Is non-union of tibial shaft fractures due to nonculturable bacterial pathogens? A clinical investigation using PCR and culture techniques.
ABSTRACT BACKGROUND: Non-union continues to be one of the orthopedist's greatest challenges. Despite effective culture methods, the detection of low-grade infection in patients with non-union following tibial fracture still presents a challenge. We investigated whether "aseptic" tibial non-union can be the result of an unrecognized infection. METHODS: A total of 23 patients with non-union following tibial shaft fractures without clinical signs of infection were investigated. Intraoperative biopsy samples obtained from the non-union site were examined by means of routine culture methods and by polymerase chain reaction (PCR) for the detection of 16 S ribosomal RNA (rRNA). Control subjects included 12 patients with tibial shaft fractures. RESULTS: 23 patients (8 women and 15 men; mean age: 47.4 years) were included into this study. Preoperative C-reactive protein levels (mean: 20.8 mg/l) and WBC counts (mean: 8,359/ul) in the study group were not significantly higher than in the control group. None of the samples of non-union routine cultures yielded microorganism growth. Bacterial isolates were found by conventional culturing methods in only 1 case of an open fracture from the control group. In this case, PCR yielded negative results. 16 S rRNA was detected in tissue specimens from 2 patients (8.7%) with non-union. The analysis of these variable speciesspecific sequences enabled the identification of specific microorganisms (1x Methylobacterium species, 1x Staphylococcus species). Both PCR-positive patients were culture-negative. CONCLUSIONS: The combination of microbiological culture and broad-range PCR seems to substantially add to the number of microbiological diagnoses obtained and may improve the clinican's ability to tailor therapy to the individual patient's needs.
- [Show abstract] [Hide abstract]
ABSTRACT: OBJECTIVE:: To review the results of a single stage treatment protocol for presumptive aseptic diaphyseal nonunion with a well healed wound and no infection history. DESIGN:: Retrospective comparative study SETTING:: Tertiary referral center PATIENTS AND METHODS:: We retrospectively reviewed all presumptive aseptic diaphyseal nonunions treated by this single stage treatment. There were 104 patients who met the inclusion criteria. Eighty-seven patients were available for follow-up through to complete healing (83.7% follow-up rate). INTERVENTION:: The protocol entails withholding preoperative antibiotics, implant removal, debridement or canal reaming, five cultures of the nonunion or reamings, followed by antibiotic administration and revision open reduction and internal fixation or exchange nailing. If intra-operative cultures are positive, long-term antibiotics are begun specific to organism sensitivities. MAIN OUTCOME MEASUREMENTS:: To analyze the rate of positive cultures and to compare the rate of secondary surgery to promote healing in positive and negative culture groups. RESULTS:: Intraoperative cultures were positive in 28.7% (25/87) of patients with complete follow-up. The overall rate of secondary surgery for persistence of nonunion was 12.6% (11/87). In patients with positive intraoperative cultures, rate of secondary surgery was 28% (7/25) versus 6.4% (4 /62) in the group without positive intraoperative cultures (p=0.01, Fischer's exact test). CONCLUSION:: A single stage treatment protocol for presumptive aseptic diaphyseal nonunions was effective in obviating further revision surgery in 93.6% of truly aseptic cases and in 72% of positive culture cases and is still employed at our institution. LEVEL OF EVIDENCE:: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.Journal of orthopaedic trauma 02/2013; · 1.78 Impact Factor
This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted
PDF and full text (HTML) versions will be made available soon.
Is non-union of tibial shaft fractures due to nonculturable bacterial pathogens?
A clinical investigation using PCR and culture techniques
Journal of Orthopaedic Surgery and Research 2012, 7:20 doi:10.1186/1749-799X-7-20
Justus Gille (firstname.lastname@example.org)
Steffen Wallstabe (email@example.com)
Arndt-Peter Schulz (firstname.lastname@example.org)
Andreas Paech (Andreas.Paech@uk-sh.de)
Ulf Gerlach (email@example.com)
23 December 2010
20 May 2012
20 May 2012
This peer-reviewed article was published immediately upon acceptance. It can be downloaded,
printed and distributed freely for any purposes (see copyright notice below).
