RESUMEN
Hepatitis C virus (HCV) genotypes and subtypes were determined in hemodialysis patients in the Federal District, Brazil, by sequencing of the 5' noncoding (NC) and nonstructural 5B (NS5B) regions. From 761 patients, 66 anti-HCV-positive samples were tested for HCV RNA. All 51 HCV RNA-positive samples by PCR of the 5' NC region were genotyped as genotypes 1 (90.2%) and 3 (9.8%). Subtype 1a (82.3%) was the most prevalent, followed by subtypes 3a (9.8%), 1b (5.9%) and 1a/1b (2.0%). Forty-two samples could be amplified and genotyped in the NS5B region: 38 (90.5%) as genotype 1, subtypes 1a, and 8 (9.5%) as genotype 3, subtype 3a. For the 42 samples sequenced in both regions, the genotypes and subtypes determined were concordant in 100% and 95.2% of cases, respectively. Two samples presented discrepant results, with the 5' NC region not distinguishing correctly the subtypes 1a and 1b. These findings indicate that the HCV genotype 1, subtype 1a, is the most prevalent among hemodialysis patients in the Federal District, Brazil.
Asunto(s)
Regiones no Traducidas 5'/genética , Hepacivirus/genética , Hepatitis C/virología , ARN Viral/análisis , Proteínas no Estructurales Virales/genética , Secuencia de Bases , Genotipo , Hepacivirus/clasificación , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Diálisis RenalRESUMEN
Hepatitis C virus (HCV) genotypes and subtypes were determined in hemodialysis patients in the Federal District, Brazil, by sequencing of the 5' noncoding (NC) and nonstructural 5B (NS5B) regions. From 761 patients, 66 anti-HCV-positive samples were tested for HCV RNA. All 51 HCV RNA-positive samples by PCR of the 5' NC region were genotyped as genotypes 1 (90.2 percent) and 3 (9.8 percent). Subtype 1a (82.3 percent) was the most prevalent, followed by subtypes 3a (9.8 percent), 1b (5.9 percent) and 1a/1b (2.0 percent). Forty-two samples could be amplified and genotyped in the NS5B region: 38 (90.5 percent) as genotype 1, subtypes 1a, and 8 (9.5 percent) as genotype 3, subtype 3a. For the 42 samples sequenced in both regions, the genotypes and subtypes determined were concordant in 100 percent and 95.2 percent of cases, respectively. Two samples presented discrepant results, with the 5' NC region not distinguishing correctly the subtypes 1a and 1b. These findings indicate that the HCV genotype 1, subtype 1a, is the most prevalent among hemodialysis patients in the Federal District, Brazil.
Os genótipos e subtipos do vírus da hepatite C (HCV) foram determinados em pacientes em hemodiálise no Distrito Federal, Brasil, pelo sequenciamento das regiões 5' não codificante (NC) e não estrutural 5B (NS5B). De 761 pacientes, 66 amostras anti-HCV positivas foram testadas para RNA-HCV. Todas as 51 amostras RNA-HCV positivas por PCR para a região 5' NC foram genotipadas como dos genótipos 1 (90,2 por cento) e 3 (9,8 por cento). O subtipo 1a (82,3 por cento) foi o mais prevalente, seguido pelos subtipos 3a (9,8 por cento), 1b (5,9 por cento) e 1a/1b (2,0 por cento). Quarenta e duas amostras puderam ser amplificadas e genotipadas na região NS5B: 38 (90,5 por cento) como genótipo 1, subtipo 1a, e 8 (9,5 por cento) como genótipo 3, subtipo 3a. Para as 42 amostras sequenciadas nas duas regiões, os genótipos e subtipos determinados foram concordantes em 100 por cento e 95,2 por cento dos casos, respectivamente. Duas amostras apresentaram resultados discrepantes, sendo que a região 5' NC não diferenciou corretamente os subtipos 1a e 1b. Estes achados indicam que o genótipo 1, subtipo 1a, do HCV é o mais prevalente em pacientes em hemodiálise no Distrito Federal, Brasil.
