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Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection Cover

Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection

Polish Journal of Microbiology
Volume 72 (2023): Issue 4 (December 2023)
By: Hassan A. Hemeg,  Hamzah O. Albulushi,  Hani A. Ozbak,  Hamza M. Ali,  Emad K. Alahmadi,  Yahya A. Almutawif,  Sari T. Alhuofie,  Rana A. Alaeq,  Areej A. Alhazmi,  Mustafa A. Najim and  Ahmed M. Hanafy  
Open Access
|Dec 2023

Figures & Tables

Fig. 1.

Polymerase chain reaction amplification of the specific 130 bp fragments with the IS6110 primer pair (panel a) and the specific 152 bp fragments with the mtp40 primer pair (panel b). Lanes 2–36 of (panel a) are representative amplified PCR products with the IS6110 primers from selected sputum samples. Lanes 5, 11, 12, 16, 17, 20, 23, 24, 25, 29, and 34 of (panel b) are representative amplified PCR products with the mtp40 primers from selected sputum samples. Lanes 2, 14, 26, 27, 28, and 30 represent the samples not amplified by the mtp40 primer pair. Lanes M (panels a and b) represent the 100-bp DNA ladder.
Polymerase chain reaction amplification of the specific 130 bp fragments with the IS6110 primer pair (panel a) and the specific 152 bp fragments with the mtp40 primer pair (panel b). Lanes 2–36 of (panel a) are representative amplified PCR products with the IS6110 primers from selected sputum samples. Lanes 5, 11, 12, 16, 17, 20, 23, 24, 25, 29, and 34 of (panel b) are representative amplified PCR products with the mtp40 primers from selected sputum samples. Lanes 2, 14, 26, 27, 28, and 30 represent the samples not amplified by the mtp40 primer pair. Lanes M (panels a and b) represent the 100-bp DNA ladder.

Fig. 2.

Multiplex-PCR results in 2% agarose gel: the 392 bp product corresponds to embB codon (306); the 335 bp to katG (315) codon, the 270 bp to (–15) promoter region of mab A -inh A; the 218 bp, 185 bp, and 170 bp products correspond to rpoB codons (516, 526 and 531), respectively. Results show a nonspecific small-size product that links to the amplification of the 170 bp fragment. Lanes M (panels a and b) 100-bp DNA ladder. Lanes 2, 5, 11, 27, 28, and 29 represent sputum samples’ DNA with no mutation at the codons under study. Lanes 12-26 (panel a) and lane 48 (panel b) show embB codon 306 mutation (ethambutol). Lanes 30, 34, 36 (panel a) show rpoB codon 526 mutation (rifampin). Lanes 37, 41, 42, 44, 46 and 47 (panel b) show mutations with both the rpoB codon 531 (rifampin) and embB codon 306. Lanes 40 and 43 show mutation in rpoB codon 531 (rifampin) only.
Multiplex-PCR results in 2% agarose gel: the 392 bp product corresponds to embB codon (306); the 335 bp to katG (315) codon, the 270 bp to (–15) promoter region of mab A -inh A; the 218 bp, 185 bp, and 170 bp products correspond to rpoB codons (516, 526 and 531), respectively. Results show a nonspecific small-size product that links to the amplification of the 170 bp fragment. Lanes M (panels a and b) 100-bp DNA ladder. Lanes 2, 5, 11, 27, 28, and 29 represent sputum samples’ DNA with no mutation at the codons under study. Lanes 12-26 (panel a) and lane 48 (panel b) show embB codon 306 mutation (ethambutol). Lanes 30, 34, 36 (panel a) show rpoB codon 526 mutation (rifampin). Lanes 37, 41, 42, 44, 46 and 47 (panel b) show mutations with both the rpoB codon 531 (rifampin) and embB codon 306. Lanes 40 and 43 show mutation in rpoB codon 531 (rifampin) only.

Fig. 3.

Rifampin and ethambutol resistance correlated with mutations and their frequency.
Rifampin and ethambutol resistance correlated with mutations and their frequency.

Fig. 4.