Articles in Journal of Orthopaedic Surgery and Research are listed in PubMed and archived at
For information about publishing your research in Journal of Orthopaedic Surgery and Research or
any BioMed Central journal, go to
For information about other BioMed Central publications go to
Journal of Orthopaedic Surgery
© 2012 Gille et al. ; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Is non-union of tibial shaft fractures due to
nonculturable bacterial pathogens? A clinical
investigation using PCR and culture techniques
1 Department of Trauma and Reconstructive Surgery, University of Schleswig-
Holstein, Campus Luebeck, Luebeck 23538, Germany
2 Department of Trauma Surgery and Sportsmedicine, BG-Traumahospital
Hamburg, Hamburg 21033, Germany
* Corresponding author. Department of Trauma and Reconstructive Surgery,
University of Schleswig-Holstein, Campus Luebeck, Luebeck 23538, Germany
Non-union continues to be one of the orthopedist’s greatest challenges. Despite effective
culture methods, the detection of low-grade infection in patients with non-union following
tibial fracture still presents a challenge. We investigated whether “aseptic” tibial non-union
can be the result of an unrecognized infection.
A total of 23 patients with non-union following tibial shaft fractures without clinical signs of
infection were investigated. Intraoperative biopsy samples obtained from the non-union site
were examined by means of routine culture methods and by polymerase chain reaction (PCR)
for the detection of 16 S ribosomal RNA (rRNA). Control subjects included 12 patients with
tibial shaft fractures.
23 patients (8 women and 15 men; mean age: 47.4 years) were included into this study.
Preoperative C-reactive protein levels (mean: 20.8 mg/l) and WBC counts (mean: 8,359/µl)
in the study group were not significantly higher than in the control group. None of the
samples of non-union routine cultures yielded microorganism growth. Bacterial isolates were
found by conventional culturing methods in only 1 case of an open fracture from the control
group. In this case, PCR yielded negative results. 16 S rRNA was detected in tissue
specimens from 2 patients (8.7%) with non-union. The analysis of these variable species-
specific sequences enabled the identification of specific microorganisms (1x
Methylobacterium species, 1x Staphylococcus species). Both PCR-positive patients were
The combination of microbiological culture and broad-range PCR seems to substantially add
to the number of microbiological diagnoses obtained and may improve the clinican’s ability
to tailor therapy to the individual patient’s needs.
Non-union, Tibial fracture, Low-grade infection, Molecular diagnosis, PCR technique
We investigated whether “aseptic” tibial non-union is related to unrecognized infection. 23
patients (8 women and 15 men; mean age: 47.4 years) were included in the study. 16 S rRNA
was detected in 2 cases (8.7%) of non-union. PCR-positive patients were culture-negative in
Diaphyseal tibia fractures are the most common lower limb fractures worldwide . Despite
advances in management, tibia fractures remain vulnerable to many complications, which
often require secondary surgery. Potential complications include delayed union, non-union,
malunion, compartment syndrome and infection . A recent study on open and closed
diaphyseal tibia fractures treated by all modalities reported an overall revision rate of 22.4%
, which was often the result of non-unions.
In about 10% of cases the healing process is delayed ; for certain at-risk patients, it can
affect over 30% . Established causes of delayed union and non-union of tibial fractures are
systemic deficits, e.g. advanced patient age  and diabetes mellitus ; prior local
impairment of the extremity, e.g. chronic impairment of the soft tissues or blood circulation
; and characteristics involving the traumatic impact itself, e.g. fracture localization ,
degree of soft tissue damage  and bacterial contamination [1,11].
Non-union, especially when infected, continues to be one of the greatest challenges in
orthopedic surgery. After open reduction and internal fixation of tibial shaft fractures, the rate
of superficial infection is up to 22% and deep infections occur in up to 15% , the latter of
which can potentially lead to septic nonunion of the tibia. Clinical signs, laboratory
investigations of infection parameters and microbiological findings are often insufficient for
detecting infection. Distinguishing between infection and aseptic non-union is essential for
determining the proper clinical course of action . The standard analyses for detecting
microorganisms – gram staining (for microscopic investigation) and culturing of tissue
biopsy specimens obtained during surgical revision – are reported to have poor sensitivity
. The hypothesis is that evidence of bacterial infection is supported further by the
detection of bacterial DNA, which suggests bacterial persistence in the area of non-union
despite negative culture results. PCR targeting highly conserved regions of the bacterial
genome (e.g. the 16 S rRNA gene) has been used successfully to detect nonculturable
bacteria that cause a variety of infections, including septic arthritis  and meningitis .