Asunto(s)
Humanos , /genética , Hepacivirus/genética , Hepatitis C/virología , ARN Viral/análisis , Proteínas no Estructurales Virales/genética , Secuencia de Bases , Genotipo , Hepacivirus/clasificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Diálisis RenalRESUMEN
This study analyzed the genes pol and env to determine the genetic variability of HIV-1 in Central Brazil. Forty-one isolates of HIV-1-infected individuals had protease, reverse transcriptase, and C2C3/ env amplified by nested PCR and sequenced. The subtype was determined by the program REGA and phylogenetic analyses. The samples identified as putative recombinant forms were analyzed by SimPlot. A high prevalence of subtype B (95.1%) was observed, followed by mosaic viruses B/F (4.9%). The amino acid sequences from 30 HIV-1 isolates were analyzed for the antigenic intrasubtype diversity. The most prevalent gp120 V3 loop motif was the GPGR (United States/Europe) (43.3%), described in B and F subtypes, followed by the GPGK tetrapeptide (10%). The Brazilian variant B" (GWGR), GFGR, and GLGR tetrapeptides were found in 6.7%. Other V3 variants were found in eight isolates (26.7%). Phylogenetic tree analysis was also performed in order to verify the relationship of the HIV-1 samples from Central Brazil with other HIV-1 sequences that circulate in Brazil. The subtype B sequences from Central Brazil formed a polyphyletic cluster in the tree, indicating that these strains are similar to those from other geographic regions. These results contribute to the understanding of HIV in Brazil, and may prove useful for the development of vaccine candidates.
Asunto(s)
Genes env , Genes pol , Variación Genética , Infecciones por VIH/virología , VIH-1/genética , Secuencia de Aminoácidos , Brasil/epidemiología , Infecciones por VIH/epidemiología , Humanos , Datos de Secuencia Molecular , Filogenia , Recombinación GenéticaRESUMEN
In order to investigate the hepatitis C virus (HCV) genotypes in mid-west region of Brazil, 250 anti-HCV positive blood donors were studied. Among them, the anti-HCV serological status was confirmed in 205 (82%). HCV RNA was detected in 165 samples, which were genotyped. HCV types 1, 2 and 3 were found in 67.9%, 3% and 29.1% of the donors, respectively. In Goiás state, subtype 1a (50%) was the most prevalent, followed by subtypes 3a (30.9%) and 1b (16.7%). In Mato Grosso state, subtype 1a was also predominant (41%), followed by subtypes 1b (29.5%) and 3a (25%). In Mato Grosso do Sul state, subtypes 1a and 1b were detected equally (36.8%), followed by 3a (21.1%). Subtype 2b was rare (2.4%, 4.5% and 5.3%, respectively). In Distrito Federal, subtype 3a (39%) was more frequent than 1a (31.7%) and the remaining (29.3%) belonged to subtype 1b.
Asunto(s)
Donantes de Sangre , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/sangre , Hepatitis C/virología , Regiones no Traducidas 5'/genética , Adolescente , Adulto , Brasil/epidemiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Genotipo , Hepacivirus/inmunología , Hepatitis C/diagnóstico , Hepatitis C/epidemiología , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/análisisRESUMEN
Com objetivo de determinar os genótipos do vírus da hepatite C (HCV) circulantes na Região Centro-Oeste do Brasil, 250 doadores de sangue anti-HCV positivos foram estudados. Dentre eles, a positividade para anti-HCV foi confirmada em 205 (82%). O RNA-HCV foi detectado em 165 amostras, as quais foram genotipadas. Os tipos 1, 2 e 3 do HCV foram encontrados em 67,9%, 3% e 29,1% dos doadores, respectivamente. No Estado de Goiás, o subtipo 1a (50%) foi o mais prevalente, seguido pelos subtipos 3a (30,9%) e 1b (16,7%). No Estado de Mato Grosso, o subtipo 1a (41%) foi também predominante, seguido pelos subtipos 1b (29,5%) e 3a (25%). No Estado de Mato Grosso do Sul, os subtipos 1a e 1b foram igualmente detectados (36,8%), seguidos por 3a (21,1%). O subtipo 2b foi raro (2,4%, 4,5% e 5,3%, respectivamente). No Distrito Federal, o subtipo 3a (39%) foi mais freqüente que 1a (31,7%), sendo o restante (29,3%) identificado como subtipo 1b.
