Evaluation of a chemiluminescent probe assay for identification of Histoplasma capsulatum isolates.

Fifty-three of 54 isolates of fungi were correctly identified with an acridinium ester-labelled probe for Histoplasma capsulatum (Accuprobe; Gen-Probe, San Diego, Calif.). One isolate of Aspergillus niger was incorrectly identified as H. capsulatum. Age of the culture, medium for isolation, and morphologic state did not affect the results.     

Comparative evaluation of a chemiluminescent DNA probe and an exoantigen test for rapid identification of Histoplasma capsulatum.

A chemiluminescent DNA probe (Accuprobe) assay developed by Gen Probe, Inc., for the rapid identification of Histoplasma capsulatum was evaluated and compared with the exoantigen test by using 162 coded cultures including Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farciminosum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and morphologically related saprobic fungi. Each test uses a chemiluminescent, acridinium ester-labeled, single-stranded DNA probe that is complementary to the rRNA of the target organism. Lysates of the test cultures were prepared by sonication with glass beads and heat treated. After the rRNA was released from the target organism, the labeled DNA probe combined with the target H. capsulatum rRNA to form a stable DNA-RNA hybrid. A hybridization protection assay was used, and the chemiluminescence of hybrids was measured initially with a Leader 1 luminometer as relative light units and later during the investigation with a probe assay luminometer as probe light units. Of the 162 coded mycelial cultures tested by the Accuprobe assay, 105 were identified as H. capsulatum. The test could be performed with an inoculum of a few square millimeters (1 to 2 mm2) of growth. In the primary evaluation, the Accuprobe identified 103 of the 105 cultures as H. capsulatum within 2 h. The remaining two cultures, contaminated with bacteria, had to be purified before the Accuprobe assay identified them correctly as H. capsulatum. Since each coded culture was concurrently tested for H. capsulatum, B. dermatitidis, and C. immitis exoantigens, the identification of all three dimorphic pathogens was provided simultaneously. Of the 162 coded cultures tested, 105 were identified by the exoantigen test as H. capsulatum, 12 were identified as B. dermatitidis, 13 were identified as C. immitis, and 32 were negative for H. capsulatum, B. dermatitidis, and C. immitis. The bacterial contamination in two isolates did not interfere with the exoantigen testing. The exoantigen test required 7- to 10-day-old colonies and required 48 to 72 h of incubation before definitive identification was obtained.     

Histoplasma capsulatum sinusitis.

Sinusitis is commonly reported in patients with AIDS. In addition to the usual bacterial pathogens isolated from immunocompetent patients, sinusitis in patients with AIDS may be caused by a variety of unusual bacteria, viruses, fungi, parasites, and mycobacteria. Histoplasma capsulatum has not typically been associated with sinusitis in either group of patients. We report a case of sinusitis caused by H. capsulatum in a patient with AIDS.     

Hydroxamate siderophores of Histoplasma capsulatum

The zoopathogenic fungus Histoplasma capsulatum, like other eukaryotic aerobic microorganisms, requires iron for growth. Under conditions of low iron availability, the fungus secretes hydroxamates that function as siderophores (iron chelators). The experiments to be reported were designed to gather further information on the hydroxamate siderophores of H. capsulatum. The fungus was grown in a synthetic medium deferrated with the cationic exchange resin Chelex 100. Siderophores were detected after 4 days of incubation at 37 degrees C in media containing 0.3 to 1.0 microM iron. The secretion was suppressed by 10 microM iron. The hydroxamates were purified by reverse-phase and size-exclusion chromatography. On the basis of ions observed during electrospray mass spectroscopy, five hydroxamate siderophores were tentatively identified: dimerum acid, acetyl dimerum acid, coprogen B, methyl coprogen B, and fusarinine (monomeric). A polyclonal antibody to dimerum acid was generated. This reagent cross-reacted with coprogen B and fusarinine. Thus, the antibody detects hydroxamates in all three families of siderophores excreted by H. capsulatum.     

