Application of Allele-Specific Polymerase Chain Reaction for Rapid Differentiation of Mycobacterium Leprae from Mycobacterium Tuberculosis

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1 Case Report Application of Allele-Specific Polymerase Chain Reaction for Rapid Differentiation of Mycobacterium Leprae from Mycobacterium Tuberculosis Yu-Ta Yen Yu-Wen Cheng Wei-Ming Wu Leprosy (Hansen s disease) is a chronic granulomatous infection caused by Mycobacterium leprae affecting primarily skin and nerves. Leprosy is an important clinical problem and is also a challenging diagnostic problem for clinicians. Clinically, leprosy might at times be difficult to be distinguished from lupus vulgaris. We report a 70-year-old woman who showed a well-demarcated erythematous enlarging plaque on left peri-ocular region suggesting leprosy resembling lupus vulgaris. Skin biopsy was performed and mycobacterial infection was identified. However, it was uncertain to differentiate between tuberculosis and leprosy. To solve the problem, we design an allele-specific polymerase chain reaction based on two-nucleotide difference between bacilli of TB and leprosy. Our result showed allele-specific polymerase chain reaction can rapidly differentiate the infection caused by M leprae or by M tuberculosis. (Dermatol Sinica 27: , 2009) Key words: Allele-specific PCR, Leprosy, Lupus vulgaris INTRODUCTION Leprosy is a chronic granulomatous infection caused by M leprae affecting primarily skin and nerves. The dermatological and pathological presentations varied according to the host immune status. On the other hand, lupus vulgaris is a chronic and progressive form of tuberculosis of the skin caused by M tuberculosis primarily involving the head and neck in 90 percent of patients. In this article, we report the application of allele-specific polymerase chain reaction (PCR) for rapid differential diagnosis of a 70-year-old woman presenting clinically as lupus vulgaris or leprosy on face as well. CASE REPORT A 70-year-old Taiwanese female had an erythematous enlarging infiltrated mild painful non-itching scaly plaque over left peri-orbital region for more than 6 months. She also developed erythematous papules and plaques on trunk and limbs in the later time (Fig. 1). She has been lived in Kaohsiung, Taiwan since childhood without history of traveling abroad. She was diagnosed as herpes zoster at other medical agent and the lesion exacerbated after treatment. She didn t have symptoms of fever, recent body weight loss, numbness, nor any decreased sensation From the Department of Dermatology, Chang Gung Memorial Hospital - Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan Corresponding author: Wei-Ming Wu, Department of Dermatology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Ta-Pei Road 123, Niao-Sung Hsiang, Kaohsiung, Taiwan TEL: ext FAX: weimin1970@cgmh.org.tw Funding source: none Conflict of interest: none declared Received: August 04, 2008 Revised: November 20, 2008 Accepted: March 21,

2 PCR for M Leprae and M Tuberculosis Fig. 1 This is a 70-year-old female. (A) An infiltrated erythematous plaque over the left periorbital area with gradual enlargement. (B, C) Erythematous papules and nodules over limbs and trunk respectively. (D) 5 months after treatment, facial lesions showed erosion and mild central atrophy of the plaque, with darkening of peri-oral papules. (E) 1 year and 5 months later, the lesions became flattened hyperpigmented patch as well as macules. There was also obvious retraction of left peri-orbital eyelids leading to ectropion. over the cutaneous lesion. Clinical examination with KOH did not show evidence of superficial fungus infection. A skin biopsy was arranged under the impression of lupus vulgaris, deep mycosis, tinea incognito, or lymphoma. Sections of hematoxylin & eosin stain showed extensive granulomatous inflammation and dense lymphocytic infiltration in dermis. No obvious epidermal change suggested a presumptive diagnosis of lupus vulgaris. Subsequent special stains revealed positive staining in Fite s stain but negative Fig. 2 (A) Histopathology shows diffuse granulomatous tubercles infiltrating in the dermis. There was no obvious epidermal change, and no nerve invasion was observed. (H&E, original magnification x40) (B) Tubercles with central histiocytes aggregation and peripheral lymphocytes infiltrate with vesiculated cells. (H&E, original magnification x100) (C) Ziehl-Neelson stain was negative. (x400) (D) Fite stain showed numerous acid-fast bacilli in vesiculated cells forming globi. (x1000) in Ziehl-Neelsen stain (Fig. 2). Tissue culture showed no growth of any mycobacterium. To reconfirm the diagnosis and establish the rapid differential diagnostic method for the possible forecoming confusing cases of leprosy and lupus vulgaris in our hospital, we designed the primers specifically amplifying the sequence for succinate dehydrogenase of M leprae and M tuberculosis respectively. The primers were as follows: for leprosy, AACGCGCATCGCTTCATT and CGCTTT- GGTCACCTCTATGTTG; for tuberculosis, AACGCGCACCGCTTCATC and CGCTTTGGTCACCTCAATGCCC (Fig. 3). DNA from paraffin-embedded samples was extracted with Gentra Puregene Tissue Kit (QIAGEN) as directed by manufacturer. PCR was carried out as follows: 95 C for 5 minutes, 36 cycles of 94 C for 20 seconds, 58 C for 60 seconds, and 72 C for 30 seconds. PCR were conducted with samples of lesions from the patient s face. For positive control, we used specimens with confirmed Dermatol Sinica, Sep