Asunto(s)
Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Donantes de Sangre , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/sangre , Hepatitis C/virología , /genética , Brasil/epidemiología , Ensayo de Inmunoadsorción Enzimática , Genotipo , Hepacivirus/inmunología , Hepatitis C/diagnóstico , Hepatitis C/epidemiología , ARN Viral/análisisRESUMEN
In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasília, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96 percent of the samples clustered with subtype B and the remaining 4 percent with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38 percent of the samples. Minor mutations were also found at the protease gene: L10I/V (7 percent), K20M (2 percent), M36I (11 percent), L63P (20 percent), A71T (2 percent), and V77I (7 percent). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11 percent), E44D (4 percent), D67N (11 percent), T69D (2 percent), K70R (11 percent), L74V (2 percent), L100I (4 percent), K103N (18 percent), V118I (9 percent), Y181C (11 percent), M184V (18 percent), G190A (4 percent), T215Y (4 percent), and K219E (4 percent). This study has shown that 84 percent of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38 percent), the NNRTIs delavirdine, nevirapine (31 percent), and efavirenz (24 percent), and the NRTIs lamivudine (18 percent), abacavir, and zidovudine (13 percent).
Asunto(s)
Humanos , Fármacos Anti-VIH , Farmacorresistencia Viral , Infecciones por VIH , VIH-1 , Terapia Antirretroviral Altamente Activa , Brasil , Variación Genética , Genotipo , Transcriptasa Inversa del VIH , Mutación , Reacción en Cadena de la Polimerasa , ARN Viral , Carga ViralRESUMEN
The objective of this study was to characterize hepatitis C virus (HCV) genotypes in blood donors from the Federal District, Central Brazil, and to compare HCV screening by serological assays and reverse transcriptase polymerase chain reaction (RT-PCR). Plasma samples from 57 individuals with reactive or indeterminate results in serological anti-HCV screening assays (ELISA or EIA) were tested for HCV RNA by RT-PCR. The results from a confirmatory LIA serological assay were also evaluated. The 5' non-coding region of the HCV genome was amplified from 41 PCR positive samples (71.9 percent), which were further characterized by nucleotide sequencing analysis. Of these, 60.9 percent were of HCV genotype 1 and 39.1 percent of genotype 3.
Asunto(s)
Humanos , Masculino , Femenino , Adolescente , Adulto , Persona de Mediana Edad , Donantes de Sangre , Hepacivirus , Hepatitis C , Anticuerpos contra la Hepatitis C , Brasil , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Genotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We report the prevalence of human papillomavirus type 16 (HPV-16) variants in women with cervical lesions from the Federal District, Central Brazil. We analyzed 34 HPV-16 samples, identifying the sequence variations of E6 and L1 genes and correlating variant frequency with disease status. The most prevalent HPV-16 variant was the European (50%), followed by Asian-American (41.2%), African-1 (5.9%), and African-2 (2.9%). European and non-European variants appeared in equal frequencies among the cytological types of lesions - atypical squamous or glandular cells of undetermined significance, cytological alterations suggesting HPV infection, cervical intraepithelial neoplasias, squamous cell carcinoma, and adenocarcinoma.
Asunto(s)
Variación Genética , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Enfermedades del Cuello del Útero/virología , Secuencia de Aminoácidos , Brasil/epidemiología , Femenino , Humanos , Datos de Secuencia Molecular , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/epidemiología , Reacción en Cadena de la Polimerasa , Prevalencia , Índice de Severidad de la Enfermedad , Enfermedades del Cuello del Útero/epidemiologíaRESUMEN
We report the prevalence of human papillomavirus type 16 (HPV-16) variants in women with cervical lesions from the Federal District, Central Brazil. We analyzed 34 HPV-16 samples, identifying the sequence variations of E6 and L1 genes and correlating variant frequency with disease status. The most prevalent HPV-16 variant was the European (50 percent), followed by Asian-American (41.2 percent), African-1 (5.9 percent), and African-2 (2.9 percent). European and non-European variants appeared in equal frequencies among the cytological types of lesions - atypical squamous or glandular cells of undetermined significance, cytological alterations suggesting HPV infection, cervical intraepithelial neoplasias, squamous cell carcinoma, and adenocarcinoma.
