Michelle Durkin

Aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients

Background. The clinical utility of Platelia Aspergillus enzyme immunoassay (EIA) for galactomannan (GM) antigen detection in bronchoalveolar lavage (BAL) for the diagnosis of invasive aspergillosis (IA) in lung transplant recipients is not known. Methods. BAL fluid samples from consecutive lung transplant recipients who underwent bronchoscopy were prospectively analyzed for GM. Results. A total of 333 BAL samples from 116 patients were tested. Invasive aspergillosis was documented in 5.2% (6/116) of the patients. Samples analyzed included 9 BALs from two patients with proven IA, 19 BALs from four patients with probable IA, and 305 BALs from 110 patients without IA. At the index cutoff value of ≥0.5, the sensitivity was 60%; specificity was 95%, with positive and negative likelihood ratios of 14 and 0.41, respectively. Increasing the index cutoff value to ≥1.0 yielded a sensitivity of 60%, a specificity of 98%, and the positive and negative likelihood ratios of 28 and 0.40, respectively. Two of six patients with IA receiving antifungal prophylaxis had false-negative results. Conclusions. A Platelia EIA index cut-off ≥1.0 in the BAL fluid in a lung transplant recipient with a compatible clinical illness may be considered as suggestive of IA.

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.

Histoplasmosis-Associated Cross-Reactivity in the BioRad Platelia Aspergillus Enzyme Immunoassay

ABSTRACT We observed false-positive results in the Platelia Aspergillus enzyme-linked immunoassay (EIA) for specimens from patients with histoplasmosis and mice with experimental infection. Platelia Aspergillus EIA-positive specimens were negative in the second-generation Histoplasma antigen EIA. Care must be taken to exclude histoplasmosis for patients with positive Platelia Aspergillus EIA results.

Comparison of the echinocandin caspofungin with amphotericin B for treatment of histoplasmosis following pulmonary challenge in a murine model.

ABSTRACT Twenty clinical isolates of Histoplasma capsulatum were tested for their in vitro susceptibilities to caspofungin in comparison to those to amphotericin B by following National Committee for Clinical Laboratory Standards guidelines for yeasts. The mean MICs were 16.6 μg/ml (range, 8 to 32 μg/ml) for caspofungin and 0.56 μg/ml (range, 0.5 to 1.0 μg/ml) for amphotericin B. Survival experiments used a 105 dose in a pulmonary challenge model with B6C3F1 mice. All mice that received amphotericin B at 2 mg/kg of body weight every other day (q.o.d.), 30% of mice that received caspofungin at 8 mg/kg/day, and 20% of mice that received caspofungin at 4 mg/kg/day survived to day 15, while mice that received caspofungin at 2 mg/kg/day and all control mice that received the vehicle died by day 14. Amphotericin B at 2 mg/kg q.o.d. markedly reduced the fungal burden in the lungs and spleens, as measured byHistoplasma antigen detection techniques and quantitative cultures, for each comparison. Caspofungin at 10 mg/kg twice a day (b.i.d.) did not reduce the fungal burden, as measured by antigen detection techniques, but slightly reduced the levels of fungi in both the lungs and spleens, as determined by quantitative cultures. Caspofungin at 5 mg/kg b.i.d. did not affect fungal burden. Overall, caspofungin had only a slight effect on survival or fungal burden.

Diagnosis of Coccidioidomycosis with Use of the Coccidioides Antigen Enzyme Immunoassay

BACKGROUND We have previously shown antigenuria in patients with coccidioidomycosis through use of the Histoplasma antigen enzyme immunoassay (EIA), and now we have developed a specific Coccidioides antigen EIA. METHODS The Coccidioides EIA uses antibodies to Coccidioides galactomannan. The sensitivity of the Coccidioides and Histoplasma EIAs was evaluated in patients with more-severe coccidioidomycosis, and the specificity of these EIAs was evaluated in patients with nonfungal infections, in patients with other endemic mycoses, and in healthy individuals. RESULTS Among patients in the present study, antigenuria was detected in 70.8% of patients with coccidioidomycosis with use of the Coccidioides EIA and in 58.3% of patients with use of the Histoplasma EIA. Antigenuria was absent in 99.4% of healthy individuals, patients with nonfungal infections, and patients with noninfectious conditions. Cross-reactions with other endemic mycoses were observed in 10.7% of patients. CONCLUSIONS The Coccidioides EIA has potential to be useful in the rapid diagnosis of more-severe forms of coccidioidomycosis.

Performance Characteristics of the Platelia Aspergillus Enzyme Immunoassay for Detection of Aspergillus Galactomannan Antigen in Bronchoalveolar Lavage Fluid

ABSTRACT 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.

