Evaluation of initial virological response to adefovir and development of adefovir-resistant mutations in patients with chronic hepatitis B.

The aims of the present study were to assess initial virological response (IVR) to adefovir (ADV) treatment for chronic hepatitis B, to identify patients with suboptimal response and to determine the incidence of ADV-resistant mutants. All patients treated with ADV for at least 12 months were evaluated for virological response and ADV resistance. IVR was defined as a reduction > or = 4 log10 IU/mL in hepatitis B virus (HBV)-DNA at month 6. Forty-two patients were analysed. Mean treatment duration was 23 +/- 7 months; 50% had prior lamivudine (LAM) therapy (LAM resistance 62%); 88% were hepatitis B e antigen (HBeAg)-negative; and 76% carried genotype D. IVR was seen in 40.5% of patients. Higher baseline ALT level was the only factor associated with IVR (P = 0.043). Patients with IVR achieved undetectable HBV-DNA at month 12 in 77% of cases compared with only 5% of those without IVR (P < 0.001). Five (12%) patients developed ADV-resistant mutations: rtN236T in four cases and one case with an rtV207L change, which has not been previously reported. This mutation was accompanied by viral rebound and alanine aminotransferase (ALT) flare. The cumulative probability of ADV-resistant mutations at 12 and 24 months was 5% and 17% respectively. IVR defined as a reduction > or = 4 log10 IU/mL in HBV-DNA at month 6 is a useful tool to predict virological response at month 12 and to identify patients with suboptimal response to ADV. Cumulative probability of ADV resistance is higher than previously reported for nucleos(t)ide-naïve patients.

[Prophylactic and pre-emptive therapy for cytomegalovirus infection in kidney transplant patients using oral valganciclovir]. / Tratamiento profiláctico y anticipado de la infección por citomegalovirus en pacientes trasplantados renales mediante valganciclovir oral.

UNLABELLED: Prophylactic and pre-emptive therapy with oral valganciclovir for cytomegalovirus infection in renal transplant recipients. BACKGROUND: Cytomegalovirus infection is a very important health problem in solid organ transplant recipients (SOT). Once-daily valganciclovir has been shown to be as clinically effective and well tolerated as oral ganciclovir tid in the prevention of CMV infection in high risk SOT recipients. METHODS: The aim of the present study was to evaluate the incidence and severity of CMV disease in 150 renal transplant recipients that received either prophylactic [high risk group (HR), N = 66] or pre-emptive [low risk group (LR), N = 84] therapy with oral valganciclovir (900 mg/day vo) for three months according to their basal risk. Patients were monitored for signs and symptoms of CMV disease and CMV plasma viral load was assessed weekly. RESULTS: A total of 31 patients (47%) of the HR and 26 patients (31%) of the LR presented a positive CMV PCR result. Twelve patients (14.3%) in the LR that had a high viral load (CMV PCR > 1,000 copies/mL) but remained asymptomatic received pre-emptive therapy. Four patients (4.7%) in the LR, after an average time of 35 days after transplant and two patients (4.5%) in the HR, after prophylactic treatment was completed, developed CMV disease. The disease was mild-moderate in most of the cases. Those patients that developed CMV disease responded to treatment with iv ganciclovir for 14 days followed by treatment with oral valganciclovir for up to three months. CONCLUSION: Prophylactic treatment with oral valganciclovir for CMV prevention is only required in high risk solid organ transplant recipients.

Two unusual hepatitis C virus subtypes, 2j and 2q, in Spain: Identification by nested-PCR and sequencing of a NS5B region.

