The diagnostic value of gyrb RFLP PCR. Mycobacteria in patients with clinical. in Mazandaran

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2 Mazandaran University of Medical Sciences The diagnostic value of gyrb RFLP PCR test t in differentiation between pathogenic Mycobacteria in patients with clinical suspicions spicions of tuberculosis in Mazandaran BY: Maryam Pourhajibagher Msc of Medical Microbiology

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4 Tuberculosis (TB) is one of the leading causes of death due to infectious agents. Ninety-five percent of cases occur in the developing countries in Asia, Africa, Middle East and Latin American, where few resources are available for diagnosis and treatment. The rate of death 3 million cases 1million children 2 million adults

5 9.4 million new TB cases in million cases were infectious to TB in 2003 WHO has estimated The resurgence of tuberculosis and the appearance of multidrug resistant strains of Mycobacterium tuberculosis have intensified the need for the increased use of rapid methods for isolating and identifying clinically encountered mycobacteria.

6 wild and domestic mammals human voles human human Mycobacterium bovis Mycobacterium microti Mycobacterium tuberculosis MTBC human Mycobacterium africanum

7 The aim of this study Is differentiation between pathogenic mycobacteria with specific concern on gyrb RFLP PCR in patients that referred to health care center in Mazandaran province.

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9 Selection Study population Region: Mazandaran province Time: from July 2010 to June 2011 Place: Health care center in Mazandaran province Population: Patients with clinical suspicions of tuberculosis

10 enter criteria cough for more than 3 weeks cheste ache sputum weight loss enter criteria coughing up blood appetite loss fever

11 Steps Collection of specimens Prepare the specimens for culture and staining Microscopic examination of smears Culture on Lowenstein-Jensen medium DNA extraction PCR for differentiation of MTBC from NTM PCR for identifying Mycobacterium tuberculosis RFLP PCR for identifying M. bovis, M. microti, M. africanum af ica u

12 Collection of specimens Collection of sputum Collection in container made of unbreakable transparent plastic Write the name of person

13 Prepare the specimens 1. liquefied and decontaminated with 4% NaOH 2. Vortexing for 15 minute. 3. centrifugation at 3,000 g for 20 minute. 4. Then the sediments e were e resuspended. 5. Added phenol-phetalein 1% 6. Addedd HCl 1N (%3.2)

14 Microscopic examination of smears Hemogenize Put the Add Heat slides by wooden specimen Fixing carbol from applicator on the slide fuchsin underneath Rinse slides gently with water Decolourising with acidalcohol Rinse slides gently with water Add methylene blue Wash, dry, observe

15 Culture on Lowenstein-Jensen 0.1 to 0.2 ml of sediment of the processed specimens were inoculated on Löwenstein-Jensen medium (Bahar Afshan, Iran) and incubated at 37 ºC for 4-6 weeks. Because of proven high sensitivity of culture, this technique was p g y, q used as the gold standard in this study.

16 DNA extraction µl of sample homogenized and mixed with 400 µl of DNG TM -Plus (CinnaGen Co., Iran, #DN8118C) 2. vortexed for seconds µl of Isopropanol added and mixed by vortexing 4. centrifuged at 12,000 rpm for 10 minutes 5. Decanted by gently inverting of tube and placing the tube on tissue paper for 2-3 sec downward 6. 1 ml %75 Ethanol added to pellet, mixed by 3-5 sec vortexing and centrifuged at 12,000 rpm for 5 min 7. Step 6 was repeated once more 8. The Ethanol poured off completely and the pellet dried at 65 ºC for 5 min 9. The DNA pellet dissolved in 50 µl of sterile distilled water by gentle shaking and placed at 65 ºC for 5 min.

