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.

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.

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.

[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 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.

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 / Remission of severe disease induced by cmv and lymphoproliferative posttransplant disease after changing the immunosuppression regime

Se describe el caso de un receptor de trasplante renal con sobreinmunosupresión inducida por la interacción de tacrolimus y fluconazol que desarrolló dos enfermedades severas producidas por dos virus diferentes del grupo herpes: enfermedad por citomegalovirus (CMV) y enfermedad linfoproliferativa post-trasplante (ELPT). La detección del genoma del virus de Epstein-Barr (VEB) en sangre periférica precede al diagnóstico de ELPT. Ambas enfermedades remitieron con el cambio del régimen inmunosupresor y tratamiento con ganciclovir. Debido a que la infección por CMV es un factor de riesgo para desarrollar ELPT y a que las manifestaciones clínicas y endoscópicas de ambas enfermedades pueden confundirse, en los pacientes con enfermedad por CMV se debe descartar la presencia de una ELPT concomitante, sobre todo si estos pacientes son seronegativos para el virus de Epstein-Barr. La detección del genoma del VEB en sangre periférica puede ser de gran ayuda en el diagnóstico precoz de ELPT en estos pacientes. (AU)

[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.

[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.

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.

Control of cytomegalovirus disease in renal transplant patients treated with prednisone, azathioprine and cyclosporine using intensive monitoring and decreased immunosuppression.

BACKGROUND: The aim of this trial was to study the effectiveness of intensive monitoring, together with an early decrease in immunosuppression, in reducing the prevalence of CMV disease in renal transplant recipients treated with prednisone, azathioprine and cyclosporine. METHODS: From 1/95 to 11/97 a prospective, longitudinal study was conducted among 146 consecutive, unselected, renal transplant patients in our unit. Only 96 patients whose immunosuppressive regimens consisted of prednisone, azathioprine and cyclosporine and whose follow-up period was greater than 4 months were included in the study. Preemptive therapy was administered to 27 high-risk patients. CMV antigenemia (CMV-AG) and other virological tests were performed weekly for the first 4 posttransplant months. The immunosuppression was decreased when the first positive CMV-AG was detected. Azathioprine was completely withdrawn when the CMV-AG count was greater than 10 cells per 10(5) PBLs. The cyclosporine dose was gradually decreased in the next 4 weeks, but it was not withdrawn in any patient. The prednisone dose was modified according to the immunosuppressive protocol. RESULTS: 53% (51/96) of the patients had positive CMV-AG on at least one occasion. The dose of azathioprine was decreased after CMV-AG detection in 41/51 (80.4%) patients and it was completely withdrawn in 23 of these (45%). The mean decrease in the dose of azathioprine was 73 +/- 31 (25-175) mg, a mean percentage decrease of 76 +/- 27% (25-100%). The dose of cyclosporine was progressively decreased during the 4 weeks after detection of the first CMV-AG (mean cyclosporine levels: 210 +/- 66, 196 +/- 54 and 164 +/- 36 ng/ml at the time of first CMV-AG detection, 2 and 4 weeks respectively, p < 0.0001, repeated measures analysis of variance). None of the 45 patients without CMV-AG and only 2 of 51 (3.9%) patients with CMV-AG developed symptomatic CMV disease (2% of the total). CMV disease was of moderate intensity in both patients. Only 3/51 (5.8%) patients developed acute rejection after the first CMV-AG detection in the 4 posttransplant months. CONCLUSION: The results of this study suggest that intensive monitoring and an early reduction of immunosuppression, together with preemptive therapy in high-risk patients, is effective in diminishing the prevalence and severity of CMV disease.

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.

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.

Rapid screening tests for determining in vitro susceptibility of herpes simplex virus clinical isolates.

