Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing

Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

High genetic diversity in human immunodeficiency virus: type 1 in Jamaica / Elevada diversidad genética del virus de la inmunodeficiencia humana: tipo 1 en Jamaica

The subtypes of the human immunodeficiency virus - type 1 (HIV-1) strains from 54 HIV-1 - infected persons including 44 strains which were typed previously by heteroduplex mobility assay (HMA) were determined by DNA sequencing and phylogenetic analysis. Of 54 HIV- infected persons, 92.5% were infected with HIV-1 subtype B and 7.5% with other HIV-1 subtypes including subtypes D (3.7%), A (1.9%) and J (1.9%). In the phylogenetic analysis, the subtype A virus found in the sample clustered with subtype A reference strains and a circulating recombinant form (CRF) reference strain which originates in Central Africa and is circulating in Cuba indicating a close relationship between these viruses. There was 86% concordance between HMA and DNA sequencing in assigning subtype B viruses. For the non-B subtype viruses, there was less concordance between the two methods (67%). The results confirm the predominance of HIV-1 subtype B strains and the high genetic diversity of HIV-1 strains in circulation in Jamaica. The efficacies and some limitations of the HMA as a method of HIV-1 subtyping also were noted. It is important that the HIV/AIDS epidemic in Jamaica be monitored meticulously for possible expansions in non-B subtypes and the emergence of inter-subtype recombinant forms. We recommend that the more expensive DNA sequencing and phylogenetic analysis, including HIV-1 genotyping for antiretroviral drug resistance testing, be used as an adjunct to the more cost-effective HMA to track the HIV/AIDS epidemic in Jamaica.

Los subtipos de cepas de virus de la inmunodeficiencia humana-tipo-1 de 54 personas infectadas con el VIH-1, que incluyeron 44 cepas previamente clasificadas según su tipo mediante ensayo de movilidad de heterodúplex (HMA), fueron determinados mediante secuenciación de ADN y análisis filogenético. De 54 personas infectados con VIH, 92.5% estaban infectadas con VIH-1 subtipo B y 7.5% con otros subtipos de VIH-1 incluidos los subtipos D (3.7%), A (1.9%), J (1.9%). En el análisis filogenético, el virus de subtipo A hallado en la muestra, se agrupa con las cepas de referencias del subtipo A y una cepa de referencia de forma recombinante circulante (CRF), que tienesu origen en África Central y está circulando en Cuba, lo que indica una estrecha relación entre estos virus. Hubo un 86% de concordancia entre el HMA y la secuenciación del DNA en la asignación de virus de subtipo B. Para los virus de subtipo no B, hubo menos concordancia entre los dos métodos (67%). Los resultados confirman el predominio de las cepas del subtipo B del VIH-1, y la alta diversidad genética de las cepas del VIH-1 en circulación en Jamaica. También se señalaron las eficacias y algunas limitaciones del HMA como método de clasificación del VIH-1 en subtipos. Es importante monitorear meticulosamente la epidemia de VIH/SIDA en Jamaica, a fin de detectar posibles expansiones de subtipos no B y la aparición de formas recombinantes inter-subtipos. Recomendamos que por ser ambos métodos más costosos, tanto la secuenciación de ADN como el análisis filogenético - incluyendo el genotipado del VIH-1 para probar la resistencia antiretroviral del medicamento - sean usados como complementos del HMA, el cual es más costo-efectivo, para seguir de cerca el rastro de la epidemia VIH/SIDA en Jamaica.

Diagnóstico de linfomas cutáneos mediante detección de clonalidad por PCR-heterodúplex / Diagnosis of cutaneous lymphomas by clonicity detection through PCR-heteroduplex technique

La detección de clonalidad en los síndromes linfoproliferativos mediante el estudio del reordenamiento de los genes de las inmuglobulinas y del receptor de células T, es utilizada para esclarecer si una proliferación o infiltrado de linfocitos es maligno o no. Este tipo de estudio es de particular utilidad en presencia de lesiones cutáneas cuyo origen linfoide o dermatológico resulta difícil de definir. Mediante la técnica de PCR-heterodúplex se estudiaron los genes de la cadena pesada de las inmunoglobulinas y de la cadena gamma del receptor de las células T, en 10 pacientes que presentaban manifestaciones dermatológicas atribuibles a algún tipo de linfoma cutáneo. Se observó reordenamiento clonal en 7 pacientes, lo cual permitió confirmar el diagnóstico de micosis fungoide y otros tipos de linfomas cutáneos. En 3 pacientes que no mostraron reordenamiento clonal, no fue posible confirmar por esta técnica un proceso linfoide de carácter maligno. Se demostró la utilidad del estudio cuando en presencia de una afección en la piel, es difícil diferenciar un proceso dermatológico de un síndrome linfoproliferativo con manifestaciones en piel.

