Clonal hematopoiesis of a novel dic(18;20) clone following allogeneic hematopoietic stem cell transplantation.

A 55-year-old man in first complete remission of acute myeloid leukemia with a normal karyotype underwent allogeneic hematopoietic stem cell transplantation from a human-leukocyte-antigen-matched sibling. Bone marrow examination on day 28 confirmed complete remission, but G-banding analysis revealed a novel chromosomal abnormality, including dic(18;20)(p11.2;q11.2). The patient developed moderate chronic graft-versus-host disease on day 174, and the abnormal clones identified by dic(18;20) significantly increased after that point. Chimerism testing repeatedly confirmed complete donor type. Although next-generation sequencing showed no clonal hematopoiesis-related gene mutations, copy number analysis of the donor and the recipient revealed copy number deletion of 18p, 18q, and 20q. The patient has maintained remission for more than 2 years to date without developing a hematologic neoplasm or cytopenia. The distinctive clonal hematopoiesis with a dicentric chromosome seemed to have undergone the breakage-fusion-bridge cycle, which could cause the complex events of deletion, amplification, and inversion. These copy number alterations might have increased the number of clones with growth advantage, and the highly inflammatory environment in the recipient due to graft-versus-host disease might have contributed to the clonal selection.

Three cases of suspected pleuroparenchymal fibroelastosis after allogeneic hematopoietic cell transplantation.

This article reports the clinical course and imaging findings of three cases of suspected pleuroparenchymal fibroelastosis (PPFE) after allogeneic hematopoietic cell transplantation (HCT). All patients complained of dyspnea more than 5 years after HCT, had progressive restrictive deficits on respiratory function tests, and presented with pneumothorax, pleural thickening, or exacerbation of consolidation in the upper lobe of the lung. Though lung biopsy was not done in all three cases, the clinical findings and results of spirometry were compatible with those of PPFE. PPFE has been sporadically reported as a pulmonary complication of allogeneic HCT; however, clinical diagnostic criteria other than histological diagnosis and treatment methods have not yet been established. The accumulation of more cases is necessary to improve the prognosis of PPFE complications.

Antibody response after third dose of COVID-19 mRNA vaccination in allogeneic hematopoietic stem cell transplant recipients is comparable to that in healthy counterparts.

To determine the efficacy of SARS-CoV-2 mRNA vaccination for allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients, we measured antibody titer serially in 92 allo-HSCT patients. Among the evaluable 87 patients, median age at vaccination was 53 years (range, 18-75). The average time between allo-HSCT and vaccination was 3.3 years (range, 0.5-15.7). One month after the second dose, 70 patients (80.5%) had a positive response, whereas 17 patients (19.5%) had a negative response (< 20 U/mL). Only patients older than 44 years had a negative response. Low IgM level was the only significant predictor of vaccine failure in elderly patients. When antibody response before and after the third vaccination was examined in 47 patients, antibodies increased significantly from a median of 18.3 U/mL to 312.6 U/mL (P < 0.01). The median antibody titer after the third vaccination of healthy individuals (n = 203) was 426.4 U/mL, which was comparable to that of patients (P = 0.2). The antibody titer after the third mRNA vaccination increased even in patients whose first two mRNA vaccinations failed. These findings suggest that allo-HSCT recipients should receive the mRNA vaccine regularly.

[Intestinal pneumatosis developed during the treatment of severe gastrointestinal graft-versus-host disease after cord blood transplantation].

A 62-year-old female developed stage4 gastrointestinal graft-versus-host disease (GVHD) on day 109 following an allogeneic cord blood transplant for relapsed refractory angioimmunoblastic T-cell lymphoma. GVHD went into remission 4 weeks after receiving the steroid (mPSL 1 mg/kg), but abdominal bloating started to emerge at the same time. A diagnosis of intestinal pneumatosis was made on day 158 after a CT scan revealed submucosal and serosal pneumatosis in the entire colon, and intestinal pneumatosis was identified as the cause. Fasting and reducing steroid use have helped. the abdominal symptoms, and the pneumatosis disappeared on day 175. No more flare-ups occurred, and the steroid was successfully stopped. After allogeneic transplantation, intestinal pneumatosis is a rather uncommon complications. Its pathogenesis is thought to be influenced by GVHD or steroids. Treatments for the disease may be incompatible with one another, and the response in individual cases needs to be studied in detail.

[Complete remission after standard induction therapy in a patient with acute myeloid leukemia associated with double-minute chromosomes].

Acute myeloid leukemia (AML) associated with double-minute chromosomes (dmin) is a rare condition and has a poor prognosis. A 68-year-old man with leukocytosis and thrombocytopenia was admitted to our hospital. Bone marrow aspiration showed that 79.5% of myeloblasts were positive for myeloperoxidase. The patient was diagnosed with acute myeloid leukemia (French-American-British classification: M2, World Health Organization classification: AML, not otherwise specified, AML with maturation). Chromosomal analysis revealed the presence of dmin: 45, X, -Y, 5-33 dmin. Fluorescence in situ hybridization revealed multiple MYC signals, and spectral karyotyping showed that dmin was derived from chromosome 8. These findings indicated resistance to chemotherapy alone. After the standard induction therapy with daunorubicin and cytarabine, the number of myeloblasts in the bone marrow decreased, and the amplified MYC signals disappeared. Then, the patient achieved complete remission. Reportedly, most patients with AML correlated with dmin have a complex karyotype, except for this case. Owing to the absence of a complex karyotype, the patient had good sensitivity to chemotherapy. Further studies with a larger population of patients with AML associated with dmin, but without complex karyotypes, should be conducted to accurately predict prognosis in such cases.

Severe tumor lysis syndrome during the induction therapy for the treatment of blastic plasmacytoid dendritic cell neoplasm arising from myelodysplastic/myeloproliferative neoplasms.

BPDCN shows clinically heterogeneous characteristics. And as other hematological malignancies, symptoms of BPDCN suggesting a high tumor burden, such as high white blood cell count or splenomegaly, should be carefully considered to prevent TLS.

Intrapatient variability in concentration/dose ratio of tacrolimus predicts transplant-associated thrombotic microangiopathy.

Tacrolimus (TAC) is essential for prophylaxis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic cell transplantation (allo-HSCT). We have sometimes observed large fluctuations in TAC concentration. However, links between the variability in the concentration or the concentration/dose (C/D) ratio of TAC and clinical complications remain ambiguous. To clarify relationships between various parameters of TAC and early complications such as pre-engraftment immune reactions/engraftment syndrome, aGVHD, and transplant-associated thrombotic microangiopathy (TA-TMA), a total of 146 patients who underwent allo-HSCT were included. Intrapatient variabilities in the concentration and C/D ratio of TAC were estimated by intrapatient mean absolute deviation (iMAD). The mean concentration and C/D ratio of TAC were not significantly different between with and without complications. A strong association was observed between greater iMAD for TAC C/D ratio from days 15 to 21 and the development of TA-TMA. iMAD values for TAC C/D ratio of 11.4 or greater was a risk factor for TA-TMA and the cumulative incidence of nonrelapse mortality (NRM) was significantly higher in patients with iMAD values for TAC C/D ratio of 11.4 or greater. Intrapatient variability in the C/D ratio of TAC was associated with the incidence of TA-TMA and NRM and might be useful for predicting TA-TMA.

Clinical usefulness of diagnostic criteria for transplant-associated thrombotic microangiopathy.

One major cause of treatment-related death is transplant-associated thrombotic microangiopathy (TA-TMA). Because of difficulties with diagnosis, many criteria for TA-TMA have been defined. Some patients clinically suspected as TA-TMA have been treated as TA-TMA regardless of TA-TMA criteria fulfillment (clinical-TMA). To examine sensitivities of TA-TMA criteria for clinical-TMA, we retrospectively evaluated 160 patients undergoing allogeneic hematopoietic stem cell transplantation by five major TA-TMA criteria and compared them with clinical-TMA. Cumulative incidences of TA-TMA and non-relapse mortality (NRM) were widely diverse between criteria. Thirty-eight patients fulfilled one or more TA-TMA criteria (total-TMA), and 12 of them fulfilled only one criterion. In patients with total-TMA, thrombocythemia, serum creatinine > 1.5 × baseline, and proteinuria were especially repeatedly observed among TA-TMA criteria. Ninety-two percent of clinical-TMA patients were classified as patients with total-TMA, and high NRM incidences were exhibited in patients with total-TMA even without clinical-TMA. Hematopoietic cell transplant-comorbidity index ≥ 3, nutritional risk index < 83.5, and grade II-IV acute graft-versus-host disease were extracted as independent risk factors for total-TMA. TA-TMA summation criteria that can cover most of clinical-TMA patients and high-risk patients of NRM were useful in clinical settings, and items of TA-TMA criteria previously described might be triggers for applying TA-TMA criteria.