The aim of this study was to investigate whether”aseptic” nonunion after tibial shaft fractures
is due to nonculturable bacteria by means of PCR amplification of 16 S rRNA genes and to
compare the efficiency of PCR with that of standard culture techniques.
Materials and methods
All patients participating in the present study were informed in detail about the surgical
technique and all alternative procedures with their respective advantages and disadvantages,
and all participants chose to undergo the index surgical procedure. All patients signed
informed consent forms to participate in the study. The study was performed in accordance
with the local ethical review board.
From November 2009 through March 2010, a consecutive series of 23 patients with non-
union following tibial shaft fractures were investigated. Normal healing was defined as union
within 4 months, delayed union as healing between 4 and 6 months and non-union was
defined as the absence of healing after 6 months . Only patients without clinical signs of
infection were included in the study. Exemplary x-rays of one case from the treatment group
are shown in Figure 1. Control subjects included 12 patients undergoing open reduction and
internal fixation for acute tibial shaft fractures.
Figure 1 Consecutive x-rays of a 30-year-old male with tibial non-union and valgus
malalignment following stabilization with external fixator for 6 months (Figures a,b).
Postoperative findings after excision of necrotic tissue and re-osteosynthesis with a
multidirectional locking plate (tifix®-tibia-plate, Litos, Hamburg, Germany) in combination
with reconstruction of skeletal defects by implantation of autologous bone removed from the
iliac crest (Figures c,d). Conventional and molecular bacteria detection methods were both
A stage-adapted treatment algorithm for tibial non-union has been established, as previously
published . Based on contemporary evidence among the recent literature, we do not
routinely administer antibiotics beyond 5 days postoperatively . We favor oral antibiotic
administration, because our experience has shown us that the route of antibiotic
administration (oral versus parenteral) does not affect the rate of disease remission if the
bacteria are sensitive to the antibiotic used.
All patients received standard preoperative care. Skin was decontaminated with Cutasept H
(Bode Chemie, Germany). Two grams of cefazolin (Basocef, Deltaselect GmbH, Germany)
were administered for perioperative prophylaxis subsequent to taking the biopsies.
Intraoperative biopsy samples (at least 3, each measuring 1 cm3) obtained from the non-union
were all divided into 2 portions. Specimens were placed into separate sterile tubes without
additional substrates. Specimens obtained for PCR were stored at −70°C. Samples were then
examined by means of routine culture methods and by polymerase chain reaction (PCR) for
the detection of 16 S ribosomal RNA (rRNA) in the laboratory. These procedures permitted
the independent examination and interpretation of the results. Histopathologic findings were
not recorded because of poor sensitivity, especially in cases of low-grade infection [18,19].
Bacterial isolation and standard culture methods
All specimens were incubated in brain-heart infusion broth (bioMérieux, Nuertingen,
Germany), TVLS medium , a medium described by Lodenkaemper and Stinen , on
Columbia blood plates (aerobic 5% CO2), and then on Brucella agar plates (anaerobic)
(bioMérieux, Nuertingen, Germany), as previously described . Samples were incubated
for 14 days. Susceptibility testing was performed according to the German Institutional
Standard Nr. 58940 .
Tissue samples were immediately stored at −70°C, and DNA was purified from homogenized
specimens after proteinase K digestion and column extraction with the NucleoSpin DNA kit
(Macherey-Nagel, Dueren, Germany). All DNA procedures before and after PCR were
performed in separate designated rooms with separate pipetting devices to avoid
contamination of the samples with foreign DNA. Master-mixture water controls were used
for every sample that was processed.
The sequences of the universal primers (16rRNA gene) and primers of the control gene
(glyceraldehydes-3-phosphate dehydrogenase; GAPDH) are indicated in Table 1.
