RESUMEN
A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.
Asunto(s)
Trasplante de Médula Ósea , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/genética , ADN Viral/aislamiento & purificación , Amplificación de Genes , Sondas de Oligonucleótidos , Adolescente , Adulto , Donantes de Sangre , Citomegalovirus/crecimiento & desarrollo , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/microbiología , ADN Polimerasa Dirigida por ADN , Femenino , Humanos , Masculino , Técnicas de Amplificación de Ácido Nucleico , Hibridación de Ácido Nucleico , Fibrosis Pulmonar/etiología , Polimerasa TaqRESUMEN
During 1989, 316 members of a cohort of homosexual men were tested for HIV-specific DNA by the polymerase chain reaction (PCR) using a pair of gag-region primers. Of 125 HIV-seronegative subjects, 123 (98.4%) were PCR-negative while 158 (82.7%) of 191 HIV-seropositive subjects were PCR-positive. Fewer of the 33 subjects who were seropositive and PCR-negative were at Centers for Disease Control (CDC) stage IV than the seropositive, PCR-positive subjects (6 versus 25%; P = 0.030). The seropositive, PCR-negative group had higher mean CD4 counts (640 versus 490 x 10(6) cells/l; P = 0.006), higher CD4: CD8 ratios (0.92 versus 0.64; P = 0.004), lower immunoglobulin (Ig) G levels (1290 versus 1645 mg/dl; P = 0.002), lower IgA levels (168 versus 251 mg/dl; P less than 0.001), and lower C1q binding activity (8 versus 14%; P = 0.010) than the seropositive, PCR-positive subjects. The median rate of CD4 cell decline in the 3 years preceding the PCR sample was less marked in the seropositive, PCR-negative group than the seropositive, PCR-positive group (-58 versus -77 x 10(6) cells/l per year; P = 0.028). To control for duration of infection, we restricted the analysis to the subgroups of 11 seropositive, PCR-negative subjects and 34 seropositive, PCR-positive subjects who had seroconverted earlier in the cohort study. Both subgroups had similar durations of infection, yet the same pattern of differences persisted.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
ADN Viral/análisis , Infecciones por VIH/inmunología , Seropositividad para VIH/inmunología , VIH-1/genética , Provirus/genética , Síndrome de Inmunodeficiencia Adquirida/inmunología , Linfocitos T CD4-Positivos/inmunología , Complemento C1q/inmunología , Anticuerpos Anti-VIH/análisis , Infecciones por VIH/genética , Infecciones por VIH/microbiología , Seropositividad para VIH/genética , Seropositividad para VIH/microbiología , Humanos , Inmunoglobulinas/inmunología , Recuento de Leucocitos , Masculino , Reacción en Cadena de la Polimerasa , Linfocitos T Reguladores/inmunologíaRESUMEN
OBJECTIVE: To derive reliable estimates of the sensitivity of HIV-1 DNA polymerase chain reaction (PCR) in the neonatal period and to quantify the relative contributions of intra-uterine and intra-partum transmission. METHODS: After reviewing studies on the early diagnosis of HIV-1 infection, investigators were asked to provide published and unpublished PCR test results on prospectively followed, non-breastfed, vertically infected children. Age-specific estimates of the sensitivity of PCR were derived using distribution-free methods for interval-censored data. RESULTS: Data on 271 infected children were combined for analysis. PCR detected HIV-1 DNA in an estimated 38% [90% confidence interval (CI), 29-46] of HIV-infected children tested on the day of, or day after, birth. Sensitivity was observed to rise rapidly in the second week of life, reaching 93% (90% CI, 76-97) by 14 days of age. CONCLUSION: The sensitivity of PCR in the neonatal period is higher than previously reported. This affects the clinical interpretation of an early negative test result and encourages the use of PCR as an endpoint for trials to evaluate interventions to reduce vertical transmission in non-breastfed populations. Approximately one-third of vertically acquired HIV-1 infection could be attributable to intra-uterine transmission.
