Real-time PCR assay for robust detection and global surveillance of the Mycobacterium tuberculosis Haarlem genotype

The Haarlem genotype is a significant yet understudied part of the Euro-American lineage of Mycobacterium tuberculosis, characterized by unique pathogenetic features. Spoligotyping is a primary method for its detection, but it is not suitable for isolates with long blocks of deleted spacers. We have developed a simple and robust method to detect the Haarlem genotype. The real-time PCR (RT-PCR) assay with LNA probes was designed to detect the Rv0282 211 C > T mutation that was shown to be specific for the Haarlem genotype. The developed RT-PCR assay was optimized with 68 isolates with known whole-genome sequencing (WGS) data and applied to the geographically and genetically diverse collection of 428 isolates. As a result, 396 isolates were concordantly assigned to either Haarlem or non-Haarlem genotypes by both methods, whereas 32 isolates were discrepant cases. Twenty-two isolates of "unknown genotype" (Russia, Belarus, and Poland) were assigned to Haarlem by SNP-based assay. WGS of these isolates confirmed the results of the RT-PCR assay. To conclude, the developed RT-PCR method provides a reliable detection of the Haarlem genotype in retrospective collections and under prospective epidemiological surveillance. Its actual proportion in M. tuberculosis populations in certain world regions is higher than previously thought. Abridged spoligoprofiles with large deleted blocks of spacers require caution in interpretation.

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Publication Details

Journal
Scientific Reports
Published
2025-12-11
DOI
https://doi.org/10.1038/s41598-025-27556-y
Primary Topic
Tuberculosis Research and Epidemiology
Type
article
Field-Weighted Citation Impact
0.00
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article

Real-time PCR assay for robust detection and global surveillance of the Mycobacterium tuberculosis Haarlem genotype

Valentin Dzagoev, Natalia Solovieva, О. Б. Огарков, Valentine B. Wuyep et al.
Scientific Reports
Tuberculosis Research and Epidemiology
article

Real-time PCR assay for robust detection and global surveillance of the Mycobacterium tuberculosis Haarlem genotype

Valentin Dzagoev, Natalia Solovieva, О. Б. Огарков, Valentine B. Wuyep, Igor Mokrousov, Irina Kostyukova, Urvashi B. Singh, David Couvin, G. N. Sergeev, Viacheslav Zhuravlev, И. Г. Кондратов, Platon Eliseev, О. А. Пасечник, Tomasz Jagielski, Ya. Sh. Schwartz, Zofia Bakuła, Anna Vyazovaya, С. Н. Жданова, Yuriy A. Skiba, Dmitrii E. Polev, Sergei A. Chekrygin, N. V. Kobesov, Ivan Gorokhov, Adong Shen, Andrei Mariandyshev, Violeta Valcheva, Douglas Tkachuk, Weiwei Jiao, Eugene Ikeh, Maria Badleeva, Mikhail V. Bezruchko, Dinara Nayzabaeva, Alena Chilikina
article en

Abstract

The Haarlem genotype is a significant yet understudied part of the Euro-American lineage of Mycobacterium tuberculosis, characterized by unique pathogenetic features. Spoligotyping is a primary method for its detection, but it is not suitable for isolates with long blocks of deleted spacers. We have developed a simple and robust method to detect the Haarlem genotype. The real-time PCR (RT-PCR) assay with LNA probes was designed to detect the Rv0282 211 C > T mutation that was shown to be specific for the Haarlem genotype. The developed RT-PCR assay was optimized with 68 isolates with known whole-genome sequencing (WGS) data and applied to the geographically and genetically diverse collection of 428 isolates. As a result, 396 isolates were concordantly assigned to either Haarlem or non-Haarlem genotypes by both methods, whereas 32 isolates were discrepant cases. Twenty-two isolates of "unknown genotype" (Russia, Belarus, and Poland) were assigned to Haarlem by SNP-based assay. WGS of these isolates confirmed the results of the RT-PCR assay. To conclude, the developed RT-PCR method provides a reliable detection of the Haarlem genotype in retrospective collections and under prospective epidemiological surveillance. Its actual proportion in M. tuberculosis populations in certain world regions is higher than previously thought. Abridged spoligoprofiles with large deleted blocks of spacers require caution in interpretation.

Scientific ReportsVol. 15(1)
Northern (Arctic) Federal University (RU), University of Jos (NG), Al-Farabi Kazakh National University (KZ), Omsk State Medical University (RU), Buryat State University (RU), Bulgarian Academy of Sciences (BG), Plateau State University (NG), North Ossetian State University (RU), Budgetary Health Care Institution of the Omsk Region "Clinical Oncology Center" (RU), Novosibirsk Tuberculosis Research Institute (RU), National Institute of Medical Statistics (IN), Institute of Plant Biology and Biotechnology (KZ), National Center for Biotechnology (KZ), Saint Petersburg Pasteur Institute (RU), Stephan Angeloff Institute of Microbiology (BG), Zhengzhou Children's Hospital (CN), Scientific Center of Family Health Problems and Human Reproduction (RU), Beijing Children’s Hospital (CN), Ural research Institute of Phthisiopulmonology (RU), Saint-Petersburg Research Institute of Phthisiopulmonology (RU), Institut Pasteur de la Guadeloupe (GP), Northern State Medical University (RU), University of Warsaw (PL), All India Institute of Medical Sciences (IN)
Openalex Percentile: Top 54%
Tuberculosis Research and Epidemiology
DOIOpen Access PDFOpenAlex
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