RESUMEN

It has been suggested that human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes could be used as a marker for viral virulence in patients with AIDS. The present study was designed to evaluate a possible association between specific gB genotypes, the presence of HCMV retinitis, and the HCMV viral load. Fifty-four blood samples were obtained from 54 HIV- and HCMV-infected patients. Twenty-seven of these patients were asymptomatic for HCMV, whereas the other 27 patients had been diagnosed recently with HCMV retinitis. HCMV gB genotyping was carried out by using restriction enzyme analysis of PCR-amplified PMNL extracts. Determination of the HCMV viral load in the same specimens was carried out using a quantitative-PCR. HCMV gB genotype 2 was found more frequently than other genotypes in PCR-amplified polymorphonuclear leukocytes (PMNL) of patients with AIDS (P < 0.05) but not more frequently in samples from patients with HCMV retinitis. No significant association was found between any HCMV gB genotypes and the viral load in blood. In conclusion, the actual HCMV gB genotyping system using PMNL provides no additional benefit over the viral load in blood for identification of HIV-infected subjects at risk of HCMV disease.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/virología , Retinitis por Citomegalovirus/virología , Citomegalovirus/genética , Proteínas del Envoltorio Viral/sangre , Recuento de Linfocito CD4 , Citomegalovirus/aislamiento & purificación , Retinitis por Citomegalovirus/sangre , Retinitis por Citomegalovirus/complicaciones , ADN Viral/sangre , Femenino , Genotipo , Humanos , Leucocitos Mononucleares/virología , Masculino , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Proteínas del Envoltorio Viral/genética

RESUMEN

The expression of a late cytomegalovirus (CMV) transcript (pp150) was sought in peripheral blood leukocytes (PBL) of subjects with AIDS and correlated with the amounts of CMV DNA in PBL and plasma, by means of quantitative polymerase chain reaction (PCR). The detection of the late CMV transcript was associated with a high number of CMV DNA copies in PBL (P=.0015) and with a positive CMV PCR assay in plasma (P<.001). Expression of CMV pp150 mRNA was best predicted by viral DNA thresholds corresponding to 7058 and 30 copies in PBL and plasma, respectively. The detection of CMV pp150 mRNA was associated with the presence of CMV disease in a univariate analysis but not in a multivariate analysis after controlling for the viral DNA load in PBL. Thus, active viral replication as determined by a high CMV DNA load in PBL is reflected by expression of the late CMV transcript in the same cells and by the presence of CMV DNA in plasma.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/virología , Infecciones por Citomegalovirus/virología , Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Leucocitos Mononucleares/virología , Fosfoproteínas , Proteínas de la Matriz Viral/genética , Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Antivirales/uso terapéutico , Citomegalovirus/genética , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/tratamiento farmacológico , Foscarnet/uso terapéutico , Ganciclovir/uso terapéutico , Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/sangre , ARN Mensajero/genética , ARN Viral/sangre , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Carga Viral , Proteínas de la Matriz Viral/biosíntesis , Viremia/virología

RESUMEN

Specific human herpesvirus 8 (HHV-8) DNA sequences were found in leukocytes of 12 of 29 (41.4%) AIDS subjects with Kaposi's sarcoma (KS), whereas they were found in 4 of 43 (9.3%) AIDS subjects without KS (P = 0.003), although the peak HHV-8 DNA load in PCR-positive subjects with KS (mean, 425 copies per 0.2 microgram of DNA) did not significantly differ from the one found in PCR-positive patients without KS (mean, 218 copies). The use of intravenous ganciclovir or foscarnet therapy to treat cytomegalovirus disease did not affect the HHV-8 DNA load in seven patients for whom serial samples were analyzed.

Asunto(s)

Infecciones por Citomegalovirus , Infecciones por VIH/complicaciones , Herpesvirus Humano 8/fisiología , Leucocitos/virología , Sarcoma de Kaposi , Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Infecciones Oportunistas Relacionadas con el SIDA/virología , Antivirales/uso terapéutico , Infecciones por Citomegalovirus/tratamiento farmacológico , Foscarnet/uso terapéutico , Ganciclovir/uso terapéutico , Infecciones por VIH/virología , Herpesvirus Humano 8/efectos de los fármacos , Humanos , Leucocitos/efectos de los fármacos , Sarcoma de Kaposi/virología , Carga Viral