Detailed Venn diagrams showing the number of shared and unique RIF-sensitive MTBC-positive samples (panel a) and RIF-resistant MTBC-positive samples (panel b) among the GeneXpert MTB/RIF, phenotypic culture-based DST, and multiplex PCR assays, and EMB-sensitive MTBC-positive samples (panel c) and EMB-resistant MTBC-positive samples (panel d) among phenotypic culture-based DST and multiplex PCR methods. The numbers under each method in brackets represent the totals detected using that method.
Detailed Venn diagrams showing the number of shared and unique RIF-sensitive MTBC-positive samples (panel a) and RIF-resistant MTBC-positive samples (panel b) among the GeneXpert MTB/RIF, phenotypic culture-based DST, and multiplex PCR assays, and EMB-sensitive MTBC-positive samples (panel c) and EMB-resistant MTBC-positive samples (panel d) among phenotypic culture-based DST and multiplex PCR methods. The numbers under each method in brackets represent the totals detected using that method.

The distribution of mutations associated with rifampin, ethambutol, and isoniazid resistance among MTBC DNA-positive sputum samples_

GenesrpoBembBkatGinhArpoB + embB
Mutation codons516526531306315(-15)526531
No. of isolates038190036
Frequency (%)011.129.670.40011.122.2

Comparisons between GeneXpert MTB/RIF and culture-based DST with MAS-PCR in detecting drug sensitivity and resistance among MTBC DNA-positive clinical samples_

Sample numberMAS-PCRGeneXpertCult-Bas-DST
RIFEMBINHRIFRIFEMBINH
  2SSSSSSS
  5SSSSSSS
11SSSSSSS
12SRSSSSS
17SRSSSSS
20SRSSSSS
23SRSSSSS
24SRSSSSS
25SRSSSSS
26SRSSSSS
27SSSSSSS
28SSSSSSS
29SSSSSSS
30RSSSSSS
34RSSRRSS
36RSSSSSS
37RRSSSSS
40RRSRRRS
41RRSSSSS
42RRSSSSS
43RRSSSSS
44RRSSSSS
46RRSRRRS
47RRSRRRS
48SRSSSRS

PCR primers for amplifying Mycobacterium tuberculosis complex DNA and MAS-PCR primers for the detection of INH, RIF, and EMB resistance mutations in M_ tuberculosis_

GenePrimer sequence (5’-3’)PCR product sizePurpose
IS6110F: CCTGCGAGCGTAGGCGTCGG123 bpM. tuberculosis complex detection (Sinha et al. 2017)
R: CTCGTCCAGCGCCGCTTCGG
mtp40F: CTGGTCGAATTCGGTGGAGT152 bp
R: ATGGTCTCCGACACGTTCGAC
katG 315(OF) F: ATACGACCTCGATGCCGC335 bpIsoniazid resistance detection (Yang et al. 2005; Sinha et al. 2019)
(5R) R: GCAGATGGGGCTGATCTACG
inhA(P15) F: GCGCGGTCAGTTCCACA270 bp
(PF2) R: CACCCCGACAACCTATCG
rpoB(516) F: CAGCTGAGCCAATTCATGGA218 bp185 bp170 bpRifampin resistance detection (Yang et al. 2005; Sinha et al. 2019)
(526) F: CTGTCGGGGTTGACCCA
(531) F: CACAAGCGCCGACTG TC
RIRm (–) R: TTG ACCCGCGCGTACAC
embBembB306 F: GGCTACATCCTGGGCATG392 bpEthambutol resistance detection (Yang et al. 2005; Sinha et al. 2019)
embBR2 R: GAGCCGAGCGCGATGAT
DOI: https://doi.org/10.33073/pjm-2023-040 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331
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Language: English
Page range: 421 - 431
Submitted on: Jun 20, 2023
Accepted on: Oct 4, 2023
Published on: Dec 16, 2023
Published by: Polish Society of Microbiologists
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
MTB-complex,
IS,
drug resistance,
mutations,
multiplex-PCR
Related subjects:
Life sciences,
Microbiology and virology

© 2023 Hassan A. Hemeg, Hamzah O. Albulushi, Hani A. Ozbak, Hamza M. Ali, Emad K. Alahmadi, Yahya A. Almutawif, Sari T. Alhuofie, Rana A. Alaeq, Areej A. Alhazmi, Mustafa A. Najim, Ahmed M. Hanafy, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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