Effect of Superimposed Infections on Viral Replication in Human Immunodeficiency Virus Type 1-Infected Children

Plasma HIV-1 genomic RNA is a direct measure of viral replication, and its elevation seems to be an important predictor of disease progression in both HIV-1 infected adults and children.1, 2 In addition results of clinical trials have shown that changes in plasma HIV-1 RNA are related to treatment effectiveness and clinical outcome.3 Consequently the measurement of plasma HIV-1 RNA is now considered to be a strong surrogate marker for monitoring HIV-1 disease and for assessing the efficacy of antiretroviral therapy.4 Activation of the immune system in response to antigenic stimulation enhances viral replication. Evidence exists that plasma HIV-1 RNA content increases with the use of cytokine therapies5 and after immunizations.6, 7 In HIV-1-infected adults opportunistic infections have been associated with a transient but consistent increase in viral load of variable magnitude.8, 9 It has also been postulated that the increased rate of disease progression as well as the greater susceptibility to infection after exposure to HIV-1 in sub-Saharan Africa is caused, at least in part, by the persistent immune activation associated with chronic parasitic infestation and frequent infections with other pathogens.10 In HIV-1-infected children the immune system is persistently activated by common viral and bacterial infections, by AIDS-related opportunistic infections and by immunizations.11 The aim of this study was to quantify plasma HIV-1 RNA in HIV-1-infected children who have superimposed viral or bacterial infections to determine the impact of these intercurrent illnesses on HIV-1 replication. Patients and methods.Patients and sample collection. Among a longitudinal cohort of 50 HIV-1-infected patients periodically followed up at Pediatric Department 4 of the University of Milan, Italy, we selected 11 children who met the following entry criteria: (1) diagnosis of superimposed common viral or bacterial infection; (2) neither HIV-1-related opportunistic infection nor HIV-1 disease progression during the study interval; (3) no immunization throughout the sampling period; and(4) stable antiretroviral therapy for at least 6 months before enrollment. The diagnosis of superimposed infections was based on clinical findings and on serologic evidence or microbiologic isolation of pathogens, according to standardized conventional methods, in specimens obtained directly from the site of disease.12-14 Table 1 summarizes the general characteristics of the subjects. All the patients acquired HIV-1 infection through vertical transmission. Patients were ages 1.3 to 12.5 years (median, 6.5 years). Seven patients were females and four were males. The diagnosis of HIV-1 infection and the clinical and immunologic status of the children met the criteria of the 1994 CDC HIV classification for children.15 Informed consent was obtained from the parents or legal guardians of all the children.TABLE 1: Demographic and clinical characteristics of the study population Blood samples were obtained during the acute phase and 1 to 2 months after the superimposed infection had resolved. Plasma HIV-1 RNA levels and CD4+ cell counts were subsequently compared with those obtained previously (1 to 3 months before) in the same subjects during routine follow-up controls. Peripheral blood was collected by venipuncture in Vacutainer® tubes containing preservative-free EDTA (Becton Dickinson, Rutherford, NJ). HIV-1 RNA assay was performed in plasma separated within 2 h of collection by centrifugation at 3.000 × g for 15 min at 4°C and then frozen and kept at−80°C until assayed. Lymphocyte subsets were assessed in fresh blood samples. Quantitation of HIV-1 RNA. Plasma HIV-1 RNA was measured in parallel samples with a branched chain signal amplification assay (Quantiplex® HIV-1 RNA assay kit, Version 2.0; Chiron Diagnostic Laboratories, Emeryville, CA), according to the manufacturer's instructions. The test has a limit of sensitivity of 500 copies/ml. Lymphocyte subsets. Diagnostic monoclonal antibodies were used to evaluate lymphocyte subsets by means of fluorocytometry with Coulter (Coulter Electronics, Inc., Miami Lakes, FL) or Ortho (Ortho Diagnostic Systems) fluorocytometers. Statistical analysis. Differences in numerical data were performed for significance using the nonparametric Wilcoxon matched pairs signed ranks test. Results. Plasma HIV-1 RNA changes were studied in 11 children with diagnosed superimposed common viral or bacterial infections. The superimposed infections diagnosed in the enrolled patients are shown in Table 2. The median interval between the prediagnosis sampling and the acute phase sampling was 81 days (range, 30 to 90 days). The median interval between the acute phase and the recovery sampling was 35 days (range, 27 to 60 days).TABLE 2: Viral replication in HIV-1-infected children before, during and after superimposed infections During the study period none of the Class N children was receiving antiretroviral therapy. Children in Classes A and B were treated with zidovudine 480 mg/m2/day and didanosine 180 mg/m2/day. Among those in Class