Y Gillet

O Dumitrescu

A Tristan

E Javouhey

D Floret

F Vandenesch

J Etienne

G Lina

Pragmatic management of Panton-Valentine leukocidin-associated staphylococcal diseases

Keywords:

A c c e p t e d M a n u s c r i p t E-mail address: gerard.lina@chu-lyon.fr (G. Lina).

A c c e p t e d M a n u s c r i p t ABSTRACT Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus is associated with a broad spectrum of diseases, ranging from common uncomplicated soft tissue infections to severe diseases such as complicated soft tissue infections, extensive bone and joint infections, and necrotizing pneumonia. Specialised management of infection based on the presence of PVL may not be required for mild infections, whereas it could be lifesaving in other settings. Moreover, most severe PVL diseases are recently identified entities and a 'gold standard' treatment from comparatives studies of different therapeutic options is lacking. Thus, recommendations are based on expert opinions, which are elaborated based on theory, in vitro data and analogies with other toxin-mediated diseases. In this review, we consider the potential need for specialised PVL-based management and, if required, which tools should be used to achieve optimal management.

Introduction

Panton-Valentine leukocidin (PVL) is a bicomponent, pore-forming toxin produced by several strains of Staphylococcus aureus. PVL was initially associated with necrotic and recurrent skin and soft-tissue infections (SSTIs). Its role in more severe disease, such as PVL-associated staphylococcal necrotizing pneumonia, was not described until the beginning of the 21st century [START_REF] Gillet | Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients[END_REF][START_REF] Lina | Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia[END_REF][START_REF] Prevost | Epidemiological data on Staphylococcus aureus strains producing synergohymenotropic toxins[END_REF]. PVL is also a predictive marker for the severity of bone and joint infections (BJIs) [START_REF] Dohin | Pediatric bone and joint infections caused by Panton-Valentine leukocidin-positive Staphylococcus aureus[END_REF], deep-seated abscesses and complicated SSTIs (cSSTIs) [START_REF] Miller | Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles[END_REF][START_REF] Perbet | Multifocal community-acquired necrotizing fasciitis caused by a Panton-Valentine leukocidin-producing methicillin-sensitive Staphylococcus aureus[END_REF]. The concomitant emergence of severe infections due to PVL-producing S. aureus and community-acquired meticillin-resistant S. aureus (CA-MRSA) initially led to a certain degree of confusion and controversy. The PVL gene, whose product has known necrotic and pro-inflammatory properties, was present in most of the described clones of CA-MRSA [START_REF] Genestier | Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils[END_REF], yet PVL was not considered a key virulence factor by several authors, who instead focused mainly on meticillin resistance [START_REF] Voyich | Is Panton-Valentine leukocidin the major virulence determinant in communityassociated methicillin-resistant Staphylococcus aureus disease?[END_REF]. Nevertheless, various clinical studies and animal models of BJIs and necrotizing pneumonia have confirmed the role of PVL, and it is now accepted that PVL is associated with the severity of disease, independent of meticillin resistance [START_REF] Crémieux | Panton-Valentine leukocidin enhances the severity of community-associated methicillin-resistant Staphylococcus aureus rabbit osteomyelitis[END_REF][START_REF] Diep | Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury[END_REF].

When considering clinical management of PVL-associated infections, it is important to assess the broad spectrum of diseases, ranging from common uncomplicated SSTIs to rare but life-threatening entities such as necrotizing pneumonia [START_REF] Gillet | Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients[END_REF][START_REF] Lina | Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia[END_REF][START_REF] Dohin | Pediatric bone and joint infections caused by Panton-Valentine leukocidin-positive Staphylococcus aureus[END_REF][START_REF] Miller | Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles[END_REF][START_REF] Perbet | Multifocal community-acquired necrotizing fasciitis caused by a Panton-Valentine leukocidin-producing methicillin-sensitive Staphylococcus aureus[END_REF][START_REF] Del Giudice | Primary skin abscesses are mainly caused by Panton-Valentine leukocidinpositive Staphylococcus aureus strains[END_REF]. Hence, specific management based on the presence of PVL may not be required for mild infections, whereas it could be life-saving for more severe disease.

Moreover, although recommendations for the management of PVL-associated disease and/or for CA-MRSA infections have been published by public health A c c e p t e d M a n u s c r i p t authorities in several countries [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF][START_REF] Liu | Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary[END_REF][START_REF]Guidelines for the management and prevention of skin infections due to community-acquired meticillin-resistant Staphylococcus aureus[END_REF], most of these diseases are recently identified entities and a 'gold standard' treatment based on comparatives studies of different therapeutic options is lacking. Thus, recommendations are based on expert opinion, which are based on theory, in vitro data and analogies with other toxin-mediated diseases.

In this review, we evaluate the potential need for specific PVL-based management for each disease and, if required, which tools should be used to achieve optimal management.

When is Panton-Valentine leukocidin-based management needed?

It is difficult to determine whether PVL-based management is needed because of the lack of comparative studies. Nevertheless, we can assess whether alternatives should be considered when standard management is insufficient or when PVL leads to specific disease defined by a significant difference in symptoms or severity from those induced by non-toxic strains. Thus, the first step should be the clinical identification of PVL-associated infection, followed by identification of the PVLproducing strain, especially because PVL remains rare in the general population in most Western European countries. for CA-MRSA in the USA. These include compromised skin integrity; all situations of skin-to-skin contact, including contact sports (e.g. wrestling, judo, rugby, and American and Australian football); and sharing of contaminated items (towels) [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF][START_REF] Hawkes | Communityassociated MRSA: superbug at our doorstep[END_REF][START_REF] Stryjewski | Skin and soft-tissue infections caused by community-acquired methicillin-resistant Staphylococcus aureus[END_REF]. Living in close and crowded communities is also a risk factor; outbreaks have occurred in prisons, military camps and colleges [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF]. Nevertheless, all of these risk factors are for SSTIs, whereas no risk factors have been identified for more severe disease, including necrotizing pneumonia and BJIs. Close contact with persons who have purulent skin infections prior to the development of infection may indicate the presence of PVL-producing bacteria.