The clonicity detection in the lymphoproliferative syndromes by studying the rearrangement of the immunoglobulin genes and of the T-receptor cells is used to make clear if a proliferation or infiltrate of lymphocytes is malignant or not. This type of study is particularly useful in the presence of cutaneous lesions whose lymphoid or dermatological origin is difficult to define. By the PCR-heteroduplex technique, the genes of the immunoglobulin heavy chain and of the T-cell receptor chain were studied in 10 patients that presented dermatological manifestations attributable to some kind of cutaneous lymphoma. Clonal rearrangement was observed in 7 patients, which allowed to confirm the diagnosis of mycosis fungoides and other types of cutaneous lymphomas. It was not possible to confirm a lymphoid process of malignant character by this technique in 3 patients who did not show clonal rearrangement. The usefulness of the study was proved when in the presence of a skin affection, it was difficult to differentiate a dermatological process from a proliferative syndrome with cutaneous manifestations.

Human immunodeficiency virus type- 1 subtypes of infected patients in Espírito Santo, Brazil

Genetic variability of human immunodeficiency virus type - 1(HIV-1) is a potential threat for both diagnosis and treatment of HIV/AIDS, as well as the development of effective vaccines. Up to now, HIV subtypes circulating among HIV-positive patients in the state of Espírito Santo were not known. In the present study, blood samples from 100 therapy-naïve HIV-1 infected patients were collected and the HIV subtype was determined through the Heteroduplex Mobility Assay (HMA). Ninety-seven out of 100 studied samples were subtyped by HMA, 73 samples (75.2 percent) were from subtype B, 9 (9.3 percent) from subtype F, 3 (3.1 percent) from subtype C, 6 (6.2 percent) Benv/Fgag, and another 6 (6.2 percent) Fenv/Bgag, what suggests that recombinant viruses were present in the studied samples. Twenty-eight percent of the subtype B samples were represented by the Brazilian B" subtype, which were identified by RFLP with Fok I. Data presented here demonstrate that the epidemiological characteristics of the HIV epidemic in the state of Espírito Santo are similar to those from the other Southeastern states and helped to better understand the genetic polymorphism of HIV in Brazil.

PCR-heterodúplex por agrupamiento: implementación de un método de identificación de portadores de la mutación más común causal de fibrosis quística en Colombia / PCR-heteroduplex by grouping: rapid screening carrier method for cystic fibrosis F508del mutation in Colombia

Introducción: La fibrosis quística (FQ) es una enfermedad autosómica recesiva frecuente, con una incidencia de 1 en 2,500 recién nacidos. La causan más de 1,300 mutaciones distintas en el gen regulador de la conductancia transmembranal de la fibrosis quística (CFTR). Sin embargo, la mutación F508del es la más común en la mayoría de las poblaciones. Objetivos: Desarrollo de una técnica rápida, de bajo costo y confiable que permita filtrar con rapidez a los portadores o afectados por esta mutación que mediante el asesoramiento genético, contribuya a disminuir la aparición de nuevos casos y a un diagnóstico temprano de los enfermos y así lograr un descenso en la morbilidad y la mortalidad asociadas con la fibrosis quística en Colombia. Metodología: En el presente estudio se aplicó la técnica PCR-heterodúplex por agrupamientos, gracias al análisis de 400 muestras de sangre en papel filtro obtenidas de individuos asintomáticos para la FQ. Resultados: En las pruebas de validación de la técnica PCR-heterodúplex por agrupamiento se obtuvo una eficiencia, reproducibilidad y especificidad de 100% y una sensibilidad de 92%. Conclusiones: Se demostró la sensibilidad y reproducibilidad de la técnica PCR Directa-heterodúplex por agrupamientos de hasta 10 muestras, que se pueden emplear en programas para filtrar heterocigotos y afectados de F508del.

