Detection of rifampicin resistance in sputum samples by PCR-ELISA

Introduction Tuberculosis caused by Mycobacterium tuberculosis shows resistance to anti-tuberculosis drugs like Rifampicin and Isoniazid generating a public health problem. Using molecular techniques, such as PCR-ELISA, is an alternative that will detect resistance to RIF easily and economically. Objective Optimize a PCR-ELISA that can detect resistance to RIF in sputum samples. Method A PCR-ELISA was standardized to amplify a 255bp of rpoB gene that encodes resistance to RIF. Different parameters were optimized: hybridization temperatures, and probe concentration, among others. The technique’s analytical sensitivity, specificity, and concordance against GenotypeMTBDRplus v2 were evaluated using 20 samples in a pilot assay. Results A PCR-ELISA was optimized for the detection of resistance mutations; the analytical sensitivity of the PCR-ELISA was 4ng of PCR product for the H445D and H445Y probes while for the S450L probe the sensitivity was 0.4 ng of PCR product respectively. The technique has a specificity of 100%. Two mutations, S450W and L452P, were not detected by our system. In the pilot assay, a “good agreement” (k=0.737) was obtained between both techniques. Conclusions A PCR-ELISA was standardized for the detection of the 3 more frequent mutations in Peru associated with resistance to RIF simultaneously, the most frequent being S450L.


INTRODUCTION
44 Tuberculosis (TB) is caused by the bacillus Mycobacterium tuberculosis (MTb), and is 45 transmitted when sick people expulse bacteria into the air (e.g., by coughing) (1) The disease 46 typically affects the lungs (pulmonary TB) but can affect other organs of the body. 47 TB is a communicable, curable, and preventable disease; approximately 85% of people who 48 develop the disease can be successfully treated (2). However, social determinants such as 49 poverty, malnutrition, HIV infection, smoking, and diabetes aggravate this disease (2).  Drug-resistant TB remains a public health problem of great concern due to simultaneous 57 resistance to isoniazid (INH) and rifampicin (RIF) the two most effective first-line drugs for TB 58 treatment, which is called Multidrug-resistant TB (MDR-TB). Extensive drug-resistant (XDR-TB) 59 tuberculosis which defined as a case of MDR-TB that is resistant to either fluoroquinolone and 60 one of the second-line injectable drugs (capreomycin, kanamycin, or amikacin) (4) also 61 contribute to worsening the problem. RIF-resistant TB (RR-TB), MDR-TB, and XDR-TB require 62 longer treatment with second-line drugs causing more side effects when patients are treated 63 (2). 64 Peru leads MDR-TB incidence cases in the Americas region unlike Brazil, which has a higher 65 incidence of TB and HIV-TB cases (3). methods and that can mainly be implemented in primary care hospitals or less complex health 114 centers. In this sense, this work aimed to optimize and evaluate, in the laboratory, a PCR-ELISA 115 for the detection of H445D, H445Y, and S450L mutations conferring RIF resistance in sputum 116 samples from tuberculosis patients.  120 The following strains were used for this work: ATCC N° 27294 (H37Rv) which was used as a 121 control sensitive to all drugs, ATCC N° 35838 used as a control for resistance to RIF with the 122 S450L mutation, and 2 strains (RIF-8853 and RIF-4469) of Mycobacterium tuberculosis which 123 were used as controls for the H445D and H445Y mutations, from the National Reference  The sputum samples were provided by the LRNM and had the following characteristics: a 131 bacilloscopy (BK+) ranging from 1+ to 3+ and a storage time in refrigeration (2 -8°C) between 132 6 and 30 days since they were collected. The sensitive sputum was used for the CUT-OFF  The aqueous phase was transferred to another sterile 1.5 ml tube taking care not to drag the 143 interphase, 500 μL of ice-cold 100% Isopropanol (-20°C) was added, mixed by inversion 20 144 times, and then centrifuged for 15 min at 14000 RPM and the supernatant was removed. To 145 the precipitate, 1 mL of ice-cold 70% ethanol was added, mixed by inversion 20 times, and 146 centrifuged for 5 minutes at 14000 RPM, the supernatant was discarded, and the tubes were 147 dried at room temperature. Finally, the DNA was resuspended in 200 µL of TE buffer and stored 148 at -20°C until use.    To determine the analytical sensitivity of the PCR-ELISA, 2 dilutions of PCR product were 182 performed: 1/10 and 1/100, starting from a concentration of 4ng for the 3 resistant probes.