Clinical symptoms are usually characterised by their severity and the presence of necrosis, abscesses and tissue destruction, and are marked by local and general inflammatory symptoms. Lesions are always painful and general reactions such as fever and asthenia are frequent, even when the infection is localised. In severe deepseated infections, leukoneutropenia may be observed [START_REF] Gillet | Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients[END_REF][START_REF] Kallen | Staphylococcus aureus community-acquired pneumonia during the 2006 to 2007 influenza season[END_REF]. The discordance between high levels of biological inflammation markers (C-reactive protein or procalcitonin) and a normal or low leukocyte count is highly suggestive of PVL bacteria [START_REF] Dohin | Pediatric bone and joint infections caused by Panton-Valentine leukocidin-positive Staphylococcus aureus[END_REF].

A c c e p t e d M a n u s c r i p t

Primary skin infection of the hair follicle is the most frequent PVL-associated disease [START_REF] Del Giudice | Primary skin abscesses are mainly caused by Panton-Valentine leukocidinpositive Staphylococcus aureus strains[END_REF][START_REF] Del Giudice | Panton-Valentine leukocidin-positive Staphylococcus aureus strains are associated with follicular skin infections[END_REF] and usually presents as large skin abscesses, boils or furuncles with large erythema, without an inoculation wound. The sores are often multiple, which may occur through self-inoculation [START_REF] Yamasaki | The association between Staphylococcus aureus strains carrying Panton-Valentine leukocidin genes and the development of deep-seated follicular infection[END_REF]. Necrosis ranging from local to necrotizing fasciitis has been observed, and secondary deep-seated localisations appear to be frequent.

Deep abscesses may be localised in subcutaneous tissue, muscle or in various organs such as the kidneys, lungs or bones, and they are often complicated by deep venous thrombosis. PVL-associated infection should be considered as the cause of BJI in severe cases presenting with high fever, painful lesions and signs of sepsis [START_REF] Dohin | Pediatric bone and joint infections caused by Panton-Valentine leukocidin-positive Staphylococcus aureus[END_REF][START_REF] Bocchini | Panton-Valentine leukocidin genes are associated with enhanced inflammatory response and local disease in acute hematogenous Staphylococcus aureus osteomyelitis in children[END_REF]. Multiple sites of infection at admission and aggravation, despite appropriate antibiotics, that lead to local extension (subperiosteal abscesses and/or soft tissue extensions) and metastatic abscesses may indicate infection by PVL bacteria.

Severe diseases such as septic shock or purpura fulminans may be indistinguishable from other bacterial infections [START_REF] Kravitz | Purpura fulminans due to Staphylococcus aureus[END_REF], and PVL may only be suspected if there is a context of preceding furunculosis in the patient or his close contacts and if bacteriological cultures grow S. aureus [START_REF] Adam | Fatal case of post-influenza, communityassociated MRSA pneumonia in an Ontario teenager with subsequent familial transmission[END_REF][START_REF] Dickson | A case of rapidly progressive necrotizing pneumonia caused by community-acquired methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Jones | Family outbreaks of invasive community-associated methicillin-resistant Staphylococcus aureus infection[END_REF][START_REF] Thomas | Breast milk transmission of a Panton-Valentine leukocidin-producing Staphylococcus aureus strain causing infantile pneumonia[END_REF][START_REF] Osterlund | Intrafamilial spread of highly virulent Staphylococcus aureus strains carrying the gene for Panton-Valentine leukocidin[END_REF].

Staphylococcal necrotizing pneumonia should be evoked before severe extensive pneumonia; it is usually multilobar and preceded by an influenza-like syndrome [START_REF] Gillet | Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients[END_REF].

Prompt progression to septic shock with acute respiratory distress syndrome (ARDS), initial leukopenia and signs of airway haemorrhage are highly suggestive of A c c e p t e d M a n u s c r i p t infection with PVL bacteria and are strongly associated with lethality [START_REF] Gillet | Factors predicting mortality in necrotizing community-acquired pneumonia caused by Staphylococcus aureus containing Panton-Valentine leukocidin[END_REF]. Therefore, specific, aggressive management should be instigated as soon as the diagnosis is suspected, even before confirmation of PVL.

Microbiological diagnosis

To date, detection of PVL-producing S. aureus strains is mainly performed with homemade polymerase chain reaction (PCR) on DNA from bacterial colonies. From the 13 sets of primers that have been described, 8 sets target genes in the nonpolymorphic region [START_REF] Lina | Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia[END_REF][START_REF] Fey | Comparative molecular analysis of community-or hospital-acquired methicillinresistant Staphylococcus aureus[END_REF][START_REF] Francois | A novel multiplex real-time PCR assay for rapid typing of major staphylococcal cassette chromosome mec elements[END_REF][START_REF] Jarraud | Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease[END_REF][START_REF] Miyashita | Destructive pulmonary embolism in a patient with community-acquired staphylococcal bacteremia[END_REF][START_REF] Roberts | A real-time PCR assay to detect the Panton Valentine leukocidin toxin in staphylococci: screening Staphylococcus schleiferi subspecies coagulans strains from companion animals[END_REF][START_REF] Strommenger | Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing[END_REF]. The manufactured molecular kits for detecting PVL genes include the GenoType ® Staphylococcus test (Biocentric) and the DNA microarray S. aureus (ARLE). The problem with molecular methods is that they do not reflect PVL production. PVL production can be detected with experimental latex agglutination assays, enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic tests [START_REF] Badiou | Rapid detection of Staphylococcus aureus Panton-Valentine leukocidin in clinical specimens by enzyme-linked immunosorbent assay and immunochromatographic tests[END_REF][START_REF] Oishi | A latex agglutination assay for specific detection of Panton-Valentine leukocidin[END_REF]. These two last tests can even be performed directly on clinical specimens.

