DTI Drug Target Insights 2022; 16: 88-96ISSN 1177-3928 | DOI: 10.33393/dti.2022.2522REVIEW Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti © 2022 The Authors. This article is published by AboutScience and licensed under Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). Commercial use is not permitted and is subject to Publisher’s permissions. Full information is available at www.aboutscience.eu Current molecular approach for diagnosis of MRSA: a meta-narrative review Sim Yi Xing1, Lee Qiao Wei2, Aisha Abushelaibi3, Kok Song Lai3, Swee Hua Erin Lim3, Sathiya Maran1 1School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, Selangor - Malaysia 2School of Science, Monash University Malaysia, Jalan Lagoon Selatan, Selangor - Malaysia 3Health Sciences Division, Abu Dhabi Women’s College, Higher Colleges of Technology, Abu Dhabi - United Arab Emirates Swee Hua Erin Lim and Sathiya Maran contributed equally to this study. ABSTRACT Introduction: Detection and diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) are important in ensuring a correct and effective treatment, further reducing its spread. A wide range of molecular approaches has been used for the diagnosis of antimicrobial resistance (AMR) in MRSA. This review aims to study and appraise widely used molecular diagnostic methods for detecting MRSA. Methods: This meta-narrative review was performed by searching PubMed using the following search terms: (molecular diagnosis) AND (antimicrobial resistance) AND (methicillin-resistant Staphylococcus aureus). Studies using molecular diagnostic techniques for the detection of MRSA were included, while non-English language, duplicates and non-article studies were excluded. After reviewing the libraries and a further manual search, 20 studies were included in this article. RAMESES publication standard for narrative reviews was used for this syn- thesis. Results: A total of 20 full papers were reviewed and appraised in this synthesis, consisting of PCR technique (n = 7), deoxyribonucleic acid (DNA) Microarray (n = 1), DNA sequencing (n = 2), Xpert MRSA/SA BC assay (n = 2), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) (n = 2), MLST (n = 4), SCCmec typing (n = 1) and GENECUBE (n = 1). Discussion: Different diagnostic methods used to diagnose MRSA have been studied in this review. This study concludes that PCR has been extensively used due to its higher sensitivity and cost-effectiveness in the past five years Keywords: Antimicrobial resistance, Molecular diagnosis, MRSA Received: November 17, 2022 Accepted: December 31, 2022 Published online: December 31, 2022 Corresponding author: Sathiya Maran Building 2, Level 5, Room 08 (2-5-08) Monash University Malaysia Jalan Lagoon Selatan 47500 Bandar Sunway Selangor Darul Ehsan - Malaysia sathiya.maran@monash.edu adapt to human hosts and healthcare environments, caus- ing detrimental effects to healthcare-associated infections such as bloodstream infections (2). AMR is reported as the world’s biggest 21st-century health threat, and the World Health Organization (WHO) is calling for immediate action. As AMR spreads, common infections are becoming incur- able. Reports state that over 700,000 die yearly due to drug- resistant illnesses; by 2050, the number is predicted to rise to 10 million (3). A major issue pertaining to AMR is the excessive and injudicious use of antibiotics that have led to widespread resistant bacteria and dissemination of their antimicrobial resistant genes (ARGs) (4). It is concerning that the AMR rates are predicted to increase if measures are not taken. One way to overcome this is through early detection, which enables effective management, allowing efficient identification and detection of microbes such that the patient can be treated with the appropriate drug in time. Introduction Antimicrobial resistance (AMR) is defined as changes in bacteria that result in the drug being used for its treat- ment becoming inefficacious (1). Staphylococcus aureus is an opportunistic pathogen with a tremendous capacity to https://doi.org/dti.2022.2522 https://creativecommons.org/licenses/by-nc/4.0/legalcode Yi Xing