ABSTRACT
We report a fast (less than 3 h) and cost-effective melting temperature assay method for the detection of single-nucleotide polymorphisms in the MBL2 gene. The protocol, which is based on the Corbett Rotor Gene real time PCR platform and SYBR Green I chemistry, yielded, in the cohorts studied, sensitive (100 percent) and specific (100 percent) PCR amplification without the use of costly fluorophore-labeled probes or post-PCR manipulation. At the end of the PCR, the dissociation protocol included a slow heating from 60° to 95°C in 0.2°C steps, with an 8-s interval between steps. Melting curve profiles were obtained using the dissociation software of the Rotor Gene-3000 apparatus. Samples were analyzed in duplicate and in different PCR runs to test the reproducibility of this technique. No supplementary data handling is required to determine the MBL2 genotype. MBL2 genotyping performed on a cohort of 164 HIV-1-positive Brazilian children and 150 healthy controls, matched for age and sex and ethnic origin, yielded reproducible results confirmed by direct sequencing of the amplicon performed in blind. The three MBL2 variants (Arg52Cys, Gly54Asp, Gly57Glu) were grouped together and called allele 0, while the combination of three wild-type alleles was called allele A. The frequency of the A/A homozygotes was significantly higher among healthy controls (0.68) than in HIV-infected children (0.55; P = 0.0234) and the frequency of MBL2 0/0 homozygotes was higher among HIV-1-infected children than healthy controls (P = 0.0296). The 0 allele was significantly more frequent among the 164 HIV-1-infected children (0.29) than among the 150 healthy controls (0.18; P = 0.0032). Our data confirm the association between the presence of the mutated MBL2 allele (allele 0) and HIV-1 infection in perinatally exposed children. Our results are in agreement with the literature data which indicate that the presence of the allele 0 confers a relative risk of 1.37 for HIV-1 infection through vertical transmission.
Subject(s)
Child , Humans , HIV Infections/genetics , Mannose-Binding Lectin/genetics , Polymorphism, Single Nucleotide , Polymerase Chain Reaction/methods , Case-Control Studies , Cost-Benefit Analysis , Gene Frequency , HIV Infections/transmission , Polymerase Chain Reaction/economics , Reproducibility of Results , TemperatureABSTRACT
Foram estudados 50 pacientes com AIDS, todos estes pacientes apresentavam anticorpos ant-HIV1 (ELISA) e preenchiam os critérios de pontuaçao OPAS/Caracas de definiçao de casos de AIDS em adultos. A análise do liquido cefalorraqueano (LCR) incluiu: pressao; citologia (número de células e aspectos citomorfológicos); proteína total e eletroforese; concentraçoes de glicose, cloretos e testes imunológicos para sífilis, toxoplasmose e infecçoes virais (citomegalovírus, varicela-zoster, Herpes simplex, e HIV1). Investigaçoes bacteriológicas e micológicas (pesquisa direta e cultura), além de teste de aglutinaçao (látex) paracryptococcus foram também realizados. Os testes imunológicos usados foram fixaçao do complemento, imunofluorescência indireta, hemaglutinaçao passiva e/ou ELISA. Todos os LCR foram analisados no mesmo laboratório seguindo sempre a mesma metodologia. O LCR esteve alterado em 45 pacientes (90,0 por cento) dos 50 pacientes estudados. As principais alteraçoes encontradas no LCR foram: aumento de gamaglobulina em 25 casos (55,5 por cento); aumento da proteína total em 23 (51,1 por cento); hipercitose em 22 (48,9 por cento) e diminuiçao dos cloretos em 18(40,0 por cento). A detecçao de anticorpos anti- HIV1 estiveram presentes em 42 pacientes (93,3 por cento). Toxoplasmose isolada ou associada a outros agentes foi a infecçao oportunista mais frequente, detectada em 26 casos (57,7 por cento). O LCR deverá ser sempre analisado em todos os pacientes com AIDS, com ou sem sintomas neurológicos.