Many studies have reported the use of the NS5B gene to subtype hepatitis C virus (HCV). Other HCV genes, such as HCV-5' UTR, Core (C) and E1, have also been used. In some studies, NS5B have been used together with 5'-UTR or C genes to improve genotyping results obtained using commercial procedures. Only two studies in Spain have compared molecular techniques versus commercial procedures regarding the efficacy of HCV subtyping. The aim of this study was to determine whether nested PCR and sequencing of a NS5B region was more reliable than commercial procedures to subtype HCV. We analyzed the results of HCV genotyping in [726] serum specimens collected from 2001 to 2013. From 2001 to 2011, we used PCR and INNO-LiPA hybridization or its new version Versant HCV Genotype 2.0 assay (471 samples). From 2012 to 2013, we used nested PCR and sequencing of a NS5B region (255 cases). This method used two pairs of primers to amplify the RNA of the sample converted to DNA by retrotranscription. The amplification product of 270 base pairs was further sequenced. To identify the subtype, the sequences obtained were compared to those in the international database: http://hcv.lanl.gov./content/sequence/, HCV/ToolsOutline.html and Geno2pheno[hcv] http://hcv.bioinf.mpi-inf.mpg.de/index.php. Nested PCR of a NS5B region and sequencing identified all but one subtype (0.4%, 1/255), differentiated all 1a subtypes from 1b subtypes, and characterized all HCV 2-4 subtypes. This approach also distinguished two subtypes, 2j and 2q, that had rarely been detected previously in Spain. However, commercial procedures failed to subtype 12.7% (60/471) of samples and to genotype 0.6% of specimens (3/471). Nested PCR and sequencing of a NS5B region improved the subtyping of HCV in comparison with classical procedures and identified two rare subtypes in Spain: 2j and 2q. However, full length genome sequencing is recommended to confirm HCV 2j and 2q subtypes.

Antiviral susceptibility of Herpes simplex viruses and its clinical correlates: a single center's experience.

The in vitro susceptibility to acyclovir of 204 herpes simplex virus isolates from 165 immunocompromised patients treated at our hospital was determined by the cytopathic effect reduction assay. Approximately 95% of herpes simplex virus 1 and 73% of herpes simplex virus 2 isolates were inhibited by acyclovir at concentrations of <2 microgram/mL. From 8 patients (5%), an isolate with low susceptibility to acyclovir (50% inhibitory dose, >3 microgram/mL) was recovered. Medical records of 83 patients were reviewed. Lesions resolved in most of the patients, independent of treatment. Treatment failures were not always associated with isolation of an in vitro-resistant virus. On the contrary, when a virus with low susceptibility to acyclovir was isolated, resolution of the lesion was the rule. In 9 of 10 patients with subsequent recurrent episodes of disease, the susceptibility of the viruses isolated was similar to that of the first episode. Routine susceptibility testing in our geographic area is not encouraged because of the low incidence of acyclovir-resistant herpes simplex viruses.

Detection of Mycobacterium tuberculosis DNA in lobular granulomatous panniculitis (erythema induratum-nodular vasculitis).

OBJECTIVE: To determine, using polymerase chain reaction (PCR) amplification, if Mycobacterium tuberculosis complex DNA is present in the skin biopsy specimens of lobular granulomatous panniculitis. DESIGN: A retrospective descriptive study. SETTING: A university-based hospital. PATIENTS: From the 65 patients included in the study, we examined 72 paraffin-embedded skin biopsy specimens with a histologic diagnosis of erythema induratum or nodular vasculitis. The biopsy specimens were from the histopathological archives of the Departments of Dermatology and Pathology of the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, from 1976 to 1994. Twenty-two biopsy specimens were excluded from the final analysis because we could not amplify the internal control. MAIN OUTCOME MEASURES: Detection of a 123-base pair fragment of the IS6110 insertion sequence specific for M tuberculosis complex. RESULTS: The results of PCR amplification were positive for M tuberculosis complex DNA in 77% of the skin biopsy specimens. No significant difference could be detected with respect to the age of the patients, ulceration of the nodules, reactivity to purified protein derivative, abnormal results of a chest x-ray examination, personal and family history of tuberculosis, and PCR results. The presence and degree of necrosis on histologic examination were significantly higher in the PCR-positive group (P = .04). None of the following variables were associated with PCR results: presence of vasculitis, degree of granulomatous infiltrates, number of giant cells, and presence of well-organized granulomas. CONCLUSIONS: The DNA of M tuberculosis can be detected in a considerable number of skin biopsy specimens of lobular granulomatous panniculitis. None of the clinical and histologic variables evaluated could accurately predict the results of PCR amplification.

[Clinical utility of susceptibility testing of herpes simplex virus to acyclovir]. / Utilidad clínica del estudio de la sensibilidad al aciclovir de los virus herpes simple.

