RESUMEN
Mycobacterium tuberculosis is a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyte Mycobacterium smegmatis. In this work we investigated the role of sigH in the M. tuberculosis response to heat and oxidative stress. We determined that a M. tuberculosis sigH mutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genes trxB2/trxC and a gene of unknown function, Rv2466c, is regulated by sigH via expression from promoters directly recognized by SigH. We also determined that the sigH mutant is more susceptible to heat stress and that inducible expression of the heat shock genes dnaK and clpB is positively regulated by sigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role of sigH in the M. tuberculosis stress response, we investigated the regulation of the stress-responsive sigma factor genes sigE and sigB. We determined that inducible expression of sigE is regulated by sigH and that basal and inducible expression of sigB is dependent on sigE and sigH. These data indicate that sigH plays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important in M. tuberculosis pathogenesis.
Asunto(s)
Mycobacterium tuberculosis/fisiología , Estrés Oxidativo/fisiología , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Calor , Mycobacterium tuberculosis/patogenicidad , Regiones Promotoras Genéticas , Unión Proteica , Factor sigma/genética , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/genética , Transcripción GenéticaRESUMEN
The Mycobacterium avium complex (MAC) is an important cause of cervical lymphadenitis in children, and its incidence appears to be increasing in the United States and elsewhere. In areas where Mycobacterium tuberculosis is not prevalent, M. avium causes the vast majority of cases of mycobacterial lymphadenitis, although several other nontuberculous mycobacterial species have been reported as etiologic agents. This report describes the case of a child with cervical lymphadenitis caused by a nontuberculous mycobacterium that could not be identified using standard methods, including biochemical reactions and genetic probes. Direct 16S ribosomal DNA sequencing showed greater than 99% homology with Mycobacterium triplex, but sequence analysis of the 283-bp 16S-23S internal transcribed spacer (ITS) sequence showed only 95% identity, suggesting that it is a novel species or subspecies within a complex of organisms that includes M. triplex. Mycolic acid high-performance liquid chromatography analysis also identified this isolate as distinct from M. triplex, and differences in susceptibility to streptomycin and rifampin between this strain and M. triplex were also observed. These data support the value of further testing of clinical isolates that test negative with the MAC nucleic acid probes and suggest that standard methods used for the identification of mycobacteria may underestimate the complexity of the genus Mycobacterium. ITS sequence analysis may be useful in this setting because it is easy to perform and is able to distinguish closely related species and subspecies. This level of discrimination may have significant clinical ramifications, as closely related organisms may have different antibiotic susceptibility patterns.
Asunto(s)
ADN Espaciador Ribosómico/genética , Linfadenitis/microbiología , Infecciones por Mycobacterium/microbiología , Mycobacterium/clasificación , Mycobacterium/genética , Secuencia de Bases , Preescolar , ADN Bacteriano/genética , Femenino , Humanos , Datos de Secuencia Molecular , Mycobacterium/aislamiento & purificación , Cuello , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Análisis de Secuencia de ADNRESUMEN
Sequence analysis of the ribosomal internal transcribed spacer of 56 Mycobacterium avium complex isolates from pediatric patients with AIDS or lymphadenitis revealed (similar to the situation in adults) that the closely related Mav-B and Mav-A sequevars caused the vast majority of disease. IS1245 restriction fragment-polymorphism analysis and pulsed-field gel electrophoresis revealed sets of isolates with closely related patterns among strains from patients in the Boston area and among isolates from Los Angeles and Miami patients. The finding of related strains that cause disease in epidemiologically unrelated patients is most consistent with one of two hypotheses: (1) a limited subset of M. avium strains is more virulent and therefore more likely to cause disease in humans, or (2) pathogenic strains are more prevalent in the environment.