Methicillin sensitive and resistant Staphylococcus aureus (MSSA/MRSA) are aetiological agents of chronic/recurrent skin and soft tissue infections (SSTI). The spectrum of infections which may be community or hospital acquired range from impetigo and folliculitis to more serious surgical site infections. Global data indicates an increasing trend of incidence and severity of SSTIs caused by MRSA isolates (Olaniyi et al., 2017). Panton Valentine leukocidin (PVL) is a virulence factor which is associated with S. aureus strains causing SSTI and severe forms of community acquired pneumonia (Gosbell, 2005; Wannet et al., 2005; Yamasaki et al., 2005; Marazza et al., 2007; Masiuk et al., 2010; Gillet et al., 2021). PVL consists of two distinct components which form polymeric pores in the membranes of white blood cells which leads to cell death. This might explain the higher risk of complicated SSTI infection associated with PVL positive S. aureus strains (Szmigielski et al., 1998; Kaneko and Kamio, 2004). The pvl genes, lukS-PV and lukF-PV, are phage borne (Narita et al., 2001; Boakes et al., 2011), and since they are mobile, they can be found in different, unrelated lineages of S. aureus (Monecke et al., 2007; Monecke et al., 2011a).

Detection of pvl genes in S. aureus is usually done by molecular methods and is not routinely offered by most diagnostic laboratories. Findings from a meta-analysis report suggests that PVL-producing S. aureus are associated with SSTIs with increased risk for surgical intervention (Shallcross et al., 2013). In light of this, a rapid and affordable method for the identification of PVL in S. aureus strains isolated from SSTI, which can be carried out without the need for expensive molecular assays is desirable for the diagnostic microbiology laboratory.

This is particularly pertinent in settings such as the Arabian Gulf region with high prevalence of pvl ^+ve S. aureus (Senok et al., 2016b; Senok et al., 2019b; Senok et al., 2020a). The countries of the Arabian Gulf region are a global hub for travel and commerce with tourists, pilgrims, and expatriate workers from all across the world. This includes regions from which nothing is known on MSSA/MRSA prevalence and S. aureus population structure. Indeed, with the cosmopolitan character of the United Arab Emirates (UAE) and reported high biodiversity of MSSA and MRSA including high prevalence of strains with pvl genes, it represents a suitable study setting with regard to S. aureus toxins and typing (Senok et al., 2020a; Senok et al., 2020b).

In this study, we describe the application of a novel lateral flow immunoassay for rapid PVL detection in S. aureus directly from bacterial cultures and in addition, we present data on the molecular characterization of a collection of MSSA/MRSA strains associated with SSTI.

A total of 135 patients were recruited with mean age ( ± SD) 27.7 ( ± 17.9) years and majority (n=71) were females. Most of the patients (n=130) were from the outpatient setting and 20.7% (n/N=28/135) gave a history of recurrent skin infections. None of the patients reported similar infections in family members. Clinical diagnoses among the patients were mostly cutaneous abscesses (n=41); folliculitis/furuncle/impetigo (n=35) and secondary infections of underlying dermatological conditions (n=35). The information regarding history of animal contact was available for only 25 participants, and all of them reported that they had no animal contact. Sixty-six patients were treated with antibiotics with a majority (n/N=36/66) receiving beta lactam drugs mainly amoxicillin-clavulanic acid (n=26), cefuroxime (n=6) or ceftriaxone (n=4). Fifteen patients received topical fusidic acid. Table 1 shows the details of the demographic and clinical profiles of the patients.

We present the first report on the application of a novel lateral flow immunoassay for PVL detection in S. aureus isolates in a setting with wide MSSA/MRSA clonal diversity and high prevalence of pvl ^+ve strains. Using well-established DNA microarray-based assays for molecular detection of pvl genes as the gold standard, the findings show that the lateral flow immunoassay demonstrates excellent diagnostic accuracy indices. There was full concordance in pvl detection by the lateral flow immunoassay and the molecular confirmatory method used. As previously reported, the in vitro expression of PVL varies widely depending on CC affiliation of the S. aureus strain and the presence of other virulence genes (Stieber et al., 2014). This could explain the weak positive signals observed for four isolates. The 100% diagnostic sensitivity and specificity we have documented is of significance as this study was carried out in the context of a setting characterized by high prevalence of pvl ^+ve strains. Two similar assays have previously been reported that yielded similar high diagnostic accuracy indices using strains obtained from various countries (Badiou et al., 2010; Monecke et al., 2013). Unfortunately, these assays were eventually not marketed by the respective companies so the need for an easy, non-molecular culture confirmation test remained unaddressed. Our findings show that despite the remarkable evolution of S. aureus genotypes in recent years, the lateral flow immunoassay for PVL detection still yields excellent diagnostic results. This could be attributed to the rare occurrence of sequence variations in pvl genes. As these genes face a selective pressure to maintain their function in presence of the host´s defenses, a significant degree of conservation is to be expected (O’hara et al., 2008; Boakes et al., 2011). The lateral flow immunoassay is rapid, easy to use and interpret, and is highly economical making it ideal for diagnostic microbiology laboratories worldwide. It will be particularly beneficial in diagnostic laboratories serving primary care facilities where majority of patients with SSTIs are first seen (Ramakrishnan et al., 2015). Several countries in the developing world have reported a high prevalence of pvl ^+ve S. aureus strains and this lateral flow assay will be optimal in such resource limited settings where access to molecular assays may be lacking (Rasigade et al., 2010; Monecke et al., 2014; Abdulgader et al., 2015; Rasigade et al., 2015). With the potential threat of poor clinical outcomes associated with pvl ^+ve S. aureus strains, their early identification is crucial particularly in settings such as ours where S. aureus strains harboring pvl genes are circulating widely (Szmigielski et al., 1998; Kaneko and Kamio, 2004; Shallcross et al., 2013; Senok et al., 2020a). In this study, most of the patients had presentations consistent with mild SSTIs (Ramakrishnan et al., 2015) and majority received antibiotics with beta lactams and fusidic acid being the most prescribed antibiotics. According to current guidelines, mild purulent SSTIs in easily accessible sites with minimal cellulitis can be treated by incision and drainage alone, without a need for antibiotics (Public Health England, 2008; Gillet et al., 2011; Ramakrishnan et al., 2015). However, an antimicrobial coverage to mitigate against a potential threat of complications occurs commonly thus contributing to antibiotic misuse. The widespread utilization of beta lactams and fusidic acid in SSTI is a likely contributory factor to the emergence of resistance to these antibiotics resulting in the high occurrence of MRSA strains with SCCmec + SCCfusC composite elements in the Arabian Gulf region (Senok et al., 2020a; Monecke et al., 2021). The continued evolution and upward trend of MRSA in the region has been linked to strains harboring these composite elements (Senok et al., 2020a; Monecke et al., 2021). Hence, the high prevalence of MRSA isolates associated with SSTI, as well as the rate of detection of isolates harboring pvl and fusC genes is worrisome. The presence of co-carriage of pvl and fusC genes was found more commonly in MRSA compared to MSSA strains which is in keeping with reports of other studies from the same geographic region (Monecke et al., 2012b; Senok et al., 2016b; Boswihi et al., 2018; Senok et al., 2019b; Senok et al., 2020a). Therefore, it is envisaged that the incorporation of this lateral flow immunoassay in the diagnostic workflow will enable microbiology laboratories to provide clinicians with information on the PVL status of the MSSA/MRSA isolates identified from bacterial cultures of SSTIs which would be helpful in guiding physicians towards rational choices that support judicious use of antibiotics.

It is noteworthy that in contrast to other studies, the findings from this study provide a unique insight into the molecular epidemiology of S. aureus strains causing SSTIs in our setting. These strains harbor a diversity of antibiotic resistance genes including mecA, fusC, aacA-aphD and in addition to pvl they also carry important toxin virulence genes notably tst-1, edinB, etA and various enterotoxin genes. In the UAE and countries of the Arabian Gulf region, MRSA contributes significantly to the burden of clinical infections and most of the MRSA strains detected in this study are those that have previously been observed in the region (Senok et al., 2016a; Boswihi et al., 2018; Senok et al., 2020a). Although wide clonal and strain diversity was evident with 19 different MRSA strains belonging to 14 different clonal complexes identified, unlike other reports no novel variants were found in this study. Some of the strains identified have also been detected in other parts of the world which suggests the role of a dynamic population movement in facilitating either importation into the UAE, or dissemination to other countries by returnees.

Our findings demonstrate the contribution of well-known pandemic and regionally restricted as well as rare MRSA strains to these infections. These strains include CC1-MRSA-[IV+fusC+ccrAB1] which has previously been described in Western Australia as well as in the UAE and Saudi Arabia (Coombs et al., 2006; Raji et al., 2016; Senok et al., 2020a). It has also been detected in Pakistan (unpublished observation/personal communication, Dr B. Jamil, BJ Micro Lab (SMC Private) Limited, Pakistan and Dr. A. Syed, University of Haripur, Pakistan), a country with dynamic population movement to Arabian Gulf countries. The CC5 strains include CC5-MRSA-IV (PVL+/edinA+) which has been reported in the Arabian Gulf as well as in Australia, where it is, dubbed WA MRSA-121 (Riley and Rouse, 1995; Dailey et al., 2005; Bloomfield et al., 2020). The CC5-MRSA-[V/VT+fusC] was found four times; one isolate was subtyped and yielded SCCmec V rather than VT. This strain which appears to be common in this region was sporadically found in Australia (WA MRSA-109) (Coombs et al., 2012; Senok et al., 2020a). The CC5-MRSA-[V/VT+fusC] (PVL+) has been identified sporadically at the Gulf and in Australia (Senok et al., 2020a). The CC6-MRSA-IV is common in the Middle East and has also been found in Europe among refugees of Middle Eastern origin (Monecke et al., 2011a; Monecke et al., 2012b; Kossow et al., 2018; Bartels et al., 2021) as well as in Australia.

CC8-MRSA-[IV+ACME] (PVL+), USA300 is a well-known community-acquired pandemic MRSA strain which is abundant in North America and sporadic in Western Europe but was recently observed in Pakistan and Afghanistan (Monecke et al., 2011a). In the UAE, novel variants of this MRSA strain and putative PVL-deletion mutants have been reported (Senok et al., 2020a). For CC15-MRSA-[V+fusC] the ancestral CC15-MSSA is a common nasal colonizer in the Middle East (Sarkar et al., 2016; Senok et al., 2018). However, in recent years, CC15-MRSA has emerged among humans and livestock (camels) in the UAE, Kuwait and Saudi Arabia (Raji et al., 2016; Senok et al., 2017; Boswihi et al., 2018; Senok et al., 2020a). CC22-MRSA are prevalent in the Arabian Gulf region and six distinct ‘strains’ of CC22-MRSA-IV have been reported (Senok et al., 2016b). Whilst CC22-MRSA-IV (PVL+) are common in the Middle East, they occur sporadically in other regions. CC22-MRSA-IV (PVL+/tst+) was apparently first observed in Iran (Goudarzi et al., 2017) and spread to other Gulf countries as well as far as to Nepal (Roberts et al., 2018; Roberts et al., 2020). The recent identification of CC22-MRSA-[IV + fusC + ccrAA/(C) which harbored a novel SCCmec element is indicative of the expansion of the diversity of CC22-MRSA in the region (Senok et al., 2020a). CC30-MRSA-IV (PVL+) also known as “WSPP/Southwest Pacific Clone” was first described from Australia and Pacific islands (Monecke et al., 2011a). However, there are different variants with different SCCmec subtypes, and this might indicate a polyphyletic origin, i.e., multiple acquisitions of SCCmec elements by ancestral CC30 MSSA. This is not fully understood and might warrant further study. However, the CC30-MRSA-[VI+fusC] (PVL+) detected in this study appears to be uncommon and restricted to the Arabian Gulf region (Senok et al., 2019a).