3 Yu-Ta Yen, et al diagnosis of tuberculosis and leprosy. DNA of normal skin was used as negative controls. The results showing positive bands in patient s DNA reacted with leprosy primers, but negative with TB primers (Fig. 4), confirmed the diagnosis of leprosy. For the treatment of the multibacillary disease, we use the combination of rifampin 600 mg daily and dapsone 100 mg daily. The patient was then transferred to Lo-Sheng sanatorium, the government-run leprosy-specific institution, for further management. The central area of the plaque became atrophic after treatment for 5 months, while it became flattened with hyperpigmentation and retraction of eyelids after treatment for 1 year and 5 months (Fig. 1). DISCUSSION Leprosy is caused by M leprae and predominantly affects the skin and peripheral nerves. The disease is endemic in many tropical and subtropical countries in the Indian subcontinent, Southeast Asia, sub-saharan countries in Africa, and Brazil. 1 The mode of transmission of leprosy is unknown, but it is probably fransmifted by inhalation of bacilli, 2 implanted from organisms in the soil, 3 or direct person-to-person infection by means of skin contact. 4 In the present case, the patient had an erythematous well-demarcated mildtender plaque on left peri-orbital region with gradual enlargement, and certain smaller erythematous papules and plaques on trunk and limbs. The histopathology showed dense granulomatous inflammation and dense lymphocytic infiltration in dermis, and absence of pseudoepitheliomatous hyperplasia suggesting mycobacterial infection. The acid fast bacilli were found on Fite stain but not in Ziehl-Neelsen stain, so borderline lepromatous leprosy was a favorable diagnosis. Cutaneous leprosy lesions must be distinguished from lesions of sarcoidosis, lymphocytic lymphoma, lymphocytoma cutis, lupus erythematosus, secondary syphilis, blastomycosis, leishmaniasis and other deep mycotic infections. Sarcoidosis could be excluded by the presence of bacilli on acid fast stain, detection of mycobacterium DNA in the skin specimen by PCR, as well as negative pulmonary picture of sarcoidosis. Certain other mycobacterioses in immunosuppressed pa- Fig. 3 Alignment of nucleotide sequences of succinate dehydrogenase protein subunit in M tuberculosis, M avium, M marinum, M abscessus, M ulcerans, and M laprae, respectively. The dash represents identical nucleotide within the corresponding allele, and the star indicates missed nucleotide. The dashed arrows represent primers of M tuberculosis, and the arrows represent primers of M laprae. By using the designed primers, M tuberculosis and M laprae can be differentiated. 172 Dermatol Sinica, Sep 2009

4 PCR for M Leprae and M Tuberculosis Fig. 4 Allele-specific polymerase chain reaction. DNA from the patient s lesional skin was strongly amplified by the leprosy primer. L: DNA ladder; 1, 2: DNA from the face reacted with primers of TB and leprosy respectively. 3, 4: DNA from specimen of confirmed leprosy-infected patient reacted with primers of TB and leprosy respectively. 5, 6: DNA from normal specimen for negative control reacted with primers of TB and leprosy respectively. 7, 8: DNA of tuberculosis reacted with primers of TB and leprosy respectively. tients, such as M avium-intracellulare, may produce histoid multibacillary lesions 5 without nerve involvement, necessitate further study for their molecular differentiation. For differential diagnosis of M leprae and M tuberculosis, we used allele-specific PCR by designing primers selectively amplifying 147bp sequences at the locus for succinate dehydrogenase of M leprae and M tuberculosis, respectively. Succinate dehydrogenase is an iron-containing flavoprotein enzyme that catalyzes the dehydrogenation of succinic acid to fumaric acid in the Krebs cycle, and it is widely distributed in animal tissues, bacteria, and yeast. Three different allele-specific oligonucleotides were located in the 3 end primers to prevent mismatch, and the specificity was confirmed in our control samples (Fig. 4). An allele-specific oligonucleotide will only anneal to sequences that match it perfectly, and a single mismatch is sufficient to prevent hybridization. Taq polymerase is used in allele-specific PCR for the discrimination of two alleles that differ by a single nucleotide because it lacks a 3-5 exonuclease activity. The robustness of the method was strongly improved by the introduction a mismatch adjacent to the 3 end of the allele-specific PCR primer, which was supported from other studies. 13 Fig. 3 shows the alignment of nucleotide sequences of M tuberculosis, M avium, M marinum, M abscessus, M ulcerans, and M laprae, respectively. The identities of them ranged from 100% to 87% when compared with the sequences of M tuberculosis in BLAST (Basic Local Alignment Search Tool, NCBI). Leprosy is sometimes difficult in diagnosis due to the varied clinical and pathological presentation and limitation of conventional diagnostic methods. PCR is a useful, rapid method that has become available in recent years in the diagnosis of leprosy. 6 Katoch AM et al. reported 40% to 50% of cases missed on histologic evaluation can be detected. 7 PCR had been found to be sensitive to 1 to 10 organisms and is positive in almost all borderline lepromatous/lepromatous cases. 6 In spite of high sensitivity and specificity, PCR has several limitations including low sensitivity for smear-negative specimens or paucibacillary samples. 8 This may be speculated by the loss of DNA during extraction, failure to sample target DNA on sectioning, or the existence of inhibitory substances. Fixative has also been reported to diminish the PCR signal, particularly when the fixation time is prolonged. 9 Efforts had been made to increase the sensitivity and specificity of PCR in diagnosing paucibacillary leprosy by one-tube nested PCR, 10 by real-time PCR 11 or by PCR using a shorter amplicon. 8 PCR also has a pitfall that it detects the DNA from the dead bacilli. However, this can be resolved Dermatol Sinica, Sep

