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.     

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.     

Production and evaluation of reagents for detection of Histoplasma capsulatum antigenuria by enzyme immunoassay

The detection of urinary Histoplasma capsulatum polysaccharide antigen (HPA) by enzyme immunoassay (EIA) has proven useful for the presumptive diagnosis of histoplasmosis in AIDS patients. Assay limitations include (i) detection of a largely uncharacterized antigen and (ii) difficulty in reproducibly generating antibodies for use in the EIA. To improve antibody production for use in this test and to better understand the antigen being detected, we compared rabbit antibodies elicited using various immunization schedules, routes, and H. capsulatum-derived antigens. Antibodies were evaluated by EIA for their ability to detect purified H. capsulatum C antigen (C-Ag) and antigenuria. Reported as enzyme immunoassay (EI) units (the A(450) with antigen divided by the A(450) without antigen), results demonstrated that intravenous immunization of rabbits with whole, killed yeast-phase cells (yeast-i.v. regimen) produced antibodies giving the highest EI values in the C-Ag EIA (mean EI units +/- standard deviation, 14.9 +/- 0.6 versus 6.4 +/- 0.4 for rabbits immunized with C-Ag versus 2.4 +/- 0.3 for all other regimens combined). Yeast-i.v. antibodies were highly sensitive for the detection of antigenuria in patients with histoplasmosis, as shown by the following results: 12/12 patients compared to 10/12, 6/12, 3/12, and 3/12, respectively, for antibodies from rabbits immunized with (i) C-Ag; (ii) whole, killed yeast-phase cells administered subcutaneously and intramuscularly; (iii) yeast-phase culture filtrates; and (iv) HPA-positive urine. Rabbits immunized using the yeast-i.v. regimen also gave higher peak antibody titers than rabbits immunized by any other regimen (P < 0.03), and their antibodies were most comparable in reactivity to antibodies produced for use in the standard HPA-EIA test (P < 0.001). Therefore, rabbits immunized using the yeast-i.v. regimen produced the most sensitive antibodies with the highest titers for detection of C-Ag and antigenuria in histoplasmosis patients.     

Comparison of enzyme immunoassay with radioimmunoassay for the detection of hepatitis B surface antigen

A direct solid-phase enzyme immunoassay (Auszyme I) and a direct solid-phase radioimmunoassay (Austria II) for detection of hepatitis B surface antigen (HBsAg) were compared in tests with a panel of 347 human sera. Compared with RIA, EIA showed a sensitivity of 98% with 153 HBsAg-positive sera and a specificity of 99% with 194 HBsAg-negative sera. Sera that gave false negative and false positive results by EIA were re-examined by both RIA and EIA to confirm the initial result. Use of less than the recommended volume of serum for EIA produced results inconsistent with RIA in four of 27 sera examined. Quantitative correlation between RIA and EIA was low (r = 0.691). Positive controls used for EIA showed considerable variation from day to day, although intra-assay variation was much less. The sensitivity of the EIA method examined compares favourably with previously published EIA studies and with the RIA used in this study. Auszyme I EIA is a sensitive and specific third generation test for HBsAg that offers several advantages over currently used RIA techniques.     

Enzyme immunoassay and enzyme-linked fluoroimmunoassay for detection ofChlamydia trachomatis antigen

An enzyme immunoassay (EIA) and an enzymelinked fluoroimmunoassay (ELFIA) utilizing monoclonal antibody to major outer membrane protein of Chlamydia trachomatis L2 were developed for rapid detection of chlamydial antigen in clinical samples. The EIA and ELFIA could detect levels of purified chlamydial outer membrane protein as low as 1.0 and 0.2 ng/ml respectively. However, when EIA and ELFIA were compared to chlamydial isolation using 160 patient samples, the sensitivity rate was 68 % and 85% respectively. The sensitivity of the antigen detection method might be increased by simply using less diluted samples than in the present study. Chlamydial antigen was also demonstrated by EIA and ELFIA in 15% and 23% of culture-negative samples. The reason for these false-positive findings remains undetermined.     

