Insight into Population Structure and Drug Resistance of Pediatric Tuberculosis Strains from China and Russia Gained through Whole-Genome Sequencing.

This study aimed to determine phenotypic and genotypic drug resistance patterns of Mycobacterium tuberculosis strains from children with tuberculosis (TB) in China and Russia, two high-burden countries for multi/extensively-drug resistant (MDR/XDR) TB. Whole-genome sequencing data of M. tuberculosis isolates from China (n = 137) and Russia (n = 60) were analyzed for phylogenetic markers and drug-resistance mutations, followed by comparison with phenotypic susceptibility data. The Beijing genotype was detected in 126 Chinese and 50 Russian isolates. The Euro-American lineage was detected in 10 Russian and 11 Chinese isolates. In the Russian collection, the Beijing genotype and Beijing B0/W148-cluster were dominated by MDR strains (68% and 94%, respectively). Ninety percent of B0/W148 strains were phenotypically pre-XDR. In the Chinese collection, neither of the Beijing sublineages was associated with MDR/pre-XDR status. MDR was mostly caused by low fitness cost mutations (rpoB S450L, katG S315T, rpsL K43R). Chinese rifampicin-resistant strains demonstrated a higher diversity of resistance mutations than Russian isolates (p = 0.003). The rifampicin and isoniazid resistance compensatory mutations were detected in some MDR strains, but they were not widespread. The molecular mechanisms of M. tuberculosis adaptation to anti-TB treatment are not unique to the pediatric strains, but they reflect the general situation with TB in Russia and China.

Nonbacterial and bacterial osteomyelitis in children: a case-control retrospective study.

Purpose: Osteomyelitis is a group of bone infectious (bacterial osteomyeilitis-BO) and noninfectious inflammatory diseases (nonbacterial osteomyelitis-NBO) with similar clinical, radiology, and laboratory features. Many patients with NBO are misdiagnosed as BO and receive unnecessary antibiotics and surgery. Our study aimed to compare clinical and laboratory features of NBO and BO in children, to define key discriminative criteria, and to create an NBO diagnostic score (NBODS). Methods: The retrospective multicenter cohort study included clinical, laboratory, and instrumental information about histologically confirmed NBO (n = 91) and BO (n = 31). The variables allowed us to differentiate both conditions used to construct and validate the NBO DS. Results: The main differences between NBO and BO are as follows: onset age-7.3 (2.5; 10.6) vs. 10.5 (6.5; 12.7) years (p = 0.03), frequency of fever (34.1% vs. 90.6%, p = 0.0000001), symptomatic arthritis (67% vs. 28.1%, p = 0.0001), monofocal involvement (28.6% vs. 100%, p = 0.0000001), spine (32% vs. 6%, p = 0.004), femur (41% vs. 13%, p = 0.004), foot bones (40% vs. 13%, p = 0.005), clavicula (11% vs. 0%, p = 0.05), and sternum (11% vs. 0%, p = 0.039) involvement. The following four criteria are included in the NBO DS: CRP ≤ 55 mg/l (56 points), multifocal involvement (27 points), femur involvement (17 points), and neutrophil bands ≤ 220 cell/µl (15 points). The sum > 17 points allowed to differentiate NBO from BO with a sensitivity of 89.0% and a specificity of 96.9%. Conclusion: The diagnostic criteria may help discriminate NBO and BO and avoid excessive antibacterial treatment and surgery.

Cytokine profile in patients with chronic non-bacterial osteomyelitis, juvenile idiopathic arthritis, and insulin-dependent diabetes mellitus.

OBJECTIVES: Our study aimed to evaluate the cytokine levels in pediatric chronic non-bacterial osteomyelitis (CNO) patients and compare these with other immune-mediated diseases and healthy controls. METHODS: In this prospective study, we included 42 children with CNO, 28 patients with non-systemic juvenile idiopathic arthritis (JIA), 17 children with insulin-dependent diabetes mellitus (IDDM), and 30 healthy age-matched controls. In each of the CNO patients and comparison groups, the levels of 14-3-3-η protein, S100A8/A9 protein, interleukin-4 (IL-4), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) were measured by ELISA assay. RESULTS: All studied cytokines in the CNO patients were significantly higher than controls, and IDDM, 14-3-3-η protein, IL-18, IL-4, IL-17, IL-1ß, and TNF-α were less than in JIA patients. In the discriminant analysis, ESR, 14-3-3 protein, S100A8/A9, IL-18, IL-4, and TNF-α can discriminate CNO from JIA, and 14-3-3 protein, S100A8/A9, IL-18, IL-17, IL-4, and TNF-α can distinguish CNO from other diseases and HC. CONCLUSION: The increased level of pro-inflammatory cytokines confirms the role of monocyte-driven inflammation in CNO patients. Cytokines may prove valuable as biomarkers and potential therapeutic targets for CNO.

First insight into the whole-genome sequence variations in Mycobacterium bovis BCG-1 (Russia) vaccine seed lots and their progeny clinical isolates from children with BCG-induced adverse events.

