Ann M. LeMonte

Antigen Assay with the Potential To Aid in Diagnosis of Blastomycosis

ABSTRACT We report results of an immunoassay for Blastomyces dermatitidis antigenuria. Sensitivity was 92.9%, and specificity was 79.3%. Cross-reactions occurred in 96.3% of patients with histoplasmosis, 100% of patients with paracoccidioidomycosis, 70% of patients with penicilliosis marneffei, 2.9% of patients with cryptococcosis, and 1.1% of patients with aspergillosis. Reproducibility was 96.3%. These findings support a potential role for antigen testing in blastomycosis.

Detection of Histoplasma Antigen by a Quantitative Enzyme Immunoassay

ABSTRACT The second-generation Histoplasma antigen immunoassay is semiquantitative, expressing results as a comparison to a negative control, which requires repeat testing of the prior specimen with the current specimen to accurately determine a change in antigen. Reporting results in this manner often is confusing to the ordering physician and laboratory. Development of a quantitative assay could improve accuracy, reduce interassay variability, and eliminate the need to test the prior sample with the current sample in the same assay. Calibrators with known concentrations of Histoplasma antigen were used to quantitate antigen in specimens from patients with histoplasmosis and from controls. Samples from cases of disseminated histoplasmosis or other mycoses and controls were tested to evaluate the performance characteristics of the quantitative assay. Paired specimens were evaluated to determine if quantitation eliminated the need to test the current and prior specimens in the same assay to assess a change in antigen. The sensitivity in samples from patients with AIDS and disseminated histoplasmosis was 100% in urine and 92.3% in serum. Cross-reactions occurred in 70% of other endemic mycoses, but not in aspergillosis. Specificity was 99% in controls with community-acquired pneumonia, medical conditions in which histoplasmosis was excluded, or healthy subjects. A change in antigen level categorized as an increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in nanograms/milliliter determined from testing current and prior specimens in different assays. Sensitivity, specificity, and interassay precision are excellent in the new third-generation quantitative Histoplasma antigen immunoassay.

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.

Improved Detection of Histoplasma Antigenemia following Dissociation of Immune Complexes

ABSTRACT The sensitivity for detection of Histoplasma antigen is lower in serum than in urine. In other antigen assays, treatment of serum at 104°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.

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.

Antifungal therapy for central nervous system histoplasmosis, using a newly developed intracranial model of infection

The outcome of central nervous system (CNS) histoplasmosis is often unfavorable. Although fluconazole plays an integral role in treatment of fungal meningitis, its role in the treatment of histoplasmosis is hampered by reduced activity and potential development of resistance. A murine model of CNS histoplasmosis was used to evaluate the hypothesis that a combination of amphotericin B and fluconazole therapy would be superior to amphotericin B monotherapy. Groups of B6C3F(1) mice were infected by injection of Histoplasma capsulatum into the subarachnoid space. The addition of fluconazole hindered the antifungal effect of amphotericin B, as determined by measurement of fungal burden, suggesting antagonism in the brain. Fluconazole was less effective as a single agent than was amphotericin B, despite the greater penetration of fluconazole into brain tissues. The hypothesis that amphotericin B-fluconazole combination therapy would be superior to amphotericin B monotherapy for treatment of CNS histoplasmosis was not supported by this study.

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 AmBs 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 AmBs 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.

Evaluation of the IMMY ALPHA Histoplasma Antigen Enzyme Immunoassay for Diagnosis of Histoplasmosis Marked by Antigenuria

ABSTRACT The sensitivity of detection of antigenuria in patients positive by the MiraVista Diagnostics Histoplasma enzyme immunoassay (MVD EIA) was 44% with the IMMY ALPHA Histoplasma antigen EIA. The specificity was 84% with the IMMY EIA versus 98% with the MVD EIA. The correlation between assays for positive cases was weak (r2 = 0.430).

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.

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.