Generally, pDST is the most conventional resistant detection method used worldwide. However, efforts are being made to replace pDST with a more time-efficient method, of which Xpert is a candidate. Xpert has a high level of sensitivity and specificity, its rapid diagnosis of two hours playing a large role in determining TB cases and RIF resistance. This study verified Xpert’s suitability in being the alternative to pDST as a diagnostic tool in NK.
Due to the steadfast reliability of pDST, some people might suggest that it be performed concurrently to Xpert to decrease the chances of cases in false-susceptibility. While pDST is the standard DST method in many countries, to perform the test safely, proficient medical technicians and a working facility following international bio-safety standards are needed, both which NK lacks. Of course, there also needs to be a sufficient supply of anti-TB drugs, and we tentatively make the assumption that NK, as a developing country, faces the highest difficulty in meeting the large demand for anti-TB drugs when none exist within the country.
Since EBF reported in using Xpert as a primary source of diagnosis, the molecular test has been analyzed further in this study to determine its aptitude in replacing pDST as the primary TB diagnostic tool. Xpert's simplicity and rapidness in decision, alongside simultaneous detection of RIF-resistance with its general detection of active TB cases should be taken advantage of in the field.
The Xpert and pDST results were compared in categories of consistency, sensitivity, and specificity, using RRDR sequencing as a gold standard. All 103 samples were target sequenced at RRDR, and as a result we were able to observe mutations in 78 samples (Table 4). When compared to RRDR sequencing, several inaccurate Xpert results with notable mutations in specimens were discovered: three falsely resistant cases (FR1, FR2 and FR3) and four falsely susceptible cases (FS1 to FS4).
Xpert has five targets which binds to a section of ropB; if any one of the five targets do not hybridize, the specimen indicates a ‘resistant’ result (Figure 2). The three specimens FR1, FR2, FR3 were cases where at least one or more probes A, C, D, and E were not hybridized to the specimen. These specimens were sequenced and revealed as wild types yet did not bind with the Xpert probes (Table 2). Of the four specimens FS1 to FS4, FS1 had mutations within the Xpert target zone and FS2 to FS4 had mutations outside the RRDR (Figure 2).
The FS1 mutation at the D516Y (GAC/TAC) and FS2 mutation at the S522Q (TCG/CAG) were cases where Xpert's probes should not have been hybridized to the location of these mutations, yet probe B and C’s signals were detected. A similar case has been reported [8]. In addition to S522Q, FS2 had a mutation at V547V (GTC/GTT) which was located outside the Xpert probe region, a synonymous mutation without a change in amino acid. FS3 and FS4 (I572F, ATC/TTC) were cases where the mutation was located outside the Xpert target region. I572F has been reported in several papers noting that it caused RIF resistance at 8-16 ㎎/㎖, but V547V is a novel mutation located within the RRDR but outside the Xpert target zone (Table 5) [11].
Besides these mutations, there were several other rare and novel mutations of ropB in this study (Table 5).
Table 5. Rare or novel mutations found in North Korea’s Mycobacterium. tuberculosis isolates
No.
|
Mutations
|
Nucleotide change
|
Amino-acid change
|
Description
[References]
|
1
|
T480A
|
ACC → GCC
|
Thr → Ala
|
Reported in other genus [12]
|
2
|
Q513K
|
CAA → AAA
|
Gln → Lys
|
Rare mutation
[13-15]
|
3
|
N518D
|
AAC → GAC
|
Asn → Asp
|
Similar case reported [16]
|
4
|
P535L
|
CCC → CTC
|
Pro → Leu
|
Not been reported
|
5
|
V547V
|
GTC → GTT
|
Val → Val
|
Not been reported
|
6
|
Pheinsertion between 513 – 514
|
TTCinsertion
|
Pheinsertion
|
Similar case
Reported [17]
|
7
|
S512Q513F514deletion
|
GAGCCAATTdeletion
|
SerGlnPhedeletion
|
Not been reported
|
T480A has not been reported in M. tb but there has been reports of rifampicin resistance in rpoB in Staphylococcus aureus [12]. Also, mutations Q513E, Q513K, Q513L, Q513P were found just as in previous studies, which destabilizes the RIF binding site and affects RIF susceptibility [13-15]. In this study we found a mutation on the Q513K, one that is not common, but a rare occurrence.
Additionally, this study discovered a N518D mutation, a novel point mutation. A similar case was reported from the MDR-TB isolates from Brazil, where four codons were deleted at 515 – 518 [16]. The novel mutations at P535L and V547V are point mutations that have not been reported.
This study also found that the insertion of phenylalanine between 513 and 514, which is the sequence for the RIF binding site. Another study similarly synthetically inserted amino acid phenylalanine (TTC) between codons 514 and 515, which caused its RIF MIC to be greater than 32 ㎍/㎖ [17].
Finally, this study discovered a three amino acid (Serine, Glutamine, and Phenylalanine) deletion located from 512 to 514. This mutation has not been reported, but the area is widely known to be related to RIF resistance.