5 Yu-Ta Yen, et al by using reverse transcriptase PCR detecting 16SrRNA, for this molecule degrades soon after the death of bacilli. The worldwide incidence of leprosy had declined continuously, and the declining trend is expected to continue. 12 Initial diagnosis of leprosy could be difficult as still no culture system exists for M leprae. By allelespecific PCR, we rapidly differentiated M leprae from M tuberculosis. This method may add in the diagnosis when conventional methods fail to identify these diseases in selected clinical situations. REFERENCES 1. Noordeen SK: Eliminating leprosy as a public health problem; why the optimism is justified. Int J Lepr Other Mycobact Dis 63: , Patrocinio LG, Goulart IM, Goulart LR, et al.: Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction. FEMS Immunol Med Microbiol 44: , Lavania M, Katoch K, Sachan P, et al.: Detection of Mycobacterium leprae DNA from soil samples by PCR targeting RLEP sequences. J Commun Dis 38: , Santos AR, De Miranda AB, Sarno EN, et al.: Use of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy. J Med Microbiol 39: , Wood C, Nickoloff BJ, Todes-Taylor NR: Pseudotumor resulting from atypical mycobacterial infection: a histoid variety of Mycobacterium avium-intracellulare complex infection. Am J Clin Pathol 83: , Katoch VM: Molecular techniques for leprosy: present applications and future perspectives. Indian J Lepr 71: 45-59, Katoch VM: Advances in the diagnosis and treatment of leprosy. Expert Rev Mol Med 4: 1-14, Goulart IM, Cardoso AM, Santos MS, et al.: Detection of Mycobacterium leprae DNA in skin lesions of leprosy patients by PCR may be affected by amplicon size. Arch Dermatol Res 299: , Benavides J, Garcia-Pariente C, Gelmetti D, et al.: Effects of fixative type and fixation time on the detection of Maedi Visna virus by PCR and immunohistochemistry in paraffin-embedded ovine lung samples. J Virol Methods 137: , Jamil S, Wilson SM, Hacket M, et al.: A colorimetric PCR method for the detection of M. leprae in skin biopsies from leprosy patients. Int J Lepr Other Mycobact Dis 62: , Martinez AN, Britto CF, Nery JA, et al.: Evaluation of real-time and conventional PCR targeting complex 85 genes for detection of Mycobacterium leprae DNA in skin biopsy samples from patients diagnosed with leprosy. J Clin Microbiol 44: , World Health Organization: Global leprosy situation, Wkly Epidemiol Rec 81: , Ayyadevara S, Thaden JJ, Shmookler Reis RJ: Discrimination of primer 3 -nucleotide mismatch by Taq DNA polymerase during polymerase chain reaction. Anal Biochem 284: 11-18, Dermatol Sinica, Sep 2009

6 PCR for M Leprae and M Tuberculosis 使用對偶基因特異性聚合連鎖反應快速辨別麻風分枝桿菌及結核分枝桿菌感染 顏育達鄭裕文吳唯銘高雄長庚紀念醫院皮膚科長庚大學醫學院 麻風 ( 漢生病 ) 是一種由麻風分枝桿菌所引起之慢性肉芽性感染, 主要侵犯皮膚及神經 麻風是一種重要的臨床課題, 對於臨床醫師而言此疾病的診斷也是一項挑戰 有時麻風與結核菌感染之尋常性狼瘡不易做鑑別診斷, 特別當感染發生於臉部時 我們報告一位 70 歲女性在左側眼部周圍有界限明顯逐漸增大的紅色斑塊, 疑似麻風或尋常性狼瘡 在皮膚切片及施行對偶基因特異性聚合連鎖反應後確認麻風的診斷 使用對偶基因特異性聚合連鎖反應可以快速正確鑑別診斷麻風及結核感染 ( 中華皮誌 :27: , 2009) Dermatol Sinica, Sep