Asunto(s)
Humanos , Femenino , Variación Genética , Papillomaviridae , Enfermedades del Cuello del Útero , Secuencia de Aminoácidos , Brasil , Datos de Secuencia Molecular , Papillomaviridae , Reacción en Cadena de la Polimerasa , Prevalencia , Índice de Severidad de la Enfermedad , Enfermedades del Cuello del ÚteroRESUMEN
In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasilia, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96% of the samples clustered with subtype B and the remaining 4% with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38% of the samples. Minor mutations were also found at the protease gene: L10I/V (7%), K20M (2%), M36I (11%), L63P (20%), A71T (2%), and V77I (7%). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11%), E44D (4%), D67N (11%), T69D (2%), K70R (11%), L74V (2%), L100I (4%), K103N (18%), V118I (9%), Y181C (11%), M184V (18%), G190A (4%), T215Y (4%), and K219E (4%). This study has shown that 84% of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38%), the NNRTIs delavirdine, nevirapine (31%), and efavirenz (24%), and the NRTIs lamivudine (18%), abacavir, and zidovudine (13%).
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Terapia Antirretroviral Altamente Activa , Brasil , Variación Genética , Genotipo , Infecciones por VIH/virología , Transcriptasa Inversa del VIH , VIH-1/efectos de los fármacos , Humanos , Mutación , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Carga ViralRESUMEN
The objective of this study was to characterize hepatitis C virus (HCV) genotypes in blood donors from the Federal District, Central Brazil, and to compare HCV screening by serological assays and reverse transcriptase polymerase chain reaction (RT-PCR). Plasma samples from 57 individuals with reactive or indeterminate results in serological anti-HCV screening assays (ELISA or EIA) were tested for HCV RNA by RT-PCR. The results from a confirmatory LIA serological assay were also evaluated. The 5' non-coding region of the HCV genome was amplified from 41 PCR positive samples (71.9%), which were further characterized by nucleotide sequencing analysis. Of these, 60.9% were of HCV genotype 1 and 39.1% of genotype 3.
Asunto(s)
Donantes de Sangre , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/sangre , Hepatitis C/virología , Adolescente , Adulto , Brasil , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Genotipo , Hepacivirus/inmunología , Hepatitis C/sangre , Hepatitis C/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
As a contribution to the public health authorities in planning prophylactic and therapeutic vaccine strategies, we describe the prevalence of human papillomavirus (HPV) types in women presenting abnormal cytological results in Pap smear screening tests in the Federal District, Central Brazil. We studied 129 cervical scraping samples from women whose cytological tests showed either pre-neoplastic or neoplastic lesions. Amplification of HPV DNA was performed by polymerase chain reaction using consensus primers MY09 and MY11 followed by identification of isolates by restriction fragment length polymorphism. We detected HPV DNA in 62 percent of the samples, including HPV-16 in 43.8 percent, HPV-58 in 12.5 percent, HPV-31 in 10 percent, HPV-53 in 6.3 percent, each of HPV-18 and HPV-33 in 3.8 percent of the isolates. Other types (HPV-35, -52, -66, -CP8304, -6, -11, and -CP8061) were less frequent (= or < 2.5 percent each). The prevalence of HPV-58 was relatively higher in this population than in data in South America, but similar to results obtained in other studies in Latin America, Europe, and Eastern Asia. Case-control studies need to be carried out to establish the association between the prevalence of HPV types specially the less frequent high-risk types and cervical cancer.
Asunto(s)
Humanos , Femenino , Adulto , Persona de Mediana Edad , Papillomaviridae , Infecciones Tumorales por Virus , Neoplasias del Cuello Uterino , Brasil , ADN Viral , Genotipo , Papillomaviridae , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Prevalencia , Factores de Riesgo , Infecciones Tumorales por Virus , Enfermedades del Cuello del Útero , Neoplasias del Cuello Uterino , Frotis VaginalRESUMEN
As a contribution to the public health authorities in planning prophylactic and therapeutic vaccine strategies, we describe the prevalence of human papillomavirus (HPV) types in women presenting abnormal cytological results in Pap smear screening tests in the Federal District, Central Brazil. We studied 129 cervical scraping samples from women whose cytological tests showed either pre-neoplastic or neoplastic lesions. Amplification of HPV DNA was performed by polymerase chain reaction using consensus primers MY09 and MY11 followed by identification of isolates by restriction fragment length polymorphism. We detected HPV DNA in 62% of the samples, including HPV-16 in 43.8%, HPV-58 in 12.5%, HPV-31 in 10%, HPV-53 in 6.3%, each of HPV-18 and HPV-33 in 3.8% of the isolates. Other types (HPV-35, -52, -66, -CP8304, -6, -11, and -CP8061) were less frequent (= or < 2.5% each). The prevalence of HPV-58 was relatively higher in this population than in data in South America, but similar to results obtained in other studies in Latin America, Europe, and Eastern Asia. Case-control studies need to be carried out to establish the association between the prevalence of HPV types specially the less frequent high-risk types and cervical cancer.
