Evelyn Heintschel von Heinegg, Jörg Steinmann, Jan Buer, Peter-Michael Rath
Fungi are carbon heterotrophic eukaryotic organisms. They decompose dead organic matter and, therefore, play an essential role in the ecological system. Only a fraction of the approximately 1.5 million fungal species estimated to exist have been recorded by taxonomic classification. About 200 species are pathogenic to humans /, , , , , , , , /.
The taxonomic nomenclature of fungi is difficult to comprehend for non mycologists. Classical identification of fungal species is based on the macro and micromorphology of their reproductive structures or, in yeast species, their biochemical performance. Fungi can reproduce asexually by mycelial fragmentation or spores as well as sexually.
While the sexual stage is the leading taxonomic criterion for classification of an isolate, historically established species names based on the morphology of asexual reproductive structures continue being used. Thus, a species may happen to have two names. Molecular biological methods have been increasingly used for identification and taxonomic classification, causing the nomenclature of fungi to be reconsidered.
The simple DYMD (dermatophytes, yeasts, molds and dimorphic fungi) scheme, which is considered incorrect from the biological perspective, has become established based on relevance of the fungi in human medicine. The description in this chapter also adheres to this scheme, but only discusses species capable of causing life threatening, invasive infections (synonyms: deep mycoses, systemic mycoses, endomycoses), with the exception of Pneumocystis jirovecii (carinii) , a pathogen just recently classified under fungi. Typically, replication of this pathogen is non invasive in alveoli (i.e., invasive), systemic infections are rare. For information on dermatophytes, which only invade dead, keratin containing cells, and for injury mycoses (exogenous infections), please refer to the relevant literature.
Most fungi relevant in human medicine become infectious due to interaction with the biotic or abiotic environment. Person-to-person transmission is rare, except regarding dermatophytes and Pneumocystis. Endogenous infections are only caused by Candida species, which are components of the transient or permanent, mucosal flora in the human body.
The relevant, human pathogenic fungi occurring in Central Europe only cause systemic (invasive) infections in the presence of local or systemic immune system malfunctions. Essential risk factors include congenital or acquired malfunctions of the cellular defense mechanism. Thus, such fungal infections must be classified as opportunistic infections. In Germany, the only occurrences of mycoses caused by dimorphic fungi are imported. They are considered to be obligate pathogenic. However, in immunocompetent patients, most of these infections are mild or even subclinical. None of the discussed pathogens or diseases are subject to mandatory reporting in compliance with the German Infection Protection Act (IfSG).
The diagnosis of invasive mycosis is based on the histological and/or cultural detection in primarily sterile specimens. Cultural detection should always be sought for species identification, on one hand, and for determination of the sensitivity to antimycotic agents, on the other. Microscopic or cultural detection in primarily non sterile specimens (e.g., from the respiratory tract) does not allow to distinguish between contamination, colonization and infection. Culture independent methods such as antigen or antibody detection usually have a lower diagnostic sensitivity and specificity and therefore can only serve as an indication of the presence of infection, except for the highly sensitive and specific antigen detection methods in cryptococcosis. Molecular biological detection methods in mycology are still in the development and validation phase. In most cases, such methods have only been established in specialized laboratories.
Every microbiological laboratory should be capable of detecting fungi relevant to human medicine. The basic spectrum of methods constantly available for diagnostic investigation should always include microscopy and culturing on universal and selective media. It should be possible to identify the most frequently occurring Candida and Aspergillus species to species level based on morphology and/or biochemical performance. For application to cerebrospinal fluid specimens, an antigen assay and culturing media should always be available to assist the diagnosis of cryptococcosis. At least one staining technique should be established for the diagnosis of Pneumocystis. Cooperation with a consultant or reference laboratory is recommended because each fungal isolate from primarily sterile specimens should be identified to species level and also be subjected to a sensitivity test.
Additional techniques can be established in the laboratory depending on the rate of infection and patient specimens received: antigen assays for Candida and Aspergillus, antibody assays, antimycotic agent sensitivity tests, molecular biological methods for pathogen detection and species identification. Diagnostic investigation of extra European systemic mycoses caused by dimorphic fungi should be left to specialized laboratories, the more so since the pathogens of these infections are classified under laboratory biosafety level L3 according to the Ordinance on Biological Agents. Participation in corresponding inter laboratory surveys is mandatory for all laboratories involved in mycological diagnostics.
Candida species can cause local (e.g., thrush, diaper dermatitis) and systemic (invasive) infections in humans. The medically relevant species C. albicans is the most frequently encountered cause of mucosal and organ mycoses. However, infections with C. glabrata, C. tropicalis, C. krusei, C. guilliermondii and other species are observed at an increasing rate. C. albicans is the only species to use the human orogastrointestinal tract as a natural habitat /, , , , , /.
Routes of infection
The majority of Candida infections arise endogenously (i.e., the normally commensally occurring yeast like organisms gain pathogenic relevance in case of disturbances of the defense mechanisms). Entry ports for the development of Candida mycoses mainly include the orobronchial tract, intravascular catheters or the gastrointestinal tract such as in epithelial lesions due to cytotoxic chemotherapy. Exogenous infections, for example due to contaminated infusion solutions or transmission by medical personnel, have been described.
Prevalence and incidence
Oral and esophageal candidiasis are very common in HIV infected patients, with up to 90% patients estimated to be affected in the pre-HAART (highly active anti-retroviral therapy) era. The rate of infection in tumor patients or patients who received bone marrow transplants is 25–35%. The infection is caused by C. albicans in most cases.
The prevalence of candidemia varies between individual countries and individual hospitals. Relative prevalences of 5–15% in positive blood cultures have been described. In risk groups such as patients with sepsis, a prevalence of 17% has been shown by a German study, and a prevalence between 7% and 30% has been reported in patients with leukemia.
The majority of systemic infections are caused by C. albicans (45–60%); other species are not as common (C. glabrata 15–30%, C. tropicalis 10–30%). However, the percentage distribution of pathogens depends on the patient’s predisposition: For instance, C. parapsilosis is more frequently detected in premature infants and C. glabrata is more common in old, multi morbid patients. An increase in non Candida albicans species has generally been reported in all studies regarding pathogen distribution. The mortality rate in systemic Candida infections is between 15% and 30%.
Candida infections manifest themselves as local mucocutaneous infections in most cases. Clinical symptoms include oral thrush, atrophic oral candidiasis, thrush esophagitis, vaginal infections and balanitis, as well as skin surface (diaper thrush) and nail infections. All of these manifestations have local or systemic disturbances of the defense mechanisms in common. Systemic factors include age extremes, metabolic diseases such as diabetes mellitus and congenital or acquired T cell defects (AIDS, malignant tumors and antineoplastic therapy, systemic steroid therapy, graft-versus-host disease). Other causes are malnutrition, long term administration of antibiotics (> 2 weeks), intravascular catheters, mandatory ventilation, hemodialysis, parenteral nutrition, gastrointestinal perforation and pancreatitis. Local factors include radiotherapy, steroid sprays or artificial dentures. In many cases, long term antibiosis, especially in patients in intensive care, causes colonization of the lower respiratory tract; however, invasive infections exclusively involving the lungs are rare.
Invasive candidiasis is the most common disease among hospitalized patients in the developed world and is usually preceded by mucocutaneous colonization. Deep seated candidiasis arises from either hematogenous dissemination or direct inoculation of candida species to a sterile site, such as the peritoneal cavity. Mortality among patients with invasive candidiasis is as high as 40%, even when patients receive antifungal therapy. Any inner organ can be affected due to hematogenous or lymphatic dissemination. Hepatosplenic candidiasis and infections in low-perfusion tissue such as the eye (enophthalmitis) or the skeleton (e.g., spondylodiscitis) represent diagnostic and therapeutic challenges.
Invasive candidiasis is characterized by nonspecific clinical symptoms: fever and elevated inflammatory markers are common and multiple organ failure and septic shock are also encountered frequently. Ophthalmologic detection of “cotton-wool” lesions in the retina is typical, but not always possible. Radiological diagnostic examinations usually show a nonspecific infection; abscesses in internal organs or typical lesions in the liver and spleen have been detected in hepatosplenic candidiasis in individual cases.
Microscopic and cultural detection of organisms
Specimens suitable for the diagnosis of Candida endomycosis are primarily samples from normally sterile areas such as blood, cerebrospinal fluid as well as aspirated fluid and biopsy material. Respiratory secretions, urine, feces and smears from mucosal surfaces do not allow to differentiate between colonization and clinically overt infection even in the case of quantitative yeast count determinations. Blastospores and possibly pseudo hyphae are recognizable in unstained wet mounts as well as in Gram stained and histological slides /, /.
