[Determination of hepatitis B virus genotype and detection of lamivudine-resistance mutations].
OBJECTIVE
To determine hepatitis B virus (HBV) genotypes in southern Seville (Spain) and investigate the development of lamivudine-resistance mutations by using a hybridization technique with specific probes and by comparing the results with those of the direct sequencing technique. To evaluate the temporal relationship between variations in the level of HBV-DNA and detection of mutant variants. To analyze the influence of several genotypes on the pattern of mutations developed and on values of viral load and alanine aminotransferase (ALT) after their development.


METHODS
In 37 patients with chronic HBV infection, HBV genotype was determined using the LiPA technique. In 10 of these patients undergoing lamivudine treatment for a mean of 19.2 months, the development of lamivudine-resistant mutations was investigated. In these 10 patients, the LiPA technique was compared with direct sequencing. During lamivudine treatment, we determined HBV-DNA by polymerase chain reaction (PCR) and ALT every 3-6 months.


RESULTS
The most frequent genotypes were D (45.9%) and A (18.9%); 2 patients were genotype B while 18.9% had mixed genotypes. Sequencing showed identical results except in one mixed genotype. Mutations were found in 60% of the cases. The results of sequencing were in agreement, except in the detection of mixed populations composed of mutants and wild-type (WT). Patients with genotype A showed the pattern M204I+WT in the first 12 months and those with genotype D showed the pattern L180M+M204V with or without WT at 18 months. In 5/6 cases, an increase of > 1 log10 in HBV-DNA was observed 3-8 months before the mutation was detected by LiPA. In patients with genotype B, levels of HBV-DNA and ALT after the development of mutations was lower than basal levels and was also lower than those in patients with genotypes A and D.


CONCLUSIONS
The LiPA technique for determination of HBV genotype and detection of lamivudine-resistance mutations shows excellent correlation with the most complex sequencing technique. Genotype D predominates in southern Seville. During lamivudine treatment, an increase in the level of HBV-DNA detected by PCR predicts the development of mutations before these are demonstrated by LiPA.
Nogales
M C
MC
Servicio de Microbiología, Hospital Universitario de Valme, Sevilla, Spain. mariac.nogales.sspa@juntadeandalucia.es
Serrano
M C
MC
Suárez
E
E
Corpas
R
R
Pérez
L
L
Claro
R
R
Jarana
R
R
Romero-Gómez
M
M
Martín-Mazuelos
E
E
D000328:Adult; D004279:DNA, Viral / Q000032:analysis; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D005821:Genetic Techniques; D005838:Genotype; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000235:genetics*; D006801:Humans; D019259:Lamivudine / Q000627:therapeutic use*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use*; D019562:Viral Load
D004279:DNA, Viral; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine