Besides conventional microscopy, cultivation and immunological techniques, the diagnosis of clinically significant microorganisms increasingly relies on molecular biological tests. Their main advantage is the speed of sample processing and also the considerable sensitivity and specificity.
Because of the fact that similar histologic images can be caused by a variety of infectious agents, molecular - genetic examination of inflammation with histiocytic or directly with a granulomatous reaction has an important meaning for more accurate histopathological diagnosis and mainly for treatment of patients.
According to WHO about 1/3 of the world's population is infected with Mycobcterium tuberculosis and about 3 million patients die from it annualy. Thus Mycobacterium tuberculosis is the most frequent lethal infection worldwide.
Tuberculosis has the highest occurence in poor countries but in relation to the migration of people from countries of the third world, HIV infection and tourism, the incidence is significantly rising also in developed countries.
The progression of the disease is variable and usually rather slow. Different organs can be affected. The most common is the pulmonary form (85 %), further then it affects lymphatic nodes (5 - 10 %), pleura, bones, kidneys, central nervous system, gastrointestinal tract, genitals etc. The typical histopathological report is granulomatous inflammation with caseous necrosis.
Our laboratory uses two different PCR techniques for detection of mycobacteria. The first method is nested PCR which is used to detect different species and strains of genus Mycobacterium. It is used to amplify a part of the gene encoding the heat shock protein with the molecular weight of 65kDa, which is highly conserved within different strains of genus Mycobacterium.
The second method is also nested PCR. It amplifies the insertion sequence IS6110. This insertion sequence is present in multiple copies and only within species complex of Mycobacterium tuberculosis (M. tuberculosis, M. bovis, M. africanum, M. microti). The method is used to distinguish the complex Mycobacterium tuberculosis from others species of mycobacteria. Both of the PRC techniques can be used for investigation of fresh material (tissue, sputum) and also for the examination of the Formalin-Fixed Paraffin-Embedded (FFPE) tissues.
For more detailed discussion of the molecular biology of Borrelia see the "Lyme borreliosis and its causal agent".
Borrelia burgdorferi is a bacterial species of the spirochete. It is typically transmitted by ticks (rarely also by the blood-sucking insect) and causes the zoonotic disease named lyme borreliosis. However, an infected tick bite does not always causes the disease and on the other hand, among some clearly infected patients there is no tick bite proven. Lyme borreliosis is a multisystem disease affecting mainly the skin, joints, muscles, central nervous system and possibly also heart and other organs. The major cutaneous manifestations of lyme borreliosis are erythema migrans, acrodermatitis chronica atropicans and borrelia lymphocytoma or pseudolymphomas (the predilection sites such as the area of nipples, ear lobe, nose or scrotal area). However, skin lesions may occur also quite solitarily without damaging other organs. Infection by borrelia was repeatedly demonstrated also among cutaneous lymfomas. For the emergence of lymphoproliferation in skin, antigenic stimulus by infection is important. This is analogous to the role of Helicobacter pylori and the emergence of gastritis and MALT lymphoma of the stomach. The detection of borrelia has a huge impact on the pathologic diagnosis of cutaneous lymphoproliferations because antibiotics (e.g. doxycyclin) often cures the lesion completely.
The method used for detection of borrelia is nested PCR amplifying a portion of the gene encoding 23S rRNA.
Infecion by B. henselae causes the cat scratch disease. Symptoms of the disease may by diverse, however, the most common manifestation is granulomatous - purulent lymphadenitis. Nonetheless, this morphological image is not pathognomonic and can be caused by many other agents. Accurate diagnosis therefore depends on the sensitive laboratory tests. Sometimes it is possible to demonstrate B. henselae in histopathological sections by the characteristic appearance after the impregnation with silver. Detection of bacterial DNA in diagnosing is of invaluable assistance.
Detection of Bartonella henselae is performed by PCR amplifying the part of the region linking the genes for 16S and 23rRNA. The method allows for distinguishing the following species of the genus Bartonella by the size of the final PCR product: B. henselae, B. bacilliformis, B. clarridgeiae, B. elizabethae, B. quintana a B. vinsonii subsp. berkhoffi.
Syphilis is the sexually transmitted systemic disease caused by spirochetes T. pallidum subsp. pallidum. When left untreated it typically progresses in three stages: primary stage occurs about 3 weeks after the infection and includes ulcers in the area of the infection occurence and regional lymphadenopathy. Secondary stage (2 - 10 weeks after the primary) is characteristic by the diffuse exanthema and enanthema, fever, lymphadenopathy, headache and arthritis. Also these lesions can cure spontaneously. Tertiary stage comes after years when the active inflammation destroy mainly thr aorta, heart and central nervous system. Even though there are already several decades of effective treatment, the disease remains a global problem. Diagnosis of syphilis is complicated because cultivation of T. pallidum under the laboratory conditions is not possible. However, identification of infected persons and their treatment depends primarily on early detection of the disease.
Method detecting Treponema pallidum is single-round PCR amplifying the part of the gene tmpA. Specificity of the amplified product can be confirmed by the restriction digestion with the enzyme Smal.
Tularemia is zootonic disease caused by bactaria F. tularensis. It is transmitted to humans from an infected animal. The disease may take place in several clinical forms: ulceroglandular (possibly oculoglandular), lung (pneumonic) called typhoid (non-specific febrile illness, possibly up to endotoxin shock), pharyngeal and rarely as endocarditis and osteomyelitis. Disability of lymph nodes is frequent. Species F. tularensis is possible to distinguish into two types. F. tularensis biovar tularensis, so far described just from North America and F. tularensis biovar palearctica occuring mostly in Asia and Europae Diagnosis of tularemia is most often based on the detection of the specific antibodies by ELISA or hemagglutination test, however, they were also demonstrated by the cross - reactivity with other relatively common bacteria.
Detection of F. tularensis by means of the methods of molecular biology is based on PRC amplifying the part of the genome encoding the protein FopA.
Brucellosis is a worldwide reaching disease with several endemic bearings in the Mediterranean region. It is typically transmitted either by contact with infected animals or as an occupational desease. It is also possible to be transferred from untreated milk or dairy products. The clinical course of the disease is quite variable, basically acute or chronic. Its diagnosis is therefore dependent on precise laboratory tests. Cultivation of Brucella is difficult and also dangerous for laboratory personnel. Not even serological tests produce the satisfactory results.
Detection of Brucella abortus is performed by PCR amplifying the part of the insertion sequence IS711.
Whipple's disease is a rare multisystem disease caused by the inracellular bacteria Tropheryma whipplei. Clinical manifestations are diverse, the most often it is malabsorption of the small intestine, weight loss, diarrhea, enlarged mesenteric lymph nodes, arthritis and skin pigmentation.
Diagnosis of Whipple's disease is based of the detection PAS - positive foamy macrophages in tissue biopsies, accompanied by a PCR evidence of infection of T. whipplei. Method of detecting T. whipplei is a single-round PCR amplifying the part of the gene encoding 16S rRNA.
Y.enterolitica and Y.pseudotuberculosis are gramm-negative facultative anaerobic rod-shaped bacteria. Primary hosts of Y.enterociltica are warm-blooded mammals but also fish and reptiles. For the transmission to humans the most important is their presence in domestic animals. The main reservoir are pigs. Y.pseudotuberculosis is widespread like a parasite of many species of birds and mammals. The presence of both Yersinia was detected through fecal contamination in water and vegetables.
Transmission to humans is performed by fecal oral route either directly or through contamination of food. There was also described nosocomial transmission and transmission by infected blood. Acute infection manifests as enterocolitis. Accute mesenteric lymphadenitis is common with the character of appendicitis (pseudoappendicitis), fever, headache, pharyngitis, anorexia, vomiting, skin ulcers, osteomyelitis and septicemia. For infection of Y. enterocolitica manifestations of gastroenterocolitis are typical and for Y. pseudotubreculosis abdominal pains. In immunosupressed patients there is a higher number of fatal infections. Under normal circumstances the disease is succesfuly treated with antibiotics, nonetheless, often with Yersinia resistance to penicillin and its synthetic derivatives.
In terms of molecular biology, particularly PCR methodologies, substantial for distinguishing individual species of Yersinia is the sequence homology. Y. enterolitica is well distinguishable but Y. pseudotuberculosis exhibits significant sequence homology with the genome of Y. pestis. According to some authors Y. pestis appeared recently (its emergence caused the first wave of plague coming from east coast of Africa) as a clone of Y. pseudotuberculosis differing by two plasmids by which its pathogenity and high virulence is given.
- Y. enterolitica - detected using conventional PCR. Two independent pairs of primers are designed in the region of the gene ail and amplify segments of the size 170 or 114 base pairs.
- Y. pseudotuberculosis - detected using conventional PCR. Two independent pairs of primers amplify the region of the gene inv of the size 295 respectively 114 pb. One pair of primers was designed for the area of the gene ail and amplifies the segment of the size 123 bp.
Results are confirmed by sequenation.
Chlamydia trachomatis are gram-negative intracellular bacteria in the perceptive cells of mucosas. Usually it is transmitted by sexual intercourse and in both, men and women, can cause serious health complications including urethritis, proctitis, inflammation of prostate and epididymis in men, inflammation of cervix and pelvis minor in women, and infertility. In women it can cause ectopic pregnancy and in newborns infected at birth pulmonary complications and infection of the eye - trachoma which is only a problem in developing countries. Diagnosis is usualy based on the detection of specific antibodies by the method ELISA, however, there may occur a cross - reactivity with other species of Chlamidia.
Cultivation is difficult due to the necessity of using tissue cultures. For the direct detection in the tissues and body liquids molecular methods are used.