Oligonucleotides used in this study were provided by Eurofins MWG Operon, Ebersberg,
Germany. DNA was amplified in a 25µL-reaction mixture consisting of ready-to-go PCR
beads (up to 23µL; Amersham Pharmacia Biotech, Muenchen, Germany), 0.5µL of each
primer (100 pmol/mL), and 1µL of the sample. After amplification, 5µL of the amplified
product was analyzed in a 2% agarose gel. Amplification products were sequenced by
SeqLab and then analyzed using the National Center for Biotechnology Investigation Blast
Table 1 Nucleotide sequences of primers used to determine agents responsible for non-
union following tibial shaft fractures
Amplification of the GAPDH control gene was performed using real-time PCR (Light Cycler
Detection System; Roche Molecular Biochemicals, Mannheim, Germany) with the FastStart
DNA Master SYBR kit (Roche Molecular Biochemicals). The PCR protocol included the
following work stages: 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, 60°C for 5 s,
and 72°C for 10s. In the dissociation protocol, single peaks were confirmed to exclude
Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS
15.0, Chicago, IL, USA) for descriptive statistics with a level of significance set at p < 0.05.
The non-parametric Wilcoxon’s signed rank test was used to analyze the data.
The experimental group of patients with tibial non-union consisted of 8 women and 15 men,
with a mean age of 47.4 years (range: 20–82 years). The mean age of the control group was
49.3 years (range: 17–73 years), including 2 females and 10 males.
The mean interval of the fracture to the index operation due to non-union was 10.2 months
(range: 6–34 months). At the time of the initial trauma, 8 cases (34.8%) were rated as open
fractures due to severe soft tissue injuries according to the classification by Gustilo et al. .
Four open fractures (33.3%) were counted in the control group. Up to 8 revision surgeries
(mean: 1.9; range: 1–8) had been performed in 18 cases (78.3%) prior to the index operation.
For all patients in the experimental group, clinical pathological data – especially C-reactive
protein levels (mean: 20.8 mg/l; range: 1.6–92.7) and WBC counts (8,359/µl; range: 4,360–
15,130) were not significantly higher compared to the control group (CRP: mean: 25.7 mg/l;
range: 2.9–133, WBC: mean: 11,234/µl; range: 6,110–17,210).
None of the non-union culture samples showed evidence of microorganism growth. Bacterial
isolates were found by conventional culturing methods in only 1 case of an open fracture
from the control group (Figure 2). The isolated pathogens were Streptococcus suis and
Enterococcus species. Tissue from the fracture gap in this case after initial débridement
yielded negative results by PCR.
Figure 2 38-year-old male with an open tibial fracture (control group). This figure
demonstrates the findings before the initial treatment. The isolated pathogens in culture were
Streptococcus suis and Enterococcus species. Tissue from the fracture gap after initial
débridement yielded negative results by PCR
16 S rRNA was detected in tissue specimens from 2 cases (8.7%) of non-union. Further
analysis of these variable species-specific sequences enabled the identification of specific
microorganisms; in one case Methylobacterium species and in the other sample
Staphylococcus species were identified. The PCR-positive patients were both culture-
To the best of our knowledge, this is the first study on conventional and molecular bacteria
detection methods in non-unions following tibia shaft fractures to validate the hypothesis that
non-culturable microorganisms are a potential source of non-union.
Diaphyseal tibia fractures are the most common lower limb fractures worldwide . There is
controversy among the literature regarding the best way to manage open tibial shaft fractures.
Recently, a metaanalysis of randomized prospective studies was performed comparing
external fixators and unreamed IM nails. There was no statistically significant difference
between the two methods of stabilization with respect to union, delayed union, deep infection
and chronic osteomyelitis . Non-union continues to be one of the orthopedist’s greatest
challenges, especially in its septic form.
As for many other infectious processes, early detection can often alter the natural course of
the disease and ultimately improve long-term outcomes for patients . There might be
clinical signs highly suggestive of infection, but diagnosis can be a difficult task, particularly
in the case of late and/or chronic infections .
Laboratory markers such as C-reactive protein, erythrocyte sedimentation rate, and white
blood count are sensitive markers of inflammation and plausible infection, but they are
unable to localize the exact site of infection and they are associated with low specificity .
