**2.10 Non-tuberculosis mycobacteriosis**

Non-tuberculosis mycobacteriosis is an inflammation caused by mycobacteria not belonging to the *Mycobacterium tuberculosis* family; those are *M. avium, M. fortuitum, M. gordonae, M. kansasii, M. xenopi*, and *M. marinum*, also designated as MAC complex. Unlike *Mycobacterium tuberculosis*, these mycobacteria can be detected intracellularly in macrophages (histiocytes), and they can be numerous in immunocompromised individuals. The diagnosis is made based on acid-fast stain, cultural or molecular biological tests. As mentioned above, the histological changes are often similar to those in tuberculosis. Non-necrotizing granulomas, histiocytic granulomas, and granulomas consisting of foamy and granular macrophages containing mycobacteria can also be detected. Solovieva et al. have described the following spectrum of histological changes in mycobacteriosis:


The MAC-hypersensitivity-like disease has also been described (or "hot tub lung"); it is caused by mycobacteria of the MAC complex, and associated with the use of sauna and showers, which leads to the aerosol inhalation. The histological features of this disease are similar to the hypersensitivity pneumonitis changes [22].

## **2.11 Differential diagnostics of granulomatous lung diseases**

A variety of diseases leading to granulomatosis determine certain difficulties in conducting the differential diagnostics even when resectional (surgical, videoassisted) biopsies are performed, which allows obtaining a sufficient amount of material for histological evaluation [23]. However, it is not always possible to establish the cause of granulomatous inflammation. According to Ulbright and Katzenstein, who analyzed 86 solitary lung granulomas detected by X-ray, the infection caused by acid-resistant mycobacteria or fungi was confirmed in 70%. In 25 cases, the infectious etiology was not proven, while two patients were diagnosed with hyalinized granuloma, one patient—with polyangiitis with granulomatosis, and in 22 cases, it was not possible to classify the process. Also, a significant similarity of histological changes in infectious granulomas and polyangiitis with granulomatosis was found; it may be possible that the latter was a reflection of the immune response disorders to an infectious agent which could no longer be found in the tissue samples. This means that polyangiitis with granulomatosis and other lung angiitis diagnoses should be made with extreme caution in cases with solitary nodes while no damage to other organs is detected. In such cases, a thorough examination of patients and follow-up should be recommended [24].

Mukhopadhyay et al. conducted a multicenter retrospective study of 500 biopsies from 10 clinics in the United States, Britain, Austria, Brazil, Japan, Turkey, and India with pulmonary granulomatosis. During the biopsy analysis, a specific diagnosis was established in 58% of the cases: most commonly sarcoidosis (27%) and mycobacterial infection (25%) were detected. Mycobacterial infection was proved in 18% outside the USA versus 8% in the USA; on the contrary, fungal infection amounted to 19% in the USA (most often histoplasmosis) versus 4% in other countries. Fungi were commonly detected by histological examination, while mycobacterial infection was confirmed in culture. In 42% of the cases, the etiological factor of granulomatosis was not established.

This study, in our opinion, is extremely interesting: First of all, it indicates the predominance of sarcoidosis and infectious granulomatous inflammation in the structure of granulomatous diseases according to histological analysis conducted in different countries and geographical regions. Fungal infection more often caused granulomatous inflammation in the United States, while mycobacterial infection was more often diagnosed in other countries, which is a reflection of the infection

**61**

**Figure 16.**

*Pathology of Sarcoidosis and Differential Diagnostics of other Granulomatous Diseases*

endemicity. It is crucial to send the specimen simultaneously to the histological and microbiological laboratory in all cases when a granulomatous disease is suspected, that will definitely improve the quality of the etiological diagnosis. According to this study, the cause of granulomatosis was not established in more than a third of

The frequency of infectious granulomatosis is high. When all other causes based on the clinical history, clinical syndromes, laboratory tests, and specific morphological features mentioned above are excluded, the remaining granulomatous diseases

An important issue in the differential diagnosis of the infectious granulomatosis diseases is the detection of an infectious agent in microscopical slides. To achieve this, it is necessary and obligatory to perform additional stains. One can detect fungal infection, first of all, by carefully evaluating hematoxylin- and eosin-stained (H&E) slides. Most fungi, such as *Cryptococcus*, *Blastomyces*, *Cocidioides*, and *Aspergillus*, can be found in H&E slides, more often in necrosis areas than in the adjacent lung tissue. The pathologist should select slides with necrosis present when ordering additional stains. Grocott's methenamine silver stain and the PAS reaction are the most commonly used for the fungal infection diagnosis confirmation, also alcian blue (Mouri), the basic brown stains (Shubich's method), or the combined

Ziehl-Neelsen stain is used to diagnose mycobacterial infection; however, mycobacteria are usually few in number, and their search is quite time-consuming. Alternative stains with auramine or auramine/rhodamine increase the sensitivity of the method, but these techniques require fluorescence microscopy (**Figure 16a** and **b**). To increase the detection of mycobacteria, Ulbright and Katzenstein recommend performing staining in at least two blocks [24]. Gomori silver or Warthin-Starry stains are recom-

One of the open questions is whether there are additional opportunities, modern techniques for diagnosing infectious granulomatosis. Immunohistochemistry is available, but it has several limitations, namely cross-reactivity, as well as the antibody accessibility, especially for rare microorganisms. *In situ* hybridization may

*Acid-fast mycobacteria: (a) Ziehl-Neelsen stain and (b) auramine/rhodamine (fluorescence).*

*DOI: http://dx.doi.org/10.5772/intechopen.90693*

clinical cases even after histological examination [22].

are most likely to be attributed to infectious.

stain of PAS with alcian blue [14].

mended for syphilis diagnosis [9].