CC80-MRSA-IV (PVL+) was frequently dubbed the “European Clone” of community-acquired MRSA but recently it emerged in the Maghreb countries (Algeria, Tunisia) and Lebanon from where the possibility of dissemination to Arabian Gulf countries is feasible due frequent travel (Kechrid et al., 2011; Djoudi et al., 2013; Basset et al., 2015).

CC97-MRSA-[V +fusC] is a Middle Eastern strain observed in Saudi Arabia and the UAE (Senok et al., 2016a; Senok et al., 2020a). However, sporadic cases from Western Europe have been reported as well as an outbreak in Ireland which was linked to an Egyptian connection (Mcmanus et al., 2021). CC121-MRSA-V/VT (PVL+) are rare in the UAE (Senok et al., 2020a) and elsewhere. Another rare strain is CC152-MRSA-IV (PVL+) which had previously been observed in Kuwait (Boswihi et al., 2018). CC398-MRSA-V/VT (PVL+) is related to the European livestock strain. However, the PVL-positive lineage has no livestock connection, and it is known to occur in South-East Asia (Yu et al., 2008; Moller et al., 2019). Indeed, cases of infections and outbreaks associated with this CC398-MRSA in Europe were linked to South-East Asia (Moller et al., 2019; Nurjadi et al., 2019). The first identification of this strain in the Middle East region was recently reported in the UAE, possibly introduced via South-East Asia or Europe (Senok et al., 2020a). CC772-MRSA-V (PVL+), “Bengal Bay Clone” is a pandemic, multi-resistant and virulent strain emerging from the Indian subcontinent (i.e., India, Bangladesh, Pakistan) (Ellington et al., 2010; Steinig et al., 2019). Given the high number of people from these countries working in the region, its occurrence is not surprising, and indeed, it has been observed in many studies from the region (Monecke et al., 2012a; Udo et al., 2014; Boswihi et al., 2016; Senok et al., 2020b). CC1153-MRSA-[V/VT+fusC] (PVL+) and CC1153-MRSA-PseudoSCCmec [class B+fusC+ccrAB1] (PVL+) are both strains that are known to occur mainly in Gulf countries (Monecke et al., 2021).

With regards to MSSA, a wide variety of different strains was found including some rare and poorly characterized lineages such as CC1156, CC2531, CC2867, CC2990 and S. argenteus CC2596. The most common pvl ^+ve MSSA strain was CC152-MSSA (7 isolates), a lineage previously known to occur in Western Africa and Trinidad & Tobago (Ruimy et al., 2008; Monecke et al., 2014; Okuda et al., 2016; Obasuyi et al., 2020).

In conclusion, we demonstrate excellent diagnostic accuracy indices for a novel lateral flow immunoassay for rapid identification of PVL in MSSA/MRSA identified from cultures of SSTI in a setting with high prevalence of pvl ^+ve strains. These findings support the introduction of this test for routine use in the diagnostic microbiology laboratory especially in settings where facilities for molecular assays are lacking. The prevalence and diversity of MRSA strains associated with SSTIs as well as the high occurrence of pvl and fusC genes in these strains is of concern.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Mohammed Bin Rashid University and Mediclinic Hospitals ethics committees (MBRU-IRB-2020-003 and MCME.CR.98.MCIT.2020). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Conceptualization, AS, SM, and RE. Data curation, AS, SM, RN, HC, ST, EM, and RE. Formal analysis, AS, SM, and RE. Investigation, RN, HC, ST, and EM. Methodology, AS, SM, RN, HC, ST, EM, and RE. Project administration, AS, SM, and RN. Resources, AS, SM, and RE. Writing – original draft, AS, SM, and RE. All authors contributed to the article and approved the submitted version.

We acknowledge the German Federal Ministry for Economic Affairs and Energy for supporting the development of the PVL antibodies and of the rapid assay within the framework of the INNO-KOM project (49MF180153).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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