Candida species will grow at 25–37 °C within 2–7 days on the usual bacteriologic and mycologic culture media. The diagnostic sensitivity of blood cultures is 21% to 71% . Whereas blood cultures may establish a diagnosis when Candida resides in the blood stream, cultures of blood obtained from patients with hematogenous, deep seated infections often yield negative results because Candida has been cleared from the blood stream at the time the blood sample is collected . Blood cultures are further limited by slow turn around times and by the fact that a positive result may revealed only late in the course of disease . Identification is achieved based on morphologic criteria and biochemically by sugar fermentation as well as sugar assimilation. MALDI-TOF mass spectroscopy is also a reliable method for species identification. Molecular biological methods have not been established in every laboratory.
Molecular biological detection
A number of in-house assays with a diagnostic sensitivity of 88–98% and a specificity of 88–95% have been described for the molecular biological detection (PCR) of Candida DNA in the blood or serum. PCR systems are also commercially available (multiplex PCRs for the detection of sepsis pathogens with the inclusion of most common Candida species) /, /.
Latex agglutination for detecting the heat labile antigen
Antibodies obtained by immunization of rabbits with heat destroyed blastospores of Candida albicans are adsorbed to latex particles. Heat labile antigen in the sample binds to the fixed antibodies and thus causes agglutination of the test mixture within 10 minutes.
It is recommended to repeat the test after heating the sample at 56 °C for 15 min. in order to verify that it was indeed the heat labile antigen and not an undiscovered thermoresistant serum factor which reacted in the test. The test can be performed qualitatively using a 1 : 2 sample dilution or quantitatively using serial sample dilutions. Threshold titer is 1 : 4. Diagnostic sensitivity is 30–77% at a specificity of 70–88%.
Latex agglutination for detecting the mannan antigen
The test mixture is composed of latex particles to which monoclonal mannan antibodies (EB-CA1) are adsorbed. The patient serum is heated to 100 °C and centrifuged in order to dissociate immune complexes and destroy rheumatoid factors. If mannan antigen is present in the sample thus prepared, the test mixture will agglutinate within 10 minutes.
A commercially available sandwich ELISA uses the same antibody. The two test systems have similar diagnostic specificity (70–80%), but the sensitivity of ELISA is higher (42–98%).
Various assays are commercially available for the detection of (1,3)-β-D-glucan, a cell wall component of many fungi. The assays differ in their setup and threshold values. The principle of the assay is the activation of co agglutination in the amebocytes of horseshoe crab hemolymph, similarly to the Limulus assays for endotoxin detection, but following a different metabolic pathway (e.g., using factor G instead of factors B and C). The serine protease inhibitors occurring in serum must be deactivated before the test mixture is prepared. Depending on the assay manufacturer, this is achieved by alkali pretreatment or by adding triton X and heating to 70 °C. Any endotoxin present must be removed by adding factor C or polymyxin.
A multitude of different antigen preparations and probably every serological method have been used for the detection of serum Candida antibodies /, , /. Three commonly used test procedures are described in the following. Since neither the production of antigens nor the test techniques are standardized, the specified threshold titers vary from laboratory to laboratory. It has been reported that both diagnostic sensitivity and specificity of antibody detection tests for the diagnosis of invasive Candida infection are low.
Indirect hemagglutination test
Poly saccharides obtained by phenol water extraction are adsorbed to sheep erythrocytes. Interaction between these sensitized erythrocytes and antibodies, mainly immunoglobulin class M (IgM), results in macroscopically visible agglutination. The test can also be used as a micro-method.
Indirect immunofluorescence test
Blastospores fixed on slides are used as antigen. On the fluorescent microscopic view, the presence of antibodies is revealed by fluorescence of the entire cells or cell contours. The immunoglobulin classes (IgG, IgM, IgA) of the antibodies may be differentiated by using heavy chain specific antisera against human immunoglobulin as indicator reagents.
Candida antigens are used which are fixed to polystyrene micro titration plates either directly (protein antigens) or by means of specific rabbit antibodies (polysaccharide antigens). Antibodies bound to antigens are detected using enzyme labeled anti-human serum. The immunoglobulin classes of the antibodies may be differentiated by using antisera against human immunoglobulin heavy chains.
Cultural detection based on primarily sterile specimens should always be sought in suspected invasive Candida infection. The diagnosis is considered to be confirmed if at least one of the following criteria are met:
- Repeated detection in blood cultures obtained on different days
- Cultural and/or histological detection in tissue biopsy samples
- Microscopic and/or cultural detection in normally sterile fluids (e.g., cerebrospinal fluid, joint fluid).
In many cases, however, such confirmation of diagnosis by laboratory findings is not attainable under clinical conditions. Instead, usually a probable diagnosis can only be made based on a review of predisposing factors, the course of the disease, the response to therapy as well as the laboratory diagnostic findings. A conclusive validation of molecular biological tests has not been available to date. Therefore, such tests should be left to specialized laboratories.
Assessments of the diagnostic value of the heat labile antigen differ tremendously. To measure diagnostically conclusive, high antigen titers, repeat serum examinations are recommended. High antigen titers may suggest invasive infection although they may also be observed in conjunction with superficial Candida colonization. False positive reactions can be caused by rheumatoid factors and occur in the presence of high creatinine concentrations. Some authors do not recommend heat labile antigen tests on account of their low diagnostic sensitivity and specificity.
In some studies, Candida mannan detection has been ascribed a higher clinical specificity for the exclusion of invasive candidiasis. Diagnostic sensitivity, however, is only moderate. Regular monitoring of patients at risk may, however, provide crucial clues.
Only a few and inconsistent validation results are available regarding test methods for the detection of (1,3)-β-D-glucans. Positive results are to be expected in invasive Candida and mold infections (not in zygomycoses and cryptococcosis). The antigen is also detectable in serum in pneumocystosis. Diagnostic sensitivities of 58–100% and specificities of 88–98% have been described regarding the detection of invasive mycosis. This test has also been reported to yield a number of false positive results (e.g., due to intravenously administered antibiotics) use of gauze, in hemodialysis, intravenous administration of albumin or β-globulin and in bacteremia. Whether this test is helpful for diagnosis has not been sufficiently documented to date.
The detection of serum Candida antibodies has not produced decisive, clinically useful progress in the diagnosis of Candida endomycoses. No procedure is suited for differentiating with adequate probability between invasive infection, superficial mucocutaneous mycosis or Candida colonization. Despite these limitations, the determination of Candida antibodies has become established for the purposes of monitoring patients at risk of infection. Monitoring must be performed regularly, however (at least once a week). In patients with intact antibody synthesis, a significant increase in the Candida antibody titer (at least fourfold i.e., two titer steps) suggests the presence of enhanced Candida exposure whose clinical relevance must be evaluated by additional tests.
In patients with impaired antibody synthesis, a significant increase in antibodies may not be found despite clinically overt Candida infection. Depending on changes in immunity status, antibody titers may vary considerably.
Monitoring of the antibody levels thus is thought to be of some value in immunocompetent patients (e.g., during the course of intensive care) as an additional diagnostic criterion. In immunocompromised patients, who in particular bear the highest risk for invasive Candida infection, antibody monitoring is the least conclusive. It has been found in several studies that a combination of various test methods will increase diagnostic conclusiveness. This aspect may be elucidated by larger scale studies yet to be performed.
The genus Cryptococcus comprises 19 species, most of which typically occur as encapsulated yeasts. Besides infections with C. neoformans (teleomorph stage: Filobasidiella neoformans), infections with C. gattii and, more rarely, with C. albidus, C. laurentii and C. adeliensis have also been described /, , /.
The gelatinous capsule of C. neoformans and its ability to grow at 37 °C and produce melanin are classical pathogenicity factors.
C. neoformans occurs worldwide. It is frequently found in the feces of birds, in particular of pigeons and parrots, and in bird nesting areas but also on tropical and subtropical plants and fruit. C. gattii, by contrast, has never been found in birds, but primarily on eucalyptus trees and in sewage. The infection affects not only humans, but may also occur in pets, especially cats.
Routes of infection
The infection is spread by inhalation of pathogen contaminated dust. The incubation period may be up to several weeks. Transmission from animals (including infected pets) to humans or from person to person has not been proven.
Prevalence and incidence
Cryptococcosis acquired in Europe is usually caused by C. neoformans. Exposure to C. neoformans apparently occurs often and usually results in latent (asymptomatic) infection. Clinically overt infection is primarily seen in immunocompromised patients with T cell defects. AIDS is the underlying disease in 2.9–13.3% of cryptococcosis cases; however, this rate has markedly decreased since the introduction of the HAART (highly active antiretroviral therapy) in HIV infected patients. Other risk groups include patients following organ transplantation and patients receiving chemotherapy in malignant tumors or on long term steroid medication. An increasing number of infections in non predisposed patients has been reported.
C. gattii infections are restricted to tropical/subtropical regions. However, C. gattii has also been detected in eucalyptus plantations in Mediterranean countries. Due to its higher virulence, it affects immunocompetent individuals more frequently than C. neoformans does.
