Leucopenia resulting from a drug interaction between azathioprine or 6-mercaptopurine and mesalamine, sulphasalazine, or balsalazide.

AIM: We evaluated the effect of coadministration of sulphasalazine, mesalamine, and balsalazide on the pharmacokinetics and pharmacodynamics of azathioprine and 6-mercaptopurine. METHODS: Thirty four patients with Crohn's disease receiving azathioprine or 6-mercaptopurine were enrolled in an eight week non-randomised parallel group drug interaction study and treated with mesalamine 4 g/day, sulphasalazine 4 g/day, or balsalazide 6.75 g/day. The primary outcome measure was the occurrence of clinically important leucopenia during the study, defined separately as total leucocyte counts < 3.0 x 10(9)/l and < or = 3.5 x 10(9)/l. Whole blood 6-thioguanine nucleotide concentrations were determined. RESULTS: Three patients could not be evaluated for the primary outcome measure. In the remaining 31 patients, the frequency of total leucocyte counts < 3.0 and < or = 3.5 were: 1/10 and 5/10 in the mesalamine group; 1/11 and 6/11 in the sulphasalazine group; and 0/10 and 2/10 in the balsalazide group. There were significant increases in mean whole blood 6-thioguanine nucleotide concentrations from baseline at most time points in the mesalamine and sulphasalazine groups but not in the balsalazide group. CONCLUSIONS: In patients with Crohn's disease receiving azathioprine or 6-mercaptopurine, coadministration of mesalamine, sulphasalazine, and possibly balsalazide results in an increase in whole blood 6-thioguanine nucleotide concentrations and a high frequency of leucopenia.

Measurement of thiopurine methyltransferase activity and azathioprine metabolites in patients with inflammatory bowel disease.

BACKGROUND: Measurement of 6-thioguanine nucleotide concentrations may be useful for optimising treatment with azathioprine and 6-mercaptopurine. METHODS: We conducted a study of 170 patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine to determine the relationship between 6-thioguanine nucleotide concentrations and both disease activity, as measured by the inflammatory bowel disease questionnaire (active disease < 170, remission > or = 170) and leucopenia. Blood was submitted for whole blood 6-thioguanine nucleotide concentration and leucocyte count. RESULTS: Mean (SD) inflammatory bowel disease questionnaire score was 176 (32). There was no correlation between inflammatory bowel disease questionnaire scores and 6-thioguanine nucleotide concentrations (r(s) = -0.09, p = 0.24). Median 6-thioguanine nucleotide concentrations in 56 patients with active disease and 114 patients in remission were similar (139 v 131 pmol/8 x 10(8) red blood cells; p = 0.26). There was no correlation between 6-thioguanine nucleotide concentrations and leucocyte counts. CONCLUSIONS: In patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine, 6-thioguanine nucleotide concentrations did not correlate with disease activity, as measured by the inflammatory bowel disease questionnaire, or leucocyte count. These findings are discrepant with most previous studies, possibly due to selection of responding patients who tolerated the medications. A prospective, randomised, dose optimisation trial using 6-thioguanine nucleotide concentrations is warranted.

Improved methods for determining the concentration of 6-thioguanine nucleotides and 6-methylmercaptopurine nucleotides in blood.

The conversion of the cytotoxic and immunosuppressive 6-mercaptopurine (6MP) to the active 6-thioguanine nucleotides (6TGN) is necessary for clinical efficacy of 6MP and its prodrug azathioprine. Another metabolite, 6-methylmercaptopurine nucleotide (6MMPN), is formed via a competing pathway by thiopurine methyl transferase. The concentrations of 6TGN and 6MMPN are measured in washed erythrocytes as a surrogate to the intracellular levels of these metabolites in the target tissues. Analysis of 6TGN and 6MMPN in multi-center clinical studies is more complicated because of the requirement to wash erythrocytes. In this investigation, we found no differences in the concentrations of 6TGN and 6MMPN in blood versus washed erythrocytes in samples obtained from patients taking therapeutic doses of oral 6MP or azathioprine for inflammatory bowel disease. We concluded that whole blood could be used for the analysis of these analytes, thus saving sample preparation time. We also found that the erythrocyte 6TGN concentration in blood at ambient temperature declined 2-4% per day, a loss that can be avoided by shipping blood samples frozen. The loss of 6TGN in blood stored at approximately -80 degrees C was 1% after 1 week and 12% after 24 weeks, indicating the analyte was moderately stable. 6MMPN in blood did not significantly change after 24 weeks of storage at approximately -80 degrees C. In addition, the sensitivity of the 6TGN assay was improved by modifying the HPLC conditions, which made the method more suitable for quantifying low levels of 6TGN in human intestinal biopsy samples and blood.

