Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

BackgroundA multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program.AimsThe objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA.MethodsSeven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets.ResultsMost of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10² fg/μl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR₂₂₀) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol.ConclusionsAll laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.     

Rapid Identification of Histoplasma capsulatum Directly from Cultures by Multiplex PCR

The multiplex PCR developed from a suspension of the yeast fungi correctly identified fifty-one clinical of H. capsulatum var. capsulatum strains isolated from clinical samples and soil specimens. The multiplex PCR was developed by combining two pairs of primers, one of them was specific to the H. capsulatum and the other one, universal for fungi, turned out to be specific to H. capsulatum, regardless of the fungus isolate studied. Primers designed to amplify a region of about 390-bp (Hc I–Hc II) and a region of approximately 600-bp (ITS1–ITS4) were used to identify a yeast isolated as H. capsulatum when both regions could be amplified. Absolute agreement (100?% sensitivity) could be shown between this assay and the cultures of H. capsulatum according to their morphological characteristics. Failure to amplify the target DNA sequence by PCR with primers Hc I–Hc II in the presence of the ITS1–ITS4 amplicon in isolates of P. brasiliensis, Cryptococcus neoformans, Trichosporon spp, Candida glabrata, C. albicans, C. tropicalis, C. parapsilosis, C. krusei, or Penicillium marneffei was an unequivocal sign of the high specificity of this assay. The assay specificity was also found to be 100?%. Incipient yeast forms obtained from clinical samples were identified as H. capsulatum by the PCR assay described before the morphological characteristics were registered shortening the time of diagnosis.     

Usefulness of molecular markers in the diagnosis of occupational and recreational histoplasmosis outbreaks

Histoplasmosis is considered the most important systemic mycosis in Mexico, and its diagnosis requires fast and reliable methodologies. The present study evaluated the usefulness of PCR using Hcp100 and 1281–1283₍₂₂₀₎ molecular markers in detecting Histoplasma capsulatum in occupational and recreational outbreaks. Seven clinical serum samples of infected individuals from three different histoplasmosis outbreaks were processed by enzyme-linked immunosorbent assay (ELISA) to titre anti-H. capsulatum antibodies and to extract DNA. Fourteen environmental samples were also processed for H. capsulatum isolation and DNA extraction. Both clinical and environmental DNA samples were analysed by PCR with Hcp100 and 1281–1283₍₂₂₀₎ markers. Antibodies to H. capsulatum were detected by ELISA in all serum samples using specific antigens, and in six of these samples, the PCR products of both molecular markers were amplified. Four environmental samples amplified one of the two markers, but only one sample amplified both markers and an isolate of H. capsulatum was cultured from this sample. All PCR products were sequenced, and the sequences for each marker were analysed using the Basic Local Alignment Search Tool (BLASTn), which revealed 95–98 and 98–100 % similarities with the reference sequences deposited in the GenBank for Hcp100 and 1281–1283₍₂₂₀₎, respectively. Both molecular markers proved to be useful in studying histoplasmosis outbreaks because they are matched for pathogen detection in either clinical or environmental samples.     

Detection of Environmental Sources of Histoplasma capsulatum in Chiang Mai, Thailand, by Nested PCR

Histoplasmosis is a systemic mycosis caused by inhaling spores of Histoplasma capsulatum, a dimorphic fungus. This fungus grows in soil contaminated with bat and avian excreta. Each year, patients with disseminated histoplasmosis have been diagnosed in Chiang Mai, northern Thailand. No published information is currently available on the environmental sources of this fungus in Chiang Mai or anywhere else in Thailand. The aim of this study was to detect H. capsulatum in soil samples contaminated with bat guano and avian droppings by nested PCR. Two hundred and sixty-five samples were collected from the following three sources: soil contaminated with bat guano, 88 samples; soil contaminated with bird droppings, 86 samples; and soil contaminated with chicken droppings, 91 samples. Genomic DNA was directly extracted from each sample, and H. capsulatum was detected by nested PCR using a primer set specific to a gene encoding 100-kDa-like protein (HcI, HcII and HcIII, HcIV). Histoplasma capsulatum was detected in seven of 88 soil samples contaminated with bat guano, one of 21 soil samples contaminated with pigeon droppings and 10 of 91 soil samples contaminated with chicken droppings. The results indicate the possibility of the association of bat guano and chicken droppings with H. capsulatum in this area of Thailand.     