Permanent Impairment-Free, Relapse-Free Survival: A Novel Composite Endpoint to Evaluate Long-Term Success in Allogeneic Transplantation.

Permanent impairment (PI) of vital organs is one of the transplantation-related health problems affecting the quality of life and morbidity even in patients who do not develop graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT), but no data are available on PI of multiple organs. This retrospective study aimed to estimate a novel composite endpoint of PI-free, relapse-free survival (PIRFS) in 164 allo-HCT recipients. We defined PI as >26% to 30% impairment of the whole person in 6 vital organs using the whole person impairment rating. Conventional GVHD-free/relapse-free survival (GRFS) and PIRFS at 5 years were 33.8% (95% confidence interval [CI], 26.5% to 41.3%) and 40.6% (95% CI, 32.6% to 48.4%), respectively. In the whole cohort, PIRFS was higher than GRFS at any time after allo-HCT. However, PIRFS was lower than GRFS after day 397 post-transplantation in patients who underwent umbilical cord blood transplantation (UCBT). In UCBT recipients, 5-year GRFS and PIRFS were 47.6% (95% CI, 34.3% to 59.7%) and 39.2% (95% CI, 26.6% to 51.5%), respectively. The cumulative incidence of PI after 5 years was 20.9% (95% CI, 13.7% to 29.0%) in patients surviving for ≥6 months without relapse. The multivariate analysis revealed that high disease risk (hazard ratio [HR], 1.91; 95% CI, 1.26 to 2.88; P < .01) and Karnofsky Performance Status score ≤90% at transplantation (HR, 1.73; 95% CI, 1.14 to 2.63; P = .01) were correlated with the lower PIRFS, whereas UCBT (HR, 2.35; 95% CI, 1.11 to 4.99; P = .03), grade III-IV acute GVHD by day 180 (HR, 3.59; 95% CI, 1.04 to 12.4; P = .04), and thrombotic microangiopathy by day 180 (HR, 2.74; 95% CI, 1.10 to 6.87; P = .03) were significantly correlated with a higher incidence of PI. More than 20% of long-term survivors had PI. Our data suggest that PIRFS is a useful endpoint for assessing long-term transplantation success from a different perspective than has been established previously.

Day 0 bone marrow pathology of allogeneic hematopoietic stem cell transplantation is a novel prognostic factor in myeloid malignancies.

Residual disease (RD) is one of the risk factors for relapse after hematopoietic stem cell transplantation (HSCT) in hematological malignancies. Although recent advances in the technology for detecting minimal/measurable RD, such as multiparameter flow cytometry and quantitative PCR, enable risk stratifications of disease relapse, these examinations still have limitations in routine clinical practice. In this study, we assessed RD in bone marrow (BM) specimens on day 0 of allogeneic HSCT by immunostaining of case-specific leukemic blast markers and analyzed the relationship between day 0 BM status and HSCT outcomes. We analyzed 82 adult HSCT recipients with myeloid malignancies. BM histology of day 0 revealed almost empty marrow with a small number of residual BM cells. However, residual blasts could be detected by immunostaining even for only a few cells. When patients were divided into two groups according to the existence of RD on day 0, those with positive RD showed significantly lower overall survival rate (27% vs. 73%, P<0.001) and higher cumulative incidence of relapse (46% vs. 9%, P=0.006) at one year compared to those with negative RD. Furthermore, even if they were not in remission at the point of the pre-conditioning evaluation, the patients who achieved negative RD on day 0 showed comparable prognosis with those who maintained remission before conditioning. This study shows the efficacy of day 0 BM pathology of allogeneic HSCT as a prognostic factor that can contribute to clinical decisions on post-transplant strategies.

Impact of a Nutritional Risk Index on Clinical Outcomes after Allogeneic Hematopoietic Cell Transplantation.