Pulmonary C. neoformans infections occurring after inhalation of the pathogen show an asymptomatic course or uncharacteristic symptoms. Practically any organ can be affected by hematogenous or lymphatic dissemination. Because of the pathogen’s neurotropism, meningitis or meningoencephalitis are the major manifestations of cryptococcosis. Nonspecific neurological symptoms such as headache, paresthesia, psychiatric abnormalities or focal deficits can indicate the need for adequate diagnostic investigation. The course is acute or chronic, and primary cutaneous infections are also possible.
Laboratory diagnostic investigation of cryptococcosis should include cultural detection methods performed in parallel with microscopic and serological antigen diagnostics. Such investigation should always be approached as emergency diagnostics .
Microscopic and cultural detection of organisms
Cerebrospinal fluid (CSF) is primarily used for the microscopic detection of the pathogen. CSF should be centrifuged for 10 min. at 3,500 × g to use the sediment for Cryptococcus antigen detection. The sediment is resuspended with 0.5 mL of CSF for use in microscopy and culture analysis. Respiratory specimens can be used for pathogen detection in the case of pulmonary manifestation /, , /.
CSF Gram stains and, in particular, Gram stained slides of respiratory secretions can show artifacts which can easily be mistaken for Cryptococcus cells. Therefore, ink preparations should be used in suspected cryptococcosis. Using ink mounts, the typical budding cells with double refractile cell walls, granulated cytoplasm and thick capsules are detected in 25–50% of patients with cryptococcal meningitis. Every microscopic finding, therefore, must be confirmed by culturing the pathogen.
Culture is also indicated in the case of a negative microscopic result. CSF, respiratory secretions such as broncho alveolar lavage fluid, blood, urine and tissue samples should be used as specimens.
Molecular biological detection
C. neoformans capsular poly saccharides (glucurono-xylomannan) appear mostly in serum and CSF, and more rarely in urine and other body fluids. Polyclonal and monoclonal antibodies are used for the detection of these antigens. Rheumatoid factors are eliminated and immune complexes are split by pretreatment of the samples using pronase. Thus, false positive and false negative reactions and the occurrence of a prozone phenomenon are reduced. Infections with C. gattii are also reliably detected with the antigen test. Enzyme immunoassay and latex agglutination test show equivalent diagnostic sensitivity and specificity in antigen detection.
Latex agglutination test
The test mixture consists of latex particles to which Cryptococcus antibodies are bound. Agglutination occurs in the presence of antigens in the sample. The lower detection limit in serum is 50 ng/mL. In the case of CSF, no pretreatment is necessary.
Polyclonal Cryptococcus antibodies which are adsorbed to wells of micro titer plates bind Cryptococcus antigens in the sample. Labeled monoclonal Cryptococcus antibodies are used to detect the antigen-antibody binding. The test can be performed quantitatively. The enzyme immunoassay is also suited for the reliable detection of C. gattii antigen.
The use of monoclonal C. neoformans antibodies, however, may yield false negative test results in C. gattii infections.
The two test methods have a diagnostic sensitivity of 98–100% and a clinical specificity of 98–99%.
Cryptococcus antigen can be detected in serum, plasma, cerebrospinal fluid (CSF) or urine at high diagnostic sensitivity and specificity using rapid immunochromatographic tests (lateral flow assay) . The test strips are coated with 4 different Cryptococcus antigens. These tests have the advantage of being easy to handle.
The culture method should always be sought for pathogen detection. CSF ink preparations are suited for the rapid detection of the pathogen in suspected Cryptococcus meningitis. However, false negative results have to be anticipated in up to 2/3 of patients with this method. Molecular biological methods have only been established as in-house tests in specialized laboratories /, /.
The detection of C. neoformans antigens in the CSF is considered to be a sensitive and specific sign of Cryptococcus meningitis; in half of the cases, the antigens are also found in serum and urine. Antigen detection in serum alone indicates extra meningeal infection with a high level of probability even though the detection limit of the test is lower in these cases. Repeat examinations increase the degree of clinical certainty and are valuable for the purpose of monitoring. Rising antigen titers are interpreted as an indicator for progression of the infection and thus are associated with poor prognosis. If antifungal therapy is successful, a decrease in the antigen titer will ensue.
False positive serum reactions have been described in disseminated Trichosporon asahii (beigelii) infection and false positive CSF reactions have sporadically been seen in patients with bacteremia and/or malignancy. Nonspecific agglutination of the latex test mixture was observed in samples of CSF which were contaminated with agar and/or powder from latex gloves. Nonspecific agglutination can also occur, if the loop was already used to spread the sample on culture media and is again immersed into the sample. Moreover, the storage of the specimens and enzyme mixture for sample pretreatment in siliconized tubes can cause nonspecific agglutination. Cryptococcus antigen detection is not widespread in diagnostic laboratories and no test systems are commercially available.
Molds of the genus Aspergillus occur worldwide. They are ubiquitous in the soil, air and water and, in particular, in dead and rotting organic material. Most species reproduce asexually (conidiophores and conidia); for some species, however, a teleomorph stage (sexual reproduction) has been described justifying their taxonomic classification under the phylum ascomycota. Thirty-three of the roughly 180 species described are human pathogens. A. fumigatus, less frequently A. flavus, A. terreus or A. nidulans cause life threatening infections with a high mortality rate (30–87%) in immunocompromised patients. Other clinically relevant conditions include aspergilloma and allergy symptoms.
Routes of infection
Transmission is primarily airborne and the lungs are the primary organ affected by invasive infection. Aspergillus spores (conidia) are omnipresent in the ambient air. They are present in especially high concentrations near compost heaps. Construction work in hospitals and contaminated air conditioning systems may also cause increased air pollution with Aspergillus spores. Therefore, plant cultures in potting soil should not be placed near immunocompromised risk patients. HEPA (high-efficiency particulate air) filtration helps to provide spore free indoor air. The sporadic detection of Aspergillus sp. in respiratory specimens is insignificant because of its ubiquitous occurrence, unless there is a risk constellation for infection or clinical symptoms are present. Contaminated food, particularly nuts and cereals, may also be a source of infection, although gastrointestinal infection is very rare. Person-to-person transmission is very rarely encountered.
Prevalence and incidence
Allergic bronchopulmonary aspergillosis occurs in approximately 1–2% of asthma patients and a mean of 7% mucoviscidosis patients. Preformed cavities that may, for example, occur in patients with chronic pulmonary disease (e.g., bullous emphysema, sarcoidosis, tuberculosis, bacterial lung abscess) predispose to the development of aspergilloma.
Exact data on the prevalence and incidence of Aspergillus infections are not available. An incidence of 1–23% has been reported depending on the investigating center and the constellation risk. In cases of aspergillosis occurring during prolonged neutropenia of more than 14 days duration (below 1.0 × 109/L), one of the main risk factors for the disease, the incidence of the invasive form of infection in patients with hemato-oncological diseases may be 5–20%. Other risk groups include patients following organ transplantation (1–20%), AIDS patients, tumor patients receiving immunosuppressants, patients with chronic obstructive pulmonary disease and patients with autoimmune disease receiving high doses of corticosteroids.
According to a German postmortem study covering the years 2001 to 2005, approximately 20% of patients with hematologic neoplasia were found to have invasive mycosis. Two thirds of the invasive mycoses were only diagnosed by autopsy. Histologically, two thirds of the cases were aspergilloses. The mortality rate is 30–80% depending on the underlying disease.
Allergic bronchopulmonary aspergillosis is characterized by pulmonary deterioration in the presence of an underlying disease (asthma, mucoviscidosis), transient pulmonary infiltrates and positive IgE antibodies. Aspergillomas are usually asymptomatic. In the presence of vascular erosion, however, life threatening hemoptysis may occur Surgical intervention is the treatment of choice. In case the para nasal sinuses are involved, the clinical symptoms correspond to those of chronic sinusitis.
Due to the predominantly airborne route of infection, invasive types usually manifest as pulmonary aspergilloses with nonspecific symptoms: cough, dyspnea, antibiotic resistant fever, hemoptyses. Risk patients with therapy refractory fever should be examined by CT imaging of the lungs at an early stage. The pathogens can disseminate via the blood stream or continuously. Cerebral aspergillosis is associated with extremely high mortality.
Microscopic and cultural detection of organisms
Invasive Aspergillus infection is confirmed by pathogen detection in tissue samples by microscopy (characteristic septate hyphae branching at angles of 45°). The detection procedure is based on Grocott-Gomori silver or PAS staining, but does not allow species identification. Microscopic detection using primarily non sterile specimens such as respiratory secretions is not as conclusive. However, the detection of Aspergillus sp. in a risk patient may indicate the presence of infection. Staining with optical brightening agents such as calcofluor white is recommended as a more sensitive detection method. The fluorescent stains bind to poly saccharides of the fungal cell wall. The hyphae are difficult to detect by the common staining method according to Gram.