Molecular detection of Histoplasma capsulatum indoors: A public health approach

BackgroundHistoplasmosis, caused by the dimorphic fungus Histoplasma capsulatum, represents an important public health problem, especially in urban environments where bats and humans cohabit indoors.AimsTo detect the presence of H. capsulatum indoors, using samples of bat droppings collected in roost sites inside houses.MethodsA Real-Time TaqMan PCR assay targeting the ITS1 region of the ribosomal DNA of H. capsulatum was carried out.ResultsFifty-nine sampling points in the municipality of São Paulo were inspected, all of them located at inhabited places. H. capsulatum was isolated from nine samples.ConclusionsThe rapid identification and monitoring of sites where the fungus is present may contribute to make a more reliable database of H. capsulatum distribution.     

Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Background

Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility.

Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum

Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited.Histoplasmosis is a serious community-acquired infection in the United States (28) and in certain countries of Latin America, where it is an especially significant problem in patients with AIDS (14). This disease is one of the most common systemic mycoses in Brazil, where epidemiological surveys carried out using the histoplasmin skin test have indicated that it is endemic in all areas surveyed (15). Data suggest that the numbers of cases of histoplasmosis in Brazil may be underestimated and that the areas where it is endemic are more widespread than previously thought.Histoplasma capsulatum is a dimorphic fungus that grows as a mold and produces aerial hyphae at 25 to 30°C, but it undergoes morphogenesis to a yeast phase at 37°C. The filamentous phase of this organism is usually found in soil enriched with several compounds, such as nitrogen and phosphate compounds. When conidial or hyphal fragments are inhaled by humans or animals, H. capsulatum changes to the yeast form and continues to replicate as a yeast. Although H. capsulatum has been recognized as an important fungal pathogen in immunocompromised hosts, particularly AIDS patients (27), there are significant gaps in our knowledge of this species'' epidemiology and pathogenesis. For instance, systemic histoplasmosis has been found in patients with AIDS who do not reside in regions where it is endemic (29), leading to the suggestion that the disease can result from reactivation of a previously acquired H. capsulatum infection. The clinical manifestations of histoplasmosis range from asymptomatic infections, mild flu-like symptoms, or pneumonia to a systemic disease involving the skin, brain, intestine, adrenal glands, and/or bone marrow (6). Importantly, diverse strains of H. capsulatum have been identified worldwide, and the strains vary in virulence. In addition to classical biochemical assays, distinctions between strains may be based on colony morphology or polymorphism of the genome (19).Multiple typing methods have been developed to study the epidemiology of H. capsulatum. These methods are based on phenotypic characteristics, such as antigenic profiles (13) and multilocus enzyme electrophoresis results (2), or on DNA-based analysis. Most recently, typing has been accomplished by analysis of fatty acid profiles of H. capsulatum (34). Molecular typing methods are generally considered to have advantages over phenotypic methods in terms of the stability of genomic markers and greater levels of typeability. Several genotype-based methods, such as hybridization of target genes (probes), chromosomal DNA typing, restriction fragment length polymorphism (RFLP) analysis, random amplified polymorphic DNA (RAPD) analysis, and sequencing, have been described for H. capsulatum (4, 5, 7, 8, 11, 17, 19). Despite the abundance of previously developed molecular techniques, there is limited information concerning comparisons of the results obtained with different methods using the same set of isolates. In H. capsulatum, no single approach based on DNA assays has been the dominant method.The current study was done to explore the diversity of H. capsulatum in Brazil and to determine the correlation between the results of three different molecular typing techniques. For this analysis, we used M13 PCR fingerprinting, PCR-RFLP analysis of the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the rDNA gene, and analysis of the nucleotide sequence polymorphism of four partial genes. M13 PCR fingerprinting (25) is based on generation of multiple PCR products with different electrophoretic mobilities. PCR fingerprinting primers are typically designed using repetitive DNA sequences (31), and the products facilitate detection of two types of genetic variations: (i) differences in the length of DNA and (ii) alterations in the sequence of the priming regions. PCR-RFLP analysis of the ITS1-5.8S-ITS2 region of the rDNA gene (9) involves use of a gene-specific PCR in combination with restriction digestion in order to generate highly stable and reproducible markers. Analysis of the nucleotide sequence polymorphism is based on the sequences of four partial protein-encoding genes (Arf, the H-anti gene, Ole, Tub1) (4). Additionally, to assess the utility of an assay to study the global epidemiology of the fungus, we performed a DNA sequencing analysis of the ITS1-5.8S-ITS2 region to compare the Brazilian H. capsulatum ITS1-5.8S-ITS2 sequences with sequences obtained for H. capsulatum strains isolated in other countries.     

