Epstein-Barr virus–induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell–depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/μg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had ≥40,000 copies of EBV DNA/μg DNA, all of whom were recipients of T-cell–depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with ≥40,000 copies EBV/μg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.

Of 495 patients reported in a large urban histoplasmosis outbreak, we studied 276 whose serologic tests were done in a single laboratory. Serologic test results were positive in 96% of these patients (compared with less than 5% of controls from an endemic area), cultures were positive in 22%, and special stains in 19%. The immunodiffusion test results were negative in 13% of patients who had positive findings by complement fixation, and 1% had positive results only by immunodiffusion. The complement fixation test was almost twice as sensitive as the immunodiffusion test in patients with subclinical infection. The serologic response differed significantly among the clinical syndromes with higher titers in cavitary and lower titers in disseminated disease. Factors associated with titers of 1:64 or greater to both antigens were black race and immunocompetence. High mycelial titers were also associated with more intense exposure, and high yeast titers were associated with age less than 36 years. No prognostic significance could be proved for fourfold titer rises or falls or persistence of precipitins.

Radioimmunoassays for IgM and IgG histoplasmal antibodies were developed and proved to be specific for their respective immunoglobulin classes, sensitive, and reproducible. Elevated IgM antibodies were detected in 59.8% of patients with histoplasmosis and 7.9% of control subjects. Elevated IgG antibodies occurred in 80.4% of patients with histoplasmosis but in only 12.9% of control subjects. Radioimmunoassay was nearly twice as sensitive as complement fixation for identifying patients with mild, presumably asymptomatic, infection. Of 13 patients with serologic follow-up at least 1 yr later, elevated IgM antibodies cleared, whereas IgG antibodies persisted in 7. In an epidemiologic investigation of a recurrent histoplasmosis outbreak, only the radioimmunoassay was able to prove the hypothesis that construction for a swimming complex was the source of exposure. These assays promise to be useful for clinical and epidemiologic investigations.

Donor lymphocyte infusions (DLI) following allogeneic stem cell transplantation are known to mediate graft-versusleukemia effect (GVL). A major side effect of these immunotherapies is the development of graft-versus-host diseases (GVHD). One promising approach to prevent GVHD is to genetically modify donor T cells with a suicide mechanism that can be induced in the case of GVHD. Here we report on a retroviral vector containing the death effector domain (DED) of the human Fas-associated protein with death domain (FADD). The DED was fused to two copies of an FKBP506-binding protein and a truncated version of the human low-affinity receptor for nerve growth factor (LNGFR). Activation of the death signal pathway can be triggered upon the addition of chemical inducers of dimerization. This construct was functionally compared to an optimized HSV-TK vector in which a hypersensitive mutant of the herpes simplex virus thymidine kinase gene (TK39) was fused to a cytoplasmic truncated version of the cell surface antigen CD34. A direct comparison between both vectors in primary T lymphocytes showed that the number of T cells transduced with vectors containing the DED was significantly reduced within 24 h of drug administration whereas ganciclovir treatment of TK39-transduced T cells showed a delay in cell death of approximately 3-4 days. Our results indicate that constructs containing the DED may prove to be the most efficient mechanism to quickly eliminate alloreactive T cells.

Detection of a Histoplasma capsulatum polysaccharide antigen (HPA) has proved a useful approach to diagnosis of histoplasmosis. Two sandwich enzyme-linked immunosorbent assays (ELISAs) using alkaline phosphatase (AP)-or horseradish peroxidase (HRP)-conjugated antibodies were compared with solid-phase radioimmunoassay (RIA) for detection of HPA. The AP-ELISA and HRP-ELISA were each positive in 17 (89.5%) of 19 urine specimens from patients with disseminated histoplasmosis, while the RIA was positive in 18 (94.7%) of 19. Specimens from patients with nondisseminated histoplasmosis were positive by AP-ELISA in 8 of 32, by HRP-ELISA in 4 of 25, and by RIA in 12 of 25. Of control specimens from patients with other fungal infections, the AP-ELISA was negative in 22 (91.7%) of 24, the HRP-ELISA in 23 (95.8%) of 24, and the RIA in 23 (95.8%) of 24. Reproducibilities AP-ELISA and HRP-ELISA were 95.1% and 95.1%, respectively. Thus, AP-ELISA and HRP-ELISA appear less sensitive than RIA and may be falsely negative in specimens containing low levels of HPA.

