2.1 Distribution of mycobacteria detected
A total of 320 patients infected with mycobacterium were collected, of which 249 (77.81%) were identified as mycobacterium tuberculosis complex group and 71 (22.19%) were identified as non-mycobacterium tuberculosis. Among the non-tuberculous mycobacteria, 22 cases (6.88%), 19 cases (5.94%) of Mycobacterium avium complex group, 13 cases (4.06%) of Mycobacterium abscess, 4 cases (1.25%) of Mycobacterium Gordon, 3 cases (0.94%) of intracellular mycobacterium, 3 cases (0.94%) of Mycobacterium toad and Mycobacterium kansans were detected according to the frequency There were 3 cases (0.94%), 2 cases (0.63%), 2 cases (0.63%), 2 cases (0.63%), 1 case (0.63%), 1 case (0.63%), 1 case (0.63%), 1 case (0.63%), 1 case (0.63%), and 1 case (0.63%). Among them, 4 cases were infected with both mycobacterium tuberculosis and non-mycobacterium tuberculosis, showing a complex infection state (Table 1).
Table 1.Frequency of detection of non-tuberculous mycobacteria
Strain types
|
Number
|
Percentage
|
Mycobacterium tuberculosis complex
|
249
|
77.81%
|
Mycobacterium tuberculosis
|
22
|
6.88%
|
Mycobacterium avium complex (biology)
|
19
|
5.94%
|
Mycobacterium abscessus
|
13
|
4.06%
|
Mycobacterium gordonii (bacterium causing gut infection)
|
4
|
1.25%
|
Mycobacterium intracellulare
|
3
|
0.94%
|
Mycobacterium avium subspecies (zoology)
|
3
|
0.94%
|
Mycobacterium kansasensis (bacterium causing gut infection)
|
3
|
0.94%
|
Mycobacterium avium subspecies (genus of saccharomyces cerevisiae)
|
2
|
0.63%
|
Mycobacterium avium subspecies (genus of saccharomyces cerevisiae)
|
2
|
0.63%
|
Mycobacterium bovis (bacterium causing gut infection)
|
1
|
0.31%
|
Mycobacterium avium incidentalum
|
1
|
0.31%
|
Mycobacterium pubis
|
1
|
0.31%
|
Mycobacterium margaretii (bacterium causing gut infection)
|
1
|
0.31%
|
Total
|
320
|
100.00%
|
Mixed infections (TB & Non-TB)
|
4
|
1.25%
|
2.2 Clinical distribution characteristics of mycobacterium infected persons
The proportion of male patients (36 cases, 50.70%, mean age 56.77±19.21) and female patients (35 cases, 49.30%, mean age 57.48±19.28) infected with non-tuberculosis mycobacterium was consistent. Among the infected patients, there were 44 cases (61.97%) of elderly patients (≥55 years old), more than young patients, and only 26 cases (36.62%) of young patients (< 55 years old). The main types of specimens were alveolar lavage fluid (48 cases, 67.61%) and paraffin tissue (16 cases, 22.54%) (Table 2). Compared with the infection characteristics of mycobacterium tuberculosis, the number of male patients (156 cases, 62.65%) was much higher than that of female patients (93 cases, 37.35%), and the average age of male patients (59.77±19.21) was higher than that of female patients (56.77±19.21). The number of elderly patients (≥55 years old, 174 cases, 69.88%) was higher than that of young patients (< 55 years old, 74 cases, 29.72%). Specimens submitted for examination were of various types, including alveolar lavage fluid (183 cases, 73.49%), paraffin tissue (31 cases, 12.45%), peripheral blood (10 cases, 4.02%), abscess (7 cases, 2.81%), sputum (6 cases, 2.41%), cerebrospinal fluid (6 cases, 2.41%) and pleural fluid (3 cases, 1.2%) (Table 3).