et al Drug Target Insights 2022; 16: 89 © 2022 The Authors. Published by AboutScience - www.aboutscience.eu Over the years, great leaps have been made in the diag- nosis of AMR and diagnostic tests are reported to be an essential tool in early diagnosis, hence it is a robust strategy against AMR (4). To enhance existing approaches, this review aims to sum- marize new and current molecular techniques and technolo- gies used to identify AMR using a systematic meta-narrative approach, with a focus on the key benefits and drawbacks. Furthermore, a critical overview of recently developed mole- cular approaches and an informed assessment of future direction will also be discussed. Methodology Study design and inclusion criteria This systematic review was carried out in a meta- narrative framework. This study qualitatively appraised dif- ferent molecular methods used in the recent 5 years for the diagnosis of methicillin-resistant Staphylococcus aureus (MRSA). This study protocol was created according to the RAMESES (Realist And Meta-narrative Evidence Syntheses: Evolving Standards) meta-narrative review publication guide- lines (5). Articles that satisfied the following requirements were considered for the review: (i) original articles written in English that were published between January 2017 and May 2022, (ii) cross-sectional or cohort studies that assessed the technical performance of molecular methods (sensitivity, specificity, accuracy or concordance) for diagnosing MRSA. Articles were excluded if they were: (i) case reports; (ii) review articles, commentary articles, and short communications. Search strategies Articles were searched using PubMed. Search keywords were (((((molecular diagnosis) AND (antimicrobial resis- tance)) NOT (review [publication type)) NOT (systematic review [publication type)) NOT (meta-analysis [publication type)) AND (methicillin-resistant staphylococcus aureus). Selection and appraisal of articles Two independent reviewers (Lee and Sim) screened the titles and abstracts. Articles with abstracts indicating the use of a molecular approach to diagnose MRSA were read in full. A final consensus was discussed between the two reviewers, and disagreements were resolved with discussion from the third reviewer (SM). EndNote Version 20 was used for article duplicate removal and archives. All the studies reviewed and appraised in this synthesis are summarized in Table I. TABLE I - Summaries of studies appraised in this review No Author Year Country Condition/patients Sample Study design Molecular diagnosis methods Reference 1 Moutaouakkil et al 2022 China Children diagnosed with Staphylococcus aureus OAI Blood cultures, articular fluids, synovial tissues and/or bone fragments Prospective study Multiplex polymerase chain reaction (6) 2 Jin et al 2022 China 1,952 MSSA strains isolated from blood across 17 provinces MSSA-PENS isolated from invasive BSIs Retrospective study Whole-genome sequencing (2) 3 Senok et al 2021 United Arab Emirates 135 patients with a clinical diagnosis of severe skin and soft-tissue infections S. aureus isolates associated with SSTI were tested for PVL detection n/a DNA microarray assays (7) 4 Reddy and Whitelaw 2021 South Africa 231 samples 2,822 patients with positive blood cultures exclusively showing GPCC on Gram stain were included Prospective study Xpert MRSA/ SA BC assay (8) 5 Choi et al 2021 South Korea 26 children aged <15 years diagnosed with SSSS Involved area of the skin, the presence of Nikolsky’s sign, and the status of desquamation n/a PCR (9) 6 Anafo et al 2021 Ghana 300 diabetes patients and 106 non-diabetic individuals Anterior nasal swabs Cross-sectional PCR (10) 7 Verdú- Expósito et al 2020 Ethiopia 80 S. aureus strains isolated from human patients with SSTIs Human samples n/a MALDI-TOF and PCR (11) 8 Tang et al 2020 China MRSE strains from the dental plaque of a normal, healthy human population Dental plaque specimens n/a PCR (12) Molecular diagnosis of MRSA90 © 2022 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti No Author Year Country Condition/patients Sample Study design Molecular diagnosis methods Reference 