High resolution melting assay as a reliable method for diagnosing drug-resistant TB cases: a systematic review and meta-analysis

A century after the discovery of Mycobacterium tuberculosis (Mtb) as the etiological agent of tuberculosis (TB) by Robert Koch, the disease is still one of the leading causes of death (after AIDS) worldwide [1, 2]. According to the World Health Organization (WHO) report in 2020, approximately 10 million (range, 8.9–11.0 million) people became infected with Mtb in 2019; of these, approximately 1.2 million (range, 1.1–1.3 million) deaths occurred among HIV-negative people [3]. Furthermore 208,000 deaths (range, 177,000–242,000) were related to HIV-positive individuals [4]. According to the use of comprehensive treatment programs, statistics show that the number of TB deaths among HIV-positive and HIV-negative people fell by 31% and 69% between 2000 and 2019, respectively [4]. Drug-resistant tuberculosis (DR-TB) is a global concern for TB control programs, and close to half a million people have been diagnosed with rifampicin-resistant TB (RR-TB); of which 78% had MDR-TB (resistant to both rifampicin and isoniazid). Three countries, India (27%), China (14%), and Russian Federation (8%) had the largest share of the global MDR-TB burden, respectively [4, 5]. Therefore, prompt diagnosis and appropriate treatment of both TB and DR-TB cases are considered as key factors in reducing mortality from this disease [6, 7]. According to WHO guidelines, diagnosis of both resistance and susceptible isolates requires methods such as molecular techniques, cultivation, sequencing, and bacteriological confirmation; although methods e.g. culture (in LJ, 7H10, 7H11 and MGIT media) and phenotypic drug susceptibility testing (DST) are considered as gold standard methods for diagnosis of this bacterium, however, these methods are time-consuming, laborious, and sometimes ambiguous results are obtained [4, 8]. Therefore, reducing the detection time leads to reduce the risk of transmission of resistant strains [9]. In recent years, several rapid methods for diagnosing TB or DR-TB isolates such as PCR, real-time PCR, line probe assay (LPA), Xpert MTB/RIF assay, and BACTEC MGIT 960 liquid culture have been introduced [8]. Xpert MTB/RIF assay is applicable in high-prevalence and low-income settings; although this method has recently been approved by WHO for rapid detection of both Mtb and RR-TB isolates, it has several disadvantages, for example, it is only used for rifampin and is not affordable in all regions of developing countries [8‐11]. BACTEC system is based on the generation of radioactive CO₂ from substrate palmitic acid; this system is high specific and helpful in distinguishing Mtb from other mycobacteria, as well as is used as a comparative method versus DST. In general, in this system growth can be detected in 5–10 days; however, the detection time of bacteria in this method is faster than the conventional culture method (more than two weeks), but it takes more time to detect than molecular methods [12]. Overall, molecular methods are more rapid compared to other methods, nevertheless, these methods are costly, and also require standardization and a multi-step process; moreover, the use of these methods increases the risk of cross-contamination and misdiagnosis [13]. High resolution melting (HRM) assay is a new method to detect point mutations, single nucleotide polymorphism (SNP), and internal tandem duplications [14, 15]. HRM technique is based on differences in the melting profiles of test and reference DNAs; in this method, using real-time PCR assay, first, immunofluorescent dyes bind to double-stranded DNA, and during the denaturation step of PCR amplicons, differences in melting curves indicate that a mutation occurs in the test DNAs compared to reference DNA [16]. The recent method is relatively inexpensive and requires only unlabeled primers and dsDNA binding dyes; the process of detection is too fast, and due to the reaction in a closed tube, the risk of contamination is low; unlike other molecular methods, this method has no post-PCR process [17, 18]. To date, various studies have examined the sensitivity and specificity of HRM, and the aim of this meta-analysis was to determine the overall accuracy of HRM in identifying DR-TB cases.

In recent years, the emergence of DR-TB strains is accounted a serious threat to TB control worldwide [45]. Diagnosis and screening of patients infected with MDR and extensively drug-resistant (XDR) strains is an important strategy to monitor and prevent the geographical spread of DR-TB strains [46]. Although conventional drug susceptibility testing such as proportional method, absolute concentration method, and resistance ratio method are known as reference methods, these are time-concuming and laborious [47]. To date, conventional drug susceptibility testing by liquid culture system has been introduced as colorimetric redox-indicator method, which in turn reduces the detection time to less than a week, but this system requires expensive tools and cannot be used in all developing countries [48, 49]. The Xpert MTB/RIF assay is a relatively new method recommended by WHO, but it should be noted that this method is only able to detect rifampin-resistant cases [50]. Among the molecular methods that can be used to determine TB antibiotic resistance, HRM is a fast, inexpensive, and simple method, which can detect different mutations using a small number of probes or even without a special probe [51]. In their meta-analysis study (using 7 articles), Yin et al. showed that the sensitivity and specificity of HRM in the diagnosing of rifampin-resistant TB cases were 94% and 99%, respectively [18]. A summary of common methods for detecting drug-resistant TB are listed in Table 3.

To date, many studies have examined the accuracy of HRM for the diagnosis of DR-TB, however, no study has systematically evaluated the effectiveness of this method. In the present meta-analysis, we estimated the sensitivity and specificity of HRM in detecting DR-TB cases at 89% and 95%, respectively. According to our results, accuracy of HRM was slightly higher than other molecular methods, especially PCR-SSCP, while it was lower compared to the conventional drug susceptibility testing method. HRM method is credible method for detecting of mutations in specific genes; approximately 95% of rifampin-related mutations occur in the rifampicin resistance determining region (RRDR), while, 5% of those are outside this locus and cannot be detected by conventional molecular methods [65]. Nevertheless, some mutations such as nucleotide transversions (A:T and G:C) are difficult to distinguish, since they have very little influence on the overall thermal denaturation profile [66]. Sharma et al. showed that HRM could be a rapid and reliable method for the diagnosis of MDR-TB in cases of extrapulmonary TB [67]. In another study, Mu et al. examined this method for detecting DR-TB in formalin-fixed or paraffin‑embedded tissues; they found that the sensitivity of HRM for antibiotics such as rifampin, isoniazid, levofloxacin, and moxifloxacin was was 95.00%, 96.00%, 100%, and 100%, respectively, while its specificity was 95.15%, 95.92%, 94.69%, and 89.92%, respectively [68]. The stability of our findings are confirmed by similar studies and our results also showed that HRM is a trustworthy method for detecting resistance to some antibiotics, especially isoniazid and rifampin. In the present study, the heterogeneity was significant in some cases so that it was not eliminated by subgroup analysis; this issue requires a comprehensive program. This phenomenon may be due to differences in some factors such as study design, low sample size, references methods, examining tools, and dyes used. Despite its reliability, HRM has disadvatages including: (1) this method is performed only on cultured isolates; (2) HRM results are strongly depend on the quality of the extracted DNA; (3) researchers use a variety of protocols to set up HRM (self-design program). Our study had several limitations: (1) low sample size; (2) potential heterogeneity due to differences in study design and also unreliability of results; (3) inaccessibility to raw data for further analysis; (4) failure to assess the publication bias.

Despite the limitations of the present study, according to previous studies, we have shown that HRM is an accurate method for diagnosing and monitoring DR-TB cases. This method, together with the results of drug susceptibility testing, seems to be a suitable strategy for rapid and inexpensive diagnosis of DR-TB cases.