Comparación de tres métodos genotípicos para la detección de resistencia del VIH-1 a los antirretrovirales / Comparison of three genotyping methods for the detection of HIV-1 resistance to antiretroviral drugs

Con la aparición de nuevos antirretrovirales ha mejorado considerablemente la expectativa de vida de los pacientes infectados por el virus de la inmunodeficiencia humana (VIH), pero mutaciones en la región que codifica la transcriptasa inversa (TI) y la proteasa (P) del VIH-1 pueden producir fallos en el tratamiento, por lo cual los estudios de resistencia pueden ser de utilidad para la selección del tratamiento más eficaz. El objetivo de nuestro trabajo ha sido comparar tres métodos genotípicos de detección de resistencias para valorar cuál puede resultar más adecuado en el laboratorio. Se han estudiado un total de 90 pacientes tratados con antirretrovirales, mediante tres métodos diferentes: hibridación reversa que identifica la presencia de virus salvaje o mutante en 19 codones clave para las regiones de transcriptasa inversa y proteasa, InnoLiPA HIV-1 (Line Probe Assay, Innogenetics, Belgica), y dos de secuenciación, ViroSeq HIV-1 Genotyping System (Perkin Elmer/Applied Biosystems, California) y TrueGene HIV-1 Genotyping System (Visible Genetics, Canada). Se detectaron 408 mutaciones por InnoLiPA, 572 por TrueGene y 721 por ViroSeq. La hibridación detectó un número significativamente superior de mutaciones primarias, asociadas con los grados de resistencia más altos (p <0.001). También la hibridación detectó un mayor número de mezclas de virus salvajes y mutantes. En general existe una elevada concordancia entre los tres métodos, si bien es superior entre los de secuenciación. Esta técnica identificó un mayor número de mutaciones, pero la hibridación presentó mayor sensibilidad en la detección de mutaciones primarias y poblaciones mixtas, siendo de fácil aplicación en los laboratorios clínicos, aunque para la detección de nuevas mutaciones implicadas en la resistencia es necesario el análisis secuencial (AU)

[Comparison of three genotyping methods for the detection of HIV-1 resistance to antiretroviral drugs]. / Comparación de tres métodos genotípicos para la detección de resistencia del VIH-1 a los antirretrovirales.

Highly active antiretroviral therapy has dramatically improved the life expectancy of human immunodeficiency virus (HIV)-infected patients, but mutations in the HIV-1 reverse transcriptase (RT) and protease (P) genes confer drug failure. Evaluation of drug resistance genotyping in HIV-1 has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. The aim of this study was to evaluate the optimal procedure to determine the resistance profile in the laboratory. Plasma from 90 antiretroviral-treated patients was analyzed by reverse hybridization, which identifies the presence of wild-types or mutations at the 19 key codons for protease and RT regions, and was compared with two other methods of direct cDNA sequencing. A total of 408 mutations were detected by InnoLiPA HIV-1, (Line Probe Assay, Innogenetics, Belgium), 572 by TrueGene HIV-1 Genotyping System (Visible Genetics, Canada), and 721 by ViroSeq HIV-1 Genotyping System (Perkin Elmer/Applied Biosystems, California). Hybridization detected a significantly higher number of primary mutations which are associated with a high level of drug resistance (p <0.001). Hybridization also detected a higher number of mixtures of wild-type and mutant viruses. There was a good concordance among the three methods, although it was higher between the two sequencing methods. Sequencing determines a higher number of mutations, but hybridization better identifies primary mutations correlated with a high level of drug resistance. Hybridization is more suitable for detecting mixed populations and is easier to implement in clinical laboratories but does not eliminate the need for sequence analysis for detection of drug-resistant HIV.

[Recent transmission of tuberculosis in Madrid (Spain): usefulness of molecular techniques]. / Transmisión reciente de la tuberculosis en Madrid: utilidad de las técnicas moleculares.