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Linfadenitis/microbiología , Complejo Mycobacterium avium/clasificación , Infección por Mycobacterium avium-intracellulare/microbiología , Microbiología del Agua , Boston , Niño , ADN Ribosómico/química , Electroforesis en Gel de Campo Pulsado , Florida , Humanos , Los Angeles , Complejo Mycobacterium avium/genética , Complejo Mycobacterium avium/aislamiento & purificación , Complejo Mycobacterium avium/patogenicidad , Polimorfismo de Longitud del Fragmento de Restricción , Abastecimiento de AguaRESUMEN
BACKGROUND AND PURPOSE: Infections caused by nontuberculous mycobacteria (NTM) commonly manifest as cervicofacial adenitis in otherwise healthy children. The aim of this study was to characterize the imaging findings of NTM infection of the head and neck in immunocompetent children. METHODS: The medical records and imaging examinations (CT in 10, MR in two) were reviewed in 12 immunocompetent children with NTM infection of the head and neck. RESULTS: The usual presentation (n = 9) was of an enlarging, non-tender mass with violaceous skin discoloration, unresponsive to conventional antibiotics. The duration of symptoms was 6 days to 5 months. Imaging revealed asymmetric adenopathy with contiguous low-density ring-enhancing masses in all patients. There was cutaneous extension in 10 patients. Inflammatory stranding of the subcutaneous fat was minimal (n = 9) or absent (n = 2) in 11 patients. The masses involved the submandibular space (n = 3), the parotid space (n = 2), the cheek (n = 1), the anterior triangle of the neck (n = 2), the submandibular and parotid spaces (n = 2), the parotid space and neck (n = 1), and the neck and retropharyngeal space (n = 1). Surgical management included incision and drainage only (n = 2), incision and drainage with curettage (n = 2), excisional biopsy after incision and drainage (n = 1), excisional biopsy only (n = 5), superficial parotidectomy only (n = 1), and superficial parotidectomy with contralateral excisional biopsy (n = 1). All patients improved in response to surgery and long-term antimycobacterial antibiotics. CONCLUSION: NTM infection of the head and neck has a characteristic clinical presentation and imaging appearance. Recognition of this disease is important; appropriate treatment is excision and, in selected cases, antimycobacterial therapy.
Asunto(s)
Linfadenitis/diagnóstico , Imagen por Resonancia Magnética , Infecciones por Mycobacterium/diagnóstico , Enfermedades Otorrinolaringológicas/diagnóstico , Tomografía Computarizada por Rayos X , Niño , Preescolar , Diagnóstico Diferencial , Femenino , Humanos , Lactante , Ganglios Linfáticos/patología , Masculino , Enfermedades de las Parótidas/diagnóstico , Glándula Parótida/patología , Glándula Submandibular/patología , Enfermedades de la Glándula Submandibular/diagnósticoRESUMEN
OBJECTIVE: [corrected] To describe a case of culture-proven multidrug-resistant tuberculous (MDR-TB) meningitis, in which the patient survived long enough for clinicians to adjust antituberculous therapy to second-line therapeutic agents. DESIGN: Case report. SETTING: Tertiary care hospital. PATIENT: Twenty-one-month-old girl with MDR-TB meningitis. INTERVENTIONS: Initial standard treatment failed. Subsequent treatment with second-line therapeutic agents including ciprofloxacin, cycloserine, ethambutol, ethionamide, and rifabutin were given for approximately two years. Concentrations of these drugs were measured in serum and cerebrospinal fluid in the presence and absence of meningeal inflammation. MAIN OUTCOME MEASURES/RESULTS: The patient survived for approximately two years after initiation of second-line anti-TB therapy. During this treatment, she developed a ventriculo-peritoneal shunt tunnel tract infection secondary to MDR-TB. CONCLUSIONS: All TB meningitis isolates for which the source case antibiotic susceptibility pattern is not known should be cultured and susceptibility tested using rapid broth techniques. Measurement and subsequent adjustment of therapeutic drug concentrations may optimize therapy with second-line anti-TB drugs in TB meningitis. Better pediatric formulations and pharmacokinetic data for second-line and anti-TB therapeutic agents are needed.