In vitro susceptibility to acyclovir of 96 strains of herpes simplex virus isolated from 80 immunocompromised patients attended in our hospital was studied by the cytopathic effect reduction assay. Ninety-eight percent (61/62) of herpes simplex virus 1 strains and 91% (31/34) of herpes simplex virus 2 strains were inhibited by acyclovir concentrations lower than 3 mg/l. In 5% of the patients herpes simplex strains resistant to acyclovir (ID(50) >3 mg/l) were isolated. Ninety-eight percent of the lesions caused by herpes simplex viruses susceptible to acyclovir (ID(50) <3 mg/l) resolved independently of treatment. In two cases, the cytopathic effect reduction assay was not able to predict treatment failure and persistance of the lesions was not always associated with isolation of a resistant strain in vitro. In four cases, isolation of a strain resistant to acyclovir was not indicative of treatment failure. In conclusion, we believe there is no need to routinely test susceptibility of herpes simplex viruses to acyclovir and that susceptibility testing should be indicated only in patients in whom lesions persist and other causes have been ruled out.

[Detection of early cytomegalovirus antigen in cell culture]. / Detección de antígeno temprano de citomegalovirus en cultivo celular.

The reference technique for the diagnosis of active cytomegalovirus infection is the isolation in cellular culture. Its major drawback is the interval between the inoculation of the sample and the development of the characteristic cytopathic effect. Occasionally, this delay may be longer than four weeks. The centrifugation of the sample on the cell monolayer at the time of inoculation and the use of a fluorescein-labeled monoclonal antibody for the detection of the early antigen in cells may considerable reduce the time required for the diagnosis of cytomegalovirus infection. In the present study the technique of detection of the early antigen by immunofluorescence was compared with conventional cell culture in 258 clinical samples referred to the laboratory for study. Fifty-one of them were positive: 28 with both techniques, 12 only with cell culture and 11 only with immunofluorescence. The mean time to obtain positive results was 25 hours for immunofluorescence and 13 days for culture.

[Hemolytic-uremic syndrome caused by Escherichia coli O157:H7. Detection in direct sample of verotoxin-coding genes]. / Síndrome hemolítico-urémico causado por Escherichia coli O157:H7. Detección en muestra directa de genes codificadores de verotoxina.

Hemorrhagic colitis is an enteritis caused by verotoxigenic strains of Escherichia coli. Conventional diagnosis requires the identification of the microorganism and the demonstration of verotoxin production. The determination of toxigenicity in isolated strains and in direct stool samples by the polymerase chain reaction (PCR) technique may simplify the diagnosis. Conventional coprocultures were performed for the detection of verotoxigenic E. coli O157:H7 from three stool samples of a patient with hemorrhagic colitis and hemolytic-uremic syndrome. The production of verotoxin was determined by cell culture and the presence of VT1 and VT2 genomic sequences by PCR. Likewise, the latter technique was applied to a direct stool sample for detection of the verotoxin codiying genes. The specificity of the amplified sequences was confirmed by enzyme restriction digestion. Escherichia coli O157:H7 was isolated in two of the three samples studied. The strains were toxigenic in the cell culture test at titers higher than 1/500 and PCR showed an amplified band of 479 pb corresponding to the VT2 codifying gene. The digestion of amplified sequences with the EcoRV enzyme led to two bands of 390 and 89 pb confirming the specificity of the results. One of the two stool samples studied directly by PCR was positive for VT2 with the result being obtained 48 hours after arrival to the laboratory. The preliminary results of this study give support to the usefulness of the polymerase chain reaction technique in the detection of verotoxin from isolated strains of Escherichia coli and in direct stool samples.

[Usefulness of polymerase chain reaction for the diagnosis of Bazin erythema induratum]. / Utilidad de la reacción en cadena de la polimerasa para el diagnóstico del eritema indurado de Bazin.

