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Upping the Ante on Drug-Resistant Tuberculosis

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Upping the Ante on Drug-Resistant Tuberculosis

Commercial diagnostic tests endorsed by the World Health Organization (WHO) are failing to identify mutant strains of tuberculosis (TB) in South Africa, leading to undetected outbreaks, misdiagnoses and recurring drug resistance in this part of the continent. A study in The Lancet Infectious Diseases describes the utility of whole genome sequencing and a new multitarget assay to zero in and capture these elusive gene mutations.

Lab tests used to make clinical diagnoses or in surveillance programs often have intrinsic limitations. “Not knowing them or ignoring them, particularly in the field of microbiology, can lead to the emergence of escape mechanisms by the microbes. They are always smarter than us,” the study’s senior author, Emmanuel André, MD, PhD, a clinical microbiologist at KU Leuven/University Hospitals Leuven in Belgium, told CLN Stat. Strategies need to be put in place to detect these difficult to diagnose strains before they spread in the community, he added.

Clinicians treat TB with at least four different drugs to avoid the selection of resistant mutants. “Nevertheless, drug-resistant strains have emerged and are actively spreading around the world. One of the reasons for this spread is that it takes several weeks to culture the bacteria and subsequently to identify resistance,” André said.

Rapid molecular assays have made significant strides in diagnosing rifampicin resistance in TB. “Fast detection of resistance to rifampicin, the most important anti-tuberculosis drug, is especially crucial because it is predictive of multidrug resistance, defined as resistance to at least isoniazid and rifampicin,” explained the study authors. South Africa has deployed on a broad scale two types of assays endorsed by the WHO, the GenoType MTBDR plus version 2.0 and Xpert MTB/RIF. While these tests can identify mutations within the 81bp region of the rpoB gene—which makes up the majority of known rifampicin-resistance mutations in TB strains—they were not designed to capture lesser-known mutations occurring outside this region. “Two of them have been regularly identified: one on position 170 and one on position 491 of the rpoB gene,” André said.

Rapid molecular test developers excluded these two mutations because identifying them would have made the tests more complex to design. “Although their existence was known, these were thought to be very infrequent. I believe this was a mistake,” André said. In 2015, a study revealed that 30% of multidrug-resistant (MDR) strains taken from a 2009 national survey in eSwatini (formerly known as Swaziland) contained the Ile491Phe mutation in the rpoB gene, a mutation known to elude detection by commercial assays, and leads to poor treatment outcomes with rifampicin.

To assess whether a similar situation existed in South Africa, the researchers screened more than 37,000 Mycobacterium TB-positive cultures from four South African provinces that were diagnosed at the Dr George Mukhari Tertiary Laboratory in Pretoria. They sought to detect isolates with rifampicin sensitivity and isoniazid resistance, using the Xpert MTB/RIF and GenoType MTBDR plus assays and culturing with the BACTEC mycobacteria growth indicator tube MGIT 960 automated detection system.

The researchers randomly selected 227 out of 1,823 isolates with rifampicin sensitivity and isoniazid resistance, screening them for Ile491Phe with multiplex allele-specific polymerase chain reaction (MAS-PCR) and Sanger sequencing of rpoB. Developed by André and his team as a rapid and low-cost screening test, the MAS-PCR specifically targets the 491 mutation. “Whole genome sequencing is the reference technique which allows us to capture the entirety of the rpoB gene, including the 81bp region and the 170 and 491 codons,” he said.

In an additional step, the researchers used whole genome sequencing and a new commercial assay based on next-generation sequencing technology known as Deeplex MycTB to further evaluate rpoB Ile491Phe positive cases. According to André, “We wanted to investigate resistance to other first- and second-line drugs,” and determine whether these strains were in any way linked to the outbreak in eSwatini.

Investigators found the Ile491Phe mutation in 37 or 15% of 249 TB isolates with valid MAS-PCR and sequencing results, reclassifying this subset of isolates as MDR. In addition, the Deeplex-MycTB identified all 37 isolates as genotypically resistant to all first-line drugs. “Consistent with Deeplex-MycTB genotypic profiles, whole-genome sequencing revealed concurrent silent spread in South Africa of a [multidrug-resistant] tuberculosis strain lineage extending from the eSwatini outbreak and at least another independently emerged Ile491Phe-bearing lineage,” the authors summarized.

The findings suggest that diagnostic assays in South Africa are missing a significant number of MDR TB cases that include the Ile491Phe mutation in the rpoB gene. Consequently, many patients infected with MDR-TB strains aren’t getting proper diagnoses or treatments, according to André. The study researchers discovered that in the South African outbreak, strains had been circulating undetected for at least 5 years. “We are further concerned because many patients lived in areas where international mining workers congregate. We therefore need to extensively investigate the situation not only in South Africa but also in other countries of the region,” André said.

Closing these gaps means employing newer technologies such as whole-genome sequencing or the Deeplex-MycTB multitarget assay in drug-resistance surveys, the study authors recommended.

Pub Time : 2018-11-23 11:38:55 >> News list
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