Main characteristics and diagnostic performance of commercial assays for detecting
Early diagnosis of histoplasmosis is essential to establish a suitable antifungal therapy and reduce morbidity and mortality rates. However, laboratory diagnosis remains challenging due to the low availability of proper methods and the lack of clinical suspicion. Conventional diagnosis is still largely used even though limitations are well known. Isolating the fungus is time consuming and requires manipulation in BSL3 facilities, while direct visualization and histopathology techniques show low sensitivity and need skilled personnel. New approaches based on the detection of antibodies and antigens have been developed and commercialized last years. Although sensitivity and specificity of these methods is variable, antigen detection has been recently listed as an essential diagnostic test for AIDS patients due to its excellent performance. DNA detection methods are recognized as promising tools but there is still a lack of consensus among laboratories and there are not commercial tests available. Not all methods are widely available, thus most laboratories combine classical and other tests in order to overcome aforementioned limitations. In this chapter, we review the diagnostic pipeline currently available for the diagnosis of histoplasmosis in microbiological laboratories, from conventional to new developed tests. Most recent approaches are introduced and future perspectives are discussed.
Histoplasmosis is caused by the thermally dimorphic fungi
2. Conventional diagnostic methods
Conventional diagnostic methods are still widely used for the diagnosis of histoplasmosis. The definite diagnosis is based on the isolation of the fungus in culture or the visualization of intracellular yeasts in tissues or other clinical samples. Thus, despite its known limitations, these methods are very useful and continue to be used in many laboratories, especially in low-resources settings.
The isolation of
The sensitivity of cultures depends on the clinical status of the patient and the origin of the sample. Cultures are negative in most cases of asymptomatic and mild disease, however they are useful in disseminated and chronic pulmonary histoplasmosis, although their sensitivity varies from 50 to 85% [2, 15].
The main limitation of cultures is the long turnaround time needed to reach a definite diagnosis. Moreover, the microscopical observation of the grown fungus requires further confirmation by other methods. Molecular identification by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA is a powerful tool to confirm the presence of
Since aforementioned limitations make culture useless for an early diagnosis of histoplasmosis, a great effort has been made for developing alternative diagnostic methods that could be used alone or in combination with cultures.
2.2 Histopathology and direct visualization
Direct examination of clinical samples or performing histopathological studies on tissues to detect yeast after staining with Gomori methenamine silver (GMS) or periodic acid-Schiff (PAS), are techniques largely used for the diagnosis of histoplasmosis.
3. Antigen detection
The detection of
|MV||Serum, plasma, urine, CSF, BALF, other body fluids||ELISA||Urine, BAL: <24 h|
Serum, plasma, CSF: 24 h
MiraVista’s test presents great efficiency in serum and urine samples from patients with disseminated infection, but it is reduced in patients with pulmonary forms of the disease , in which BALF samples are more suitable for diagnosis . This test is only performed in MVista’s facilities then is not accessible out of USA limiting their use. On the other hand, IMMY has recently released an
In last years, point-of-care (POC) testing has been emerged as a new diagnostic methodology and immuno-chromatographic assays performed in lateral-flow devices (LFD) are good examples. These “pregnancy tests-like” assays are easy to use, have low turnaround time (less than an hour) and require minimal laboratory equipment which facilitate its implementation in low- and middle-income countries [32, 33]. This technology was first developed for
4. Antibody detection
The main advantages of antibody detection tests are the requirement of minimally invasive samples and the achievement of results when culture is still negative reducing the need of handling potentially infectious fungi . Serological techniques such as complement fixation or immunodiffusion are useful when testing samples from travelers coming from endemic regions for the first time. Both techniques are commercially available (Immuno Mycologics, Norman, OK, USA) and the sensitivity of these tests in acute and subacute pulmonary histoplasmosis has been reported as 95% . However, sensitivity is very limited in immunosuppressed patients due to low or absent antibody titers. A recent meta-analysis described sensitivity and specificity values of 58% and 100%, respectively, in samples from HIV patients . Finally, since seropositivity remains long time after disease, interpretation of serological results could be challenging [39, 40]. Despite the limitations previously described, antibody detection tests are still considered as valuable diagnostic tools and have been demonstrated to improve diagnostic yield when combined with other diagnostic methods [41, 42].