17 PCR for differentiation of MTBC from NTM Primers used in identifying Mycobacterium tuberculosis complex Gene MTUB F MTUB R Primer sequences 5 TCGGACGCGTATGCGATATC ACATACAGTTCGGACTTGCG 3 PCR product 1,020 bp Mycobacterium tuberculosis H37Rv gyrase B (gyrb) gene TCGGACGCGTATGCGATATCTGGTGGTCTGCACGGCGTCGGCGTGTCGGTGGTTAACGCGCTATCCACCCGGCTCGA AGTCGAGATCAAGCGCGACGGGTACGAGTGGTCTCAGGTTTATGAGAAGTCGGAACCCCTGGGCCTCAAGCAAGGGG CGCCGACCAAGAAGACGGGGTCAACGGTGCGGTTCTGGGCCGACCCCGCTGTTTTCGAACCCACGGAATACGACTTCG AAGCCGTCGCCCGCCGGCTGCAAGAGATGGCGTTCCTCAACAAGGGGCTGACCATCAACCTGACCGACGAGAGGGTG ACCCAAGACGAGGTCGTCGACGAAGTGGTCAGCGACGTCGCCGAGGCGCCGAAGTCGGCAAGTGAACGCGCAGCCG AATCCACTGCACCGCACAAAGTTAAGAGCCGCACCTTTCACTATCCGGGTGGCCCGGTGGACTTCGTGAAACACATCA ACCGCACCAAGAACGCGATTCATAGCAGCATCGTGGACTTTTCCGGCAAGGGCACCGGGCACGAGGTGGAGATCGCGA TGCAATGGAACGCCGGGTATTCGGAGTCGGTGCACACCTTCGCCAACACCATCAACACCCACGAGGGCGGGCGGCAC GAAGAGGGCTTCCGCAGCGCGCTGACGTCGGTGGTGAACAAGTACGCCAAGGACCGCAAGCTACTGAAGGACAAGG ACCCCAACCTCACCGGTGACGATATCCGGGAAGGCCTGGCCGCTGTGATCTCGGTGAAGGTCAGCGAACCGCAGTTCG AGGGCCAGACCAAGACCAAGTTGGGCAACACCGAGGTCAAATCGTTTGTGCAGAAGGTCTGTAACGAACAGCTGACC CACTGGTTTGAAGCCAACCCCACCGACGCGAAAGTCGTTGTGAACAAGGCTGTGTCCTCGGCGCAAGCCCGTATCGCG GCACGTAAGGCACGAGAGTTGGTGCGGCGTAAGAGCGCCACCGACATCGGTGGATTGCCCGGCAAGCTGGCCGATTGC CGTTCCACGGATCCGCGCAAGTCCGAACTGTATGT

18 PCR for identifying Mycobacterium tuberculosis Primers used in identifying Mycobacterium tuberculosis Gene MTUB 756 GF MTUB 1450 CR Primer sequences 5 GAAGACGGGGTCAACGGTG 3 5 CCTTGTTCACAACGACTTTCGC 3 PCR product 734 bp Mycobacterium tuberculosis H37Rv gyrase B (gyrb) gene TCGGACGCGTATGCGATATCTGGTGGTCTGCACGGCGTCGGCGTGTCGGTGGTTAACGCGCTATCCACCCGGCTCGAAG TCGAGATCAAGCGCGACGGGTACGAGTGGTCTCAGGTTTATGAGAAGTCGGAACCCCTGGGCCTCAAGCAAGGGGCG CCGACCAAGAAGACGGGGTCAACGGTGCGGTTCTGGGCCGACCCCGCTGTTTTCGAACCCACGGAATACGACTTCG AAGCCGTCGCCCGCCGGCTGCAAGAGATGGCGTTCCTCAACAAGGGGCTGACCATCAACCTGACCGACGAGAGGGTG ACCCAAGACGAGGTCGTCGACGAAGTGGTCAGCGACGTCGCCGAGGCGCCGAAGTCGGCAAGTGAACGCGCAGCCG AATCCACTGCACCGCACAAAGTTAAGAGCCGCACCTTTCACTATCCGGGTGGCCCGGTGGACTTCGTGAAACACATCA ACCGCACCAAGAACGCGATTCATAGCAGCATCGTGGACTTTTCCGGCAAGGGCACCGGGCACGAGGTGGAGATCGCGA TGCAATGGAACGCCGGGTATTCGGAGTCGGTGCACACCTTCGCCAACACCATCAACACCCACGAGGGCGGGCGGCAC GAAGAGGGCTTCCGCAGCGCGCTGACGTCGGTGGTGAACAAGTACGCCAAGGACCGCAAGCTACTGAAGGACAAGG ACCCCAACCTCACCGGTGACGATATCCGGGAAGGCCTGGCCGCTGTGATCTCGGTGAAGGTCAGCGAACCGCAGTTCG AGGGCCAGACCAAGACCAAGTTGGGCAACACCGAGGTCAAATCGTTTGTGCAGAAGGTCTGTAACGAACAGCTGACC CACTGGTTTGAAGCCAACCCCACCGACGCGAAAGTCGTTGTGAACAAGGCTGTGTCCTCGGCGCAAGCCCGTATCG CGGCACGTAAGGCACGAGAGTTGGTGCGGCGTAAGAGCGCCACCGACATCGGTGGATTGCCCGGCAAGCTGGCCGATT GCCGTTCCACGGATCCGCGCAAGTCCGAACTGTATGT

