Preferred specimens for diagnosis according the histoplasmosis syndromes [1, 2].
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Barely three months into the new year and we are happy to announce a monumental milestone reached - 150 million downloads.
\n\nThis achievement solidifies IntechOpen’s place as a pioneer in Open Access publishing and the home to some of the most relevant scientific research available through Open Access.
\n\nWe are so proud to have worked with so many bright minds throughout the years who have helped us spread knowledge through the power of Open Access and we look forward to continuing to support some of the greatest thinkers of our day.
\n\nThank you for making IntechOpen your place of learning, sharing, and discovery, and here’s to 150 million more!
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This is a thermally dimorphic fungus presenting as a yeast at body temperature and as a hyaline mold in the natural environment. Human histoplasmosis is due to the two varieties of the pathogen:
Histoplasmosis can be challenging to diagnose because it is laborious and requires special characteristics for its revealing. According to the criteria recommended by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) and those of the Council of State and Territorial Epidemiologists (CSTE) [7, 8], an integrated approach including clinical, radiographic, and laboratory evidence is required. For laboratory diagnosis, several techniques are available as microbiology, histopathology, and immune-serological assays, depending on the clinical context and laboratory capabilities. Since 1978, the introduction of the
This review describes the current diagnostics for the laboratory identification of
A pulmonary histoplasmosis presenting as acute, subacute, chronic, or pulmonary nodules.
A progressive disseminated histoplasmosis (PDH) that mainly concerns some particular populations (extreme age groups, immunosuppressed people including AIDS/HIV, iatrogenic origin). However, immunocompetent people defined as without obviously immunodeficiency can develop such syndrome when important inoculum.
A cerebral nervous system (CNS) histoplasmosis.
Other clinical findings as mediastinal histoplasmosis (adenitis, granuloma, mediastinitis).
For
All types of clinical specimens can be processed according to symptomatology [1, 2, 4, 6, 7, 8]:
Bronchoalveolar lavage (BAL), sputum, and lung biopsy should be performed for patients with pulmonary symptoms
Bone marrow aspiration
Punctures of lymph node, pus, or exudates
Organ biopsies (lymph node, digestive tract, liver, skin, oral mucous)
Peripheral blood (EDTA or fungal blood culture vacutainers)
Cerebrospinal fluid (CSF)
Different microbiological approaches can be performed as on one hand mycological examination (direct, culture) and molecular assays (qPCR) and, on the other hand, histopathology. The performances depend on the specimens tested and are much more details on paragraph 5.4 (Table 1).
PHD | Acute pulmonary syndrom | Subacute pulmonary syndrom | Chronic pulmonary syndrom | Pulmonary nodules | Mediastinal histoplasmosis | CNS histoplasmosis | Other manifestations (pericarditis, rheumatological), | ||
---|---|---|---|---|---|---|---|---|---|
Culture | Pulmonary tissue or secretions (LBA, sputum) | x | x | x | x | (x): differential diagnosis | |||
Bone marrow | x | x (if associated PHD) | |||||||
Lymph nodes | x | (x) mediastinal | |||||||
Exudates | x | (x) Non viable yeasts | (x) Non viable yeasts | ||||||
Organ biopsy | (x) | x Non viable yeasts | |||||||
Peripheral blood | x | x (if associated PHD) | |||||||
Cerebrospinal fluid | x | ||||||||
histopathology | x | x Cytopathologic examination of BAL (50%) | x Non viable yeasts | (x) | |||||
Serology | (x) | x | x | X (low sensitivity) | x | X | x | ||
Antigen | x serum, urine +/-LBA to repeat | x (83%): serum, urine +/-LBA or if associated PDH | (x) up to 40% (urine) | (x) serum, urine +/-LBA | x |
Examination of liquid smears or fine-needle aspiration and/or tissue apposition on slides from all types of samples is carried out by staining with May-Grünwald-Giemsa (MGG). Using 10% KOH with or without calcofluoric acid, a chitin-binding fluorescent stain for the fungal cell wall, the yeasts can be easily visualized between slide and slip cover. For some specimens as the peripherical blood, cerebrospinal fluid, bronchoalveolar fluid, and cytocentrifugation may be required [3, 13, 14].
Diagnosis is possible according to the morphology of the yeasts.
Yeasts are spherical, small (2–5 μm in diameter), intensely violet colored, and surrounded by a clear halo. These yeasts are narrow-based budding yeast cells, usually intracellular (macrophage, histiocyte, etc.), and do not produce any filaments (Figure 1). The differential diagnosis includes
Yeasts cells are oval and large (8–15 μm by 4–6 μm), with a “hourglass” or “figure eight” budding form, a thick wall and a narrow budding. They may have inside fat droplets. These yeasts are intra- or extracellular, sometimes arranged in short chains of 2 or 3 [3, 6, 13, 14].
Specimens as pus from abscess, draining sinuses and bone marrow lesions, colic biopsy, or BAL smears are relatively contributive to the diagnosis [4, 6, 15] in disseminated histoplasmosis.
Direct examination is inexpensive, and according to the relatively typical appearance of the levuriform elements, it allows to quickly and easily provide presumptive evidence for histoplasmosis. However, doubts may remain, and then it requires trained personnel and further investigations. Indeed confusion can concern
Histopathology consists in the research for elements evocative of the yeast form of
This concept of juggling between different stains and the description of morphologies of the microorganism (shape, size variation, cell disposition) and the tissue (cell response) permit to distinguish other pathogens as
Size (μm) | Cell disposition | Characteristic | Tissue response | Specific coloration | |
---|---|---|---|---|---|
2 - 5 | IC | Spherical with halo Narrow-based budding Grouped in clusters into macrophage | Granulamatous tissue response, necrosis | GMS, PAS | |
6 - 12 | IC | Oval with halo | Granulamatous tissue response | GMS, PAS | |
3 - 8 | Facultative IC | Spherical with characteristic halo (thick capsule) Narrow-based budding | Predominantly granulomatous inflammation, necrosis, +/- fibrosis | Mucicarmin (capsulated yeast) Fontana-Masson (uncapsulated yeast) | |
> 15 | EC | Round, Thick retractile wall Broad-based budding | Mixed suppurative and granulomatous inflammation | GMS, PAS, H&E | |
1 - 4 | EC | Oval to round No pseudohyphal production | Suppurative tissue response | GMS, PAS, H&E | |
2 - 5 | IC/ EC | spherical Confusion when endospores are outside spherules or young spherules without endospores | Mixed suppurative and granulomatous inflammation, (Splendore-Hoëppli phenomenon likely) | GMS, H&E, +/-PAS | |
5-8 | EC | Cysts | Minimal reaction | GMS, PAS, H&E | |
2-5 | EC | Small oval-shaped yeast Transverse septum No bud | Mixed suppurative and granulomatous inflammation | ||
2 - 5 | IC | Oval to round kinetoplast | MGG |
Diagnosis differential of
IC: intracellular, EC: extra cellular, GMS: Gromori Methenamine Silver, PAS: Periodique Acid Schiff:, GE, MGG: May-Grunwald Giemsa.
It usually shows a granulomatous reaction with “giant cells,” containing large rounded or oval-shaped elements (2–4 μm in diameter).
The yeast phase of
Isolation of
All specimen types can be used from superficial to deep ones.
Culture is performed on Sabouraud dextrose agar with antibiotics (chloramphenicol +/− gentamicin) +/− actidione that inhibits the contaminants. Other mediums can be used: brain heart infusion (BHI), yeast extract peptone agar (YEP agar) and potato dextrose agar (PDA) [3]. Mediums are incubated at 25–30°C for 6–8 weeks.
As the rate of growth is slow, the growth of the mycelial forms usually takes 2–3 weeks but may take up to 8 weeks. Colony is initially white and smooth and then becomes brown, with a granular or cottony texture. The reverse is white, yellow, or orange (Figure 2).
Culture of
Confirmation of the cultured organism as
It allows determining microscopy morphology with identification characters [18]:
The differential diagnosis includes
The identification of the colony can be carried out by matrix-assisted laser desorption/ionization time off light (MALDI-TOF). Mass spectrometry by MALDI-TOF allows to confirm species accurately and quickly.
This identification technique is based upon the detection of highly abundant proteins in a mass range of 2–20 kDa by calculating their mass (m) to charge (z), m/z values. The spectrum thus obtained is compared with the reference spectra [19, 20].
This can be achieved using enriched media such as BHI or blood agar plates incubated at 35–37°C in a CO2-enriched atmosphere [3]. However,
The urease test allows the distinction between Hcc and Hcd as it reveals the expression of urease strongly for Hcc and weakly for Hcd at 48 hours [3, 5].
Others tests performed from the cultured fungi such as DNA hybridization using a highly specific commercially kit (AccProbe; Gen-Probe, Inc., San Diego, CA®) or the detection of specific precipitin by the exoantigen test was used in some laboratories [10, 13]. They were gradually replaced by less time-consuming method as MALDI-TOF. They will not be more detailed in this chapter.
The lysis centrifugation method as Isolator® system followed by the inoculation of the collected buffy coat [11] into an appropriate media culture has been proved to be the most efficient for detecting
The sensitivity of culture depends on the clinical manifestation, the clinical specimen, the state of immunity of the host, and the burden of disease [2, 9, 14]. Sensitivity is lower for patients with acute pulmonary histoplasmosis (40%) than for patients with disseminated histoplasmosis (75%) [2, 9, 10].
The diagnosis of disseminated histoplasmosis blood culture processed by lysis methodology and bone marrow shows higher sensitivity (60–90%). Conversely, respiratory samples presented poor sensitivity (0–60%) [2, 9, 10].
The limitations of the culture are the time frame for diagnosis, the mycological expertise required, and safety. Several weeks for growth of the fungus to establish the diagnosis are not consistent with the severity of the disease in immunosuppressed patients. Moreover,
Conversely, detection of
Several protocols do exist based on
Complement fixation (FC) | Immunodiffusion (ID) | ELISA | Western Blott | |
---|---|---|---|---|
Mechanisms | A supplemented specific Ag induces Ag-Ab complexes | Precipitins Ac-Ag (H/M) on gel agar | Indirect sandwich ELISA | Visualisation of band profiles on nitrocellulose membranes |
Type of Ag | Entire yeast or HMIN | HMIN Prot M, Prot H | Yeast cell extract, ribosome, HMIN (glycosylayte/deglycosylate) | Histoplasma antigens of 115, 91,88, 83, 70 and 38 kDa from HMIN (glycosylayte/deglycosylate) |
delay | 3 -6 weeks | 4-6 weeks IgG anti-M → IgG M+H | 2 -4 weeks | 2-4 weeks |
Duration of positive antibodies after resolution | Months to years Months to years Persistent in recurrences | Prot M: up to 3 years | Months to years | Months to years |
Prot H: 1-2 years | ||||
Sensitivity | 72-95% | 70-95% | 75-100% | 45-100% |
Specificity | 70-80% | 100% | 91-100% | 94-100% |
Histoplasmosis meningitidis | When positive (culture usually negative (63%) | When positive (culture usually negative (44%) | Specificity: 93% Sensibility: 82% (26) | No data |
Acute infection | ++ | Band M (80%) Band H (7-20%) | 66-100% (9-10, 13) | 45-94% (13, 22) |
Chronic infection | + | Band M | 90% (9-10, 13) | 94-100% (13, 22) |
Special remarks | Interference with Rheumatoid factor and cold agglutinins | Positive after skin test Histoplasmine (M +++/H+) | Ribosome antigen and ptHMIN induces better response than glycosylate HMIN antigene. Best results with ELISA using ferrous metal | Better results when using ptHMIN Can detect early in the infection |
Kits/tests | House-made tests | House-made tests | House-made tests Commercialized tests with IgG, IgM, IgA | House-made tests |
Immunodiffusion assay qualitatively detects the precipitating antibodies to antigens M and H on agar gel. The H band appears after the M band, and the presence of both bands is highly significant for histoplasmosis diagnosis. Thus, M band is detectable in most patients with acute infection and persists for long periods of time up to 3 years after disease resolution and is often present in chronic forms [9, 10, 14]. It does not distinct between active from latent or resolved infection. H band is less frequent, confirms acute infection only in 7% according [13], remains present 1–2 years after disease resolution, and indicates a more severe form of the disease. This method is simple, reliable, and inexpensive.