Cultural detection for species identification and resistance testing are the procedures of choice. Aspergillus species grow on universal and selective culture media within a few days at incubation temperatures generally used in bacteriology. Species identification is based on macroscopic and microscopic colony morphology. Specialized laboratories use molecular biology methods (DNA chip technology, ITS region sequencing). Although rarely used to date, isolates with triazole resistance have been described. Resistance testing is only helpful in isolates from invasive infections.
Molecular biological detection
A number of in-house assays with a diagnostic sensitivity of 40–100% and a specificity of 65–100% have been described for the molecular biological detection of pathogen DNA in the blood, serum, respiratory secretions or tissue samples. Commercial systems are also available (multiplex PCR for the detection of sepsis pathogens with the inclusion of A. fumigatus and/or Aspergillus specific PCR tests). However, no conclusive validation of these systems is available /, , , /.
Molecular biology methods for pathogen DNA detection should, therefore, be left to specialized laboratories. Result interpretation is only meaningful in the context of risk constellation and other laboratory and radiological findings.
ELISA is most frequently used to detect circulating serum galactomannan, a cell wall component of many hyphomycetes. The assay is not Aspergillus specific and does not detect mucoromycotina (zygomycetes). It is based on a monoclonal antibody directed against the β-D-(1,5)-galactofuranoside side chain. The antigen is detected by a second, peroxidase labeled antibody. The specimen must be incubated at 100 °C prior to analysis for immunocomplex dissociation. A non dimensional index is calculated based on the absorption values of the specimen. The detection limit is 1 ng/mL of galactomannan.
Another method available includes various tests for the detection of (1,3)-β-D-glucan, a cell wall component of many fungi. The underlying principle of this method is described in Section 45.2.
Indirect hemagglutination tests or ELISA are commercially available for the detection of antibodies. Other serological methods include tests based on immunoblot, immunodiffusion, immunoelectrophoresis or immunofluorescence /, /.
A positive antibody test can be helpful for diagnosing immunocompetent patients and in cases of suspected aspergilloma or allergic bronchopulmonary aspergillosis. However, the specificity of these test methods is limited due to the high number of cross-reacting antigens. Test results in immunocompromised patients are not very conclusive. Clinical sensitivities of 14–36% and specificities of 72–99% have been described in hemato-oncological patients with suspected invasive aspergillosis. Because of their poor clinical sensitivity, antibody diagnostics are not helpful in such patients.
The cultural detection of Aspergillus species in primarily sterile specimens confirms the diagnosis of invasive infection and allows species identification and resistance testing.
Microscopic or cultural detection in primarily non sterile respiratory specimens (sputum, respiratory secretion, lavage fluids) does not allow to differentiate between contamination, colonization or infection. However, the detection in patients with a risk constellation may indicate the need for further clinical and radiological investigation. Microscopic or cultural detection is practically never successful in blood and CSF samples.
Only a small number of commercially available, direct molecular genetic detection tests have been validated. Such tests should be left to specialized laboratories. Result interpretation is only meaningful in the context of risk constellation and other laboratory and radiological findings.
The galactomannan ELISA is the most frequently used for antigen detection. It has only been adequately validated for hemato-oncological patients. In these patients, the diagnostic sensitivity for the detection of invasive infection is 70–92% and the specificity is approximately 90%. The diagnostic sensitivity in patients under antimycotic therapy has been improved by reducing the index from 1.5 to 0.5. Little information is available on other groups of risk patients (e.g., patients following organ transplantation or intensive care patients). However, it indicates a markedly lower diagnostic sensitivity, probably based on the rapid degradation of circulating galactomannan in undisturbed cell poiesis.
False positive results can be caused by β-lactam antibiotics, nutritional solutions or translocation of fungal antigens from the gastrointestinal tract. Therefore, a positive result only indicates the presence of an infection and should be confirmed by a second sample in the near term. One or two screening tests at weekly intervals appear to be justified in patients with prolonged neutropenia.
Investigations using broncho alveolar lavage specimens have a markedly higher diagnostic sensitivity compared to serum specimens. A study showed a diagnostic sensitivity of 96% and a specificity of 88% for an ELISA index threshold value of 1.0 in patients with hemato-oncological disease. CSF specimens can also be analyzed. However, the test is currently only approved for serum. The antigen concentration decreases rapidly under sufficient treatment. Thus, the test can be used for treatment monitoring.
Only a few and inconsistent validation results are available regarding test methods for the detection of (1,3)-β-D-glucans. Positive results are to be expected in invasive Candida and mold infections (not in mucoromycotina infections and cryptococcosis). The antigen is also detectable in serum in pneumocystosis. Diagnostic sensitivities of 58–100% and specificities of 88–98% have been described regarding the detection of invasive mycosis. False positive results are obtained due to intravenously administered antibiotics, use of OR gauze and bacteremia.
Other molds (Mucoromycotina sp., Fusarium sp., Scedosporium/Pseudallescheria sp.) rarely act as pathogens of invasive infection, but have been observed increasingly often /, /. Besides immunosuppression (neutropenia, steroid therapy), risk factors also include diabetes mellitus (Mucoromycotina sp.), hemochromatosis (Mucoromycotina sp.) or almost-drowning (Scedosporium sp., Pseudallescheria sp.). The mortality rate is 40–90% depending on the underlying disease. The diagnosis is based on histologic and cultural detection. Commercial tests for antigen or antibody detection are not available. Molecular biological detection systems have been described.
Pneumocystis jirovecii (formerly carinii ssp. humanis) is an important pathogen of pulmonary infection (pneumocystosis) in immunocompromised patients . This pathogen cannot be cultured in vitro and was considered to be a parasite for a long time. Based on molecular biological research data, however, it is now classified as a fungus (ascomycete). Pneumocystis sp. can be detected in practically all mammals, but its host specificity is high. It occurs in two basic structural forms: an ameboid trophozoite and a cyst with eight nuclei. The trophozoite binds to type 1 alveolar epithelial cells in a mucous extracellular matrix which impairs the gas exchange. The cysts are thought to additionally induce inflammatory response.
Routes of infection
Infection is presumably acquired by inhalation. The human is the only reservoir for the human-pathogenic Pneumocystis species P. jirovecii detectable to date /, /. The structure of the infectious pathogen has not been elucidated to date. Outbreaks in risk patients have been reported.
Prevalence and incidence
According to serological analysis, the clinically inapparent primary infection in immunocompetent individuals occurs in early childhood. Information obtained by molecular genetic investigation indicates that pneumocysts can in many cases be detected in immunocompetent individuals at obviously low microorganism concentrations without overt clinical symptoms. The main risk factors for a clinically apparent infection include a decreased CD4+T cell count (below 0.2 × 109/L) and immunosuppressive therapy. AIDS patients, hemato-oncological patients, transplantation patients and patients with autoimmune disease belong, therefore, to the risk groups. Recent investigations indicate a correlation between clinical deterioration in COPD and Pneumocysts.
Partial to global respiratory insufficiency with low fever and unproductive cough is the predominant clinical symptom. Elevated LD values are typical, but not specific to pneumocystosis. Radiological examination reveals atypical pneumonia. From a differential diagnostic point of view, Mycoplasma, Chlamydophila, Legionella infection or mycobacteriosis must be taken into consideration. Systemic infections are rare.
Detection of organisms by microscopy
A number of staining methods are available for the detection of the pathogen in respiratory specimens such as broncho alveolar lavage (BAL) and induced sputum. The Giemsa staining method will detect both trophozoites and cysts, whereas methods using Grocott-Gomori silver staining, toluidine blue O staining and staining with optical brightening agents (e.g., calcofluor white) will only detect cysts. The diagnostic specificity of these methods is higher than 99% and the sensitivity in BAL is 70–80%. The sensitivity of immunofluorescence techniques is higher (90%). Since the pathogens tend to form clusters in many cases, all respiratory specimens should be liquefied and several preparations be examined.
Molecular biological detection
Molecular biological methods are also commercially available. Real-time PCR allows to detect pneumocysts from respiratory tract specimens even if microscopy yielded a negative result. BAL specimens have the highest sensitivity for detection; PCR can, however, also be used to analyze tracheal secretion and induced sputum.
Test methods for the detection of (1,3)-β-D-glucans seem to be helpful for laboratory diagnostics in patients with clinically relevant P. jirovecii infection. A recent study showed a diagnostic sensitivity of 92% and a specificity of 65%. Refer to .
Microscopy is the gold standard for the laboratory detection of Pneumocystis pneumonia. Positive results correlate well with the clinical picture. The choice of staining method depends on the experience of the laboratory. Commercially available molecular biological procedures have not yet been adequately validated. Since the level of infection is obviously high, also in the healthy population, a positive test result in the presence of a negative microscopy result should be interpreted with reservation regarding the need for treatment because microscopy does not allow to differentiate between colonization and an infection requiring treatment.
The serum detection of (1,3)-β-D-glucans has a diagnostic sensitivity > 90%. However, the specificity is limited because the test also covers many other fungi (e.g., Candida, Aspergillus) and yields false positive findings in many cases. This test method is commendable for therapy monitoring.
