Epidemiological and clinical features of Panton-Valentine Leukocidin positive Staphylococcus aureus bacteremia: A case-control study.

BACKGROUND: Panton-Valentine Leukocidin (PVL) toxin in Staphylococcus aureus has been associated with both severe pneumonia and skin and soft tissue infections. However, there are only limited data on how this virulence factor may influence the clinical course or complications of bacteremic S. aureus infections.
METHODS: Between September 2016 and March 2018, S. aureus isolates from clinical cultures from hospitals in an academic medical center underwent comprehensive genomic sequencing. Four hundred sixty-nine (29%) of 1681 S. aureus sequenced isolates were identified as containing the genes that encode for PVL. Case patients with one or more positive blood cultures for PVL were randomly matched with control patients having positive blood cultures with lukF/lukS-PV negative (PVL strains from a retrospective chart review).
RESULTS: 51 case and 56 control patients were analyzed. Case patients were more likely to have a history of injection drug use, while controls more likely to undergo hemodialysis. Isolates from 78.4% of case patients were methicillin resistant as compared to 28.6% from control patients. Case patients had a higher incidence of pneumonia and skin and soft tissue infection and longer duration of fever without differences in length of bacteremia. Clinical cure or expiration was comparable.
CONCLUSIONS: These results are consistent with prior observations associating the PVL toxin with both community-acquired MRSA strains as well as severe staphylococcal pneumonia. The presence of the PVL toxin does not appear to otherwise influence the natural history of bacteremic S. aureus disease other than in prolonging the duration of fever.

Introduction

Staphylococcus aureus bacteremia (SAB) is a common and serious infection with an incidence of 10 to 30 per 100,000 person-years in the industrialized world and a 10–50% case fatality rate among [1]. Both host and pathogen factors likely contribute to the development of SAB, and contributing host factors including age, male gender, ethnicity, injection drug use, hemodialysis, the presence of intravenous catheters, neutrophil dysfunction, iron overload, diabetes, cancer, corticosteroid therapy, and S. aureus colonization.[1-5]

Pathogen factors contributing to bacteremia have been less well defined, although both methicillin-resistance and the presence of the Staphylococcal enterotoxin P gene in methicillin-resistant S. aureus (MRSA) strains have recently been identified as risk factors for SAB. [2, 6] Of note, there has been a significant increase in the relative incidence of infections due to the community associated MRSA strain ST8-USA300 in North America [7] and Europe [1]. For instance, in Geneva, ST8-USA300 has been linked with transmission from the Americas [8]. An important virulence factor of this strain is the Panton-Valentine Leukocidin (PVL), a binary exotoxin, encoded by lukF-PV and lukS-PV, that forms pores in leukocyte membranes. [9-11] First identified in 1932, the toxin was initially associated with the development of abscesses, and more recently with severe, necrotizing pneumonia whether present in methicillin-susceptible or MRSA isolates [9, 12–16]

Given the association of PVL with these severe infections, it would seem possible that the presence of the toxin would impact the clinical manifestations of SAB. However, the limited available literature has not found significant differences in clinical outcomes (e.g., mortality) or severity (e.g., sepsis, septic shock) between PVL positive bloodstream isolates and PVL negative isolates, although some studies suggest an association between PVL and prolonged duration of bacteremia, and greater sites of metastatic infection.[2, 17–20]

The purpose of this study was to further assess the potential impact of PVL on the clinical characteristics of SAB comparing the clinical manifestations of bacteremic episodes due to S. aureus isolates that did or did not have the lukF-PV and lukS-PV genes encoding PVL.

Methods

This study was approved by the University of Massachusetts Medical School institutional review board H00014429_2. Consent was not obtained as this is a retrospective study deemed minimal risk to subjects. Requirement for informed consent was waived by the UMass Chan Medical School IRB.

Between September 2016 and December 2019, a convenience sample of 1681 S. aureus isolates obtained from the UMass Memorial Medical Center clinical microbiology laboratory underwent comprehensive genomic sequencing as part of a proof-of-concept study on the utility of genomic sequencing in infection control surveillance [21].

