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.

Divergent strains of human immunodeficiency virus type 1 circulating in India, subtyped by heteroduplex mobility assay.

Genomic variations in HIV-1 represent a major problem in understanding disease progression, studying drug resistance and developing effective vaccines. Heteroduplex Mobility Assay (HMA) was used for analyzing HIV-1 subtypes resulting from genetic similarity or divergence of C2 -V3 -V5 region of envelope gene between HIV-1 strains obtained from clinical samples in a tertiary care center at Pune. DNA from the PBMCs of infected individuals was amplified by nested PCR. Heteroduplexes were then formed by denaturing DNA from the unknowns with DNA from the reference strains. The results were analyzed by polyacrylamide gel electrophoresis. Out of 177 samples analyzed, 170 were of subtype C (96%). Four samples were found to be of subtype B (2.2%); in three samples, no definitive assignment of subtype was possible by HMA and these perhaps could be circulating recombinant forms (CRFs) of HIV-1. These findings may have significant implications toward development of a candidate vaccine for India.

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.

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.

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay.

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.

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