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Abstract
The epidemiology of acute paediatric osteoarticular infections (OAI) has recently evolved, mainly due to the improvement of microbio-
logical diagnosis. We conducted a prospective study to analyse the recent epidemiology and the clinical evolution of paediatric OAI in
order to validate the adequacy of our probabilistic first-line antibiotic treatment (intraveinous cefamandole + gentamicin). All children
suspected of community acquired OAI were included and followed-up for 3 years. The etiologic diagnosis was based on blood cultures,
joint aspirations and bone punctures. All osteoarticular (OA) samples were systematically inoculated into blood culture bottles. Real-
time universal 16S rRNA and PCR targeted on Staphylococcus aureus, Kingella kingae, Streptococcus pneumoniae and Streptococcus pyogenes
were performed twice a week. From 17 March 2007 to 26 February 2009, 98 septic arthritis, 70 osteomyelitis, 23 osteoarthritis and
six spondylodiscitis were analysed. A portal of entry was suspected in 44% of cases, including 55% of otorhinolaryngological infections.
C reactive protein was the most sensitive inflammatory marker. PCR increased by 54% the performance of bacteriological diagnosis.
Among the patients completely investigated (blood culture and OAI samples), there were 63% documented OAI. The main pathogens
found were K. kingae (52%), S. aureus (28%), S. pyogenes (7%), S. pneumoniae (3%) and Streptococcus agalactiae (2%). All isolated bacteria
were sensitive to the probabilist treatment and outcome was favorable. PCR has significantly improved the performance and the delay
of IOA diagnosis in children, for which K. kingae turned out to be the first causative agent. The probabilistic treatment was active
against the main bacteria responsible for paediatric OAI.
probabilist antibiotic treatment, which comprised a combina- All the patients were evaluated systematically at day 10,
tion of cefamandole and gentamicin. Three- or 8-day intrave- 1 month, 2 months, 6 months and every year after the onset.
nous treatment was conducted depending on clinical
outcome and results of blood cultures. This treatment was Techniques used in the laboratory of microbiology
followed by a probabilistic combination of oral amoxicillin/cla- A portion of the OA samples was frozen at )80C for sub-
vulanate and rifampicin if OAI was not documented. We car- sequent diagnosis by molecular biology, particularly in the
ried out a clinical and biological prospective study to assess case of negative culture. Blood cultures and enrichment cul-
the current epidemiology of children with OAI using the tures of AF were incubated in a continually monitored
molecular techniques currently available. This knowledge was instrument (Bact/Alert 3D, bioMérieux) for 10 days. A por-
used to evaluate the adequacy of the probabilistic treatment. tion of bone and synovial homogenates was inoculated into
aerobic and anaerobic blood culture bottles and introduced
in the Bact/Alert 3D. The AF and the remaining bone homo-
Material and Methods
genates were seeded directly on rich solid media (chocolate
and blood agar) in CO2-enriched air and an anaerobic atmo-
Patients sphere, for 10 days.
We prospectively included all children (<15 years of age) with The bacterial species identification was performed by
presumed or documented acute septic arthritis (A), osteoar- MALDI-TOF mass spectrometry from bacterial colonies or
thritis (OA), osteomyelitis (OM) and spondylodiscitis. Immu- directly from enrichment broth [6]. The antibiotic suscepti-
nocompromised patients or those with sickle cell anaemia bility testing was performed as recommended by the diffu-
were excluded. On admission, clinical symptoms and imaging sion method, according to the recommendations of the
data (X-ray, technetium-99 scintigraphy, ultrasonography, European Committee on Antimicrobial Susceptibility Testing.
magnetic resonance imaging and CT scan) were recorded. Real-time PCR was performed once or twice a week.
Inflammatory markers (C-reactive protein, white blood cells DNA was extracted from specimens with the automated
and polymorphonuclear blood cell count) and bacteriological MagNa Pure system, using the kit LC DNA Isolation III
data (blood cultures, joint fluids, synovial biopsies and bone (Roche Diagnostica, Meylan, France) according to the manu-
punctures) were collected. Anti-inflammatory and antibiotic facturer’s recommendations. A universal real-time PCR
treatment given before hospitalization, antibiotic treatments RNAr 16S followed by sequencing was performed as previ-
for OAI and potential portals of entry were documented. ously described [2]. In addition, four specific PCRs targeted
For each patient with suspicion of OAI, blood cultures towards S. aureus, K. kingae, S. pneumoniae and S. pyogenes
were performed in aerobic and anaerobic vials (Bact alert, were performed. The mixture reagents contained 10 lL
bioMerieux, Marcy l’Etoile, France). In cases of A or OA, the DNA, 1.5 lL of each primer at 10 lM, 2 lL of probe at
articular fluid (AF) was withdrawn by syringe and part of the 5 lM, and 25 lL of qPCR master mix Plus Quick Gold
fluid was inoculated by the surgeon into an aerobic blood StarTM (Eurogentec, Seraing, Belgium) in a final volume of
culture bottle (bioMerieux). The remaining AF, the bone 50 lL. PCR was performed in an ABI PRISM 7300 Seq detec-
biopsies (BB) and synovial biopsies (SB) were sent directly to tor system thermocycler (Applied Biosystems, Foster City,
the laboratory in sterile vials. CA, USA) with an initial step of 2 min at 50C and 10 min at
Bone puncture was performed only in the case of subpe- 95C, followed by 45 cycles of 15 s at 95C, and 1 min at
riosteal collection or abnormal X-ray at entry to hospital. 60C. Table 1 shows the primers and probes used for uni-
Documented OAIs were defined by the presence (by cul- versal and specific PCRs.
ture or PCR) of a pathogenic bacteria in the AF, BB, SB or
blood culture. The diagnosis of presumed OAI was assigned in Statistical analysis
cases where joint-fluid or bone sample had not been obtained R (Language and Environment for Statistical Computing, R
or were negative, if clinical signs, abnormal biological parame- Foundation for Statistical Computing, Vienna, Austria) was
ters and radiological imaging demonstrated conclusive signs of used for statistical analysis. The levels of CRP were com-
infection and if there was a clinical improvement after 48 h of pared using the Student t-test, and proportions of abnormal
antibiotic treatment. The diagnosis of OAI was excluded when vs. normal values (white blood cell count and polymorphonu-
there was no abnormal radiological imaging, and/or improve- clear cell count, T) were compared by Fisher’s exact test.
ment without treatment or another cause of limb movement A p <0.05 was defined as statistically significant for all cal-
limitation, or any of these conditions. culations.
16S RNA Primers Neck1 5¢- TCA AAK GAA TTG ACG GGG GC-3¢ This study
Neck2 5¢- GCC CGG GAA CGT ATT CAC-3¢
Primers P13p 5¢- AGG CCC GGG AAC GTA TTC AC -3¢ [1]
PL06 5¢- GGT TAA GTC CCG CAA CGA GCG C -3¢
LytA S. pneumoniae Primers LytA F 5¢ACG CAA TCT AGC AGA TGA AGC A-3¢ [7]
LytA R 5¢ TCG TGC GTT TTA ATT CCA GCT-3¢
Probe LytA T 5¢ Fam GCC GAA AAC GCT TGA TAC AGG GAG-3¢Tamra
Nuc S. aureus Primers LT NUC F 5¢AAA TTA CAT AAA GAA CCT GCG ACA-3¢ [8]
LT NUC R 5¢ GAA TGT CAT TGG TTG AAC TTT GTA-3¢
Probe LT NUC HP1 5¢ Fam AAT TTA ACC GTA TCA CCA TCA ATC GTC-3¢Tamra
Cpn60 K. kingae Primers 105 F 5¢- TTT GGT TGG CGA ATT GGC-3¢ This study
341 R 5¢- ACG AAA TAA GGC GAC AAG TAG CC-3¢
Probe 141 T 5¢ Fam -CGA AAC ATA CGA GCA AAT -MGB-3¢
Lambda (extraction Primers Lambda F 5¢-TTG TGG CTT GGC TCT GCT AAC-3¢ This study
and inhibition control) Lambda R 5¢-AAT CAT TCA ACA CCC GCA CTA TC-3¢
Probe Lambda T 5¢VIC- ACC AGC CAG CCG CA- MGB-3¢
N N
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10 15 1 2 FIG. 2. Imaging explorations with their contri-
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TABLE 2. Pathogens isolated in the 83 children with proven TABLE 3. Respective contributions of standard cultures in
osteoarticular infection solid media, enrichment in blood culture bottles, PCR and
*16S PCR does not allow accurate species identification among Enterobacteriacae
of cases, is helpful in identifying a particular pathogen, partic-
ularly K. kingae in the case of ORL infection.
As bone puncture samples were very limited, the bacteri- The WBC count was abnormal in <25% of all OAI.
ological results of OM constitute a bias. If bone punctures This low diagnostic value has already been shown in two
had been performed routinely, K. kingae would be probably previous studies, where the frequency of abnormal WBC
the first microorganism responsible not only for arthritis, count was 10% and 5%, respectively, in K. kingae infections
but also for spondylodiscitis and osteomyelitis in children [5,10]. The CRP and body temperature remain the best
<4 years. As the high performance of bone biopsy in OM sensitive means for the initial diagnosis. In K. kingae infec-
contrasts with the low efficiency of blood cultures, there is tions, the CRP level was lower than in OAI due to other
therefore a need to look for other diagnostic markers of bacteria. Mild biological and clinical signs for K. kingae
bacterial OM, such as optimization of PCR in blood or blood infections have already been reported in other clinical
cultures. A portal of entry, found in our study in almost 45% studies [5,13–15].
X-ray is not very useful in the earlier stages of OAI, as Transparency Declaration
confirmed by this study, but helpful for long-term follow-up.
Indeed, the radiographic signs are rarely present before the
The authors declare no conflicts of interest.
second week after clinical onset of infection. Ultrasound
studies were very sensitive in arthritis or osteoarthritis. The
scintigraphy is very useful to locate the infection, particularly References
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