Kanishka Senaratha, C. L. Goonasekarab , C. D. Wijayarathnaa A NOVEL MOLECULAR ASSAY FOR DIAGNOSIS OF TUBERCULOSIS: PROBLEMS IN ESTABLISHMENT Kanishka Senaratha, C. L. Goonasekarab , C. D. Wijayarathnaa aDepartment of Chemistry, Faculty of Science, University of Colombo, Sri Lanka. bFaculty of Medicine, General Sir John Kotelawala Defence University, Sri Lanka.

Objectives of the project To apply LAMP method for TB diagnosis. To assess its performance as a simple, rapid, accurate and cost-effective diagnostic tool, suitable for Sri Lankan settings.

Introduction Tuberculosis (TB) is an infectious disease caused by the bacillus, Mycobacterium tuberculosis (MTB). It typically affects the lungs, causing pulmonary TB. But it can affect other sites like pleural space, genitourinary tract, intra-abdominal organs, central nervous system and pericardium as well which is known as extra-pulmonary TB. Major health problem in developing countries.

Parsons, L. M. et al., J, Clin. Microbiol. Rev., 2011, 24, 314-350

The status of TB in Sri Lanka The Ministry of Healthcare and Nutrition has launched a National Program for Tuberculosis Control and Chest Diseases (NPTCCD), in order to make Sri Lanka TB free. According to NPTCCD data, about 9000 new cases of TB are notified every year, showing that TB continues to be a major public health concern. Therefore, rapid and accurate laboratory diagnostics are necessary to control TB in Sri Lanka. The status of TB in Sri Lanka

Why TB control is difficult ? Late diagnosis of the disease leads to the emergence of drug-resistant strains, which are resistant to standardized first-line antimicrobial drugs, which ultimately causes multi-drug resistant TB (MDR-TB). Therefore, the ability to rapidly detect Mytcobacterium tuberculosis in clinical specimens is essential for the appropriate treatment of TB patients and the prevention of spread of TB.

Current TB diagnostic methods in Sri Lanka Sputum smear microscopy Rapid Simple Mycobacterium under light microscope Identification is possible only up to the genus level Expert in observing specimen http://www.bikupan.se/tuberculosis/tuberc.html http://ww.bikupan.se/tuberculosis/tuberc.html

Distinctive clusters of colorless Mycobacterium tuberculosis 2. Microbial culture Distinctive clusters of colorless Mycobacterium tuberculosis http://en.wikipedia.org/wiki/File:TB_Culture.jpg Highly sensitive Specific Time consuming

Rapid reaction Simple operation Easy detection LAMP method

Introduction to LAMP "LAMP" Loop-mediated Isothermal Amplification. Developed by Notomi et al. in 2000. Employs Bacillus stearothermophilus (Bst) DNA polymerase, which has a high strand displacement activity. The whole amplification reaction takes place at a single constant temperature (650C), with in a short time (45 – 6o minutes).

LAMP is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions on the target gene. Primer design of the LAMP reaction Tomita, N., Mori, Y., Kanda, H., Notomi, T., Nat. Protoc., 2008, 3, 877-882

Final products of LAMP reaction are stem-loop structures with several inverted repeats of the target sequence. Cauliflower-like structures with multiple loops are also being formed by annealing between alternative inverted repeats of target sequence in the same strand. Cauliflower-like structures Final products of LAMP reaction Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., Nucleic Acids Res., 2000, 28, E63.

LAMP reaction products using agarose gel electrophoresis Amplified products of LAMP can be visualized by gel electrophoresis of a small aliquot from the final reaction mixture. LAMP products produce a characteristic smeared ladder-like pattern with multiple bands. LAMP reaction products using agarose gel electrophoresis Tomita, N., Mori, Y., Kanda, H., Notomi, T., Nat. Protoc., 2008, 3, 877-882

Disease diagnosis by LAMP African trypanosomes (Kuboki et al., 2003) Severe acute respiratory syndrome (Thai et al., 2004) West Nile virus (Parida et al., 2004) Dengue virus (Parida et al., 2005) Plasmodium falciparum (Poon et al., 2006), etc.