Test procedures for antibody detection are not commercially available and are unnecessary for diagnosis because of the high seroprevalence, on the one hand, and immunosuppression of affected patients, on the other.
Histoplasma capsulatum var. capsulatum (teleomorph stage: Ajellomyces capsulatus) is an obligate pathogenic, dimorphic fungus. The distinction of the H. capsulatum varieties (H. capsulatum var. duboisii and H. capsulatum var. farcinimosum) is no longer valid because of recent phylogenetic findings. Certain genotypes of H. capsulatum occur at different frequencies. In natural environments and in vitro at 25–30 °C, the fungus grows in the saprophytic mycelial phase, forming infectious microconidia (2–5 μm) and characteristic tuberculate macroconidia (8–15 μm). In vitro at 37 °C, the parasitic yeast phase develops which is composed of globose and oval budding cells with a diameter of 2–4 μm.
The American histoplasmosis is endemic to the Midwest regions of the USA, in particular to the valleys of the Mississippi, Ohio and Missouri, and to parts of Central and South America. Sporadic cases have been reported worldwide. The large cell variant of H. capsulatum (formerly H. capsulatum var. duboisii) is endemic to Central and West Africa. In a study on histoplasmoses in Europe, 46 cases were reported in Germany between 1995 and 1999. The majority of patients came from endemic regions.
Routes of infection
The natural reservoir of H. capsulatum consists of soil with high nitrate contents (including bird and bat feces which are a particularly potent growth enhancer) in warm and humid regions. The infection is acquired by inhalation of dust containing hyphal fragments and conidia. Direct person-to-person or bird-to-person transmission does not occur. Birds and bats can be intestinally colonized by Histoplasma, but only bats become diseased because of their lower body temperature.
Prevalence and incidence
According to studies in endemic regions (Tennessee, Kentucky) using the histoplasmin skin test, the prevalence of infection can amount up to 90%. Men and women are exposed equally frequently. However, apparent infection is more frequently encountered in men. In the USA, an estimated number of 500,000 individuals are infected each year.
Depending on the location, differentiation is made between acute pulmonary, chronic pulmonary and disseminated types of histoplasmosis as well as between primary and reactivation induced types. The mean incubation period is 10 days. In 90% of cases, the primary type of the disease is asymptomatic or produces influenza like symptoms.
The course and severity of the disease depend on the infection dose and the immune status of the infected individual. Complex courses can lead to lobar pneumonia or pericarditis.
After infection, calcifications of the hilar and/or mediastinal lymph nodes or pulmonary lesions (so-called coin lesions) can remain.
Chronic pulmonary histoplasmosis usually occurs in older patients (above 50 years) with preexisting pulmonary emphysema, manifests as fibrosing or cavitary pulmonary disease and takes a clinical course resembling tuberculosis.
The disseminated type (1 in 2,000–5,000 clinically manifest infections) occurs primarily in immunocompromised patients and especially in those with AIDS. Besides the lung, there is involvement of the lymph nodes, liver, spleen, bone marrow, meninges, gastrointestinal tract, skin and mucosal surfaces. Chronic types of the disease are characterized by nonspecific symptoms over the course of many years. Disseminated histoplasmosis is an AIDS defining disease. From the differential diagnosis point of view, pulmonary histoplasmosis must be distinguished from sarcoidosis, tuberculosis, atypical mycobacteriosis, blastomycosis as well as paracoccidioidosis and coccidioidosis.
In cases of African histoplasmosis, pulmonary involvement is the exception. Instead, subcutaneous foci of infection, granulomatous alterations within the oral cavity and osseous lesions are predominant.
Microscopic and cultural detection of organisms
The microscopic detection of the organism is most likely accomplished by examining histological slides, biopsy samples and bone marrow smears using the Giemsa staining or the Grocott-Gomori silver staining technique. The intracellular location (macrophages) of the budding cells is characteristic /, /.
Blood, bone marrow, respiratory secretions and tissue samples are suited to detect the pathogen by culture. Standard culture media such as Sabouraud glucose agar can be used. An incubation period of 4–6 weeks at 30 °C should be allowed due to the slow growth of the organism. The yeast stages are cultured at 37 °C on agars containing blood and cystine.
Successful cultural detection can primarily be expected in disseminated and chronic pulmonary histoplasmosis. Even in cases of disseminated histoplasmosis in patients with AIDS, positive cultures of the organism are found in approximately 90% of cases. The organism can be identified based on the above mentioned micromorphological characteristics of the macro and microconidia.
The time consuming cultural detection of dual phase growth has been replaced by highly specific, commercially available gene probes. Specific handling of the pathogen is only permitted in laboratories under laboratory biosafety level L3 (Ordinance on Biological Agents) because of the risk of laboratory infection.
H. capsulatum antigen (heat-stable polysaccharide) can be detected in urine, serum, cerebrospinal fluid and broncho alveolar lavage fluid. The commercially available test is suited to diagnose disseminated histoplasmosis. Diagnostic sensitivities are /, /:
- Approximately 90% in disseminated histoplasmosis
- Approximately 40% in chronic pulmonary histoplasmosis
- Approximately 20% in acute pulmonary histoplasmosis.
A diagnostic specificity of 98% has been reported for urine samples.
False positive reactions can occur in coccidioidomycosis, para coccidioidomycosis or blastomycosis. Rheumatoid factors and treatment with rabbit thymoglobulin can cause false positive reactions in serum. Antigen detection is suited as a marker for disease and therapy monitoring in AIDS patients.
Antibody tests play an important role in the diagnosis of the various types of histoplasmosis. Filtrates of H. capsulatum mycelial cultures (histoplasmin) and extracts of the yeast phase are used as antigen preparations.
Complement fixation test
The test is performed using serum and the customary complement fixation technique. A four-fold increase in the titer confirms the suspicion of an active infection process. Cross reactions with coccidioidomycosis, blastomycosis and para coccidioidomycosis are possible.
The test is performed in agarose according to the Ouchterlony technique. Differentiation is made between two diagnostically relevant precipitate bands. The M-band may occur during the early stage of the disease and shows prolonged persistence even in inactive processes. An additional H-band is considered to be an indicator for active infection. Immunodiffusion tests are less sensitive (80%) than the complement fixation tests but superior as far as clinical specificity is concerned.
Latex agglutination test
Latex particles coated with histoplasmin are agglutinated by antibodies in the serum. IgM antibodies are primarily detected. Therefore, the test is positive mostly in acute infections.
This test uses yeast phase antigen. The diagnostic sensitivity is 97% and the specificity is 84%. False positive reactions are seen in the presence of invasive aspergillosis, blastomycosis and nonspecific pulmonary diseases.
Antibodies occur 2–4 weeks after exposure and may persist for a long time. Depending on the type of the disease, 60–80% of the cases can be seroreactive. Seroconversion may not occur in immunocompromised patients (particularly AIDS patients). Cross reactions with other fungi have been observed. Compared to the standard methods (complement fixation and immunodiffusion tests), the latex agglutination test and the enzyme immunoassay do not achieve any improvement in serodiagnostics.
The histoplasmin skin test plays a significant role in epidemiological investigations in endemic regions. It is, however, not recommended for diagnostic purposes.
PCR detection of histoplasma DNA has been described, but is not yet commercially available.
45.8 Coccidioides immitis, Coccidioides posadasii (coccidioidomycosis, Valley fever, San Joaquin fever, Desert rheumatism)
C. immitis and C. posadasii are obligate pathogenic, dimorphic fungi causing coccidioidomycosis . In the saprophytic mycelial phase, they form infectious arthroconidia (2–5 μm) which are propelled by the slightest movements of the air. Arthroconidia are defined as a genetically programmed disarticulation of septate hyphae to produce a chain of conidia. Arthroconidia are produced by dermatophytes, both in culture and in lesions, under certain predisposing conditions. In their host, the arthroconidia transform into round structures with thick walls, so-called spherules (10–80 μm), containing up to 600 endospores (2–5 μm). Following the rupture of spherules, each endospore may give rise to the development of a new spherule.
Coccidioidomycosis is endemic to deserts, semi deserts, and plains (arid regions) of the American continent, especially in the Southwest of the USA (California, Arizona, Nevada, New Mexico, Utah, Texas) and the adjacent regions of northern Mexico.
Routes of infection
The natural reservoir of C. immitis and C. posadasii is dry, sandy and dusty soil containing the dwellings of rodents which provide especially suitable conditions for the development of the organism. Infection is acquired by inhalation of arthroconidia which are spread, in particular, by sand storms and construction work.
It is not necessary to isolate individuals afflicted by this disease since the tissue forms of the organism are not infectious. Excreted endospores may, however, grow into mycelia and infectious arthroconidia, thus necessitating disinfection of body secretions or specimens contaminated with them.
Prevalence and incidence
In the USA, the estimated numbers are approximately 100,000 infected individuals/year and approximately 500–5000 individuals afflicted by disease/year. Most infections occur in the late summer or early fall.