9 Khawaja et al 2020 Pakistan 105 samples Human samples Descriptive cross-sectional study PCR (13) 10 Jin et al 2020 China 65-Year-old healthy man with a history of leprosy Isolate was obtained from the patient’s blood, and identified as an ST9-MRSA strain n/a Whole-genome sequencing (14) 11 Geng et al 2020 China 536 neonates Nasal swabs Prospective surveillance study Staphylococcal chromosomal cassette (and) type, spa type, MLST (15) 12 Crandall et al 2020 USA 357 children with invasive S. aureus infections Pleural fluid and/or blood Prospective study PCR, MLST, SCCmec typing (16) 13 Bouza et al 2020 Spain 155 adult inpatients diagnosed with skin and soft-tissue infection Microbiological samples Prospective study Gram stain plus GeneXpert® MSSA/MRSA SSTI (17) 14 Yang et al 2019 China 269 nonduplicate S. aureus clinical isolates were isolated from children Steril specimens and non-STERIL specimen using VITEK MS system n/a MALDI-TOF (18) 15 Mutonga et al 2019 Kenya 83 adult patients diagnosed with diabetic foot ulcers Wound swab cultures Cross-sectional study Real-time PCR (19) 16 Latour et al 2019 Belgium 1,447 residents from nursing homes Pooled sampling of nose, throat and perineum Cross-sectional prevalence survey Triplex PCR and MLST (20) 17 Hida et al 2019 Japan 263 patients suspected of having staphylococcal bacteremia Fresh and frozen blood culture samples n/a GENECUBE mecA (21) 18 Luo et al 2018 China 275 isolates of S. aureus, including 148 isolates from patients, 127 from ready-to- eat food samples Secretions, blood, phlegm, cerebrospinal fluid, transudation, urine, fresh meat, meat product, cereal products, fruits and vegetables n/a PCR, multiplex PCR (22) 19 Lin et al 2018 Taiwan 106 hemodialysis patients diagnosed with MRSA Blood cultures Retrospective study PCR and MLST (23) 20 Yang et al 2017 China 104 children diagnosed with MRSA Sputum, bronchioalveolar lavage fluid, skin and soft tissues, pus, secretions, secretions of omphalitis, blood, joint effusion, pleural effusion n/a MLST (24) BSI = bloodstream infection; DNA = deoxyribonucleic acid; MALDI-TOF = matrix-assisted laser desorption/ionization-time of flight; MLST = multilocus sequence typing; MRSA = methicillin-resistant Staphylococcus aureus; MRSE = methicillin-resistant Staphylococcus epidermidis; MSSA = methicillin-sensitive Staphylococcus aureus; n/a = not available; PCR = polymerase chain reaction; SCCmec = staphylococcal cassette chromosome mec; spa = staphylococcal protein A. GPCC = Gram positive cocci in clusters; MSSA-PENS = methicillin-sensitive S. aureus – penicillin-susceptible; OAI = osteoarticular infections; SSSS = Staphylo- coccal scalded skin syndrome ; SSTI = skin and soft tissue infections; PVL = Panton Valentine leukocidin Results The dataset includes 20 different authors from Asia (n = 13), Africa (n = 5), Europe (n = 1) and America (n = 1). A total of 20 studies were included in this synthesis: seven studies employed polymerase chain reaction (PCR) for diag- nosing MRSA (6,9,10,12,13,19,22), one study employed deoxyribonucleic acid (DNA) Microarray (7), two stud- ies used DNA sequencing (2,14), Xpert MRSA/SA BC assay (n = 2) (8,17), matrix-assisted laser desorption/ionization-time Yi Xing et al Drug Target Insights 2022; 16: 91 © 2022 The Authors. Published by AboutScience - www.aboutscience.eu of flight (MALDI-TOF; n = 2) (11,18), multilocus sequence typ- ing (MLST; n = 4) (15,20,23,24), GENECUBE (n = 1) (21) and staphylococcal cassette chromosome mec (SCCmec) typing (n = 1) (16). Figure 1 is the diagrammatic flow of the study selection and list of techniques appraised in this review. Recent molecular methods for diagnosis of MRSA Polymerase chain reaction PCR approaches have been commonly used for the effective diagnosis of MRSA, and the rapid emergence of MRSA has led to a series of PCR approaches that have been developed for the identification of MRSA (25). PCR approach identifies S. aureus based on a single-base-pair mismatch in the staphylococcal 16S ribosomal RNA gene sequence (26). Recent researchers have also cited the use of the PCR approach for mecA gene detection as the gold