Background: Cystic fibrosis (CF) is the most frequent autosomal recessive disorder in the Caucasian population with an incidence of 1 in 2,500 newborns. More than 1,300 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that causes CF have been described. However, mutation F508del is the most common mutation in different populations around the world. Objective: To develop a fast, reliable and low-cost technique to screen carriers and affected individuals for the F508del mutation. This kind of analysis will have an impact on genetic counselling to decrease the incidence of new cases, in the early diagnosis and instauration of appropriate treatment to decrease morbidity and mortality associated to CF in Colombia. Methods: The reliability of the PCR-heteroduplex by grouping technique by analysis of 400 blood spot samples from asymptomatic CF patients was defined. Results: Using PCR-heteroduplex by grouping technique 100% efficiency, reproducibility and specificity and 92%sensitivity were found.Conclusions: The sensitivity and reproducibility of the PCR-heteroduplex by grouping technique up to pooling of 10 samples were demonstrated. This kind of analysis could be used in heterozygotes and affected screening programs.

Detection of genetic polymorphism among and within Echinococcus granulosus strains by heteroduplex analysis of a microsatellite from the U1 snRNA genes

Polymerase chain reaction of a pentanucleotide microsatellite in the U1 snRNA gene complex generated a multiple band pattern due to the priming of paralogous sequences. Denaturation and slow renaturation of polymerase chain reaction products allow the formation of heteroduplex DNA that can be detected by its differential mobility in polyacrylamide gel electrophoresis. Heteroduplex analysis was used to determine if the U1 snRNA microsatellite could be a useful genetic marker in Echinococcus granulosus. A U1 snRNA microsatellite fragment from E. granulosus was isolated and characterized by Southern blot and sequencing. Four E. granulosus strains were analyzed: sheep, Tasmanian sheep, cattle, and camel strains. The former two showed polymorphism and shared three of the six patterns found for sheep strain. The cattle strain displayed two patterns, and the camel strain was monomorphic. The electrophoretic profiles were used for statistical analysis in order to determine genetic distance and the relationship among strains. Heteroduplex analysis can be helpful in genotyping E. granulosus strains and is useful in detecting polymorphism within strains.

HIV-1 genetic diversity in Argentina and early diagnosis of perinatal infection

HIV-1 diagnosis of perinatally exposed children is usually performed by molecular biology-based methods, allowing the direct detection of the virus. Thus, HIV-1 genomic variability within and across strains plays a major role in relation to the sensitivity of these tests, often leading to misdiagnosis. We describe the performance of an in-house multiplex nested PCR (nPCR) for early detection of HIV-1 infection in perinatally exposed children born in Argentina, where the percentage of diverse BF recombinants is as high as 80%. After evaluation of 1316 HIV-1 perinatally exposed children collected over a 7-year period, the specificity and sensitivity of the diagnostic nPCR was of 100% and 99.2% respectively, with only two false negative cases indicating a good performance of the diagnostic nPCR in the Argentine pediatric cohort. In search of unusual HIV-1 subtypes among 22 HIV-1 infected cases presenting partial or complete HIV-1 gene amplification failure, we performed phylogenetic and recombination analysis of a vpu-env fragment in addition to gag and env Heteroduplex Mobility Assay screening. The most unusual findings included two subtypes A and a novel BC recombinant, while the majority of the strains were a variety of different BF recombinants. These results indicate the presence of novel and heterogeneous genotypes in our country and the need of continuous viral surveillance not only for diagnostic test optimization but also for the eventual implementation of a successful vaccine.

Morphological and molecular characterization of three species of Anastrepha Schiner and of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

Four species of Tephritoidea, three from genus Anastrepha: A. obliqua (Macquart), A. sororcula Zucchi and A. serpentina (Wiedemann), and one from genus Ceratitis, C. capitata (Wiedemann) were compared based on puparium morphology and application of the Heteroduplex Mobility Assay (HMA). Puparia were characterized for the first time using the spiracular posterior plate morphology. Application of HMA allowed the detection of variability in the D2 domain from 28S rRNA gene in all four species (confirmed by sequencing). This is a fast, simple, sensitive and inexpensive assay that was used for the first time in the analysis of two Tephritoidea genera. The detected variability suggests that the tecnique has great potential for rapid determination of infestations with two or more species in the same host fruit.

Foram comparadas com base na morfologia do pupário e na técnica de HMA (Heteroduplex Mobility Assay)) quatro espécies de Tephritoidea, três delas pertencentes ao gênero Anastrepha, A. obliqua (Macquart), A. sororcula Zucchi e A. serpentina (Wiedemann) e uma do gênero Ceratitis, C. capitata (Wiedemann). Os pupários foram caracterizados pela primeira vez com base na morfologia da placa espiracular posterior. A aplicação da técnica de HMA possibilitou a detecção de variabilidade no segmento D2 do gene 28S rRNA nas quatro espécies (confirmada por seqüenciamento). Esta é uma técnica rápida, simples, sensível e barata que é aplicada pela primeira vez na análise de dois gêneros de Tephritoidea. A variabilidade observada sugere grande potencial da técnica no caso da determinação da infestação por duas ou mais espécies num mesmo fruto hospedeiro.

