Detection of cytomegalovirus in bronchoalveolar lavage fluid from immunocompromised patients with pneumonitis by viral culture and DNA quantification.

PURPOSE: To compare the detection of human cytomegalovirus (HCMV) in bronchoalveolar lavage (BAL) fluid by viral culture and quantitative polymerase chain reaction (qPCR), and to establish a viral load threshold that can identify cases of HCMV replication indicative of pneumonitis. There is currently no universal viral load cut-off to differentiate between patients with and without pneumonitis, and the interpretation of qPCR results is challenging. METHODS: 176 consecutive BAL samples from immunosuppressed hosts with signs and/or symptoms of respiratory infection were prospectively studied by viral culture and qPCR. RESULTS: Concordant results were obtained in 81.25% of the BAL samples. The rest were discordant, as only 34% of the qPCR-positive BAL samples were positive by culture. The median HCMV load was significantly higher in culture-positive than in culture-negative BAL samples (5038 vs 178 IU/mL). Using a cut-off value of 1258 IU/mL of HCMV in BAL, pneumonia was diagnosed with a sensitivity of 76%, a specificity of 100%, a VPP of 100% and VPN of 98%, and HCMV was isolated in 100% of the BAL cultures. CONCLUSION: We found that a qPCR-negative was a quick and reliable way of ruling out HCMV pneumonitis, but a positive result did not always indicate clinically significant replication in the lung. However, an HCMV load in BAL fluid of ≥ 1258 IU/mL was always associated with disease, whereas < 200 IU/mL rarely so.

A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Impact of Epstein Barr virus-related complications after high-risk allo-SCT in the era of pre-emptive rituximab.

We monitored 133 high-risk allo-SCT recipients for 6 months after transplant for EBV reactivation by quantitative real-time PCR. Rituximab was given as pre-emptive therapy for viremia >1000 copies/mL. The 1-year cumulative incidence of EBV reactivation was 29.4% (95% confidence interval (CI): 18-40) in patients monitored due to initial high-risk characteristics (n=93) and 31.8% (95% CI: 19.7-44) in those followed because of the development of refractory GVHD (n=40). Overall response rate to Rituximab was 83%. Nine patients (9.6%) developed post-transplant lymphoproliferative disorder (PTLD) at a median of +62 days after SCT. Eight of them showed a concomitant CMV reactivation. Second SCT was the only risk factor associated with EBV infection and PTLD in multivariate analysis (hazard ratio (HR) 2.6 (95% CI: 1.1-6.4; P=0.04) and HR 6.4 (95%CI: 1.3-32; P=0.02)). The development of EBV reactivation was not associated with non-relapse mortality or OS (P=0.97 and P=0.84, respectively).

Viral infections of the central nervous system in Spain: a prospective study.

The aim of the study was to determine the incidence of viruses causing aseptic meningitis, meningoencephalitis, and encephalitis in Spain. This was a prospective study, in collaboration with 17 Spanish hospitals, including 581 cases (CSF from all and sera from 280): meningitis (340), meningoencephalitis (91), encephalitis (76), febrile syndrome (7), other neurological disorders (32), and 35 cases without clinical information. CSF were assayed by PCR for enterovirus (EV), herpesvirus (herpes simplex [HSV], varicella-zoster [VZV], cytomegalovirus [CMV], Epstein-Barr [EBV], and human herpes virus-6 [HHV-6]), mumps (MV), Toscana virus (TOSV), adenovirus (HAdV), lymphocytic choriomeningitis virus (LCMV), West Nile virus (WNV), and rabies. Serology was undertaken when methodology was available. Amongst meningitis cases, 57.1% were characterized; EV was the most frequent (76.8%), followed by VZV (10.3%) and HSV (3.1%; HSV-1: 1.6%; HSV-2: 1.0%, HSV non-typed: 0.5%). Cases due to CMV, EBV, HHV-6, MV, TOSV, HAdV, and LCMV were also detected. For meningoencephalitis, 40.7% of cases were diagnosed, HSV-1 (43.2%) and VZV (27.0%) being the most frequent agents, while cases associated with HSV-2, EV, CMV, MV, and LCMV were also detected. For encephalitis, 27.6% of cases were caused by HSV-1 (71.4%), VZV (19.1%), or EV (9.5%). Other positive neurological syndromes included cerebellitis (EV and HAdV), seizures (HSV), demyelinating disease (HSV-1 and HHV-6), myelopathy (VZV), and polyradiculoneuritis (HSV). No rabies or WNV cases were identified. EVs are the most frequent cause of meningitis, as is HSV for meningoencephalitis and encephalitis. A significant number of cases (42.9% meningitis, 59.3% meningoencephalitis, 72.4% encephalitis) still have no etiological diagnosis.

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.