Asunto(s)
Antituberculosos/uso terapéutico , Resistencia a Múltiples Medicamentos , Tuberculosis Meníngea/tratamiento farmacológico , Antituberculosos/sangre , Antituberculosos/líquido cefalorraquídeo , Farmacorresistencia Microbiana , Femenino , Humanos , Lactante , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Meníngea/microbiologíaRESUMEN
Extracytoplasmic function (ECF) sigma factors are a heterogeneous group of alternative sigma factors that regulate gene expression in response to a variety of conditions, including stress. We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses. A gene encoding a closely related sigma factor, sigH, was cloned from Mycobacterium tuberculosis and Mycobacterium smegmatis. A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including M. fortuitum and M. avium. While the M. tuberculosis and M. smegmatis sigH genes encode highly similar proteins, there are multiple differences in adjacent genes. The single in vivo transcriptional start site identified in M. smegmatis and one of two identified in M. bovis BCG were found to have -35 promoter sequences that match the ECF-dependent -35 promoter consensus. Expression from these promoters was strongly induced by 50 degrees C heat shock. In comparison to the wild type, an M. smegmatis sigH mutant was found to be more susceptible to cumene hydroperoxide stress but to be similar in logarithmic growth, stationary-phase survival, and survival following several other stresses. Survival of an M. smegmatis sigH sigE double mutant was found to be markedly decreased following 53 degrees C heat shock and following exposure to cumene hydroperoxide. Expression of the second gene in the sigH operon is required for complementation of the sigH stress phenotypes. SigH is an alternative sigma factor that plays a role in the mycobacterial stress response.
Asunto(s)
Proteínas Bacterianas , Respuesta al Choque Térmico , Mycobacterium smegmatis/genética , Mycobacterium/genética , Estrés Oxidativo , Factor sigma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Western Blotting , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mycobacterium/metabolismo , Mycobacterium/fisiología , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Mycobacterium bovis/fisiología , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/fisiología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiología , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Factor sigma/química , Factor sigma/metabolismo , Transcripción GenéticaRESUMEN
mariner family transposons are widespread among eukaryotic organisms. These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence of added cofactors. Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo. We have developed simple transposition systems by using minitransposons, made up of short inverted repeats flanking antibiotic resistance markers. These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter. We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene. With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E. coli and Mycobacterium smegmatis. mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.
Asunto(s)
Elementos Transponibles de ADN , Escherichia coli/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Bacteriófago lambda/genética , Secuencia de Bases , Conjugación Genética , Cianobacterias/genética , Proteínas de Unión al ADN , Mutagénesis Insercional , Sistemas de Lectura Abierta , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Rhodobacter capsulatus/genética , Streptomyces/genética , TransposasasRESUMEN
The most common manifestation of infection due to nontuberculous mycobacteria (NTM) in children is cervical lymphadenitis in an otherwise healthy patient. We identified and reviewed 19 cases of proven or presumptive lymphadenitis due to NTM seen at our hospital over the course of 13 months. Nine patients underwent initial surgical excision of involved lymph nodes. Ten children did not have involved lymph nodes excised initially and were treated with macrolide-containing antibiotic regimens. Of these patients, five required subsequent surgical excision and five were cured with combination chemotherapy. Six patients underwent radiographic imaging of the head and neck that revealed asymmetrical adenopathy with ring-enhancing masses but minimal inflammatory stranding of the subcutaneous fat, a finding that may distinguish adenitis caused by NTM from staphylococcal and streptococcal adenitis. Our data suggest that if surgical excision is not considered feasible, antimicrobial therapy for adenitis due to NTM may be beneficial for some patients.
