[Determination of hepatitis B virus genotype and detection of lamivudine-resistance mutations]. / Determinación del genotipo del virus de la hepatitis B y detección de mutaciones de resistencia al tratamiento con lamivudina.

OBJECTIVES: 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. PATIENTS AND METHOD: 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.

Intrahepatic hepatitis C virus replication is increased in patients with regular alcohol consumption.

AIMS: To assess clinical significance of liver hepatitis C virus RNA levels and their relationship with epidemiological, biochemical and histological factors. METHODS: A total of 50 patients (mean age 35.5+/-7 years) with biopsy-proven chronic hepatitis C infection were recruited. Risk factors were drug abuse (n=21), transfusion (n=16), other parental routes (n=8; surgery=3, tattooing=5), and idiopathic (n=5). Duration of infection was 16+/-9 years. All patients showed abnormal alanine aminotransferase levels and positive serum hepatitis C virus RNA. Hepatitis C virus genotype was assessed by Inno-Lipa. Liver biopsy was performed for histology and for hepatitis C virus RNA quantification by Amplicor-HCV-Monitor Daily alcohol consumption was recorded on two occasions by anamnesis. Inflammation grade was mild (n=31) or severe (n=19). Fibrosis was early stage (n=42) or advanced (n=8). RESULTS: Mean hepatitis C virus RNA levels were 9.4x10(5)+/-1.5x10(6) copies/microg of total RNA in liver tissue, and 9.1x10(5)+/-1.3x10(6) copies/ml in serum. Viral load in liver was positively correlated with that in serum (r=0.51, p<0.001) and there was a significant relationship between daily alcohol consumption and intrahepatic hepatitis C virus burden (r=0.53; p<0.001). Patients infected with genotype 3a showed lower intrahepatic hepatitis C virus load than patients infected with genotype 1b; albeit without reaching statistical significance (0.49x10(6)+/-0.89x10(6) vs 1.44x10(6)+/-1.9x10(6) copies/microg of total RNA; p=NS). No relationships were observed between liver viral burden and age, risk factor status, duration of infection, ferritin and alanine aminotransferase levels or with grading and staging. CONCLUSIONS: Hepatitis C virus load in serum is a mirror of intrahepatic hepatitis C virus levels. Chronic alcohol consumption enhances intrahepatic hepatitis C virus concentration.

[Influence of pregnancy in chronic hepatitis C virus infection]. / Influencia del embarazo en la infección crónica por el virus C de la hepatitis.

BACKGROUND: There is scarce information about the influence of pregnancy in patients with chronic hepatitis C virus infection is little know. PATIENTS AND METHODS: 6,556 pregnant women were screened for anti-HCV (ELISA II). We determine ALT, HCV-RNA by PCR (Amplicor Roche) and HCV viraemia (Amplicor-HCV-Monitor Roche) in the third trimester of pregnancy and after 6 months of delivery. HBsAg, anti-HIV and HCV serotype (Murex 1-3) were also determined. STATISTICAL ANALYSIS: Fisher test, paired-t and U Mann Whitney. RESULTS: Anti-HCV was positive in 59 out of 6,556 (0.9%). Mean (SD) age: 27 (9) years (range, 18-40). Drug users: 34 (57%), post-transfusion: 10 (18%) and unknown: 15 (25%). HIV positive 11 (19%). Serotype 1, 30 (51%), setotype 3, 7 (20%), and nontypeable, 22 (37%). We studied HCV-RNA before and after delivery in 35 women, 8 out of 35 (23%) had HCV-RNA negative in both analysis. ALT was normal in 88% of women during pregnancy and in 42% after delivery. ALT levels in pregnancy were 32.6 (39.5) and in postpartum 64.5 (53.4) U/l (p < 0.005). 6 women were RNA-VHC negative during pregnancy and positive in postpartum. HCV viraemia during pregnancy and postpartum was 503 (1,203) and 1,014 (1,907) thousand copies/ml (p < 0.05). No relation was found among ALT or HCV viraemia with risk factors, serotype or coinfection with HIV. CONCLUSIONS: The prevalence of anti-HCV in pregnant women is 0.9%. ALT is usually normal in pregnancy. A quarter of women were HCV-RNA negative in pregnancy and positive after delivery. The viraemia was lower in pregnancy than after delivery, which is consistent with the fact of the low mother-to-infant HCV transmission rate.

Outbreak of Brucella melitensis among microbiology laboratory workers.

We report on an outbreak of laboratory-acquired brucellosis involving four technicians working at a microbiology laboratory. All cases occurred in a period of 4 months. Blood cultures and the Rose Bengal test were positive for Brucella spp. in all cases. Microagglutination was positive for Brucella spp. at titers of between 1/40 and 1/160. All patients were cured after treatment.

[An evaluation of the blood culture in disseminated mycobacteriosis]. / Valoración del hemocultivo en la micobacteriosis diseminada.