Therapeutic tools for Panton-Valentine leukocidin-associated infections

To determine specific treatments for PVL-associated infections, it is necessary to postulate that PVL is directly or indirectly (pro-inflammatory response) responsible for the lesions and tissue damage that leads to clinical symptoms. Hence, the main goal of treatment should be not only to eradicate the PVL-producing S. aureus but also to diminish the effects of PVL by (i) removing PVL from the patient, (ii) inhibiting PVL production by S. aureus and (iii) blocking the toxic effects of PVL after its production.

A c c e p t e d M a n u s c r i p t

PVL removal can only be achieved by complete drainage, surgical or spontaneous, of all PVL-containing suppuration [START_REF] Badiou | Panton-Valentine leukocidin is expressed at toxic levels in human skin abscesses[END_REF]. Therefore, one of the key rules for management of PVL-associated disease should be to perform surgical drainage whenever possible and as soon as possible.

Inhibition of PVL production may be promptly achieved with all antistaphylococcal antibiotics, but only if the drug concentration is far above the minimum inhibitory concentration (MIC) at the site of infection. This may be very difficult in PVLassociated infections because intense necrosis leads to a very low concentration of antibiotics in the pus. Dumitrescu et al. [START_REF] Dumitrescu | Effect of antibiotics, alone and in combination, on Panton-Valentine leukocidin production by a Staphylococcus aureus reference strain[END_REF][START_REF] Dumitrescu | Effect of antibiotics on Staphylococcus aureus producing Panton-Valentine leukocidin[END_REF] have shown experimentally in vitro and in experimental infection of animals that if the concentration of -lactams and, to a lesser extent, vancomycin is below the MIC, PVL secretion may be enhanced, which may aggravate symptoms. Hence, a treatment targeting PVL should include molecules able to reduce bacterial protein synthesis of PVL, even at suboptimal concentrations. Clindamycin, linezolid and rifampicin have been shown to be able to achieve this goal [START_REF] Dumitrescu | Effect of antibiotics on Staphylococcus aureus producing Panton-Valentine leukocidin[END_REF] and these antitoxic effects persist even when these molecules are associated with -lactams or vancomycin [START_REF] Dumitrescu | Effect of antibiotics, alone and in combination, on Panton-Valentine leukocidin production by a Staphylococcus aureus reference strain[END_REF].

Neutralisation of the PVL effect in vivo requires specific antibodies. It is proven that deep infection with a PVL-positive S. aureus induces the production of significant amount of neutralising antibodies [START_REF] Croze | Serum antibodies against Panton-Valentine leukocidin in a normal population and during Staphylococcus aureus infection[END_REF]. Moreover, Gauduchon et al. [START_REF] Gauduchon | Neutralization of Staphylococcus aureus Panton Valentine leukocidin by intravenous immunoglobulin in vitro[END_REF] demonstrated that polyvalent human intravenous immunoglobulin (IVIg) may inhibit the cytotoxicity of PVL from S. aureus on polymorphonuclear cells in a concentrationdependent manner. This inhibition starts at a concentration above 2 mg/L and is A c c e p t e d M a n u s c r i p t complete at 10 mg/L. These concentrations are easily achieved using a high-dose regimen of IVIg (2 g/kg/day) in humans. The inhibitory effect of IVIg was tested in various batches of commercially available preparations of IVIg and was confirmed in all cases (personal data). There is no clinical study on the in vivo efficacy of IVIg, but several case reports have shown dramatic improvement in severe PVL-associated infections after the use of IVIg [START_REF] Libert | Antitoxin treatments for necrotizing pneumonia due to Panton-Valentine leukocidin-secreting Staphylococcus aureus [in French][END_REF][START_REF] Lorenz | Severe pyomyositis caused by Panton-Valentine leucocidin-positive methicillin-sensitive Staphylococcus aureus complicating a pilonidal cyst[END_REF][START_REF] Rouzic | Prompt and successful toxin-targeting treatment of three patients with necrotizing pneumonia[END_REF].

Indications

Indications and therapeutic options for the different types of PVL-associated diseases are summarised in Table 1.

Uncomplicated skin and soft-tissue infection

SSTIs are the most frequent PVL-associated diseases, but in uncomplicated cases PVL-specific management is probably not required because studies have not demonstrates a link between PVL and worse outcome [44]. In most cases, SSTIs result in collection of pus; in such situations surgical drainage is recommended. In a large series of 422 uncomplicated SSTIs, Moran et al. [START_REF] Moran | EMERGEncy ID Net Study Group. Methicillin-resistant S. aureus infections among patients in the emergency department[END_REF] found that outcomes were usually favourable (in >90% of cases), even if antibiotics were inappropriate (i.e. lactams for MRSA), as long drainage was performed. Most of the published guidelines do not recommend the use of systemic antibiotics in the case of cutaneous abscesses or boils [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF][START_REF] Liu | Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary[END_REF][START_REF] Moran | EMERGEncy ID Net Study Group. Methicillin-resistant S. aureus infections among patients in the emergency department[END_REF][START_REF] Stevens | Infectious Diseases Society of America. Practice guidelines for the diagnosis and management of skin and soft-tissue infections[END_REF] and there are no data suggesting that the presence of PVL may modify these guidelines. Nevertheless, antibiotics are recommended both by the UK Health Protection Agency (HPA) and the Infectious Disease Society of America (IDSA) in particular conditions [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF][START_REF] Liu | Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary[END_REF]. The HPA A c c e p t e d M a n u s c r i p t considers antibiotics if the diameter of the abscess is >5 cm, in cases of associated cellulitis and in cases of severe extensive disease with systemic symptoms. In the latter case, the patient should be immediately referred to a hospital [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF]. The recommendations of the IDSA are the same, but some additional indications are included in the recommendation for antibiotic treatment [START_REF] Liu | Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary[END_REF]:

 associated co-morbidities or immunosuppression;  extreme age;  abscesses in difficult to drain areas (e.g. face, hands and genitalia);  associated septic phlebitis; and

 lack of response to incision and drainage (A-III).