About 60% of infected individuals have an asymptomatic course of the infection which manifests by transient, influenza like symptoms. In approximately 25% of infected individuals, acute infection of the lower respiratory tract develops 1–3 weeks after exposure and may be associated with arthralgias (desert rheumatism), erythema multiforme or erythema nodosum. This acute, pulmonary (primary) form of the disease usually heals spontaneously in immunocompetent individuals after a few weeks. In rare cases, there is a transition from the acute to a chronic pulmonary form of the disease with cavity formation, pleural empyema and bronchopleural fistulae. Extrapulmonary dissemination, which may occur both in the acute and chronic forms of the disease, is frequently encountered in immunocompromised individuals (approximately 0.5% of infected patients). The granulomatous or abscess like lesions mostly affect the skin, soft tissues, bones and meninges. Coccidioidomycosis closely resembles tuberculosis but other nonspecific pulmonary infections, sporotrichosis, blastomycosis, leishmaniasis, paracoccidioidomycosis and malignant tumors also need to be considered.
Microscopic and cultural detection of organisms
Sputum, purulent secretions, aspirated fluid, cerebrospinal fluid and tissue samples are primarily used as specimens. Silver staining according to Grocott-Gomori or labeling with optical brightening agents allow the detection of the characteristic spherules (20 to 70 μm) with endospores (2–5 μm). Cultural detection is achieved on customary nutrient culture media within an incubation period of 1 week at 20–30 °C (mycelial phase). Identification of the species necessitates transformation into the yeast phase using special media or animal tests.
A commercially available test for the specific detection of C. immitis DNA by culture yields results within one day. Specific handling of C. immitis and C. posadasii is only permitted in laboratories under laboratory biosafety level L3 (Ordinance on Biological Agents) because of the risk of laboratory infection.
Autolysates and cultural filtrates of the mycelial phase (i.e., so-called coccidioidin) are used as antigen preparations. Serum and cerebrospinal fluid (CSF) are primarily suited as specimens.
Complement fixation test
Diluted, inactivated (56 °C, 30 min.) serum or CSF is incubated with coccidioidin solution and a defined amount of complement for 2 h at 37 °C (alternatively for 18 h at 4 °C) followed by another incubation period of 1 h at 37 °C after the addition of hemolysin sensitized erythrocytes. A lack of hemolysis indicates the presence of antibodies. The test detects IgG antibodies. Threshold titer is 1 : 8 in serum and 1 : 2 in CSF. Titers lower than this require confirmation by means of immunodiffusion testing. Data concerning diagnostic sensitivity and specificity are not available.
Using the Ouchterlony technique, application sites punched out in agarose gel are filled with serum or CSF on the one side and with coccidioidin solution on the opposite side. Pre diffusion of the test fluid prior to the application of antigen is beneficial. After incubation, an antigen-antibody reaction is revealed by the occurrence of precipitate bands. If heated (60 °C, 30 min.) coccidioidin is used, IgM antibodies will react as they do in the tube immunoprecipitation test. This modification of immunodiffusion (ID) is therefore referred to as IDTP. If non heated antigens are used, IgG antibodies, in particular, will react. This ID modification is called IDCF. Diagnostic sensitivity is higher than in the complement fixation reaction.
Latex agglutination test
Latex particles coated with heated coccidioidin serve as the test mixture. The test primarily detects IgM antibodies. The test is considered to be a sensitive screening method but it has the disadvantage of false positive reactions at a rate of approximately 15%. Positive reactions, therefore, must be confirmed by the more specific immunodiffusion tests.
Wells of micro titer plates are coated with coccidioidin. Bound antibodies in diluted serum samples are detected by monovalent IgM or IgG specific conjugates. Diagnostic sensitivity is 100% in the case of joint evaluation of IgM and IgG reactions. Diagnostic specificity is 96%. False positive reactions are observed in the presence of blastomycosis and pulmonary infections of other causes.
The Coccidioides galactomannan is detectable in the serum and urine of patients with a severe course of the disease. The test can also be helpful for diagnosing recently acquired infections when antibody production cannot yet be detected. Antigen detection is also a surrogate marker of fungal load in disseminated infection. In a study , it was shown that EDTA heat pretreatment of samples improves the diagnostic sensitivity to 73% in serum samples and to 50% in urine samples. The test achieved a high specificity in healthy individuals, but cross reactions were seen in 22% of patients with histoplasmosis or blastomycosis /, /.
Coccidioidin and spherulin are used as test antigens. Performance and interpretation are analogous to the tuberculin skin test. The skin test does not allow to differentiate between latent and overt infection. Anergy can occur in massive dissemination and progressive course of the disease. The test is used to determine the level of infection in endemic regions, but is not commercially available /, , /.
IgM antibodies in the serum (tube immunoprecipitation, IDTP, latex agglutination) are detectable within 2–3 weeks after the occurrence of symptoms. They are usually no longer present after 6 months but may appear again and/or persist in the case of relapse and dissemination. IgG antibodies (complement fixation, IDCF) usually do not occur until 2–3 months after the onset of the disease. A complement fixation titer above 16 is considered to be a sign of florid infection and raises the suspicion of extra pulmonary dissemination. Complement fixation examinations at 3–4 week intervals are recommended for monitoring. A significant titer decrease under therapy is interpreted as a favorable prognostic sign. Detection of IgM antibodies in cerebrospinal fluid, in the case of meningitis, is the exception rather than the rule whereas IgG antibodies are usually detectable although sometimes not until several weeks after the onset of the disease /, , , , /.
Blastomyces dermatitidis (teleomorph stage: Ajellomyces dermatitidis) is a temperature dependent, dimorphic fungus. B. dermatitidis grows in its natural environment and in vitro at 25–30 °C in the mycelial phase and at 37 °C in the yeast phase. The yeast cells can vary in size (8–15 μm), are thick walled and characteristically bear one daughter cell with a wide bud base.
No precise information is available about the incidence and epidemiology of blastomycosis because the pathogen is difficult to detect in the environment and no sensitive skin test is available. Case reports have suggested that the distribution of blastomycosis is similar to that of histoplasmosis. Blastomycosis is endemic to the USA, especially to the regions of the Mississippi and Ohio Rivers and the Midwest, well as the Canadian regions bordering on the Great Lakes and along the St. Lawrence River. Sporadic cases have been reported from Hawaii, Central and South America, Africa and the Middle East. No case of blastomycosis acquired in Germany has been reported to date.
Routes of infection
Moist soil containing decomposing plants and decaying wood (river banks, beaver dams) is the natural reservoir of the fungus. The infection is most commonly acquired by conidia inhalation. Direct infections due to skin lesions during outdoor activity or after a dog bite have also been described. Person-to-person transmission is a rarity.
Prevalence and incidence
Blastomycosis is a rare disease. In a study from Wisconsin covering the years 2000 and 2004, the rate of clinically overt infections in urban US areas such as Milwaukee was estimated to be 1–3/100,000 inhabitants. The incidence in individuals living near rivers was 74/100,000 inhabitants.
Blastomycosis can be asymptomatic, manifest as acute or chronic pneumonia or take a disseminated form of the disease. Acute infection of the lung manifests after a 30–45 day incubation period with uncharacteristic, influenza like symptoms. If untreated, blastomycosis in many cases assumes a chronic form manifesting in the lungs as granulomatous, abscess like lesions and resembling the clinical picture of tuberculosis. Extra pulmonary manifestations involve the skin (verrucous or ulcerative lesions) in 40–80% of cases, bone (osteomyelitis) in 10–15% of cases, urogenital tract (primarily prostatitis, epididymitis in men) and, more rarely, the central nervous system.
Direct microscopic and cultural detection of organisms
Respiratory secretions, exudates, tissue samples and urine are suitable specimens for examination . The budding cells with a wide bud based daughter cell are characteristic findings in the direct microscopic preparation (native or with optical brightening agent). Cultural detection is performed at 25–30 °C and at 37 °C on Sabouraud glucose agar and blood enriched brain-heart infusion agar for a time period of at least 14 days. Culturing from primarily non sterile specimens should be performed on culture medium containing inhibitors such as chloramphenicol or cycloheximide agar.
The detection of antibodies to Blastomyces dermatitidis antigens is possible by immunodiffusion (Ouchterlouny) as well as by enzyme immunoassay and complement fixation test. Immunodiffusion is the only commercially available test. Extracts from the yeast phase are used as antigens in immunodiffusion testing. In the agarose gel technique according to Ouchterlony, antibodies in the serum react by forming precipitin lines (A and B bands). The diagnostic sensitivity in pulmonary blastomycosis is 65–80% at a specificity of approximately 30% /, , , /
Antigen detection, molecular biological detection
A test kit for urine Blastomyces antigen detection is commercially available. Clinical sensitivity is 93% at low specificity. The test yields a positive result even in the presence of blastomycosis, histoplasmosis, para coccidioidomycosis and penicillinosis. A test kit for pathogen detection by PCR is not commercially available. Broncho alveolar lavage, urine, blood and other body fluid samples can be sent to the consultant laboratory for pathogens of extra European systemic mycoses at the Robert-Koch Institute for immunological and molecular biological analyses /, , /.