standard method for the detection and identification of the prevalence of MRSA (27,28). In this synthesis, a total of seven studies have employed PCR for the detection and diagnosis of MRSA. A study conducted by Moutaouakkil and colleagues among patients suspected of S. aureus hospital- ized in pediatric orthopedic clinic reported the detection of mecA using PCR (6). This study also utilized different bio- logical samples such as blood cultures, articular fluids, syno- vial tissues and bone fragments for the detection of MRSA. Another study showed that the fluorescence signal of real- time (RT)-PCR could display the quantity of products formed and increases exponentially, enabling a user-friendly diag- nostic (29). Furthermore, Mutonga and colleagues (2019) have demonstrated that the sensitivity of RT-PCR for MRSA is 100% (19). Multiplex PCR amplifies multiple DNA sequences simul- taneously, which gives an advantage over conventional PCR (30). The detection of target sequences, such as the nuc and coaA or elements necessary for methicillin resistance, such as femA, or femB, has provided the basis for PCR identifica- tion of S. aureus. It uses two pairs of primers specific to the staphylococcal nuc and mecA for PCR amplification of a 280- bp nuc-based fragment and a 533-bp mecA-based fragment (31). Tsai and colleagues (2019) reported mecA gene (mecA-F and mecA-R) is amplified and can be used to diagnose MRSA (32). Chikkala and colleagues showed that it exhibits 97% of specificity and 90% sensitivity (33). DNA sequencing DNA sequencing allows the detection of single- nucleotide polymorphisms (SNPs) and known resistance- associated genes and their variations (34). The availability of bacterial genomes in public databases facilitates the use of whole-genome sequencing for MRSA detection. It enables high-resolution characterization of antibiotic resistance (35). Whole-genome sequencing has a definite edge over conventional Sanger sequencing because it may produce millions of reads that are roughly 35 to 700 bp in length (36). There is growing evidence on the effectiveness of bacterial Records identi�ed from databases (n = 28) Records removed before screening: Duplicate records removed (n = 0) Records screened (n = 28) Reports sought for retrieval (n = 20) Reports assessed for eligibility (n = 20) Studies included in this review (n = 20) PCR (n = 7)In cl u d ed Sc re en in g Id en ti �c at io n DNA sequencing (n = 2) Records excluded (n = 8) Reports not retrieved (n = 0) MLST (n = 4) SCCmec typing (n = 1) DNA Microarray (n = 1) Xpert MRSA/SA BC assay (n = 2) GENECUBE (n = 1) MALDI-TOF (n = 2) Fig. 1 - Diagrammatic flow of the study selection and list of techniques appraised in this review. Molecular diagnosis of MRSA92 © 2022 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti whole-genome sequencing in controlling outbreaks. Whole- genome analysis, such as DNA microarray, simultaneously identifies relative concentration of different nucleic acid sequence (37). It allows a bulk number of nucleic acid sequences in a mixture to be tested and analyzed. The study by Jin and colleagues (2,14) used StaphyType DNA micro- array (Abbott [Alere Technologies GmbH], Jena, Germany) and the INTER-ARRAY Genotyping Kit S. aureus (Inter- Array GmbH, Bad Langensalza, Germany) for the detection of MRSA. The study by Senok and colleagues (2021) also reported that DNA microarray exhibited 100% specificity and sensitivity (7). In a study done by Ma and fellow col- leagues, Illumina’s Nextera DNA library preparation kit was used to create whole-genome sequencing libraries, which were then sequenced on an Illumina MiSeq using the 500 cycle V2 protocol (38). Xpert MRSA/SA BC assay Xpert MRSA/SA Blood Culture is an in vitro diagnostic test for S. aureus and MRSA. The targeted DNA is amplified using automated RT-PCR and Fluorogenic target-specific hybridization, providing real-time detection of specific genes of MRSA and S. aureus. A study by Buchan and colleagues (39) reported the use of blood cultures for the detection of Staphylococcus protein A (spa) sequences, gene that encodes for methicillin resistance (mecA) and SCCmec. A study by Reddy and colleagues has shown the performance of the Xpert MRSA/SA BC assay to be 100% in specificity and sen- sitivity. It shows a failure rate for an interpretable result of just 1.7% (8). However, it is notable that the microbiological sampling should be of high quality to ensure rapid and accu- rate results, despite the significance of Xpert MRSA system. MALDI-TOF MALDI-TOF mass spectrometry (MS) has become a widely used technique for the rapid and accurate identification of bacteria (40). Despite the efficiency and sensitivity of MALDI- TOF, this method’s limitation is that new isolates can only be detected if the spectral database contains peptide mass fin- gerprints (PMFs) of the type strains of specific genera/spe- cies/subspecies/strains. This method identifies microbes by comparing the PMF of unknown organisms with the PMFs deposited in the database or matching the masses of bio- markers with the proteome database. A recent study by Tang and colleagues (41) reported that MALDI-TOF MS on intact bacteria combined with a refined analysis framework allows accurate classification of methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. Esener and colleagues showed that MALDI-TOF has a sensitivity of 99.93% ± 0.25%, specific- ity of 95.04% ± 3.83%, and accuracy = 97.54% ± 1.91% (42). MALDI-TOF is low in cost, and analysis can be conducted within a short time, allowing rapid microbial resistance to be detected. Latour and colleagues employed MALDI BioTyper database for bacterial identification of suspected colonies (20). A study by Chen and colleagues has shown that MLST has been used for the past decades for MRSA epidemiological typing (43). However, it is only based on the sequences of seven house-keeping genes’ internal fragments to identify individual isolate lineages. MLST MLST is a technique that distinguishes between isolates of bacteria species by utilizing sequences of internal frag- ment house-keeping genes (44). The strands are sequenced on both side by using an automated DNA sequencer. Different sequences of house-keeping genes found in bacterial spe- cies are characterized as distinct alleles. In contrast, seven loci alleles address each isolate’s allelic profile or sequence type. Hence, species isolates are unambiguously character- ized by a series of seven integers which label the alleles at the seven house-keeping genes. The seven house-keeping genes used in MLST for S. aureus are the Carbamate kinase (arcC), Shikimate dehydrogenase (aroE), glycerol kinase (glpF), Guanylate kinase (gmk), Phosphate acetyltransferase (pta), Triosephosphate isomerase (tpi), acetyl coenzyme A acetyl- transferase (18,24,45). SPA typing Spa is an important gene virulence factor that allows S. aureus to avoid host immune responses (46). It codes for protein A, which is found in the cell wall of S. aureus (47). SPA genes were replicated using PCR followed by DNA sequenc- ing (48). This method identifies the polymorphic X region of the protein A gene (spa). Based Upon Repeat Pattern (BURP) algorithm was used, and spa types with more than five repeats were clustered into different groups, with the calculated cost between group members being less than or equal to 6 (49). Spa typing is evidently reproducible and provides inter- changeable information. However, a disadvantage of this method is that it requires additional targets such as SCCmec, lineage-specific virulence or resistance genes or alternative polymorphic regions of the S. aureus chromosome. Studies included in this synthesis employed Ridom Staph Database and SPA typer tool (http://spatyper.fortinbras.us/) (24,50,51). Reports cited that spa type of t437 was more prevalent in MRSA (24). A study by Luo and colleagues showed that the most prominent spa type was t030, reported to be 15.64% (43/275) (22). GENECUBE assays GENECUBE (TOYOBO Co., Ltd., Osaka, Japan) is a fully automated genetic analyzer that uses PCR to amplify a tar- get gene (21). This tool can evaluate up to eight samples simultaneously. The target DNA is amplified, and fluores- cently labeled oligonucleotides are used to hybridize