In this series, no significant difference was obvious between the treatment and control group
according to inflammatory laboratory markers, although the mean values in both groups
diverged from the norm. Elevations in patients from the control group might have been due to
trauma, which often leads to increased levels of laboratory markers in the absence of
The standard analyses for detecting microorganisms – gram staining (for microscopic
investigation) and culturing of tissue biopsy specimens obtained during surgical revision –
are reported to have poor sensitivity . The sensitivity seems to be related to the amount of
biopsies taken . Antibiotics administered for perioperative prophylaxis, as well as
extended transportation time, inadequate quantities of vital bacteria and preservation of
specimens before processing may all lead to negative culture results .
Although there is a large body of evidence on wound bacteriology after open fractures 
with positive culture results in up to 83% , the literature regarding infection in closed
fracture gaps that affect healing is lacking . In our series, none of the closed fractures in
the control group were culture-positive.
Many molecular tools for bacterial DNA detection from clinical samples have been
developed. One of the most significant contributions thus far has been amplification-based
techniques (PCR), since some studies have confirmed its excellent sensitivity and specificity
. Moreover, the PCR technique takes less than 5 hours to complete, which is significantly
shorter than the couple of days required for routine cultures. In a prospective study
comparing PCR and culture techniques in the diagnosis of prosthetic joint infection, Gallo et
al. demonstrated a significantly higher sensitivity, accuracy and negative predictive value for
PCR versus culture . There was 83% concordance between the results of intraoperative
culture and PCR detection of causative bacteria . This is in accordance with Hoeffel et
al., who reported a PCR sensitivity and specificity of 71% and 49%, respectively, and a
positive predictive value of 22% and a negative predictive value of 7%, when compared with
culture methods . The authors concluded that the PCR methods should not serve as
screening tests for musculoskeletal infections, but they could be useful to confirm infections,
especially after initiating antibiotic treatment. Shortcomings of the PCR technique compared
to routine cultures are higher costs, false-positive results and problems with interpretation
In our series, 16 S rRNA was detected in tissue specimens from 2 cases (8.7%) of non-union.
In contrast to our results, Szczèsny et al. report the presence of bacteria in the callus of closed
fractured bones in up to 42% . Both viable bacteria and their DNA were detected.
Interestingly, the majority of isolates were not detected in the fracture gap tissue but in the
subcutaneous tissue and muscles. In patients with nonalignment, S. epidermidis and aureus
were detected in 4 out of 24 patients, whereas in the delayed healing group bacterial isolates
were found in 15 out of 43 patients . The reasons for the diversity between their results
and ours remain unclear, although one can pinpoint the fact that in the present study, tissue
from non-unions was investigated. In addition, the results of 16 S PCR are known to be very
susceptible for contamination. Our tests were performed under sterile conditions in a
laboratory room designated for RNA isolation and identification only. Sodium chloride
solution used for tissue samples was checked for the presence of 16 S rRNA and was found
to be negative for all samples. Further, most authors consider the positive cultures of deep
tissue specimens to be contamination from external sources . Contamination of
specimens could be precluded from being the source of detected isolates.
There are two limitations that need to be acknowledged and addressed regarding the present
study. The first limitation concerns the heterogeneous patient population, which reflects the
situation of patients with tibial non-union. The second limitation involves the extent to which
the findings can be generalized beyond the cases studied. The number of cases is too small
for broad generalizations. However, these limitations can be seen as fruitful avenues for
future research along the same lines.
This is the first study comparing routine cultures and molecular bacterial DNA detection in
tibial non-union. In summary, nonculturable pathogens seem to play a causative role in tibial
non-unions. The combination of microbiological culture and broad-range PCR seems to
substantially add to the number of microbiological diagnoses obtained and may improve the
clinican’s ability to tailor therapy to the individual patient’s needs.
The authors declare that they have no competing interests. None of the authors received
1. Conception and design of the study: JG, UG 2, Analysis and interpretation of data: SW,
AP, AS, JG 3, Collection and assembly of data: SW, AP, UG, AS, JG 4, Drafting of the
article: JG, UG. All authors read and approved the final manuscript.