Complement fixation method quantitatively measures the presence of complex antigen antibodies in a patient’s serum against the entire yeast form or mycelial antigen (HMIN) from 3 to 6 weeks following infection [13]. Interlaboratory results vary as it is entirely strain-dependent for the antigen preparation. It is quite more sensitive bus less specific than the ID. Indeed, it presents numerous cross-reactions with other fungi and interference with rheumatoid factor and cold agglutinins [13]. A threshold defined as up to 1:32 or titer a 4-fold rise indicates an active infection.
This method does not allow differentiation between an active infection and an old infection. Indeed, antibodies require 4–8 weeks to become detectable in peripheral blood. Serology is unreliable in patients with a reduced ability to produce antibodies, and then it is often negative in immunocompromised patients [13, 14]. Antibody testing is most useful for subacute and chronic forms of histoplasmosis [2].
Western blot immunoassay uses Ag proteins of different kDa that will complex with the specific
Interferon gamma release assay (IGRA): this test is based on the quantification of lymphocyts-released interferon (IFN) -γ after restimulation in vitro of the cell-mediated immunity with the same specific antigens. Recently, Rubio-Carrasquilla et al. [23] considered it as a promising screening method to detect individuals with latent Hc infection, even decades after the primary infection.
Latex agglutination tests were developed as a commercial kit, but false positives results were found in patients with tuberculosis. It was compared as more sensitive than CF [13].
A hemagglutination test was available but failed to differentiate
A recent meta-analysis [9] interested in the global sensitivity of antibody detection for disseminated histoplasmosis and showed a low sensitivity of 58% in contrast with high specificity (100%). But there is a real distinction between the different assays. WB and ELISA methods have the highest analytical performance, with sensitivity of up to 90% when used in ptHMIN. This can explain that it is relevant to associate different methods in order to improve the diagnosis.
Cross-reactivity with granulomatous disease, tuberculosis, and sarcoidosis can occur with immunodiffusion and complement fixation tests. Moreover, serologic cross-reaction can occur with other common fungal pathogens like
The presence of antibodies in the CSF allows making the diagnosis of Histoplasma meningitis [2, 26].
Circulating specific-
This polysaccharide mostly found in the cell wall of
This noninvasive method can be performed in urine, serum, and other body fluids as LBA [2, 9, 10, 13, 14] and is the simplest diagnostic method, easily implemented in low- and middle-income countries.
It allows a rapid diagnosis especially for immunosuppressed patients with severe acute or disseminated histoplasmosis. It is less sensitive than serology for the diagnosis of subacute and chronic pulmonary histoplasmosis [2, 9].
Antigen detection in urine is more sensitive than in serum for the diagnosis of disseminated histoplasmosis (95% versus 86% for HIV-infected patient) [9, 10, 11]. However, antigen detection in urine is less useful for pulmonary forms or chronic histoplasmosis.
This method has also been applied to other body fluids, including BAL for patient with pulmonary symptomatology and CSF for
If useful for the diagnosis, it can also monitor the antigen clearance, particularly in serum and thus appears as an useful marker for treatment response [32].
Moreover, it has proven its applicability to infected animals that may act as potential reservoirs for humans [28, 29].
Cross-reactivity of antigen testing occurs in patients who have other fungal infections, including infections by
Recently, a lateral flow assay (LFA) has been developed and evaluated for rapid diagnosis of histoplasmosis. This point of care antigen detection in serum allows rapid results with high analytical performance. It is based on the using of rabbit polyclonal antibody that recognizes galactomannan antigen of
Molecular methods can improve and accelerate the histoplasmosis diagnosis with a high analytical sensibility and specificity [9, 10, 34, 35, 36]. This combined with turnaround times shorter than those of other diagnostics. It is much more safety and reduces the risk of laboratory acquired histoplasmosis when handle its positive culture. Several applications can be linked to the use of PCR based techniques as (i) human diagnosis from different samples, with an increasingly use for the imported cases diagnosis in non-endemic areas [9, 10, 11, 13], (ii) environmental exploration for revealing the histoplasmosis reservoir [35], (iii) and prevention policies and strategies for exposure risk [2].