Combination therapy with oral tacrolimus (FK506) and azathioprine or 6-mercaptopurine for treatment-refractory Crohn's disease perianal fistulae.

Our aim was to report the clinical experience with combination treatment using tacrolimus and either azathioprine (AZA) or 6-mercaptopurine (6MP) in patients with Crohn's disease (CD) perianal fistulae. The medical records of all patients with Crohn's disease perianal fistulae seen at the Mayo Clinic from 1996-1998 who were treated with tacrolimus were reviewed. Clinical response was classified as: complete response, partial response, and nonresponse. Eleven patients were treated with oral tacrolimus for a mean duration of 22 weeks. The initial oral dose of tacrolimus ranged from 0.15 to 0.31 mg/kg/day. Azathioprine or 6MP was continued in combination with tacrolimus in seven patients and initiated simultaneously with tacrolimus in four patients. All patients improved clinically, seven had a complete response, and four had a partial response. The mean time to initial improvement was 2.4 weeks, and the mean time to complete response was 12.2 weeks. The most frequent adverse events were nausea, paresthesias, nephrotoxicity, and tremor. Patients with nephrotoxicity had a significantly higher mean initial tacrolimus dose (0.31 mg/kg/day) compared with patients who did not have nephrotoxicity (0.25 mg/kg/day) (p = 0.035); however, there was not a statistically significant association between the starting dose or mean blood level and clinical response. Combination therapy with oral tacrolimus and AZA or 6MP may be effective treatment for CD perianal fistulae. Higher initial tacrolimus doses increase the risk of nephrotoxicity without improving clinical response.

Brain tumors in the elderly: recent trends in a Minnesota cohort study.

OBJECTIVE: To compare the clinical presentation, time elapsed to diagnosis, and survival of elderly patients (> or = 65 years) with that in younger patients with malignant primary brain tumors. DESIGN: Retrospective cohort study. SETTING: Four hospitals in Minneapolis, Minn. PATIENTS: Seven hundred fourteen patients diagnosed as having and treated for primary malignant brain tumors between 1980 and 1995; 230 (32%) were 65 years or older. MAIN OUTCOME MEASURES: The type and duration of the chief presenting symptom, the time elapsed to diagnosis, the treatment modalities used, and patient survival were analyzed. RESULTS: Time elapsed from onset of symptom to diagnosis was not longer for elderly patients than younger ones, with the exception of patients aged 18 to 24 years, who had a significantly longer delay in diagnosis (P = .004). Elderly patients were significantly less likely to present with headache or seizure (P<.001), and more likely to present with confusion, aphasia, or memory loss (for each, P<.001). With the single exception of confusion, the duration of all other presenting symptoms was not significantly longer for patients 65 years and older compared with younger patients. Survival is significantly reduced in older patients, and appears to worsen significantly in patients 45 years and older (P<.001). A significantly higher proportion of patients 65 years and older with glioblastoma multiforme received no treatment (P = .004) if diagnosed after 1990. CONCLUSIONS: Elderly patients (> or = 65 years) with malignant brain tumors are diagnosed as promptly as younger patients, although they have a markedly different constellation of symptoms. Since diagnosis of brain tumors continues to improve in the elderly, it may be more difficult to ascribe the steady increase in incidence to artifactual factors.

Cellular immune responses to four doses of percutaneous bacille Calmette-Guérin in healthy adults.

To explore the hypothesis that low-dose immunization might induce preferential Th1 cell immunity, 76 adults were vaccinated with one of four doses of bacille Calmette-Guérin (BCG): The doses contained very low (1.6 x 10(5) cfu), low (3.2 x 10(6) cfu), standard (1.6 x 10(8) cfu), or high (3.2 x 10(8) cfu) levels of BCG. Delayed-type hypersensitivity responses occurred 8 weeks after vaccination in 10% of persons given very low or low doses of BCG, compared with 95% and 100% of persons given standard or high doses, respectively. Lymphoproliferative responses, which were increased only for high-dose vaccinees, peaked 2 weeks after vaccination and were directed chiefly against Mycobacterium tuberculosis-secreted proteins, particularly the antigen 85 complex. Significant increases in mycobacteria-specific interferon-gamma expression were present 16 weeks after vaccination only for persons given standard or high doses of BCG. Percutaneous BCG appears capable of inducing a temporary Th1-like immune response, but standard or higher dosages are required.

