Use of specific rRNA oligonucleotide probes for microscopic detection of Mycobacterium avium complex organisms in tissue

Abstract

Members of the Mycobacterium avium complex (MAC) are important environmental pathogens that are implicated in several chronic, idiopathic diseases. Diagnosis of MAC-based diseases is compromised by the need to cultivate these fastidious and slowly growing organisms in order to identify which mycobacterial species are present. Detection is particularly difficult when MAC is intracellular or embedded within mammalian tissues. We report on the development of culture-independent, in situ hybridization (ISH) assays for the detection of MAC in culture, sputum, and tissue. This assay includes a highly reliable technique for the permeabilization of mycobacterial cells within culture and tissues. We describe a set of rRNA-based oligonucleotide probes that specifically detect either M. intracellulare, the two M. avium subspecies associated with human disease, or all members of MAC. The results call into question the validity of ISH results derived by the use of other gene loci, such as IS900.

FIG. 1.

Specificities of the MAC-specific probes with cultures. (A) M. avium subsp. avium culture hybridized with MAVP187ssu-Cy3 and MAVP515lsu-Cy3 (red) probes; (B) M. avium subsp. paratuberculosis culture hybridized with MAVP187ssu-Cy3 and MAVP515lsu-Cy3 (red) probes; (C and D) M. intracellulare dually probed with the EUB338-FAM probe (green; C) and the MAVP187ssu-Cy3 and MAVP515lsu-Cy3 probes (red; D) in the same field; (E and F) M. tuberculosis dually probed with the EUB338-FAM probe (green; E) and the MAVP187ssu-Cy3 and MAVP515lsu-Cy3 probes (red; F) in the same field; (G and H) M. avium culture dually probed with the EUB338-FAM probe (green; G) and the MAC2543-Cy3 probe (red; H) in the same field; (I and J) M. intracellulare culture dually hybridized with the EUB338-FAM probe (green; I) plus the MAC2543-Cy3 probe (red; J) in the same field; (K and L) M. tuberculosis dually probed with the EUB338-FAM probe (green; K) and the MAC2543-Cy3 probe (red; L) in the same field; (M and N) M. avium subsp. avium culture dually probed with the EUB338-FAM probe (green; M) and the MIN351lsu-Cy3 and MIN1586lsu-Cy3 probes (red; N) in the same field; (O and P) M. intracellulare culture dually hybridized with the EUB338-FAM probe (green; O) and the MIN351lsu-Cy3and MIN1586lsu-Cy3 probes (red; P) in the same field; (Q and R) M. tuberculosis dually probed with the EUB338-FAM probe (green; Q) and the MIN351lsu-Cy3 and MIN1586lsu-Cy3 probes (red; R) in the same field. Bars, 1 μm. Magnifications, ×1,000.

FIG. 2.

Representative application of the MAVP187ssu-Cy3 and MAVP515lsu-Cy3 probes to bovine tissue. (A) Negative control MLNs probed with MAVP187ssu-Cy3 and MAVP515lsu-Cy3; (B) positive control MLNs probed with MAVP187ssu-Cy3 and MAVP515lsu-Cy3. Bars, 1 μm. Magnifications, ×1,000. (C) Confocal laser scanning microscopy picture of positive control MLNs stained with DAPI and probed with MAVP187ssu-Cy3. Bar, 1 μm. Magnification, ×630. (D) Bright-field microscopy picture of positive control ileum tissue hybridized with MAVP187ssu-FAM and MAVP515lsu-FAM probes and visualized with antifluorescein-AP antibodies and the INT-BCIP substrate. Bar, 1 μm. Magnification, ×1,000. Arrows indicate bacilli of the expected morphology.

FIG. 3.

Representative applications of different MAC-specific oligonucleotide probes to various human tissue specimens. (A) Resected lung from a patient with an M. avium subsp. avium infection probed with MAVP187ssu-FAM and MAVP515lsu-FAM; the signal was visualized by using antifluorescein-AP Fab fragments and INT-BCIP; (B) MAVP187ssu-Cy3 and MAVP515lsu-Cy3 probes detect red fluorescent M. avium subsp. avium bacilli in a section of tissue from the hand of a patient with tenosynovitis; (C) MAVP187ssu-Cy3 and MAVP515lsu-Cy3 probes detect red fluorescent bacilli in a tissue section of the duodenum from a pediatric CD patient; (D) a small-bowel biopsy specimen from the same pediatric CD patient described for panel C was probed with MAVP187ssu-FAM and MAVP515lsu-FAM; the signal was visualized by using antifluorescein-AP Fab fragments and INT-BCIP; (E) a lung tissue section from a patient with a pulmonary M. intracellulare infection was probed with MIN351lsu-FAM and MIN1586lsu-FAM and visualized with antifluorescein-AP antibodies and INT-BCIP substrate; (F) sputum (stained with DAPI; blue) from a chronic smoker with a possible mycobacterial infection probed with MAC2543lsu-FAM; the signal was visualized with antifluorescein-AP antibodies and INT-BCIP substrate, and the micrograph represents a merged images obtained by bright-field and epifluorescence microscopy. Bars, 1 μm. Magnifications, ×1,000. Arrows indicate bacilli of the expected morphology.

FIG. 4.

Representative applications of IS900-biotin ISH or MAVP187ssu-FAM ISH with negative and positive control MLN tissue specimens. (A) Negative control MLN tissue stained by the Ziehl-Neelsen acid-fast procedure; (B) positive control MLN tissue stained by the Ziehl-Neelsen acid-fast procedure; (C) negative control MLN tissue hybridized with MAVP187ssu-FAM by ISH; (D) positive control MLN tissue hybridized with MAVP187ssu-FAM by ISH; (E) negative control tissue hybridized with IS900-biotin; (F) positive control tissue from a cow with Johne's disease hybridized with IS900-biotin; (G) negative control tissue hybridized with IS900-fluoroscein; (H) positive control tissue from a cow with Johne's disease hybridized with IS900-fluoroscein. Bars, 1 μm. Magnifications, ×1,000. Arrows indicate signals positive for bacilli in panels A, B, C, and D and amorphous nonspecific signals in panels E, F, G, and H. Note that for technical reasons it was necessary to probe different tissue sections of the same sample block separately for each panel.

FIG. 5.

Representative applications of IS900 ISH with pure cultures. (A) B. subtilis dually probed with IS900-fluorescein (green) and EUB338-Cy3 (red); (B) a Rhodococcus sp. probed with IS900-biotin by ISH; (C) M. kansasii probed with IS900-biotin by ISH; (D) M. avium subsp. avium hybridized with EUB338-FAM by ISH. Bars, 1 μm. Magnifications, ×1,000. Arrows indicate amorphous nonspecific signals in panels A, B, and C and a signal positive for bacilli in panel D.