Comparison of an established antibody sandwich method with an inhibition method of Histoplasma capsulatum antigen detection

The Histoplasma antigen immunoassay utilizes an antibody sandwich method that provides a rapid and reliable means of diagnosing the more severe forms of histoplasmosis. Inhibition assays have been developed for antigen detection and offer at least one potential advantage, namely, reduced antibody requirements. We have developed an inhibition assay using the polyclonal antibody employed in our standard sandwich assay. Urine and serum specimens from patients with culture-proven histoplasmosis and controls were tested using both methods. The two methods had similar sensitivities for detection of antigen in urine (antibody sandwich = 92.5% versus inhibition = 87.5%, P = 0.500) and serum (82.5% versus 80.0%, P = 1. 000). With serum, the specificities of both methods were similar (antibody sandwich assay = 95.0% versus inhibition assay = 92.5%, P = 1.000), and with urine, the specificity of the antibody sandwich method was superior (97.5% versus 80.0%, P = 0.039). While the overall reproducibility of both methods was excellent (with urine, antibody sandwich assay intraclass correlation coefficient = 0.9975 and with serum = 0.9949; correlation coefficient of the inhibition assay with urine = 0.9736 and with serum = 0.9850), that of the inhibition method was only fair to poor for the controls: urine = -0. 0152, serum = 0.5595. Reproducibility was good for the controls using the sandwich method: urine = 0.7717, serum = 0.9470. Cross-reactivity was observed in specimens from patients infected with Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Penicillium marneffei. In conclusion, the decreased specificity and inferior reproducibility with control specimens suggest that the inhibition assay has poorer precision toward the lower end of the detection range.     

A complementary tool for management of disseminated Histoplasma capsulatum var. capsulatum infections in AIDS patients

In South America, disseminated histoplasmosis due to Histoplasma capsulatum var. capsulatum (H. capsulatum), is a severe and frequent opportunistic infection in AIDS patients. In areas outside the USA where specific-Histoplasma antigen detection is not available, the diagnosis is difficult. With the galactomannan antigen (GM) detection, a test commonly used for invasive aspergillosis diagnosis, there is a cross-reactivity with H. capsulatum that can be helpful for the diagnosis of histoplasmosis. The aim of this study was to evaluate the GM detection for the diagnosis of disseminated histoplasmosis in AIDS patients. The performance of the GM detection was evaluated with serum collected in French Guiana where H. capsulatum is highly endemic. Sera from AIDS patients with disseminated histoplasmosis occurring from 2002 to 2009 and from control HIV-positive patients without histoplasmosis were tested with the GM detection and Histoplasma-specific antibody detection (IEP). In 39 AIDS patients with proven disseminated histoplasmosis, the sensitivity of the Histoplasma IEP was only 35.9% and was linked to the TCD4+ lymphocyte level. For the GM detection, the sensitivity (Se) was 76.9% and specificity (Sp) was 100% with the recommended threshold for aspergillosis diagnosis (0.5). The test was more efficient with a threshold of 0.4 (Se: 0.82 [95% CI: 0.66–0.92], Sp: 1.00 [95% CI: 0.86–1.00], LR+: >10, LR−: 0.18). This study confirms that the GM detection can be a surrogate marker for the diagnosis of disseminated histoplasmosis in AIDS patients in endemic areas where Histoplasma EIA is not available.     

Diagnosis of disseminated histoplasmosis by detection of Histoplasma capsulatum antigen in serum and urine specimens

The diagnosis of Histoplasma capsulatum infection by serologic testing for the presence of antibodies is limited by a high rate of false positive and false negative results and by the requirement that the patient have a normal immune response. We have developed a radioimmunoassay for the detection of H. capsulatum antigen in urine and serum specimens. Antigenuria was noted in 20 of 22 episodes of disseminated histoplasmosis that occurred in 16 patients, in 6 of 32 patients with self-limited infection, in 2 of 32 patients with cavitary histoplasmosis, and in 4 of 8 patients with a sarcoid-like illness caused by H. capsulatum. The detection of antigen in urine was reproducible in 38 of 41 (93 percent) retests of specimens. H. capsulatum antigen was also detected in the serum during 11 of the 22 episodes of disseminated histoplasmosis, in none of the 12 episodes of other types of histoplasmosis in patients with antigenuria, in 1 of the 33 patients with histoplasmosis who lacked the urinary antigen, and in none of the 50 controls. Antigenemia and antigenuria decreased after initiation of antifungal therapy and recurred in patients who had a relapse. We conclude that this radioimmunoassay for H. capsulatum antigen represents a useful new method for the rapid diagnosis of disseminated histoplasmosis.     