Japanese encephalitis among hospitalized pediatric and adult patients with acute encephalitis syndrome in Hanoi, Vietnam 1995.

The etiologic spectrum of acute encephalitis syndrome (AES) has not been well defined in Vietnam. Cohort and case-control studies were performed on all adult and pediatric AES patients admitted to the Neurology Service of Bach Mai Hospital between June 5 and August 3, 1995. Among pediatric AES patients, 31 (67%) of 46 had acute Japanese encephalitis (JE), compared with only two (6%) of 33 adult AES patients (P < 0.0001). For confirmed JE cases, serum specimens obtained 15-21 days after symptom onset had the highest mean anti-JE IgM signal-to-noise (P/N) ratios (8.08 + 1.09 SE). A serosurvey of adult household members did not reveal any cases of recent subclinical JE infection, although 26% had evidence of past JE infection. The use of bed netting was nearly universal but did not appear to reduce the risk of AES or JE. Given the high incidence of JE, particularly among children, Vietnam seems well suited for the development of a targeted JE vaccination strategy.

Tuberculosis among Tibetan immigrants from India and Nepal in Minnesota, 1992-1995.

OBJECTIVE: To study screening outcomes among a group of Tibetan immigrants at high risk for developing active tuberculosis (TB) after arrival in Minnesota. DESIGN: Retrospective cohort study. PARTICIPANTS: A total of 191 Tibetan immigrants undergoing medical screening. MAIN OUTCOME MEASURES: Occurrence and treatment outcomes of active TB. SETTING: A health maintenance organization and a public TB clinic in Minneapolis, Minn. RESULTS: Positive (induration, > or =10 mm) tuberculin skin test results were documented in 98% of Tibetans, compared with 44% of Vietnamese, 10% of Hmong, and 51% of Russian refugees in Minnesota (P<.001 for each group). Sixteen active cases (8.4%) were confirmed by isolation of Mycobacterium tuberculosis; however, 5 (31%) were culture-negative on initial screening in Minnesota. Seven cases (44%) were diagnosed during initial screening efforts, and 9 cases (56%) were diagnosed a mean of 19 months (range, 10-27 months) after their initial medical evaluation. Of these 9 cases, 6 (38% of all Tibetan cases) had isolates resistant to 1 or more antituberculous drugs, and 3 (19% of all Tibetan cases) were multidrug resistant (MDR TB). All 3 MDR TB cases were culture-negative on initial screening; these cases constituted 75% of the MDR TB isolates in Minnesota in 1994. The presence of MDR TB was associated with a known history of active TB in Asia (P<.02). Any abnormality on chest radiograph noted either during the Immigration and Naturalization Service screening evaluation in India (relative risk [RR], 5.2; P=.006) or on arrival in Minnesota (RR, 6.8; P=.005) was associated with an increased risk of subsequent active TB. CONCLUSIONS: Tuberculosis infection is nearly universal among Tibetans settling in Minnesota. A single screening evaluation failed to detect the majority of TB cases among Tibetans. Even in the face of negative M tuberculosis cultures, persons with a history of active TB require particularly close follow-up.

The synthesis and immunogenicity of varicella-zoster virus glycoprotein E and immediate-early protein (IE62) expressed in recombinant herpes simplex virus-1.

In order to evaluate the conditions for optimal expression and immunogenicity of varicella-zoster virus (VZV) proteins in a herpes simplex virus-1 (HSV-1) vector, we selected the VZV glycoprotein E (gE), encoded by ORF 68 and the VZV product of ORF 62, an immediate-early major tegument protein (IE62). Three HSV/VZV recombinants were generated: (1) VZV gE protein coding sequences along with the promoter region were inserted into the thymidine kinase (TK) gene of HSV-1 strain KOS; (2) VZV gE expressed from the HSV-1 ICP4 promoter was inserted into the glycoprotein C (gC) gene of HSV-1 strain F; and (3) VZV IE62 protein coding sequences under the control of the HSV-1 ICP4 promoter were inserted into the gC gene of HSV-1 strain F. Immunoblot analysis and immunoperoxidase staining of infected cell monolayers demonstrated vector expression of VZV proteins. Following intracranial inoculation in mice, both VZV gE-HSV (TK) and VZV IE62-HSV (gC) induced an IgG response against VZV gE or VZV IE62. When tested in cytotoxicity assays using T-lymphocytes from VZV immune human donors, the range of precursor frequencies for T-lymphocytes that recognized VZV gE or VZV IE62 was similar whether these proteins were expressed by HSV-1 or a vaccinia vector. These experiments demonstrate that HSV-1 is a competent vector for expression of these VZV proteins and support the feasibility of engineering a combined vaccine for these closely related alpha-herpesviruses.