Nutritional status is an important component of cancer care, and malnutrition itself can cause death in 10% to 20% of cancer patients. A nutritional risk index (NRI) is a useful tool for nutritional assessment of cancer patients. This study aimed to evaluate the impact of pretransplant NRI values on outcomes of allogeneic hematopoietic cell transplantation (allo-HSCT). One hundred sixty patients who underwent allo-HSCT between January 2008 and July 2017 at Konan Kosei Hospital were included in this single-center, retrospective analysis. NRI was calculated at the beginning of the conditioning regimen. The patients were divided into high NRI (NRI ≥ 97.5) and low NRI (NRI < 97.5) groups, and overall survival (OS), nonrelapse mortality (NRM), and cumulative incidences of acute and chronic graft-versus-host disease (GVHD) were evaluated. Two-year OS rates were 76% (95% confidence interval [CI], 63% to 83%) and 50.4% (95% CI, 38% to 62%) in the high NRI and low NRI groups, respectively (P < .001). One-year cumulative incidences of NRM were 7.9% (95% CI, 3.5% to 15%) and 23% (95% CI, 14% to 33%; P = .014) and 2-year cumulative relapse rates were 17% (95% CI, 10% to 26%) and 32% (95% CI, 21% to 43%; P = .10) in the high NRI and low NRI groups, respectively. The multivariate analysis indicated low NRI was a significant risk factor for OS and NRM. Conversely, high NRI was associated with increased incidences of grades II to IV acute GVHD and chronic GVHD. Additionally, the subgroup analysis according to stem cell source revealed a significant benefit of higher NRI on survival only in umbilical cord blood recipients. Overall, these results suggest that pretransplant NRI might predict OS and NRM after allo-HSCT.

Patterns of onset and outcome of cryptogenic organizing pneumonia after allogeneic hematopoietic stem cell transplantation.

Cryptogenic organizing pneumonia (COP) after allogeneic hematopoietic stem cell transplantation (HSCT) is characterized by frequent recurrence. Few studies have examined onset and recurrence patterns of COP after HSCT. We investigated the clinical features of COP after HSCT in a single-center retrospective study including 165 consecutive patients who underwent allogeneic HSCT. Eighteen patients (11%) developed COP after HSCT. Hypoxemia and pleural effusion at the onset of COP were significantly associated with umbilical cord blood transplantation (P = 0.002 and P = 0.002, respectively). Recurrence of COP was observed in six patients and significantly associated with the presence of chronic graft-versus-host disease (cGVHD; P = 0.013) and stem cell sources other than umbilical cord blood (P = 0.038). Four patients with COP died of pulmonary failure after recurrence of COP. No patients who underwent umbilical cord blood transplantation experienced recurrence of COP. These findings suggest that the clinical features at the onset of COP may depend on stem cell sources. Moreover, both stem cell source and the absence or presence of cGVHD may affect COP recurrence, indicating the need to develop treatment strategies against COP according to stem cell source and risk of cGVHD.

Optimal dosage of methotrexate for GVHD prophylaxis in umbilical cord blood transplantation.

The combination of methotrexate (MTX) and a calcineurin inhibitor is widely used for GVHD prophylaxis in umbilical cord blood transplantation (UCBT). However, the optimal MTX dosage for GVHD prophylaxis in UCBT remains unclear. In the present study, we investigated the impact of MTX dosage on clinical outcomes following UCBT in a single-center retrospective study. Of 70 UCBT recipients included in this study, 37 received MTX at doses of 10 mg/m2 on day 1, and 7 mg/m2 on days 3 and 6 (low-dose MTX: LD-MTX), and 33 received MTX at doses of 15 mg/m2 on day 1, and 10 mg/m2 on days 3 and 6 (standard-dose MTX: SD-MTX), in addition to tacrolimus (TAC). Grade 3-4 acute GVHD and/or transplant-related complications with endothelial cell damage were considered severe transplant-related complications. Univariate analysis findings revealed that the risk of grade 3-4 acute GVHD was significantly lower in the SD-MTX group than in the LD-MTX group (P = 0.013). Multivariate analysis findings revealed that SD-MTX was significantly associated with a lower incidence of severe transplant-related complications (HR = 0.25; 95% CI, 0.07-0.87; P = 0.029). We conclude that SD-MTX in combination with tacrolimus is optimal for GVHD prophylaxis in UCBT.

Decline of forced expiratory volume in 1 s after allogeneic hematopoietic cell transplantation is a good indicator for pulmonary damage and is associated with busulfan use.