The susceptibility of human herpes simplex virus (HSV) to acyclovir (ACV) was determined with the use of a single dose of the drug (1 and 2 micrograms of ACV per ml for HSV-1 and HSV-2, respectively) in two rapid assays: a rapid cytopathic effect inhibitory assay (Rapid CIA) and a rapid dye uptake assay (Rapid DUA). These tests allow the simultaneous determination of virus titer and susceptibility to ACV at a determined viral concentration (100 50% tissue culture infective doses and 100 50% dye uptake units). These tests were compared with a conventional susceptibility assay (dye uptake assay) and showed similar results. Indeterminate results with the Rapid CIA appeared in 3 of 30 samples. With the use of both Rapid CIA and Rapid DUA, we were able to determine the susceptibility of 100% of the isolates. The rapid tests, unlike conventional assays, are able to provide susceptibility results within 3 days after the virus has been isolated from a clinical specimen and could thus play a direct role in therapeutic decisions.

Diagnosis of herpetic keratoconjunctivitis by nested polymerase chain reaction in human tear film.

A study was performed to evaluate nested PCR (nPCR) versus viral cultures as method and tear film versus corneal scrapings as specimen in the diagnosis of viral keratoconjunctivitis. Tear film specimens were taken from both eyes and corneal scrapings from the affected eye only in 17 patients with suspected viral keratoconjunctivitis. In 15 of the 17 patients the viral agent of the infection could be detected: 11 patients had herpes simplex virus type 1, two varicella-zoster virus, one both herpes simplex virus type 1 and varicella-zoster virus, and one adenovirus. Overall there was no significant difference between the detection rate for corneal scrapings (85%) and tear film (75%). In both types of specimens nPCR showed a higher detection rate than viral cultures (corneal scrapings: 87.5% vs 31.25%; tear film: 75% vs 12.5%; P 0.05). For the diagnosis of keratoconjunctivitis nPCR is superior to viral culture and tear film is an adequate sample that is easier to collect, causing the patient less discomfort.

[Diagnosis of meningoencephalitis caused by herpes simplex virus type 1 using nested PCR in CSF samples]. / Diagnóstico de meningoencefalitis por virus herpes simple tipo 1 mediante PCR anidada en muestras de LCR.

BACKGROUND: The aim of this study was to achieve the early diagnosis of the neurologic alteration caused by the Herpes Simplex virus type 1 (HSV-1) with the nested PCR technique in CSF. PATIENTS AND METHODS: From January, 1994 to October, 1995, 140 CSF from 140 patients were studied in our laboratory. Ninety-five were diagnosed with viral meningoencephalitis (Group A) and 45 with other neurologic diseases (Group B). Nested PCR of HSV-1 and conventional viral cultures were carried out in all the cases. RESULTS: Laboratory diagnosis was achieved in 13 (13.68%) of Group A patients: in 12 (12.63%) HSV-1 genome was detected by nested PCR and in one patient adenovirus was isolated. In Group B, the HSV-1 was detected by nested PCR in 2 patients (4.44%). CONCLUSIONS: The results obtained demonstrate the usefulness of nested PCR in HSV-1 infection for the diagnosis of herpetic meningoencephalitis in initial stages of the disease, from a single CSF sample.

Cytomegalovirus preemptive therapy with ganciclovir in renal transplant patients treated with OKT3.

In an attempt to decrease the prevalence and severity of cytomegalovirus (CMV) disease, preemptive therapy with ganciclovir was administered to all renal transplant patients treated with OKT3 between February 1993 and December 1994 (26 patients). The results were compared with those of a historical group treated with OKT3 but not with ganciclovir (29 patients). Both groups were similar in age, sex, number of previous transplants, number of rejections, serological status of donor and recipient and OKT3 dose. Ganciclovir was administered during the period of treatment with OKT3. Only 2 (7.7%) treated patients developed CMV disease versus 11 (37.9%) of the control group (p = 0.01). In the control group the intensity of the disease was severe in 7 (63.6%) cases, whereas in the treated patients it was always of slight intensity (p = 0.01). In conclusion, preemptive therapy with ganciclovir during treatment with OKT3 decreases the prevalence and severity of CMV disease.