Objective. To determine the role of Helicobacter pylori infection in children with recurrent abdominal pain and the usefulness of serologic tests in screening H pylori infection and monitoring treatment of H pylori-associated gastritis. Methods. During a 3 year period, we investigated the presence of serum immunoglobulin G (IgG) antibody to H pylori in 456 children using the high-molecular-weight cell-associated protein H pylori enzyme immunoassay kit. Among the 456 children studied, 218 (age range, 3 to 18 years; mean age, 9.5 years) had symptoms of recurrent abdominal pain (RAP syndrome) with or without vomiting, and the remaining 238 (age range, 3 to 18 years; mean age, 9.8 years) had no RAP (non-RAP syndrome). We performed upper gastrointestinal endoscopy on 111 consecutive children of the 218 with RAP syndrome and obtained mucosal biopsies for culture, histologic analysis, CLO test (Delta West, Perth, Australia), and H pylori detection by polymerase chain reaction. Results. Thirty-eight (17.4%) of 218 children in the RAP group and 25 (10.5%) of 238 children in the non-RAP group were seropositive for H pylori. Of the 111 children endoscoped, 95 were found to be negative, and 12 were positive by all five assays. Specimens from 2 children were negative by culture and the CLO test but positive by the other three assays. Specimens from 1 child were negative by histologic analysis but positive by all other tests. The remaining child was positive for anti-H pylori IgG but negative by all of the other four assays. Upper gastrointestinal endoscopy detected 14 children with peptic ulcer disease (9 duodenal ulcer and 5 gastric ulcer) and 12 with antral nodular gastritis. Only 4 of the 14 diagnosed with peptic ulcer were H pylori positive by all five assays, whereas all 12 children with antral nodular gastritis were H pylori positive. Nine of the 12 H pylori-positive children were treated with a combination of bismuth subsalicylate, amoxicillin, and metronidazole for 2 weeks. Sera obtained at 2, 4, and 6 months after treatment from all 9 children showed a decrease in anti-H pylori IgG titer. Three H pylori-infected children who did not receive any treatment served as control children, and their IgG levels remained elevated or increased over time. Conclusion. The results from our study indicate that screening for the serum IgG antibody to H pylori is a practical method for diagnosing H pylori infection in children, and that serial measurements of the H pylori IgG antibody are useful for monitoring treatment of H pylori because of its high sensitivity and ease of performance. Only 4 of the 14 children diagnosed with peptic ulcer disease were confirmed to be infected with H pylori, whereas all 12 children with antral nodular gastritis were found to be infected by H pylori. These observations suggest that H pylori infection is more frequently associated with gastritis than with peptic ulcer disease in children, and that H pylori gastritis is a cause of RAP syndrome in children.

Epstein-Barr virus–induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell–depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/μg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had ≥40,000 copies of EBV DNA/μg DNA, all of whom were recipients of T-cell–depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with ≥40,000 copies EBV/μg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.

The purpose of this study was to determine whether measurement of immunoglobulin M (IgM) antibodies against Legionella pneumophila serogroup 1 can aid in the diagnosis of Legionnaires disease. On the basis of measurements of antibody levels in 1,942 control sera, we used an IgM titer of 1:256, observed in 2.3% of the controls, as presumptive evidence of Legionnaires disease. Measurement of IgM titers permitted us to presumptively or definitively diagnose Legionnaires disease in 13 of 34 patients earlier than we would have if only IgG titers had been measured. Of the 13 patients, 5 were diagnosed serologically only by IgM antibody determination. IgM titers were presumptively diagnostic in week 1 of clinical symptoms in 4 of the 13 patients. We conclude that conjugates used for antilegionella indirect fluorescent-antibody tests should be capable of detecting IgM antibodies so that the value of serological results in diagnosing and managing Legionnaires disease will be maximized.