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To determine the specificity of the PCR-ELISA, DNA from 7 Non-tuberculosis mycobacteria  Samples that showed "inferred resistance" by GenotypeMTBDRplus v2 were also sequenced 202 to identify mutations conferring resistance to RIF.  The CUT-OFF was determined using SPSSv25 software which allowed the calculation of the 209 cut-off limit of the average of the absorbances of the 15 sensitive and H37Rv samples plus 3 210 times standard deviation (SD) for the H445Y and H445D probes and 2 times the SD for the 211 S450L probe. A value above the CUT-OFF was considered resistant (presence of the mutation) 212 and below was considered sensitive (absence of the mutation).
Once the cut-off values were obtained, the pilot assay was performed in blind (without knowing 214 the resistance profile) using 20 sputum samples, whose resistance was previously determined 215 by GenotypeMTBDRplus v2. DNA was extracted from these samples and PCR-ELISA was 216 performed using the 3 probes simultaneously. The PCR-ELISA results were compared with the 217 results obtained against GenotypeMTBDRplus v2 after revealing the blind.  (Table 2). 235 The PCR profile was established: Initial denaturation at 95°C for 10 min (1 cycle), denaturation 236 at 95°C for 30 sec, annealing at 60°C for 45 sec and extension at 72°C for 30 sec (35 cycles).

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Final extension at 72°C for 5 min (1 cycle). PCR products were stored at -20°C until use.

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Standardization of all PCR-ELISA conditions allowed to establish the following parameters: 239 hybridization temperature at 50°C, SSPE hybridization buffer at low stringency (3X), 240 streptavidin concentration at 10 μg/mL, probe concentration at 20 pmol/mL, PCR product 241 concentration at 4ng and conjugate concentration at 150mU (1/1000) ( Table 2). The analytical sensitivity of the PCR-ELISA was 4ng of PCR product for the H445D and H445Y 245 probes while for the S450L probe the sensitivity was 0.4 ng of PCR product (Table S1).  For the samples that did amplify, the PCR-ELISA gave negative results, determining that there 253 was no hybridization in the NTMs nor the negative control (Table S2) (Table S3). Only for the H445D probe, it was decided to  (Table 3).

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The PCR-ELISA system presented a sensitivity of 86.7% which corresponded to a successful 293 concordance of 13 out of 15 samples. The value of Cohen's Kappa index was 0.737, which 294 denotes a "good agreement" between GenotypeMDRTBplus v2 and PCR-ELISA (Table 4).

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This work presents the standardization and pilot evaluation of the PCR-ELISA technique for the 299 detection of RIF resistance using biotinylated probes for which several parameters were 300 optimized including DNA extraction.

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In the present work, sputum samples and strains from which DNA was extracted by the CTAB-302 lyz method were used; this method proved to be adequate for direct extraction of DNA from 303 sputum compared to other extraction protocols (29-31).    The analytical sensitivity of PCR-ELISA for the H445D and H445Y mutations was 4 ng of PCR 328 product (Table S1), while for the S450L mutation a sensitivity of 0.4ng was detected because 329 the latter tends to have high absorbance values, this behavior coincides with that reported by  However, for the NTMs that did amplify when the ELISA was performed, they did not hybridize, 341 therefore, the PCR-ELISA has 100% specificity (Table S2)   BK between (1 to 2 +) and with a storage time in refrigeration between 6 to 20 days (Table 3), 356 despite having a higher concentration of DNA there was no amplification of PCR product, 357 probably because prolonged refrigeration times cause lysis in cells and MTB bacilli causing the 358 DNA concentration detected during quantification to be more human DNA than bacillus DNA in 359 these samples. Therefore, the PCR-ELISA analysis was performed on 17 samples (Table 3).  , Table S4.A), and for sample 13, the nucleotide change T → C was found at position 1356 which generated 369 the variation of the amino acid Leucine for Proline (L452P , Table S4.B).

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On the other hand, the samples (M-9 and M-14) were discordant in the detection of resistance. 371 these samples had many days of refrigeration (approx. 24 days) and a bacilloscopy lower than 372 3+, this situation caused a low concentration of extracted DNA (<1ng/µL) which generated a 373 faint amplification, poor hybridization, and therefore a false sensitive result in the PCR-ELISA 374 (Table 3). 375 The concordance (kappa index "k") between GenotypeMTBDRplus v2 and the PCR-ELISA 376 technique was calculated. A value k = 0.737 was found (Table 4), which corresponded to a 377 "good agreement" between both techniques.