Because of the emergence of CA-MRSA, empirical therapy should be adapted based on the bacteriological culture [START_REF] Stryjewski | Skin and soft-tissue infections caused by community-acquired methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Grayson | The treatment triangle for staphylococcal infections[END_REF] of any cutaneous exudates, or blood cultures in the case of systemic symptoms. The choice of molecule, if indicated, is based on the incidence of CA-MRSA in the community. In areas with low-to-moderate incidence, antistaphylococcal -lactams (flucloxacillin) remain the first choice, but when available the combination of amoxicillin and clavulanic acid may be better because of increased coverage of -haemolytic streptococci, especially in case of associated cellulitis [START_REF] Stevens | Infectious Diseases Society of America. Practice guidelines for the diagnosis and management of skin and soft-tissue infections[END_REF][START_REF] Moulin | Managing children skin and soft tissue infections [in French][END_REF]. In high-incidence areas, such as North America, CA-MRSA should be systematically covered in the case of a purulent lesion. For empirical coverage of CA-MRSA in outpatients with SSTI, oral antibiotic options include the following: clindamycin (A-II); trimethoprim/sulfamethoxazole (TMP/SMX) (A-II); a tetracycline (doxycycline or minocycline) (A-II); and linezolid (A-II). If coverage of both haemolytic streptococci and CA-MRSA is desired, options include the following:

A c c e p t e d M a n u s c r i p t clindamycin alone (A-II); TMP/SMX (A-II); a tetracycline in combination with a lactam (e.g. amoxicillin) (A-II); or linezolid alone (A-II) [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF][START_REF] Liu | Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary[END_REF][START_REF]Guidelines for the management and prevention of skin infections due to community-acquired meticillin-resistant Staphylococcus aureus[END_REF]. There are no studies supporting significant differences between these molecules or a recommendation for using specific treatments for antitoxic activity, even in cases of documented PVLassociated infection.

Severe skin and soft-tissue infections

The severity of SSTIs associated with PVL could be due to local extension, metastatic localisations, or association with septic or toxic shock syndrome. Severe local extensions lead to necrosis of cutaneous and deep subcutaneous tissue and are clinically undistinguishable from streptococcal necrotizing fasciitis before bacteriological documentation. Moreover, monomicrobial or polymicrobial necrotizing skin infections containing PVL-producing S. aureus may exist. In this case, the role of PVL may be less important, and PVL-targeted management is probably not required.

Because of its rarity, the optimal management of monomicrobial staphylococcal necrotizing fasciitis is unknown and there are no data supporting major differences in the management versus streptococcal cases, except for the adaptation of antibiotics.

Hence, penicillin and ampicillin, which are the first-choice -lactams for streptococcal infections [START_REF] Stevens | Infectious Diseases Society of America. Practice guidelines for the diagnosis and management of skin and soft-tissue infections[END_REF], should be switched to penicillinase-resistant semisynthetic penicillin in meticillin-susceptible S. aureus (MSSA) cases. If MRSA is found or strongly suspected because of local epidemiology, therapeutic options recommended by the IDSA and HPA include intravenous (i.v.) vancomycin (A-I), oral or i.v. linezolid (A-I), i.v. daptomycin (A-I), i.v. telavancin (A-I) or i.v. clindamycin (A-III). As with uncomplicated SSTIs, there are no data regarding significant differences between outcomes when patients are treated with these molecules. Nevertheless, the A c c e p t e d M a n u s c r i p t usefulness of treatment with antitoxins has been demonstrated for streptococcal necrotizing fasciitis, especially with clindamycin [START_REF] Mulla | Treatment options in the management of necrotising fasciitis caused by group A Streptococcus[END_REF][START_REF] Mulla | Invasive group A streptococcal infections in Florida[END_REF][START_REF] Stevens | In vitro antimicrobial effects of various combinations of penicillin and clindamycin against four strains of Streptococcus pyogenes[END_REF][START_REF] Zimbelman | Improved outcome of clindamycin compared with -lactam antibiotic treatment for invasive Streptococcus pyogenes infection[END_REF]. Even if no comparable data for S. aureus necrotizing fasciitis exist, we believe that such treatment should be considered systematically for severe SSTIs. If clindamycin is used, clinicians must be aware of the possible discrepancy between clinical efficacy and susceptibility testing, which is due to inducible resistance when the strain is not susceptible to erythromycin. In such cases, a D-zone test procedure is warranted [START_REF] Lewis | Inducible clindamycin resistance in staphylococci: should clinicians and microbiologists be concerned?[END_REF]. Some experts recommend bactericidal activity in the case of intravascular infection, which is a common feature in severe SSTIs, and none of the antitoxic treatments have shown such activity. Thus, considering the risk of inducible resistance and the need for bactericidal activity, we recommend a combination of clindamycin and -lactam for MSSA or vancomycin for MRSA. Linezolid alone is approved for the treatment of cSSTIs but, to our knowledge, there is no study regarding its effects on necrotizing fasciitis.