BACKGROUND: Recent studies suggest that many tuberculosis cases in urban areas result from recent transmission. The aim of this study was to determine patterns of tuberculosis transmission in Madrid. PATIENTS AND METHODS: A prospective population-based molecular epidemiological study of patients diagnosed of tuberculosis was conducted in three urban districts of Madrid (455.050 inhabitants) during 1997-1998. Clinical, demographic and epidemiological data were reviewed. Patients were included in clusters when their isolates contained: a) six or more IS6110 bands in an identical pattern, or b) five or fewer IS6110 bands that matched identically and had an identical spoligotyping pattern. RESULTS: Of 207 positive-culture patients, 148 (71,5%) were DNA fingerprinted. A total of 18 clusters which included 62 patients (41,9%) were identified. Clusters contained between 2 and 12 cases. Risk factors for clustering included: age < 35 years (OR = 4,1, 95% CI: 1,9-8,9), injection drug use (OR = 4,7, 95% CI: 1,6-14,8), HIV infection (OR = 2,7, 95% CI: 1,1-6,8), and a history of imprisonment (OR = 2,9, 95% CI: 1,2-7,2). The epidemiological investigation identified connections among 27% of clustered patients. CONCLUSIONS: A high proportion of cases of tuberculosis in urban Madrid result from recent transmission. Molecular epidemiology studies give valuable information for urban tuberculosis control.

Incidence and clinical impact of fluoroquinolone-resistant Escherichia coli in the faecal flora of cancer patients treated with high dose chemotherapy and ciprofloxacin prophylaxis.

This study evaluated the susceptibility of Escherichia coli to quinolones in 72 stool samples collected from 31 patients with solid tumours who had undergone high dose chemotherapy (HDC) and peripheral blood stem-cell (PBSC) rescue with ciprofloxacin prophylaxis. Samples were obtained at admission, after completing prophylaxis and three months later. All E. coli strains isolated from baseline samples were susceptible to quinolones. Fluoroquinolone-resistant E. coli strains were isolated in 10 (32%) patients in the second sample. In eight of these patients isolates were susceptible 3 months later. No patient developed infection due to fluorquinolone-resistant E. coli. No differences were observed in outcome between patients with susceptible and resistant flora.

[Prospective randomized trial of meropenem versus cefotaxime and metronidazole in the treatment of intraabdominal infections]. / Comparación prospectiva, aleatoria y abierta de meropenem frente a cefotaxima y metronidazol en el tratamiento de las infecciones intraabdominales.

BACKGROUND: The empiric antibiotic treatment of intraabdominal infections is in constant evolution. Monotherapy appears to be a desirable goal because of the simplicity of its administration, lack of toxic effects and wide spectrum. PATIENTS AND METHODS: A multicentre, prospective, randomized, open study was carried out to compare two antibiotic regimens in the treatment of intraabdominal infections in patients undergoing surgery. Ninety-eight consecutive patients were randomly allocated into two groups. One group (GM, n = 51) received meropenem (1 g/8 h) and the other (GCM, n = 47) a combination of cefotaxime (2 g/8 h) plus metronidazol (0.5 g/8 h). Clinical and bacteriological responses were assessed at the end of treatment and at 2-4 weeks. RESULTS: The severity of patients as assessed by the APACHE II score was similar in both groups (GM: 7.2 and GCM: 8.1). Three patients in each group could not be evaluated due to premature interruption of treatment or deviation from the protocol. The mean duration of treatment was 7.4 days in GM and 7.9 days in GCM. A satisfactory clinical response was obtained in 95% of patients in both groups. 31 patients (61%) in GM and 26 patients (55%) in GCM were bacteriologically evaluable. Bacteriological erradication was achieved in 94% of patients in GM and in 92% of patients in GCM. CONCLUSION: Meropenem is a good alternative for single antibiotic therapy in intraabdominal infections of moderate severity.

[Invasive fungal infections in liver transplant recipients: analysis of 21 cases]. / Infección fúngica profunda en receptores de un trasplante de hígado: análisis de 21 casos.