Table 2. Clinical features of non-tuberculous Mycobacterium infected persons
Sex
|
Number
|
Percentage
|
`x ± SD
|
Male
|
36
|
50.70%
|
56.77±19.21
|
Women
|
35
|
49.30%
|
57.48±19.28
|
Age
|
|
|
|
≥55
|
44
|
61.97%
|
|
<55
|
26
|
36.62%
|
|
uncharted
|
1
|
1.41%
|
|
Sample Types
|
|
|
|
Alveolar lavage fluid
|
47
|
66.20%
|
|
paraffin tissue section
|
16
|
22.54%
|
|
Sputum
|
4
|
5.63%
|
|
Peripheral blood
|
2
|
2.82%
|
|
Hydrothorax
|
1
|
1.41%
|
|
Urine
|
1
|
1.41%
|
|
Total
|
71
|
100.00%
|
|
Table 3. Clinical characteristics of Mycobacterium tuberculosis infected patients
Sex
|
Number
|
Percentage
|
x ± SD
|
Male
|
156
|
62.65%
|
59.74±15.76
|
Women
|
93
|
37.35%
|
55.91±16.06
|
Age
|
|
|
|
≥55
|
174
|
69.88%
|
|
<55
|
74
|
29.72%
|
|
uncharted
|
1
|
0.40%
|
|
Sample Type
|
|
|
|
alveolar lavage fluid
|
183
|
73.49%
|
|
paraffin tissue section
|
31
|
12.45%
|
|
peripheral blood
|
10
|
4.02%
|
|
abscess
|
7
|
2.81%
|
|
sputum
|
6
|
2.41%
|
|
cerebrospinal fluid (CSF)
|
6
|
2.41%
|
|
hydrothorax
|
3
|
1.20%
|
|
joint effusion
|
1
|
0.40%
|
|
ascitic fluid
|
1
|
0.40%
|
|
pericardial effusion
|
1
|
0.40%
|
|
add up the total
|
249
|
100.00%
|
|
2.3 Differences in morphological characteristics between Mycobacterium tuberculosis and non-mycobacterium tuberculosis
In the patients with mycobacterium tuberculosis detected by macrogene and whose tissues were sent for pathological diagnosis at the same time, the detected pathogens were classified, and the tissues were stained with hematoxylin-eosin, and the histological morphological differences between mycobacterium tuberculosis and non-mycobacterium tuberculosis infections were analyzed. Typical nodules were found to contain areas of caseous necrosis (Fig.1A-1), epithelioid cell proliferation (Fig.1A-2), and inflammatory response (Fig.1A-3). The specific granuloma composed of peripheral local aggregation of lymphocytes and a small number of reactive proliferative fibroblasts is a characteristic lesion of tuberculosis (Fig.1-B). In the caseous necrotic area, cells and histology disappear, with only a small number of cell fragments remaining (Fig.1-C). In the center of the tubercle of tuberculosis are multinucleated giant cells derived from giant cells, also known as Langerhans giant cells. Multiple nuclei are annular or horseshoe shaped, surrounded by epithelioid cells (Fig.1-D), and acid-fast bacillus staining is positive (Fig.3-B). Compared with M. tuberculosis infection, it is difficult to see the typical granulomatous nodular changes in the morphology of non-tuberculous mycobacteria. Most mycobacterial infections only show the proliferative changes of epithelioid cell proliferation with extensive inflammatory cell infiltration. No typical caseous necrosis or definite Langerhans giant cells have been found. Most of the samples of non-mycobacterium tuberculosis infected tissues were lung tissue, and a few were found in bone tissue, intestinal mucosa, and subcutaneous soft tissue. Among them, a large number of lymphocyte infiltrates can be seen in the tissues of patients infected with M. teremys, M. avium, and M. abscess, and the inflammatory cells are evenly distributed and uniform in texture (Fig.2A-C). The morphological characteristics of M. Gordon and M. intracellularis are mainly hyperplasia of epidermoid cells (Fig. 2D-E). The morphological characteristics of Mycobacterium bufoi, Mycobacterium Kansensis, and Mycobacterium Aubris were lymphocyte infiltration with epitheloid cell proliferation and crowded cell growth, but no definite necrosis or multinucleate giant cell production was observed (Fig.2F-H). The non-tuberculous mycobacteria detected above were negative for acid-fast staining and fungal fluorescence staining (Fig.3-A, C).
Fig 1. Morphological features of HE staining of nodules. A: 1: area of caseous necrosis; 2: area of epithelioid cell proliferation; 3: area of inflammatory cell infiltration (100 ×); B: area of epithelioid cells (200 ×); C: area of caseous necrosis (200 ×); D: Langerhans' giant cell megacells
Fig 2. Histopathological and morphological characteristics of non-tuberculous mycobacteria. A. Mycobacterium turtle (100×), a. (200×); B. Mycobacterium avium (100×), b. (200×); C. Mycobacterium abscessus (100×), c. (200×); D. Mycobacterium gordonii (100×), d. (200×); E. Mycobacterium intracellulare (100×), e. (200×);
F. Mycobacterium toadii (100×), f. (200×); G. Mycobacterium kansasensis (100×), g. (200×); H. Mycobacterium obtusum (100×), h. (200×).
Fig 3. A: Negative antacid staining (200×), B: Positive antacid staining (200×); C: Negative fungal staining (200×).
2.4 Distribution characteristics of pathogen species associated with concurrent infection with mycobacterium
Other pathogens were detected along with mycobacterium tuberculosis, including candida albicans, human herpesvirus, pneumocystis yerinii, Enterococcus faecium, Klebsiella pneumoniae, pseudomonas aeruginosa and Aspergillus fumigatus. In addition to human symbiotic bacteria, fungi, molds and viruses are pathogens with high detection rates, suggesting that the resistance of tuberculosis bacteria is decreased after infection, and the pathogenicity of opportunistic pathogens is enhanced (Table 4). The concomitant pathogens of non-tuberculous mycobacterium are completely different. In the analysis of concomitant infections, it was found that staphylococcus, acinetobacter baumannii, actinomyces, Klebsiella pneumoniae, Escherichia intestinalis and so on were commonly associated with non-tuberculous mycobacterium, among which opportunistic pathogens were not common (Table 5).
Table 4. Pathogens detected simultaneously with Mycobacterium tuberculosis
genus (taxonomy)
|
frequency
|
genus (taxonomy)
|
frequency
|
genus (taxonomy)
|
frequency
|
tubercle bacillus
|
249
|
Aspergillus flavus / Aspergillus oryzae
|
4
|
Novel cryptococcus Grubii var.
|
1
|
Pseudohyphae
|
26
|
2019 Novel Coronavirus
|
4
|
rootlet mold (botany)
|
1
|
human herpes virus (HSV) type 1 (med.)
|
24
|
Weillonella pallidum (genus of sac fungus, a plant pathogen)
|
3
|
streptococcus salivarius
|
1
|
Human herpesvirus 4 (EBV)
|
23
|
Corynebacterium striatum
|
3
|
human herpesvirus 6B (med.)
|
1
|
Human herpesvirus 5 (CMV)
|
19
|
tropical pseudofilament yeast
|
3
|
human Boca virus
|
1
|
Pneumocystis jiroveci
|
15
|
Staphylococcus aureus
|
3
|
Human coronavirus OC43
|
1
|
Enterococcus faecalis (genus Enterococcus)
|
12
|
Smooth Pseudofilament Yeast
|
3
|
human parainfluenza virus 1 (HPV)
|
1
|
Human herpesvirus 7
|
12
|
streptococcus pneumoniae
|
3
