Proven Histoplasmosis Research Articles

Diagnostic accuracy of a novel lateral flow assay for histoplasmosis.

Antigen testing is an important diagnostic tool for histoplasmosis but has limited availability globally. We evaluated the OIDx urine lateral flow antigen assay among 204 persons suspected to have histoplasmosis. Among patients with proven histoplasmosis, sensitivity was 33.3% (3/9, 95% CI 7.5%-70.1%) and specificity 80.5% (157/195, 95% CI 74.3%-85.8%). The MiraVista urine antigen test had better specificity (96.9%) and equal sensitivity. The OIDx test demonstrated 33.3% (3/9) positive agreement and 84.0% (163/194) negative agreement with the MiraVista test. These results should be considered in the context of our low HIV prevalence population with a mixture of pulmonary and disseminated disease.

Application of Real-Time PCR Assays for the Diagnosis of Histoplasmosis in Human FFPE Tissues Using Three Molecular Targets.

Histoplasmosis is a fungal infection caused by the thermally dimorphic fungus Histoplasma capsulatum. This infection causes significant morbidity and mortality in people living with HIV/AIDS, especially in countries with limited resources. Currently used diagnostic tests rely on culture and serology but with some limitations. No molecular assays are commercially available and the results from different reports have been variable. We aimed to evaluate quantitative real-time PCR (qPCR) targeting three protein-coding genes of Histoplasma capsulatum (100-kDa, H and M antigens) for detection of this fungus in formalin-fixed paraffin-embedded (FFPE) samples from patients with proven histoplasmosis. The sensitivity of 100-kDa, H and M qPCR assays were 93.9%, 91% and 57%, respectively. The specificity of 100-kDa qPCR was 93% when compared against samples from patients with other mycoses and other infections, and 100% when samples from patients with non-infectious diseases were used as controls. Our findings demonstrate that real-time PCR assays targeting 100-kDa and H antigen showed the most reliable results and can be successfully used for diagnosing this mycosis when testing FFPE samples.

Comparison of Indirect Fungal Diagnostic Tests in Patients With Proven Histoplasmosis.

Histoplasmosis is a common cause of invasive fungal infection in endemic regions and accurate diagnosis is difficult without direct tissue culture or pathology. Indirect fungal antigen testing for various fungal pathogens are typically performed to assist with diagnostic workup, though cross-reaction can lead to difficulty in interpreting results. We aimed to compare indirect fungal diagnostic tests and evaluate prevalence of positive antigen testing for non-Histoplasma fungal pathogens in patients with proven histoplasmosis. We performed a single-center retrospective review of adult patients with proven histoplasmosis diagnosed by fungal culture and/or cytology from January 2010 to March 2018. Patient demographics, clinical characteristics, and results of fungal antigen testing for Histoplasma, Blastomyces, Aspergillus, Cryptococcus, and (1→3)-β-D-glucan were evaluated. Two different urine Histoplasma antigen assays were used during the study period. Fifty-seven of 182 (31.3%) patients reviewed had proven histoplasmosis and presented with acute pulmonary (n = 10), chronic pulmonary (n = 7), and disseminated (n = 40) disease. Forty-one (72%) of these patients were immunosuppressed. Urine Blastomyces antigen (93%) and serum (1→3)-β-D-glucan (88%) were commonly positive in patients with histoplasmosis, whereas Aspergillus antigen was detected in 50% of patients and Cryptococcus antigenemia was rare (5%). In patients with disseminated disease, the MiraVista urine Histoplasma antigen assay had higher sensitivity than the Viracor urine Histoplasma antigen assay (86% vs 50%, respectively; P = .019). Noninvasive fungal antigen assays are helpful diagnostic tools; however, given their low specificity, clinicians must be aware of the various clinical presentations of invasive fungal infections and be aware of the limitations of these tests.

Immunoproteomics Reveals Pathogen's Antigens Involved in Homo sapiens-Histoplasma capsulatum Interaction and Specific Linear B-Cell Epitopes in Histoplasmosis.