Asunto(s)
Linfadenitis/microbiología , Linfadenitis/terapia , Infecciones por Mycobacterium no Tuberculosas/terapia , Antibacterianos/uso terapéutico , Preescolar , Femenino , Humanos , Lactante , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/cirugía , Linfadenitis/diagnóstico , Macrólidos , Masculino , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Micobacterias no Tuberculosas/aislamiento & purificación , Estudios Retrospectivos , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
BACKGROUND: Disseminated Mycobacterium avium complex (DMAC) infection occurs in HIV-infected children and adults with advanced immunosuppression. Prophylactic therapy has been shown to prevent this infection in adults with low CD4 cell counts. The United States Public Health Service and the Infectious Disease Society of America recently published guidelines (USPHS/CDC guidelines) for the prevention of opportunistic infections, including DMAC infection, in children and adults with HI infection. These guidelines incorporate age-specific CD4 counts at which DMAC prophylaxis should be used. OBJECTIVES: To determine (1) the extent to which the USPHS/CDC guidelines are being followed and (2) current practices for the prevention and management of DMAC infection in HIV-infected children. METHODS: A questionnaire was sent to 65 centers that specialize in the care of HIV-infected children. RESULTS: Forty-one of 65 centers responded to the questionnaire. A strikingly low rate of adherence to age-specific criteria for DMAC prophylaxis was found for the age groups < 1 year and 1 to 2 years (34 and 39%, respectively), in contrast to good adherence for the age groups 2 to 6 years (80%) and >6 years (93%) (<0.0001). Reasons for lack of adherence to the guidelines included a perception of low risk of DMAC in the youngest age groups and difficulty administering additional medications to these patients. The survey also documented substantial variability in DMAC prophylactic and treatment regimens and in susceptibility testing of M. avium complex isolates.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/prevención & control , Actitud del Personal de Salud , Bacteriemia/prevención & control , Infección por Mycobacterium avium-intracellulare/prevención & control , Guías de Práctica Clínica como Asunto , Prevención Primaria/normas , Adulto , Niño , Preescolar , Recolección de Datos , Femenino , Humanos , Lactante , Masculino , Prevención Primaria/métodos , Estados UnidosRESUMEN
The extracytoplasmic function (ECF) sigma factors constitute a diverse group of alternative sigma factors that have been demonstrated to regulate gene expression in response to environmental conditions in several bacterial species. Genes encoding an ECF sigma factor of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium smegmatis, designated sigE, were cloned and analyzed. Southern blot analysis demonstrated the presence of a single copy of this gene in these species and in Mycobacterium bovis BCG, Mycobacterium leprae, and Mycobacterium fortuitum. Sequence analysis showed the sigE gene to be highly conserved among M. tuberculosis, M. avium, M. smegmatis, and M. leprae. Recombinant M. tuberculosis SigE, when combined with core RNA polymerase from M. smegmatis, reconstituted specific RNA polymerase activity on sigE in vitro, demonstrating that this gene encodes a functional sigma factor. Two in vivo transcription start sites for sigE were also identified in M. smegmatis and M. bovis BCG. Comparison of wild-type M. smegmatis with a sigE mutant strain demonstrated decreased survival of the mutant under conditions of high-temperature heat shock, acidic pH, exposure to detergent, and oxidative stress. An inducible protective response to oxidative stress present in the wild type was absent in the mutant. The mycobacterial SigE protein, although nonessential for viability in vitro, appears to play a role in the ability of these organisms to withstand a variety of stresses.
Asunto(s)
Genes Bacterianos , Mycobacterium/fisiología , Factor sigma/fisiología , Factores de Transcripción/fisiología , Aclimatación , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Escherichia coli , Datos de Secuencia Molecular , Mycobacterium/genética , Mycobacterium/crecimiento & desarrollo , Mycobacterium avium/genética , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Factor sigma/biosíntesis , Factor sigma/química , Factor sigma/genética , Especificidad de la Especie , Factores de Tiempo , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
OBJECTIVE: To evaluate immunologic and virologic correlates of vertical transmission of human immunodeficiency virus type 1 (HIV-1). DESIGN: Case-control study. PATIENTS: Women who were prospectively enrolled in a natural history study of HIV-1 infection in women and infants. Sixteen HIV-1-infected women whose infants became infected were matched by CD4+ cell percentage and use of zidovudine during pregnancy with women whose infants did not become infected. MEASUREMENTS: Maternal autologous neutralizing antibody, virus load determined by RNA-polymerase chain reaction (RNA-PCR), and virus phenotype. RESULTS: Most women in both groups had low titers of autologous neutralizing antibody, and no difference in neutralizing titers was observed (range, < 4 to 181 in both groups). The HIV-1 copy number in maternal plasma was not significantly different in the two groups but was inversely correlated with maternal CD4+ cell percentage (p < 0.005). Five women in the transmitting group and four in the non-transmitting group had syncytium-inducing (SI) phenotype virus. Two infected infants had SI phenotype virus. The SI phenotype virus was associated with a greater HIV-1 copy number in maternal plasma (p < 0.05) and an increase in the mortality rate for the infants (p < 0.01). CONCLUSIONS: In women matched for CD4+ cell percentage, low titers of autologous neutralizing antibody, high virus load, and SI phenotype virus were not associated with an increased risk of transmission of HIV-1 to their infants.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/transmisión , Anticuerpos Anti-VIH/análisis , VIH-1 , Transmisión Vertical de Enfermedad Infecciosa , Síndrome de Inmunodeficiencia Adquirida/mortalidad , Recuento de Linfocito CD4 , Femenino , Proteína p24 del Núcleo del VIH/análisis , VIH-1/genética , VIH-1/inmunología , VIH-1/aislamiento & purificación , Humanos , Lactante , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Viral/análisisRESUMEN
BACKGROUND: Didanosine has demonstrated promising antiviral activity and a tolerable toxicity profile in short term studies. We describe a cohort of HIV-infected children who were treated for a prolonged period of time with didanosine. METHODS: Children (6 months to 18 years of age) with symptomatic HIV infection or an absolute CD4 count < 0.5 x 10(9) cells/L, received oral didanosine at doses between 20 mg/m2 to 180 mg/m2 every 8 hours. Clinical, immunological, and virological parameters were assessed at least every 2 months. The pharmacokinetics of didanosine were evaluated in 85 patients. RESULTS: Previously untreated children (n = 51) and children who had received prior antiretroviral therapy (n = 52) were enrolled in the study (median time on study 22.6 months; range 2 to 48). The long-term administration of didanosine was well tolerated and no new toxicities were observed. The absolute CD4 count increased by > or = .05 x 10(9) cells/L in 28 of 87 (32%) of patients after 6 months of therapy. Responses were also sustained in 41% of these children after 3 years of therapy. Children entering the study with a CD4 count > 0.1 x 10(9) cells/L (n = 51) had a marked survival advantage (P = .00002) with an estimated survival probability after 3 years of 80% compared to 39% for children with lower CD4 counts. Although the area under the curve of didanosine increased proportionally with the dose, there was considerable interpatient variability at each dose level. There was no apparent relationship between surrogate markers of clinical outcome and plasma drug concentration. CONCLUSIONS: Didanosine was well tolerated with chronic administration, and toxicities were uncommon and usually reversible. In 41% of patients, the CD4 count increased and was maintained at the higher level even after years of treatment.
Asunto(s)
Didanosina/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Adolescente , Antígenos CD4 , Niño , Preescolar , Didanosina/administración & dosificación , Didanosina/efectos adversos , Didanosina/sangre , Progresión de la Enfermedad , Femenino , Infecciones por VIH/inmunología , Humanos , Lactante , Masculino , Resultado del TratamientoRESUMEN
As part of a phase I/II trial in children infected with human immunodeficiency virus, we studied the pharmacokinetics of zidovudine and didanosine administered as single agents and in combination. Zidovudine (60 to 180 mg/m2 per dose) was given orally every 6 hours, and didanosine (60 to 180 mg/m2 per dose) every 12 hours. Pharmacokinetic samples were obtained from 54 patients and the area under the plasma concentration-time curve (AUC) was estimated by means of a previously defined limited sampling strategy. Follow-up blood samples were obtained after 4 and 12 weeks of treatment. The mean AUC for zidovudine ranged from 4.8 mumol.hr per liter at 60 mg/m2 to 11.0 mumol.hr per liter at the 180 mg/m2 level, and increased in proportion to the dose. The mean AUC for didanosine ranged from 2.8 mumol.hr per liter (60 mg/m2) to 8.0 mumol.hr per liter (180 mg/m2), with a wide interpatient variability. The AUCs of zidovudine and didanosine remained unchanged when the agents were administered in combination. There was no significant change in the AUCs of either drug after 4 and 12 weeks in comparison with those on day 3 of therapy. However, there was greater interpatient and intrapatient variability with didanosine than with zidovudine. These observations have implications for the future utility of therapeutic drug monitoring with these agents.