19 RFLP PCR for identifying i another Mycobacterium Restriction enzyme RsaI Enzyme Species Product RsaI (Fermentas) Mycobacterium bovis Mycobacterium microti Mycobacterium africanum 480, 360 bp 660, 360 bp 560, 360 bp TCGGACGCGTATGCGATATCTGGTGGTCTGCACGGCGTCGGCGTGTCGGTGGTTAACGCGCTATCCACCCGGCTCGAAG TCGAGATCAAGCGCGACGGGTACGAGTGGTCTCAGGTTTATGAGAAGTCGGAACCCCTGGGCCTCAAGCAAGGGGCG CCGACCAAGAAGACGGGGTCAACGGTGCGGTTCTGGGCCGACCCCGCTGTTTTCGAACCCACGGAATACGACTTCGA AGCCGTCGCCCGCCGGCTGCAAGAGATGGCGTTCCTCAACAAGGGGCTGACCATCAACCTGACCGACGAGAGGGTGA CCCAAGACGAGGTCGTCGACGAAGTGGTCAGCGACGTCGCCGAGGCGCCGAAGTCGGCAAGTGAACGCGCAGCCGA ATCCACTGCACCGCACAAAGTTAAGAGCCGCACCTTTCACTATCCGGGTGGCCCGGTGGACTTCGTGAAACACATCAA CCGCACCAAGAACGCGATTCATAGCAGCATCGTGGACTTTTCCGGCAAGGGCACCGGGCACGAGGTGGAGATCGCGAT GCAATGGAACGCCGGGTATTCGGAGTCGGTGCACACCTTCGCCAACACCATCAACACCCACGAGGGCGGGCGGCACG AAGAGGGCTTCCGCAGCGCGCTGACGTCGGTGGTGAACAAGTACGCCAAGGACCGCAAGCTACTGAAGGACAAGGA CCCCAACCTCACCGGTGACGATATCCGGGAAGGCCTGGCCGCTGTGATCTCGGTGAAGGTCAGCGAACCGCAGTTCGA GGGCCAGACCAAGACCAAGTTGGGCAACACCGAGGTCAAATCGTTTGTGCAGAAGGTCTGTAACGAACAGCTGACCC ACTGGTTTGAAGCCAACCCCACCGACGCGAAAGTCGTTGTGAACAAGGCTGTGTCCTCGGCGCAAGCCCGTATCGCGG CACGTAAGGCACGAGAGTTGGTGCGGCGTAAGAGCGCCACCGACATCGGTGGATTGCCCGGCAAGCTGGCCGATTGCC GTTCCACGGATCCGCGCAAGTCCGAACTGTATGT

20 Statistical Analysis The χ2 and Fisher exact tests were used to compare the frequency of discrete variables. Mean, Central tendency and Dispersion were used for analyzing data with SPSS18.

