Cutaneous manifestations of systemic mycoses.

Systemic fungal diseases are primary pulmonary diseases caused by the dimorphic fungal pathogens, Blastomyces dermatitides, Coccidioides immitis. Histoplasma capsulatum, or Paracoccidioides brasiliensis. Infection occurs after inhalation of the infectious form of the fungus and may be acute, self-limited, or subclinical. Primary cutaneous infection occurs only after traumatic implantation of the fungus and is unusual. Erythema nodosum or erythema multiforme may accompany the acute form of the disease. Other cutaneous manifestations represent disseminated disease and, as such, require systemic antifungal therapy. Because cutaneous lesions have occurred coincidentally with other cutaneous pathologies, emphasis should be placed on a complete clinical history, physical examination, and diagnosis by histopathology and culture.

Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers.

A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification of DNA targets from Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2. By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR assay were compared with the results of dark-field microscopy and H. ducreyi culture on two different culture media. HSV culture results were available for 99 specimens collected during the third interval. Confirmatory PCR assays targeting different gene sequences for each of the three organisms were used to validate the M-PCR results. Specimens were resolved as positive for the determination of sensitivity if the reference diagnostic test was positive or if the results of both the M-PCR and the confirmatory PCR were positive. The resolved sensitivities of M-PCR for HSV, H. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively. The resolved sensitivities of HSV culture, H. ducreyi culture, and dark-field microscopy were 71.8, 74.2, and 81%, respectively. These results indicate that the M-PCR assay is more sensitive than standard diagnostic tests for the detection of HSV, H. ducreyi, and T. pallidum from genital ulcers.

Tuberculosis alert: an old killer returns.

Health officials predicted tuberculosis would be completely eradicated in the U.S. by the year 2010. They were wrong. Here's how to gear up the lab for its return.

Multicenter evaluation of a broth macrodilution antifungal susceptibility test for yeasts.

Thirteen laboratories collaborated to optimize interlaboratory agreement of results of a broth macrodilution procedure for testing three classes of antifungal drugs against pathogenic yeasts. The activities of amphotericin B, flucytosine, and ketoconazole were tested against 100 coded isolates of Candida albicans, Candida tropicalis, Candida parapsilosis, Candida lusitaniae, Torulopsis (Candida) glabrata, and Cryptococcus neoformans. Two starting yeast inoculum sizes (5 x 10(4) and 2.5 x 10(3) cells per ml) were compared, and readings were taken after 24 and 48 h of incubation. All other test conditions were standardized. The resultant turbidities in all tubes were estimated visually on a scale from 0 to 4+ turbidity, and MIC-0, MIC-1, and MIC-2 were defined as the lowest drug concentrations that reduced growth to 0, 1+, or 2+ turbidity, respectively. For flucytosine, agreement among laboratories varied between 57 and 87% for different inocula, times of incubation, and end point criteria. Agreement was maximized (85%) when the lower inoculum was incubated for 2 days and the MICs were defined as 1+ turbidity or less. For amphotericin B, variations in test conditions produced much smaller differences in interlaboratory agreement. For ketoconazole, interlaboratory agreement was poorer by all end point criteria. However, MIC-2 endpoints distinguished T. glabrata as resistant compared with the other species. Overall, the studies indicated that readings from the lower inoculum obtained on the second day of reading result in the greatest interlaboratory agreement. In combination with data from previous multicenter studies (National Committee for Clinical Laboratory Standards, Antifungal Susceptibility Testing: Committee Report, Vol. 5, No. 17, 1988; M. A. Pfaller, L. Burmeister, M. S. Bartlett, and M. G. Rinaldi, J. Clin. Microbiol. 26:1437-1441, 1988; M. A. Pfaller, M. G. Rinaldi, J. N. Galgiani, M. S. Bartlett, B.A. Body, A. Espinel-Ingroff, R.A. Fromtling, G.S. Hall, C.E. Hughes, F. C. Odds, and A. M. SUgar, J. Clin. Microbiol. 34:1648-1654, 1990), these findings will be used by the National Committee for Clinical Laboratory Standards to develop a standardized method for in vitro antifungal susceptibility testing for yeasts.

Collaborative investigation of variables in susceptibility testing of yeasts.

A multicenter study was performed to evaluate the effect of medium, incubation time (24 and 48 h), and temperature (30 and 35 degrees C) on intra- and interlaboratory variations in MICs of flucytosine, amphotericin B, and ketoconazole for yeasts. Testing was performed on coded isolates of Candida species (11 strains) and Cryptococcus neoformans (2 strains) by using a standard macrodilution protocol 11 laboratories. Four chemically defined media buffered to pH 7.0 with morpholinepropanesulfonic acid were evaluated, including buffered yeast nitrogen base, synthetic amino acid medium-fungal, RPMI 1640 medium, and high-resolution antifungal assay medium. Intralaboratory variability was less than or equal to fourfold for 97% of the replicate sets of data. The highest level of interlaboratory agreement, irrespective of antifungal agent or incubation conditions, was observed with RPMI 1640 medium. Intralaboratory variability was less than or equal to fourfold for 93% of the determinations with ketoconazole and 100% with flucytosine tested in RPMI 1640 medium at 35 degrees C for 24 h. Variability in amphotericin B results was less than or equal to fourfold for 81% of the determinations in RPMI 1640 medium at 35 degrees C for 48 h. The rank order of MICs within each antifungal test group was similar among the various laboratories and was generally in agreement with the reference rank order regardless of the test medium that we used.