Asunto(s)
Papillomaviridae/clasificación , Infecciones por Papillomavirus/virología , Infecciones Tumorales por Virus/virología , Neoplasias del Cuello Uterino/virología , Adolescente , Adulto , Anciano , Brasil , ADN Viral/genética , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Prueba de Papanicolaou , Papillomaviridae/genética , Infecciones por Papillomavirus/patología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Prevalencia , Factores de Riesgo , Infecciones Tumorales por Virus/patología , Enfermedades del Cuello del Útero/patología , Enfermedades del Cuello del Útero/virología , Neoplasias del Cuello Uterino/diagnóstico , Frotis VaginalRESUMEN
BACKGROUND: Current guidelines for antiretroviral (ARV) therapy recommend at least triple-drug combination, the so-called highly active antiretroviral therapy (HAART). Not all patients respond to HAART and the development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV. OBJECTIVE: In an attempt to correlate the HIV therapeutic failure with reverse transcriptase (RT) and protease resistance mutations, we describe the ARV resistance profile in patients failing HAART in Brazil. We studied 267 Brazilian HIV-1 infected patients failing HAART looking for mutations in RT and protease genes. The mutation profile of the viruses infecting these individuals were deduced and correlated to laboratorial parameters. STUDY DESIGN: Two different HIV-1 genomic regions were targeted for PCR amplification, the protease (pro) and pol RT (palm finger region) genes. The mutations related to drug resistance in RT gene was analyzed using a line probe assay (LIPA(R)) and pro amino acids positions 82 and 90 were screened through RFLP using HincII restriction digestion. RESULTS: There was strong correlation between the mutation in the pro and RT genes and therapeutic failure. The main mutation found in RT gene was the M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). In the pro gene the main mutation found was L90M (26%) followed by dual substitution in L90M and V82A (6%). All mutations profiles matched very well with the patients drug regimen. CONCLUSIONS: This study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance.
Asunto(s)
Farmacorresistencia Viral/genética , Infecciones por VIH/virología , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , VIH-1/genética , Mutación , Terapia Antirretroviral Altamente Activa , Brasil/epidemiología , Estudios Transversales , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , VIH-1/efectos de los fármacos , Humanos , Prevalencia , Insuficiencia del TratamientoRESUMEN
Introdução: O carcinoma de células escamosas corresponde a 95 porcento dos tumores penianos malignos. Sua relação com o papilomavirus humano (HPV) tem sido estudada por vários autores. Nosso estudo descreve o predomínio do DNA do HPV nos carcinomas de células escamosas penianas em Brasília, Brasil. Materiais e métodos: O DNA do papilomavirus humano foi amplificado por reação em cadeia de polimerase (RCP) utilizando os "primers" GP5+/GP6+ e pU-1M/pU-2R, após a extração do material embebido em parafina, de 59 pacientes com carcinoma peniano. HPV isolados foram classificados pela digestão com enzimas de restrição e seqüência automática. Resultados: DNA de boa qualidade foi obtido de 37 amostras das 59 selecionadas previamente. O predomínio do DNA do HPV variou de 13,5 porcento (5/37) à 64,8 porcento (19,24) quando consideramos amplificação com "primers" pU-1M/pU-2R ou GP5+/GP6+, respectivamente. O HPV-16 foi o único tipo detectado. Conclusões: Nossos dados indicam que GP5+/GP6+RPC fornecem uma boa taxa de detecção do DNA do HPV e classificação em tecidos fixados com formol e embebidos em parafina. O predomínio do DNA do HPV pode der considerado alto quando comparado aos dados de outros estudos sul-americanos. A alta freqüência do HPV-16 e a baixa ocorrência do HPV-18 estão de acordo com os nossos resultados numa população feminina da mesma região do Brasil. Investigações adicionais devem ser conduzidas para avaliar a freqüência dos tipos de HPV associados a outras anomalias epiteliais penianas e diferentes tipos de neoplasias assim como em pacientes assintomáticos da região central do Brasil.