Histoplasma : Introduction, Morphology,Life Cycle, Pathogenesis, Laboratory Diagnosis, Treatment and Prevention

Histoplasma capsulatum was discovered by Pathologist, Samuel Taylor Darling in 1906. It is  a causative agent of histoplasmosis ( systemic disease) sometimes called Darling’s disease, in honor of discoverer. It a domorphic fungus and thus it grows as filamentous molds as saprobes and in culture at 25°C; while in humans or culture at 37 °C , it transforms to a unicellular morphology ( yeast cells

Introduction of Histoplasma capsulatum

Histoplasma capsulatum was discovered by Pathologist, Samuel Taylor Darling in 1906. It is a causative agent of histoplasmosis ( systemic disease) sometimes called Darling’s disease, in honor of the discoverer. It is a dimorphic fungus and thus it grows as filamentous molds as saprobes and in the culture at 25°C; while in humans or culture at 37 °C, it transforms to a unicellular morphology ( yeast cells).

Taxonomic classification

(Darling, 1906)
Kingdom: Fungi
Phylum: Ascomycota
Subphylum: Ascomycotina
Class: Ascomycetes
Order: Onygenales
Family: Onygenaceae
Genus: Histoplasma

Species: Histoplasma capsulatum

The genus, Histoplasma contains one species, H. capsulatumH. capsulatum has two varieties: Histoplasma capsulatum var. capsulatum and H. capsulatum var. duboisii ( causative agent of African histoplasmosis)

Habitat:

Soil contaminated with bird droppings or excrement of bats is the common natural habitat for Histoplasma. Although it is claimed to exist worldwide, tropical areas are where this fungus is more frequently encountered. It is endemic in Midwestern and Central USA, along the Mississippi and Ohio river valley.

Morphology of Histoplasma

It completes in two phases i.e. macroscopic and microscopic features.

Macroscopic Features
Being a thermally dimorphic fungus, Histoplasma capsulatum grows in mould form at 25°C, and in yeast form at 37°C. Below are the macroscopic characteristics at varying temperatures and for both varieties.

At 25°C
Colonies are slow-growing and granular to cottony in appearance. The color is white initially and usually becomes buff-brown with age. The colonies are not sensitive to cycloheximide ( actidione)  in the culture media. From the reverse, a yellow or yellowish-orange color may be observed. While these features are best observed on SDA, brain heart infusion agar (BHIA) enhances growth more efficiently.

At 37°C
Creamy, slowly growing, moist, and yeast-like colonies are formed. This phase is observed in infected tissues and in vitro on enriched media, such as BHIA containing 5-10% blood.

For definitive identification of the fungus, yeast-to-mold conversion should be demonstrated.

Microscopic Features
At 25°C
Hyphae are septate and hyaline. H. capsulatum produces hyphae-like conidiophores which arise at right angles to the parent hyphae. It has both macro and microconidia. Macroconidia are tuberculate, thick-walled, round, unicellular, hyaline, large, and often have fingerlike projections on the surface. These macroconidia are also referred to as tuberculochlamydospores or macroaleurioconidia. Microconidia (microaleurioconidia) are unicellular, hyaline, and round, with a smooth or rough wall.

At 37°C
Narrow-based, ovoid, budding yeast cells are formed. Yeasts of H. capsulatum var. capsulatum are smaller than (2-4 µm) those

