Molecular identification of two genotypes of mumps virus causing two regional outbreaks in Asturias, Spain.

BACKGROUND: In spite of universal vaccination, several sporadic cases of mumps infection, which could produce outbreaks, are detected every year in different countries. OBJECTIVE: Mumps virus strains causing two regional outbreaks in Asturias (Spain) were phylogenetically characterized. STUDY DESIGN: Mumps virus strains, which were detected in samples from patients belonging to two regional outbreaks in Asturias, were characterized by sequencing of the SH gene and further alignment to homologous sequences of representative strains of the different mumps genotypes. RESULTS: Two different strains (Ast/SP02 and Ast/SP07) were isolated. Sequence analysis revealed that while Ast/SP02 belonged to genotype H, Ast/SP07 was phylogenetically close to UK02-19, a reference strain for a new genotype. Both strains belonged to different genotypes from those used in the vaccination (Jeryl-Lynn strain is genotype A). CONCLUSION: Mumps virus strains different from those used in vaccination program can cause mumps outbreaks even in vaccinated patients.

Cytomegalovirus replication and "herpesvirus burden" as risk factor of cardiovascular events in the first year after renal transplantation.

Cytomegalovirus (CMV) infection alone or in combination with other pathogens ("pathogen burden") has been postulated as a factor producing arteriosclerosis in some solid organ transplant recipients. The aim of this study was to assess whether the patients with CMV replication and/or "herpesvirus burden" experienced a greater incidence of cardiovascular events during the first year after kidney transplantation. One hundred twenty-one consecutive transplant recipients were prospectively studied for CMV replication using antigenemia and polymerase chain reaction (PCR) weekly during the 4 first months, and monthly thereafter for 1 year. Simultaneously, nested-PCR for human herpes virus (HHV)-6 and HHV-7 were performed to yield a herpesvirus burden (as determined by seropositivity), including CMV, herpes simplex virus (HSV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). The following additional parameters were analyzed: gender, age, smoking, duration of dialysis, preexistent diabetes, and preexistent cardiovascular events. After 1 year posttransplantation cardiovascular events, body mass index, arterial hypertension, number of antihypertensive drugs, use of ACE and/or ARBs inhibitors, diabetes, anemia, homocysteine, creatinine, cholesterol, HDLc, LDLc, PTH-i, proteinuria, and immunosuppression with cyclosporine or tacrolimus. CMV replication was present in 79 (65.3%) patients. Among 121 renal transplant recipients, 13 presented cardiovascular events, all associated with CMV replication (P = .004). Neither HHV-6 or HHV-7 replication influenced this complication. All patients with these events were seropositive for CMV, HSV, VZV, and EBV, as opposed to 64.8% without them (P = .009). Other factors that showed differences between patients with versus without events were as follows: preexistent events (76.9% vs 14.8%; P = .000), age (60 +/- 10 vs 49 +/- 14; P = .002), serum triglyceride value (191 +/- 82 vs 135 +/- 72; P = .02), and anemia (23.1% vs 5.6%; P = .05). Multiple logistic regression analysis for statistically significant variables only showed that preexistent events influenced the development of posttransplantation events (odds ratio, 27; 95% confidence interval, 4.7-154; P = .0005). In conclusion, cardiovascular events within 1 year after transplantation were more frequent among patients with CMV replication and seropositivity for other herpesviruses. An important risk factor was the presence of preexistent events.

Hepatitis C virus reactivation in anti-hepatitic C virus-positive renal transplant recipients.