Apart from standard antibiotic treatment, most of the recommendations for streptococcal necrotizing fasciitis can be considered useful for PVL-associated infections. The IDSA's guidelines recommend surgical intervention as the primary therapeutic modality. Aggressive surgical debridement is indicated when antibiotics fail and the SSTI worsens, despite treatment, to severe sepsis or septic shock and in cases of skin necrosis with easy dissection along the fascia during a limited surgical exploration. Considering the role of PVL in necrosis and the difficulty in achieving active concentrations of antibiotics in necrotic tissues, it is reasonable to apply these recommendations to staphylococcal necrotizing SSTIs.

A c c e p t e d M a n u s c r i p t

The use of clindamycin is recommended for streptococcal necrotizing fasciitis based on in vitro studies demonstrating both toxin suppression and modulation of cytokine [i.e. tumour necrosis factor (TNF)] production in animal studies, which demonstrate the superior efficacy of clindamycin versus penicillin [START_REF] Stevens | In vitro antimicrobial effects of various combinations of penicillin and clindamycin against four strains of Streptococcus pyogenes[END_REF]. In addition, observational studies have demonstrated that clindamycin has greater efficacy than -lactam antibiotics [START_REF] Mulla | Treatment options in the management of necrotising fasciitis caused by group A Streptococcus[END_REF][START_REF] Mulla | Invasive group A streptococcal infections in Florida[END_REF][START_REF] Zimbelman | Improved outcome of clindamycin compared with -lactam antibiotic treatment for invasive Streptococcus pyogenes infection[END_REF]. Clindamycin may protect against S. aureus infection in undocumented cases and its antitoxin properties may be useful against PVLassociated disease [START_REF] Dumitrescu | Effect of antibiotics, alone and in combination, on Panton-Valentine leukocidin production by a Staphylococcus aureus reference strain[END_REF].

The usefulness of IVIg for the treatment of necrotizing fasciitis remains controversial, despite evidence that the toxin plays a role in shock, organ failure and tissue necrosis and that variable amounts of neutralising antibodies are observed after treatment with IVIg. There are no studies on the effects of IVIg against staphylococcal necrotizing fasciitis and few data for streptococcal necrotizing fasciitis. One observational study demonstrated better outcomes for streptococcal necrotizing fasciitis in the IVIg group versus historical controls, but the IVIg patients were also more likely to have benefited from a surgical procedure and to have been treated with clindamycin [START_REF] Norrby-Teglund | Intravenous immunoglobulin adjunctive therapy in sepsis, with special emphasis on severe invasive group A streptococcal infections[END_REF][START_REF] Norrby-Teglund | Successful management of severe group A streptococcal soft tissue infections using an aggressive medical regimen including intravenous polyspecific immunoglobulin together with a conservative surgical approach[END_REF]. A second placebo-controlled study showed reduced mortality in the IVIg group but failed to demonstrate a statistically significant difference owing to the small number of patients enrolled in the study [START_REF] Darenberg | Intravenous immunoglobulin G therapy in streptococcal toxic shock syndrome: a European randomized, double-blind, placebo-controlled trial[END_REF]. Therefore, even if the in vitro data are promising, there is not enough evidence to recommend routine use of IVIg in severe PVL-associated SSTIs. Nevertheless, in cases when optimal therapy has failed, including surgical drainage, appropriate antibiotics with at least one antitoxin molecule and optimal management of septic shock, IVIg should be considered as a therapeutic option.

A c c e p t e d

M a n u s c r i p t

Complicated skin and soft-tissue infections

The most common complications of SSTIs are deep abscesses associated with deep vein thrombosis, which are caused by PVL-positive strains. The management of these complications depends on the number, size and location of the abscesses. As in any purulent collection, antibiotics alone are often insufficient and, therefore, surgical drainage is the primary therapeutic option. There are no data on treatment with antitoxin antibiotics. In cases of multiple abscesses that are not surgically accessible and that are progressing despite appropriate antibiotics, two published cases report a dramatic improvement after treatment with IVIg [START_REF] Lorenz | Severe pyomyositis caused by Panton-Valentine leucocidin-positive methicillin-sensitive Staphylococcus aureus complicating a pilonidal cyst[END_REF][START_REF] Hampson | Disseminated sepsis due to a Panton-Valentine leukocidin producing strain of community acquired meticillin resistant Staphylococcus aureus and use of intravenous immunoglobulin therapy[END_REF].

Considering the risk of developing deep venous thrombosis, prophylaxis is usually recommended, and curative treatment should be considered for acquired thrombosis.

Nevertheless, the haemorrhagic risk should be taken into account in case of pulmonary localisation because severe airway haemorrhages are common in PVLassociated necrotizing pneumonia [START_REF] Gillet | Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients[END_REF][START_REF] Gillet | Factors predicting mortality in necrotizing community-acquired pneumonia caused by Staphylococcus aureus containing Panton-Valentine leukocidin[END_REF].

Bone and joint infections

Staphylococcus aureus is the most common cause of BJI. The potential need for a specific management plan for PVL-associated BJIs is illustrated in the description of these infections: standard care (i.e. use of antistaphylococcal drugs approved for BJIs) resulted in treatment failure, as indicated by extension of the lesions in 85% of cases that were treated with active antibiotics. The extension is usually local, with abscess formation in the infected bones and local soft tissues and secondary A c c e p t e d M a n u s c r i p t bacterial metastasis in various organs. In 71% of cases additional surgery is required after the initial procedure (drainage for arthritis or puncture for osteomyelitis), with a median of three procedures per patient. This unfavourable outcome is observed in areas where MRSA is dominant [START_REF] Bocchini | Panton-Valentine leukocidin genes are associated with enhanced inflammatory response and local disease in acute hematogenous Staphylococcus aureus osteomyelitis in children[END_REF] and in Europe, where the majority of cases are due to MSSA [START_REF] Dohin | Pediatric bone and joint infections caused by Panton-Valentine leukocidin-positive Staphylococcus aureus[END_REF]. Unfortunately, comparisons were made with historical controls and no comparative studies for different therapeutic options have been reported.