Reduced pulmonary function is commonly observed after allogeneic hematopoietic stem cell transplantation (HSCT); however, its relationship with the development of noninfectious pulmonary complications (NIPCs) is unclear, and the impact of changes in pulmonary function test (PFT) values on HSCT outcome remains controversial. We conducted a retrospective study including 150 patients to investigate changes in PFTs and impact on clinical outcome. PFT data at around 1 year after HSCT were available in 84 patients, and showed a significant time-dependent decline in percentage predicted forced expiratory volume in 1 s and other parameters. We focused on %FEV1, calculated decline of %FEV1 from pretransplant to around 1 year after HSCT (Δ%FEV1), and divided patients into good-Δ%FEV1 or poor-Δ%FEV1 groups, using a cut-off point of 20% decline of Δ%FEV1. In the poor-Δ%FEV1 group, half of the patients developed NIPCs. In the good-Δ%FEV1 group, PFT values were maintained, whereas those of the poor-Δ%FEV1 group declined significantly. Multivariate analysis showed that busulfan use was a risk factor for %FEV1 decline, and poor-Δ%FEV1 was a risk factor for overall survival. These data indicate that decline of %FEV1 may be a useful indicator of pulmonary damage after HSCT, and is strongly associated with busulfan use.

Promising Outcome of Umbilical Cord Blood Transplantation in Patients with Multiple Comorbidities.

The hematopoietic cell transplantation-specific comorbidity index (HCT-CI) has been recently proposed to predict the probability of nonrelapse mortality (NRM) and overall survival (OS) in allogeneic hematopoietic stem cell transplantation (HSCT). However, the usefulness of the HCT-CI in single-unit umbilical cord blood transplantation (UCBT) remains unclear. We investigated the impact of the HCT-CI on the clinical outcomes of allogeneic HSCT in a single-center retrospective study including 53 recipients of UCBT (UCBT group) and 90 recipients of other HSCT (non-UCBT group). In the non-UCBT group 2-year OS rates for HCT-CI score < 3 and ≥3 were 67% (n = 74; 95% confidence interval [CI], 54% to 78%) and 26% (n = 16; 95% CI, 7% to 51%), respectively (P = .001). In the UCBT group these rates were 66% (n = 39; 95% CI, 48% to 79%) and 69% (n = 14; 95% CI, 36% to 87%), respectively (P = .73). In the non-UCBT group 1-year NRM rates for HCT-CI score < 3 and ≥3 were 14% (95% CI, 6.4% to 22%) and 37% (95% CI, 14% to 61%), respectively (P = .02). In the UCBT group these rates were 6.1% (95% CI, 3.4% to 24%) and 7.7% (95% CI, .4% to 29%), respectively (P = .78). Using multivariate analysis we showed that HCT-CI score ≥ 3 was significantly associated with lower OS (hazard ratio, 3.06; 95% CI, 1.47 to 6.38; P = .003) and higher NRM (hazard ratio, 2.87; 95% CI, 1.18 to 6.96; P = .02) for the non-UCBT group. UCBT showed good OS with low incidence of NRM, even in patients with high HCT-CI scores. Altogether, we propose single-unit UCB to be a promising stem cell source for improving survival in patients with multiple comorbidities.

Acute Megakaryoblastic Leukemia Developing as Donor Cell Leukemia after Umbilical Cord Blood Transplantation.

A 64-year-old man with acute myeloid leukemia underwent umbilical cord blood transplantation (UCBT). After 11 months of complete remission (CR) following UCBT, the bone marrow showed 7.5% myeloblasts. CR was obtained after a single course of azacitidine monotherapy, but the myeloblasts gradually increased in the blood. We made a diagnosis of acute megakaryoblastic leukemia derived from donor cell with a fluorescence in situ hybridization (FISH) analysis of the sex chromosomes and an immunophenotypic analysis. Azacitidine was administered again and produced a therapeutic effect of stable disease. This case suggests that azacitidine may be a useful therapy for patients with acute megakaryoblastic leukemia in situations in which intensive chemotherapy and transplantation are not indicated.

Small-molecule Hedgehog inhibitor attenuates the leukemia-initiation potential of acute myeloid leukemia cells.