Enteroviruses in Spain over the decade 1998-2007: virological and epidemiological studies.

BACKGROUND: Human enteroviruses (HEV) are the commonest cause of viral meningitis as well as other pathologies, therefore HEV characterization is important both in patient management and epidemiological investigation. OBJECTIVES: A 10-year study of patients with enteroviral infection was carried out in Spain to determine the underlying etiology. STUDY DESIGN: HEV were fully typed by microneutralisation tests and/or molecular methods. RESULTS: A collection of 86404 clinical samples were studied in several Spanish laboratories. These were collected from patients with different syndromes, mainly aseptic meningitis (AM), fever, respiratory diseases and acute flaccid paralysis. Of these, 6867 HEV were obtained. At the National Poliovirus Laboratory 2814 were serotypically characterised. Among non-polio enteroviruses, the eight main serotypes were Echovirus 30 (25%), Echovirus 6 (12.4%), Echovirus 13 (8.3%), Echovirus 11 (7.4%) and Echovirus 9 (4.7%), followed by Coxsackievirus B5 (4.2%) and Echovirus 7 and Coxsackievirus A9 (3.7%) each. In AM cases, Echovirus 30 was identified in 39% of them, followed by Echovirus 6 (14%). However, Echovirus 6 was mainly associated with respiratory disease (17%), followed by Echovirus 11 (10%). On the other hand, Echovirus 30, Echovirus 11 and Echovirus 6 contributed equally with 12% of each serotype in the cases of fever. CONCLUSIONS: The present report complements previous data (Trallero et al.(13)), with the results of HEV incidence in Spain from 1998 to 2007. The surveillance described in this study provided valuable information as to which serotypes are in circulation, the emergence of new HEV and association with clinical manifestations.

Lower respiratory tract respiratory virus infections increase the risk of invasive aspergillosis after a reduced-intensity allogeneic hematopoietic SCT.

We have analyzed the incidence and risk factors for the occurrence of invasive aspergillosis (IA) among 219 consecutive recipients of an allogeneic hematopoietic SCT after a reduced-intensity conditioning regimen (Allo-RIC). Twenty-seven patients developed an IA at a median of 218 days (range 24-2051) post-Allo-RIC, for a 4-year incidence of 13% (95% confidence interval 4-24%). In multivariate analysis, risk factors for developing IA were steroid therapy for moderate-to-severe graft vs host disease (GVHD) (Hazard Ratio (HR) 2.9, P=0.03), occurrence of a lower respiratory tract infection (LRTI) by a respiratory virus (RV) (HR 4.3, P<0.01) and CMV disease (HR 2.8, P=0.03). Variables that decreased survival after Allo-RIC were advanced disease phase (HR 1.9, P=0.02), steroid therapy for moderate-to-severe GVHD (HR 2.2, P<0.01), not developing chronic GVHD (HR 4.3, P<0.01), occurrence of LRTI by an RV (HR 3.4, P<0.01) and CMV disease (HR 2, P=0.01), whereas occurrence of IA had no effect on survival (P=0.5). Our results show that IA is a common infectious complication after an Allo-RIC, which occurs late post-transplant and may not have a strong effect on survival. An important observation is the possible role of LRTI by conventional RVs as risk factors for IA.

[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.

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

Antecedentes: La enfermedad por citomegalovirus (CMV) es un problema sanitario muy importante en receptores de trasplante de órgano sólido (TOS). Una dosis diaria de valganciclovirha demostrado ser tan clínicamente efectiva y bien tolerada como ganciclovir oral dos veces al día en la prevención de la infección por CMV en los receptores de TOS de alto riesgo. Métodos: El objetivo del presente estudio fue evaluar la incidencia y severidad de la enfermedad por CMV en 150 receptores de trasplante renal que recibieron tratamiento profiláctico(grupo de alto riesgo, N = 66) o anticipado (grupo de bajo riesgo, N = 84) con valganciclovir oral (900 mg/día)durante tres meses según el riesgo basal de sufrir la misma. Se hizo un seguimiento de los síntomas clínicos de la enfermedad por CMV en los pacientes y la carga viral de CMV en plasma fue monitorizada semanalmente. Resultados: Un total de 31 pacientes (47%) del grupo de alto riesgo y 26 pacientes (31%) del grupo de riesgo estándar presentaron un resultado de PCR-CMV positivo. Doce pacientes (14,3%) del grupo de riesgo estándard que presentaron una elevada carga viral (PCR-CMV > 1.000 copias/mL) pero que permanecieron asintomáticos recibieron tratamiento anticipado. Cuatro pacientes (4,7%) del grupo de alto riesgo, en un tiempo medio de 35 días después del trasplante y dos pacientes (4,5%) del grupo de alto riesgo, tras completar el tratamiento profiláctico, desarrollaron la enfermedad por CMV, que fue de intensidad media a moderada en la mayoría de los casos. Aquellos pacientes que desarrollaron la enfermedad respondieron al tratamiento con ganciclovir ev durante 14 días seguido de valganciclovir oral hasta tres meses. Conclusión: El tratamiento profiláctico con valgancicloviroral para la prevención de CMV sólo es requerida en receptores de TOS de alto riesgo (AU)

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 (AU)

Upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies.