Comparison of a new triazole, posaconazole, with itraconazole and amphotericin B for treatment of histoplasmosis following pulmonary challenge in immunocompromised mice

ABSTRACT A murine model of intratracheally induced histoplasmosis in immunocompromised B6C3F1 mice was used to evaluate a new triazole antifungal agent, posaconazole. This compound was previously shown to be comparable to amphotericin B and superior to itraconazole for the treatment of histoplasmosis in immunocompetent mice. The current study used mice that were depleted of T lymphocytes by intraperitoneal injection of anti-CD4 and anti-CD8 monoclonal antibodies beginning 2 days before infection and continuing at 5-day intervals until completion of the study. Groups of B6C3F1mice that were depleted of CD4 and CD8 T cells were infected with an inoculum of 104Histoplasma capsulatum yeasts. All mice receiving posaconazole at 1 or 0.1 mg/kg of body weight/day, amphotericin B at 2 mg/kg every other day (qod), or itraconazole at 75 mg/kg/day survived to day 29. Only 60% of mice receiving itraconazole at 10 mg/kg/day and none receiving amphotericin B at 0.2 mg/kg qod survived to that date. Fungal burdens were determined at day 14 of infection, 1 day after discontinuation of therapy. Quantitative colony counts and Histoplasma antigen levels in lung and spleen tissues declined following treatment with amphotericin B at 2 mg/kg qod, posaconazole at 5 and 1 mg/kg/day, and itraconazole at 75 mg/kg/day but not in mice treated with amphotericin B at 0.2 mg/kg qod or itraconazole at 10 mg/kg/day. Posaconazole at 0.1 mg/kg/day reduced fungal colony counts and antigen levels in spleens but not in lungs. This study shows posaconazole activity for the treatment of histoplasmosis in immunosuppressed animals.

Activity of clindamycin with primaquine against Pneumocystis carinii in vitro and in vivo.

The combination of primaquine with clindamycin is effective in both in vitro and in vivo models of Pneumocystis infection. Primaquine alone at concentrations from 10 to 300 micrograms/ml reduced the numbers of organisms in cultures to less than 7% of control. Significant inhibition was observed down to 0.1 microgram/ml. Clindamycin at 5 micrograms/ml was ineffective alone. Combinations of clindamycin and primaquine in culture at various concentrations were effective, but there was no evidence of true synergy. In rats with established Pneumocystis pneumonia, clindamycin alone at 5 or 225 mg/kg was ineffective. Primaquine alone at 0.5 or 2 mg/kg did not significantly affect the numbers of organisms remaining. The combination of 0.5 mg of primaquine per kg and 225 mg of clindamycin per kg was effective for therapy, lowering the numbers of organisms in the lungs by about 90%. The combination of 2 mg of primaquine per kg and 225 mg of clindamycin per kg was more effective, lowering the numbers of organisms by almost 98%. In the in vivo prophylaxis model, primaquine at 0.1 or 0.2 mg/kg did not prevent the development of Pneumocystis pneumonia in immune-suppressed rats. Clindamycin at 50 mg/kg had a modest effect alone, but at 5 mg/kg all animals became heavily infected. At 0.5 mg/kg, primaquine alone reduced the severity of infection, but seven of eight rats were still infected. In contrast, the combination of 5 mg of clindamycin per kg and 0.5 mg of primaquine per kg prevented infection in 8 of 10 rats; 2 rats had minimal infection. These studies suggest that the combination of clindamycin and primaquine should be tested in therapy or prophylaxis of Pneumocystis infections in humans. Images

Diagnosis of Coccidioidomycosis by Antigen Detection Using Cross-Reaction with a Histoplasma Antigen

BACKGROUND In 2005, patients with coccidioidomycosis were observed to have positive Histoplasma antigen test results. METHODS We performed a review of the records of patients with coccidioidomycosis who were under our care who underwent testing for Histoplasma antigen to determine the value of this test in the diagnosis of coccidioidomycosis. Many of the patients were immunosuppressed and critically ill. RESULTS The Histoplasma antigen test had positive results when urine samples from 11 (58%) of 19 patients who had acute or chronic coccidioidomycosis were tested. The sensitivity was highest for patients who had acute coccidioidomycosis, and antigenuria was detected in 11 (79%) of 14 patients. One patient who had chronic coccidioidomycosis but who had a negative result when a urine sample was tested had antigen detected in bronchoalveolar lavage fluid. CONCLUSIONS Physicians should be alerted that infections with Coccidioides species may cause positive Histoplasma antigen test results. There is potential for the use of this test in the diagnosis of coccidioidomycosis by taking advantage of this observed cross-reactivity. The greatest benefit appears to be in the population of seriously ill patients with acute pneumonia who live in areas that are endemic for Coccidioides infection.