From 1992 to 2001 hepatitis C virus (HCV) viremia was studied in 53 renal transplant recipients anti-HCV+ with at least 3 months follow-up posttransplant using a quantitative retrotranscriptase-PCR method. HCV-RNA was detected in 45 (85%): 29 of the 34 recipients treated with azathioprine-based therapy and 15 of 18 treated with mycophenolate mofetil. Immunosuppressive therapy type did not affect HCV replication. Three different patterns of HCV-RNA evolution were detected: 13 (28.8%) patients with high RNA-HCV levels; 21 (46.7%) patients with low levels; and 11 (24.4%) patients with viremia elevation. In 10 (90%) of 11 of the last group, HCV viremia was detected before 15 days posttransplantation, significantly earlier than in the other two groups. Thus, replication during the first 15 days after transplantation leads to a high RNA-HCV viral load. No clinical symptoms were related to HCV.

Influence of ganciclovir prophylaxis on citomegalovirus, human herpesvirus 6, and human herpesvirus 7 viremia in renal transplant recipients.

In order to know the influence of ganciclovir (GCV) prophylaxis on cytomegalovirus (CMV) human herpesvirus (HHV)-6 and HHV-7 replication in renal transplant recipients, three groups were studies: 54 patients without GCV; 29, with short-term GCV prophylaxis (less than 30 days); and 51, with long-term GCV prophylaxis (more than 60 days). CMV viremia was more prevalent in the first group (74%, 55%, and 29%, respectively), but CMV replication was also found in 14 patients during therapy, in the other two groups. The antiviral did not affect the prevalence of HHV-6 (67.2%) or HHV-7 (76%), but HHV-6 viremia appeared later (42 +/- 31 vs 21 +/- 25/38 +/- 29 days posttransplant) and was shorter (29 +/- 30 vs 62 +/- 34/41 +/- 33 days) among patients with long-term GCV prophylaxis. On the other hand, CMV viremia was longer when HHV-6 replication was present (40 +/- 25 days vs 18 +/- 16 days). In addition, HHV-7 DNA was detected in all patients with CMV disease.

[Parvovirus B9 infection in a renal transplant recipient. Diagnosis by detection of viral genome in peripheral blood]. / Infección por parvovirus B19 en trasplante renal. Diagnóstico por detección del genoma viral en sangre periférica.

Parvovirus B19 can produce a picture known as pure red blood aplasia in recipients of solid organ. Occasionally the viruses cause decrease of the other blood cells, and various extra-hematologic manifestations. Common diagnosis is realised by bone marrow examination. The diagnostic value of the viral genome in the blood stream is not well defined. We reported the case of a male of 17 years of age, whose diagnosis was done by repeated determinations of the viral parvovirus B19 genome in peripheral blood. It was confirmed by a biopsy of the iliac crest. The patient was treated with unspecific IgG immunoglobulins, with complete recovery from the symptoms and signs. It did not have any recurrence of the disease. This case suggests that the realisation of PCR of Parvovirus B19 in renal transplant patients with pure red cell aplasia could be of greater interest in the diagnosis and monitoring of the disease. The detection of the viral genome could avoid the administration of unnecessary blood transfusions, and possibly the realization of bone marrow biopsy.

Cytomegalovirus antigenemia surveillance in the treatment of cytomegalovirus disease in AIDS patients.

OBJECTIVE: Surveillance of quantitative cytomegalovirus (CMV) antigenemia among AIDS patients with CMV treated complications in order to determine its value in assessing the response to treatment and survival. METHODS: A longitudinal follow-up of antigenemia measurement at diagnosis, after induction therapy with ganciclovir or foscarnet, and every 3 months during maintenance therapy was carried out in 25 patients with CMV retinitis and in 8 with extraocular CMV disease. Positive antigenemia was defined as the presence of any amount of immunofluorescent pp65-positive leukocytes/10(5) cells. RESULTS: Mean antigenemia values were: 77+/-148/10(5) leukocytes at retinitis diagnosis; 45+/-114 after induction therapy; and 7+/-18 and 1.5+/-4 after 6 months and one year of therapy, respectively. Patients achieving undetectable antigenemia increased from 44% at baseline to 68% at postinduction and 80% during follow-up. Seven patients (28%) who remained free of relapses presented significant minor baseline antigenemias and became negative after induction therapy. Patients with extraocular disease showed erratic antigenemia values and absent therapeutic response. CMV blood cultures before and after induction therapy were positive in 39% and 21% of patients, respectively. Kaplan-Meier analysis revealed a significantly longer survival for patients with retinitis when compared to those with extraocular complications, and for patients with negative antigenemia after induction in comparison with those who failed to achieve it. CONCLUSIONS: Low basal antigenemia and antigenemia clearance after induction therapy are variables directly related to good response to treatment and survival. Continuous surveillance of antigenemia during treatment could permit designing of individual strategies to obtain a better response.