Detection of Chlamydia trachomatis is performed by unaccredited Touchdown Enzyme Time Release-PCR with primers amplifying the part of the gene for 16S rRNA. This PCR is approximately 10x more sensitive than conventional PCR methods.
Chlamydophila pisttaci is primarily an animal pathogen but can be also transmitted to humans by contaminated droppings or feathers. In human it can cause the disease ongoing under the image of atypical pneumonia (bird strains) or placentitidy (mammalian strains). It is also associated with adnexal MALT lymphoma. This infection is subject to mandatory reporting. Diagnosis is usually based on the detection of specific antibodies by the method ELISA, however, there may occur a cross - reactivity with other species of Chlamydia. Cultivation is difficult due to the necessity of using tissue cultures. For the direct detection in the tissues the method PCR is used.
Detection of Chlamydophila psittaci is performed by unaccredited Touchdown Enzyme Time Release-PCR with primers amplifying the part linking the genes for 16S rRNA and 23S rRNA. This PCR is approximately 10x more sensitive than conventional PCR methods.
- Schwartz JJ, Gazumyan A, Schwartz I. rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi. J Bacteriol. 1992, 174 (11): 3757-65.
- Inagaki H, Kawai T, Miyata M, Nagaya S, Tateyama H, Eimoto T. Gastric Syphilis: Polymerase chain reaction detection of treponemal DNA in pseudolymphomatous lesions. Hum Pathol. 1996;27:761-65.
- Scott MA, McCurley TL, Vnencak-Jones CL, Hager C, McCoy JA, Anderson B, Collins RD, Edwards KM. Cat scratch disease: detection of Bartonella henselae DNA in archival biopsies from patients with clinically, serologically, and histologically defined disease. Am J Pathol. 1996;149(6):2161-7.
- Ramzan NN, Loftus E Jr, Burgart LJ, Rooney M, Batts KP, Wiesner RH, Fredricks DN, Relman DA, Persing DH. Diagnosis and monitoring of Whipple disease by polymerase chain reaction. Ann Intern Med. 1997;126(7):520-7.
- Marchetti G, Gori A, Catozzi L, Vago L, Nebuloni M, Rossi MC, Esposti AD, Bandera A, Franzetti F. Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin fixed, paraffin embedded tissues: comparison of four amplification assays. J Clin Microbiol. 1998;36:1512-17.
- Jensen WA, Fall MZ, Rooney J, Kordick DL, Breitschwerdt EB. Rapid identification and differentiation of Bartonella species using a single-step PCR assay. J Clin Microbiol. 2000;38(5):1717-22.
- Wicki R, Sauter P, Mettler C, Natsch A, Enzler T, Pusterla N, Kuhnert P, Egli G, Bernasconi M, Lienhard R, Lutz H, Leutenegger CM. Swiss Army Survey in Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus in ticks. Eur J Clin Microbiol Infect Dis. 2000;19(6):427-32.
- Al Nakkas AF, Wright SG, Mustafa AS, Wilson S. Single-tube, nested PCR for the diagnosis of human brucellosis in Kuwait. Ann Trop Med Parasitol. 2002;96(4):397-403.
- Hercik K, Melter O, Janecek J, Branny P. In situ detection of Bartonella henselae cells. Mol Cell Probes. 2002;16(1):49-56.
- Achtman M, Zurth K, Morelli G, Torrea G, Guiyoule A, Carniel E. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):14043-8. Erratum in: Proc Natl Acad Sci U S A 2000 Jul 5;97(14):8192.
- Lamps LW, Madhusudhan KT, Greenson JK, Pierce RH, Massoll NA, Chiles MC, Dean PJ, Scott MA. The role of Yersinia enterocolitica and Yersinia pseudotuberculosis in granulomatous appendicitis: a histologic and molecular study. Am J Surg Pathol. 2001 Apr;25(4):508-15.
- Lamps LW, Madhusudhan KT, Havens JM, Greenson JK, Bronner MP, Chiles MC, Dean PJ, Scott MA. Pathogenic Yersinia DNA is detected in bowel and mesenteric lymph nodes from patients with Crohn's disease. Am J Surg Pathol. 2003 Feb;27(2):220-7.
- Madico G, Quinn TC, Boman J, Gaydos CA. Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S-23S spacer rRNA genes. J Clin Microbiol. 2000, 38(3):1085-1093.
- Chanudet E, Zhou Y, Bacon CM, Wotherspoon AC, Müller-Hermelink HK, Adam P, Dong HY, de Jong D, Li Y, Wei R, Gong X, Wu Q, Ranaldi R, Goteri G, Pileri SA, Ye H, Hamoudi RA, Liu H, Radford J, Du MQ. Chlamydia psittaci is variably associated with ocular adnexal MALT lymphoma in different geographical regions. J Pathol. 2006, 209(3):344-351.