5. DNA based detection methods
PCR methods based on the detection of fungal DNA directly from clinical samples are currently implemented in the routine of several laboratories for the diagnosis of main fungal infections, but there are considerably fewer PCR tests for the diagnosis of histoplasmosis. Their advantages rely on their simplicity, high specificity and short turnaround time with the bonus that real-time PCR (qPCR) formats allow for determining the fungal burden in patients by using non-specific DNA-binding dyes or fluorescently labeled probes [36, 43]. However, this technique also has some limitations as the moderate amount of DNA in low invasive samples, the lack of standardization and the low availability of widely validated commercial systems [44, 45]. Recently, PCR based methods have been included in the EORTC/MSG criteria for the diagnosis of some fungal infections such as invasive aspergillosis or candidiasis but not for endemic mycoses .
The majority of PCR tests for the diagnosis of histoplasmosis have been developed in house and none of them has been commercialized. They have been recently reviewed in several reports with different purposes [31, 38, 46]. Most developed methods targeted specific multicopy regions of the ribosomal DNA or the single-copy Hcp100 gene and were performed by using conventional, nested or qPCR formats. The sensitivity and specificity of these assays depends on the type of sample analyzed, the clinical characteristics of patients and the PCR format used for DNA detection. These tests showed an excellent analytical performance (overall sensitivity of 95% and specificity of 99%) when testing samples of HIV patients , but sensitivity decreased when testing blood and serum samples from immunocompetent patients [31, 46]. Panfungal or broad-range PCRs are used when there is not a clear suspicion of the fungus involved in the infection, since universal primers are used to detect any fungal DNA in the clinical sample. Although studies are scarce, several reports achieved the detection of
Non-PCR based methods are also able to amplify and detect
6. Conclusions and future perspectives
Early diagnosis of histoplasmosis is essential to establish a suitable antifungal therapy, which results in the reduction of mortality rates [55, 56]. This becomes especially important in certain hyper-endemic regions since they usually are disfavored areas where patients develop the disease in its disseminated form. While culture and histopathological examination are considered the gold standards methods for histoplasmosis diagnosis, these techniques show moderate sensitivity. In addition, culture is time consuming, requiring handling fungi in BSL-3 facilities. New approaches as MALDI-ToF MS technology allow for a rapid identification, but studies are still scarce. Antibody and antigen detection are useful tools for an early detection of the pathogen in low invasive clinical samples such as serum and urine. Despite limitations concerning sensitivity in certain populations and specificity have been widely reported,
All these problems have gained attention thanks to different initiatives coming from researchers from hyper-endemic regions  or international foundations as the Global Action Fund for Fungal Infections (GAFFI). As a result, proposals such as the Manaus declaration have been launched, specifically to get access to rapid testing for histoplasmosis in the Americas and Caribe until 2025 (https://www.gaffi.org/the-manaus-declaration-on-histoplasmosis-in-the-americas-and-caribbean-100-by-2025/). However, in addition to these excellent initiatives further work is required to improve diagnosis. In this sense, novel techniques as next generation sequencing (NGS) have been found to be useful in the diagnosis of several infections. Sequences obtained from clinical samples through NGS can be compared against reference databases enabling their identification to the genus and species level . This technique has been used recently to identify
In summary, the aim of this chapter was to summarize the diagnostic pipeline currently available for the diagnosis of histoplasmosis in microbiological laboratories (Figure 2). Although so much progress has been made in the area, much certainly remains to be done to improve the early diagnosis of histoplasmosis, allowing the establishment of a prompt antifungal therapy and consequently reducing morbidity and mortality rates of this infection.
Conflict of interest
The authors declare no conflict of interest.