Detection of herpes simplex virus-1 by nested PCR. An experimental model.

Nested polymerase chain reaction (nested PCR) was performed using a reaction mix batch-prepared and kept frozen in single reaction tubes at -20 degrees C until use. Twenty-one New Zealand white rabbits were infected with herpes simplex virus type 1 (HSV-1). Eleven animals were killed on day seven and the other ten were sacrificed on day 21. Viral culture and nested PCR was used to determine the presence of HSV-1 in samples from the tongue, HSV-1 was detected in 90.47% of the animals; in 84.21% by nested PCR and in 52.63% by culture. Nested PCR assay had greater sensitivity than culture in animals sacrificed on day seven with significative difference (p < 0.05). Higher sensitivity and faster results were obtained with this method, so we found it reliable and useful in the setting of a clinical laboratory dealing with diagnosis of herpes virus infections.

[Remission of severe disease induced by CMV and lymphoproliferative post-transplant disease after changing the immunosuppressive regime]. / Remisión de enfermedad severa por citomegalovirus y enfermedad linfoproliferativa post-trasplante en un receptor de trasplante renal tras terapia con ganciclovir y cambio en la medicación inmunosupresora.

We describe a renal transplant recipient, with overimmunosuppression induced by the interaction of tacrolimus and fluconazole, who developed two severe diseases produced by two different viruses of the herpes group (cytomegalovirus [CMV] disease and posttransplant lymphoproliferative [PTLD] disease EBV-related). Detection of Epstein-Barr virus (EBV) DNA in the blood preceded the histological diagnosis of PTLD. Both diseases improved after changes in the immunosuppressive regime and treatment with ganciclovir. Because CMV infection is a risk factor in developing PTLD, and the clinical and endoscopic manifestations of both diseases could be become confused, PTLD should be excluded in EBV seronegative patients that develop CMV disease. The detection of the EBV genome in blood could help in the early diagnosis of PTLD in these patients.

[Replication indexes of the human immunodeficiency virus: predictive value of viral culture and blood antigens]. / Indices de replicación del virus de la inmunodeficiencia humana: valor predictivo de los cultivos virales y de la antigenemia.

BACKGROUND: To investigate the relation between markers of load and replication of the HIV [viral culture in plasma and in mononuclear cells of peripheral blood (MCPB) and antigen p24 (p24Ag) with the number of CD4+ cells and the prognosis of the patients. METHODS: A retrospective study was performed in 188 patients who were analyzed and followed over a mean period of 431 days. The criteria of clinical progression (AIDS related complex, and new opportunistic infections), immunologic progression (CD4+ < 0.1 and < 0.05 + 10(9)/l) and death. Cocultures of HIV in free plasma and in MCPB were performed with the detection of complete AgHIV in the supernatant of the culture being used for analysis. Circulating p24Ag was determined by an ELISA technique without previous dissociation of the immunocomplexes. RESULTS: HIV cultures in plasma, in MCPB and p24Ag were positive in 27, 48 and 33% of the patients, respectively. The sensitivity of the indexes increased in agreement with the clinical progression of the patients and was inversely proportional to the depletion of the CD4+ lymphocytes (79% of the patients with CD4+ lymphocytes < 0.05 x 10(9)/l presented positive HIV culture in plasma). Viremia in plasma and to a lesser measure p24Ag correlated with variables recognized as bad prognosis and were found to be predictive of unfavorable evolution. Multivariate analysis demonstrated that pertenence to a symptomatic group and the presentation of a number of CD4+ lymphocytes of less than 0.2 x 10(9)/l were independent factors associated to the positivity of the viral culture in plasma and p24Ag. The culture positive in MCPB was principally related with the volume of blood analyzed. The risk of death was 6.38 fold greater in the presence of a positive plasma culture and 2.02 fold greater in the presence of positive p24Ag. In contrast, the unquantified positive HIV culture in MCPB showed no statistical significance in relation with patient survival. CONCLUSIONS: Positive HIV culture in plasma was the greatest prognostic index in patients with a number of CD4+ lymphocytes less than 0.2 x 10(9)/l. Unquantified cell culture had no predictive significance. To establish the prognosis of patients, the indexes of viral replication should not be used in isolation.