The impact of human enterovirus (HEV) and human rhinovirus (HRV) respiratory tract infections in adult patients with hematological malignancies has been infrequently reported. We retrospectively studied 31 patients with an upper or lower respiratory tract infection (URTI/LRTI) by HEV (n = 18) or HRV (n = 15). At onset, a LRTI was present in 6 (33%) and 2 (13%) episodes of HEV and HRV infections, respectively, with or without an URTI. Progression to LRTI (pneumonia) from prior URTI was seen in 1 (6%) and 2 (13%) HEV and HRV infections, respectively. The presence of lymphocytopenia (<0.5 x 10(9)/l) was higher in LRTI by HEV: 4/5 (80%) versus 2/10 (20%) by HRV. Eight of 18 (44%) patients with immunosuppression versus 3/14 (21%) patients with no immunosuppression at the onset of respiratory infection developed a LRTI. Thirteen per cent of patients had associated respiratory infections from bacteria, aspergillus, or CMV. Pulmonary aspergillosis was diagnosed in 20% of HRV infections. Three of 11 patients (27%) with a LRTI died, but pulmonary copathogens were also involved in all cases. In conclusion, HEV and HRV can be associated with LRTI in immunocompromised patients, although their direct impact on mortality is uncertain.

Relation between pp65 antigenemia, RT-PCR and viruria for cytomegalovirus detection in kidney transplant recipients.

INTRODUCTION: We compared the efficiency of detection using pp65 antigenemia, reverse transcription-polymerase chain reaction (RT-PCR), and viruria for the diagnosis of cytomegalovirus (CMV) infections after kidney transplantation. PATIENTS AND METHODS: We evaluated 40 renal transplant recipients between weeks 5 and 12 after transplantation, including 179 blood and 181 urine specimens. RESULTS: All positive samples by antigenemia were also positive by PCR. However, in 52 cases only PCR was positive (kappa = 0.134 [P < .001]). Viruria was positive in 66 cases, but only 26 were CMV PCR positive. In 34 cases, viruria was negative and PCR positive (P = .192). CONCLUSION: Detection of DNA in serum is a more sensitive method than antigenemia for the diagnosis of CMV infection. Viruria was not related to the presence of CMV in blood.

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.

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.

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

Se ha estudiado la sensibilidad in vitro al aciclovir de 96 cepas de virus herpes simple, aisladas de 80 pacientes inmunodeprimidos atendidos en nuestro hospital, mediante la técnica de reducción del efecto citopático. El 98 por ciento (61/62) de las cepas de virus herpes simple tipo 1 y el 91 por ciento (31/34) de las de virus herpes simple tipo 2 eran sensibles a una concentración de aciclovir inferior a 3 mg/l. En un 5 por ciento de los pacientes estudiados se aislaron cepas de virus herpes simple resistentes al aciclovir (DI50 >3 mg/l). El 98 por ciento de las lesiones causadas por virus herpes simple sensibles in vitro al aciclovir (DI50 <3 mg/l) se resolvieron independientemente del tratamiento. En dos casos la técnica de reducción del efecto citopático no pudo predecir el fallo del tratamiento y la persistencia de las lesiones no se asoció con el aislamiento de una cepa resistente in vitro. En cuatro casos el aislamiento de una cepa resistente al aciclovir no fue indicativo de fallo terapéutico. En conclusión, creemos que no es necesario determinar la sensibilidad in vitro de los virus herpes simple al aciclovir de forma sistemática, y que este estudio debe reservarse para aquellos casos en que persistan las lesiones y se hayan descartado otras circunstancias como posible causa de la falta de respuesta clínica. (AU)

[In vitro susceptibility to ganciclovir and foscarnet of cytomegaloviruses]. / Sensibilidad in vitro de citomegalovirus al ganciclovir y al foscarnet.