Improved version of the exoantigen test for identification of Coccidioides immitis and Histoplasma capsulatum cultures.

A rapid and simple method for extracting specific cell-free antigens of Coccidioides immitis and Histoplasma capsulatum from agar slant cultures was developed. The extracts were analyzed in immunodiffusion tests for the presence of C. immitis or H. capsulatum specific antigens. These extracts were compared with culture filtrates of brain heart infusion broth subcultures in tests with 32 isolates of C. immitis and H. capsulatum and 13 other fungi which might be morphologically confused with them. The studies showed that the slant extracts were as useful as the culture filtrates and allowed more rapid identification of C. immitis and H. capsulatum. In every case, when an identification was made by conventional morphological methods, the immunological test yielded correlating results. The immunological tests were positive for two isolates that were converted to their yeast forms only after 4 months of study by conventional tests. The new procedure permits the identification of C. immitis and H. capsulatum 2 days after receipt of a pure mycelial-form culture. The test is recommended for the presumptive identification of C. immitis and H. capsulatum cultures.     

A serendipitous isolate of Histoplasma capsulatum

This is a report of an unexpected laboratory diagnosis of Histoplasma capsulatum. The fungus was isolated from an acute cellulitic lesion on the forearm of an elderly male patient with a functioning renal transplant. The patient resides within the environs of Brisbane and has not travelled outside Australia. We consider the isolation of H. capsulatum from a rare site in a patient resident in a non-endemic area indicative of a latent opportunistic infection in an immunocompromised patient.     

An aberrant variant of Histoplasma capsulatum var. capsulatum.

We studied an aberrant culture of Histoplasma capsulatum var. capsulatum isolated from synovial fluid collected from the right elbow of a patient from Kansas. Colonies on Sabouraud glucose agar and other routine mycological media were glabrous to soft, moist, heaped, deeply folded or convoluted, and orange-brown with a white, irregular margin. Microscopically, hyphae were hyaline, septate, and branched and remained totally devoid of conidiation over a period of 2 years on all mycological media. Conversion to the yeast form was achieved on Pine's medium at 37 degrees C. Colonies at early stages of growth were smooth, moist, pasty, shiny, and orange-brown but soon became wrinkled and slightly raised and produced oval, thin-walled cells measuring 2 to 3 by to 4.5 microns which multiplied by polar budding. The identity of the isolate was further confirmed by utilizing the Accuprobe DNA and the exoantigen test for H. capsulatum var. capsulatum.     

Fosmid-based physical mapping of the Histoplasma capsulatum genome

A fosmid library representing 10-fold coverage of the Histoplasma capsulatum G217B genome was used to construct a restriction-based physical map. The data obtained from three restriction endonuclease fingerprints, generated from each clone using BamHI, HindIII, and PstI endonucleases, were combined and used in FPC for automatic and manual contig assembly builds. Concomitantly, a whole-genome shotgun (WGS) sequencing of paired-end reads from plasmids and fosmids were assembled with PCAP, providing a predicted genome size of up to 43.5 Mbp and 17% repetitive DNA. Fosmid paired-end sequences in the WGS assembly provide anchoring information to the physical map and result in joining of existing physical map contigs into 84 clusters containing 9551 fosmid clones. Here, we detail mapping the Histoplasma capsulatum genome comprehensively in fosmids, resulting in an efficient paradigm for de novo sequencing that uses a map-assisted whole genome shotgun approach.     

Studies on the catalase of Histoplasma capsulatum.

Factors which control the levels of catalase within yeast cells of Histoplasma capsulatum were studied. Only a small fraction of the total catalase activity could be detected in whole cells. The bulk of the activity was revealed in cell-free extracts or in cells permeabilized with acetone. The formation of the enzyme was regulated by glucose and by oxygen. There were large, consistent differences in the levels of catalase among strains of H. capsulatum. The sensitivity of the strains to H2O2 toxicity also varied remarkably. Peroxidase activity could not be detected in cell-free extracts of the strains. Resistance to H2O2 did not correspond to levels of catalase. There was no obvious correlation of H2O2 sensitivity and virulence among the strains.     