The pathogen P. brasiliensis is a temperature dependent, dimorphic fungus. It grows in cultures at 37 °C and in vivo in the yeast form with characteristic round or oval cells (up to 60 μm) surrounded by multiple peripheral buds (2–10 μm) also referred to as pilot wheel. At low temperatures, a mycelium is formed from thin septate hyphae with differently shaped conidia (3.5–5 μm).
Routes of infection
The natural reservoir of P. brasiliensis is not exactly known. To date, the fungus has only been identified twice in soil samples. Epidemiological and experimental data suggest that the ecological niches are located within humid, warm and forested areas. Fish, amphibians and armadillos possibly play a role in the life cycle of the fungus. The infection is probably acquired by inhaling conidia present in the environment. Person-to-person transmission does not occur /, , /.
Prevalence and incidence
Para coccidioidomycosis is endemic to regions from Mexico to Argentina. Approximately 80% of recorded infections occurred in Brazil, followed by Colombia and Venezuela. All clinically overt infections diagnosed outside these regions, in many cases after years of latency, can be traced back to stays in endemic regions. Infections are usually inapparent in immunocompetent individuals. In many cases, patients aged 30–50 years are affected. The disease occurs 15 times more often in men than in women. In endemic regions, approximately 5 clinically overt cases of the disease per 1 million people occur each year. The infection rate in the population is estimated to be 10–20%.
The clinical picture of this chronic progressive disease resembles that of tuberculosis. Differentiation is made between a juvenile form of the disease with an acute or subacute course (3–5% of the cases) and an adult form with a chronic course (> 90% of cases). In the juvenile, rapidly generalizing form, pulmonary dissemination into the spleen, liver, lymph nodes and bone marrow is the main characteristic. The adult form of the disease, which develops over the course of months and years, only affects the lung in approximately 25% of the cases (unifocal form). It leads to emphysema and fibrous abnormalities in the lung. Dissemination (multi focal form) occurs mostly to the oral and nasal mucosa, skin, lymph nodes and adrenal glands.
Microscopic and cultural detection
Sputum, broncho alveolar lavage fluid, exudates, cerebrospinal fluid and tissue samples can be used as specimens. Microscopic examination is performed using native preparations (potassium hydroxide solution) and histologically prepared slides using various staining techniques (e.g., Grocott-Gomorri staining). These methods allow to detect typical yeast cells with multiple budding.
Sabouraud agar is suitable to culture the organism. An incubation period of 10–15 days at 37 °C (yeast form) and of 20–30 days at 25 °C (mycelial form) is necessary for colonies to occur. The diagnostic sensitivity of microscopic and of cultural detection are reported as 85–100% and 86–100%, respectively.
However, these methods often show cross reactions. More recently, a glycoprotein (GP43) was identified, whose peptide component is a carrier of epitopes specific for P. brasiliensis.
According to the Ouchterlony technique, application wells are punched out in agarose gel and filled with antigen solution (e.g., 1 μl of GP43, and/or serum). The diagnostic sensitivity is approximately 90% and the specificity is 100%.
Indirect hemagglutination test
Antigen GP43 adsorbed to sheep erythrocytes is incubated with diluted serum for 1 h at room temperature. Antibodies cause agglutination of the loaded erythrocytes. The diagnostic sensitivity and specificity are 100%.
The available information allows the conclusion that the detection of antibodies to the GP43 antigen clearly indicates the presence of manifest para coccidioidomycosis. The level of antibody titers or titer changes might be helpful to assess disease activity and the success of therapy.
Numerous different antigen preparations (para coccidioidins) have been used for skin tests . By using a polysaccharide antigen, positive reactions were obtained in 67% of confirmed cases of the disease but also in 87% of the references, indicating the presence of subclinical or cross-reacting infections. Skin tests are generally not helpful for diagnosis due to their unspecificity. Cross reactions with histoplasmin have been reported.
The pathogen Sporothrix schenckii is a temperature-dependent, dimorphic fungus . In rich and liquid cultures and in vivo at 37 °C, it grows in the yeast phase, forming characteristic oval or tuberculate daughter cells. At low temperatures, it forms mycelium from septate hyphae with conidia of various shapes that develop a brown/black pigment (melanin) when incubated for an extended period of time.
Routes of infection
Sporotrichosis occurs worldwide. However, most cases have been reported from the tropical and subtropical regions in America. S. schenkii can be found in its natural environment on plants and in the soil. For this reason, sporotrichosis affects farmers, florists, nursery workers, landscapers, greenhouse workers, leisure gardeners (i.e., individuals working with plants, peat or soil). S. schenckii invades the subcutaneous tissue through puncture injuries caused by splinters or thorns. Transmissions from animals to humans have been reported.
Prevalence and incidence
Systematic information has not been available to date because of the low specificity of the reactivity of skin tests using sporotrichin or antibody detection tests. The disease occurs in humans of any age, preferably in young individuals aged between 10 and 40 years.
Infections are usually sporadic. They have often been described in connection with forest or peat work. Epidemic outbreaks following feline contact, infection after armadillo hunting and person-to-person transmission have been reported. Epidemic outbreaks in pets such as dogs and cats have also been described.
An ulcerative, verrucous or reddish lesion, in some cases associated with lymphangitis, develops at the site of invasion.
After inhalation of the spores, the fungus can cause granulomatous, often bullous, pneumonia with symptoms resembling tuberculosis. Hematogenous dissemination via central nervous, articular and ocular infiltrate has been reported. Disseminated sporotrichosis has been described to occur in immunocompromised individuals.
Microscopic and cultural detection of organisms
Cultural detection is the gold standard for the detection of sporotrichosis. Biopsy samples or tissue scrapings from skin lesions, respiratory secretions and cerebrospinal fluid are suited as specimens for culturing and analysis. Culturing should at first be performed at room temperature and subsequently at 37 °C to confirm the identity of the biphasic fungus /, , /.
The detection of antibodies by immunodiffusion and/or immunoelectrophoresis has already been used successfully for the disseminated form of sporotrichosis. However, because of its low sensitivity to the most frequently encountered cutaneous form of the disease, antibody detection has not been established in routine diagnostics. Reliable diagnosis of the cutaneous form of sporotrichosis has been achieved by using an antigen (SsCBF) derived from a peptido-rhamnomannan component of the cell wall. Based on this antigen, an ELISA was developed showing a diagnostic sensitivity of 90% and a specificity of 86% in various forms of sporotrichosis. However, the usability of this ELISA is limited because of the difficult antigen preparation and the low robustness of the test. At the same time, an antibody assay was developed which is based on the easily obtained exoantigens of the mycelial phase. These antigens do not show cross reactivity in the presence of coccidioidomycosis, histoplasmosis or paracoccidioidomycosis and/or chromoblastomycosis and leishmaniosis. However, cross reactions with numerous molds have been observed in antigen preparations.
Sporotrichin is the yeast form of a cell wall glycopeptide already used since the early days of sporotrichosis diagnostics. In many cases, however, it showed false positive as well as false negative reactions. Therefore, the skin test should not be applied for routine serodiagnosis .
8. Haase G, Borg-von Zepelin M, Bernhardt H, Fegeler W, Harmsen D, Kappe R, et al. Qualitätsstandards in der mikrobiologisch-infektiologischen Diagnostik. Pilzinfektionen Teil I (MiQ 14), Teil II (MiQ 15), 2001. München Jena Urban & Fischer.
15. Clancy CJ, Nguyen MH. Finding the missing 50% of invasive candidiasis: how nonculture diagnostics will improve understanding of disease spectrum and transform patient care. Clin Infect Dis 2013; 56: 1284–92.
19. Prella M, Bille J, Pugnale M, Duvoisin B, Cavassini M, Calandra T, et al. Early diagnosis of invasive candidiasis with mannan antigenemia and antimannan antibodies. Diag Microbiol Infect Dis 2005; 51: 95–101.
20. Sendid B, Poirot JL, Tabouret M, Bonnin A, Caillot D, Camus D, et al. Combined detection of mannanaemia and anti-mannan antibodies as a strategy for the diagnosis of systemic infection caused by pathogenic Candida species. J Med Microbiol 2002; 51: 433–42.
22. Miyazaki T, Kohno S, Mitsutake K, Maesaki S, Tanak K-I, Ishikawa N, et al. Plasma (1–3)-β-D-glucan and fungal antigenemia in patients with candidemia, aspergillosis, and cryptococcosis. J Clin Microbiol 1995; 33: 3115–8.
23. Ostrowsky-Zeichner L, Alexander BD, Kett DH, Vazquez J, Pappas PG, Saeki F, et al. Multicenter clinical evaluation of the (1–3) β-D-Glucan assay as an aid to diagnosis of fungal infections in humans. Clin Infect Dis 2005; 41: 654–9.