Life cycle of Histoplasma

Histoplasma spores circulate in the air after contaminated soil is disturbed. The spores are too small to see without a microscope. When people breathe in the spores, they are at risk of developing histoplasmosis. After the spores enter the lungs, the person’s body temperature allows the spores to transform into yeast. The yeast can then travel to lymph nodes and can spread to other parts of the body through the bloodstream. Histoplasmosis is not contagious but is contracted by inhalation of the spores from disturbed soil or guano. The inoculum is represented principally by microconidia. These are inhaled and reach the alveoli. In the alveoli, macrophages ingest these microconidia. They survive inside the phagosome.  As the fungus is thermally dimorphic, these microconidia are transformed into yeast. They grow and multiply inside the phagosome. The macrophages travel in lymphatic circulation and spread the disease to different organs.  Within the phagosome, the fungus has an absolute requirement for thiamine. Cell-mediated immunity for histoplasmosis develops within 2 weeks. If the patient has strong cellular immunity, macrophages, epithelial cells, and lymphocytes surround the organisms and contain them, and eventually calcify. Fibrosis around the cavities in the lung creates dense, round, flattened areas that are described as “coin lesions”.  In immunocompromised individuals, the organisms disseminate to different organs such as bone, spleen, liver, adrenal glands, and mucocutaneous membranes, resulting in progressive disseminated histoplasmosis. Chronic lung disease can manifest.

Pathogenesis of Histoplasma

Histoplasmosis is a systemic intracellular mycotic disease caused by dimorphic fungus, H. capsulatum. It usually occurs by the inhalation of microconidia by the host, deposit in alveoli, and rapidly convert to a parasitic yeast form in tissues. This germination and conversion can occur prior to or after ingestion by pulmonary macrophages. Conidia and yeasts are ingested by macrophages and reticuloendothelial cells where the organism can survive within phagolysosomes. Once within the macrophage, the yeast multiply and travel to hilar and mediastinal lymph nodes where they gain access to the blood circulation for dissemination to various organs. It is also known as Cave disease or Darling’s disease or Ohio valley disease or reticuloendotheliosis or spelunker’s lung and caver’s disease. It is common among AIDS patients because of their suppressed immunity and approximately 30% of HIV/AIDS patients diagnosed with histoplasmosis die from it. In immunocompetent individuals, past infection results in partial protection against ill effects if reinfected.

Types of Histoplasmosis

It  may be divided into the following types: 

  1. Primary pulmonary histoplasmosis
  2. Progressive disseminated histoplasmosis
  3. Primary cutaneous histoplasmosis
  4. African histoplasmosis

Clinical Findings

The clinical findings of histoplasmosis range from asymptomatic infection to disseminated sepsis. These manifestations depend mainly on the magnitude of exposure (i.e. the number of fungal particles inhaled), the immunological status of the host, and the virulence of the infective strain, indicating that environmental and genetic factors control the manifestation of disease. Additionally, in the setting of severely immunocompromised patients, such as individuals with AIDS, H. capsulatum strains previously not considered virulent have been able to cause fatal disease. In the patients who are clinically ill, histoplasmosis generally occurs in one of three forms-acute pulmonary,  chronic pulmonary, and disseminated. There is generally a complete recovery from the acute pulmonary form (another “flu-like” illness).  However, if untreated, the disseminated form of the disease is usually fatal.  Patients will first notice shortness of breath and a cough which becomes productive. If symptoms of histoplasmosis infection occur, they will start within 3 to 17 days after exposure; the average is 12–14 days.  Most affected individuals have clinically silent manifestations and show no apparent ill effects. The acute phase of histoplasmosis is characterized by non-specific respiratory symptoms, often cough or flu-like.  Chest X-ray findings are normal in 40–70% of cases.

Chronic histoplasmosis cases can resemble tuberculosis.

  • The sputum may be purulent or bloody.
  • Patients will become anorexic and lose weight.
  • Complain of fever and  chills
  • Night sweats
  • Fatigue (extreme tiredness)
  • Headache  and body aches
  • Chest pain

This again sounds like tuberculosis, and the lung X-ray also looks like tuberculosis, but today radiologists can distinguish between these diseases on the chest film (histoplasmosis usually appears as bilateral interstitial infiltrates.)The skin test is NOT used for diagnostic purposes, because it interferes with serological tests.

While histoplasmosis is the most common cause of mediastinitis, this remains a relatively rare disease. Severe infections can cause hepatosplenomegaly, lymphadenopathy, and adrenal enlargement. Lesions have a tendency to calcify as they heal.

Laboratory Diagnosis of Histoplasma

To diagnose histoplasmosis medical and travel history, Signs and symptoms, physical examinations, and laboratory tests are useful.