|
Human polyomavirus type 4
|
1
|
Klebsiella pnenmoniae
|
10
|
Rhinovirus A
|
3
|
Human polyomavirus type 2 (JC polyomavirus)
|
1
|
Pseudomonas aeruginosa
|
8
|
Anopheles stephensi
|
3
|
Aspergillus pyrogallus
|
1
|
Aspergillus fumigatus
|
7
|
Mycoplasma hominis
|
2
|
Ornithococcus (genus of sac fungus, a plant pathogen)
|
1
|
Human coronavirus 229E
|
7
|
human metapneumovirus (HPV)
|
2
|
Phytolacca myrtillus (root fungus)
|
1
|
Acinetobacter baumannii (immobile bacteria)
|
6
|
Catamorpha
|
2
|
ureaplasma urealyticum
|
1
|
hemophilus influenzae
|
5
|
streptococcus suis
|
2
|
Mycobacterium avium subspecies heparinum
|
1
|
oral streptococcus
|
5
|
Aspergillus niger
|
2
|
Streptococcus pseudopneumoniae
|
1
|
Rhinovirus C
|
5
|
Escherichia coli (E. coli)
|
2
|
streptococcus parahaemolyticus
|
1
|
Stenotrophomonas maltophilia
|
4
|
Rochesteria (genus of bacteria)
|
1
|
Fusarium oxysporum
|
1
|
Legionella pneumophila (bacterium causing legionnaires' disease)
|
4
|
Enterobacter cloacae complex
|
1
|
Enterococcus faecalis (genus Enterococcus)
|
1
|
human parainfluenza virus 3 (HPV3)
|
4
|
influenza B virus
|
1
|
Mycoplasma pneumoniae
|
1
|
influenza A virus
|
4
|
Chlamydia (genus of intracellular parasitic bacteria)
|
1
|
|
|
Table 5 . Pathogens detected simultaneously with non-tuberculous mycobacteria
Genus (taxonomy)
|
Number
|
Genus (taxonomy)
|
Number
|
Genus (taxonomy)
|
Number
|
Mycobacterium tuberculosis
|
22
|
Artobacterium gingivalis
|
2
|
Corynebacterium congestans
|
2
|
Mycobacterium avium subspecies complex
|
19
|
hemoccococcus
|
2
|
Porphyromonas solani
|
1
|
Mycobacterium abscessus
|
13
|
Microcystis aeruginosa
|
2
|
maltophilic spirochete
|
1
|
Staphylococcus epidermidis
|
12
|
Neisseria pacificus
|
2
|
Sputum-generating carbon dioxide fibrophilic bacteria
|
1
|
Human herpesvirus 4 (EBV)
|
11
|
streptococcus salivarius
|
2
|
Sphingomonas oligoclonalis (a type of bacteria)
|
1
|
Propionibacterium acnes (the bacterium that causes acne)
|
11
|
Prevotella tamorum
|
2
|
human metapneumovirus (HPV)
|
1
|
Human herpesvirus 7
|
10
|
Lactobacillus rhamnosus
|
2
|
human herpesvirus 6B (med.)
|
1
|
Acinetobacter baumannii (immobile bacteria)
|
10
|
Streptococcus suis
|
2
|
Human coronavirus OC43
|
1
|
Pseudohyphae
|
10
|
the bacillus subtilis organism (botany)
|
2
|
human parainfluenza virus 3 (HPV3)
|
1
|
Restricted Malassezia
|
9
|
human staphylococcus
|
2
|
tropical pseudofilament yeast
|
1
|
Pseudomonas aeruginosa
|
9
|
Rösslerella caries
|
2
|
Rhizobium perfringens
|
1
|
Moraxella catarrhalis (genus of bacteria)
|
9
|
Trichoderma reesei (Trichoderma harzianum)
|
2
|
Agrobacterium tumefaciens
|
1
|
Weillonella pallidum (genus of sac fungus, a plant pathogen)
|
8
|
hemophilus influenzae
|
2
|
Mycobacterium chimerae
|
1
|
human herpes virus (HSV) type 1 (med.)
