Fluorescence In Situ Hybridization (FISH) Tests for Identifying Protozoan and Bacterial Pathogens in Infectious Diseases

Abstract

Diagnosing and treating many infectious diseases depends on correctly identifying the causative pathogen. Characterization of pathogen-specific nucleic acid sequences by PCR is the most sensitive and specific method available for this purpose, although it is restricted to laboratories that have the necessary infrastructure and finance. Microscopy, rapid immunochromatographic tests for antigens, and immunoassays for detecting pathogen-specific antibodies are alternative and useful diagnostic methods with different advantages and disadvantages. Detection of ribosomal RNA molecules in the cytoplasm of bacterial and protozoan pathogens by fluorescence in-situ hybridization (FISH) using sequence-specific fluorescently labelled DNA probes, is cheaper than PCR and requires minimal equipment and infrastructure. A LED light source attached to most laboratory light microscopes can be used in place of a fluorescence microscope with a UV lamp for FISH. A FISH test hybridization can be completed in 30 min at 37 °C and the whole test in less than two hours. FISH tests can therefore be rapidly performed in both well-equipped and poorly-resourced laboratories. Highly sensitive and specific FISH tests for identifying many bacterial and protozoan pathogens that cause disease in humans, livestock and pets are reviewed, with particular reference to parasites causing malaria and babesiosis, and mycobacteria responsible for tuberculosis.

Keywords: Babesia duncani; Babesia microti; FISH tests; LED fluorescence microscopy; Mycobacterium avium; Mycobacterium tuberculosis; Plasmodium falciparum; Plasmodium knowlesi; Plasmodium vivax; diagnostic tests; fluorescence in situ hybridization; pathogen identification; ribosomal RNA.

Figure 1

Plasmodium genus-specific FISH test identifying all human malaria parasites. Photographs showing Plasmodium genus-specific FISH test results with blood smears from patients with confirmed P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi infections. Fluorescence was viewed in (1)—fluorescence microscope with a UV light source, and (2)—microscope with LED light source illustrated in Figure S1. Green fluorescence demonstrated the presence of Plasmodium rRNA. (A,A1) P. falciparum including a crescent shaped gametocyte; (B,B1) P. vivax; (C,C1) P. knowlesi; (D) P. ovale; (E) P. malariae; and (N,N1) negative controls. Alexa 488 green was used to label the Plasmodium genus-specific probe in the FISH test. Only Plasmodium parasites fluoresce green in the assay. Scale bars represent approximately 5 µm. Figure reproduced with permission under the creative commons license from Reference [39].

Figure 2

P. falciparum-specific PF-FISH test. Photographs showing results from the PF-FISH test performed on thin blood smears in patients from Peru and Kenya infected with: (A) P. falciparum; (B) 1. P. malariae and 2. P. falciparum; (C) P. ovale; (D) P. vivax. The P. falciparum-specific probe and Plasmodium genus-specific probe fluoresce green and red, respectively, in the same field when viewed with appropriate light filters. The scale bars represent approximately 5 µm. Figure reproduced with permission under the creative commons license from Reference [39].

Figure 3

P. vivax-specific FISH test.Photographs showing PV-FISH test results on patient blood samples with independently confirmed malaria infections from Peru (A), India (B) and Kenya (C–E). Patient blood positive for (A,B) P. vivax; (C) P. ovale; (D) P. malariae; (E) P. falciparum. Green and red fluorescence are due to hybridization with the P. vivax-specific probe and Plasmodium genus-specific probe, respectively, in the same field when viewed with appropriate light filters. The scale bars represent approximately 5 µm. Figure reproduced with permission under the creative commons license from Reference [39].

Figure 4

Specificity of the PK-FISH test for P. knowlesi. Photographs showing PK-FISH test results with the P. knowlesi-specific probe (green fluorescence) and the Plasmodium genus-specific probe (orange fluorescence) in blood smears containing P. knowlesi from monkey blood (Pk), and from human blood with confirmed infections of P. falciparum (Pf), P. malariae (Pm), P. ovale (Po) and P. vivax (Pv). Each set of paired photographs shows fluorescence in the same field when viewed in a LED fluorescence microscope with appropriate light filters (Figure S1). The scale bars represent approximately 5 µm. Reproduced with permission under the creative commons license from Reference [42].

Figure 5

Detection of ring, trophozoite and schizont stages of P. knowlesi in the PK-FISH test. Photographs showing results from the PK-FISH test with the P. knowlesi-specific probe (green fluorescence) and the Plasmodium genus-specific probe (orange fluorescence) on R—rings; T—trophozoites; S—schizonts. Dual colour fluorescence in the same field is shown in paired photographs R1 and R2, T1 and T2, and S1 and S2. Fluorescence was viewed in a LED fluorescence microscope with pertinent light filters (Figure S1). The ring, trophozoite and schizont-stage parasites were produced from synchronised in vitro cultures of P. knowlesi. Parasites stained with Giemsa from smears prepared in parallel to the corresponding smears used in the PK-FISH test are shown in R3, T3 and S3 respectively. The scale bars represent approximately 5 µm. Reproduced with permission under the creative commons license from Reference [42].

Figure 6

Babesia genus-specific FISH test on different Babesia species. Parasites stained with Giemsa in smears used for FISH tests are shown in the case of (A1) B. microti; (B1) B. duncani; and (C1) B. divergens. Fluorescence observed in FISH tests on corresponding smears from the same preparations are shown in (A2) B. microti (from hamster blood); (B2) B. duncani (from hamster blood); (C2) B. divergens (from culture). Other FISH test results on smears of (D) B. bovis (from bovine blood); (E) B. bigemina (from bovine blood) are also shown. Fluorescence in FISH tests on blood smears from two patients with the infecting species confirmed by DNA sequencing are shown in (A3) for B. microti and (B3) for B. duncani. Scale bars represent approximately 5 μm. Reproduced with permission under the creative commons license from Reference [43].

Figure 7

Dual color fluorescence reactivity of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii in the MN Genus-MTBC FISH and MTBC-MAC FISH tests. Paired photographs showing dual colour fluorescence in the same microscopic field with A-MN Genus- specific probe (green fluorescence) and B-MTBC-specific probe (orange fluorescence) in the MN Genus-MTBC FISH test; and C-MTBC- specific probe (green fluorescence) and D-MAC-specific probe (orange fluorescence) in the MTBC-MAC FISH test. Mycobacteria used in the FISH tests were reference cultures of M. tuberculosis, M. avium, and M. kansasii, as well as an artificially mixed culture of M. tuberculosis and M. avium. Scale bars represent approximately 50 µm. Reproduced with permission under the creative commons license from Reference [28].

Figure 8

Sputum smears in the MN Genus-MTBC FISH test. Photographs showing dual colour fluorescence reactivity of sputum smears from patients with Mycobacterium tuberculosis and Mycobacterium abscessus infections in the MN Genus-MTBC FISH test. A shows Ziehl–Neelsen staining for acid-fast bacilli in sputum from a patient with Mycobacterium tuberculosis infection. MN Genus-MTBC FISH test results with the MN Genus- and MTBC-specific probes on the sputum of the same patient are shown in B (green fluorescence) and C (orange fluorescence), respectively. Reactions with the MN Genus- and MTBC-specific probes in the same field in a sputum smear from another patient with Mycobacterium abscessus infection are shown in D (green fluorescence) and E (orange fluorescence), respectively. Scale bars represent approximately 5 µm. Reproduced with permission under the creative commons license from Reference [29].