26. Sendid B, Caillot D, Baccouch-Humbert B, Klingspor L, Grandjean M, Bonnin A, et al. Contribution of the Platelia Candida-Specific Antibody and Antigen Tests to early diagnosis of systemic Candida tropicalis infection in neutropenic patients. J Clin Microbiol 2003; 41: 4551–8.
27. Clancy CJ, Nguyen M-L, Cheng S, Huang H, Fan G, et al. Immunoglobulin G responses to a panel of Candida albicans antigens as accurate and early markers for the presence of systemic candidiasis. J Clin Microbiol 2008; 46: 1647–54.
30. Perfect JR. Cryptococcus neoformans. In: Mandell, Douglas and Bennett’s Principles and Practice of Infectious Disease. Mandell GL, Bennett JE, Dolin R eds. Philadelphia; Churchill Livingstone 2010. p. 3287–3301.
33. Bialek R, Weiss M, Bekure-Nemariam K, Najvar LK, Alberdi MB, Graybill JR, et al. Detection of Cryptococcus neoformans DNA in tissue samples by nested and real-time PCR assays. Clin Diag Lab Immunol 2002; 9: 461–9.
34. Lindsay MD, Mekha N, Baggett HC, Surinthong Y, Autthateinchai R, Sawatwong P, et al. Evaluation of a newly developed lateral flow immunoassay for the diagnosis of cryptococcosis. Clin Infect Dis 2011; 53: 321–5.
36. Patterson TF. Aspergillus species. In: Mandell, Douglas and Bennett’s Principles and Practice of Infectious Disease. Mandell GL, Bennett JE, Dolin R eds. Philadelphia; Churchill Livingstone 2010. p. 3241–55.
39. Buchheidt D, Baust C, Skladny H, Ritter J, Suedhoff T, Baldus M, et al. Detection of Aspergillus species in blood and bronchoalveolar lavage samples from immunocompromised patients by means of 2-step polymerase chain reaction: Clinical results. Clin Infect Dis 2001; 33: 428–35.
40. Chen SC-A, Kontoyiannis DP. New molecular and surrogate biomarker-based tests in the diagnosis of bacterial and fungal infection in febrile neutropenic patients. Curr Opin Infect Dis 2010; 23: 567–77.
41. Tuon FF. A systematic literature review on the diagnosis of invasive aspergillosis using polymerase chain reaction (PCR) from bronchoalveolar lavage clinical samples. Rev Iberoam Micolog 2007; 24: 89–94.
42. Spies B, Seifarth W, Hummel M, Frank O, Fabarius A, Zheng C, et al. DNA Microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients. J Clin Microbiol 2007; 45: 3743–53.
43. Maertens J, Maertens V, Theunissen K, Meersseman W, Meersseman P, Meers S, et al. Bronchoalveolar lavage fluid galactomannan for the diagnosis of invasive pulmonary aspergillosis in patients with hematologic diseases. Clin Infect Dis 2009; 49: 1688–93.
44. Marr KA, Balajee SA, McLaughlin L, Tabouret M, Bentsen C, Walsh TJ. Detection of galactomannan antigenemia by enzyme immunoassay for the diagnosis of invasive aspergillosis: variables that affect performance. J Infect Dis 2004; 190: 641–9.
45. Mennink-Kersten MASH, Ruegebrink D, Klont RR, Warris A, Blijlevens NAM et al. Improved detection of circulating Aspergillus antigen by use of a modified pretreatment procedure. J Clin Microbiol 2008; 46: 1391–97.
48. Chamilos G, Luna M, Lewis RE, Bodey GP, Chemaly R, Tarrand JJ, et al. Invasive fungal infections in patients with hematologic malignancies in a tertiary care cancer center: an autopsy study over a 15-year period (1989–2003). Haematologica 2006; 91: 986–9.
51. Rath PM, Schmid EN, Ansorg R. Vergleich zwischen mikroskopischen Methoden und Polymerasekettenreaktion zum Nachweis von Pneumocystis carinii in bronchoalveolären Lavage-Flüssigkeiten. J Lab Med 1996; 20: 553–9.
52. Sax PE, Komarow L, Finkelman MA, Grant PM, Andersen J, Scully E, et al. Blood (1–3)-β-D-Glucan as a diagnostic test for HIV-related Pneumocystis jirovecii pneumonia. Clin Infect Dis 2011; 53: 197–202.
55. Ashbee HR, Evans EGV, Ashbee HR, Evans EGV, Viviani MA, Dupont B, et al. Histoplasmosis in Europe: Report on an epidemiological survey from the European Confederation of Medical Mycology Working Group. Med Mycol 2008; 46: 57–65.
56. Durkin MM, Connolly PA, Karimi K, Wheat E, Schnizlein-Bick C, Allen SD, et al. Pathogenic differences between North American and Latin American strains of Histoplasma capsulatum var. capsulatum in experimentally infected mice. J Clin Microbiol 2004; 42: 4370–3.
57. Karimi K, Wheat LJ, Connolly P, Cloud G, Hajjeh R, Wheat E, et al. Differences in histoplasmosis in patients with acquired immunodeficiency syndrome in the United States and Brazil. J Infect Dis 2002; 186: 1655–60.
58. Spitzer ED, Keath EJ, Travis SJ, Painter AA, Kobayashi GS, Medoff G. Temperature-sensitive variants of Histoplasma capsulatum isolated from patients with acquired immunodeficiency syndrome. J Infect Dis 1990; 162: 258–61.
61. Sekhon AS, Kaufman L, Kobayashi GS, Moledina N, Jalbert M, Notenboom RH. Comparative evaluation of the Premier enzyme immunoassay, micro-immunodiffusion and complement fixation tests for the detection of Histoplasma capsulatum var. capsulatum antibodies. Mycoses 1994; 37: 313–6.
63. Galgiani JN. Coccidioides species. In: Mandell, Douglas and Bennett’s Principles and Practice of Infectious Disease. Mandell GL, Bennett JE,Dolin R eds. Philadelphia; Churchill Livingstone 2010. p. 3333–44.
65. Durkin M, Estok L, Hospenthal D, Crum-Cianflone N, Swartzentruber S, Hackett E, et al. Detection of Coccidioides antigenemia following dissociation of immune complexes. Clin Vaccine Immunol 2009; 16: 1453–6.
66. Binnicker MJ, Popa AS, Catania J, Alexov M, Tsaras G, Lloyd F, et al. Meningeal coccidioidomycosis diagnosed by real-time polymerase chain reaction analysis of cerebrospinal fluid. Mycopathologia 2011; 171: 285–9.
67. Kaufman L, Sekhon AS, Moledina N, Jalbert M, Pappagianis D. Comparative evaluation of commercial premier EIA and microimmunodiffusion and complement fixation tests for Coccidioides immitis antibodies. J Clin Microbiol 1995; 33: 618–9.
71. Baumgardner DJ, Knavel EM, Steber D, Swain GR. Geographic distribution of human blastomycosis cases in Milwaukee, Wisconsin, USA: association with urban watersheds. Mycopathologia 2006; 161: 275–82.
74. Bariola JR, Hage CA, Durkin M, Bensadoun E, Gubbins PO, Wheat LJ, et al. Detection of Blastomyces dermatitidis antigen in patients with newly diagnosed blastomycosis. Diag Microbiol Infect Dis 2011; 69: 187–91.
76. Restrepo A, Tobon AM. Paracoccidioides brasiliensis. In: Mandell, Douglas and Bennett’s Principles and Practice of Infectious Disease. Mandell GL, Bennett JE,Dolin R editors. 7th ed. Philadelphia; Churchill Livingstone 2010. p. 3357–3366.
82. Rex JH, Okhuysen PC. Sporothrix schenckii. In: Mandell, Douglas and Bennett’s Principles and Practice of Infectious Disease. Mandell GL, Bennett JE,Dolin R editors. 7th ed. Philadelphia; Churchill Livingstone 2010. p. 3357–3366.
83. Almeida-Paes R, Pimenta MA, Pizzini CV, Monteiro PCF, Peralta JM, Nosanchuk JD, et al. Use of mycelial-phase Sporothrix schenckii exoantigens in an enzyme-linked immunosorbent assay for diagnosis of sporotrichosis by antibody detection. Clin Vaccine Immunol 2007; 14: 244–9.
84. Bernardes-Engemann AR, Orofino Costa RC, Miguens BP, Penha CVL, Neves E, Pereira BAS, et al. Development of an enzyme-linked immunosorbent assay for the serodiagnosis of several clinical forms of sporotrichosis. Med Mycol 2005; 43: 487–93.
85. Fernandes GF, Lopes-Bezerra LM, Bernardes-Engemann AR, Schubach TMP, Dias MAG, Pereira SA, et al. Serodiagnosis of sporotrichosis infection in cats by enzyme-linked immunosorbent assay using a specific antigen, SsCBF, and crude exoantigens. Vet Microbiol 2011; 147: 445–9.