Immunization with the immediate-early tegument protein (open reading frame 62) of varicella-zoster virus protects guinea pigs against virus challenge.

The IE62 protein, the primary regulatory protein of varicella-zoster virus (VZV) and the major component of the virion tegument, was an effective immunogen in the guinea pig model of VZV infection, whereas the ORF 29 gene product, a nonstructural DNA replication protein, did not elicit protection. All animals immunized with the ORF 29 protein had cell-associated viremia compared with 2 of 11 guinea pigs given the IE62 protein (P = 0.005). VZV was detected in ganglia from 38% of the animals given the ORF 29 protein and 44% of the control animals compared with 9% of the animals immunized with the IE62 protein (P = 0.04). In contrast to the IE62 protein, immunization with the ORF 29 protein did not prime the animals for an enhanced T-cell response upon challenge with infectious virus. The VZV IE62 protein has potential value as a vaccine component.

Nosocomial legionellosis: a review of pulmonary and extrapulmonary syndromes.

Surgical patients appear to be at highest risk for acquisition of nosocomial Legionella pneumonia; most appear to become infected during respiratory tract manipulation and mechanical ventilation. Although the lungs are the most common site of nosocomial Legionella infection, an important subset of patients have infection at extrapulmonary sites. We describe 22 cases of extrapulmonary legionellosis reported in the literature. Most of these patients were surgical patients; more than half did not have serious underlying illnesses, and only five (23%) were receiving immunosuppressive agents. A total of 13 extrapulmonary sites of infection were reported, many in the absence of clinical pneumonia; these infections included sinusitis, hip wound infection, and prosthetic valve endocarditis. Five patients (23%) had fatal infections; in four of these cases diagnosis of Legionella infection was made after death, underscoring the need for a high index of clinical suspicion. A large percentage of extrapulmonary Legionella infections may result from direct topical exposure of susceptible tissue to contaminated tap water. Use of tap water must be carefully monitored, particularly in dressing changes and bathing of surgical patients.

Investigation of the pathogenesis of varicella-zoster virus infection in guinea pigs by using polymerase chain reaction.

The polymerase chain reaction method (PCR) was used to investigate events in the pathogenesis of varicella-zoster virus (VZV) infection in strain 2, Hartley, and euthymic hairless guinea pigs. VZV was detected in peripheral blood mononuclear cells (PBMC) obtained 2-5 days after infection in 8 (50%) of 16 strain 2, 4 (40%) of 10 hairless, and 10 (34%) of 29 Hartley guinea pigs. The frequency of VZV-infected PBMC was estimated to be at least 1/200,000, which is comparable to that observed in human infection. When VZV PCR was used to test ganglia from hairless guinea pigs, samples from 6 of 8 animals were positive. Of 45 VZV-infected guinea pigs that were tested for cellular immunity by VZV T lymphocyte proliferation assay, 44 developed a stimulation index > 2.0. Control animals had no detectable virus by PCR and did not develop cellular immunity to VZV. These experiments showed that viremia was detectable by PCR during primary VZV infection of guinea pigs in about half of the animals regardless of the strain of guinea pig. Acquisition of cellular immunity provided a consistent marker of infection in all guinea pig strains. PCR was also useful for demonstrating VZV in guinea pig ganglia tissue, with VZV gene sequences being detectable for at least 80 days after infection. With the combination of PCR and immunologic assays, various guinea pig strains should be useful for studies of VZV pathogenesis and for the evaluation of antiviral agents and vaccine strategies.

Immunity in strain 2 guinea-pigs inoculated with vaccinia virus recombinants expressing varicella-zoster virus glycoproteins I, IV, V or the protein product of the immediate early gene 62.