[The clinical significance of culturing Toxoplasma gondii on blood and other organic media]. / Significado clínico del cultivo de Toxoplasma gondii en sangre y en otros medios orgánicos.

BACKGROUND: The aim of this study was to determine the value of the Toxoplasma gondii culture in blood and in other organic fluids in HIV positive and negative patients. METHODS: Retrospective analysis (October 1990-May 1992) was carried out including all patients with positive cultures for T. gondii admitted to the Hospital Central of Asturias. The parasite was identified by monoclonal antibodies against the tachyzoite membrane. All patients with positive cultures were treated with pyrimethamine and sulphadiazine. RESULTS: Three hundred two samples from 256 patients, seropositive and seronegative for HIV, were analyzed. Of the seropositive group 8/45 (18%) had positive cultures for T. gondii versus 9/211 (4.3%) of the seronegative group (p = 0.002). Of the 19 positive samples, 15 were from blood, 3 from bronchoalveolar lavage and one from the vitreous fluid. Four out of 9 patients (44%) with AIDS and encephalic toxoplasmosis (ET) had blood cultures positive for T. gondii. Another 4 patients with AIDS presented toxoplasmenia without visceral involvement. Of the 9 HIV seronegative patients (3 immunodepressed patients), 4 had pulmonary toxoplasmosis, one ocular toxoplasmosis, and other clinical forms of toxoplasmosis were seen in the remaining 4. All the patients evolved to cure except 2 cases coinfected by cytomegalovirus who died. CONCLUSIONS: The identification of Toxoplasma gondii may be performed by blood cultures in half of the patients with AIDS and encephalic toxoplasmosis and in an undetermined percentage of the other clinical forms both in immunocompetent and immunodepressed subjects. In addition, toxoplasmemia has been registered in AIDS patients preceding any other organic seating of the parasite. Early antitoxoplasma therapy may, therefore, be effective.

[The prognostic value of cytomegalovirus antigenemia and viremia for the development of cytomegalovirus disease and the survival of AIDS patients]. / Valor predictivo de la antigenemia y la viremia por citomegalovirus para el desarrollo de enfermedad por citomegalovirus y la supervivencia en pacientes con SIDA.

OBJECTIVES: To analyse risk factors for morbidity and survival associated with blood cytomegalovirus (CMV) detection with the antigenemia method among AIDS patients. PATIENTS AND METHODS: CMV antigenemia and CMV blood cultures in 277 AIDS patients IgG-CMV sero-positive with a CD4 level lower than 200 x 10(6)/l under antiretroviral monotherapy were analysed. We consider cases the 116 patients with one or more positive blood samples tested for pp65 antigenemia or CMV culture. They were matched with 161 control patients with negative antigenemia or viremia. RESULTS: Multivariate analysis pointed out a significant positive association for blood CMV reactivation with the following variables: CMV disease development and CMV urine detection, sex-acquired HIV infection, CD4+ < 50 x 10(6)/l and matched time from AIDS diagnosis to CMV blood culture correlated with positive antigenemias. Quantitative antigenemia title showed predictive value for risk of CMV disease although 23% of retinitis patients had persistent undetectable antigenemia. CMV invasive disease developed in 48% of cases and 11% of controls (relative risk [RR]: 7.9; 95% confidence interval [CI]: 4.2-14.7). Mortality after 12 months of follow-up was 73% vs 52% respectively (p < 0.001). Time survival curves after CD4+ count adjusting remained significantly lower for case patients (median, 127 days vs 355 days; p < 0.01 by log-rank test). Increased death rate was found in patients with CMV disease (74%), followed by patients with CMV antigenemia but no disease (70%) and patients without antigenemia or CMV disease (mortality 49%). CONCLUSIONS: CMV blood detection in AIDS patients may be considered as a bad prognosis marker for CMV morbidity and survival. This risk increases with higher CMV antigenemias. Therefore, pre-emptive anti-CMV therapy should be considered in this restricted population.