Relevant aspects of the Hcp100 molecular marker of Histoplasma capsulatum and its potential therapeutic use in histoplasmosis

BackgroundFungal pathogens have developed strategies, involving genes expression that favors their persistence and multiplication in the host. The absence of molecules encoded by these genes could interfere with the growth and death of these fungi. In the past, a coactivator protein coding gene (Hcp100) of the fungus Histoplasma capsulatum was reported, which is overexpressed after 1 h of contact between fungal yeast-cells and murine macrophages. The product of this gene, a protein of 100 kDa (Hcp100) of H. capsulatum, is probably a regulatory protein involved in the processes required for fungal adaptation and its survival in the intracellular hostile conditions of the macrophages. A 210-bp fragment of the Hcp100 marker has proved to be an excellent tool for H. capsulatum molecular detection in clinical samples. The potential use of this gene as a therapeutic target in Plasmodium falciparum has been explored through the inhibition of both, the gene and the protein p100 of the parasite, by blocking its growth.MethodsBased on the above mentioned antecedents, we believe that the Hcp100 has an important role in the development and maintenance of the H. capsulatum yeast cells within macrophages.Results and conclusionsTo study the probable function of Hcp100 in the yeast-phase of this fungal pathogen is relevant to understand its activity and to propose it as a therapeutic target for histoplasmosis treatment.     

Histoplasma capsulatum in the environment of sporadic histoplasmosis cases

Conclusions 1. Acute pulmonary histoplasmosis in adults is demonstrated to be associated with exogenous sources of infection in one-half of a series of cases. 2. No exogenous sources of infection were found in chronic pulmonary histoplasmosis in adults by methods comparable to those used in acute pulmonary histoplasmosis. 3. The significance of these findings for the pathogenesis of the three clinical forms of histoplasmosis is discussed. 4. In acute pulmonary histoplasmosis in adults, the isolation ofH. capsulatum from the environment at sites of exposure aided in specific diagnosis.Two acute pulmonary histoplasmosis cases were reported previously (2–3).     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

Histoplasmosis in Africa: Current perspectives,knowledge gaps,and research priorities

BackgroundHistoplasmosis is a chronic granulomatous disease caused by the thermally dimorphic fungus Histoplasma capsulatum. The 2 variants Histoplasma capsulatum var. capsulatum (Hcc) and Histoplasma capsulatum var. duboisii (Hcd) causes infection in humans and commonly termed classical or American histoplasmosis and African histoplasmosis, respectively. Histoplasma capsulatum var. farciminosum (Hcf) affects equines. In recent times, there have been heightened sensitization on fungal infections such as histoplasmosis in Africa, aimed at improving awareness among relevant stakeholders, particularly healthcare workers. This effort is expected to be paralleled with increased detection of both classical and African histoplasmosis, which has remained underdiagnosed over the years. In this narrative review, we describe the current perspectives of histoplasmosis in Africa, identify knowledge gaps, and suggest research priorities.MethodsA PubMed, Google Scholar, and Africa Journal Online (AJOL) literature search was conducted for studies on histoplasmosis in Africa between 2000 and 2020. Histoplasmosis essays in medical mycology textbooks were also consulted. This narrative review was prepared from the data gathered.FindingsIn the past 2 decades, histoplasmosis in general has seen a relative increase in case detection in some Africa countries, probably attributable to the gradually increasing medical mycology advocacy efforts in Africa. Histoplasmosis cases are dominated by African histoplasmosis mostly in Western and Central Africa, while classical histoplasmosis is more common in Southern and Northern Africa. Although both classical and African histoplasmosis are common in Africa, the latter is more restricted to Africa, and cases outside the continent usually have a travel history to the continent. Despite the clinical and laboratory difference between African histoplasmosis and classical histoplasmosis, it is not straightforward to distinguish them. The typical manifestation of African histoplasmosis is the appearance of lesions affecting the skin, bones, and lymph nodes and unusually linked to human immunodeficiency virus (HIV)/AIDS. By contrast, classical histoplasmosis mostly affects the lungs and is often associated with immunosuppression, mainly HIV/AIDS. The present perspectives of histoplasmosis in Africa highlight unclear details on the true burden, strain diversity, infection route and genetic basis of African histoplasmosis, availability of specie-specific diagnostic tools, and compliance with recommended antifungal therapy. These knowledge gaps represent research questions that require scientific exploration.ConclusionsDespite a subtle increase in identifying histoplasmosis cases in Africa, it remains underdiagnosed and neglected in some parts of the continent. Increasing awareness and training among healthcare workers, bridging diagnostic and therapeutic gaps, and encouraging more research in Africa are crucial to improve the current perspectives of histoplasmosis in Africa.     