Molecular epidemiology of human immunodeficiency virus-1 in the state of Ceará, Northeast, Brazil

We analyzed, by env and gag heteroduplex mobility assay, 149 human immunodeficiency virus (HIV-1) positive samples collected in Ceará during the year 2000. The prevalence of subtype B was 81.2 percent and the prevalence of subtype F and B/F recombinants were both 2.7 percent. Eight (5.4 percent) and 12 (8 percent) out of 149 samples showed indeterminate results in the env and gag analysis respectively. By FokI restriction fragment length polymorphism, 34 percent of the subtype B samples were identified as the typical Brazilian subtype B.In the present study, we identified HIV-1 subtype F and B/F in Ceará for the first time. Our results contribute to the understanding of HIV in Brazil, and may prove useful for the development of vaccine candidates

Molecular epidemiology of HIV-1 in Rio Grande, RS, Brazil

We conducted a molecular epidemiological study to investigate HIV-1 strains in Rio Grande, southern Brazil, searching for an association with transmission mode and risk behavior. Patients (185) identified at an AIDS treatment reference Hospital, from 1994 to 1997, were included; from which 107 blood samples were obtained. Nested PCR was realized once for each sample; for amplified samples (69) HIV subtypes were classified using the heteroduplex mobility assay. Subtypes identified were B (75 percent), C (22 percent) and F (3 percent). All infections with C were diagnosed after 1994. Comparing patients with B and C, no differences were detected regarding demographic, clinical and laboratory characteristics; survival analysis did not reveal differences in HIV to AIDS evolution. A higher proportion of injecting drug users, IDU (not significant, p<.07) was found among those with C. This suggests that C may have been introduced in this area through IDU, and is being spread, probably by their sexual partners, to persons with other risk practices

HIV-1 subtypes among intravenous drug users from two neighboring cities in São Paulo State, Brazil

In order to assess the molecular epidemiology of HIV-1 in two neighboring cities located near the epicenter of the HIV-1 epidemics in Brazil (Santos and São Paulo), we investigated 83 HIV-1 strains obtained from samples collected in 1995 from intravenous drug users. The V3 through V5 region of the envelope of gp 120 was analyzed by heteroduplex mobility analysis. Of the 95 samples, 12 (12.6 percent) were PCR negative (6 samples from each group); low DNA concentration was the reason for non-amplification in half of these cases. Of the 42 typed cases from São Paulo, 34 (81 percent, 95 percent confidence limits 74.9 to 87.0 percent) were B and 8 (19 percent, 95 percent confidence limits 12.9 to 25.0 percent) were F, whereas of the 41 typed cases from Santos, 39 (95 percent, 95 percent confidence limits 91.6 to 98.4 percent) were B and 2 (5 percent, 95 percent confidence limits 1.6 to 8.4 percent) were C. We therefore confirm the relationship between clade F and intravenous drug use in São Paulo, and the presence of clade C in Santos. The fact that different genetic subtypes of HIV-1 are co-circulating indicates a need for continuous surveillance for these subtypes as well as for recombinant viruses in Brazil

Uso de una reacción de polimerasa en cadena simple combinada con un ensayo de movilidad de heteroduplexes en la caracterización de la región 5ï no codificante del virus de la hepatitis C / Use of a simple polymerase chain reaction combined with a heteroduplex mobility assay to characterize the non coding 5ï region of hepatitis C virus

Background: One of the most used methods for the characterization of hepatitis C virus strains is the use of a nested polymerase chain reaction (PCR) with a restriction fragment length polymorphism (RFLP) assay. Sometimes, RFLP results do not differentiate new strains. There are other more complex methods and only the sequencing of the PCR fragment allows a correct characterization of the strain. Aim: To report the detection of hepatitis C virus using a single PCR assay of the 5' non codifying region. Material and methods: Thirty five serum samples coming from patients with chronic hepatitis or blood donors were assayed for hepatitis C virus. Results: The reported method increases the PCR sensitivity through the combination of polyacrylamide gel electrophoresis and silver staining of amplified products. This allowed the semi quantitative estimation of viral load and the characterization of amplified products through their electrophoretic motility. These PCR products were used in a heteroduplex motility assay, allowing the discrimination between sequences of different genotypes. Conclusions: Heteroduplex assays can be used to characterize the 5' non codifying region of the hepatitis C virus for routine laboratory purposes