BACKGROUND: Invasive fungal infections (IFI) are one of the most important causes of mortality in liver transplant (LT) recipients. The aim of this study was to describe the characteristics of IFI in the LT program of our institution with an special emphasis in the differences between Candida infections (CI) and that caused by other fungi (NCI). PATIENTS AND METHODS: Retrospective analysis of the hospital charts of 21 patients who underwent a LT from February 1987 to December 1995. The diagnosis of IFI required the histological evidence of tissue invasion or a positive culture in a tissue sample or in an usually sterile fluid. Esophageal candidiasis was not considered as IFI. Antifungal prophylaxis was performed either with nystatin or fluconazole. RESULTS: Twenty-one of 356 patients (6%) developed a total of 23 episodes of IFI. Pathogens were Candida spp. (n = 10), Aspergillus (n = 8), Zygomicetes (n = 4) and Cryptococcus (n = 1). Fifty-seven percent of the episodes of IFI (80% of those caused by Candida and 38% of those produced by other fungi; p < 0.05) developed in the first 3 months after transplantation and only 5 episodes appeared after the sixth month. The diagnosis of IFI was done at autopsy in 6 patients (29%). Overall, NCI (13 episodes) predominated over CI (10 episodes), being the later the cause of the 54% of the episodes in the first 178 recipients but only the 30% in the last 178 patients (p = 0.09). No differences were found in the distribution of the risk factors amongst those patients with CI or NCI. Seventeen of the 21 patients (71%) died and 15 of these deaths (72%) were attributable to fungi; 15 patients who died either did not receive amphotericin (n = 6) or received a cumulative dose lower than 500 mg. Six patients received a cumulative dose of more than 1.5 g (mean, 3.2 g) and four of them were cured. Mortality in the nonfungal infection group was 26% (p < 0.001). CONCLUSIONS: IFI was a rare but severe complication in our LT recipients. The relative frequency of CI was progressively decreasing during the study period, being NCI the predominant infections. Amphotericin therapy was effective only when a high cumulative dose could be administered.

[Bipolaris australiensis in a Spanish patient with allergic chronic sinusitis]. / Bipolaris australiensis en un enfermo Español con sinusitis alérgica crónica.

Allergic fungal sinusitis is a disease that results from hypersensitivity reaction of the host against fungi colonizing the paranasal sinuses. A 36 years old Spanish man with no history of travel abroad had a history of asthma, nasal polyps, allergic rhinitis and a chronic sinusitis with nasal congestion and headaches. Computed tomography showed pansinusitis and opacification of the paranasal sinuses. A pure culture of Bipolaris australiensis was grown from sinus tissues. Infections caused by Bipolaris spp. and treatment regimes are discussed.

A double-blind, randomized comparative trial: flutrimazole 1% solution versus bifonazole 1% solution once daily in dermatomycoses.

In a double-blind, randomized study the efficacy and tolerance of flutrimazole 1% solution were compared with bifonazole 1% solution, applied once daily for 4 weeks, in 40 patients with culturally proven dermatophytosis or cutaneous candidosis. Forty patients with mycologically proven pityriasis versicolor were treated with once-daily application for 1 week. The four groups of patients and distribution of target lesions were similar, although in the flutrimazole group more patients had cutaneous candidosis (n = 8 versus n = 1). The distribution of the sum of clinical scores was also similar in both groups. At the end of therapy the proportion of patients with negative microscopy and culture was 85% in the flutrimazole group and 65% in the bifonazole group. There was a significant difference (P = 0.022) in terms of efficacy, since 80% of patients in the flutrimazole group versus 40% in the bifonazole group were judged to have received effective treatment. At the assessment 6 weeks after the end of therapy the percentages of flutrimazole- and bifonazole-treated patients with negative mycology were 75% and 65% respectively. There were two relapses (one in each group), which represents a 5% rate. Fifteen flutrimazole-treated patients (75%) compared with 12-bifonazole-treated patients (60%) had overall effective therapy. Two patients treated with bifonazole (10%) and one treated with flutrimazole (5%) had a premature termination due to adverse events attributable to the medication. On assessment 3 weeks after the end of treatment, the patients with pityriasis versicolor were all clinically and mycologically healed with negative fluorescence, including the patients who withdrew from the full course of treatment (one in each group). Nine weeks after the end of therapy all the patients remained cured, with no relapses. The overall incidence of adverse events (mild local reactions such as irritation, burning and itching) was one and seven cases for bifonazole and flutrimazole respectively. One patient in each group had to abandon treatment owing to severe intolerance.

Topical treatment of tinea corporis and tinea cruris with eberconazole (WAS 2160) cream 1% and 2%: a phase II dose-finding pilot study.

In a phase II pilot dose-finding study 60 patients with mycologically proven tinea corporis and tinea cruris were treated with eberconazole cream 1% once daily (group A, 15 patients), 1% twice daily (group B, 15 patients), 2% once daily (group C, 15 patients) or 2% twice daily (group D, 15 patients). Treatment was continued for 2 weeks after clinical cure; the maximum duration of treatment was limited to 6 weeks. The characteristics of the four groups of patients, distribution of the target lesions, clinical sum of baseline scores and infecting organisms were similar. Statistical examination showed that the mean time of appearance in weeks of negative microscopy and culture was similar in the four groups. There was no significant difference between the groups in terms of the range and mean duration of treatment. By the end of the study, treatment was effective in 13 patients (87%) in group A, 14 (93%) in group B and 11 (73%) in both groups C and D (mycological cure and clinical cure or residual minimal signs and symptoms). One patient in group A did not respond to treatment and two patients in group C had to withdraw because of side-effects. No undesirable effects or significant changes were seen in the blood tests. At the assessment 6 weeks post therapy, eberconazole was judged to have been effective in 93% of patients in group A, 100% of patients in groups B and D and 61% of patients in group C. Although not statistically significant, a trend towards more favourable results was seen in group B when considering the mean time of appearance of clinical cure and negative KOH and culture.