The authors thank Jan Rupp, PhD, for his support and the opportunity to perform the PCR in
We gratefully acknowledge the skilled technical assistance of Ms. Angela Gravenhorst and
Mr. Stefan Bark.
None of the authors received financial support.
1. Harris I, Lyons M: Reoperation rate in diaphyseal tibia fractures. ANZ J Surg 2005,
2. Littenberg B, Weinstein LP, McCarren M, Mead T, Swiontkowski MF, Rudicel SA, Heck
D: Closed fractures of the tibial shaft. A meta-analysis of three methods of treatment. J
Bone Joint Surg Am 1998, 80:174–183.
3. Bhandari M, Guyatt GH, Swiontkowski MF, Schemitsch EH: Treatment of open
fractures of the shaft of the tibia. J Bone Joint Surg Br 2001, 83:62–68.
4. Einhorn TA: The cell and molecular biology of fracture healing. Clin Orthop Relat Res
5. Rothman RH, Klemek JS, Toton JJ: The effect of iron deficiency anemia on fracture
healing. Clin Orthop Relat Res 1971, 77:276–283.
6. Tonnesen PA, Heerfordt J, Pers M: 150 open fractures of the tibial shaft–the relation
between necrosis of the skin and delayed union. Acta Orthop Scand 1975, 46:823–835.
7. Macey LR, Kana SM, Jingushi S, Terek RM, Borretos J, Bolander ME: Defects of early
fracture-healing in experimental diabetes. J Bone Joint Surg Am 1989, 71:722–733.
8. Spector JA, Mehrara BJ, Greenwald JA, Saadeh PB, Steinbrech DS, Bouletreau PJ, Smith
LP, Longaker MT: Osteoblast expression of vascular endothelial growth factor is
modulated by the extracellular microenvironment. Am J Physiol Cell Physiol 2001,
9. Nelson GE Jr, Kelly PJ, Peterson LF, Janes JM: Blood supply of the human tibia. J Bone
Joint Surg Am 1960, 42-A:625–636.
10. Brinker MR, Bailey DE Jr: Fracture healing in tibia fractures with an associated
vascular injury. J Trauma 1997, 42:11–19.
11. Kienast B, Kiene J, Gille J, Thietie R, Gerlach U, Schulz AP: Posttraumatic severe
infection of the ankle joint - long term results of the treatment with resection
arthrodesis in 133 cases. Eur J Med Res 2010, 15:54–58.
12. Ince A, Rupp J, Frommelt L, Katzer A, Gille J, Lohr JF: Is “aseptic” loosening of the
prosthetic cup after total hip replacement due to nonculturable bacterial pathogens in
patients with low-grade infection? Clin Infect Dis 2004, 39:1599–1603.
13. Gallo J, Kolar M, Dendis M, Loveckova Y, Sauer P, Zapletalova J, Koukalova D:
Culture and PCR analysis of joint fluid in the diagnosis of prosthetic joint infection.
New Microbiol 2008, 31:97–104.
14. Ince A, Tiemer B, Gille J, Boos C, Russlies M: Total knee arthroplasty infection due to
Abiotrophia defectiva. J Med Microbiol 2002, 51:899–902.
15. Ni H, Knight AI, Cartwright K, Palmer WH, McFadden J: Polymerase chain reaction
for diagnosis of meningococcal meningitis. Lancet 1992, 340:1432–1434.
16. Meiners J, Gerlach U, Magerlein S, Jurgens C, Faschingbauer M: Pseudo-arthroses.
Chirurg 2009, 80:979–986.
17. Conterno LO, da Silva Filho CR: Antibiotics for treating chronic osteomyelitis in
adults. Cochrane Database Syst Rev 2009, :CD004439.
18. Frommelt L: Aspiration of joint fluid for detection of the pathogen in periprosthetic
infection. Orthopade 2008, 37:1027–1034. quiz 1035–1026.
19. Milgram JW: Nonunion and pseudarthrosis of fracture healing. A histopathologic
study of 95 human specimens. Clin Orthop Relat Res 1991, 268:203–213.
20. Caselitz FHFV: Halbflüssiges Kombinationsmedium zur Züchtung anaerober
Bakterien. Aerztl Lab 1969, 15:426–430.