Immunological studies on Histoplasma capsulatum.

Alveolar macrophages freshly harvested from normal and immunized rabbits were parasitized with yeast cells and protoplasts of Histoplasma capsulatum. Macrophages obtained from either normal or sensitized rabbits failed to phagocytize protoplasts, whereas, the yeast cells were actively ingested. There was no detectable intracellular killing by macrophages. A serological similarity was found between the whole yeast cell, the purified isolated cell wall, and the protoplasts of the fungus. Aprecipitin test of the protoplasts of the fungus gave a postive band, whereas immunodiffusion in agar was negative. Addition of immune sera activated phagocytosis, the immune sera against cell walls being the most active.     

Evaluation of two nested PCR assays for detection of Histoplasma capsulatum DNA in human tissue

In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.     

Morphogenesis and pathogenicity of Histoplasma capsulatum.

The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma capsulatum, G184A and G222B, when the temperature of incubation was raised to 37 degrees C, and the block persisted even after the cultures were washed free of PCMS. Instead of transforming to yeast cells, PCMS-treated mycelia continued to grow as mycelia at the elevated temperatures. A less virulent strain (Downs) was more temperature sensitive, but it showed a similar irreversible effect at 34 degrees C. Therefore, the mycelium-to-yeast transition of H. capsulatum is not required for the adaptation of mycelia to elevated temperatures but probably results from the temperature-dependent activation of yeast-specific genes. The transition to yeast is inferred to be obligate for pathogenicity in mice because PCMS-treated mycelia failed to cause infection, and no fungi were seen in tissues after PCMS-treated mycelia were injected into mice.     

Molecular detection of Histoplasma capsulatum var. capsulatum in human clinical samples

We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.     

Histoplasma capsulatum infection in nude mice.

Congenitally athymic nude (nu/nu) mice, when injected intraperitoneally with Histoplasma capsulatum, developed a rapidly fatal disseminated infection characterized by heavy parasitization of reticuloendothelial tissues. In contrast, their heterozygous (nu/X) littermates, which possessed a functioning thymus, developed only a low-grade infection which was apparently self-limited and rarely fatal. Transplantation of thymic tissue into nu/nu mice diminished greatly the severity of infection and reduced mortality by about 50%. These studies emphasize the importance of cell-mediated immunity in host defense against histoplasmosis and suggest that the nude mouse may be a valuable model for the study of this chronic intracellular infection.     

Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples

Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the organism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n = 6) collected from HIV-positive individuals with culture- and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n = 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was ≤6 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of antigen-positive urine specimens (4/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. capsulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to detect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted.     

DNA probe for identification of Streptococcus pneumoniae

A total of 287 clinical isolates of Streptococcus pneumoniae (pneumococcus) were tested for their ability to undergo autolysis when treated with sodium deoxycholate. The test was positive for all but one isolate, strain DOC-1. This autolysis required the activity of an enzyme which is unique and characteristic of S. pneumoniae: a choline-dependent N-acetylmuramoyl-L-alanine amidase, the gene product of the lytA gene. We used lytA as a DNA probe to test the distribution of the autolysin gene among clinical isolates of S. pneumoniae. In dot blot hybridization experiments our probe reacted with the DNA of 60 of 60 strains tested, including the autolysis-deficient clinical isolate DOC-1. No hybridization occurred when strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus pyogenes, Streptococcus (Enterococcus) faecalis, Streptococcus (Enterococcus) faecium, Streptococcus agalactiae, and Streptococcus bovis were tested. The lytA gene appears to be an ideal candidate for use as a DNA probe for the identification of S. pneumoniae.