Asunto(s)
ADN Viral/genética , Infecciones por VIH/transmisión , VIH-1/genética , Transmisión Vertical de Enfermedad Infecciosa , Reacción en Cadena de la Polimerasa , Complicaciones Infecciosas del Embarazo/diagnóstico , Femenino , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , Humanos , Recién Nacido , Valor Predictivo de las Pruebas , Embarazo , Complicaciones Infecciosas del Embarazo/virología , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To describe persons with HIV infection and AIDS but with persistently negative HIV antibody enzyme immunoassay (EIA) results. DESIGN: Surveillance for persons meeting a case definition for HIV-1-seronegative AIDS. SETTING: United States and Canada. PATIENTS: A total of eight patients with seronegative AIDS identified from July 1995 through September 1997. MAIN OUTCOME MEASURES: Clinical history of HIV disease, history of HIV test results, and CD4 cell counts from medical record review; results of testing with a panel of EIA for antibodies to HIV-1, and HIV-1 p24 antigen; and viral subtype. RESULTS: Negative HIV EIA results occurred at CD4 cell counts of 0-230 x 10(6)/l, and at HIV RNA concentrations of 105,000-7,943,000 copies/ml. Using a panel of HIV EIA on sera from three patients, none of the HIV EIA detected infection with HIV-1, and signal-to-cut-off ratios were < or = 0.8 or all test kits evaluated. Sera from five patients showed weak reactivity in some HIV EIA, but were non-reactive in other HIV EIA. All patients were infected with HIV-1 subtype B. CONCLUSIONS: Rarely, results of EIA tests for antibodies to HIV-1 may be persistently negative in some HIV-1 subtype B-infected persons with AIDS. Physicians treating patients with illnesses or CD4 cell counts suggestive of HIV infection, but for whom results of HIV EIA are negative, should consider p24 antigen, nucleic acid amplification, or viral culture testing to document the presence of HIV.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Técnicas para Inmunoenzimas , Síndrome de Inmunodeficiencia Adquirida/sangre , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Adolescente , Adulto , Reacciones Falso Negativas , Femenino , Infecciones por VIH/sangre , Humanos , MasculinoRESUMEN
We have used the polymerase chain reaction (PCR) to detect HIV proviral sequences in minute amounts of peripheral blood collected onto newborn screening blotters. Forty-three newborns, infants, and children of HIV-infected mothers were serially studied: dried blood spot (DBS) specimens were processed for PCR; serum was assayed for HIV antibodies, p24 antigen, and immunoglobulins; mononuclear cells were cultured and CD4 cells were quantitated by immunofluorescence. There was excellent agreement between the results of blood spot PCR, viral culture, and clinical and immunological indicators of HIV infection. Eighteen of 19 infected children tested positive by both PCR and culture, including six asymptomatic infants who were less than 10 weeks of age. As expected, p24 antigen capture assays were insensitive, detecting only 13 of the 19 infected children. One infected infant tested positive by PCR, but negative by culture and antigen. This infant was seropositive at 27 months and had pronounced hypergammaglobulinemia in association with non-specific symptoms. Twenty-four of the 43 infants were asymptomatic with normal immune profiles, declining antibody levels and no evidence of infection. These children tested repeatedly negative by PCR, culture, and p24 antigen assays. Our results indicate that DBS PCR is a sensitive, specific, and cost-effective alternative to viral culture for the early diagnosis (or exclusion) of perinatal HIV infection. DBS sampling opens the way for large-scale prospective studies to determine the exact rates of vertical HIV transmission in industrialized, as well as, nonindustrialized countries.
Asunto(s)
Infecciones por VIH/diagnóstico , VIH-1/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Preescolar , Estudios de Cohortes , ADN Viral/sangre , Femenino , Infecciones por VIH/sangre , Infecciones por VIH/transmisión , VIH-1/genética , Humanos , Lactante , Recién Nacido , Estudios Longitudinales , Masculino , Madres , Estudios ProspectivosRESUMEN
The ability to measure accurately viral RNA in the plasma (1-3) and intracellular (4-7) compartments of HIV-1-infected persons has led to a dramatic improvement in our understanding of the natural history of HIV-1/AIDS. A number of recent studies have convincingly demonstrated that high levels of viral replication occur at all stages of disease (8-10), and that changes in viral RNA load are predictive of disease outcome (11,12) and response to therapy (13,14). These findings, combined with the introduction of potent new antivirals (15,16), have stimulated a growing interest in viral load monitoring, both as a function of disease status, and as a predictor of disease progression and therapeutic efficacy.