Asunto(s)
Didanosina/farmacocinética , Infecciones por VIH/tratamiento farmacológico , Zidovudina/farmacocinética , Adolescente , Adulto , Niño , Preescolar , Didanosina/administración & dosificación , Didanosina/uso terapéutico , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Monitoreo de Drogas , Quimioterapia Combinada , Femenino , Infecciones por VIH/sangre , Humanos , Lactante , Masculino , Zidovudina/administración & dosificación , Zidovudina/uso terapéuticoRESUMEN
OBJECTIVE: To determine the safety, tolerance, pharmacokinetics, and antimycobacterial activity of orally administered clarithromycin in children with acquired immunodeficiency syndrome and disseminated Mycobacterium avium complex (MAC) infection. DESIGN: Phase I study with a 10-day pharmacokinetic phase followed by a 12-week continuation therapy phase. PATIENTS: Twenty-five patients with a median age of 8.3 years were enrolled. Ten were receiving zidovudine and 13 were receiving didanosine at the time of enrollment. INTERVENTION: Clarithromycin suspension was administered to each patient at one of three dose levels: 3.75, 7.5, and 15 mg/kg per dose every 12 hours. Clarithromycin and antiretroviral pharmacokinetics were measured during single-drug and concurrent-drug administration. Clinical and laboratory monitoring was performed biweekly. MEASUREMENTS AND MAIN RESULTS: Clarithromycin was well tolerated at all dose levels. Plasma clarithromycin concentrations increased proportionately with increasing doses, and significant pharmacokinetic interactions were not observed during concurrent administration with zidovudine or didanosine. Decreases in mycobacterial load in blood were observed only at the highest clarithromycin dose level. Decreased susceptibility to clarithromycin developed rapidly (within 12 to 16 weeks) in the majority of MAC strains isolated from study patients.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Claritromicina/uso terapéutico , Complejo Mycobacterium avium , Infección por Mycobacterium avium-intracellulare/tratamiento farmacológico , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Administración Oral , Adolescente , Niño , Preescolar , Claritromicina/efectos adversos , Claritromicina/farmacocinética , Femenino , Humanos , Lactante , Masculino , Pruebas de Sensibilidad Microbiana , Complejo Mycobacterium avium/efectos de los fármacos , Complejo Mycobacterium avium/aislamiento & purificación , Infección por Mycobacterium avium-intracellulare/microbiología , RecurrenciaRESUMEN
OBJECTIVE: Zidovudine and didanosine are both beneficial for the treatment of human immunodeficiency virus (HIV) infection in children. Because disease progression and toxicity often limit their long-term use as single agents, new approaches to using nucleoside analogues are necessary to improve current antiretroviral therapy. DESIGN: We conducted a phase I-II study to evaluate the tolerance, pharmacokinetics, and antiviral activity of the combination of zidovudine and didanosine in children with HIV infection. Sixty-eight children who were either previously untreated or who had manifested hematologic toxicity on full-dose zidovudine were enrolled. Eight dose combinations were studied in the previously untreated children, with doses of zidovudine ranging from 90 to 180 mg/m2 every 6 hours and doses of didanosine ranging from 90 to 180 mg/m2 every 12 hours. RESULTS: Fifty-four previously untreated HIV-infected children were enrolled in this part of the study, of whom 49 remained in the study for a minimum of 24 weeks. For children with previous zidovudine-related hematologic toxicity, three dose levels with zidovudine at 60 mg/m2 every 6 hours orally and didanosine ranging from 90 to 180 mg/m2 every 12 hours orally were used. A total of 14 children were enrolled in this part of the study, and 12 remained on therapy for at least 24 weeks. No evidence of new or enhanced toxicity was observed in either group. After 24 weeks, the median CD4 cell count for all patients increased from 331 to 556 cells/mm3 (P = .01). For the previously untreated group, the median increase in CD4 counts was from 386 to 726 cells/mm3 (P = .003). The median p24 antigen concentration (in those with a detectable level at baseline) decreased from 95 to < 31 pg/mL (p < .001). The geometric mean titer of HIV in plasma decreased from 83.1 to 2.7 tissue culture infectious doses/mL (P = .001). CONCLUSIONS: The combination of zidovudine and didanosine was well-tolerated at doses as high as those used in single agent therapy. Potent in vivo antiviral activity was observed. Combination therapy with nucleoside analogues may be an important approach to optimizing the use of these agents in the treatment of HIV infection.