C one was receiving zidovudine 480 mg/m2/day and didanosine 180 mg/m2/day, two were receiving zidovudine 480 mg/m2/day and zalcitabine 0.03 mg/kg/day, whereas two were treated with stavudine 2 mg/kg/day, lamivudine 8 mg/kg/day and indinavir 1500 mg/m2/day. Results of the plasma HIV-1 RNA assays and the CD4+ cell counts before, during and after the superimposed infections are shown in Table 2. Two patients showed no detectable HIV-1 RNA before, during and after the acute phase of disease. Among the nine children with detectable HIV-1 RNA, there was always an increase in plasma HIV-1 RNA during superimposed infections; the magnitude of the increases in plasma viremia ranged from a factor of 2- to 7-fold. In all nine children there was a decline in plasma HIV-1 RNA with recovery from the disease; the magnitude of the decreases in plasma viremia ranged from a factor of 1- to 13-fold. The difference in HIV-1 RNA quantity between prediagnosis and acute phase sampling was statistically significant (P = 0.007), as well as the difference in HIV-1 RNA between acute phase and recovery sampling (P= 0.007). The difference between HIV-1 RNA content before and after superimposed infections was not significant (P = 0.9). Considering all the 11 children, a decrease in the absolute CD4+ cell count from a median of 522 cells/μl before superimposed infections to 391 cells/μl during the acute phase of intercurrent illnesses was observed (P = 0.05). The median absolute CD4+ cell count then rebounded slightly to 500 cells/μl with recovery from the disease (P = 0.30). However, the overall change in the absolute CD4+ cell count from prediagnosis to recovery samples was significant (P = 0.03). Discussion. We showed that superimposed common infections in HIV-1-infected children can be associated with a transient but consistent increase in HIV-1 RNA of variable magnitude. Our data are in agreement with previous studies which demonstrated that the activation of the immune system by an antigen-specific response to an exogenous stimulus can enhance viral replication.5-10 To our knowledge this is the first report to show that superimposed infections in pediatric age can have an effect on in vivo replication of HIV-1. The sample size in this study is, however, too small to allow any correlation between the severity of the infection and the likelihood of having an increased viral load. Among the nine clinical events that occurred in children with detectable HIV-1 RNA, four were associated with at least a 6-fold rise in HIV-1 RNA concentrations during the acute period of illness. A rise of 3-fold was observed in two patients, whereas a smaller increase (2 times the baseline level) was detected in three children. In addition in all the children plasma HIV-1 RNA returned to or near the baseline values after the superimposed infections resolved. Interestingly, the two children who had undetectable viral loads that did not change with infection were on triple therapy with a protease inhibitor. As regards the other surrogate marker tested, the CD4+ cell count, we observed a decrease of more than 250 and 100 cells/μl, respectively, in five and two patients during the acute phase of superimposed infections. In three patients CD4+ cell counts remained stable, and in one it increased. Among the seven children who showed a significant decrease of CD4+ cell count with the onset of infection, only three had an increase to or near baseline levels after the recovery from the disease, whereas four showed a persistent decrease. Our data support the concept that caution should be used when interpreting plasma HIV-1 RNA quantity in HIV-1-infected children. A burst of plasma HIV-1 RNA must be evaluated by taking into account the patient's clinical situation because it may represent a transient condition resulting from an antigenic stimulation, possibly by an increasing number of activated CD4+ T cells that produce virus and spread the infection. To minimize misleading results it may be advisable to postpone the testing of viral load at least 1 month after recovery from a superimposed infection and to refrain from therapeutic decisions based on viral load during bouts of intercurrent illnesses. The potential pathogenic consequences of repeated superimposed common infections in HIV-1-infected children must be considered. In this study the differences between HIV-1 RNA before and after superimposed infections were not statistically significant. On the contrary the differences between CD4+ cell counts before and after superimposed infections were statistically significant. It is possible that multiple bursts in HIV-1 production caused by intercurrent illnesses contribute to HIV-1 disease progression and thereby exacerbate immune decline; if so, aggressive suppression or avoidance of these infections and their associated HIV-1 replication bursts might have an important clinical impact on the health of HIV-1-infected children. Paola Marchisio, M.D. Susanna Esposito, M.D. Nadia Zanchetta, M.D. Raffaella Tornaghi, M.D. Maria Rita Gismondo, M.D. Nicola Principi, M.D. Pediatric Department 4 (PM, SE, RT, NP) and Microbiology Clinic (NZ, MRG); University of Milan; Medical School; Milan, Italy