#### *Pathology of Sarcoidosis and Differential Diagnostics of other Granulomatous Diseases DOI: http://dx.doi.org/10.5772/intechopen.90693*

endemicity. It is crucial to send the specimen simultaneously to the histological and microbiological laboratory in all cases when a granulomatous disease is suspected, that will definitely improve the quality of the etiological diagnosis. According to this study, the cause of granulomatosis was not established in more than a third of clinical cases even after histological examination [22].

The frequency of infectious granulomatosis is high. When all other causes based on the clinical history, clinical syndromes, laboratory tests, and specific morphological features mentioned above are excluded, the remaining granulomatous diseases are most likely to be attributed to infectious.

An important issue in the differential diagnosis of the infectious granulomatosis diseases is the detection of an infectious agent in microscopical slides. To achieve this, it is necessary and obligatory to perform additional stains. One can detect fungal infection, first of all, by carefully evaluating hematoxylin- and eosin-stained (H&E) slides. Most fungi, such as *Cryptococcus*, *Blastomyces*, *Cocidioides*, and *Aspergillus*, can be found in H&E slides, more often in necrosis areas than in the adjacent lung tissue. The pathologist should select slides with necrosis present when ordering additional stains. Grocott's methenamine silver stain and the PAS reaction are the most commonly used for the fungal infection diagnosis confirmation, also alcian blue (Mouri), the basic brown stains (Shubich's method), or the combined stain of PAS with alcian blue [14].

Ziehl-Neelsen stain is used to diagnose mycobacterial infection; however, mycobacteria are usually few in number, and their search is quite time-consuming. Alternative stains with auramine or auramine/rhodamine increase the sensitivity of the method, but these techniques require fluorescence microscopy (**Figure 16a** and **b**). To increase the detection of mycobacteria, Ulbright and Katzenstein recommend performing staining in at least two blocks [24]. Gomori silver or Warthin-Starry stains are recommended for syphilis diagnosis [9].

One of the open questions is whether there are additional opportunities, modern techniques for diagnosing infectious granulomatosis. Immunohistochemistry is available, but it has several limitations, namely cross-reactivity, as well as the antibody accessibility, especially for rare microorganisms. *In situ* hybridization may

**Figure 16.** *Acid-fast mycobacteria: (a) Ziehl-Neelsen stain and (b) auramine/rhodamine (fluorescence).*

*Sarcoidosis and Granulomatosis - Diagnosis and Management*

lular abundance of mycobacteria, no necrosis;

intracellular abundance of mycobacteria;

abundance of mycobacteria;

• nonspecific granulation tissue;

• acute purulent abscess [21].

• multibacillary histiocytosis—diffuse macrophage infiltration with an intracel-

• multibacillary minimal histiocytosis—a mild inflammatory reaction with an

• histoid lesion—nodular clusters of spindle-shaped macrophages with an

The MAC-hypersensitivity-like disease has also been described (or "hot tub lung"); it is caused by mycobacteria of the MAC complex, and associated with the use of sauna and showers, which leads to the aerosol inhalation. The histological features of this disease are similar to the hypersensitivity pneumonitis changes [22].

A variety of diseases leading to granulomatosis determine certain difficulties in conducting the differential diagnostics even when resectional (surgical, videoassisted) biopsies are performed, which allows obtaining a sufficient amount of material for histological evaluation [23]. However, it is not always possible to establish the cause of granulomatous inflammation. According to Ulbright and Katzenstein, who analyzed 86 solitary lung granulomas detected by X-ray, the infection caused by acid-resistant mycobacteria or fungi was confirmed in 70%. In 25 cases, the infectious etiology was not proven, while two patients were diagnosed with hyalinized granuloma, one patient—with polyangiitis with granulomatosis, and in 22 cases, it was not possible to classify the process. Also, a significant similarity of histological changes in infectious granulomas and polyangiitis with granulomatosis was found; it may be possible that the latter was a reflection of the immune response disorders to an infectious agent which could no longer be found in the tissue samples. This means that polyangiitis with granulomatosis and other lung angiitis diagnoses should be made with extreme caution in cases with solitary nodes while no damage to other organs is detected. In such cases, a thorough examination