RESUMEN

The AMPLICOR cytomegalovirus (CMV) test, a new qualitative assay for the detection of CMV DNA in plasma, was compared to conventional methods and quantitative PCR (Q-PCR) assays by using leukocytes and plasma from 179 blood samples from subjects with AIDS. For the diagnosis of CMV disease, cell-based assays such as a Q-PCR with polymorphonuclear leukocytes (Q-PCR-PMNL) and a pp65 antigenemia assay had the highest sensitivities but suffered from a lack of specificity. The best agreement between the results of the Q-PCR-PMNL assay and those of the AMPLICOR test was found when a threshold diagnostic value of 690 copies per 10(5) cells was selected for the Q-PCR-PMNL assay. In that context, the AMPLICOR CMV test had a sensitivity of 96.4% and a specificity of 95.3% when results were compared to results of the cell-based PCR assay. This threshold was close to the one described as associated with the best sensitivity and specificity for the diagnosis of CMV disease in a recently published study (4). Blood samples that tested positive by the Q-PCR-PMNL assay but negative by the AMPLICOR CMV test were associated with viral loads (mean, 785 copies, median, 96 copies per 10(5) leukocytes) lower than the viral loads of blood samples that tested positive by both assays (mean, 21,452 copies; median, 9,784 copies per 10(5) leukocytes) (P = 0.003). The AMPLICOR CMV test gave positive results at least 48 days before the development of symptomatic CMV disease in a longitudinal analysis of a limited subset of patients (n = 6) from whom sequential specimens were available for testing. In conclusion, the AMPLICOR CMV test is a very convenient assay combining rapidity, simplicity, and the possibility of batch testing. A positive result by this test seems particularly important since this implies, in most instances, the presence or the imminence of CMV disease, although a negative test result does not rule out disease.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Seropositividad para VIH/complicaciones , Reacción en Cadena de la Polimerasa/métodos , Infecciones Oportunistas Relacionadas con el SIDA/sangre , Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Antígenos Virales/sangre , Recuento de Linfocito CD4 , Células Cultivadas , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/complicaciones , Seropositividad para VIH/inmunología , Seropositividad para VIH/virología , Humanos , Estudios Longitudinales , Masculino

RESUMEN

OBJECTIVES: To evaluate the prevalence of the most common cytomegalovirus (CMV) UL97 mutations associated with ganciclovir resistance directly in polymorphonuclear leukocytes (PMNL) of patients with AIDS and CMV retinitis. Also to correlate the presence (or absence) of these mutations with the systemic CMV viral load and the ophthalmologic outcome of these subjects. METHODS: Monthly blood samples were obtained from 19 patients with AIDS and CMV retinitis who had been treated with systemic ganciclovir for > or = 2 months. Detection of CMV UL97 mutations was done using nested PCR amplification followed by restriction enzyme analysis. The viral load was assessed with a polymerase chain reaction-based assay and non-isotopic hybridization detection. RESULTS: CMV UL97 mutations were detected in PMNL of four of 13 (30.8%) patients who had been treated with ganciclovir for > or = 3 months but in none of six patients who had been treated for < 3 months. All four patients with detectable UL97 mutations were presenting evidence of retinitis progression at the time those mutations were first detected (mean, 145.7 days of ganciclovir) and three of four patients had a viral DNA load > 10000 copies per 10(5) PMNL contrasting with the copy numbers in the 15 subjects without mutations (mean, 492.9 copies per 10(5) PMNL after a mean of 146.8 days of ganciclovir). CONCLUSIONS: The prevalence of the most common CMV UL97 mutations associated with ganciclovir resistance in PMNL of patients with AIDS treated for > or = 3 months (30.8%) appears to be higher than the rate of emergence of ganciclovir-resistant CMV isolates as previously reported using phenotypic assays (about 8%). Moreover, the detection of these mutations is associated with a considerable increase in the CMV DNA load in the blood as well as with progression of CMV retinitis during ganciclovir therapy.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Antivirales/farmacología , Retinitis por Citomegalovirus/tratamiento farmacológico , Citomegalovirus/efectos de los fármacos , Citomegalovirus/genética , Ganciclovir/farmacología , Mutación , Infecciones Oportunistas Relacionadas con el SIDA/virología , Antivirales/uso terapéutico , Retinitis por Citomegalovirus/virología , ADN Viral , Progresión de la Enfermedad , Farmacorresistencia Microbiana/genética , Ganciclovir/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Neutrófilos/virología , Reacción en Cadena de la Polimerasa , Carga Viral

RESUMEN

The cytomegalovirus (CMV) DNA load was determined in polymorphonuclear leukocytes (PMNL) and plasma samples from 106 human immunodeficiency virus-infected subjects at risk of developing CMV disease (group 1) and from 27 AIDS patients with documented CMV disease (group 2). For both groups, the number of CMV copies in PMNL was significantly higher than in plasma when results were derived from an equivalent blood volume (P < .001, PMNL vs. plasma). Additionally, group 2 (symptomatic) patients had a greater viral DNA load than group 1 (asymptomatic) subjects (P < .001 for both PMNL and plasma). The sensitivity, specificity, and positive and negative predictive values of qualitative polymerase chain reaction using PMNL (PCR-PMNL) for the presence of CMV disease were 100%, 58%, 38%, and 100%, respectively, compared with 70%, 93%, 74%, and 92% for qualitative PCR-plasma and 93%, 92%, 76%, and 98% for quantitative PCR-PMNL using a cutoff of 16,000 copies/mL. Thus, the best strategy for diagnosing CMV disease in these individuals relies on quantitative assessment of the viral DNA load in PMNL.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/virología , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/aislamiento & purificación , ADN Viral/aislamiento & purificación , Neutrófilos/virología , Carga Viral/métodos , Infecciones Oportunistas Relacionadas con el SIDA/sangre , Infecciones por Citomegalovirus/sangre , Humanos , Estudios Longitudinales , Reacción en Cadena de la Polimerasa/métodos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad

RESUMEN

The nucleotide sequence of the ponA gene encoding the high molecular-mass penicillin-binding protein 1A (PBP1A) of Pseudomonas aeruginosa (Pa) PAO1 was determined and characterized. The predicted PBP1A protein of 822 amino acids (aa) has a calculated molecular mass of 91.2 kDa corresponding to the size of the protein expressed in vitro and in vivo. A penicillin-binding (PB) assay showed that the Pa ponA gene product covalently binds penicillin. The deduced PBP1A aa sequence has features typical of class-A high-molecular-mass PBPs: a highly hydrophobic N-terminus portion containing a potential transmembrane segment which might anchor the protein to the cytoplasmic membrane; an N-terminal module with the conserved boxes 1 (E86D(DN)F(AN)H(Y)G), 2 (G117GS(T)I(TM)Q), 3 (R139K(IN)E(ILL)AL) and 4 (R221R(NW)IL); a PB module with the conserved boxes 5 (S461SFK), (S520RN) and (K695TG); an internal extension at aa 297-407 between the N-terminal and PB modules; and a C-extension at the end of the PB module at aa 742 to 822. The highest percentage of similarity (62.8%) was found with the class A high-molecular-mass PBP1A of Escherichia coli (Ec) and Haemophilus influenzae. The observed extensive homology in the modular design of the Pa PBP1A with the bifunctional Ec PBP1A suggests structural and functional relationships between these proteins and refutes the proposed correspondence between Pa PBP1A and Ec PBP1B.

Asunto(s)

Proteínas Bacterianas , Proteínas Portadoras , Proteínas de Escherichia coli , Genes Bacterianos/genética , Hexosiltransferasas/genética , Complejos Multienzimáticos/genética , Muramoilpentapéptido Carboxipeptidasa , Peptidoglicano Glicosiltransferasa , Peptidil Transferasas/genética , Pseudomonas aeruginosa/genética , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Sistemas de Lectura Abierta/genética , Proteínas de Unión a las Penicilinas , Penicilinas/metabolismo , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Unión Proteica , Proteínas Recombinantes , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

OBJECTIVE: To study the temporal relationships between cytomegalovirus (CMV) viral load and specific UL97 mutations in polymorphonuclear leukocytes (PMNL) and plasma samples from a patient with AIDS who developed ganciclovir-resistant CMV retinitis. METHODS: Sequential PMNL and plasma samples were analysed for determination of the CMV viral load using non-molecular methods and a quantitative polymerase chain reaction (PCR) assay. Screening of the same samples for the most common mutations conferring ganciclovir resistance was performed using nested PCR and restriction enzyme analysis. RESULTS: At the time of progression of CMV retinitis (after 6 months of ganciclovir), a rapid increase in the CMV DNA load was found in both PMNL and plasma samples. This increase paralleled the emergence of a specific mutation (V594) in the same samples and recovery of ganciclovir-resistant blood isolates. In this patient, however, the only tests that substantially predicted the progression of CMV disease were the quantitative PCR assay using PMNL and to a lesser extent the pp65 antigenemia assay. CONCLUSIONS: Quantitative evaluation of the CMV viral load in PMNL using sensitive assays such as PCR appears to be a promising approach for monitoring antiviral therapy in subjects with AIDS. In addition, common mutations conferring ganciclovir resistance can be detected directly in PMNL and plasma samples.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/virología , Antivirales/uso terapéutico , Retinitis por Citomegalovirus/virología , Ganciclovir/uso terapéutico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Infecciones Oportunistas Relacionadas con el SIDA/sangre , Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Adulto , Citomegalovirus/efectos de los fármacos , Citomegalovirus/genética , Retinitis por Citomegalovirus/sangre , Retinitis por Citomegalovirus/complicaciones , Retinitis por Citomegalovirus/tratamiento farmacológico , Farmacorresistencia Microbiana/genética , Genotipo , Humanos , Estudios Longitudinales , Masculino , Mutación , Neutrófilos/virología , Fenotipo , Reacción en Cadena de la Polimerasa , Carga Viral

RESUMEN

We report the development of a simple and rapid PCR assay for quantitation of the cytomegalovirus (CMV) DNA load in polymorphonuclear leukocytes. Using this system, a very good correlation was found between a high number of CMV copies in the blood and the presence of CMV disease in subjects with AIDS.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/virología , Infecciones por Citomegalovirus/virología , Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Neutrófilos/virología , Reacción en Cadena de la Polimerasa/métodos , Antígenos Virales/sangre , Citomegalovirus/genética , Infecciones por VIH/virología , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad