Improved detection of Histoplasma antigenemia following dissociation of immune complexes.

The sensitivity for detection of Histoplasma antigen is lower in serum than in urine. In other antigen assays, treatment of serum at 104 degrees C in the presence of EDTA was required for detection of antigenemia. Sensitivity and specificity for detection of Histoplasma antigenemia were examined with or without EDTA heat treatment of the serum using the MVista Histoplasma antigen enzyme immunoassay. A total of 94.6% of serum specimens from patients with AIDS and histoplasmosis that were negative untreated were positive after EDTA-heat treatment. Two-thirds of the negative serum specimens from patients with probable histoplasmosis, based upon clinical suspicion and Histoplasma antigenuria, were positive after heat treatment. Specificity was 99.0% in controls, including healthy subjects and patients in whom histoplasmosis or blastomycosis, were excluded. Precision and reproducibility were good and excellent, respectively. These findings demonstrate improvement in sensitivity without reduction in specificity, precision, or reproducibility after heat-EDTA treatment.

Performance characteristics of the platelia Aspergillus enzyme immunoassay for detection of Aspergillus galactomannan antigen in bronchoalveolar lavage fluid.

We have evaluated the Platelia Aspergillus enzyme immunoassay for detection of galactomannan in bronchoalveolar lavage (BAL) specimens in solid organ transplant patients with aspergillosis. The precision and reproducibility in serum or BAL to which galactomannan was added were similar. Sensitivity was 81.8% in patients with aspergillosis, and specificity was 95.8% in lung transplant patients who underwent BAL for surveillance for infection or rejection. Among transplant controls, positive results were more common in patients (i) who underwent diagnostic BAL performed for evaluation of symptoms or chest computed tomographic abnormalities, (ii) who had undergone lung transplantation, or (iii) who were colonized with Aspergillus. Galactomannan testing in BAL is useful for diagnosis of aspergillosis in transplant patients. The significance of positive results in patients without confirmed aspergillosis requires further evaluation.

Effects of CD4 and CD8 T lymphocyte depletion on the course of histoplasmosis following pulmonary challenge.

Use of a pulmonary model of histoplasmosis in CD4/CD8 lymphocyte depleted animals offers an opportunity to study pathogenesis in a setting resembling AIDS. Flow cytometric analysis demonstrated that administration of anti-CD4 and anti-CD8 antibodies reduced CD4 and CD8 T cell subsets in the lungs, lymph nodes and spleen. Depletion of these cells transformed the infection from a self-limited course in normal mice to a progressive, fatal course in CD4/CD8 depleted mice. CD4 depletion alone had a lesser effect on survival, but increased fungal burden, while CD4/CD8 depletion had the greatest effect. Histopathologic studies showed marked differences in the inflammatory response between the dually depleted animals and the non-depleted controls. In conclusion, the course of histoplasmosis in CD4/CD8 depleted animals is progressive and fatal, resembling that observed in immunosuppressed patients. This model appears suitable for investigation of immunity to H. capsulatum, and should be useful for evaluation of treatment in the immunocompromised host.

Chronic infection and reactivation in a pulmonary challenge model of histoplasmosis.

Reactivation may be a mechanism for the development of histoplasmosis in AIDS. In this study, histoplasmosis was reactivated by the depletion of CD4 and CD8 lymphocytes in mice. CD4 and/or CD8 depletion beginning 1 month after intratracheal infection and continuing for 2 months caused reactivation with a 2.1 log/g increase in the lungs and a 1.5 log increase in the spleen of B6C3F1 mice. Because control animals showed persistent infection, a subsequent experiment sought to determine the long-term outcome in competent mice. Twelve of 32 immunocompetent mice died at weeks 26-52 of infection, and 4 survivors appeared to be clinically ill; all ill mice had high fungus burdens, whereas cultures were sterile in the healthy mice. Eight of the surviving healthy-appearing mice underwent autopsy 2 years after infection, and cultures were sterile. Thus, 16 of 32 immunocompetent mice exhibited progressive infection. CD4 and/or CD8 depletion exacerbated infection, but a chronic progressive and ultimately fatal infection occurred in half the immunocompetent mice.

Amphotericin B combined with itraconazole or fluconazole for treatment of histoplasmosis.

To investigate the efficacy of combined treatment with fluconazole (Flu) and amphotericin B (AmB) for Histoplasma capsulatum meningitis, MICs were determined for 10 clinical isolates, following National Committee for Clinical Laboratory Standards guidelines. Weak synergy was observed for 6 of the 10 isolates. For the in vivo models, mice either were sham treated or were given Flu (75 mg/kg/day), AmB (2 mg/kg every other day), itraconazole (Itra; 75 mg/kg/day), AmB+Flu, or AmB+Itra. Following infection with 5x105 yeasts, Flu antagonized AmB's reduction of fungal burden without reducing its effect on survival. When in vivo antagonism was reproduced following infection with 1x104 yeasts, a higher fungal burden was observed in the lungs. Itra had no effect on AmB's activity and was more effective than Flu for clearance of fungal burden. These findings caution against use of AmB+Flu for treatment of histoplasmosis, but studies of the effect of treatment on the fungal burden in the brain are needed to assess combination therapy for meningitis.

Does long-term itraconazole prophylaxis result in in vitro azole resistance in mucosal Candida albicans isolates from persons with advanced human immunodeficiency virus infection? The National Institute of Allergy and Infectious Diseases Mycoses study group.

The effects of prolonged itraconazole exposure on the susceptibility of Candida albicans isolates to itraconazole and fluconazole have not been well characterized. A recent placebo-controlled study of long-term itraconazole antifungal prophylaxis in persons with advanced human immunodeficiency virus infection afforded the opportunity to address this question. Mucosal Candida sp. isolates were obtained from subjects who developed oropharyngeal or esophageal candidiasis, and in vitro susceptibilities of the last isolate obtained at removal from the study as a prophylaxis failure were compared in itraconazole and placebo recipients. More subjects in the placebo group (74 of 146 [51%]) than in the itraconazole group (51 of 149 [34%]) developed mucosal candidiasis (P = 0.004). A total of 112 isolates were recovered from 56 of the 74 (76%) subjects with mucosal candidiasis assigned to the placebo group, compared to 97 isolates from 45 of the 51 (88%) subjects in the itraconazole group. C. albicans accounted for 98% of isolates in the placebo group and 89% of isolates in the itraconazole group. The itraconazole MIC at which 50% of the isolates tested were inhibited (MIC(50)) for last-episode isolates from the itraconazole group was 0.125 microg/ml compared to 0.015 microg/ml for the placebo group subjects, P = 0.0001. The MIC(50) of fluconazole for the last isolates from the itraconazole group was 1.5 microg/ml compared to 0.5 microg/ml for the placebo subjects (P = 0.005). A lower proportion of isolates recovered from subjects on itraconazole therapy were classified as susceptible to itraconazole (63%) compared to isolates from the placebo group (96%) (P = 0.001). Similarly, a lower proportion of C. albicans isolates from subjects on itraconazole therapy were susceptible to fluconazole (78%) compared to isolates from the placebo group (96%) (P = 0.01). Also, the proportion of isolates that were not fully susceptible to itraconazole or fluconazole was greater in patients assigned to the itraconazole group than the placebo group (itraconazole susceptibility, 37 and 4%, respectively (P = 0.001); fluconazole susceptibility, 23 and 4%, respectively (P = 0.01). In conclusion, long-term itraconazole prophylaxis in patients with AIDS is associated with reduction in susceptibility to itraconazole and cross-resistance to fluconazole.

DNA typing and control of methicillin-resistant Staphylococcus aureus at two affiliated hospitals.

OBJECTIVE: To describe control of endemic and outbreak-related methicillin-resistant Staphylococcus aureus (MRSA) at two affiliated hospitals. DESIGN: Prospective surveillance of patients with MRSA. Disposable gloves were used by all staff having direct contact with the affected patient or his immediate environment, and patient isolates were typed by pulsed-field gel electrophoresis (PFGE) of genomic DNA. Surveillance and PFGE typing were used concurrently to identify possible nosocomial outbreaks, confirm or refute cross-infection, and support a need for additional outbreak control interventions. SETTING: A university hospital (Hospital A) and a university-affiliated public hospital (Hospital B). PARTICIPANTS: Patients with MRSA colonization or infection over an 18-month interval (June 1993-November 1994). INTERVENTION: Proper handwashing and gloving practices were reemphasized with staff following confirmation of outbreaks. RESULTS: Hospital A had 60 community-acquired and 48 nosocomial cases of MRSA. Two small outbreaks (affecting a total of seven patients) and two pseudo-outbreaks were identified. Hospital B had 36 community-acquired and 22 nosocomial cases of MRSA. Only one outbreak affecting five patients occurred. All outbreaks ended shortly after staff meetings that emphasized ongoing and extremely careful handwashing and gloving when caring for identified patients. The majority of nosocomial cases at both hospitals were not related epidemiologically or had isolates with unique PFGE types. Pseudo-outbreaks were confirmed by demonstrating that isolates from epidemiologically related cases (by time and clinical service or hospital unit) had different PFGE types. Hospital A cases had 39 different PFGE types, and Hospital B cases had 31 different PFGE types. CONCLUSION: MRSA in hospitals, including outbreaks identified by prospective surveillance and confirmed by PFGE typing, can be controlled by minimal special precautions and interventions. This is possible despite the continuous admission of patients with MRSA from the community. PFGE typing is useful to confirm outbreaks and pseudo-outbreaks, demonstrate differences among epidemiologically unrelated isolates, and substantiate the efficacy of MRSA control programs within hospitals.

Characterization of DFA-negative, probe-positive Neisseria gonorrhoeae by pulsed field gel electrophoresis.

Eight Neisseria gonorrhoeae isolates, negative by direct fluorescent antibody (DFA) but positive by a DNA probe, were characterized by pulsed field gel electrophoresis and compared to eight DFA-positive, probe-positive isolates. Results indicate that DFA-negative, probe-positive Neisseria gonorrhoeae isolates may be clonal.

Typing of sequential bacterial isolates by pulsed-field gel electrophoresis.

We typed 39 sets of multiple bacterial isolates of the same species from patients by pulsed-field gel electrophoresis of genomic DNA (PFGE). Isolates were cultured from different sites or over a 2-week or longer interval. Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae were tested. Excluding E. cloacae, 28 of 32 sets of isolates (87%) demonstrated only identical or highly related PFGE types. Four of the seven sets of E. cloacae showed different types. For species other than E. cloacae, our results suggest that patients are usually colonized and infected with a single strain of these bacterial pathogens. Unlike all of the other tested species, E. cloacae PFGE typing differences suggested the presence of multiple strains causing colonization and infection.

Control of methicillin-resistant Staphylococcus aureus in a hospital and an intensive care unit.

OBJECTIVE: To describe methicillin-resistant Staphylococcus aureus (MRSA) control in a hospital, including a surgical intensive care unit (SICU) outbreak. DESIGN: Prospective surveillance of newly identified patients with MRSA. Barrier isolation (disposable gloves for direct contact with patient or immediate environment) was used for the routine care of hospitalized MRSA patients as of October 1991. Beginning in 1992, MRSA isolates were typed by restriction endonuclease enzyme analysis of plasmid DNA (REAP) and/or pulsed-field gel electrophoresis of genomic DNA (PFGE). Surveillance information and MRSA typing were used concurrently to identify nosocomial case clustering, confirm cross-infection, and support a need for additional outbreak control interventions. SETTING: University-affiliated public hospital. PARTICIPANTS: Patients with newly identified MRSA colonization or infection from 1991 through 1993 and epidemiologically associated staff providing care to eight SICU patients in an outbreak. INTERVENTIONS: Barrier isolation for affected and unaffected patients in and admitted to the SICU institution when the outbreak was identified and cross-infection confirmed. Anterior nares cultures of staff in contact with outbreak cases for detection of MRSA colonization. RESULTS: Fifty-six hospitalized patients with community-acquired MRSA and 80 patients with nosocomial MRSA colonization or infection were identified during the 3 years. After the introduction of barrier isolation, the annual frequency of new nosocomial MRSA cases decreased and only one outbreak (eight cases in the SICU) caused by type-related isolates occurred. The other 35 nosocomial cases of MRSA during 1992 and 1993 were not epidemiologically related or were caused by isolates with different types. The SICU outbreak ended after instituting barrier isolation for all patients (with and without MRSA) in and admitted to the unit. Six colonized SICU staff were identified. All outbreak cases had identical or related MRSA types by PFGE and REAP. Staff isolates were different from case isolates by typing, and staff were not restricted and not given treatment for colonization. After more than 6 months of follow up, no further outbreaks of MRSA in the SICU or elsewhere in the hospital occurred despite returning to barrier isolation for affected patients only. CONCLUSION: MRSA in hospitals and outbreaks of MRSA in ICUs can be controlled by surveillance and minimal barrier interventions. REAP or PFGE typing of MRSA can be used to support or refute the presence of cross-transmission. Typing also may be helpful when planning and assessing the effectiveness of interventions directed at endemic, as well as outbreak, MRSA control.