21. Lodenkamper H, Stienen G: Therapy of anaerobic infections. Dtsch Med Wochenschr
22. Edelmann A, Pietzcker T, Wellinghausen N: Comparison of direct disk diffusion and
standard microtitre broth dilution susceptibility testing of blood culture isolates. J Med
Microbiol 2007, 56:202–207.
23. Bednarski AE, Elgin SC, Pakrasi HB: An inquiry into protein structure and genetic
disease: introducing undergraduates to bioinformatics in a large introductory course.
Cell Biol Educ 2005, 4:207–220.
24. Thompson SM: Constructing and refining multiple sequence alignments with PileUp,
SeqLab, and the GCG suite.
doi:10.1002/0471250953.bi0306s00. Chapter 3:Unit 36.
Curr Protoc Bioinformatics 2003,
25. Gustilo RB, Anderson JT: JSBS classics. Prevention of infection in the treatment of
one thousand and twenty-five open fractures of long bones. Retrospective and
prospective analyses. J Bone Joint Surg Am 2002, 84-A:682.
26. Giannoudis PV, Papakostidis C, Roberts C: A review of the management of open
fractures of the tibia and femur. J Bone Joint Surg Br 2006, 88:281–289.
27. Gallo J, Raska M, Dendis M, Florschutz AV, Kolar M: Molecular diagnosis of
prosthetic joint infection. A review of evidence. Biomed Pap Med Fac Univ Palacky
Olomouc Czech Repub 2004, 148:123–129.
28. Schulz AP, Faschingbauer M, Seide K, Schuemann U, Mayer M, Jurgens C, Wenzl M: Is
the Wave Plate Still a Salvage Procedure for Femoral Non-union? Results of 75 Cases
Treated with a Locked Wave Plate. Eur J Trauma Emergency Surgery 2009, 35:127–131.
29. Virolainen P, Lahteenmaki H, Hiltunen A, Sipola E, Meurman O, Nelimarkka O: The
reliability of diagnosis of infection during revision arthroplasties. Scand J Surg 2002,
30. Amara U, Kalbitz M, Perl M, Flierl MA, Rittirsch D, Weiss M, Schneider M, Gebhard F,
Huber-Lang M: Early expression changes of complement regulatory proteins (CRegs)
and C5a receptor (CD88) on leukocytes after multiple injury in humans. 2009, Shock.
31. Knesek MJ, Litinas E, Adiguzel C, Hopkinson W, Hoppensteadt D, Lassen M, Fareed J:
Inflammatory biomarker profiling in elderly patients with acute hip fracture treated
with heparins. Clin Appl Thromb Hemost, 16:42–50.
32. Oheim R, Gille J, Schoop R, Magerlein S, Grimme CH, Jurgens C, Gerlach UJ: Surgical
therapy of hip-joint empyema. Is the Girdlestone arthroplasty still up to date? Int
33. Trampuz A, Osmon DR, Hanssen AD, Steckelberg JM, Patel R: Molecular and
antibiofilm approaches to prosthetic joint infection. Clin Orthop Relat Res 2003, :69–88.
34. Robinson D, On E, Hadas N, Halperin N, Hofman S, Boldur I: Microbiologic flora
contaminating open fractures: its significance in the choice of primary antibiotic agents
and the likelihood of deep wound infection. J Orthop Trauma 1989, 3:283–286.
35. Swinson BD, Morrison CM, Sinclair JS: Pyoderma gangrensum–a complication of
breast biopsy. Ulster Med J 2002, 71:66–67.
36. Hoeffel DP, Hinrichs SH, Garvin KL: Molecular diagnostics for the detection of
musculoskeletal infection. Clin Orthop Relat Res 1999, 360:37–46.
37. Szczesny G, Interewicz B, Swoboda-Kopec E, Olszewski WL, Gorecki A, Wasilewski P:
Bacteriology of callus of closed fractures of tibia and femur. J Trauma 2008, 65:837–842.
38. Vehmeyer SB, Slooff AR, Bloem RM, Petit PL: Bacterial contamination of femoral
head allografts from living donors. Acta Orthop Scand 2002, 73:165–169.