Validation of the isothermal Schistosoma haematobium Recombinase Polymerase Amplification (RPA) assay,coupled with simplified sample preparation,for diagnosing female genital schistosomiasis using cervicovaginal lavage and vaginal self-swab samples

BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (QIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings.     

Agentes de micosis endémicas en un área rural de Argentina: estudio seroepidemiológico en perros

BackgroundThree fungal species causing human disease, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides sp., are endemic in different areas of Argentina. Rates of infection in domestic dogs have been used in other Latin American countries as indicators of the presence of these pathogens in a given area. We used such an approach to investigate the epidemiological relevance of paracoccidiodomycosis, histoplasmosis and coccidioidomycosis in our country.AimTo investigate the presence of P. brasiliensis, H. capsulatum and Coccidioides sp. in a rural area of Argentina called Interfluvio Teuco-Bermejito, located in Chaco province.MethodsWe applied Western Blotting to determine the presence of specific antibodies in sera from 89 domestic dogs inhabiting the area. Antibodies against the following extra-cellular fungal antigens were investigated: gP43 of P. brasiliensis, H/M of H. capsulatum and 120, 82 and 48 kDa antigen bands of Coccidioides sp.ResultsSpecific antibodies against H. capsulatum were found in 9/89 (10%) sera: 8 reacted against both H and M antigens and 1 only reacted against antigen M. Of these 9 sera, one showed additional anti-gp43 activity and another reacted against all the fungal antigens tested.ConclusionsThis is the first study using dog infection to assess the presence of endemic fungal pathogens in Argentina. Our results suggest that H. capsulatum is the main dimorphic fungal pathogen in the Interfluvio Teuco-Bermejito area. Therefore, the diagnosis of histoplasmosis should be taken into account in patients living in this geographic region who show pulmonary or mucocutaneous symptoms compatible with the disease.     

Fluorescent antibody techniques for identification of Coccidioides immitis and Histoplasma capsulatum

This is a review of practical uses of immunofluorescence in detection of the two fungi in host and environment and in identification of their cultures, as well as in serologic case finding. Reagents directed at the yeast phase ofHistoplasma capsulatum have been fairly successful in differentiating this species from others, the main difficulty being the tendency to cross-react withBlastomyces dermatitidis andH. duboisii. Conjugates for the mycelial phase ofH. capsulatum tend to cross-react withSepedonium andChrysosporium, but careful absorption may yield specific reagents. Anti-yeast-phase conjugates are a valuable adjunct to cultural and clinical methods when used to detect and identifyH. capsulatum in sputum and other clinical specimens. Conjugates specific for the spherules or tissue phase ofCoccidioides immitis have yielded false negative results when applied to clinical specimens. The fluorescent-inhibition procedure is useful for serologic case finding in histoplasmosis and the same technique has shown fairly good agreement in coccidioidomycosis with complement-fixation and tube-precipitin methods. Immunofluorescence reagents for the two species have been useful in screening surgical and autopsy specimens, animal tissues, and soils.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

High prevalence and mortality due to Histoplasma capsulatum in the Brazilian Amazon: An autopsy study

BackgroundHistoplasmosis is acquired by inhalation of spores of the dimorphic fungus Histoplasma spp. Although this pathogen is distributed worldwide, it is more prevalent in the Americas. However, the real burden of histoplasmosis remains undefined in many endemic regions.MethodologyWe conducted a series of 61 autopsies to individuals who died in a hospital in the Brazilian Amazon focused on infectious diseases. We performed a detailed histological and microbiological evaluation with genetic characterization of Histoplasma strains with the aim to evaluate the contribution of histoplasmosis to morbidity and mortality. Additionally, we assessed the clinicopathological correlation.Principal findingsEvidence of Histoplasma infection was detected in 21 patients (34%). Eight cases were disseminated infections, all of them occurred in HIV-positive patients. Six cases were localized histoplasmosis, limited to the lungs. In seven patients Histoplasma DNA was detected by PCR in patients with no histological lesions. Histoplasma infection was detected in 38% of HIV-positive patients and was a major contributor to death in 22% of them. Lungs, liver and spleen were affected in all cases of disseminated histoplasmosis. Phylogenetic analysis of the strains suggested a high diversity of Histoplasma species circulating in the Brazilian Amazon. Histoplasmosis was clinically missed in 75% of the disseminated infections.ConclusionsThe high incidence of histoplasmosis, the low index of clinical suspicion, and the severity of the disseminated disease highlight the need of proactively implementing sensitive routine screening methods for this pathogen in endemic areas. Antifungal prophylaxis against Histoplasma should be encouraged in the severely immunocompromised HIV patients in these areas. In conclusion, substantial mortality is associated with disseminated histoplasmosis among HIV-positive patients in the Brazilian Amazon.     