Specimen

Clinical specimens depend on the nature of the infection. e.g. Sputum or Bronchial alveolar lavage:  if it is a pulmonary disease, or  Skin scrapping from the edges of ulcer. Biopsy material from the diseased organ. Aspirated Pus from lymph nodes and subcutaneous abscess. Bone marrow is an excellent source of fungus, which tends to grow.  Peripheral blood is also a source of visualizing the organism histologically. The yeast is usually found in monocytes or in PMN’s. In peripheral blood, H. capsulatum appears as a small yeast about 5-6 µ in diameter. (Blastomyces is 12 to 15 µ). Gastric washings are also a source of H. capsulatum as people with pulmonary disease produce sputum and frequently swallow their sputum.

CSF and Urine for culture

Microscopy

H. capsulatum is most likely to be found within cells instead of free in the fluid. Staining – Wright’s method, Giemsa stain, or Calcofluor white and special stains: Papanicolaou stain or H & E stain for tissue specimen. Mucus and other thick substances should be treated with N-acetyl-L-cysteine before the wet mount is prepared. KOH wet mount prepared from clinical samples shows tiny yeast cells.

Culture

Blood agar or Modified SDA with antimicrobials and actidione ( cycloheximide)

BHIA containing 5-10 % blood is strongly recommended for specimens from sites that are sterile.

Yeast extract phosphate agar with ammonium hydroxide: It encourages conidiation and inhibits Candida species. Potato dextrose agar (PDA): to encourage conidia production.

All cultures should be examined daily for 1 week. When it is grown on modified Sabouraud dextrose agar (SDA) with antimicrobial at 25ºC, it appears as a white, cottony mycelium after 2 to 3 weeks.  Later they become woolly, as aerial hyphae develop.  The mold phase of H. capsulatum is characterized by thin, branching, septate hyphae, 1-2 µm, that produce microconidia (2-5µm). Macroconidia develop directly on the hyphae or on short, slender conidiophores that develop at right angles to vegetative hyphae. The macroconidia are hyaline, unicellular, and relatively large 8-14 µm, spherical to pyriform shape.  As the macroconidia age, they become tuberculate macroconidium, they form finger-like extensions of the thick wall of conidium.

Mature macroconidia sometimes described

Grown at 37ºC the budding yeast form appears after 10-15 days. It is a white to the tan and mucoid colony, with a rough membranous texture.  The yeast cell is 5-6 µ in diameter and slightly oval in shape and found exclusively within macrophages.  To confirm the diagnosis, one must convert the organism from mycelium to yeast or use the DNA probe.  Primary cultures for H. capsulatum should be held for 10-12 weeks before being discarded as no Growth. Culture of H. capsulatum on SDA showing a white suede-like colony with a pale yellow-brown. Microscopic morphology of the saprophytic or mycelial form of H. capsulatum showing characteristic large, rounded, single-celled, tuberculate macroconidia formed on short, hyaline, undifferentiated conidiophores. Yeast-like culture of H.  capsulatum on blood agar incubated at 37°C.

Non-culture based diagnosis of histoplasmosis

These tests include antibody and antigen detection as well as newer
techniques that have been developed in molecular diagnosis for the improved identification of H. capsulatum in clinical specimens.

Antibody detection

The two routine methodologies are complement fixation and immunodiffusion. Latex agglutination can also be used.

Immunodiffusion Assay: The Histoplasma immunodiffusion assay utilizes 2 different antigens, the H and the M proteins, and we can therefore detect up to 2 different precipitating bands in the agar plate. Antibodies to the M antigen, appear shortly the following exposure and can remain detectable by immunodiffusion for up to 3 years following disease resolution. Therefore, the presence of the M band alone cannot be used to discriminate between acute or remote infection. On the other hand, the presence of an H band either alone or in combination with the M band is indicative of active or recent histoplasmosis because the H band of the immunodiffusion test is usually present for only 4 to 6 weeks after exposure.  Importantly, a negative immunodiffusion result should not be used to exclude histoplasmosis as the specimen may have been collected early following exposure and prior to the development of detectable precipitating antibodies.  Specificity >95%  and variable sensitivity 80- 100 %.