BACKGROUND: Erythema induratum of Bazin (BEI), is included in the group of cutaneous granulomatous lobulillar panniculitis. The aethiopathogenic association between EI and tuberculosis can not rely on the clinicohistological features of these panniculitis and M. tuberculosis has never been isolated from BEI lesions. Detection of the mycobacterial DNA by PCR on cutaneous biopsy samples would allow to confirm this association. PATIENTS AND METHODS: Fourteen patients with clinical BEI were chosen retrospectively. Seventeen lesional biopsy samples were obtained, stained with the Kinyoun carbolfuchsin acid-fast technique and haematoxylin and eosin and tested by PCR. A fragment of the IS6110 insertion sequence specific of M. tuberculosis was amplified and confirmed by digestion with Sal I restriction endonuclease. The efficiency of the procedure, the presence of inhibitory substances and the preservation of DNA were checked by PCR of the beta-actin gene. RESULTS: M. tuberculosis DNA was detected in 12 of the 17 samples tested (70.5%) which corresponded to 10 of the 14 patients (71.4%). According to beta-actin PCR results, the rate of extracted DNA was inadequate on four of the five negative biopsies. CONCLUSIONS: The results of these series suggest the probable involvement of M. tuberculosis on the BEI pathogenesis and give support to the usefulness of the PCR in the diagnosis of this pathology concerning the need of specific treatment.

[Haemophilus influenzae type b invasive disease in Catalonia (1996)]. / Enfermedad invasiva por Haemophilus influenzae tipo b en Cataluña (1996).

BACKGROUND: The purpose of this study was to find out the incidence and characteristics of H. influenzae type b invasive disease (HibID) in Catalonia, Spain. MATERIAL AND METHODS: An active surveillance of H. influenzae isolated from normally sterile sites was carried out during 1996. Microbiology laboratories of hospitals of Catalonia were periodically contacted by telephone. The serotype of all the strains was studied. RESULTS: The incidence of H. influenzae invasive disease (HIID) was 7.1 per 100,000 in children under 5 years and 1.0 per 100,000 in those over 5 years. The incidence of serotype b was 6.4 per 100,000 children under 5 years and 0.2 above this age. Only three strains belonged to types other than b (d, e and f). CONCLUSIONS: The incidence of HIbID is uncommon in Catalonia, lower than that registered in the prevaccine era in other countries and regions of the same geographical area.

[Gastro-hemorrhagic Escherichia coli]. / Escherichia coli enterohemorrágica.

Groups of Escherichia coli enteropathogen are described, with special attention to Escherichia coli enterohaemorragic. Some serotypes of Escherichia coli verocitotoxin-producing are able to produce haemorrhagic enteritis, which can develop a complication with hemolityc uraemic syndrome. This complication is most frequent in children and has a high mortality rate. The transmission takes place via food and its capacity to cause epidemic outbreaks together with the seriousness of the complications caused by enteritys make this microorganism of great importance to Public Health. The epidemiology of this microorganism in Spain is reviewed.

[Mucocutaneous manifestations in acute HIV infection. 3 case reports]. / Manifestaciones mucocutáneas en la infección aguda por el VIH. Presentación de tres casos.

We report three patients who developed a generalized rash with oral, genital or perianal ulcerations as a result of acute infection due to HIV. The primary infection was diagnosed by seroconversion (by means of EIA and Western blot techniques). Definitive diagnosis was established on days 52, 85 and 97 after the appearance of the rash. The p24 protein of the HIV was only detected in the early phase of the disorder in the two cases in which this study was carried out.

Cytomegalovirus infection and disease after reduced intensity conditioning allogeneic stem cell transplantation: single-centre experience.

The aim of this study was to analyse the incidence and risk factors for cytomegalovirus infection (CMV-I) and disease (CMV-D) after a reduced intensity conditioning allogeneic hematopoietic stem cell transplantation (alloHSCT-RIC). We included 186 consecutive alloHSCT-RIC adult patients at risk for CMV reactivation (patient and/or donor CMV seropositivity). Conditioning regimen was based on fludarabine plus an alkylating agent. For guiding pre-emptive anti-CMV therapy, Pp65 Antigenemia (pp65Ag) (n=116) or quantitative polymerase chain reaction (quantPCR) (n=70) were used. The 2-year incidence of CMV-I and/or CMV-D was 36% (11% for CMV-D). Of note, 12/14 (86%) episodes of CMV-D in the pp65Ag group had lung involvement compared with only 3/15 (20%) in the quantPCR group (P=0.01). Importantly, the number of patients who developed CMV pneumonia with prior negative screening tests was unusually high (67% overall). Multivariate analysis of risk factors for CMV-D identified two risk factors: (i) steroid therapy for moderate-to-severe graft-vs-host disease (GVHD) (hazard ratio 4.7, P=0.02); and (ii) alternative donors (non-HLA-identical siblings) [hazard ratio 2.7, P=0.002]. Our findings suggest that CMV is still a major concern in alloHSCT-RIC. Variables associated with poor anti-CMV T-cell recovery (that is, GVHD and donor type) are helpful in identifying patients at higher risk for CMV-D in the alloHSCT-RIC setting.

