Panton-Valentine Leukocidin Genes Are Associated With Enhanced Inflammatory Response and Local Disease in Acute Hematogenous Staphylococcus aureus Osteomyelitis in Children

ARTICLE

Panton-Valentine Leukocidin Genes Are Associated

With Enhanced Inflammatory Response and Local

Disease in Acute Hematogenous

Staphylococcus aureus

Osteomyelitis in Children

Claire E. Bocchini, MD, Kristina G. Hulten, PhD, Edward O. Mason, Jr, PhD, Blanca E. Gonzalez, MD, Wendy A. Hammerman, RN, Sheldon L. Kaplan, MD

Department of Pediatrics, Baylor College of Medicine, Houston, Texas; Texas Children’s Hospital, Houston, Texas

Financial Disclosure: Dr Kaplan has received a grant from Pfizer.

ABSTRACT

BACKGROUND.Staphylococcus aureusstrains carrying the genes encoding Panton-Val-entine leukocidin (pvl-positive [pvl⫹]) are associated with more febrile days and higher complication rates of osteomyelitis in children than arepvl-negative (pvl⫺) strains.

OBJECTIVES.Selected clinical, laboratory, and radiographic findings in children with osteomyelitis caused bypvl⫹andpvl⫺S aureusstrains were compared.

METHODS.The demographics, selected clinical features, laboratory values, and radio-graphic findings of children with community-acquired S aureus osteomyelitis prospectively identified at Texas Children’s Hospital between August 2001 and July 2004 were reviewed. Polymerase chain reaction was performed to detect the genes forpvl(luk-S-PVandluk-F-PV) and fibronectin-binding protein (fnbB) inS aureusisolates.␹2_{, 2-samplettest, and multiple logistic regression were used for} statistical analysis.

RESULTS.Methicillin-susceptible and methicillin-resistant S aureus (MSSA and MRSA, respectively) caused osteomyelitis in 33 and 56 children, respectively. Twenty-six isolates werepvl⫺(26 MSSA), 59 werepvl⫹(3 MSSA, 56 MRSA), and 4 were not available for analysis (4 MSSA). On univariate analysis, patients with

pvl⫹S aureusisolates had significantly higher erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) level both at presentation and as a maximum value during hospitalization and were more likely to have a blood culture positive forS aureusduring their admission. Patients withpvl⫹S aureusisolates were significantly more likely to have concomitant myositis or pyomyositis compared with patients withpvl⫺S aureusisolates on MRI. In a multivariate analysispvlremained signif-icantly associated with ESR and CRP levels at presentation and blood culture

www.pediatrics.org/cgi/doi/10.1542/ peds.2005-0566

doi:10.1542/peds.2005-0566

Key Words

Staphylococcus aureus,Panton-Valentine leukocidin, osteomyelitis

Abbreviations

AHO—acute hematogenous osteomyelitis MRSA—methicillin-resistantS aureus

CA— community acquired MSSA—methicillin-susceptibleS aureus

TCH—Texas Children’s Hospital PVL—Panton-Valentine leukocidin ESR— erythrocyte sedimentation rate CRP—C-reactive protein WBC—white blood cell ANC—absolute neutrophil count

Accepted for publication Apr 20, 2005 Address correspondence to Sheldon L. Kaplan, MD, Texas Children’s Hospital, Mail Code 3-2371, 6621 Fannin St, Houston, TX 77030. E-mail: [email protected]

positive forS aureus.pvl⫹status and younger age were associated with myositis on MRI.

CONCLUSIONS.Osteomyelitis caused bypvl⫹S aureusstrains were associated with more severe local disease and a greater systemic inflammatory response compared with osteomyelitis caused bypvl⫺S aureus.

S

hematogenous osteomyelitis (AHO) in otherwise healthy children, accounting for 70% to 90% of cases.1,2

Community-acquired (CA) methicillin-resistantS aureus

(MRSA) infections are now common in many areas of the United States.3–9_{At Texas Children’s Hospital (TCH),}

MRSA accounted for 76% of CA S aureusisolates from August 1, 2003, to July 31, 2004.8_{Although the}

spec-trum of illnesses caused by CA-MRSA remains primarily skin and soft tissue infections, similar to that of CA methicillin-susceptible S aureus (MSSA), over the past decade CA-MRSA has been increasingly responsible for invasive infections with substantial morbidity and mor-tality in children.5–9 _{From August 1, 2001, to July 31,}

2004, at TCH, 117 CA-MRSA isolates were recovered from children with invasive infections, the most com-mon of which was osteomyelitis.8

Multiple virulence factors have been identified as im-portant to the pathogenesis ofS aureus infections. One such factor is Panton-Valentine leukocidin (PVL), a bi-component cytotoxin that destroys leukocytes by pore-forming activity and is encoded by pvl genes,luk-S-PV

and luk-F-PV.10,11 _{PVL is a member of the}

synergohy-menotropic family of exotoxins that destroy leukocytes by creating pores in the cell membrane. In vivo studies have shown that PVL injected intradermally into rabbits caused severe inflammatory lesions including capillary dilation, chemotaxis, polymorphonuclear infiltration, polymorphonuclear karyorrhexis, and skin and tissue necrosis.12 _{Clinically, PVL has been linked to a severe}

form of necrotizing pneumonia and necrotic skin lesions such as furuncles and carbuncles.10,11,13–15

Thepvlgenes are more common in CA-MRSA isolates compared with CA-MSSA isolates,7,13,16,17 _{and a recent}

molecular study at TCH showed the presence of pvl in almost all of the CA-MRSA strains.18_{More than 90% of}

the CA-MRSA isolates at TCH belong to 1 clone, identi-cal or closely related to the USA300 clone (Centers for Disease Control and Prevention)18,19_{that currently}

pre-dominates in many areas of the United States.18–21 _The

predominant (USA300) clone at TCH carriespvl as well as fnbB encoding S aureus fibronectin-binding factor.18

Fibronectin-binding proteins FnBPA and FnBPB, en-coded byfnbAandfnbB, are involved in the adherence of

S aureus to fibronectin and have been associated with enhanced bacterial adhesion and invasion.22–25

Although there is no evidence that the presence of

PVL genes confers a greater risk of AHO, we have re-ported that children with osteomyelitis caused byS au-reus isolates carrying the pvl genes (pvl⫹) experienced more febrile days and had a greater complication rate than patients with pvl-negative (pvl⫺) isolates.7 _{To our}

knowledge, other clinical features of children with AHO caused bypvl⫹versuspvl⫺S aureushave not been char-acterized. In this study, we compared selected laboratory and radiographic findings in children with AHO caused bypvl⫹andpvl⫺S aureusand also investigated the role of

fnbBas an independent factor contributing to these find-ings.

METHODS

Patient Identification and Medical Chart Review

From August 2001 to July 2004, all S aureus strains isolated by the TCH clinical microbiology laboratory were collected as part of prospective surveillance. A standardized form including antibiotic susceptibility and patient demographic and diagnostic information was filled out for each S aureusstrain, and this information was entered into a computerized patient database. For this study, all patients who were diagnosed with AHO caused by S aureus were selected, and their medical charts were reviewed. The diagnosis of AHO was made via a consistent clinical presentation (patients had no history of penetrating trauma or overlying infection at the site of the osteomyelitis) and radiographic evidence onⱖ1 of the following: plain film, computed tomogra-phy, radioisotope scan, or MRI. All of the patients had a positive culture from bone biopsy and/or blood culture for S aureus. Only patients with CAS aureus infection were included in this study. The strains were classified as described previously8,18_{and in accordance with Centers}

for Disease Control and Prevention classifications.26

tomography scan) from the medical charts. The Baylor College of Medicine Institutional Review Board ap-proved this study.

Laboratory Methods

S aureusIdentification

Isolates were identified as S aureus by standard meth-ods.27_{Screening for methicillin resistance was performed}

by using Clinical and Laboratory Standards Institute guidelines.28

Detection of Genes EncodingS aureusVirulence Factors

DNA was extracted from all available S aureus strains, and polymerase chain reaction was completed to detect thepvl(luk-S-PVandluk-F-PV) andfnbBgenes. Primers and polymerase chain reaction conditions have been reported previously.7,18

Statistical Analysis

Student’sttest,␹2_{analysis, log-likelihood ratio test, and} multivariate analysis using logistic regression were used for statistical analysis. All analyses were 2-tailed, andP⬍.05 was considered statistically significant. Sta-tistical analysis was performed by using True Epistat, fifth edition (Epistat Services, Richardson, TX).

RESULTS

Demographic Analysis

During the study period of August 2001 to July 2004, 89 patients were identified as having AHO caused by CA

S aureus. Thirty-three of these patients had CA-MSSA, and 56 had CA-MRSA. With the exception that the proportion of cases caused by CA-MSSA was greater in Hispanic children and the proportion of cases caused by CA-MRSA infection was greater in black children (P ⫽ .007), no significant differences were found in the demographic characteristics between the children with CA-MSSA compared with CA-MRSA osteomyelitis (Table 1).

Twenty-six S aureusisolates werepvl⫺(26 MSSA, 0 MRSA), and 59 were pvl⫹ (3 MSSA, 56 MRSA; P ⬍

.000001); 4 MSSA isolates were not available for molec-ular analysis. Related to the differences in distribution of CA-MRSA versus CA-MSSA isolates among the children of different races, pvl⫹ S aureus was less likely to be isolated from Hispanic children but more likely to be isolated from black children (P⫽.01). The mean age of the children with pvl⫹isolates was significantly greater than for those whose isolates were pvl⫺ (P ⫽ .046) (Table 2).

Clinical and Laboratory Features of Patients With Osteomyelitis Caused bypvlⴙandpvlⴚS aureusIsolates

Patients with osteomyelitis caused by pvl⫹ S aureus

(15.3%) were more likely to have multiple sites of

os-teomyelitis compared with patients with pvl⫺ S aureus

(3.8%), although this difference was not statistically sig-nificant. For patients with a single site of infection, pa-tients withpvl⫹andpvl⫺osteomyelitis had similar loca-tions of infection. There was no difference between the

pvl⫹andpvl⫺patients with regard to receiving antibiotics before diagnosis or in the duration of symptoms before diagnosis. Surgical interventions (including both aspira-tion and incision-and-drainage procedures) were per-formed in a high percentage of patients in both thepvl⫹

andpvl⫺patient groups (Table 3).

pvl⫹ S aureus isolates (18.6%) were more likely to cause severe infection that required care in the ICU than were pvl⫺isolates (3.8%) (P ⫽.01). Patients withpvl⫹ S aureusisolates had a significantly higher ESR and CRP level, both at presentation (P⫽.0008 andP⬍.000002, respectively) and as a maximum value during hospital-ization (P⬍.0000001 andP⬍.0000001, respectively). Likewise,pvl⫹patients had higher WBC counts (P⫽.03) and ANCs (P ⫽ .002) on presentation and were more likely to have a blood culture positive forS aureusduring their admission (P⫽.0001) (Table 3).

TABLE 1 Demographic Characteristics of Children With CA-MSSA or CA-MRSA Osteomyelitis at TCH: August 2001 to July 2004

Demographics MRSA

(n⫽56)

MSSA (n⫽33)

P

Age NS

Mean⫾SD, mo 119.0⫾53.8 97.2⫾55.6 Median (range), mo 133.5 (9–215) 104.0 (1–182)

Gender,n(%) NS

Male 40 (71.4) 19 (57.6)

Race,n(%) .007

White 24 (42.9) 14 (42.4)

Black 19 (33.9) 3 (9.1)

Hispanic 13 (23.2) 16 (48.5)

NS indicates not significant.

TABLE 2 Demographic Characteristics of Children With

Osteomyelitis Caused bypvlⴙ_andpvlⴚ_{S aureusIsolates}

at TCH: August 2001 to July 2004 Clinical Characteristic pvl⫹

(n⫽59)

pvl⫺ (n⫽26)

P

Isolate,n(%) ⬍.000001

MRSA 56 (94.9) 0 (0.0)

MSSA 3 (5.1) 26 (100.0)

Age .046

Mean⫾SD, mo 120⫾52.8 95⫾57.6 Median (range), mo 136 (9–215) 103 (1–182)

Gender,n(%) NS

Male 41 (69.5) 15 (57.7)

Race,n(%) .01

White 25 (42.4) 11 (42.3)

Black 20 (33.9) 2 (7.7)

Hispanic 14 (23.7) 13 (50.0)

Radiographic Findings in Patients With Osteomyelitis Caused bypvlⴙandpvlⴚS aureusIsolates

Patients with osteomyelitis caused by pvl⫹ and pvl⫺ S aureus had similar radiographic studies during their initial diagnostic workup. Seventy-six patients (24pvl⫺, 48pvl⫹, 4pvl-unknown) had a plain film, 9 (2 pvl⫺, 6 pvl⫹, 1 pvl-unknown) of which revealed early stages of osteomyelitis. Twenty-seven patients (6 pvl⫺, 18 pvl⫹, 3 pvl-unknown) had a radioisotope scan, 16 (3pvl⫺, 12pvl⫹, 1pvl-unknown) of which were positive for osteomyelitis. Sixty-eight patients (19pvl⫺, 45

pvl⫹, 4pvl-unknown) had an MRI, 67 (18pvl⫺, 45pvl⫹, 4

pvl-unknown) of which had a positive study.

Of the patients who had an MRI as part of their initial diagnostic workup, the patients with pvl⫹ S aureus isolates were significantly more likely to

have concomitant myositis or pyomyositis (P ⫽ .05) and tended to have more subperiosteal or intraosseal abscesses (P⫽.06) compared with patients withpvl⫺ S aureusisolates (Table 3).

Laboratory and Radiographic Features of Patients With Osteomyelitis Isolated to the Femur, Tibia, or Fibula Caused by pvlⴙandpvlⴚS aureus

Because the patients with infections caused by pvl⫹ S aureus isolates included more children with multifocal disease and more children requiring ICU care compared with the patients withpvl⫺S aureus, patients with osteo-myelitis isolated to the femur, tibia, or fibula who did not require ICU care were analyzed separately. Fifty-one patients had osteomyelitis confined to the femur, tibia, or fibula, 35 of whom hadpvl⫹S aureusand 16 of whom

TABLE 3 Clinical, Laboratory, and Radiographic Features of Children Withpvlⴙ_andpvlⴚ_{AcuteS aureus}

Osteomyelitis

Characteristic pvl⫹

(n⫽59)

pvl⫺ (n⫽26)

P

Single site of infection,n(%) 50 (84.7) 25 (96.2) .17

Multiple sites of infection,n(%) 9 (15.3) 1 (3.8)

Location of single-site osteomyelitis,n(%)a _NS

Femur 15 (25.4) 6 (23.1)

Tibia/fibula 20 (33.9) 10 (38.5)

Otherb _{15 (25.4) 9 (34.6)}

Antibiotics prior to diagnosis,n(%) 18 (30.5) 6 (23.1) NS

Duration of symptoms prior to diagnosis, d 6.0 6.1 NS

Surgical intervention,n(%) 54 (91.5) 22 (84.6) NS

ICU treatment,n(%) 11 (18.6) 1 (3.8) .01

ESR at presentation .0008

n 56 25

Mean⫾SD, mm/h 80⫾28.7 60⫾21.8

Median (range), mm/h 88 (4–140) 62 (15–97)

Maximum ESR ⬍.0000001

n 59 26

Mean⫾SD, mm/h 104⫾27.8 68⫾26.5

Median (range), mm/h 112 (4–140) 70 (15–140)

CRP level at presentation .000002

n 49 20

Mean⫾SD, mg/dL 23.1⫾18.1 7.1⫾7.1

Median (range), mg/dL 20 (0.7–79.4) 5.7 (1.1–35.2)

Maximum CRP level ⬍.0000001

n 53 23

Mean⫾SD, mg/dL 24.6⫾16.8 7.5⫾6.7

Median (range), mg/dL 23.8 (0.8–79.4) 6.4 (1.1–35.2)

WBC count at presentation .03

n 59 26

Mean⫾SD, cells⫻103_/mm3 _{15.1⫾6.6 11.6⫾6.4}

Median (range) 13.8 (4.07–39.82) 9.5 (5.25–32.27)

ANC at presentation .002

n 59 26

Mean⫾SD, cells⫻103_/mm3 _{12.1⫾6.4 7.5⫾4.9}

Median (range) 10.9 (2.89–37.43) 5.2 (1.82–21.3)

Blood culture positive forS aureusisolates,n/N(%) 39/58 (67.2) 5/26 (19.2) .0001 Surrounding myositis/pyomyositis on MRI,n/N(%) 28/45 (62.2) 6/19 (31.6) .05 Subperiosteal/intraosseal abscess on MRI,n/N(%) 34/45 (75.6) 9/19 (47.4) .06

NS indicates not significant.

a_{Patients with unknownpvlstatus (n⫽4): pelvis (2), toe, and femur.}

had pvl⫺ S aureus. Four of these patients (all pvl⫹) re-quired ICU care and were excluded from this analysis.

The patients withpvl⫹S aureusisolates had a signifi-cantly higher ESR (P⫽.001), CRP level (P⫽.006), and ANC (P ⫽ .004) at presentation, as well as maximum ESR (P⬍.0000001) and CRP level (P⫽.0004) during hospitalization. In addition, the patients withpvl⫹S au-reus compared with patients with pvl⫺ S aureus were significantly more likely to have a blood culture positive forS aureus(70.0% vs 12.5%, respectively; P⫽.0004) and tended to have more subperiosteal/intraosseal ab-scesses on MRI (72.0% vs 38.5%; P ⫽ .1) than the patients withpvl⫺S aureusisolates. There was no signif-icant difference in the incidence of surrounding myositis on MRI between patients with pvl⫹ and pvl⫺ S aureus

isolates (64.0% vs 61.5%) or in the WBC count on admission (P⫽.08).

Laboratory and Radiographic Features of Patients With Osteomyelitis Caused byfnbBⴙandfnbBⴚS aureus

Nineteen S aureus isolates were fnbB⫺ (4 MRSA, 15 MSSA; 5pvl⫹, 14pvl⫺), and 66 isolates werefnbB⫹(52 MRSA, 14 MSSA; 54pvl⫹, 12pvl⫺). Four MSSA isolates were not available forfnbBanalysis. MRSA isolates (P⫽

.000006) and pvl⫹ isolates (P ⫽ .00002) were signifi-cantly more likely to be fnbB⫹ than MSSA and pvl⫺

isolates, respectively. There were no demographic differ-ences between patients with fnbB⫹ and fnbB⫺ isolates. Patients withfnbB⫹isolates were not significantly more likely to require ICU care than the patients withfnbB⫺

isolates (16.7% vs 5.3%, respectively).

When the laboratory and radiographic findings of patients withfnbB⫹S aureusstrains were compared with

those of the patients withfnbB⫺S aureus strains, there were no significant differences in the ESR, CRP level, WBC count, or ANC at presentation, the maximum CRP level during admission, incidence of S aureus–positive blood cultures, or radiographic evidence of myositis or subperiosteal/intraosseal abscess. Only the maximum ESR during hospital admission varied significantly by

fnbBstatus, withfnbB⫹patients having higher maximum ESR values thanfnbB⫺patients (P⫽.03) (Table 4).

Multivariate Analysis

Age, gender, race, duration of symptoms, prior antibiotic therapy, methicillin susceptibility, pvl status, and fnbB

status were included in a multivariate analysis to deter-mine which factors correlated independently with our laboratory and radiographic parameters. Only the pres-ence ofpvlremained significantly independently associ-ated with an ESR of ⬎75 mm/hour at presentation (P⫽.002) and maximum value during admission (P⫽

.000002), CRP level of ⬎10 mg/dL at presentation (P⫽.0003) and maximum value during admission (P⫽

.00002), and blood culture positive for S aureus (P ⫽

.0002).pvl⫹status (P⫽.01) and younger age (P⫽.02) were associated with myositis on MRI. Methicillin resis-tance remained significantly associated with subperios-teal/intraosseal abscesses on MRI (P⫽.01).

DISCUSSION

PVL is an S aureus exotoxin that has been linked to necrotic skin lesions and severe necrotizing pneumo-nia.10,11,13–15_{In 1999, Lina et al}13_{reported a study of 172}

clinicalS aureusisolates, only 37% of which werepvl⫹. When the isolates were categorized according to disease,

TABLE 4 Laboratory and Radiographic Features of Children WithfnbBⴙ_andfnbBⴚ_{AcuteS aureus}

Osteomyelitis

Clinical Characteristic fnbB⫹

(n⫽66)

fnbB⫺ (n⫽19)

P

ESR at presentation NS

n 63 18

Mean⫾SD, mm/h 75.7⫾28.3 68.1⫾28.1

Median (range), mm/h 74 (4–140) 69 (15–115)

Maximum ESR .03

n 66 19

Mean⫾SD, mm/h 97.1⫾31.3 79.3⫾31.4

Median (range), mm/h 106 (4–140) 85 (15–140)

CRP level at presentation NS

n 55 14

Mean⫾SD, mg/dL 19.4⫾15.8 13.6⫾22.1

Median (range), mg/dL 16.4 (0.7–72.7) 5.75 (1.1–79.4)

Maximum CRP level NS

n 59 17

Mean⫾SD, mg/dL 21.0⫾15.0 13.9⫾20.9

Median (range), mg/dL 20.3 (0.8–72.7) 6.6 (1.1–79.4)

Blood culture positive forS aureusisolates,n/N(%) 38/65 (58.5) 6/19 (31.6) NS Surrounding myositis/pyomyositis,n/N(%) 30/52 (57.7) 4/12 (33.3) NS Subperiosteal/intraosseal abscess,n/N(%) 37/52 (71.2) 6/12 (50) NS

93% of 30 necrotic skin isolates and 85% of 27 primary CA pneumonia isolates werepvl⫹. In contrast, only 23% of the 13 strains isolated from patients with osteomyeli-tis werepvl⫹.13

Although there is no proven association betweenpvl

and development of osteomyelitis, we recently reported a retrospective analysis of children with musculoskeletal

S aureusinfections, in which children with osteomyelitis caused by pvl⫹ S aureus had more febrile days and were more likely to have a complication such as a deep venous thrombosis or to develop chronic osteomyelitis than children with osteomyelitis caused by pvl⫺ S au-reus.7 _{In our current study, multivariate analysis of}

several factors including methicillin susceptibility and presence ofpvlandfnbBgenes again showed an associ-ation betweenpvl(or an unknownpvl-linked factor) and a more severe systemic inflammatory response,S aureus– positive blood cultures, and greater frequency of contig-uous myositis/pyomyositis than pvl⫺ S aureus isolates. Thus, PVL or an associated factor likely contributes to the severity of AHO caused by CAS aureus.

In patients who are undergoing treatment for AHO, the ESR and CRP values typically increase until the second or third day of therapy, after which the CRP level falls rapidly within 1 week, which is an indicator of acute response to therapy. The ESR takes weeks to nor-malize and is often used to gauge length of therapy.1,2,29,30

The WBC count frequently is not elevated in children with AHO.1,2,29,30 _{In our study, 95.5% and 95.0% of}

patients had elevated ESR and CRP values, respectively, and 53.9% of patients had an elevated WBC count of

⬎12 000/mm3 _{at admission. The patients with pvl}⫹

S aureus isolates had a significantly increased systemic inflammatory response compared with patients with

pvl⫺S aureus, as evidenced by higher ESR, CRP, WBC count, and ANC values at presentation and maximum ESR and CRP level during admission.

Roine et al31_{reported that children with higher CRP}

values on days 1 through 6 of admission and ESR values on days 4 through 7 of admission were more likely to have sequelae at their 1- to 2-month follow-up appoint-ments than children with lower CRP and ESR values. All patients who had sequelae and had an organism identi-fied as responsible for their AHO (21 of 28) hadS aureus

identified.31_{Both the higher CRP and ESR values and the}

development of sequelae were likely secondary to the development of more invasive disease and could be a consequence ofS aureusorganisms with increased viru-lence secondary to a factor such as PVL.

Overall, 30% to 50% of children with AHO caused by any organism haveS aureus–positive blood cultures.1_In

our previous study, the number of days with persistently positive blood cultures between patients with osteo-myelitis caused bypvl⫹andpvl⫺S aureuswas not differ-ent.7_{However, in our current study, patients with pvl}⫹ S aureus isolates were significantly (P ⫽ .0001) more

likely to have anS aureus–positive blood culture during admission compared with children with pvl⫺ S aureus

(67.2% vs 19.2%, respectively). Patients who required ICU care and those with multifocal osteomyelitis in our study tended to have pvl⫹ S aureusisolated more com-monly than patients who did not require ICU care or who had a single focus of disease. This, along with the increased incidence ofS aureus–positive blood cultures, indicates thatpvllikely plays some role, or is a marker for some other factor, in the increasing numbers of children who are being reported with severe systemic invasive disease, including septic shock, secondary toS aureus.32

In addition, when we analyzed the subgroup of pa-tients in our study who had osteomyelitis that was iso-lated to the femur, tibia, or fibula and did not require ICU care, the children with pvl⫹ S aureus isolates re-mained significantly (P⫽.0004) more likely to have an

S aureus–positive blood culture (70.0% vs 12.5%, re-spectively), a more severe inflammatory response (in-cluding ESR, CRP level, and ANC at presentation and maximum ESR and CRP level during admission), and more extensive local disease (with more subperiosteal/ intraosseal abscesses seen on MRI). Thus, the differ-ences between the laboratory and radiographic features of thepvl⫹andpvl⫺patients in this study are not simply secondary to the fact that thepvl⫹group contained more patients with multifocal disease and who required ICU care.

We also screened our isolates for the S aureus gene

fnbBin this study. A greater proportion of MRSA isolates carry pvl and fnbB compared with MSSA isolates, and both factors have been linked to the increased virulence of CA-MRSA. The products of fnbB mediate S aureus

adhesion to epithelial cells and are hypothesized to fa-cilitate internalization of the bacteria into these cells.22–24

When infused into mice,S aureusstrains that arefnbB⫹

are associated with induction of systemic inflammation that is characterized by interleukin-6 secretion, signifi-cant weight loss, and mortality.33_{Peacock et al}23_reported

that fnbBwas significantly more frequently detected in CA-invasive strains (responsible for endocarditis plus septic arthritis and/or osteomyelitis) compared with car-riage isolates (84.4% vs 68.4%, respectively;P⫽.02).23

The patients in our study did have a high frequency of

fnbB⫹isolates (77.6% were positive), but the presence of

fnbBwas only associated with a greater mean maximum ESR during hospitalization in univariate analysis and was not independently associated with a greater sys-temic inflammatory reaction in our multivariate analy-sis. Furthermore, patients with fnbB⫹isolates were not more likely to have a subperiosteal/intraosseal abscess or surrounding myositis/pyomyositis on MRI, and in our previous study,fnbB⫹isolates were not associated with higher complication rates in children with musculoskel-etal infections that were caused byS aureus.7

WBC count, and ANC on admission, maximum ESR and CRP level during hospitalization, incidence of blood cul-ture positive for S aureus, and subperiosteal/intraosseal abscess on MRI) except for surrounding myositis/pyo-myositis on MRI were significantly associated with methicillin resistance on univariate analysis, which in-dicates that children with MRSA isolates were more likely to have evidence of increased systemic inflamma-tory response and more extensive local disease than children with MSSA isolates. However, on multivariate analysis, methicillin resistance only remained signifi-cantly associated with subperiosteal/intraosseal ab-scesses.

CONCLUSIONS

Our study supports the hypothesis that pvl is strongly associated with the severity of AHO caused byS aureusin children. Because the USA300 clone that is predominant in our area is present in many parts of the United States,18–21_{the results of this study and the significance of} pvl are relevant to physicians across the country. If fu-ture studies determine that PVL specifically contributes to the pathophysiology of invasive staphylococcal infec-tions, therapies to combat PVL may prove beneficial.34

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DOI: 10.1542/peds.2005-0566

2006;117;433

Pediatrics

Wendy A. Hammerman and Sheldon L. Kaplan

Claire E. Bocchini, Kristina G. Hulten, Edward O. Mason, Jr, Blanca E. Gonzalez,

Osteomyelitis in Children

Staphylococcus aureus

Inflammatory Response and Local Disease in Acute Hematogenous

Panton-Valentine Leukocidin Genes Are Associated With Enhanced

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DOI: 10.1542/peds.2005-0566

2006;117;433

Pediatrics

Wendy A. Hammerman and Sheldon L. Kaplan

Claire E. Bocchini, Kristina G. Hulten, Edward O. Mason, Jr, Blanca E. Gonzalez,

Osteomyelitis in Children

Staphylococcus aureus

Inflammatory Response and Local Disease in Acute Hematogenous

Panton-Valentine Leukocidin Genes Are Associated With Enhanced

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