tar- gets based on fluorescence intensity changes (52). Data are automatically obtained on the GENECUBE monitor after completion of the assay. The advantage of this assay is that it is time efficient and easy to prepare. GENECUBE tests are anticipated to be clinically valuable for effectively identifying MRSA. Studies have reported the sensitivity and specificity http://spatyper.fortinbras.us/ Yi Xing et al Drug Target Insights 2022; 16: 93 © 2022 The Authors. Published by AboutScience - www.aboutscience.eu of the GENECUBE to be 100% (33). The system is accurate, rapid (52 minutes), and reliable; however, it does not detect the mecC gene (21). SCCmec typing SCCmec is a diagnostic method that divides SCCmec ele- ments into groups based on their structural variations (53). The mec complex, which comprises the mec gene, its regu- latory genes, the mecI and mecR1 genes, and several inser- tion sequences, confers methicillin resistance (54,55). The specific SCCmec type is determined by combining the ccr gene complex and the mec gene class. SCCmec typing pro- vides valuable information about the resistance of genes to methicillin and identifies the origin of strains. A recent study by Chongtrakool et al (56) typed SCCmec of methicillin- resistant S. aureus strains isolated in 11 Asian countries. Another study showed that 610 of 615 (99.2%) MRSA strains could be classified into four SCCmec elements: type 3A, 370 strains; type 2A, 207; type 2B, 32; type 1B, 1 strain. This study on pandemic MRSA clones in Asia reported the ST59- SCCmecIVa as the most prevalent MRSA clone (15). A study by Chen and colleagues that used the web-based SCCmecFinder reported that this technique is efficient for detecting MRSA (43). SCCmecFinder is a web-based tool for SCCmec typing using whole-genome sequences (https://cge.cbs.dtu.dk/ services/SCCmecFinder/, accessed on January 11, 2023). The SCCmecFinder website uses read data for whole-genome sequencing or preassembled genome/contigs to determine homology to the complete cassette in prediction of SCCmec types, mec complex and J regions (57). Discussion This meta-narrative review reports the commonly used molecular methods for the detection of MRSA in the past 5 years. This review has also summarized the advantages and disadvantages of each technique included in this synthesis. S. aureus is a common cause of community and hospital- acquired infection (58,59). The WHO has regarded it as one of the primary clinical concerns, due to the global recognition of MRSA as a public health issue and the antibiotic resistance pattern of MRSA (60). The primary issue with MRSA is the incidence of multidrug resistance, which remains high (61). The mecA encodes penicillin-binding protein 2a (PBP2a), which is an enzyme responsible for crosslinking peptidogly- cans in the bacterial cell wall (62). The low affinity of PBP2a for β-lactams leads to resistance to β-lactam antibiotics, including penicillins, cephalosporins (except ceftaroline and ceftobiprole) and carbapenems (63). Recent reports have reported growing resistance to clindamycin and levofloxacin, necessitating an effective treatment. The virulence factor of S. aureus is multifactorial and depends on a variety of toxins, adhesion, immune evasion and other virulence characteristics (64). Evaluation of the virulence factor is an effective method of predicting how these bacteria would behave in the host, enabling predic- tion of the onset and progression of an infection. The first stage of staphylococcal infection is when the bacterial cells connect to the host’s tissues. The surface-exposed proteins, MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), are made by S. aureus, which functions to attach to one or more host extracellular matrix (ECM) components, such as laminin, elastin, fibrinogen, fibro- nectin and collagen (65,66). The extracellular adherence pro- tein (Eap) produced by S. aureus is a member of the SERAMs (secretable expanded repertoire adhesive molecules) family, binds to ECM glycoproteins, including fibronectin, fibrinogen, sialoprotein and collagens (67). This protein is involved in the