Mukhopadhyay et al. conducted a multicenter retrospective study of 500 biopsies from 10 clinics in the United States, Britain, Austria, Brazil, Japan, Turkey, and India with pulmonary granulomatosis. During the biopsy analysis, a specific diagnosis was established in 58% of the cases: most commonly sarcoidosis (27%) and mycobacterial infection (25%) were detected. Mycobacterial infection was proved in 18% outside the USA versus 8% in the USA; on the contrary, fungal infection amounted to 19% in the USA (most often histoplasmosis) versus 4% in other countries. Fungi were commonly detected by histological examination, while mycobacterial infection was confirmed in culture. In 42% of the cases, the etiologi-

This study, in our opinion, is extremely interesting: First of all, it indicates the predominance of sarcoidosis and infectious granulomatous inflammation in the structure of granulomatous diseases according to histological analysis conducted in different countries and geographical regions. Fungal infection more often caused granulomatous inflammation in the United States, while mycobacterial infection was more often diagnosed in other countries, which is a reflection of the infection

**2.11 Differential diagnostics of granulomatous lung diseases**

of patients and follow-up should be recommended [24].

cal factor of granulomatosis was not established.

**60**

also be useful, but not for the detection, but for the identification of fungi found in traditionally stained slides. Real-time PCR can be performed on paraffin sections for tuberculosis diagnostics; the specificity of this method is 99%; however, the sensitivity amounts to only 65%. Nevertheless, when the same method is used to detect mycobacteria tuberculosis in the cerebrospinal fluid, urine, or bronchoalveolar lavage, the sensitivity is more than 90%. Thus, this technique is useful and confirms the diagnosis if mycobacteria are detected, but PCR does not exclude the tuberculosis diagnosis if the result is negative. In addition, it does not allow detecting non-tuberculous mycobacteria. According to Aubry, the cultural study remains the "gold standard" for non-tuberculous mycobacteriosis diagnostics, and, according to their research, indicates that in more than 75% of cases, the only method to confirm mycobacteriosis was in culture [10].

The etiological factor remains unclear in 30–40% of infectious granulomatosis even in leading US university clinics despite a complete histological evaluation of lung tissue slides, as well as correlations with clinical, microbiological, and serological data. In this regard, the question of diagnostic significance of these diseases arises. Ulbright and Katzenstein propose that such cases represent infectious granulomas in which the microorganism was destroyed and/or removed by means of the developed inflammatory process [24]. A retrospective analysis of necrotizing granulomas showed that patients who did not receive further specific therapy were still alive and did not demonstrate any clinical symptoms. The same hypothesis is confirmed by Aubry who notes that even if new foci appear in these patients, poor outcomes were not detected [10]. It can be recommended to pathologists to give a descriptive histological conclusion indicating the presence/absence of necrosis, the absence of detected microorganisms: "The disease etiology is most likely to be infectious, special stains for the microorganism detection are negative."

When a granulomatous disease is suspected and lung resection is performed, it is necessary to save some specimen tissue unfixed for possible cultural study, and use the quick freeze method at -70°С for subsequent DNA and RNA analyses, if available. In the differential diagnosis of granulomatous diseases, first of all, one should determine whether the granuloma is infectious, or there are signs of other diseases, including Wegener's granulomatosis. If a specific diagnosis is excluded, it is crucial to perform special stains for microorganism detection, it is preferable to stain sections from at least two blocks, while making sure that necrosis foci are present in the material. If the microorganism detection is not possible at the first glance, we recommend evaluating the slides again at a higher magnification, and also using an additional block for staining. When negative result is received, but clinical data are in favor of tuberculosis or other infections, PCR is suggested. Additional cultural and serological studies, which would be able to exclude the infectious process, should be performed in case of another negative result obtained. Nevertheless, according to this algorithm results, a certain part of granulomatous diseases appears to have an uncertain etiology.

In conclusion, we would like to emphasize that the differential diagnosis of granulomatous pulmonary diseases is not so easy for pathologists. To exclude or prove the infectious disease, the pathologist should carefully examinate special stained specimens. Noninfectious granulomatous lung disease should be proved, taking into consideration both clinical and radiological data. Finally, from the pathologic point of view, there are the situations for which a specific diagnosis cannot be made. Multidisciplinary approach sometimes is recommended for decision-making.

**63**

**Author details**

Maria V. Samsonova1

Russian Federation, Moscow, Russia

Agency of Russian Federation, Moscow, Russia

\*Address all correspondence to: samary@mail.ru

provided the original work is properly cited.

*Pathology of Sarcoidosis and Differential Diagnostics of other Granulomatous Diseases*

\* and Andrey L. Chernyaev2

1 Department of Pathology of Federal State Budgetary Institution, Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of

2 Division of Fundamental Pulmonology of Federal State Budgetary Institution, Pulmonology Scientific Research Institute under Federal Medical and Biological

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*DOI: http://dx.doi.org/10.5772/intechopen.90693*

*Pathology of Sarcoidosis and Differential Diagnostics of other Granulomatous Diseases DOI: http://dx.doi.org/10.5772/intechopen.90693*