The immunogenicity of specific varicella-zoster virus (VZV) proteins, with emphasis upon cell-mediated immune responses, was evaluated by immunizing strain 2 guinea-pigs with vaccinia virus recombinants that express gpI (vac-gpI), gpIV (vac-gpIV) and gpV (vac-gpV) or the IE-62 protein (vac-IE-62). Vac-gpI elicited the highest initial mean T cell proliferation response [stimulation index (S.I.) 3.8 +/- 0.9 S.E.M.] whereas inoculation with vac-gpV produced the lowest primary T cell response (S.I. 2.5 +/- 1.1 S.E.M.). T cell proliferation was detected for a shorter period after immunization with vac-gpV compared to vac-gpI, vac-gpIV or vac-IE-62. A comparison of the immunogenicity of vac-gpI and vac-IE-62 with the same proteins prepared by immunoaffinity purification showed that immunization with these proteins in either form elicited virus-specific IgG antibodies and T cell recognition. The presence or absence of IgG antibodies to the IE-62 protein was used to assess protection against challenge with guinea-pig cell-adapted infectious VZV in animals that had been inoculated with vac-gpI, vac-gpIV or vac-gpV. Immunization with vac-gpI and vac-gpIV restricted VZV replication but all animals given vac-gpV developed antibodies to IE-62 after challenge with infectious VZV. Priming of the T lymphocyte response was observed in all animals immunized with VZV-vaccinia virus recombinants after subsequent exposure to infectious VZV. These experiments with VZV vac-gpI, vac-gpIV and vac-gpV in guinea-pigs suggest variability in the capacity of herpesviral glycoproteins to elicit cell-mediated immunity in vivo. Induction of virus-specific immunity using IE-62 means that this major tegument protein of VZV could be a useful component for vaccine development.

Use of tap water and disinfection practices in outpatient settings. A survey of otolaryngologists.

A survey of otolaryngologists belonging to the American Academy of Otolaryngology-Head and Neck Surgery was conducted to estimate the frequency of tap water use during otologic examinations and to assess methods used for disinfection of otologic instruments in outpatient settings. Questionnaires were returned by 516 persons residing in 49 states. Tap water was used commonly for rinsing suction tips while suctioning patients even with tympanic membrane perforations (45%). Most respondents (87%) reported that their otologic instruments undergo either high-level disinfection or sterilization between patient examinations; however, only 63% to 67% of respondents reported adequate duration of treatment times (high-level disinfection, greater than or equal to 30 minutes; boiling, greater than or equal to 5 minutes; or autoclaving, greater than or equal to 20 minutes). The risk posed by the use of tap water during otologic examinations and the need for adequate disinfection of otologic instruments between patient examinations are presented.

Blood contacts during surgical procedures.

Operating room personnel are at risk for infection with blood-borne pathogens through blood contact. To describe the nature and frequency of blood contact and its risk factors, trained observers monitored a sample of operations performed by six surgical services at Grady Memorial Hospital, Atlanta, Ga, for 6 months. In 62 (30.1%) of 206 operations, at least one blood contact was observed. Of 1828 operating room person-procedures observed, 96 (5.3%) had 147 blood contacts (133 skin contacts [90%], 10 percutaneous injuries [7%], and four eye splashes [3%]). The mean number of blood contacts per 100 person-procedures was highest for surgeons (18.6). The frequency of percutaneous injury was similar among surgeons and scrub staff (mean, 1.2 per 100 worker-procedures for each group). Risk factors for surgeons' blood contacts were (1) performing a trauma, burn, or orthopedic emergency procedure (odds ratio [OR], 4.1; 95% confidence interval [CI], 2.0 to 8.7); (2) patient blood loss exceeding 250mL (OR, 2.1; 95% CI, 1.2 to 3.7); and (3) being in the operating room longer than 1 hour (OR, 3.3; 95% CI, 1.6 to 7.1). Of 110 blood contacts among surgeons, 81 (74%) were potentially preventable by additional barrier precautions, such as face shields and fluid-resistant gowns. Twenty-one (84%) of 25 blood contacts among surgeons in procedures in which all three risk factors were present were potentially preventable by additional barriers. Of 29 blood contacts among anesthesia and circulating personnel, 20 (69%) would have been prevented by glove use. For surgical procedures in which operating room personnel are at increased risk of blood contact, reevaluation of surgical technique, use of appropriate barrier precautions, and development of puncture-resistant glove materials are indicated.