Therefore, no definite conclusions can be made regarding the optimal therapy for PVL-associated BJIs. However, the use of clindamycin or linezolid as the first-line regimen appears to be rational, because both drugs are approved for treatment of BJIs and have antitoxin properties. Because clindamycin is only bacteriostatic, there are some limitations for its use alone, and considering the high incidence of sepsis in the published series of PVL-associated BJIs we recommend combining clindamycin with i.v. cloxacillin (or equivalent semisynthetic -lactamase-resistant penicillin) when the incidence of CA-MRSA is low in the community. For suspected (in a highincidence area) or proven PVL MRSA, clindamycin could be combined with vancomycin (despite the poor bone penetration associated with that drug) [START_REF] Dombrowski | Clinical failures of appropriately-treated methicillin-resistant Staphylococcus aureus infections[END_REF] or daptomycin [START_REF] Liu | Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary[END_REF], which is approved for treatment of BJIs in the US and off-label in Europe. Alternative regimens for the treatment of MRSA are linezolid alone, a combination of TMP/SMX plus rifampicin or vancomycin plus rifampicin. Like clindamycin and linezolid, rifampicin has shown interesting antitoxin properties against PVL in vitro, but there is some concern about its use as a first-line regimen owing to the risk of selecting resistant isolates with high inoculum. There are limited data about the use of linezolid alone, which appears to be effective, but may be limited by its toxicity, especially in the bone marrow. Weekly monitoring of complete blood counts is recommended if therapy exceeds 2 weeks, and an ophthalmological A c c e p t e d M a n u s c r i p t examination should be performed 1 month after initiation of therapy because optic neuritis may occur after prolonged treatment, which is usually required for BJIs.

Like in other PVL-associated diseases, the frequency of abscesses is important.

Drainage of any bone or subperiosteal abscess, surgical debridement and any associated soft-tissue abscesses is the most common therapy and should be performed whenever feasible [START_REF] Dohin | Pediatric bone and joint infections caused by Panton-Valentine leukocidin-positive Staphylococcus aureus[END_REF][START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF][START_REF] Liu | Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary[END_REF][START_REF] Bocchini | Panton-Valentine leukocidin genes are associated with enhanced inflammatory response and local disease in acute hematogenous Staphylococcus aureus osteomyelitis in children[END_REF]. Because bone abscesses are quite rare in non-PVL staphylococcal BJIs and therefore may not be immediately suspected, even in cases with unfavourable outcome we suggest performing magnetic resonance imaging (MRI) and gadolinium imaging during the week following initiation of therapy whenever a PVL-associated BJI is documented. MRI should be repeated unless a favourable outcome is achieved.

Necrotizing pneumonia

Staphylococcal necrotizing pneumonia is by far the most severe presentation of PVLassociated infections and therefore requires special management. Although lethality has diminished since the initial description in 2002, overall mortality remains high (42.9-56%) [START_REF] Gillet | Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients[END_REF][START_REF] Kallen | Staphylococcus aureus community-acquired pneumonia during the 2006 to 2007 influenza season[END_REF][START_REF] Gillet | Factors predicting mortality in necrotizing community-acquired pneumonia caused by Staphylococcus aureus containing Panton-Valentine leukocidin[END_REF][START_REF] Li | Factors associated with the outcome of life-threatening necrotizing pneumonia due to community-acquired Staphylococcus aureus in adult and adolescent patients[END_REF][START_REF] Morgan | Diagnosis and treatment of Panton-Valentine leukocidin (PVL)associated staphylococcal pneumonia[END_REF] and the median survival is only 4 days. Approximately one-half of deaths occur before PVL identification, and in some cases even before bacterial documentation. Necrotizing pneumonia is a rare disease and there are no comparative studies evaluating its management. Nevertheless, guidelines for specific management are necessary considering the severity and the fact that standard antibiotic regimens have no impact on mortality. Hence, in the 51 cases we studied, the percentage of patients who received antibiotics that were active against their A c c e p t e d M a n u s c r i p t staphylococcal strain was the same for survivors and non-survivors (76.2% vs. 76.9%, respectively) [START_REF] Gillet | Factors predicting mortality in necrotizing community-acquired pneumonia caused by Staphylococcus aureus containing Panton-Valentine leukocidin[END_REF].

Unfortunately, surgical drainage of the infected lesions is almost never feasible during the early stages of necrotizing pneumonia. In most cases there is global necrosis of the bronchial and alveolar epithelium with diffuse alveolar damage, and surgical debridement is not possible. Delimited lung abscesses may appear secondarily if the patient survives and should be drained if the clinical situation has not improved, but in most severe cases there is no individualised collection. Drainage of pleural effusion should be performed whenever significant effusion is present, but this has little influence on the evolution of lung infection.

Most necrotizing pneumonia causes symptoms that are consistent with severe sepsis or septic shock at admission or during the first hours following admission. Therefore, we recommend systematically referring patients with suspected necrotizing pneumonia to the Intensive Care Unit. Aggressive non-specific management is required, following the guidelines of the Surviving Sepsis Campaign [START_REF] Dellinger | Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008[END_REF], and artificial ventilation is needed in up to 60% of cases. Progression to ARDS is common and respiratory support should follow the standard recommendation for ARDS [START_REF] Sevransky | Mechanical ventilation in sepsis-induced acute lung injury/acute respiratory distress syndrome: an evidence-based review[END_REF]. There are no published data regarding adjunctive treatment of sepsis with activated protein C, which is associated with a high frequency of severe airway haemorrhage in necrotizing pneumonia; in our opinion, this fact contraindicates this treatment.