internalization of bacteria and the adherence of S. aureus to fibroblasts. Proteases are crucial virulence factors for S. aureus and can cleave host proteins to enable MRSA cells to change from an adhesive to an invasive phenotype. Early diagnostic and therapeutic intervention in patients with MRSA infection risk factors is essential (68). Treatment with empiric antibiotics against MRSA should not be delayed in the event that MRSA infection is diagnosed. Molecular diag- nostic tests can robustly identify staphylococcal species in clin- ical samples, thus improving antimicrobial stewardship (69). In this review, multiple molecular methods such as PCR, DNA sequencing, Xpert MRSA/SA BC array, MALDI-TOF, MLST, SPA typing and SCCmec typing, have been appraised. This review summarizes that PCR technique has been widely used for the diagnosis of MRSA within the last 5 years (2017-2022). PCR technique is frequently and commonly used to detect S. aureus and it identifies a single-base-pair mismatch in the staphylococcal 16S ribosomal RNA gene sequence for detec- tion (26). PCR assay is cost and labor effective and can be con- ducted within a short period of time (70,71). However, studies have reported that different target genes may impact the specificity and sensitivity of PCR for diagnosis. The nuc gene has a 100% success rate (25,72). Several PCR techniques such as multiplex PCR, RT-PCR and isothermal identification have been developed to identify MRSA as a result of its rapid emergence. The mecA and nuc genes are being used due to their 100% sensitivity and 97% specificity respectively with a shorter turnaround time of 48 hours (73,74). The second commonly used molecular techniques are SCCmec typing and MLST, respectively. Over the years, the structures of novel SCCmec have been identified and veri- fied by molecular cloning and traditional sequencing (75). In a study by Singh-Moodley and colleagues (76), SCCmec typing method was used to replace multiplex PCR and was employed to classify additional un-typeable SCCmec elements based on ccr and mec gene complex combinations. However, this tech- nique has been deemed highly complex because the SCCmec region is variable and newer types are permanently being developed. Another possible reason for using SCCmec typing could be its potential as a benchmark for testing for the ccr gene and mecA gene compared to other methods. MLST is well-established and assigns alleles at multiple house-keeping loci directly by DNA sequencing. Sequence type is obtained based on the alleles identified at each of the seven loci using the SA MLST database. MLST detection of MRSA is based on the sequencing of the seven house- keeping conserved genes in the bacterial chromosome (77). MLST is also widely used due to its straightforward procedure for characterizing isolates of bacterial species (78). Due to Molecular diagnosis of MRSA94 © 2022 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti numerous alleles in each of the seven loci, it is unlikely that two isolates will have the same allelic profile. Instead, isolates with the same allelic profile can be identified as belonging to the exact clone. MLST has several advantages: (1) it uses sequence data to detect changes at the DNA level; (2) it is readily reproduced and does not require specialized reagents or training; (3) it does not require high-quality genomic DNA; and (4) the data generated are fully portable (79). The dis- advantage of MLST is that it only uses seven genes, limiting its ability. DNA microarray and Xpert MRSA/SA BC assay are the least used in the last 5 years. DNA microarray contains cova- lently immobilized probes specific for about 180 genes and 300 alleles of S. aureus (80). It allows simultaneous detec- tion of the presence of numerous genomic loci. Studies have reported that DNA microarray may serve as an alternate molecular typing method, offering complementary charac- terization of the MRSA strains. However, this technique is labor and cost extensive and a single experiment could signi- ficantly increase the budget of the experiment. 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