Use of Gen-Probe and Bactec for rapid isolation and identification of mycobacteria. Correlation of probe results with growth index.

Gen-Probe culture confirmation tests (Gen-Probe, San Diego, CA) for Mycobacterium tuberculosis complex and Mycobacterium avium complex were performed on 276 mycobacterial isolates. All 138 M. tuberculosis complex isolates and 79 of 80 M. avium complex isolates were identified correctly. No falsely positive test results were obtained; 58 nontuberculous mycobacteria other than M. avium complex were negative by Gen-Probe. In a second phase of testing, Gen-Probe tests were performed using concentrates from 101 patient Bactec 12B cultures. Positive results by Gen-Probe tests were correlated with the growth index (GI) reading on the day of processing as well as the accumulated GI readings. For those 51 with high (greater than or equal to 999) final GIs, 40/40 (100%) M. tuberculosis complex isolates and 9/11 M. avium complex isolates were positive by Gen-Probe, and six other mycobacteria were negative. Of the 25 with moderate final readings (400 less than or equal to GI less than 999), 12/17 M. tuberculosis complex isolates and 1/1 M. avium complex isolates were correctly identified by Gen-Probe; seven other mycobacteria were negative. Of 25 with low readings (GI less than 400), 8/24 M. tuberculosis isolates were correctly identified by Gen-Probe, and no falsely positive test results were obtained with the other probes. All true negative tests on seven other mycobacteria (not M. tuberculosis complex or M. avium complex) had less than 2% hybridization. Of the 24 falsely negative tests on M. tuberculosis complex isolates or M. avium complex isolates, 22 had greater than 2% hybridization with their respective probes. Thus, percent hybridization greater than 2% may be a useful indicator of the need for retesting.

Cutaneous Bipolaris spicifera infection.

Bipolaris spicifera is a dematiaceous fungus that has rarely been reported to cause cutaneous infection in humans. A patient with leukemia was examined for a non-healing ulcer on her leg that developed following minor trauma. Histopathologic study revealed groups of nonpigmented, septate fungal hyphae located predominantly in the necrotic ulcer base. Cultures of a biopsy specimen yielded colonies that were gray to black with a black reverse. Microscopic examination revealed dematiaceous, straight, oblong conidia consistent with B spicifera. The ulcer was successfully treated with surgical excision, skin graft, and amphotericin B.

Cunninghamella bertholletiae and Pneumocystis carinii pneumonia as a fatal complication of chronic lymphocytic leukemia.

A unique patient with chronic lymphocytic leukemia died with pneumonia caused by both Cunninghamella bertholletiae and Pneumocystis carinii. In tissue sections, the hyphae of C bertholletiae were twisted and ribbon-like, but were smaller than those typical for zygomycetes, displayed more than occasional septa, and exhibited Y branching, making histologic distinction from Aspergillus sp difficult.

Comparison of the Quantum II, API Yeast Ident, and AutoMicrobic systems for identification of clinical yeast isolates.

The Quantum II Yeast Identification System (Abbott Laboratories) is a microprocessor-based spectrophotometric system for identification of clinical yeast isolates within 24 h. We compared the Quantum II system with the API Yeast Ident (Analytab Products) and the AutoMicrobic System Yeast Biochemical Card (AMS-YBC; Vitek Systems, Inc.) for the identification of 221 clinical yeast isolates, including 120 common clinical isolates (Candida albicans, C. tropicalis, C. parapsilosis, Torulopsis glabrata, and Cryptococcus neoformans) and 101 relatively uncommon clinical isolates. The API 20C (Analytab) was used as the reference system. The Quantum II and AMS-YBC systems correctly identified 181 (82%) and 184 (83%) isolates, respectively, whereas the Yeast Ident system correctly identified 132 (60%) isolates. Of the 120 common clinical isolates, 113 (94%) were correctly identified by Quantum II, 103 (86%) were correctly identified by AMS-YBC, and 83 (69%) were correctly identified by Yeast Ident. Of the 101 uncommon clinical isolates tested, 68 (67%) were correctly identified by Quantum II, 81 (80%) were correctly identified by AMS-YBC, and 49 (49%) were correctly identified by Yeast Ident. The overall accuracy of the Quantum II, AMS-YBC, and API Yeast Ident was not sufficient to recommend any of these systems for routine use in the clinical microbiology laboratory without substantial expansion of the respective data bases.

Comparison of the lysis centrifugation and radiometric blood culture systems for recovery of yeast.