|
8
|
Oral Prevotella
|
2
|
Serratia marcescens
|
1
|
Escherichia coli (E. coli)
|
8
|
Nearly smooth pseudofilamentous yeast
|
2
|
Mycobacterium avium incidentalum
|
1
|
Stenotrophomonas maltophilia
|
7
|
influenza A virus
|
2
|
Erwinia amylovora
|
1
|
Actinobacillus solani
|
6
|
Aspergillus flavus / Aspergillus oryzae
|
2
|
Mycobacterium margaretii (bacterium causing gut infection)
|
1
|
Rochesteria (genus of bacteria)
|
5
|
streptococcus gordonii
|
2
|
Actinobacillus marinus
|
1
|
Arbuscularcular mycobacteria (AMM)
|
5
|
Mycobacterium avium subspecies (genus of saccharomyces cerevisiae)
|
2
|
Clostridium difficile (bacterium causing gut infection)
|
1
|
Human herpesvirus 5 (CMV)
|
5
|
Fusarium oxysporum
|
2
|
Sloth and Cunning Bacteria
|
1
|
Staphylococcus aureus
|
5
|
Corynebacterium difficile (bacterium causing gut infection)
|
2
|
oral streptococcus
|
1
|
Campylobacter (genus of saccharomyces cerevisiae)
|
5
|
Mycoplasma pneumoniae
|
2
|
Rochesteria (genus of bacteria)
|
1
|
Klebsiella pnenmoniae
|
5
|
inert cohesion bacillus
|
2
|
Mycobacterium avium subspecies heparinum
|
1
|
Aspergillus fumigatus
|
4
|
Mycobacterium avium subspecies (genus of saccharomyces cerevisiae)
|
2
|
Fusarium oxysporum
|
1
|
cyclovirus
|
4
|
dense intermediate spirochete
|
1
|
Klebsiella atrophytica
|
1
|
Streptococcus granulosus
|
4
|
Armillaria swampii (botany)
|
1
|
Mycobacterium bovis (bacterium causing gut infection)
|
1
|
streptococcus suis
|
4
|
Campylobacter showa
|
1
|
Smooth Pseudofilament Yeast
|
1
|
hemophilus parainfluenzae
|
4
|
Bifidobacterium longum (bacterium causing bifidus)
|
1
|
Aspergillus oryzae (genus of fungi)
|
1
|
streptococcus pneumoniae
|
4
|
Neisseria gonorrhoeae
|
1
|
streptococcus parahaemolyticus
|
1
|
Prevotella nigra
|
4
|
Serratia marcescens
|
1
|
Enterococcus faecalis (genus Enterococcus)
|
1
|
Pneumocystis jiroveci
|
3
|
Lactobacillus mucosus
|
1
|
Atypical Verrucomicrobia (genus of atypical viruses)
|
1
|
Porphyromonas gingivalis
|
3
|
Corynebacterium mucilaginum
|
1
|
Lactobacillus inertus
|
1
|
Micrococcus garciniae
|
3
|
Yersinia pestis
|
1
|
Prevotella polymorpha
|
1
|
Enterococcus faecalis (genus Enterococcus)
|
3
|
streptococcus angina
|
1
|
Mycobacterium pubis
|
1
|
hemolytic staphylococcus
|
3
|
periodontal clostridium difficile (bacterium causing gut infection)
|
1
|
Enterovirus Group D
|
1
|
Lauteria monocytogenes
|
3
|
Carbon dioxide fibrophilic bacteria of the gums
|
1
|
Providencia alkali-producing bacteria
|
1
|
Prevotella jejuni
|
3
|
Sai Kung Piroglobulin
|
1
|
Prevotella pallidum
|
1
|
Mycobacterium kansasensis (bacterium causing gut infection)
|
3
|
Corynebacterium striatum
|
1
|
Prevotella nigra
|
1
|
Mycobacterium avium subspecies (zoology)
|
3
|
gastrointestinal streptococcus
|
1
|
somerae
|
1
|
Brucella Aspergillus (undulant fever)
|
3
|
Porphyromonas vaginalis (a.k.a. Porphyromonas viridis)
|
1
|
Porphyromonas
|
1
|
Mycobacterium intracellulare
|
3
|
Verrucomicrobium vulgare, a type of ciliate
|
1
|
leadbetteri
|
1
|
Neisseria mucus
|
2
|
Mycoplasma salivarius
|
1
|
Capnocytophaga
|
1
|
2.5 Infection characteristics of mycobacterium chelonis, mycobacterium avium and mycobacterium abscess
Among the non-tuberculous mycobacteria, the highest detection rates were Mycobacterium chelonis, Mycobacterium avium and Mycobacterium abscess. The clinical characteristics of these three infections were similar to those of tuberculosis infection, with low fever, cough and fatigue as the main clinical manifestations, but no significant differences in imaging characteristics, mostly manifested as lung shadow. Among them, the infection of M. chelonis was often accompanied by the infection of staphylococcus, Acinetobacter baumannii and Moraxella osloensis. Mycobacterium avium infection is often accompanied by Aspergillus fumigatus, pseudomonas aeruginosa, and influenza virus infection. Infection with Mycobacterium abscess is often accompanied by infection with fine cyclic virus, staphylococcus aureus, and Streptococcus bradycosus. Although the characteristics of the combined infection of these three types of non-tuberculous mycobacteria are different, they are accompanied by high frequency detection of herpes virus (Table 6), suggesting a possible reduction in immunity.
