% of specificity) support the use of this method in our laboratory as a complementary tool to carry out seroepidemiological studies on a large scale and with diagnostic ends.

[ELISA identification of agents causing light cytopathic effect isolated during the neuropathy outbreak in Cuba, 1991]. / Identificación por ELISA de agentes productores de efecto citopático ligero aislados durante la epidemia de neuropatía en Cuba, 1991.

Using as a reference the strain 44/93 isolated during the neuropathy epidemic in 1991 and characterized as a producer of a light cytopathic effect (L-CPE), it was possible the standardization of an ELISA for the fast identification of other strains with similar effect. The assay consisted in a sandwich-type ELISA where the conditions selected for each reactive (10 micrograms/mL for the coating antibody, 1 mg/mL for the antigen, and dilution 1/2,000 for the conjugate) allowed to have an adequate discrimination between the antigen and the antigen control for the reference strain used. The evaluation of a panel of reference viral strains and of other L-CPE producing strains showed a 100% of coincidence between this method and the isolation in cellular culture. The results obtained permit us to recommend the use of this assay as a more precise alternative to identify these agents.

[The standardization of an ultramicro ELISA assay for the detection of IgG antibodies to the respiratory syncytial virus]. / Normalización de un ensayo de ultramicroELISA para la detección de anticuerpos IgG al virus sincitial respiratorio.

An ultramicro ELISA assay of double antibody for the detection of IgG antibodies to the respiratory syncytial virus (RSV) was standardized. It was used a RVS antiprotein F monoclonal antibody produced by the Genetic Engineering and Biotechnology Center (GEBC) in Havana. The use of this antibody allowed to include crude antigenic preparations instead of purified fractions, which caused a significant reduction of the reactivity obtained with the antigen control. The assay conditions were determined by crossed titration. It was obtained a sensitivity of 97.2%, a coincidence of 91%, and a specificity of 83.3% of the UMELISA as regards the complement fixation. The results may be qualitatively expressed or by antibody titres using only one serum dilution (1:40) and a pattern curve.

[Evaluation of an ELISA for the serologic diagnosis of tuberculosis]. / Evaluación de un ELISA para el diagnóstico serológico de tuberculosis.

The use of an ELISA method for the serological diagnosis of tuberculosis was assessed through the study of the presence of circulating IgG antibodies to PPD in 220 serum samples. An 82% sensibility was determined in 50 serum samples from patients with pulmonary tuberculosis, and a specificity of 95.33% in 150 serum samples from apparently healthy subjects. 20 serum samples from patients with disorders other than tuberculosis were included in the study to determine possible cross reactions.

[Indirect ultramicroELISA for the detection of total adenovirus antibodies in human serum]. / UltramicroELISA indirecto para la detección de anticuerpos totales a Adenovirus en suero humano.

An indirect ultramicroELISA method was normalized for the detection of total antibodies to Adenovirus (AD) (UMELISA AD). The optimum concentration of surface antigen determined was 30 micrograms/mL; serum dilution was of 1:40; and conjugate dilution, of 1:1,000. The UMELISA AD was compared with the complement fixing and indirect immunofluorescence techniques. Results showed accordance by 88% and 96.6%, respectively. The method is valid for the screening of antibodies in seroepidemiological studies as well as for the diagnosis of the infection by paired serum samples.

[The use of ELISA and ultramicro ELISA in identifying a Coxsackie A9 strain isolated during the epidemic of neuropathy in Cuba]. / Utilización del ELISA y el ultramicroELISA en la identificación de una cepa de Coxsackie A9 aislada durante la epidemia de neuropatía en Cuba.

During 1993 an outbreak of epidemic neuropathy characterized by 2 major clinical forms: the optic neuropathy and peripheral neuropathy, occurred in our country. Although the cause of the disease was considered to be multifactorial (mainly due to neurotoxic agents and to nutritional deficiency) an Enterovirus was isolated from the cerebrospinal fluid of a patient (strain 47/93, IPK) which was subsequently classified as a Coxsackie A9 virus (Cox A9) by the neutralization test, and using pools of Lim Benyesh Melnick (LBM) serum. Results obtained during the application of ELISA and ultramicro ELISA techniques for the identification of this agent from the supernatant of a culture of infected cells are reported, as well as the detection of antibodies to the strain found within populations with high and low incidence rates of the disease.