[Detection of pathogenicity factors in strains of classical enteropathogenic Escherichia coli]. / Detección de factores de patogenicidad en cepas de Escherichia coli enteropatógena clásica.

OBJECTIVE: To determine the number of strains of classic enteropathogenic E. coli (EPEC) that have the eae gene, that is considered a pathogenicity factor. MATERIAL AND METHODS: The presence of the eae gene has been evaluated on 62 EPEC strains of ten different serogroups, isolated from children with gastroenteritis. RESULTS: Amplification of the eae gene was positive in 10 out of 62 EPEC strains analyzed (16%) corresponding to seven different serogroups. DISCUSSION: The low frequency of the detection of the eae gene on EPEC strains shows the limited correlation between the pathogenicity and the serogroup of the strains and would corroborate the need to reexamine this subject prospectively in our country.

Unmasking influenza virus infection in patients attended to in the emergency department.

BACKGROUND: Infection by the influenza virus may pass undetected in many adult patients attended to in the emergency department because its diagnosis usually relies on clinical manifestations, which can be distorted by symptoms of a preexisting disease, superposed complications or nontypical manifestations of influenza virus infection (confusing symptoms). PATIENTS AND METHODS: We performed this observational, prospective study with an antigen detection test by indirect immunofluorescence assay (IFA) to estimate the presence of influenza virus infection in such patients. No confirmatory test was performed to validate a positive or negative IFA result. Then we compared those who were antigen positive to those who were negative and also analyzed those who were positive classified by age, comorbidity and clinical presentation. We also evaluated the use of medical and hospital resources and vaccination status. Posterior pharynx swab specimens from 136 consecutive adult patients, 74 women and 62 men with a mean age of 68.7 +/- 17.9 (range: 18-97) years attended to in the emergency department of a university hospital in Barcelona during the 1999-2000 influenza epidemic were examined. Tested patients presented either a classical influenza syndrome, a deterioration of a previous condition or any abrupt onset of symptoms without an obvious cause. RESULTS: Influenza A virus antigen was detected in 99 (72.8%) of the 136 patients included in the study. Confusing symptoms were present in 86 patients with laboratory-confirmed influenza and 40 of them lacked influenza syndrome. Prostration, aching and fever out of proportion to catarrhal symptoms (disproportionate prostration) and cough were independent predictors for this diagnosis (OR = 5.14; 95% CI: 1.98-13.35, p = 0.001 and OR = 4.40, 95% CI, 1.65-11.75, p = 0.03, respectively). Among the 99 patients who tested positive, 72 were >or= 65 years of age. This older positive group compared to the 27 also positive < 65 (non-old) had a tendency to show symptoms mediated by cytokines less frequently: malaise was present in 76.4% of the older positive patients vs 92.6% in the non-old positive ones, p = 0.07. The equivalent percentages for muscle ache were: 56.9% vs 77.8%, p = 0.06; for dysthermia: 54.2% vs 70.4%, p = 0.08; for headache: 35.2% vs 66.7%, p = 0.005, and for disproportionate prostration: 47.2% vs 66.7%, p = 0.08. Cough was more frequent in the older positive group: 94.4% vs 77.8%, p = 0.02. Older positive patients were also hospitalized and received antibiotics more frequently than the non-old positive ones: 65.3% vs 40.7%, p = 0.03 and 81.9% vs 63.0%, p = 0.046, respectively. Hospitalization was independently correlated with the presence of complications (OR = 4.5, 95% IC 1.27-15.95, p = 0.02). Patients with the highest comorbidity, evaluated with the Charlson scale, were more inadequately vaccinated than those with moderate or low comorbidity. CONCLUSION: Influenza virus infection has a great and underestimated impact in the emergency department during influenza epidemics. High frequency of confusing symptoms, which overcome classical influenza syndrome in adult people with comorbidity, may explain this effect. Disproportionate prostration and cough are symptoms that independently predict its diagnosis in the global adult population, whereas in the elderly, fever and cough should arouse this suspicion whether or not they present classic symptoms. In our setting, individuals with high comorbidity are inadequately vaccinated.