Table 6. Pathogens associated with non-tuberculous mycobacteria are frequently detected
Mycobacterium chelonae
|
Mycobacterium avium
|
Mycobacterium abscessus
|
Genus (taxonomy)
|
Number
|
Genus (taxonomy)
|
Number
|
Genus (taxonomy)
|
Number
|
herpes virus (med.)
|
20
|
human herpes virus (HSV)
|
9
|
Human herpesvirus 7
|
4
|
Staphylococcus epidermidis
|
10
|
Aspergillus fumigatus
|
4
|
cyclovirus
|
2
|
Acinetobacter baumannii (immobile bacteria)
|
8
|
Pseudomonas aeruginosa
|
4
|
Staphylococcus aureus
|
2
|
Moraxella catarrhalis (genus of bacteria)
|
8
|
influenza A virus
|
2
|
streptococcus suis
|
2
|
Weillonella pallidum (genus of sac fungus, a plant pathogen)
|
7
|
Pseudohyphae
|
2
|
hemophilus parainfluenzae
|
2
|
Stenotrophomonas maltophilia
|
7
|
Pneumocystis jiroveci
|
1
|
Propionibacterium acnes (the bacterium that causes acne)
|
2
|
Escherichia coli (E. coli)
|
6
|
Porphyromonas gingivalis
|
1
|
Pseudohyphae
|
2
|
Propionibacterium acnes (the bacterium that causes acne)
|
6
|
Weillonella pallidum (genus of sac fungus, a plant pathogen)
|
1
|
Armillaria swampii (botany)
|
1
|
Pseudohyphae
|
6
|
Corynebacterium striatum
|
1
|
Serratia marcescens
|
1
|
Pseudomonas aeruginosa
|
5
|
Enterococcus faecalis (genus Enterococcus)
|
1
|
Pneumocystis jiroveci
|
1
|
Arbuscularcular mycobacteria (AMM)
|
5
|
Actinobacillus solani
|
1
|
Clostridium perfringens
|
1
|
Rochesteria (genus of bacteria)
|
4
|
human staphylococcus
|
1
|
hemoccococcus
|
1
|
Prevotella jejuni
|
4
|
human metapneumovirus (HPV)
|
1
|
Weillonella pallidum (genus of sac fungus, a plant pathogen)
|
1
|
Campylobacter (genus of saccharomyces cerevisiae)
|
4
|
tropical pseudofilament yeast
|
1
|
Restricted Malassezia
|
1
|
Klebsiella pnenmoniae
|
4
|
Mycobacterium avium
|
1
|
Neisseria pacificus
|
1
|
Prevotella nigra
|
4
|
oral streptococcus
|
1
|
Verrucomicrobium vulgare, a type of ciliate
|
1
|
Porphyromonas endodontica
|
3
|
Aspergillus flavus / Aspergillus oryzae
|
1
|
Pseudomonas aeruginosa
|
1
|
Mycobacterium avium subspecies complex
|
3
|
Mycoplasma pneumoniae
|
1
|
Micrococcus garciniae
|
1
|
streptococcus pneumoniae
|
3
|
Escherichia coli (E. coli)
|
1
|
Prevotella tamorum
|
1
|
Mycobacterium avium subspecies (zoology)
|
3
|
Propionibacterium acnes (the bacterium that causes acne)
|
1
|
human staphylococcus
|
1
|
Brucella Aspergillus (undulant fever)
|
3
|
Enterovirus Group D
|
1
|
Human coronavirus OC43
|
1
|
Neisseria mucus
|
2
|
Brucella Aspergillus (undulant fever)
|
1
|
human parainfluenza virus 3 (HPV3)
|
1
|
Staphylococcus aureus
|
2
|
Staphylococcus epidermidis
|
1
|
hemophilus influenzae
|
1
|
hemophilus parainfluenzae
|
2
|
Acinetobacter baumannii (immobile bacteria)
|
1
|
Aspergillus oryzae (genus of fungi)
|
1
|
Mycoplasma pneumoniae
|
2
|
Moraxella catarrhalis (genus of bacteria)
|
1
|
Streptococcus pneumoniae
|
1
|