Itraconazole versus griseofulvin in the treatment of tinea capitis: a double-blind randomized study in children.

BACKGROUND: Tinea capitis is a fungal infection in which topical therapy is often unsuccessful. Griseofulvin has been considered to be a first-line therapy. Other antifungal agents are the azole derivatives. Among these, itraconazole was compared with griseofulvin in children in a double-blind study. PATIENTS AND METHODS: Thirty-four children and one adult with clinical signs and symptoms of tinea capitis and with positive culture and microscopy for dermatophytes have been included in a double-blind comparison between itraconazole, 100 mg daily, and ultramicronized griseofulvin, 500 mg daily. Both drugs were given for 6 consecutive weeks. The final evaluation was made 8 weeks after the end of treatment to allow the hairs to regrow. Seventeen itraconazole- and 15 griseofulvin-treated patients received the complete 6-week treatment course. Fifteen of these 17 itraconazole patients and 14 of the 15 griseofulvin patients had infections caused by Microsporum canis. Fifteen of 17 patients were cured by itraconazole (88%) and 15 of 17 patients by griseofulvin (88%). One of the patients who discontinued griseofulvin therapy after 4 weeks was clinically and mycologically cured. Two of the original 17 griseofulvin patients discontinued therapy because of vomiting. None of the itraconazole-treated children experienced side effects. CONCLUSIONS: Itraconazole is the first azole derivate that matches griseofulvin for the treatment of tinea capitis in children. The drug also appears to be better tolerated than griseofulvin.

Experience with amorolfine in the treatment of dermatomycoses.

In a double-blind randomized comparative study, 75 patients were treated with amorolfine cream 0.125, 0.25 or 0.5%. At the end of treatment clinical cure rates of 80, 76 and 84%, respectively, and mycological cures of 72, 64 and 76% were obtained. At 2 months posttherapy follow-up relapse rates were 0, 12 and 12%, respectively. There was no significant difference between the three groups in terms of clinical and mycological response, duration of treatment or tolerance. In a double-blind parallel study, 40 patients were treated topically with either 0.5% amorolfine cream or 1% bifonazole cream. The percentages of combined clinical and mycological cures were 83.3 and 78.95%, respectively. There was no significant difference in terms of tolerance and clinical and mycological cure rates. All treatments were applied once daily. Posttreatment MIC values did not indicate development of resistance to either amorolfine or bifonazole.

Nosocomial infection by Rhizomucor pusillus in a clinical haematology unit.

Three patients suffering from acute leukaemia were treated with cytotoxic agents and broad-spectrum antibiotics and received blood transfusion and nasal packing for severe epistaxis. All developed necrosis of nasal and facial tissues, with facial swelling an oedema; two biopsies showed typical phycomycete mycelium, and Rhizomucor pusillus was grown from one biopsy. Air and surfaces in the unit and the air intake and ducting were all heavily colonized by Rh. pusillus and other phycomycetes. It is suggested that Rh. pusillus spores from the air invaded the tissues in the conditions promoted by the nasal packing in these patients with impaired defences.

In vitro sensitivity of anaerobic bacteria to fosfomycin.

The minimum inhibitory concentrations were determined in 98 strains of anaerobic bacteria that came from clinical samples against fosfomycin, penicillin, cephalothin, tetracycline, chloramphenicol, clindamycin and lincomycin. The results obtained indicate that fosfomycin is usually inactive against Bacteroides sp. and is active up to 32 mug/ml or less against 85% of Peptococcus and 95% of Peptostreptococcus, being consequently comparatively less active than the rest of the antibiotics that were tested against Bacteroides, sp., while against Peptococcus and Peptostreptococcus it is less active than penicillin, cephalothin, clindamycin and lincomycin, and somewhat more active than chloramphenicol and tetracycline.