Complement Fixation Test: Alongside immunodiffusion, there is also Histoplasma complement fixation testing. Briefly, complement fixation assays are based on the ability of antibody-antigen complexes, formed between antibodies present in the patient sample and added fungal antigens, to fix and inactivate exogenously added complement. Sensitized RBCs are added to this mix and since the complement pathway is inactivated, the red blood cells will remain intact and settle to the bottom of the well as a compact pellet, indicative of a positive reaction and the presence of specific antibodies.   In the absence of patient antibodies, however, the added complement will remain active and lead to lysis of the sensitized red blood cells.  Therefore, the presence of lysis is indicative of a negative reaction and the absence of specific antibodies.  The Histoplasma complement fixation assay is performed using 2 different Histoplasma antigens, one purified from the yeast phase and the second purified from the mycelial phase of growth. Serial dilution of patient samples, allows us to determine an endpoint titer that can be used to guide diagnosis.  For either antigen, serially increasing titers or an endpoint titer greater than or equal to 1:32 is associated with the presence of active histoplasmosis. A titer of 1:8 or 1:16 is considered positive, but only presumptive evidence of infection and additional clinical correlation is necessary. Titers less than 1:8 are not considered to be significant and therefore are not reported.  Importantly, low-level titers may be detected in individuals who reside in endemic areas and are otherwise healthy.  Finally, declining complement fixation titers over months to years have been associated with disease resolution, but should not be used to monitor response to therapy.

The complement fixation assay has a higher sensitivity for histoplasmosis as compared to immunodiffusion assays, particularly during early disease, however, their specificity is lower and for this reason, complement fixation and immunodiffusion are performed concurrently.  Also, these assays are fairly labor-intensive with long incubation times which leads to an average turnaround.  One disadvantage is that complement-fixing antibody develops late in the disease, about 2 to 3 weeks after onset.  A second disadvantage is that it cross-reacts with other mycotic infections like blastomycosis, paracoccidioidomycosis. An advantage of the C-F test is that it is quantitative, so the physician can follow the course of the disease by observing the titer of several samples.

Antigen detection

Antigen detection tests may be more effective than antibody testing for diagnosing histoplasmosis.

  • Radioimmunoassay (RIA): Recently, a radioimmunoassay can be used to measure H. capsulatum polysaccharide antigen (HPA) levels in samples of a patient’s urine, serum, and other body fluids. The test appears to meet the important need for a rapid and accurate method for early diagnosis of disseminated histoplasmosis, especially in patients with AIDS. HPA is detected in body fluid samples of most patients with disseminated infection. The antigen cross-reacts with Coccidioides immitis and  Blastomyces dermatidis. High Sensitivity and low specificity, so should be confirmed by another method. Immy exoantigen immunodiffusion test kit for the identification of H. capsulatum, C. immitis and B. dermatitidis. Exoantigen immunodiffusion plate showing positive identification of H. capsulatum.
  • ELISA

Exoantigens

Exoantigens are valuable for the immunoidentification of fungal pathogens and for resolving taxonomic problems. Most fungi produce unique antigens that allow specific identification. Exoantigens are soluble immunogenic macromolecules produced by fungi early in their development.

Molecular based methods

As described, H. capsulatum colonies grow slowly and the differentiation from other dimorphic or saprophytic fungi having similar colony or microscopic morphology is challenging. In order to confirm the diagnosis, tests for the production of exoantigens and the conversion to the yeast phase should be done, which takes at least 3 to 4 weeks. Methods for the identification of fungal isolates such as species-specifics DNA can decrease this time-consuming step while maintaining or improving the specificity, accuracy, and sensitivity. The use of chemiluminescence-labeled DNA probes for the detection of specific sequences of rRNA has been able to detect and confirm H. capsulatum clinical isolates tested. PCR Assays: A real-time PCR assay that correctly identified H. capsulatum from among a variety of fungi growing in the laboratory appears promising. Using this assay, positive identification of H. capsulatum was shown in tissue biopsies and bronchoalveolar lavage fluid from patients who had documented histoplasmosis. Two different semi-nested PCR assays have also shown promise when applied to blood and tissue scrapings obtained from patients with histoplasmosis and mice experimentally infected with H. capsulatum