There is no comparative study for antibiotics in necrotizing pneumonia. The IDSA, which focuses on MRSA and not on the severity of disease, recommends A c c e p t e d M a n u s c r i p t vancomycin, i.v. linezolid or i.v. clindamycin, whereas the HPA recommends a combination of clindamycin plus linezolid [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF][START_REF] Liu | Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary[END_REF]. As in severe SSTI, there is some concern regarding the use of only bacteriostatic molecules for such a severe condition, but a recent retrospective review of necrotizing pneumonia has shown that using therapies that inhibit toxin production is associated with better outcomes and with a significant impact on mortality [START_REF] Li | Factors associated with the outcome of life-threatening necrotizing pneumonia due to community-acquired Staphylococcus aureus in adult and adolescent patients[END_REF]. Therefore, we consider that initial antibiotic therapy should combine at least one molecule with antitoxin effects with a bactericidal antibiotic. Some recommend avoiding -lactams in cases of suspected PVL-associated disease, arguing that -lactams at suboptimal concentration enhance PVL production in vitro [START_REF]Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England[END_REF][START_REF] Morgan | Diagnosis and treatment of Panton-Valentine leukocidin (PVL)associated staphylococcal pneumonia[END_REF]. We do not fully agree with this point of view because it is shown that, under the same experimental conditions, the overproduction of PVL induced by -lactams is completely reversed when clindamycin or linezolid is added, which results in inhibition comparable with using clindamycin or linezolid alone [START_REF] Dumitrescu | Effect of antibiotics, alone and in combination, on Panton-Valentine leukocidin production by a Staphylococcus aureus reference strain[END_REF].

Moreover, it is important to consider that, at the time of treatment, the bacteria has often not been identified and the possibility of severe necrotizing pneumonia due to other bacteria, such as Streptococcus pyogenes or Streptococcus pneumoniae, cannot be excluded. Hence, we believed that a third-generation cephalosporin should be added to the initial antibiotic regimen to achieve full coverage for S. pyogenes and penicillin-resistant pneumococci. The choice to systematically cover MRSA is evident in high-incidence areas, but considering the severity and the risk of prompt degradation before full susceptibility testing, coverage of MRSA should be considered during the first-line regimen, even in low-incidence areas. Taking into account all these points, our recommendations for the first line of antibiotic treatment A c c e p t e d M a n u s c r i p t for suspected necrotizing pneumonia would be to combine a third-generation cephalosporin with vancomycin and clindamycin or linezolid at the highest acceptable doses through an i.v. route. Treatment should be adapted after susceptibility testing and should always be combined with a bactericidal antibiotic (cloxacillin for MSSA or vancomycin for MRSA) and molecules that can inhibit toxin production. As an alternative, linezolid could be used alone for the treatment of MRSA infection because the concentration of linezolid in the epithelial lining of the lung fluid is at higher levels than its concentration in plasma [START_REF] Conte | Intrapulmonary pharmacokinetics of linezolid[END_REF]. Nevertheless, the comparison between linezolid and vancomycin was performed with nosocomial MRSA ventilatorassociated pneumonia, and no study on community-acquired pneumonia has been performed [START_REF] Rubinstein | Pneumonia caused by methicillinresistant Staphylococcus aureus[END_REF][START_REF] Wunderink | Linezolid vs vancomycin: analysis of two double-blind studies of patients with methicillin-resistant Staphylococcus aureus nosocomial pneumonia[END_REF]. Adding rifampicin to the treatment regimen has been proposed to optimise diffusion in necrotic lung tissue and to achieve optimal clearance of intracellular staphylococci [START_REF] Libert | Antitoxin treatments for necrotizing pneumonia due to Panton-Valentine leukocidin-secreting Staphylococcus aureus [in French][END_REF], but its limitations in cases of high inoculum may restrict it to a second-round therapy after the initial sepsis has cleared.

Although recommended by UK health authorities, IVIg has never been studied in necrotizing pneumonia, and evidence for efficacy or optimal dosage is lacking. In fact, the use of IVIg is only supported by in vitro data and a few promising case reports [START_REF] Libert | Antitoxin treatments for necrotizing pneumonia due to Panton-Valentine leukocidin-secreting Staphylococcus aureus [in French][END_REF][START_REF] Rouzic | Prompt and successful toxin-targeting treatment of three patients with necrotizing pneumonia[END_REF]. Comparison with other severe staphylococcal toxin-mediated diseases is tempting but is not appropriate because PVL does not have superantigenic activity, and although shock is often present, only a small number of patients have toxic shock syndrome. Therefore, it is difficult to recommend systematic use of IVIg. Because of the low frequency of the disease, it will probably take years or even decades before a well-designed placebo-controlled study is performed. On the other hand, necrotizing pneumonia is a frightening disease with a A c c e p t e d M a n u s c r i p t high mortality rate, in which insufficient use of antibiotics is proven, and therefore it may be unethical to not use such a promising therapy. Moreover, rapid progression of the disease does not allow a lot of time to make the decision. The choice remains difficult, but learning more about factors associated with severity may help. All of the largest necrotizing pneumonia studies have emphasised the role of initial leukopenia and airway haemorrhages as independent factors associated with mortality [START_REF] Kallen | Staphylococcus aureus community-acquired pneumonia during the 2006 to 2007 influenza season[END_REF][START_REF] Gillet | Factors predicting mortality in necrotizing community-acquired pneumonia caused by Staphylococcus aureus containing Panton-Valentine leukocidin[END_REF][START_REF] Li | Factors associated with the outcome of life-threatening necrotizing pneumonia due to community-acquired Staphylococcus aureus in adult and adolescent patients[END_REF]. A leukocyte count <3  10 9 /L increases by 8-fold the odds of death, with an overall mortality of >80%. Airway bleeding is also associated with poor outcome, with a lethality of 80%. Hence, we could propose the systematic use of a high dose of IVIg (2 g/kg in one dose or 1 g/kg/day during two consecutive days) as soon as Staphylococcus is identified or whenever lethality-associated factors (i.e. leukopenia <3  10 9 /L and/or airway haemorrhage) are present. Moreover, when symptoms of toxic shock syndrome are present, especially skin eruption (even if the eruption is probably due to other toxins), IVIg should be used, even if the effect of IVIg on staphylococcal toxic shock syndrome is not fully proven [START_REF] Darenberg | Differences in potency of intravenous polyspecific immunoglobulin G against streptococcal and[END_REF]67].