The objective of this study was to evaluate the usefulness of blood culture in the diagnosis of disseminated mycobacteria (DMB). This prospective study included all blood cultures done for patients with fever and under suspicion of having DMB between January 1991 and July 1992. Fifty-seven blood samples from 16 patients were cultured; 14 (87.5%) patients were HIV positive and all were diagnosed as having DMB. The cultures were processed by lysis-centrifugation and identification of mycobacteria was by hybridization with a DNA probe. Mycobacterial growth was detected in 5 cultures (8.7%) from 4 patients (25%) (3 HIV positive). M. tuberculosis was isolated in 3 and M. avium in 1. Mean time until isolation was 46 days. In all cases mycobacteria were isolated in other samples before they were found in cultures: M. tuberculosis was isolated in 2 bronchial aspirates (BAS), 2 in liver tissue (L), 2 in spleen tissue (S), one in alveolar bronchial lavage, one in sputum, one in spinal fluid (SF) and one in urine. M. avium was isolated in sputum and ALB. The three patients in whom M. tuberculosis was found died 1.4 and 32 days after admission. In samples from the 12 DMB patients with negative cultures (11 HIV positive, 92%), M. tuberculosis was isolated in 100% of ganglion and S samples, 90% in urine, 69% in sputum, 67% in ABL and LB, 63% in BAS and 33% in SF. None of these patients died in hospital. We find blood culture to be of little use in the diagnosis of DMB. Analysis of other samples leads to faster diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

[The pattern of the sensitivity of Aeromonas spp. that produce extraintestinal infections]. / Patrón de sensibilidad de las Aeromonas spp. productoras de infecciones extraintestinales.

We studied the sensibility to several antimicrobics of aeromonas strains isolated in extraintestinal infections during a period of five years, in order to establish which could be the best empirical therapy in these processes. Twenty-two strains of A. hydrophila were evaluated, was isolated (8 from hemocultures, 6 from wound exudate, 2 from abscess aspirate, 2 from peritoneal fluid and 1 from urine, pleural fluid, bile and catheter). The identification was made using the GNI card System (Vitek System) and other tests. The sensibility was assessed using the GNS-BH and GNS-BI card System (Vitek), which allows to know the clinical category and CMI value in microgram/ml. All the strains presented some resistance to antibiotics. 77.8% of the strains were resistant to more than one antibiotic and 64.9%, to more than two antibiotics. We detected 21.7% strains resistant to 4 drugs. The most frequent resistance association was ampicillin, cephoxitine, cephazoline, cephalotine, with or without amoxicillin and clavulanic acid. All the strains were sensible to cephamandol, cephuroxime, cephtriaxone, cephtazidime, cephotaxime, thicarciline, aztreonam, azlociline, mezlociline, piperaciline, gentamycin, amikacine, chloranphenicol and ciprofloxacine. All the strains were resistant to ampicillin; 60.9% of the strains were sensible to the association amoxicillin and clavulonic acid. In conclusion, the treatment of extraintestinal infections does not differs basically from the treatment of the infections caused by Gram-negative bacillus. There are a broad range of effective antibiotics: aminoglycosides (gentamycin, tobramycin, amikacine), piperacyline, cephalosporines (cephuroxime, cephotaxime, cephtazidime and cephtriaxone), chloranphenicol, penemas, monobactanes of fluorquinolone.

[Lower respiratory infections caused by beta hemolytic group A Streptococcus]. / Infecciones respiratorias bajas por Streptococcus betahemolítico grupo A.

Three patients with beta-hemolytic group A streptococcal pneumonia were described. We emphasize the opportunistic pathogenic ability of the microorganism in the lower respiratory tract, and we describe the possible pathogenic mechanisms as well as the more frequent symptoms and complications.

Interaction of aminoglycosides and cephalosporins against Pseudomonas aeruginosa. Correlation between interaction index and killing curve.

Three multiresistant clinical isolates of Pseudomonas aeruginosa were evaluated, by the chequerboard and the killing curve methods, for in-vitro synergy between cephalosporins and aminoglycosides. The killing-curve method was standardized to give results comparable with those obtained with the chequerboard test (FIC index). This was achieved with combinations showing synergy by chequerboard (FIC less than or equal to 0.75) by using in the killing curves subinhibitory concentrations: half the MIC in single antibiotic assay and one eighth the MIC in the combinations. For combinations that showed indifference by chequerboard (FIC = 1) half the MIC was used for antibiotics alone and in combination. For the antagonistic combinations by chequerboard (FIC greater than 2) concentrations equal to the MIC were used with single antibiotics and combinations in the killing curve experiments. Prediction of killing curve results could then be obtained with the FIC index. The killing curve results could not be explained by pH changes or inactivation of the antibiotics.

Value of serological diagnosis in congenital syphilis. Report of nine cases.

The diagnosis of congenital syphilis is difficult since it depends mainly on the results of serological tests. The results of five serological tests (three specific and two non-specific) in nine neonates with congenital syphilis are compared with those obtained in three with passively acquired antibodies. It appeared that the serological diagnosis of congenital syphilis must be based on the finding of specific neonatal antibodies in cord serum, which give positive results to the fluorescent treponemal antibody absorption test for immunoglobulin M, together with high titres of total IgM and negative results to latex tests. The non-specific tests are useful for confirming the efficacy of treatment. The mean number of cases of congenital syphilis in Seville is 0.81/1000 live births.