RESUMEN
Since its discovery in 1981, human immunodeficiency virus type 1 (HIV-1) has rapidly emerged as one of the most devastating infectious pathogens of this century (1-3). The World Health Organization (WHO) estimates that, as of 1995, there were at least 15 million HIV- infected men, women, and children worldwide, with the vast majority of infections occurring in developing countries and isolated rural regions where specimen collection, preparation and shipment are difficult (4). Simple and improved sampling methods that can be widely applied under difficult field conditions are needed to effectively monitor the changing dynamics of the HIV-1/AIDS pandemic, track the spread of HIV-1 variants among different population groups, and ensure that research and interventive activities are directed against biologically important variants of the virus. To date, at least eight major HIV-1 subtypes, designated A through H, have been identified (5,6). More recently, a ninth subtype, I, has been detected (7), as well as several highly divergent, or "outlying" variants of HIV-1 that have been tentatively classified as group O (8,9). This subtyping is based on a relatively small number of specimens collected from a few geographic areas and the full range and distribution of HIV-1 variants remains to be established. The collection of whole blood on filter paper provides an innovative and powerful approach for the systematic and unbiased collection of large numbers of field specimens for diagnostic and surveillance purposes (10-19).
RESUMEN
The ability to accurately measure viral RNA in the plasma (1-3) and intracellular (4-7) compartments of HIV-1-infected persons has led to a dramatic improvement in the understanding of the natural history of HIV-1 and AIDS. A number of recent studies have convincingly demonstrated that high levels of viral replication occur at all stages of disease (8-10), and that changes in viral RNA load are predictive of disease outcome (11,12), and response to therapy (13,14). These findings, combined with the introduction of potent new antivirals (15,16), have stimulated a growing interest in viral load monitoring, both as a function of disease status, and as a predictor of disease progression and therapeutic efficacy.
RESUMEN
The mechanism underlying a transition of the oral cavity mucosal epithelium towards susceptibility to opportunistic infections in HIV-seropositive patients was investigated. Phenotypic markers CD1a, HLA-DR, and CD86 of oral mucosal Langerhans' cells (LCs), p17 core protein of human immunodeficiency virus (HIV), and CD45RO of memory T cells were labeled on oral hairy leukoplakia lesional biopsies and clinically normal autologous tissue of HIV-infected patients. HIV p17 protein was detected in association with mucosal LCs, mainly within the lesional epithelium. There were significant correlations between the detection of HIV p17 and the depletion of LCs, and between the depletion of LCs and the presence of hairy leukoplakia lesions. Conjugates of activated LCs and memory T cells were also evident in the submucosal area of lesional biopsies. The findings from this study support the hypothesis that oral mucosal LCs are also the target of HIV infection. Cytopathic changes of LCs caused by productive HIV infection may contribute to selective depletion of LCs, which may impair the mucosal immunologic protection against colonization by microorganisms causing HIV-associated oral mucosal lesions.
Asunto(s)
Infecciones por VIH/patología , VIH/fisiología , Células de Langerhans/virología , Mucosa Bucal/patología , Infecciones Oportunistas Relacionadas con el SIDA/patología , Antígenos CD/análisis , Antígenos CD1/análisis , Antígeno B7-2 , Efecto Citopatogénico Viral , Susceptibilidad a Enfermedades , Células Epiteliales/inmunología , Células Epiteliales/virología , VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Seronegatividad para VIH , Seropositividad para VIH/patología , Antígenos HLA-DR/análisis , Humanos , Memoria Inmunológica , Células de Langerhans/inmunología , Antígenos Comunes de Leucocito/análisis , Leucoplasia Vellosa/patología , Masculino , Glicoproteínas de Membrana/análisis , Mucosa Bucal/inmunología , Mucosa Bucal/virología , Fenotipo , Estadísticas no Paramétricas , Linfocitos T/patología , Proteínas del Núcleo Viral/análisisRESUMEN
The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.
Asunto(s)
Serodiagnóstico del SIDA/métodos , Síndrome de Inmunodeficiencia Adquirida/diagnóstico , Recolección de Muestras de Sangre , Filtración/métodos , VIH-1/genética , Papel , Vacunas contra el SIDA , Síndrome de Inmunodeficiencia Adquirida/transmisión , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Resistencia a Medicamentos , Variación Genética , Humanos , Lactante , Recién Nacido , Mutación , Reacción en Cadena de la Polimerasa , Análisis de Secuencia , ZidovudinaRESUMEN
Simple, robust approaches are needed to monitor prevalence, incidence, and mother-to-child transmission of HIV-1 in rural Africa. We have designed a method that uses antibody and viral RNA testing of dried blood spots obtained from mother-infant pairs attending routine immunisation clinics. In our study, prevalence and incidence of HIV-1 was highest in young women in their late teens and early twenties. In children born to infected mothers, prevalence increased from 14% in infants younger than 6 weeks of age to 24% at 3-6 months. The blood-spot approach is an effective method for surveillance of HIV-1 in women and children, and for early identification of incidence of this infection in women of child-bearing age.