There are no currently official approved molecular assays for H. capsulatum that are directly applicable to clinical specimens and no commercially kits are available [9, 10, 34]. However, there are numerous reports of the laboratory-developed PCR assays (in house PCR) using the different type of PCR (conventional, nested and quantitative) and a variety of molecular targets. The most relevant are the Internal Transcribed Spacer (ITS) multicopy region of the ribosomal DNA, genes encoding the M antigen or the 100-kDa-like protein [9, 34]. Most studies in the literature evaluated the performance of nested PCR in the diagnosis of PDH, this assay is associated with the increased risk of amplicon contamination, because of the manipulation of amplified products.
A multiplex approach can also be performed. For example, in a reference laboratory in Spain they developed a multiplex qPCR for
As a full tool to Histoplasmosis diagnosis, molecular assays can be performed on different types of specimens including respiratory secretions, biopsies, bone marrow, blood, or sera.
For diagnosis of disseminated histoplasmosis (PDH), sensitivity and specificity are 95% and 99%, respectively [2, 9]. Sample of blood and bone marrow have an excellent sensitivity (100%) in immunocompromised patients with disseminated histoplasmosis, whereas it is weaker in immunocompetent patients [2, 11].
Moreover, sensitivity increases by testing more than one sample per patient in cases with extra-pulmonary histoplasmosis. So, in case of high suspicion, clinicians should repeat and diversify samples [2, 9, 11, 34].
Recent findings in molecular biology permit to evaluate genetic diversity using multilocus sequence typing (MLST) OR RAPD-PCR [36]. This could be Interesting to associate genotypes and clinical presentation.
Loop-mediated isothermal amplification (LAMP) is a highly efficient, sensitive, specific and cost-effective isothermal amplification method that uses at least four primers, recognizing six different regions in the target sequence (Figure 4) and results in a self-primed DNA [37].
Principe of LAMP [
In LAMP, the target sequence is amplified at a constant temperature of 60–65°C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. The amount of DNA produced in LAMP is considerably higher than PCR-based amplification.
To increase the reaction sensitivity, the ITS region was used as target, since it is a multicopy sequence and also because it is considered an important barcoding sequence for fungal identification, being conserved among
Forward inner primer (FIP) and backward inner primer (BIP) have inverted sequences attached at the 5′ end, named F1c and B1c, which are complementary to an internal sequence from the amplified strand, forming a loop at each extremity of a single strand DNA.
The outer primers (F3 and B3) anneal upstream to the FIP and BIP, acting as a binding site for DNA polymerase, which, in the LAMP reaction, also contains the strand displacement activity.
LAMP results can be observed using several strategies with minimal ambiguity with real-time turbidimetry (magnesium pyrophosphate formation), fluorescent compounds (Sybr Green, Eva Green, SYTO, calcein), magnesium colorimetric titration, fluorescent-labeled probes, quencher-labeled primers, dye-labeled primers, and PH-sensitive dyes.
LAMP can be used on samples of whole blood or bone marrow for patients suspected of progressive disseminated histoplasmosis (PDH).
A new assay, the so-called ITS LAMP, showed no cross-reactivity when assayed with DNA from other pathogenic or environmental fungi. The assay is able to detect isolates from all geographical clades of
This method remains cost-effective with or without the extraction DNA step and did not require a thermocycler or an electrophoresis apparatus. It saves time with test performed in less than 200 minutes [35]. However, this little-known and recent method is currently underused and much more reserved for resource-limited laboratories. It should evolve in the next years to come and need further evaluation to be routinely used.
The performance for each test according the clinical context is summarized in Table 4. It considers global sensitivity without any distinction between the different protocols (antigen preparation, methods, ...).