Histoplasmosis is one of the most frequent systemic mycosis in HIV patients. In these patients, histoplasmosis has high rates of morbidity/mortality if diagnosis and treatment are delayed. Despite its relevance, there is a paucity of information concerning the interaction between Histoplasma capsulatum and the human host, especially regarding the B-cell response, which has a direct impact on the diagnosis. Culture-based “gold-standard” methods have limitations, making immunodiagnostic tests an attractive option for clinical decisions. Despite the continuous development of those tests, improving serological parameters is necessary to make these methods efficient tools for definitive diagnosis of histoplasmosis. This includes the determination of more specific and immunogenic antigens to improve specificity and sensitivity of assays. In this study, we performed a co-immunoprecipitation assay between a protein extract from the yeast form of H. capsulatum and pooled sera from patients with proven histoplasmosis, followed by shotgun mass spectrometry identification of antigenic targets. Sera from patients with other pulmonary infections or from healthy individuals living in endemic areas of histoplasmosis were also assayed to determine potentially cross-reactive proteins. The primary structures of H. capsulatum immunoprecipitated proteins were evaluated using the DNAStar Protean 7.0 software. In parallel, the online epitope prediction server, BCPREDS, was used to complement the B-epitope prediction analysis. Our approach detected 132 reactive proteins to antibodies present in histoplasmosis patients’ sera. Among these antigens, 127 were recognized also by antibodies in heterologous patients’ and/or normal healthy donors’ sera. Therefore, the only three antigens specifically recognized by antibodies of histoplasmosis patients were mapped as potential antigenic targets: the M antigen, previously demonstrated in the diagnosis of histoplasmosis, and the catalase P and YPS-3 proteins, characterized as virulence factors of H. capsulatum, with antigenic properties still unclear. The other two proteins were fragments of the YPS-3 and M antigen. Overlapping results obtained from the two aforementioned bioinformatic tools, 16 regions from these three proteins are proposed as putative B-cell epitopes exclusive to H. capsulatum. These data reveal a new role for these proteins on H. capsulatum interactions with the immune system and indicate their possible use in new methods for the diagnosis of histoplasmosis.

A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r(2) of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

Clinical Presentation and Management of Histoplasmosis in Older Adults

To describe the influence of age on clinical features of histoplasmosis. Retrospective single-center cohort study. Large tertiary care center. All individuals who met criteria for probable or proven histoplasmosis between 1998 and 2008. Participants were divided into the following categories of histoplasmosis: acute pulmonary, chronic pulmonary, asymptomatic, disseminated, and other. Correcting for immune status, the influence of age at diagnosis on presentation, diagnosis, imaging, treatment, and all-cause mortality was evaluated. In 347 participants with histoplasmosis, a number of characteristics were associated with age when evaluating participants according to diagnostic category. An age-associated increase in asymptomatic histoplasmosis was observed (P<.001). In symptomatic pulmonary histoplasmosis, older adults were less likely to present with chest pain (P<.001) and less likely to have hilar lymphadenopathy on imaging (P=.04). Lower rates of seropositivity with older age were seen in asymptomatic (P=.04) but not other forms of histoplasmosis. Cavitary disease was associated with older age in chronic pulmonary histoplasmosis (P=.05). Treatment did not change with age. All-cause mortality at 6months was 4% and was associated with older age (P=.02). Although most studied characteristics of histoplasmosis were similar, notable age-related differences were present. Chronic cavitary disease and asymptomatic histoplasmosis were more common with older age. In acute histoplasmosis, the lack of chest pain and hilar lymphadenopathy may hinder diagnosis in older adults.

Histoplasmosis and Paracoccidioidomycosis in a Non‐Endemic Area: A Review of Cases and Diagnosis

Histoplasmosis and paracoccidioidomycosis (PCM) have increased in Spain in recent years, due firstly to the migration from endemic regions and secondly to travelers returning from these regions. In non-endemic areas, diagnosis of both diseases is hampered by the lack of experience, long silent periods, and the resemblance to other diseases such as tuberculosis and sarcoidosis. A total of 39 cases of imported histoplasmosis and 6 cases of PCM diagnosed in the Spanish Mycology Reference Laboratory since 2006 were analyzed. Microbiological diagnosis was performed using classical methods and also a specific real-time polymerase chain reaction (RT-PCR) assay for each microorganism. We had 9 cases of probable histoplasmosis in travelers and 30 cases in immigrants, 29 of whom were defined as proven. Paracoccidioidomycosis (PCM) cases were either immigrants or people who had lived for a long period of time in endemic regions, all of whom were classified as proven cases. Cultures showed a good sensitivity in detecting Histoplasma capsulatum in immigrants with proven histoplasmosis (73%); however, growth was very slow. The fungus was never recovered in traveler patients. Paracoccidioides brasiliensis was isolated in a culture only in one case of the proven PCM. Serological methods were not very reliable in immunocompromised patients with histoplasmosis (40%). A PCR-based technique for histoplasmosis detected 55.5% of the cases in travelers (probable cases) and 89% of the cases in immigrants (proven). The PCR method for PCM detected 100% of the cases. These kinds of mycoses are increasingly frequent in non-endemic areas, and newer and faster techniques should be used to reach an early diagnosis. The RT-PCR techniques developed appear to be sensitive, specific, and fast and could be helpful to detect those mycoses. However, it is also essential that physicians perform differential diagnosis in individuals coming from endemic areas.