Asunto(s)
Infecciones por VIH/transmisión , VIH-1 , Transmisión Vertical de Enfermedad Infecciosa , Salud Rural , Adolescente , Adulto , Femenino , Infecciones por VIH/diagnóstico , Infecciones por VIH/epidemiología , Humanos , Incidencia , Lactante , Recién Nacido , Embarazo , Prevalencia , Sudáfrica/epidemiologíaRESUMEN
We sought to define the relation between serum and plasma HIV-1 viral RNA load in patients with primary and chronic HIV-1 disease. HIV-1 viral load was determined from 116 serum and plasma samples, including 33 matched pairs, from five patients with primary and three patients with chronic HIV disease using the Roche HIV Monitor assay. The mean +/- standard deviations of the serum and plasma viral RNA levels from the 33 matched pairs were 4.372 +/- 0.885 and 4.478 +/- 0.950 log10 (copies/ml), respectively. This -0.106 log difference between serum and plasma viral RNA levels, which equates to 21% of non-log-transformed values, was not statistically significant by the Wilcoxon sign rank test (p = 0.09). The distributions of serum and plasma viral load slopes, calculated from all available viral RNA load data for each patient, were also not statistically different (p = 0.07). The levels of HIV-1 RNA measured in the serum or plasma of HIV-seropositive patients yield equivalent biologic information.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , VIH-1/genética , ARN Viral/sangre , Recuento de Linfocito CD4 , Enfermedad Crónica , Humanos , Estudios ProspectivosRESUMEN
We report the results of a longitudinal study of RNA splicing patterns in 31 early-stage human immunodeficiency virus disease patients with an average follow-up time of 3 years. Eighteen patients showed no evidence for disease progression, whereas 13 patients either showed a > or = 50% reduction in baseline CD4 count or developed opportunistic infections. Levels of unspliced, tat, rev, and nef mRNAs in peripheral blood mononuclear cells were measured by a reverse transcriptase-quantitative, competitive PCR assay. Viral RNA was detected in all patients at all time points. All 13 rapid progressors had viral RNA loads that were > or = 1 log unit greater than those of the slow progressors. In addition, seven of the rapid progressors showed a reduction of more than threefold in the ratio of spliced to unspliced RNA over the 3 years of follow-up. Conversely, two slow progressors with intermediate levels of viral RNA showed no splicing shift. These results confirm earlier observations that viral RNA is uniformly expressed in early-stage patients. We further show that cellular RNA viral load is predictive of disease progression. Importantly, the shift from a predominately spliced or regulatory viral mRNA pattern to a predominately unspliced pattern both is associated with disease progression and adds predictive utility to measurement of either RNA class alone.
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Regulación Viral de la Expresión Génica , Infecciones por VIH/microbiología , VIH-1/genética , ARN Viral/genética , Vacunas contra el SIDA/inmunología , Secuencia de Bases , Recuento de Linfocito CD4 , Cartilla de ADN/química , Infecciones por VIH/patología , Humanos , Estudios Longitudinales , Datos de Secuencia Molecular , Pronóstico , Empalme del ARN , ARN Mensajero/genética , Factores de TiempoRESUMEN
A patient is described who rapidly progressed from primary human immunodeficiency virus (HIV) type 1 infection to death without seroconversion but with consistently high plasma viremia. His asymptomatic sex partner had been HIV-1 seropositive for >8 years prior to transmission. Analysis of viral sequences from these subjects and controls confirmed the transmission event. Although the biologic properties of the patient's virus were unremarkable, he had poor functional immune responses to HIV and an HLA haplotype associated with rapid disease progression. The disparity between immune responses and clinical course in this transmission pair, coupled with infection with an unremarkable HIV-1 isolate, underscores the crucial importance of host factors in HIV-1 pathogenesis.