To identify PVL-positive S. aureus isolates, WGS sequence data was first assembled (SPAdes [22]) and assembly metrics were applied to select isolates for inclusion (QUAST [23]; GC (%) between 30 and 33.5, contig N50 > 7500nt, # contigs (> = 1000 bp) < 500, and Total length (> = 1000 bp) between 2.5 and 3.0 Mbases. A BLAST database was created from all passing assemblies. A blastn [24] search against a data base created from all passing assemblies (parameters: -qcov_hsp_perc 80 -perc_identity 90) with a 1918nt query sequence containing the lukF-PV and lukS-PV genes (GenBank Accession CP039167; region 1513198..1515115) identified isolates as lukSF positive (PVL+).

For isolates included in the study, a core, single nucleotide variant (SNV) phylogeny was constructed. Variant calling was performed using Freebayes version v1.2.0-dirty [25] as deployed in the snippy pipeline, version 4.3.8 [26], using Staphylococcus aureus subsp. aureus NCTC 8325 (NC_007795.1) as reference. Custom scripts were used to determine core single nucleotide positions from the *snps.raw.vcf outputs and create a multi-fasta alignment. snp-sites [27] was then used to extract a core SNV alignment. FastTree2 (parameters: -gtr) version 2.1.10 [28] was used to generate a phylogenetic tree, which was then mid-point rooted.

Multi-locus sequence type assignments were determined from assemblies using mlst version 2.16.2 [29], making use of the PubMLST website (https://pubmlst.org/) developed by Keith Jolley [30] and sited at the University of Oxford. The development of that website was funded by the Wellcome Trust.

Case patients with one or more positive blood cultures for strains that were PVL+ were randomly matched with control patients having positive blood cultures for PVL-. A retrospective chart review was conducted via a review of the institution’s electronic health records assessing for demographics (age, gender), risk factors for SAB, clinical symptoms, concomitant infections (criteria per S1 File), severity of illness, laboratory markers of severity, clinical cure, all-cause mortality, loss to follow-up, length of hospitalization, duration of fever, duration of bacteremia, duration of ICU stay, and maximum temperature. Patients required post-hospitalization follow-up to be considered a clinical cure. Furthermore, determination of outcome was made based on index hospitalization records and follow up notes related to the index hospitalization.

Categorical variables were compared with chi-square or Fisher’s Exact tests. For continuous variables, Student’s t-test with Satterthwaite adjustment, when appropriate, and Wilcoxon rank-sum tests for non-normal distributions were used. To further delineate the epidemiology of these isolates, they were classified according to methicillin resistance status as a subset of antimicrobial resistance profiles, as well as analyzed per multi-locus sequence type (MLST)

This study was approved by the University of Massachusetts Medical School institutional review board H00014429_2

Results

From the pool of 1681 sequenced isolates, 56 patients with bacteremia due to PVL-negative S. aureus (controls) and 56 patients with bacteremia due to PVL-positive S. aureus (cases) had undergone analysis. Three of these case patients were removed due to multiple strains of S. aureus found in blood culture. Twelve control isolates and two case isolates that failed to meet assembly criteria were removed, and replaced randomly with twelve other control isolates, leading to a final analysis of 51 case patients matched with 56 randomly selected control patients. If a patient had multiple hospitalizations with bacteremia, the first encounter was chosen for analysis. The 107 case-control patients were comparable in age and gender, as well as most risk factors for bacteremia (Table 1). However, case patients were more likely to have a history of injection drug use, while controls more likely to undergo hemodialysis or have had indwelling IV catheters. Isolates from 78% of case patients were methicillin resistant as compared to 29% from control patients.

Table 1

Patient demographics by PVL′ Case/Control status.

	Case N = 51	Control N = 56	P-value 1	P-value 2**
Age at Collected Date (Mean ± SD, Range)	50.1 ± 23.3 (2.6–96.0)	55.3 ± 21.9 (0.1–100.1)	0.23	0.18
Methicillin Resistant	40(78.4%)	16(28.6%)	<0.001
Risk Factors (N, %)
Diabetes	5 (9.8)	15 (26.8)	0.05*
IVDU″	24 (47.1)	6 (10.7)	<0.001*
Immunosuppressed	7 (13.7)	5 (8.9)	0.54*
Immunosuppressive Medications	5 (9.8)	8 (14.3)	0.28*
Indwelling IV catheters	5 (9.8)	13 (23.2)	0.07*
Hemodialysis	2 (3.9)	11 (19.6)	0.02*

′Panton-Valentine Leukocidin.

″Intravenous Drug Use.

*P-values in P-value 1 column for categorical variables from chi-squared tests, unless indicated with an asterisk (*) where Fisher’s Exact Test was used; for continuous variables, p-values from Student’s t-test with Satterthwaite adjustment when appropriate.

**P-values in P-value 2 column from Wilcoxon rank-sum tests for non-normal distributions.

Case patients were more likely to have complained of chest pain and had more diagnoses of pneumonia and skin/soft tissue infection, with no differences seen in the incidence of endocarditis, osteomyelitis, or septic arthritis (Table 2).

Table 2

Patient symptoms and infections by PVL′ Case/Control status.

Signs & Symptoms (N, %)	Case N = 51	Control N = 56	P-value 1	P-value 2**
Fever	35 (63.4)	41 (73.2)	0.31
Chills	8 (15.4)	10 (17.8)	0.800.11<0.001
Abdominal Pain	8 (15.4)	3 (5.4)
Chest Pain	12 (23.1)	1 (2.0)
Localized Swelling	3 (5.7)	0 (0)	0.11*
Cough	4 (7.7)	5 (8.9)	0.99*
Infection Class (N, %)
Pneumonia	19 (36.5)	9 (16.1)	0.02
Endocarditis	12 (23.1)	7 (12.5)	0.21
Osteomyelitis	10 (19.2)	12 (21.4)	0.82
Septic arthritis	3 (5.8)	3 (5.4)	0.72*
Skin/soft tissue infection	22 (42.3)	13 (23.2)	0.04
Focal hepatic/splenic/renal abscess	1 (1.9)	0 (0.0)	0.48*

′Panton-Valentine Leukocidin.

*P-values in P-value 1 column for categorical variables from chi-squared tests, unless indicated with an asterisk (*) where Fisher’s Exact Test was used; for continuous variables, p-values from Student’s t-test with Satterthwaite adjustment when appropriate.

**P-values in P-value 2 column from Wilcoxon rank-sum tests for non-normal distributions.

Creatinine and alkaline phosphatase were higher among controls compared to cases (Table 3).

Table 3

Laboratory markers by PVL Case/Control Status.

Characteristic	CaseN = 51	ControlN = 56	P-value 1*	P-value 2**
White blood cell count (10^3 cells/μL), on date of culture (Mean ± SD, Range)	13.5 ± 5.3(4.9,28.3)	14.0 ± 7.3 (3.1–31.1)	0.65	0.0.97
White blood cell count (10^3 cells/μL), maximum (Mean ± SD, Range)	17. ± 7.1 (6.0–37.5)	17.3 ± 7.5 (4.3–38.4)	0.92	0.98
Creatinine (mg/dL) (Mean ± SD, Range)	1.4 ± 1.1 (0.3–5.9)	2.2 ± 2.8(0.3–16.7)	0.06	0.10
Alanine transaminase (U/L)(Mean ± SD, Range)	77.0± 253.9 (5.0–1626)	35.9 ± 41.6 (5.0–216.0)	0.31	0.56
Alkaline phosphatase (U/L) (Mean ± SD, Range)	85.6 ± 35.5 (23.0–190.0)	137.1 ± 104.4 (34.0–608.0)	0.005	0.02
T. Bili (mg/dL) (Mean ± SD, Range)	1.4 ± 2.2 (0.2–11.0)	3.7 ± 9.9(0.2–54.8)	0.19	0.12

The percentage of patients who were clinically cured or expired was comparable. No differences in length of stay nor illness severity (by markers such as sepsis or septic shock) had been found. Duration of fever was longer among cases, however, despite no difference in duration of bacteremia; controls had more positive prior cultures (Table 4).

Table 4

Clinical outcomes by PVL Case/Control Status.

Characteristic	Case N = 51	Control N = 56	P-value 1*	P-value 2**
Sepsis	5 (9.8)	17 (30.4)	16
Septic Shock	10 (19.6)	11 (19.6)
Previous positive culture (Mean ± SD, Range)	0.3 ± 0.6 (0.0–3.0)	1.5 ± 3.5 (0.0–14.0)	0.02	0.77
Duration of the bacteremia, days (Mean ± SD, Range)	3.4 ± 3.5 (1.0–15.0)	3.0 ± 2.8 (1.0–16.0)	0.57	0.73
Fever (⁰C), T MAX (Mean ± SD, Range)	39.0 ± 0.7 (37.3–41.1)	38.9 ± 0.5 (38.1–40.0)	0.91	0.64
Fever duration, days (Mean ± SD, Range)	4.8 ± 6.5 (0.0–29.0)	2.2 ± 1.9(1.0–7.0)	0.04	0.03
Length of hospital stay, days (Mean ± SD, Range)	15.4 ± 15.1 (0.0–89.0)	11.4 ± 7.7 (0.0–50.0)	0.74	0.10
Length of ICU stay, days (Mean ± SD, Range)	4.0 ± 5.8 (0.0–21.0)	3.6 ± 5.5(0.0–22.0)	0.72	0.91
Death Date Known (N, %)	8 (15.7)	10 (17.9)	0.97
Number of positive blood cultures (Mean ± SD, Range)	4.8 ± 4.5 (1.0–20.0)	4.6 ± 3.4(0.0–18.0)	0.72	0.61
Outcome of Infection (N, %)			0.59
Cured	25 (49.0	33 (58.9)
Expired	11 (21.6)	10 (17.9)
Unknown	15 (29.4)	13 (23.2)

′Panton-Valentine Leukocidin.

*P-values in P-value 1 column for categorical variables from chi-squared tests, unless indicated with an asterisk (*) where Fisher’s Exact Test was used; for continuous variables, p-values from Student’s t-test with Satterthwaite adjustment when appropriate.

**P-values in P-value 2 column from Wilcoxon rank-sum tests for non-normal distributions

The differences in proportions between cases and controls were tested among patients receiving vancomycin, vancomycin in the first five days, cefazolin or nafcillin, and cefazolin or nafcillin in the first five days using a chi-square test across the four treatment groups and one control group (Table 5). The number of cases receiving cefazolin or nafcillin were significantly less than the number of controls, padj 0.004, 95% CI [-0.53,-0.14], adjusting for multiple comparisons. The days of antibiotic therapy among cases and controls were tested as counts using a negative binomial regression model with treatment groups entered as a set of dummy (0,1) variables with the control group as the reference. No statistical difference in days of therapy was found (all p-values > 0.05).

Table 5

Antibiotics given by Case/Control status*.

	Case	Control	P-value* ‡
Average total days of antibiotic therapy	32.3	23.6	0.08‡
Average number of different antibiotics patients received	2.9	2.9	-
Number of patients receiving vancomycin	46	52	0.73*
Number of patients receiving vancomycin in first 5 days	41	39	0.29
Average Total days of vancomycin therapy	14.5	8.4	0.07 ‡
Number of patients receiving cefazolin or nafcillin	12	32	0.004 adjusted
Number of patients receiving cefazolin or nafcillin in first 5 days	8(15.7%)	13(23.2)	0.46

* The differences in proportions between cases and controls were tested among patients receiving vancomycin, vancomycin in the first five days, cefazolin or nafcillin, and cefazolin or nafcillin in the first five days using a chi-square test across the four treatment groups and one control group. The number of cases receiving cefazolin or nafcillin were significantly less than the number of controls, padj 0.004, 95% CI [-0.53,-0.14], adjusting for multiple comparisons. The days of antibiotic therapy among cases and controls were tested as counts using a negative binomial regression model‡ with treatment groups entered as a set of dummy (0,1) variables with the control group as the reference. No statistical difference in days of therapy was found (all p-values > 0.05).

Phylogenetic analysis revealed that PVL-positivity was not randomly distributed amongst the patient cohort. Staphylococcus aureus ST8 strains were overwhelmingly represented among PVL+ isolates, whereas PVL- isolates had a distribution of sequence types, with the plurality being ST5 (Fig 1).

Fig 1

Phylogenetic analysis of S. aureus strains.

Strains are identified by MicroSEQ ID_sequence type (ST). MRSA and PVL status along with selected clinical factors of interest are linked with strains. Legend: Strain/isolate numbers are listed as S###, followed by _ then sequence type (ST); PVL = Panton Valentine Leukocidin; IVDU = intravenous drug use; MRSA = methicillin resistant Staphylococcus aureus; CATH = presence of intravenous catheter; HEMO = on hemodialysis; Both PVL+ and PVL- strains included both methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) strains with the PVL+ isolates predominately MRSA. A high percentage of patients infected with strains from the larger PVL-positive cluster had a history of intravenous drug use (IVDU).

Subset analysis was performed to see if laboratory markers and clinical outcome differences (Table 6) were present between isolates from patients with a history of IVDU and those without. Though the primary outcome composite showed significant differences between IVDU and non-IVDU, the proportions of cured and expired illness, in light of the greater proportion of unknown outcomes among IVDU, were comparable.

Table 6

Patient outcomes by IVDU” History.

Characteristic	IVDU YesN = 30	IVDU NoN = 77	P-value 1	P-value 2**
Sepsis	2 (6.7)	20 (26.0)	0.41*
Septic Shock	4 (13.3)	17 (22.1)
Previous positive culture (Mean ± SD, Range)	0.8 ± 2.6(0.0–14.0)	0.9 ± 2.7(0.0–14.0)	0.30	0.84
Number of positive blood cultures (Mean ± SD, Range)	5.8 ± 4.91(0.0–20.0)	4.2 ± 3.5(1.0–18.0)	0.14	0.21
Fever (⁰C), T MAX (Mean ± SD, Range)	39.1 ± 0.8(37.9–41.1)	38.5 ± 0.5(37.3–40.1)	0.29	0.39
Fever duration, days (Mean ± SD, Range)	4.1 ± 5.4(0.0–25.0)	3.1 ± 4.4(0.0–29.0)	0.48	0.26
Length of ICU stay, days (Mean ± SD, Range)	2.8 ± 5.4(0.0–21.0)	4.2 ± 5.7(0.0–22.0)	0.790.29	0.84
Outcome of Infection
Cured	11 (36.7)	47 (61.0)	0.002
Expired	4 (13.3)	17 (22.1)		0.003
Unknown	15 (50.0)	13 (16.9)
Death Date Known (N, %)	4 (3.7)	14(13.1)	0.75	0.77

″Intravenous Drug Use

*P-values in P-value 1 column for categorical variables from chi-squared tests, unless indicated with an asterisk (*) where Fisher’s Exact Test was used; for continuous variables, p-values from Student’s t-test with Satterthwaite adjustment when appropriate.

**P-values in P-value 2 column from Wilcoxon rank-sum tests for non-normal distributions.

Those with IVDU tended to be younger, with an equal number of men and women, and have far less prevalence of diabetes, indwelling IV catheters, hemodialysis, and immunosuppression (Table 7).

Table 7

Patient demographics by IVDU” history.

Characteristic	IVDU YesN = 30	IVDU NoN = 77	P-value 1	P-value 2**
Age at Collected Date (Mean ± SD, Range)	40.2 ± 13.8(21.8–73.2)	58.8 ± 23.7(0.1–100.1)	< .0001	< .0001
Risk Factors (N, %)
Diabetes	1 (3.3)	19 (24.7)	0.02
Immunosuppressive Medications	0 (0.0)	11 (14.3)	0.02*
Indwelling IV catheters	2 (6.7)	16 (20.8)	0.14
Hemodialysis	0 (0.0)	13 (16.9)	0.02*
Genetic virulence factors (N, %)
PVL Positive (Case)	24 (80.0)	27 (35.1)	0.00003*

″Intravenous Drug Use.

*P-values in P-value 1 column for categorical variables from chi-squared tests, unless indicated with an asterisk (*) where Fisher’s Exact Test was used; for continuous variables, p-values from Student’s t-test with Satterthwaite adjustment when appropriate.

**P-values in P-value 2 column from Wilcoxon rank-sum tests for non-normal distributions.

Discussion

This retrospective, case-control study was conducted primarily to explore possible differences in clinical outcomes for SAB associated with the presence of PVL exotoxin. Secondarily, clinical and laboratory markers of severity, and epidemiologic data such as strains, comorbidities, and risk factors were compared.

Case patients were found to have longer duration of fever but there was no difference in duration of bacteremia, number of positive blood cultures, rates of cure, all-cause mortality, or rates of unknown outcomes between case and control isolates. Furthermore, there were no differences in duration of bacteremia or length of hospitalization. Deep-seated infections such as osteomyelitis, septic arthritis, and endocarditis were not found more often in the case patients, although there was approximately double the percentage of pneumonia and skin and soft tissue infections (SSTI) in case patients.

Differences in mortality (as part of the primary composite clinical outcome) were not seen in this case-control format study specifically evaluating patients with PVL-positive SAB against those with PVL-negative SAB. The data from other studies generally have not found a consistent difference in mortality, which is consistent with what has been usually observed in studies evaluating clinical outcomes and the presence of PVL.

One retrospective study in a Greek hospital [31] found no relationship between PVL and fatality in 32 isolates that spanned all types of invasive disease due to S. aureus. Another retrospective, 10-year study at a single center in the Gambia found that length of hospital stay and mortality were not associated with PVL status among 29 isolates from SAB [32]. Knudsen et al. found, among 129 PVL-positive SAB cases from a pool of 9490 total cases of SAB, no difference in all-cause 30-day mortality was seen in the unadjusted analysis [33]. Wehrhahn et al. found no difference in 30-day mortality when directly comparing PVL-positive with PVL-negative isolates from SAB [34].

On the contrary, Knudsen et al. in the same study found an association between higher 30-day mortality and PVL-positive status, after adjusting for illness severity and older age, and cited a 1.66 (1.16–2.38) adjusted hazard ratio [33]. Seybold et al. found a non-statistically significant lower crude in-hospital mortality in patients with SAB and PVL-positive status linked to USA300; mortality, however, was not seen to be lower in the univariate nor multivariate analyses. This study also did not find a difference in length of stay [35].

As there was a difference in the relative percentage of MRSA and MSSA strains between the cases and controls, a difference in the antibiotics used for SAB treatment would be anticipated and was found in our patient populations. As expected, case patients in our study had received more average days of vancomycin compared to controls, though more control patients had received vancomycin in the first five days. Despite the suggestion that vancomycin may be an inferior anti-staphylococcal antibiotic compared to semisynthetic penicillins or cephalosporins [36], case patients did not show worse outcomes compared to controls.

Case patients did tend to be younger and have IVDU compared to controls, though the subset analysis of IVDU did not suggest lower mortality associated with this risk factor. Many of these patients, compared to non-IVDU, had unknown follow up (not shown in data), rendering some degree of associated mortality and outcomes unknown.

The lack of difference in mortality may be in part attributable in part to bacterial genotypic/phenotypic factors. Hamilton et al. found varying levels of in vitro PVL toxin production from S. aureus isolates taken from patients with SSTI, pneumonia, and bacteremia; no correlation between disease severity and PVL production was found [37]. In a mouse model of S. aureus sepsis and skin abscess, infection with PVL genetic knockout strains compared to PVL+ strains did not produce differences in survival after bloodstream infection [38]. In fact, this study found that several PVL-negative strains were either more virulent or comparably lethal as genetically similar PVL positive strains, and that there was identical lysis of neutrophils in wild type and PVL knockout strains [38]. Badiou et al. found varying levels of PVL production in vitro according to sequence type, without a difference in production with regard to mecA-positivity. PVL+ isolates taken from patients with invasive infections spanning skin infections, pneumonia, and bone and joint infections were found to have 90% producing toxin concentrations deemed toxic to human leukocytes [39]. Other proteins related to virulence such as alpha-toxin, those regulated by the agr locus, the arginine catabolic mobile element (ACME) [1] have been implicated in virulence.

The predominance of MRSA compared to MSSA among the cases mirrors that found in a longitudinal, multi-year sample of about 1000 American isolates from multiple body sites, with PVL prevalence associated with MRSA being about 8 times that of MSSA, a trend seen across SSTI, bacteremia, lower respiratory tract infection, and other infections. [40] In that study, almost all isolates, regardless of methicillin resistance, were of the CC8 MLST, a combination identified as strain USA300. However, there were few bloodstream isolates. Case strains tended to be almost all ST8, compared to much greater heterogeneity of the control strains, with the plurality of the latter being ST5. This preponderance of ST8 among PVL-positive MRSA isolates reflects the successful dissemination of the USA300 MRSA strain in North America [1]; MSSA with the PVL locus have been characterized as highly diverse [41], though with genetic similarity to MRSA with PVL as well [41-43]. It has been theorized that PVL-positive CA-MRSA have arisen from PVL-positive MSSA strains [41, 44].

Strengths of this study include a case-control perspective focused on PVL exposure in SAB, the relatively high number of unique isolates, the WGS tracking of strain types. Some studies have explored PVL and clinical outcomes in the context of all invasive staphylococcal disease, with SAB as only a subset. Even among the studies that specifically evaluate PVL status and SAB, this study further evaluates clinically relevant parameters such as duration of fever, duration of bacteremia, and length of stay. Limitations include the retrospective study design, the relatively overall small sample size, the relatively high proportion of patients with unclear outcome of infection, analysis of patients from only a single academic medical center, the use of multiple different antibiotic regimens for SAB treatment, and a relatively high number of patients with active IVDA which could impact patient follow up as well as outcomes including hospital LOS. treat.

This retrospective study examining differences in risk factors and clinically relevant outcomes (ie: sepsis, death, duration of bacteremia, length of stay, duration of fever) among patients with SAB who had infection due to S. aureus isolates with and without PVL found only a longer duration of fever without differences in length of stay, duration of bacteremia, nor degree of bacteremia.

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors performed a retrospective case-control study to compare clinical characteristics and outcome of PVL+ and PVL- S. aureus bacteremia. The premise of the study is interesting, since PVL has been associated with SSTI and pneumonia but its relevance in S. aureus bacteremia (SAB) is still unclear. In total, 52 case and 56 control patients were included and analyzed. The authors concluded that the clinical cure was comparable and that the presence of PVL does not have any significant effect on the clinical course of SAB.

In order to assess the methodological quality of the paper, some missing information needs to be provided/clarified.

Major comments:

• In the selection of the case/control, was the antibiotic taken into account? The (optimal) treatment for MRSA SAB and MSSA SAB are not the same. MRSA=vancomycin and MSSA=beta-lactams/oxacillin. That being said, the proportion of MRSA/MSSA in both case and control groups were unclear. This information (and statistics) should be included in the tables. This is an essential information as vancomycin has been demonstrated to be inferior in terms of clinical cure (which is the outcome in the study) than oxacillin for treating MSSA SAB.

• In the US, most PVL+ strains are MRSA (ST8 USA300). Therefore, PVL- MRSA controls would have been the best choice of control group (or at least equal proportions of MRSA/MSSA). In the study 77% of the cases were MRSA and only 29% of the control was MRSA. Did all patients in both groups receive the same antibiotic treatment (vancomycin)?

• Tables are not intuitive. For example, table 6. How were the categories for vancomycin determined? Were these vancomycin plasma levels? If yes, units are missing and please state so. I assume that N=number of patients (e.g. for Vancomycin categories). It was unclear how the numbers in the column should add up (Total IVDU users N=30; but VAN <0.5 N=27; 1 N=38 and 2 N=5 giving a total of N=70)? Similar to outcome of infection (16+4+16 does not add up to N=30/column total). Please check your tables and numbers. Missing values and others should be included in the tables.

• These points should be clarified before other aspects of the study can be assessed.

Minor points

• Line 58: gene in italics

• Line 62: please cite the correct papers. Papers cited did not show the increase of ST8-USA300 in the community worldwide! (e.g. PMID: 30315958, 34160741, 26884428, 26464204, 33636329, etc..)

• Line 67: please cite the correct papers. Pneumonia PMID: 10524952, SSTI/Abscesses PMID: 25753191.

• Line 100: bacterial genus/species in italics

• Line 156: explain the coding of vancomycin

• Lines 211: length of stay, fever and other clinical parameters can only be assessed if information on the antibiotic therapy was provided (choice of antibiotic has an effect on the clinical course)

Reviewer #2: In the manuscript “Epidemiological and clinical features of Panton-Valentine Leukocidin positive Staphylococcus aureus bacteremia: a case-control study” authors describe the possible differences in clinical outcomes for SAB associated with the presence and absence of the PVL exotoxin. The manuscript presents interesting data, it is well structured and the writing quality is good.

Minor comments:

1. Line 39: correct percentage of MRSA in case patients

2. Please correct the order of tables (table #6 is after the table #3, and #4 is after #6)

3. Line 243: correct spelling of “however”

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Reviewer #1: Yes: Dennis Nurjadi

Reviewer #2: No

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3 Feb 2022

Dear Dr. Bae,

My coauthors and I would like to thank the editors and reviewers for their thoughtful review of our manuscript entitled “Epidemiological and clinical features of Panton-Valentine Leukocidin positive Staphylococcus aureus bacteremia: a case-control study”( PONE-D-21-28267). We have now made a number of revisions to the manuscript to address the reviewer’s concerns and have attached both a clean version of the revision as well as a red-lined version showing all the revisions made to the original text.

In addition, we would like to address each of the concerns raised by the reviewers.

Reviewer #1

Major comments:

• In the selection of the case/control, was the antibiotic taken into account? The (optimal) treatment for MRSA SAB and MSSA SAB are not the same. MRSA=vancomycin and MSSA=beta-lactams/oxacillin. That being said, the proportion of MRSA/MSSA in both case and control groups were unclear. This information (and statistics) should be included in the tables. This is an essential information as vancomycin has been demonstrated to be inferior in terms of clinical cure (which is the outcome in the study) than oxacillin for treating MSSA SAB.

In our selection of controls we did not include an adjustment for antibiotic therapy given that this was a retrospective real world review and the patient’s received multiple different antibiotic regimens initially and had multiple different subsequent modifications in antibiotic regimens. We appreciate the reviewer’s comment on the importance of providing this information, and we have now added this data to table 1and to a new table providing information on the antibiotic therapy received by the patients.

• In the US, most PVL+ strains are MRSA (ST8 USA300). Therefore, PVL- MRSA controls would have been the best choice of control group (or at least equal proportions of MRSA/MSSA). In the study 77% of the cases were MRSA and only 29% of the control was MRSA. Did all patients in both groups receive the same antibiotic treatment (vancomycin)?

We appreciate the reviewer’s suggestion about matching cases based on whether the case isolate was either MRSA or MSSA. However, our initial study plan was to solely assess the impact of PVL alone independent of the presence or absence of the mecA gene. In addition, the predominant use of vancomycin as opposed to a beta-lactam agent for MRSA infections would potentially have led to a worse clinical outcome for patients infected with a PVL+ isolate. This was not the finding of the study, and consequently we have chosen not to reselect a new control population for the study or to restrict our analysis to the PVL-/MRSA strains in our original control population. However, we have now added a description of this issue to the discussion section of our manuscript on lines 249 – 256.

• Tables are not intuitive. For example, table 6. How were the categories for vancomycin determined? Were these vancomycin plasma levels? If yes, units are missing and please state so. I assume that N=number of patients (e.g. for Vancomycin categories). It was unclear how the numbers in the column should add up (Total IVDU users N=30; but VAN <0.5 N=27; 1 N=38 and 2 N=5 giving a total of N=70)? Similar to outcome of infection (16+4+16 does not add up to N=30/column total). Please check your tables and numbers. Missing values and others should be included in the tables.

In re-reviewing the data in the tables to make certain that the results have been accurately reported we found that one there was one individual included in who had

simultaneous PVL+ and PVL- S. aureus bloodstream isolates; and this case has now been excluded from the study. This change has led to minor adjustments in results.

Data on follow up details were removed, as were vancomycin mic values; neither of these sets of data was felt to be relevant to this analysis.

Minor points

• Line 58: gene in italics This has been corrected

• Line 62: please cite the correct papers. Papers cited did not show the increase of ST8-USA300 in the community worldwide! (e.g. PMID: 30315958, 34160741, 26884428, 26464204, 33636329, etc..) Reviewing literature and adjusting citations

• Line 67: please cite the correct papers. Pneumonia PMID: 10524952, SSTI/Abscesses PMID: 25753191. Reviewing literature and adjusting citations

• Line 100: bacterial genus/species in italics This has been corrected

• Line 156: explain the coding of vancomycin Vancomycin mic no longer included in study.

• Lines 211: length of stay, fever and other clinical parameters can only be assessed if information on the antibiotic therapy was provided (choice of antibiotic has an effect on the clinical course) We have incorporated a discussion of the impact of antibiotic therapy on these outcomes, as well as a table.

Reviewer #2

In the manuscript “Epidemiological and clinical features of Panton-Valentine Leukocidin positive Staphylococcus aureus bacteremia: a case-control study” authors describe the possible differences in clinical outcomes for SAB associated with the presence and absence of the PVL exotoxin. The manuscript presents interesting data, it is well structured and the writing quality is good.

We thank the reviewer for his comments

Minor comments:

1. Line 39: correct percentage of MRSA in case patients this has been corrected

2. Please correct the order of tables (table #6 is after the table #3, and #4 is after #6) this has been corrected

3. Line 243: correct spelling of “however” this has been corrected

________________________________________

Submitted filename: Response to Reviewers draft Jan 5 2022.docx

Click here for additional data file.

3 Mar 2022

Epidemiological and clinical features of Panton-Valentine Leukocidin positive Staphylococcus aureus bacteremia: a case-control study

PONE-D-21-28267R1

Dear Dr. Qu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Carla Pegoraro

Division Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: thank you for addressing the reviewers' comments, no further comments or concerns for the revised manuscript from my side.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

10 Mar 2022

PONE-D-21-28267R1

Epidemiological and clinical features of Panton-Valentine Leukocidin positive Staphylococcus aureus bacteremia: a case-control study

Dear Dr. Qu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

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Staff Editor

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