The LAMP for detection of M.tuberculosis is simple! DNA amplification is highly robust. It does not require thermal cyclers. No expensive reagents or time-consuming steps for DNA purification. No need of down stream processing for amplicon detection.

TB DIAGNOSIS BY LAMP METHOD Three LAMP assays were set up to detect MTB specific DNA sequences. - specific for 16S ribosomal RNA (16S rRNA) gene in genus Mycobacterium - contained MTB specific primers targeting rimM and gyrB gene sequences in MTB.

Iwamoto et al, Clin. Microbiol., 2003, 41, 2616-2622

LAMP assay - + 11.5 µl Primers Thermopol buffer dNTPs Bst DNA polymerase LAMP reaction mixture Sample for LAMP assay SYBR Green I Visual detection LAMP amplification At 650C for 45 minutes - +

Results The detection of false positives was unavoidable throughout the study.

Troubleshooting Optimization of LAMP assay Several factors are thought to affect the LAMP assay. These include; - incubation time/reaction time, - enzyme concentration in the reaction mixture, - temperature during the reaction, etc. Each parameter was varied in order to establish standards to the assay.

A new set of working solutions (new aliquot of enzyme, dNTPs and buffer) and freshly prepared primers with PCR grade water, were used for the LAMP reaction, to rule out the possibility of false positives due to reagent contamination. Separate working areas were used for each step of the reaction procedure; reagent preparation/mixing, amplification and amplicon detection. The reaction mixture preparation was done under a lamina hood to avoid any aerial contamination.

Expected result for agarose gel electrophoresis of LAMP amplicons. No change between positive samples and negative controls. Size of amplicons was checked. Marker + - Expected result for agarose gel electrophoresis of LAMP amplicons. Zhu, R.-Y., Zhang, K.-X., Zhao, M.-Q., Liu, Y.-H., Xu, Y.-Y., Ju, C.-M., Li, B., Chen, J.-D., J. Microbiol. Meth., 2009, 78, 339-343.

Agarose gel electrophoresis results for LAMP products 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Agarose gel electrophoresis results for LAMP products 1:Marker, 2:Blank, 3, 6, 8, 11, 13, 15, 17, 19:LAMP products with universal primers, 4, 7:LAMP products with rimM primers, 5:LAMP control without Bst, 10:Negative control with rimM primers, 9, 12, 14, 16, 18, 20:Negative controls with universal primers.

The region being amplified by gyrB primer set. 2. Restriction digestion of purified LAMP amplified products LAMP amplicons were digested with the restriction enzyme; BsaI. The region being amplified by gyrB primer set.

Theoritically predicted sizes after BsaI digestion are 94 bp and 114 bp. The sizes of the fragments generated after the digestion of MTB positive sample were in good agreement with theoritically predicted sizes. This provides evidence for the MTB specific DNA amplification in a positive LAMP reaction and non specific amplification in the negative control.

100 bp Agarose gel electrophoresis results after the restriction digestion. 1- 100 bp marker, 2- Blank, 3- MTB positive sample before digestion, 4- MTB positive sample after digestion, 5- Negative control before digestion, 6- Negative control after digestion.

Restriction digestion results were further confirmed by sequencing LAMP amplicons of a MTB positive sample and a negative control. The sequencing results established that the false positives are not due to contamination with MTB, because the restriction digestion site (GGTCTC) is absent in the negative control. It further suggested that the false positives could be due to a self amplification of the LAMP primers.

Therefore, the LAMP assay could be recommended for the diagnosis of TB if the detection is carried out using restriction digestion of the LAMP amplicons. However, since this reaction is more expensive and time consuming, further refining of the method is necessary before it can be validated as a fast and cost effective method for TB diagnosis.