Asunto(s)
Didanosina/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Zidovudina/uso terapéutico , Adolescente , Adulto , Linfocitos T CD4-Positivos , Niño , Preescolar , Didanosina/administración & dosificación , Didanosina/efectos adversos , Esquema de Medicación , Quimioterapia Combinada , Femenino , Humanos , Lactante , Recuento de Leucocitos/efectos de los fármacos , Masculino , Resultado del Tratamiento , Zidovudina/administración & dosificación , Zidovudina/efectos adversosRESUMEN
Molecular genetic manipulation of mycobacteria would benefit from the isolation of mycobacterial genes that could serve both as genetic markers and as sequences used to target homologous integration of recombinant DNA into the genome. We isolated the Mycobacterium bovis BCG gene encoding orotidine-5'-monophosphate decarboxylase (OMP-DCase) by complementing an Escherichia coli mutant defective in this activity. The BCG OMP-DCase gene (uraA) and the flanking DNA were sequenced. The predicted BCG OMP-DCase protein sequence is closely related to the Myxococcus xanthus OMP-DCase and more distantly related to the other known prokaryotic and eukaryotic OMP-DCases. To investigate whether homologous integration can occur in M. bovis BCG, an improved protocol for transformation of BCG was developed and a linear fragment of mycobacterial DNA containing the uraA locus, marked with a kanamycin resistance gene, was introduced into BCG cells by electroporation. The kanamycin-resistant BCG transformants all contained vector DNA integrated into the genome. The marked DNA had integrated into the homologous uraA locus in approximately 20% of the transformants. These results have implications for understanding the role of mycobacterial genes in disease pathogenesis and for the genetic engineering of improved mycobacterial vaccines.