Histoplasmosis in the Republic of Congo dominated by African histoplasmosis,Histoplasma capsulatum var. duboisii

The Republic of Congo (RoC) is one of the African countries with the most histoplasmosis cases reported. This review summarizes the current status regarding epidemiology, diagnostic tools, and treatment of histoplasmosis in the RoC. A computerized search was performed from online databases Medline, PubMed, HINARI, and Google Scholar to collect literature on histoplasmosis in the RoC. We found 57 cases of histoplasmosis diagnosed between 1954 and 2019, corresponding to an incidence rate of 1–3 cases each year without significant impact of the AIDS epidemic in the country. Of the 57 cases, 54 (94.7%) were cases of Histoplasma capsulatum var. duboisii (Hcd) infection, African histoplasmosis. Three cases (5.3%) of Histoplasma capsulatum var. capsulatum infection were recorded, but all were acquired outside in the RoC. The patients’ ages ranged between 13 months to 60 years. An equal number of cases were observed in adults in the third or fourth decades (n = 14; 24.6%) and in children aged ≤15 years. Skin lesions (46.3%), lymph nodes (37%), and bone lesions (26%) were the most frequent clinical presentations. Most diagnoses were based on histopathology and distinctive large yeast forms seen in tissue. Amphotericin B (AmB) was first line therapy in 65% of the cases and itraconazole (25%) for maintenance therapy. The occurrence of African histoplasmosis in apparently normal children raises the possibility that African histoplasmosis is linked to environmental fungal exposure.     

Histoplasmosis en un varón inmunocompetente manifestada 45 años después de la infección

We report a case of a lung reactivation of a latent histoplasmosis in a 68 year-old patient without immunologic dysfunction living in Barcelona (Spain).The Histoplasma capsulatum var. capsulatum infection was probably acquired in a previous stay in Equatorial Guinea 48 years before.Diagnosis of mild chronic pulmonary histoplasmosis was performed by histopathology of lung biopsy plus antibodies detection against H. capsulatum. Treatment with oral itraconazol during three months leaded to a complete clinical cure, and antibodies disappeared after four years of follow up.     

A natural focus ofHistoplasma capsulatum var.duboisii is a bat cave

The natural reservoir ofHistoplasma capsulatum var.duboisii, the etiological agent of histoplasmosis duboisii (African histoplasmosis) is not yet known. We report the isolation ofH. capsulatum var.duboisii from soil admixed with bat guano and from the intestinal contents of a bat in a sandstone cave in a rural area, Ogbunike in Anambra State of Nigeria. Eight of 45 samples of soil admixed with bat guano yieldedH. capsulatum var.duboisii. Of the 35 bats belonging to the speciesNycteris hispida andTadirida pumila examined, only one (N. hispida) yielded this fungus from its intestinal contents. Identification of the isolates asHistoplasma was confirmed by exoantigen tests and by mating with tester strains ofH. capsulatum. In vitro conversion to large yeast from suggestive ofH. capsulatum var.duboisii was obtained on brain heart infusion agar supplemented with sheep blood and glutamine or cysteine. Pathogenicity tests with mice for all the isolates confirmed their identity by the demonstration of large yeast forms (8–15 µm in diameter) within giant cells in the infected tissues. Investigations on the possible occurrence of human infections in the area are in progress.A poster based on this work was presented at the 11th ISHAM Congress in Montreal, Canada (22–28 June 1991), La-Hoffman Roche, Basel, Switzerland kindly financed the trip of one of us (H.C.G) for the Congress.     

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats,captured in a central zone of Mexico

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.     

An unusual presentation of disseminated histoplasmosis in a non-HIV patient from Vietnam

BackgroundHistoplasmosis is a systemic infection caused by the dimorphic fungus Histoplasma capsulatum, naturally found in nitrogen-rich soil, whose main transmission route is the inhalation of conidia. Up to 95% of histoplasmosis cases are asymptomatic or transient, and the remaining 5% of cases have pathological manifestations in the lungs, bone marrow, liver, spleen, intestine, mucous membranes, and rarely on the skin. This mycosis has been reported from many endemic areas, mainly in immunosuppressed patients, such as HIV-positive patients, and its disseminated form is rarely reported.Case reportHistoplama capsulatum was isolated and identified by means of microscopy, culture characteristics and nested PCR from the cutaneous lesions of a non-HIV patient from Vietnam. The patient improved significantly with systemic itraconazole treatment.ConclusionsDisseminated histoplasmosis with cutaneous involvement in non-HIV patients is an extremely unusual presentation.