Pathogenic differences between North American and Latin American strains of Histoplasma capsulatum var. capsulatum in experimentally infected mice

Clinical differences in histoplasmosis between North America and Brazil prompted investigation of experimental infection with representative strains. Mortality was higher with Latin American strains, and lung pathology showed large necrotizing granuloma with prominent neutrophilic infiltration. Chronic disease was unique to the North American strain.     

Comparison of radioimmunoassay and enzyme immunoassay kits for detection of Legionella pneumophila serogroup 1 antigen in both concentrated and nonconcentrated urine samples.

We evaluated a commercial enzyme immunoassay (EIA) kit for detection of Legionella pneumophila serogroup 1 soluble antigen by comparing it to radioimmunoassay (RIA), using both concentrated and nonconcentrated urine samples. The sensitivity of EIA was 67.4% in nonconcentrated urine samples and 82.6% in concentrated urine samples. The sensitivity of RIA was 60.9% and 84.8% in nonconcentrated and concentrated urine samples, respectively. Our study indicates that the sensitivity and specificity of EIA are comparable to those of RIA, and that concentrating the antigen by selective ultrafiltration increases sensitivity for both EIA and RIA, with no significant decrease in specificity.     

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.     

Histoplasma capsulatum var. capsulatum occurring in an HIV-positive Ghanaian immigrant to Italy. Identification of H. capsulatum DNA by PCR from paraffin sample

Histoplasmosis, which is highly endemic in the United States, is rare in Europe, usually imported but sometimes autochthonous. In Africa, histoplasmosis capsulati coexists with "African histoplasmosis", a characteristic skin infection caused by H. capsulatum var. duboisii. Histoplamosis due to H. capsulatum is one of the 12 secondary infections listed in the surveillance definitions of AIDS. We report the case of a 36-year-old black man with acquired immunodeficiency syndrome (AIDS) who was living in Italy but originally came from Ghana. Histoplasmosis was disseminated with fever and cutaneous manifestations. The diagnosis was demonstrated morphologically based on the presence of yeast, observed by light microscopy, in skin lesions and by identification of H. capsulatum var. capsulatum DNA by nested PCR from a paraffin sample. No clinical reports of histoplamosis capsulati in Ghana have been published until now. The present case stresses the role of immigration of subjects from outside Europe who have been infected in their native country.     

Comparison of staining methods and a nested PCR assay to detect Histoplasma capsulatum in tissue sections

To optimize diagnosis of histoplasmosis in tissue sections, 30 spleen specimens from mice, experimentally infected with Histoplasma capsulatum, were examined by H&E, Grocott stain, anti-bacille Calmette-Guerin antibody immunostain, Fungiqual A fluorochrome stain (Drs Reinehr and Rembold, Kandern, Germany), and a nested polymerase chain reaction (PCR) assay. Results were compared with the tissue burden determined by quantitative culture. By applying logistic regression, the nested PCR assay was the most sensitive method, but not significantly more sensitive than the Grocott stain. The 50% quantile to achieve a positive result was determined to be 3 colony-forming units per milligram of spleen tissue for the PCR assay, 11 for the Grocott stain, 27 for the fluorochrome stain, 190 for immunostaining, and 533 for the H&E stain. The Grocott and fluorochrome stains did not differ significantly in detecting fungal elements. The PCR assay unambiguously identified H. capsulatum in tissue sections.     

Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces.

Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus, and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy.     

Comparison of the reversed passive hemagglutination with radioimmunoassay methods for hepatitis B antigen.

Radioimmunoassay for the detection of hepatitis B antigen has been accepted in many diagnostic laboratories now. The question of nonspecific positives has always been a matter of controversy. Two improved radioimmunoassay tests, namely Ausria II-125 by Abbott Laboratories and a radioimmunoassay method by Connaught Laboratories Limited (Hebria), were compared with the original Ausria 125I. Included in the comparison was the reversed passive hemagglutination test (Auscell, Abbott). Five hundred sera of clinical patients were tested. Fifty-five or 11% were found to have hepatitis B antigen. Three tests, Ausria 125I, Ausria II-125, and Hebria showed the same number of positive sera, whereas Auscell missed one. However, Ausria 125I indicated two additional false positives. Dilution experiments, however, indicated that Ausria II-125 and Hebria were the most sensitive tests, with the reversed passive hemaglutination showing the least sensitivity. Therefore, the new Ausria II-125 and the Hebria radioimmunoassay tests are preferable in view of their sensitivity and specificity.     