Asunto(s)
Genes Bacterianos , Mycobacterium bovis/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Recombinación Genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Escherichia coli/enzimología , Marcadores Genéticos , Genoma Bacteriano , Datos de Secuencia Molecular , Mycobacterium/enzimología , Mycobacterium bovis/enzimología , Sistemas de Lectura Abierta , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transformación BacterianaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) isolates from children receiving long-term therapy with an alternating regimen of zidovudine and zalcitabine, or with didanosine monotherapy, were evaluated for resistance to zidovudine, zalcitabine, and didanosine, and for mutations known to be associated with zidovudine or didanosine resistance. HIV-1 from four of six patients receiving zidovudine with zalcitabine developed high-level resistance to zidovudine. A mutation in the HIV-1 reverse transcriptase that is highly associated with zidovudine resistance was identified in all four zidovudine-resistant posttherapy isolates. In contrast, none of the HIV-1 isolates from the seven patients receiving didanosine developed high-level resistance to this agent, despite the identification of a didanosine-associated mutation in six of these posttherapy isolates, although small decreases in sensitivity to didanosine were observed. These results indicate that nucleoside analog-associated mutations in HIV-1 occur frequently in children receiving long-term antiretroviral therapy and that alternating combination therapy does not prevent the development of resistance to zidovudine. They also suggest that there may be differences in the degree of resistance conferred by mutations that result from therapy with different nucleoside analogs. These findings underscore the need for studies to define the clinical importance of these mutations, and for treatment strategies to overcome the emergence of viral resistance in vivo.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Didanosina/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Zalcitabina/antagonistas & inhibidores , Zidovudina/antagonistas & inhibidores , Síndrome de Inmunodeficiencia Adquirida/microbiología , Niño , Preescolar , ADN Viral/genética , Didanosina/administración & dosificación , Farmacorresistencia Microbiana , Quimioterapia Combinada , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Lactante , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Reacción en Cadena de la Polimerasa/métodos , Factores de Tiempo , Zalcitabina/administración & dosificación , Zidovudina/administración & dosificaciónRESUMEN
To define predictive or contributory risk factors for pancreatitis in human immunodeficiency virus-infected children receiving dideoxyinosine (ddI), the authors evaluated 95 children, 3 months to 18 years of age, who had received ddI at 60 to 540 mg/m2 per day for a mean of 56 weeks. Pancreatitis developed in 7 patients (7%) but resolved in all upon withdrawal of ddI. Neither age, sex, nor CD4 count at study entry was predictive of pancreatitis, but pancreatitis appeared more likely to develop in hemophiliacs than in other patients (4 of 23 vs 3 of 72). Pancreatitis developed only in patients who received ddI at the highest dose levels (7 of 60 patients who received ddI at a dose > or = 360 mg/m2 per day vs 0 of 35 patients who received < or = 270 mg/m2 per day). Patients in whom pancreatitis developed had received a higher mean daily dose of ddI than patients with normal amylase and lipase levels throughout the study (348 mg/m2 vs 282 mg/m2), but no relationship with the cumulative dose or the duration of ddI therapy was observed. Although a statistically significant relationship between ddI plasma concentration (area under the curve) and pancreatitis was not conclusively demonstrated, as the number of patients in whom pancreatitis actually developed was small, such a relationship may have been obscured.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Didanosina/efectos adversos , Infecciones por VIH/tratamiento farmacológico , Pancreatitis/inducido químicamente , Adolescente , Alanina Transaminasa/sangre , Amilasas/sangre , Aspartato Aminotransferasas/sangre , Niño , Preescolar , Didanosina/administración & dosificación , Didanosina/farmacocinética , Relación Dosis-Respuesta a Droga , Femenino , Infecciones por VIH/enzimología , Humanos , Incidencia , Lactante , Lipasa/sangre , Masculino , Pancreatitis/enzimología , Pancreatitis/epidemiologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) strains were isolated from nine patients before and after prolonged therapy with either an alternating regimen of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) (AZT/ddC) or 2',3'-dideoxyinosine (ddI) alone. All strains obtained from four patients who received AZT/ddC for up to 41 mo were highly insensitive to AZT in vitro. Only one strain obtained after AZT/ddC therapy showed reduced susceptibility to ddC in addition to AZT and had previously unreported amino acid substitutions in the viral polymerase-encoding pol region, whereas three other strains had one or more of the five previously reported AZT-related mutations. In five HIV-1 strains from patients who received ddI for up to 29 mo, no appreciable decrease in sensitivity to ddI was detected. Two strains isolated after ddI therapy had no significant amino acid mutations, although three strains had a mutation reportedly associated with ddI administration. These data suggest that HIV-1 develops reduced susceptibility to AZT more readily than to ddC and ddI and/or that the reduced susceptibility to ddC and ddI is modest in degree. Moreover, the present data suggest that an alternating regimen of AZT and ddC does not block the emergence of AZT-insensitive variants. It should be noted, however, that the current results do not provide a basis for concluding that AZT/ddC or ddI is inferior, equivalent, or superior to AZT as therapy of AIDS.