The performance of the Isolator (lysis centrifugation) and Bactec (radiometric) detection systems for the recovery of fungi from blood was studied prospectively by comparison of 2,188 paired cultures obtained at two geographically separated teaching hospitals. Eight-three yeast isolates were recovered from 78 (3.6%) cultures that were obtained from 43 patients. Seventy-three (88%) yeast strains were recovered using the Isolator system, and 60 (72%) were recovered in the Bactec system. The average time for recovery of yeast was 2.3 days for the Isolator system and 3.1 days for the Bactec system. Optimal recovery can be accomplished through the use of both systems.

Immunoidentification of Histoplasma capsulatum and Blastomyces dermatitidis with commercial exoantigen reagents. Effect of culture age.

We have studied the effect of the length of incubation on the reliability of commercial exoantigen test reagents (Nolan/Scott Biological Laboratories Inc, and Immuno-Mycologics Inc). Antigen extracts, tested in each commercial system, were prepared from duplicate sets of cultures of Histoplasma capsulatum (12 isolates) and of Blastomyces dermatitidis (11 isolates) after one to six weeks of growth. For isolates of H capsulatum, antigens necessary for immunoidentification were detected in most cultures in two weeks and in all cultures after four weeks of incubation for both Nolan and Immuno-Mycologics reagents, and continued to be detected for at least six weeks. Positive results could usually be obtained from exoantigen tests for H capsulatum before dimorphism could be demonstrated by conversion of the mold to yeast. Diagnostically significant antigen was not uniformly present in all cultures of B dermatitidis during the test period, but ten of 11 isolates were positive at some time during the six weeks using the Nolan reagents; antigen of only one of 11 isolates was detected by the Immuno-Mycologic reagents. Results of exoantigen tests for B dermatitidis were usually not available until after the identity had been confirmed by conversion of the mold to yeast phase on cottonseed agar.

Use of cerebrospinal fluid lactic acid concentration in the diagnosis of fungal meningitis.

A patient with a several year history of normal pressure hydrocephalus was found to have an infection owing to Cryptococcus neoformans. Cryptococcal infection was not suspected until typical cells were observed in a Wright's stained smear of cerebrospinal fluid (CSF). A review of past medical findings in this patient showed elevated CSF values for lactic acid and protein. This case prompted us to review the use of lactic acid as an indicator of fungal meningitis and compare it to other more commonly used nonspecific indicators of fungal meningitis, notably the concentrations of glucose and protein, and the number of leukocytes in CSF. In our institution, all 10 culturally proven cases of fungal meningitis, for which the lactic acid concentration in the CSF was available, were found to have an elevated lactic acid concentration (range 3.2 to 13.3 mmol per L vs normal range 0.8 to 2.8 mmol per L). No other nonspecific indicator was elevated in all 10 patients. In view of the poor sensitivity of stained smear or wet preparations and cultures, when less than five ml of CSF are used for culture, an elevated lactic acid value in a patient with or without signs of meningitis should raise the suspicion of fungal infection.

Allergic fungal sinusitis due to Curvularia lunata.

The clinical and pathologic features of allergic fungal sinusitis caused by Curvularia lunata, as seen in two patients, are described. The findings are identical to those of allergic aspergillus sinusitis. Patients have allergies, nasal polyposis, and, occasionally, eosinophilia. Radiographs show opacification of multiple sinuses without bone destruction. Surgical specimens consist of polyps and inspissated, mucoid material. Diagnostic microscopic features, termed "allergic mucin," include eosinophils, numerous Charcot-Leyden crystals, and hyphae embedded in pools of mucus. The recognition of allergic mucin may be impeded by extensive degranulation and fragmentation of the eosinophils. In addition, the fragments of mucin exhibit large, poorly stained central areas, probably due to incomplete penetration by the fixative. Eosinophils are easier to recognize in well-fixed areas. Electron microscopy, though not of diagnostic necessity, confirms the eosinophilic nature of the infiltrate. It is important that surgical pathologists recognize this distinctive clinicopathologic entity and recommend appropriate cultures.

Pulmonary disease due to Mycobacterium malmoense.

A case of pulmonary disease due to Mycobacterium malmoense was recently diagnosed in a 43-year-old man from Virginia. This organism was isolated from sputum and bronchial washings. This is the first case of documented human infection due to this organism in the United States

An improved reagent for mycobacterial nitrate reductase tests.

A new crystalline reagent for nitrate reductase tests was compared with standard liquid reagents on 437 strains of mycobacteria. The results for isolates of Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae, Mycobacterium scrofulaceum, Mycobacterium fortuitum, and Mycobacterium chelonei agreed 100% with the expected results. Of the 177 Mycobacterium tuberculosis isolates, 4 were negative by the conventional method. Two of these four isolates were positive with the new reagent. Of the positive nitrate tests carried out with liquid reagents, 42% flashed instantly or faded in color; none of the tests carried out with the new crystalline reagent flashed or faded. A stronger color reaction was seen for 28% of the positive tests with the new reagent.