[Virological diagnosis of an outbreak of fever and rashes caused by parvovirus B19, Cuba, 1995]. / Diagnóstico virológico de un brote de fiebre y rash producido por parvovirus B19, Cuba, 1995.

The results obtained in the study of an a outbreak of fever and rash occurred in Havana City in March, 1995, are reported. Dengue, measles, rubella, herpes simplex, and Epstein Barr were discarded as causal agents of the outbreak in the samples of 35 patients. Parvovirus B19 was identified as the causing agent of the outbreak by the detection of IgM antibodies and the polymerase chain reaction technique (PCR). The infection produced by this agent was confirmed in 14/18 samples (77.7%) by some of the techniques used. This study makes reference to the first outbreak of Parvovirus B19 that was proved in Cuba.

[Validation of a ultramicroELISA for detecting antibodies against hepatitis B surface antigen]. / Validación de un ultramicroELISA de detección de anticuerpos contra el antígeno de superficie de la hepatitis B.

The results of a validation study of the ultramicroanalitical assay for the detection of antibodies against the hepatitis B surface antigen (UMELISA anti-HBsAg), which was carried out by comparing the results obtained with the Hepanostika anti-HBsAg, commercial diagnosis kit are presented. For this purpose, sera from the clinical assays of the Cuban recombinant vaccine against hepatitis B were used. With the first sera group (n = 30) it was obtained, 93.1% of sensitivity, 98.5% of specificity and a concordance of 94.3%. The correlation coefficient showed a similar trend of the results (p < 0.01) and no significant differences were found in the average geometrical titre (TPG) between both assays (p > 0.05). With the second group (n = 100), whose assays were carried out at the "Pedro Kouri" Institute of Tropical Medicine (PKI) and at the Immunoassay Center (IAC) simultaneously, it was observed a sensitivity of 96.25% in both centers, a specificity of 75% at the PKI and of 90% at the IAC, and a coincidence of 92% and 95%, respectively. The correlation coefficient presented similar values and there were no significant differences between the TPG obtained by the two methods (p > 0.05). The results attained show in general the validity of the new assay and the feasibility to put it into practice either for following up the infection, or for carrying out clinical assays of vaccine evaluations.

Reemergence of dengue in Cuba: a 1997 epidemic in Santiago de Cuba.

After 15 years of absence, dengue reemerged in the municipality of Santiago de Cuba because of increasing migration to the area by people from disease-endemic regions, a high level of vector infestation, and the breakdown of eradication measures. The 1997 epidemic was detected early through an active surveillance system. Of 2,946 laboratory-confirmed cases, 205 were dengue hemorrhagic fever, and 12 were fatal. No deaths were reported in persons under 16 years of age. Now the epidemic is fully controlled.

A phase I clinical trial of a multi-epitope polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV-1 infected human volunteers.

A phase I clinical trial was performed to examine the safety and immunogenicity of a multi-epitope polypeptide comprising the central 15 amino acids of the V3 loop from six HIV-1 isolates. This protein called TAB9 was emulsified in Montanide ISA720 (Seppic, Paris) and administered intramuscularly at doses of 0, 0.2 and 1 mg to 24 healthy, HIV-1 seronegative adult males. Three immunisations were given at months 0, 1 and 6 in a randomised, double blind, placebo controlled clinical trial. The placebo was generally well tolerated. However, severe local reactions were observed in TAB9 vaccinated subjects after the second and third inoculations. Seven out of eight volunteers from the lower dose group showed moderate or severe local inflammation, while four out of eight subjects from the higher dose group developed granulomas and sterile abscesses. In general, the reactogenicity depended on the number of inoculations given and the dose of TAB9. Both doses were immunogenic, all immunised volunteers seroconverted and antibodies were broadly reactive against the V3 peptides included in the protein. All vaccine's sera reacted against gp120 in Western blot and 50% of them also neutralised at least one out of five laboratory isolates tested. No differences between doses were found. Anti TAB9 lymphoproliferative responses were observed, being more intense in the high dose group. Due to the strong local reactions that were found in this study, a change in the formulation will be required for further trials with this vaccine candidate in humans.