Histoplasmin skin test: A person can learn from a histoplasmin skin test whether he or she has been previously infected by H. capsulatum. This test, similar to a tuberculin skin test like delayed-type hypersensitivity. Skin test with intradermal injection of 0.1ml of Histoplasmin antigen, a culture filtrate of the mycelial phase of growth, in the forearm. It is useful as an epidemiological tool. A histoplasmin skin test becomes positive 2 to 4 weeks after a person is infected by H. capsulatum. The test is positive after 48 hours with induration of 5 mm. A positive skin test does not mean that a person is completely protected against ill effects, it indicates either present or past exposure to H.capsulatum. A previous infection by H. capsulatum can provide partial protection against ill effects if a person is reinfected.

Animal Pathogenicity: Dogs, guinea pigs, hamsters, rabbits, mice.  The mouse is an ideal laboratory animal for the isolation of H. capsulatum. Mycelial and yeast forms can be inoculated to establish systemic infection. Due to the discovery of newer methods, its frequency of usage has decreased.

Treatment

For some people, the symptoms of histoplasmosis will go away without treatment. However, prescription antifungal medication is needed to treat severe histoplasmosis in the lungs, chronic histoplasmosis, and infections that have spread from the lungs to other parts of the body (disseminated histoplasmosis). The drug of choice is amphotericin (avoided in a renal patients due to the risk of nephrotoxicity.  Itraconazole and Voriconazole are now also being used. Liposomal preparations of amphotericin B are more effective than deoxycholate preparations. Alternatives to itraconazole are posaconazole, voriconazole, and fluconazole. Individuals taking itraconazole are monitored for hepatic function.

Epidemiology of Histoplasma

H. capsulatum is found throughout the world. It is endemic in certain areas of the United States, particularly in states bordering the Ohio River valley, the lower Mississippi River, and the Missouri river.  The humidity and acidity patterns of soil are associated with endemicity. Incidence of histoplasmosis in adults aged 65 years and older in the U.S. to be 3.4 cases per 100,000 population.  Rates were highest in the Midwest, with an estimated 6.1 cases per 100,000 population.  Bird and bat droppings in soil promote the growth of H.capsulatum. Contact with such soil aerosolizes the microconidia, which can infect humans.  It is also common in caves in southern and East Africa. Positive histoplasmin skin tests occur in as many as 90% of the people living in areas where H. capsulatum is common, such as the eastern and central United States. In Canada, the St. Lawrence River Valley is the site of the most frequent infections, with 20-30 percent of the population testing positive. In the United States, an estimated 60% to 90% of people who live in areas surrounding the Ohio and Mississippi River valleys (where H. capsulatum  is common in the environment) have been exposed to the fungus at some point during their lifetime India is another Asian country where H. capsulatum is known to be endemic, although the true prevalence of this mycosis is still underappreciated. The first case was reported as early as 1954, and since then several cases have been published. In India, the majority of histoplasmosis cases were reported from the eastern and north-eastern parts of the country, especially from Calcutta (West Bengal) and Assam. The Gangetic West Bengal is the site of most frequent infections, with 9.4 percent of the population testing positive. H. capsulatum was isolated from the local soil proving the endemicity of histoplasmosis in West Bengal. The prevalence of histoplasmosis has not been well studied in Nepal.