Ganciclovir is the drug of choice for the treatment of acute cytomegalovirus infections. This antiviral agent is a nucleoside analog of guanine whose activity is dependent upon its intracellular phosphorylation to the triphosphate derivative. Foscarnet is used to treat immunosuppressed patients such as organ transplant recipients and AIDS patients with cytomegalovirus who do not tolerate or develop resistance to ganciclovir. Foscarnet is a pyrophosphate analog that directly inhibits viral DNA polymerase. Resistant isolates have been recovered from immunocompromised patients treated with both anticytomegalovirus compounds. The aims of this study were to prepare a plaque reduction assay to study the in vitro susceptibility of cytomegalovirus to ganciclovir and foscarnet, and to apply it to the knowledge of in vitro susceptibility values of cytomegalovirus isolated from clinical samples. Eighty isolates from patients who had never been treated with ganciclovir or foscarnet were tested for antiviral susceptibility. The plaque reduction assay took 6-8 weeks. The results are expressed as ID(50) (inhibitory dose 50), and the ID(50) values of ganciclovir were between 2.14 and 13.49 microM. The ID(50) for ganciclovir was higher that 12 microM in only two cases (2%). The molecular study of the DNA of these did not show any mutation in the UL97 gene. The ID(50) values of foscarnet were between 46.65 and 460.22 microM. In 78 cases (98%) foscarnet ID(50) was lower than 400 microM. These results were comparable with those obtained by other authors. To summarize, the frequency of cytomegalovirus strains resistant in vitro to ganciclovir and foscarnet in previously untreated patients was low and when it was present it did not involve therapeutic failure since the patients progressed favorably.

Sensibilidad in vitro de citomegalovirus al ganciclovir y al foscarnet / In vitro susceptibility to ganciclovir and foscarnet of cytomegaloviruses

El antivírico de elección en el tratamiento de las infecciones graves causadas por citomegalovirus es el ganciclovir, un análogo del nucleósido de guanina que precisa ser fosforilado a la forma ganciclovir trifosfato para actuar como antivírico. En los casos en que por alguna razón el ganciclovir no sea el tratamiento de elección, la alternativa terapéutica es el foscarnet, que se engloba en el grupo de los análogos del pirofosfato. En pacientes con sida y otras inmunodeficiencias se ha descrito enfermedad por citomegalovirus resistente al tratamiento con ganciclovir o foscarnet. Los objetivos de este trabajo han sido la puesta a punto de la técnica de reducción del número de placas para el estudio de la sensibilidad in vitro al ganciclovir y al foscarnet, y su aplicación para el conocimiento de los valores de sensibilidad in vitro de los citomegalovirus aislados de muestras clínicas. Se estudiaron 80 cepas de citomegalovirus obtenidas de pacientes que no habían recibido tratamiento previo. La realización de la técnica requirió entre seis y ocho semanas. Los resultados se han expresado como DI50 (dosis inhibitoria 50) y los valores para el ganciclovir se han situado entre 2,14 y 13,49 µM. En 78 cepas (98 por ciento) la DI50 del ganciclovir fue inferior a 12 µM. Para el foscarnet los valores de DI50 se han situado entre 46,65 y 460,22 µM. En 78 cepas (98 por ciento) la DI50 del foscarnet fue inferior a 400 µM. En dos cepas la DI50 del ganciclovir fue superior a 12 µM, y en ellas el estudio molecular de su DNA no mostró ninguna mutación del gen UL97. En otras dos cepas la DI50 del foscarnet fue superior a 400 µM. En resumen, la frecuencia de cepas de citomegalovirus resistentes in vitro al ganciclovir y al foscarnet en pacientes no tratados previamente ha sido baja, y cuando ha existido no ha sido indicativa de fallo terapéutico puesto que los pacientes de que se aislaron evolucionaron favorablemente (AU)

[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.

Thrombocytopenia associated with liver cirrhosis and hepatitis C viral infection: role of thrombopoietin.

BACKGROUND/AIMS: Thrombocytopenia in chronic liver diseases has traditionally been considered a consequence of platelet pooling and destruction in spleen. We tried to evaluate the influence of thrombopoietin, the physiological regulator of thrombopoiesis, on the origin of this thrombocytopenia. METHODOLOGY: We determined serum thrombopoietin levels by ELISA in thrombocytopenic patients with liver cirrhosis (n = 32) and with chronic hepatitis C viral infection (n = 23). A group of 43 healthy subjects was used as a control. RESULTS: Liver cirrhosis patients presented slightly, but not significantly, lower serum thrombopoietin levels (104 +/- 56 pg/mL) than controls (121 +/- 58 pg/mL) or patients infected with chronic hepatitis C virus (125 +/- 40 pg/mL). No correlations were found between serum thrombopoietin concentrations and liver tests or hematological parameters. CONCLUSIONS: We conclude that low thrombopoietin production may play a role, along with hypersplenism, in the development of thrombocytopenia in patients with liver cirrhosis. Normal thrombopoietin levels exclude a defect in thrombopoietin production as a possible etiology for the thrombocytopenia in patients with chronic hepatitis C viral infection. However, a direct viral megakaryocyte infection or an immune mechanism could explain this thrombocytopenia, according to the thrombopoietin levels detected.