Molecular cloning and characterization of a recombinant Histoplasma capsulatum antigen for antibody-based diagnosis of human histoplasmosis.

Immunological cross-reactivity among fungi has hampered the development of specific serodiagnostic assays for histoplasmosis. We report the molecular cloning and characterization of a Histoplasma capsulatum cDNA (GH17) that encodes an antigen with immunodiagnostic potential. GH17 is an 810-bp cDNA which encodes a protein of 211 amino acid residues. The GH17 sequence has almost no significant homology with other sequences in GenBank. Southern blot analysis suggests that GH17 is confined to a single location in the genomic DNA of H. capsulatum. Immunoblots indicated that the protein product of GH17 (expressed as a 140-kDa beta-galactosidase fusion protein) was recognized by antibodies in 18 of 18 sera from histoplasmosis patients, but not by antibodies in sera from patients or animals infected with other fungi. GH17 was expressed in a prokaryotic expression vector, pPROEX-1, and recombinant protein was purified by preparative electrophoresis. Antibodies raised to this protein bound to a 60-kDa native antigen in immunoblots of H. capsulatum yeast antigen extract. These results suggest that GH17 encodes an H. capsulatum antigen that may be useful for the diagnosis of histoplasmosis in humans.     

Comparison of two enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen.

Two enzyme-linked immunosorbent assays (ELISAs) for herpes simplex virus (HSV) detection were compared with culture in a prospective, blinded study with 153 patients with suspected recurrent oral or genital HSV. A subset of 15 of these subjects were studied daily until symptom resolution during a single episode of recurrent HSV. Direct-site specimens were collected and either placed in viral transport media (for Ortho ELISA and fresh inoculation into primary rabbit kidney cells) or frozen in ELISA collection media (DuPont). One hundred eighty-six culture-ELISA comparisons were analyzed. On the basis of culture positivity, the DuPont and Ortho ELISAs differed substantially with regard to sensitivity (93 versus 35%) but had similar specificities (95 versus 100%) and positive (85 versus 100%) and negative (98 versus 85%) predictive values. There were seven DuPont ELISA-positive, culture-negative samples which were confirmed positive for HSV by blocking antibody test (revised specificity, 100%; positive predictive value, 100%). Six of these discrepant samples were from previously culture-positive subjects. These results demonstrate that currently available ELISA kits vary substantially as to their sensitivities in detecting HSV antigen from direct-site specimens. In addition, antigen detection, by ELISA technology, is not always synonymous with state of viral infectivity as judged by tissue culture cytopathic effect.     

Detection of Histoplasma capsulatum antigen in Panamanian patients with disseminated histoplasmosis and AIDS

Histoplasmosis is a common endemic mycosis in the Americas, often causing severe disease in patients with AIDS. Antigen detection has become an important method for rapid diagnosis of histoplasmosis in the United States but not in Central or South America. Isolates from patients in the United States are predominantly found to be class 2 isolates when typed using the nuclear gene YPS3, while isolates from Latin America are predominantly typed as class 5 or class 6. Whether infection with these Latin American genotypes produces positive results in the Histoplasma antigen assay has not been reported. In this study, we have compared the sensitivity of antigen detection for AIDS patients from Panama who had progressive disseminated histoplasmosis to that for those in the United States. Antigenuria was detected in the MVista Histoplasma antigen enzyme immunoassay (EIA) in 95.2% of Panamanian cases versus 100% of U.S. cases. Antigenemia was detected in 94.7% of the Panamanian cases versus 92% of the U.S. cases. Two clinical isolates from Panama were typed using YPS3 and were found to be restriction fragment length polymorphism class 6. We conclude that the MVista Histoplasma antigen EIA is a sensitive method for diagnosis of histoplasmosis in Panama.