Prevention

It can be difficult to avoid breathing in H. capsulatum in areas where it’s common in the environment. In areas where Histoplasma is known to live, people who have weakened immune systems (for example, by HIV/AIDS, an organ transplant, or medications such as corticosteroids or TNF- inhibitors) should avoid doing activities that are known to be associated with getting histoplasmosis, including Disturbing material (for example, digging in the soil or chopping wood) where there are bird or bat droppings, cleaning chicken coops. Exploring caves

Key Notes on Histoplasma

  1. Bone marrow biopsy for histopathology may be the most rapid method of establishing a definitive diagnosis of invasive infection.
  2. The structure of the H. capsulatum yeasts is very similar to other pathogens(Candida glabrata, Penicillium marneffei, Pneumocystis jirovecii, Toxoplasma gondii, Leishmania donovani and Cryptococcus neoformans) and thus it is extremely important to be familiar with the morphology of these pathogens and the staining characteristics of the yeast by different methods.
  3. This African variant differs by having larger (7-15 µm) budding yeast cells in-vivo.
  4. In people who have weakened immune systems, histoplasmosis can remain hidden in the body for months or years and then cause symptoms later (also called a relapse of infection).
  5. Histoplasmosis can’t spread from the lungs between people or between people and animals. However, in extremely rare cases, the infection can be passed through an organ transplant with an infected organ.
  6. Systemic Mycoses : H. capsulatum is also a fungus that is responsible for system mycoses other than B. dermatitidis, P. barasiliensis,  C.immitis,  C. neoformans. Four of these pathogens (H. capsulatum, B dermatitidis, P. barasiliensis and C. immitis) are dimorphic. They grow as filamentous molds in the culture at 25°C and yeast forms in humans or culture at 37°C.
  7. Differential Diagnosis:  Tuberculosis,  localized pneumonitis is mistaken for Mycoplasma pneumoniae, Legionella,  Coxiella burnetii and Chlamydia pneumoniae. The uninucleate yeasts should be distinguished from other intracellular organisms like Leishmania donovani ( contain kinetoplast), Toxoplasma gondii (tachyzoites being protozoa are not stained with GMS stain). P. marneffei (dividing transverse fission cells), C. neoformans (capsulated), Candida glabrata (never found intracellularly).
  8. Sources of Histoplasmosis: H. capsulatum is found in blackbird roosts, chicken houses, and bat guano and especially from the soil with a high nitrogen content resulting from deposits of excreta from chicken, starlings, and bats. It is a significant occupational disease in bat caves in Mexico when workers harvest the guano for fertilizer. Disruption of soil from excavation or construction can release infectious elements that are inhaled and settle into the lung. In the endemic area, the majority of patients who develop histoplasmosis (95%) are asymptomatic. 5 %  of the cases have chronic progressive lung disease, chronic cutaneous or systemic disease, or an acute fulminating fatal systemic disease.  All stages of this disease may mimic tuberculosis and thus diagnosis is made from their history, serologic testing, or skin test.
  9. Most of the infections associated with histoplasmosis are asymptomatic. even though the common symptoms of acute and epidemic histoplasmosis include high fever, cough, and asthenia, and weight loss.
  10. People with compromised immune systems such as HIV/AIDS, cancer, and organ transplant recipients are at risk of developing this disease.
  11. Unlike its name; H. capsulatum is not encapsulated. The designation H. capsulatum is actually a misnomer (inaccurate name).
  12. Tuberculate macroconidium (with typical thick walls and radial, finger-like projections) is a diagnostic structure of H. capsulatum.

References

  1. A Textbook of Medical Mycology- 3rd Edition –Jagdish Chander
  2. http://www.cdc.gov/fungal/diseases/histoplasmosis /
  3. http://www.medicinenet.com/histoplasmosis_facts/ article.htm
  4. Textbook of Microbiology Ananthanarayan & Paniker -9th Edition
  5. https://en.wikipedia.org/wiki/Histoplasma_capsulatum
  6. drfungus.org/knowledge-base/histoplasma-species
  7. https://www.researchgate.net/publication/44576304_Diagnosis_of_Histoplasmosis
  8. https://www.slideshare.net/shahmanthan24/histoplasmois-mycology-epidemiology-laboratory
  9. https://www.nepjol.info/index.php/NJB/article/view/26949
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