PHD | Acute pulmonary syndrom | Subacute pulmonary syndrom | Chronic pulmonary syndrom | Mediastinal histoplasmosis | CNS histoplasmosis | |||
---|---|---|---|---|---|---|---|---|
(2) | (9) | |||||||
Ag detection | 92 | 95 | 83 | 30 | 88 | 5 | 66 | |
81 | 31 | |||||||
Serology | 75 | 58 | 64 | 95 | 83 | 83 | 59 | |
63 | 54 | |||||||
Pathology | 76 | NE | 20 | 42 | 75 | 75 | ||
culture | 74 | 77 | 42 | 54 | 67 | 67 | 38 | |
40 | 33 | |||||||
Molecular Biology | 95 |
The different performances (sensibilité %)of the methods are summarized in this table adapted to azar et al. (2), Caceres et.al (9) and Wheat et.al (26).
PDH: Progressive histoplasmosis disseminated, CNS: Central nervous system, NE: Non evaluated.
It highlights that Ag detection is the best diagnosis tool for PHD and SCN histoplasmosis, serology for the subacute/chronic form, and the culture although this is the reference method remains less sensitive.
Histoplasmosis remains a severe and neglected disease for which early diagnosis of invasive fungal infections is critical to allow a prompt patient care. Indeed the mortality rate among HIV/AIDS patients diagnosed with histoplasmosis is high: 42% mortality for disseminated histoplasmosis if treatment is delayed and 100% if antifungal therapy is not prescribed.
The gold standard for histoplasma diagnosis remains culture. It’s a time-consuming process and has limitations in sensitivity. Moreover, it requires invasive procedure and mycological expertise.
Nonculture methods have been developed to improve and accelerate diagnosis of histoplasmosis, such as histoplasma antigen detection, antibody detection, and molecular biology. It should have conjunction between the different tools of diagnosis to be reliable in the histoplasmosis management regarding the wide range of clinical features.
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\n\n\n\nBook Chapters and Monographs
\n\n\n\nCorresponding authors will receive a 25% discount on their Open Access Publication Fees (OAPF) for Open Access book chapters. A 20% discount for publishing a long-form monographs, 25% for compacts and 23% for short-form monographs.
\n\nCSIC affiliated authors can also take advantage of a central Open Access fund (amounting to 10,000 EUR) to cover up to 50% of the rest of the OAPF until it expires. Effective for chapters accepted from January 1, 2020.
\n\nCorresponding authors will receive a 25% discount on their Open Access Publication Fees (OAPF) for Open Access book chapters. A 20% discount for publishing a long-form monographs, 25% for compacts and 23% for short-form monographs.
\n\nCorresponding authors will receive a 25% discount on their Open Access Publication Fees (OAPF) for Open Access book chapters. A 20% discount for publishing a long-form monographs, 25% for compacts and 23% for short-form monographs.
\n\n\n\nCorresponding authors will receive a 25% discount on their Open Access Publication Fees (OAPF) for Open Access book chapters. A 20% discount for publishing a long-form monographs, 25% for compacts and 23% for short-form monographs.
\n\nBook Chapters and Monographs
\n\nBook Chapters and Monographs
\n\nBook Chapters and Monographs
\n\n\n\nBook Chapters and Monographs
\n\nThe Claremont Colleges are pledging funds via the Knowledge Unlatched program to ensure academics can publish Open Access content more easily.
\n\nCorresponding authors will receive a 15% discount on their Open Access Publication Fees (OAPF) for Open Access book chapters or monograph publications. To use the discount you will need to verify your institutional email address. These discounts are valid from 2020 to 2022.
\n\nThe University of Massachusetts, Amherst is pledging funds via the Knowledge Unlatched program to ensure academics can publish Open Access content more easily.
\n\nCorresponding authors will receive a 10% discount on their Open Access Publication Fees (OAPF) for Open Access book chapters or monograph publications. To use the discount you will need to verify your institutional email address. These discounts are valid from 2020 to 2022.
\n\nThe University of Surrey is pledging funds via the Knowledge Unlatched program to ensure academics can publish Open Access content more easily.
\n\nCorresponding authors will receive a 10% discount on their Open Access Publication Fees (OAPF) for Open Access book chapters or monograph publications. To use the discount you will need to verify your institutional email address. These discounts are valid from 2020 to 2022.
\n\nMonographs Only
\n\n\n\nImportant: You must be a member or grantee of the above listed institutions in order to apply for their Open Access publication funds.
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