Evaluación de una técnica de PCR cuantitativa para el diagnóstico clínico de la histoplasmosis importada

Evaluation of the usefulness of a quantitative real-time polymerase chain reaction-based (RT-PCR) technique for clinical diagnosis of histoplasmosis. Primers and probes were designed on the basis of sequences from the ITS regions of ribosomal DNA of 20 clinical strains of Histoplasma capsulatum. LightCycler procedures (Roche Applied Science) were used with probes marked by fluorescence resonance energy transfer (FRET). Reproducibility, sensitivity, and specificity were analyzed. In addition, an internal control was designed to identify false negative results by PCR inhibition. The RT-PCR assay was tested in 22 clinical samples from 14 patients with proven histoplasmosis. In addition, 30 samples from patients with febrile neutropenia or mycoses other than histoplasmosis, and from healthy volunteers were analyzed as controls. The limit of detection of the assay was 1 fg of genomic DNA per microl of sample. The PCR-based technique was reproducible and highly specific. Positive results were obtained in 11/14 (78.6%) patients and in 17/22 (77.3%) clinical samples. RT-PCR was positive in 100% of respiratory secretions and bone marrow samples, but only 70% of sera (p < 0.01). Mean fungal DNA value was 23.1 fg/microl in serum and 4.85 x 10(3) fg/microl in respiratory and bone marrow samples. RT-PCR results were positive in serum from three HIV patients for which antibody detection by immunodiffusion was negative. Specificity was 100%, since PCR results were negative for all the control samples. Thes RT-PCR technique is a sensitive, specific method for early diagnosis of histoplasmosis, particularly when respiratory secretions or bone marrow samples are analyzed. The reliability is lower in serum, but it can be used as an additional, complementary technique to culture and serology in HIV patients.

Detection of imported histoplasmosis in serum of HIV-infected patients using a real-time PCR-based assay

A new real-time PCR-based assay was used for detecting DNA of Histoplasma capsulatum in serum samples collected from four HIV-infected patients with proven histoplasmosis. The assay targeted the ITS1 region of rDNA and its in vitro sensitivity, specificity and reproducibility were evaluated. The technique detected DNA of H. capsulatum in all of the HIV-infected patients with proven histoplasmosis (4/4, 100%). The PCR result was positive for seven of the ten (70%) samples studied. The assay's specificity was determined to be 100%, since the method was negative for 25 other serum samples (10 from patients with proven aspergillosis and 15 from healthy controls). The PCR assay is a new and promising diagnostic alternative and further investigation is warranted.

Mucocutaneous Histoplasmosis

PRIMARY inoculation lesions can be caused by fungi that more commonly cause systemic mycotic disease. Such lesions have been shown with<i>Coccidioides immitis</i>^1,2and<i>Blastomyces dermatitidis</i>,³among others. Reports also appear in the literature of primary or localized infections of<i>Histoplasma capsulatum</i>occurring in the vagina,⁴eye,⁵and oropharynx,^6,7as well as on the external cutaneous surface in accidentally infected laboratory personnel.^8,9 A number of criteria have been proposed as critical in establishing the diagnosis of primary inoculation disease.^9,10Unfortunately, most of the suspected cases of such infection do not conform to all of these criteria; in particular, questions arise in regard to those cases allegedly involving the oropharynx.¹¹Most authors suggest that a case of proven histoplasmosis of the oral mucosa, despite an unusual appearance, should be regarded as an indication of systemic disease and approached as such therapeutically. We describe

Pulmonary histoplasmosis

Thirty-five patients with proven histoplasmosis and 9 patients with presumptive histoplasmosis were treated between 1st January 1957 and 31st July 1962. Four patients had acute pulmonary histoplasmosis, 21 chronic pulmonary histoplasmosis, 2 generalized disseminated disease, 3 nodular lesions in the lungs, and 2 histoplasmosis of thoracic lymph nodes. The disease of 3 patients was not classified because their X-rays could not be obtained. Four patients also had active pulmonary tuberculosis. The fungus was isolated from the sputum in 23 patients, from resected tissue in 9, from a lymph node in 1, at autopsy in 1, and from soil with which the patient had had contact in one. Complement fixation serologic tests were negative in almost half the patients on admission. Many patients later showed a significant rise. The positive tests were seen most often in patients with an exudative element to their disease. The yeast phase antigen gave more positive tests than the mycelial phase antigen. Eight patients had completely negative complement fixation tests during their entire hospitalization. Skin tests with histoplasmin were positive in 91 percent of our patients tested. Treatment consisted of bedrest only, in 6 patients, surgical measures in 8, and amphotericin in 21. Nine patients showed radiologic clearing and 7 fungus-free sputum even before amphotericin was begun. During amphotericin therapy, radiologic clearing occurred in 15 patients and sputum conversion in all the patients who were still positive except for one who died of generalized disseminated histoplasmosis shortly after the start of therapy. Most patients showed some evidence of amphotericin toxicity but in only two was this sufficiently severe to require cessation of therapy. One additional patient refused to continue the medication because of the symptoms. Two patients died during treatment, one of generalized disseminated histoplasmosis and tuberculosis, and the other of myocardial infarction.