Aberrant activation of the Hedgehog signaling pathway has been implicated in the maintenance of leukemia stem cell populations in several model systems. PF-04449913 (PF-913) is a selective, small-molecule inhibitor of Smoothened, a membrane protein that regulates the Hedgehog pathway. However, details of the proof-of-concept and mechanism of action of PF-913 following administration to patients with acute myeloid leukemia (AML) are unclear. This study examined the role of the Hedgehog signaling pathway in AML cells, and evaluated the in vitro and in vivo effects of the Smoothened inhibitor PF-913. In primary AML cells, activation of the Hedgehog signaling pathway was more pronounced in CD34+ cells than CD34- cells. In vitro treatment with PF-913 induced a decrease in the quiescent cell population accompanied by minimal cell death. In vivo treatment with PF-913 attenuated the leukemia-initiation potential of AML cells in a serial transplantation mouse model, while limiting reduction of tumor burden in a primary xenotransplant system. Comprehensive gene set enrichment analysis revealed that PF-913 modulated self-renewal signatures and cell cycle progression. Furthermore, PF-913 sensitized AML cells to cytosine arabinoside, and abrogated resistance to cytosine arabinoside in AML cells cocultured with HS-5 stromal cells. These findings imply that pharmacologic inhibition of Hedgehog signaling attenuates the leukemia-initiation potential, and also enhanced AML therapy by sensitizing dormant leukemia stem cells to chemotherapy and overcoming resistance in the bone marrow microenvironment.

Combinations of cytogenetics and international scoring system can predict poor prognosis in multiple myeloma after high-dose chemotherapy and autologous stem cell transplantation.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard therapy for newly diagnosed multiple myeloma. Combinations of recently proposed prognostic factors such as cytogenetics and international scoring system (ISS) may be useful to predict prognosis after ASCT. This study evaluated 60 consecutive patients who underwent ASCT in four institutes. The median age of patients was 57 years old. Cytogenetic analyses of bone marrow at diagnosis detected metaphase abnormalities in 9 of 51 patients and interphase abnormalities in six of 35 patients (17p13 deletion, t(4;14) and t(14;16)). Seventeen patients had ISS stage 3 at diagnosis. Twenty-five patients who had any of these risk factors were defined as high risk. All patients were conditioned with high-dose melphalan. With a median follow-up of 3.4 years, overall survival and event-free survival at 3 years were significantly worse in high-risk patients (48% vs. 97%; P = 0.0005 and 16% vs. 37%; P = 0.038, respectively) despite the higher CR plus VGPR rate among high-risk patients. In addition, survival at 1 year after progression was significantly worse in high-risk patients despite salvage chemotherapy containing thalidomide (32% vs. 100%, P = 0.0001). Combinations of cytogenetics and ISS could readily predict prognosis. Quality of response is a poor surrogate marker for ultimate outcome. High-risk patients may need more effective treatment.

Establishment of rat model of acute staphylococcal osteomyelitis: relationship between inoculation dose and development of osteomyelitis.

INTRODUCTION: Many animal models of acute and chronic osteomyelitis have been developed. In these models, osteomyelitic lesions are induced using sclerosing agents and foreign bodies with bacterial strains. In the present rat model, these sclerosing agents were not used. We assessed the relationship between inoculation dose and histological, radiological, and microbiological changes in the acute phase (1 week after inoculation) using this rat osteomyelitis model. MATERIALS AND METHODS: An experimental rat model of acute osteomyelitis was developed by direct inoculation of the virulent strain BB of Staphylococcus aureus into tibial bone without sclerosants. To examine the relationship between the inoculation dose of the bacteria and the progression of the osteomyelitis, the inoculated lesions were assessed for changes in histological, radiological, and bacteriological parameters at 1 week after infection. Serial dilutions of the bacteria [6 x 10(1) to 6 x 10(5) colony-forming units (CFU)/5 microl] suspended in saline or saline alone were inoculated into the proximal metaphysis of the tibia. RESULTS: Development of significant histological and radiological signs of osteomyelitis required an inoculum of at least 6 x 10(3) CFU/5 microl. The number of viable bacteria at the lesion reached a maximum of 6 x 10(3) CFU/5 microl. CONCLUSION: These results suggest that strain BB induces the development of acute staphylococcal osteomyelitis with clear infective destruction in the tibia, and that our model may be applied to the identification of virulence factors in studies of posttraumatic osteomyelitis.