Involvement of adenovirus in clinical mononucleosis-like syndromes in young children.

Although Epstein-Barr virus (EBV) commonly causes infectious mononucleosis (IM) or IM-like syndromes, other agents can be implicated. In this study, viral and parasitic screening was performed to determine the etiological agent of pediatric IM-like syndromes in 38 children. Adenovirus was the agent most frequently detected (47.3%), followed by EBV (31.5%) and cytomegalovirus (2.6%). Although the statistically significant difference between viral detection rates observed in patients who fulfilled clinical and hematological criteria and detection rates in those who presented clinical symptoms only (91.6% vs. 64.3%) indicates that hematological abnormalities are common in viral IM-like syndromes, the existence of syndromes of viral etiology without hematological criteria cannot be discarded. A further analysis showed an absence of lymphocytosis in adenovirus infections as well as a low number (14.3%) of EBV infections associated with increased neutrophils. These data suggest the usefulness of appropriate virological techniques for the detection of adenovirus in pediatric IM-like syndromes.

Isolation of influenza virus in human lung embryonated fibroblast cells (MRC-5) from clinical samples.

Ninety-four pharyngeal swab samples corresponding to 94 patients with suspected influenza virus infection were inoculated in Madin-Darby canine kidney (MDCK) cells, the conventional cell system for the isolation of influenza virus, and in fibroblastic human embryo lung (MRC-5) cells, a cell system less commonly used for this purpose but one frequently used in clinical virology laboratories. Both cell preparations were treated with trypsin. Influenza virus was recovered from 15% of the samples inoculated in MDCK cells and from 18% of those inoculated in MRC-5 cells. The use of MRC-5 cells can simplify the search for respiratory viruses and would assist in the rapid detection of influenza virus during new epidemics.

Disseminated herpes simplex virus infection in a renal transplant patient as possible cause of repeated urinary extravasations.

Disseminated herpes simplex virus type 2 (HSV-2) infections are infrequent in patients receiving organ transplants, but usually have a poor outcome. We describe the case of a renal transplant patient who developed a disseminated HSV-2 infection with repeated urinary extravasations. The diagnosis was carried out using a multiplex polymerase chain reaction nested assay and it suggested HSV-2 as a possible cause of repeated urinary fistulas.

Cytomegaloviraemia and T cell subpopulations in renal transplant patients.

We studied 54 consecutive recipients of renal transplants to evaluate their immunological responses to cytomegalovirus (CMV) infection. Forty-three (79.6%) patients developed CMV infection, and all of them subsequently recovered. Fourteen of these infected patients (32.6%) developed viraemia during the infectious process, four of whom then manifested the disease. The number of lymphocytes and their main subpopulations was normal before the appearance of CMV infection. During the infection there was a significant growth (P < 0.001) in the CD8+DR+ subset, corresponding to activated T suppressor/cytotoxic lymphocytes, whereas the natural killer measured subsets remained within normal limits during the whole infectious process. As all viraemic patients recovering from the infection developed CD8+DR+ activation, we conclude that this recovery is associated with the immunological activation.