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22 Of the 1345 samples of patients with clinical suspicions of tuberculosis referred to health care center of Mazandaran, 65 (4.83%) were positive culture and 57 (4.23%) were positive smear. The collected specimens were 1222 (90.8%) sputa, 12 (0.9%) urine, 15 (1.11%) pleural effusion, 1 (0.07%) aspirate cerebrospinal fluid (CSF), 8 (0.6%) acit fluid, 2 (0.14%) blood samples, 63 (4.68%) bronchial washes, 14 (1.04%) gastric washes, 1 (0.07%) skin biopsy, 1(0.07%) abscesses, 1 (0.07%) wound, 2 (0.14%) liver cystic fluids and 3 (0.22%) tissue.

23 The average of age of patients was 45.5± The minimum of age was 15 years old and the maximum of age was s79 years old. od The Frequence of tuberculosis was 3-5% with confidence interval 95% (CI95%).

24 Results of positive culture and smear according to sex and age obtained from patients of tuberculosis obtained from patients of tuberculosis

25 65 mycobacterial isolates were investigated by PCR to confirm their identity as members of the Mycobacteria. 59 (90.76%) of the isolates were identified as members of the Mycobacterium tuberculosis complex whereas 6 (9.24%) of the isolates were negative and identified as NTM. L ,020 bp 500 bp 100 bp Figure 1: PCR amplified 1,020 bp fragment of the gyrb gene. L: 100 bp ladder, Lane 1: positive control amplified from Mycobacterium tuberculosis H37Rv. Lane 2-7: DNAs from clinical isolates identified as Mycobacterium tuberculosis complex

26 The 59 MTBC isolates confirmed with gyrb PCR were further differentiated by species specific PCR using specific set of the primers MTUB-756-Gf and MTUB-1450-Cr that allowed selective amplification of the gyrb fragments from M. tuberculosis. All tested isolates produced a band of 734 bp, which is specific for M. tuberculosis. L bp 500 bp 100 bp Figure 2: Analysis of PCR amplified 734 bp fragment of the gyrb gene from M. tuberculosis. L: 100 bp ladder, Lane 1: positive control amplified from M. tuberculosis H37Rv. Lane 2-5: clinical isolates identified as Mycobacterium tuberculosis.

27 Reference strains M. tuberculosis H37Rv as well as all of 59 M. tuberculosis isolates confirmed by gyrb PCR were analyzed RFLP PCR. All M. tuberculosis isolates showed the typical RsaI pattern (230 and 480 bp) compared to reference strain. L bp 500 bp 230 bp 100 bp Figure 3: RFLP pattern of PCR products obtained by RsaI digestion. L: 100 bp ladder, Lane 1: positive control amplified from M. tuberculosis H37Rv. Lane 2-5: Mycobacterium tuberculosis clinical pattern.

28 1,020 bp In this study t d Using gyrb gene and observation of band in 1,020 bp In previous studies Abass NA, 2010 Richter E, 2004 Kasai H, 2000 Niemann S, 2000

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30 Bovine tuberculosis is the agent of M. bovis that it is between human and i common disease di b t h d bovine. gyrb gene is one of the genes that it can be used Fortunately, in present study, M. bovis did not in clinical mycobacteriology laboratories. identify of patients. patients Based on the results obtained in this study M. Microti infects voles and humans and M. and those previously published, PCR RFLP africanum are limited to human. In this study. of the gyrb gene using the commonly used None of them had been identified. enzyme RsaI, permits one to easily separate So, the agent of tuberculosis in patients the MTBC species from other atypical referred to health care center of Mazandaran Mycobacterium and replacing the more time was M. tuberculosis. consuming biochemical tests. tests By early diagnosis of TB, we can help clinicians to select appropriate drugs and reduce drug resistance and MDR. MDR

31 Acknowledgments Dr. Mohtaram Nasrollahi Dr. Dr Mohammad Ahanjan Dr. Alireza Khalilian Dr. Alireza Rafiee Dr. Mohsen Tehrani Dr. Saeed Abedian M h M fi Mahyar Mafi Seyed Reza Musavi Saeede Ghorban alipour Bahman Rahimi esboei Abuzar Ghorbani This study was supported by Mazandaran Abdolsattar PagheUniversity of Medical Sciences, Sari, Iran. Masumeh Sadeghi sarsari