Conclusion

Establishing guidelines for the management of PVL-associated diseases is challenging because of the diversity of diseases and because of the lack of knowledge regarding treatment outcomes. Nevertheless, we propose a pragmatic approach based on clinical reports and experimental studies. Our recommendation can be summarised in five key points. a Linezolid could be used alone for bone and joint infections and skin and soft-tissues infections. No studies for community-acquired pneumonia. The combination of vancomycin and linezolid is not be recommended because of potential antagonism.

A c c e p t e d M a n u s c r i p t 3 .

 3 Diagnosis of Panton-Valentine leukocidin-associated disease 3.1. Clinical suspicion In areas with a low-to-moderate incidence of PVL-secreting bacteria, PVL infection should be diagnosed on an individual clinical basis and confirmed by laboratory tests, except during outbreaks of PVL-positive S. aureus infection. Most of the cases occur in young immunocompetent patients without underlying disease or other conditions favouring staphylococcal infection. Risk factors for acquisition are the same as those



  The presence of PVL should be suspected on an individual clinical basis, independent of meticillin resistance, and every effort should be made to obtain laboratory confirmation. Standard management should be used when there are not indicators of the presence of PVL bacteria, such as in uncomplicated SSTIs. In cases where antibiotics with proven antitoxin activity (clindamycin, linezolid and rifampicin) are approved, as in complicated SSTI and BJI, they should be used as the first line of defence and should be combined with bactericidal antibiotics in the most severe cases. Surgical drainage of purulent collection is recommended when possible. In cases of severe life-threatening disease, antitoxic antibiotics and adjunctive treatment such as polyvalent IVIg should be considered systematically because there is not enough time for evidence-based medicine in many cases. Decision-making could be helped in such situations by the knowledge of severity-associated factors.

  s c r i p t due to Staphylococcus aureus strains carrying the Panton-Valentine leukocidin genes. J Clin Microbiol 2010;48:1952-5. [44] Bae IG, Tonthat GT, Stryjewski ME, Rude TH, Reilly LF, Barriere SL, et al. Presence of genes encoding the Panton-Valentine leukocidin exotoxin is not the primary determinant of outcome in patients with complicated skin and skin structure infections due to methicillin-resistant Staphylococcus aureus: results of a multinational trial. J Clin Microbiol 2009;47:3952-7.

  Treatment guidelines for Panton-Valentine leukocidin (PVL) Staphylococcus aureus infections MSSA, meticillin-susceptible Staphylococcus aureus; AMX/CLA, amoxicillin/clavulanic acid; TMP/SMX, trimethoprim/sulfamethoxazole; MRSA, meticillin-resistant S. aureus; 3GC, third-generation cephalosporin.

	Edited Table 1
	Type of infection Skin and soft-tissue infections Uncomplicated Moderate infection (abscess diameter >5 cm; cellulitis, deep thrombosis; septic shock) Bone and joint infections	Surgical drainage Yes Yes Yes	Microbiological analysis No Yes, for antimicrobial Yes, for a n u s c r i p t Antibiotics Antitoxic antibiotics No No MSSA: AMX/CLA, clindamycin, No MRSA: vancomycin, daptomycin, telavancin, linezolid MSSA: cloxacillin Yes, antimicrobial resistance and clindamycin, rifampicin, MRSA: vancomycin, M PVL status linezolid a daptomycin,	IVIg No No No
	infection; systemic symptoms; co-morbidity; extreme age) Severe or complicated infection (cutaneous necrosis; deep Necrotizing pneumonia A c c e p t e d resistance Yes Yes, for antimicrobial Not feasible Yes, for antimicrobial resistance and PVL status IVIg, intravenous immunoglobulin;	TMP/SMX, tetracycline, linezolid MRSA: vancomycin, daptomycin, telavancin, linezolid MSSA: AMX/CLA, TMP/SMX, TMP/SMX Initial: 3GC plus vancomycin MSSA: cloxacillin MRSA: vancomycin, linezolid	Yes, clindamycin, Yes, clindamycin, linezolid a Yes, clindamycin, linezolid a , rifampicin	Yes, in cases when optimal therapy has Yes, when leukopenia <3  10 9 /L and/or airway haemorrhage
	subcutaneous necrosis; deep		resistance and	tetracycline,	linezolid a	failed or toxic shock
	abscesses; deep venous		PVL status	linezolid		syndrome is present
			2

Funding

ODa, FV, JE and GL were supported by grants from the European Community (EC 222718), Pfizer, Leo Pharma, Novartis, LBF and bioMérieux.

Competing interests

GL, FV, ODu, FV and JE received financial support from Pfizer, Leo Pharma, Novartis, LBF and bioMérieux. All other authors declare no competing interests.

Ethical approval