BACKGROUND: The only licensed live Bacille Calmette-Guérin (BCG) vaccine used to prevent severe childhood tuberculosis comprises genetically divergent strains with variable protective efficacy and rates of BCG-induced adverse events. The whole-genome sequencing (WGS) allowed evaluating the genome stability of BCG strains and the impact of spontaneous heterogeneity in seed and commercial lots on the efficacy of BCG-vaccines in different countries. Our study aimed to assess sequence variations and their putative effects on genes and protein functions in the BCG-1 (Russia) seed lots compared to their progeny isolates available from immunocompetent children with BCG-induced disease (mainly, osteitis). RESULTS: Based on the WGS data, we analyzed the links between seed lots 361, 367, and 368 used for vaccine manufacture in Russia in different periods, and their nine progeny isolates recovered from immunocompetent children with BCG-induced disease. The complete catalog of variants in genes relative to the reference genome (GenBank: CP013741) included 4 synonymous and 8 nonsynonymous single nucleotide polymorphisms, and 3 frameshift deletions. Seed lot 361 shared variants with 2 of 6 descendant isolates that had higher proportions of such polymorphisms in several genes, including ppsC, eccD5, and eccA5 involved in metabolism and cell wall processes and reportedly associated with virulence in mycobacteria. One isolate preserved variants of its parent seed lot 361 without gain of further changes in the sequence profile within 14 years. CONCLUSIONS: The background genomic information allowed us for the first time to follow the BCG diversity starting from the freeze-dried seed lots to descendant clinical isolates. Sequence variations in several genes of seed lot 361 did not alter the genomic stability and viability of the vaccine and appeared accumulated in isolates during the survival in the human organism. The impact of the observed variations in the context of association with the development of BCG-induced disease should be evaluated in parallel with the immune status and host genetics. Comparative genomic studies of BCG seed lots and their descendant clinical isolates represent a beneficial approach to better understand the molecular bases of efficacy and adverse events during the long-term survival of BCG in the host organism.

How nonbacterial osteomyelitis could be discriminated from tuberculosis in the early stages: the simple algorithm.

Chronic nonbacterial osteomyelitis (CNО) and tuberculous osteomyelitis (TBO) are both primarily chronic inflammatory bone diseases with similar clinical and radiological findings, but entirely different in aetiology, pathogenesis, treatment, and outcomes. Our study aimed to evaluate the clinical and laboratory features which could discriminate the CNO and TBO. The study included 124 patients-91 with CNO and 33 with TBO. All patients underwent routine blood tests: WBC, platelets, ESR, C-reactive protein, haemoglobin, and imaging. The ability of each variable to discriminate CNO from TBO was evaluated with sensitivity and specificity analysis, AUC-ROC analysis, and calculating the odds ratio. Patients with TBO had less number of bone foci (p = 0.0000001), onset age (p = 0.00001), rarely articular involvement (p = 0.01), lower haemoglobin level (p = 0.02), higher incidence of TBO in the male subjects (p = 0.002), and higher leukocyte bands (p = 0.0000001). TBO is rarely characterized by spine (p = 0.0009), foot (p = 0.01), and clavicula (p = 0.047) involvement. The diagnostic rule: criteria allowing to differentiate NBO from TBO are negative bone microbiota tests (sensitivity-100.0%, specificity-100.0%) or major discriminative criteria or clavicula involvement alone (sensitivity-11.0%, specificity-100.0%) and at least four from the five additional criteria: number of foci > 1.0 (p = 0.00002), WBC ≤ 11.0 (p = 0.004), neutrophil bands ≤ 120.0 × 106/l (p = 0.002), lymphocytes ≤ 52% (p = 0.0005), and CRP > 0.2 mg/l (p = 0.003). All patients with monofocal CNO required bone biopsy with microbiology assessment. The created provisional criteria may help to discriminate TBO and CNO and should be used only with other known diagnostic tools. Key Points • Nonbacterial osteomyelitis and tuberculous osteomyelitis are both primarily chronic inflammatory bone diseases with similar presentations. • Nonbacterial osteomyelitis and tuberculous osteomyelitis may be associated with other immune-mediated diseases. • Only bone biopsy can confirm and discriminate both conditions. All patients with monofocal CNO required bone biopsy with microbiology assessment.

The peculiarities and treatment outcomes of the spinal form of chronic non-bacterial osteomyelitis in children: a retrospective cohort study.

Chronic non-bacterial osteomyelitis (CNO) is a group of immune-mediated diseases which appears in bone inflammation, destruction and some orthopaedic consequences, especially in the cases of spinal involvement. This study is to compare characteristics and treatment outcomes of CNO patients with spinal involvement. The retrospective cohort study included data from 91 pediatric patients with CNO. The diagnosis is based on Jannson's criteria with morphological confirmation (nonspecific chronic inflammation). Spine involvement detected by X-ray, computed tomography, magnetic resonance imaging, and bone scan in 29 (31.9%) patients. No differences in the family history, concomitant immune-mediated diseases between spinal (SpCNO) and peripheral (pCNO) forms of CNO have been revealed. Only 5 (10.2%) SpCNO patients (10.2%) had monofocal monovertebral involvement. The main risk factors of spinal involvement were female sex: RR = 2.0 (1.1; 3.9), sensitivity (Se) = 0.66, specificity (Sp) = 0.6; multifocal involvement: RR = 2.1 (0.9; 5.0), Se = 0.83, Sp = 0.37; no foot bones involvement: RR = 3.1 (1.3; 7.5), Se = 0.83, Sp = 0.5; sternum involvement RR = 2.3 (1.3; 4.1), Se = 0.24, Sp = 0.94. In the linear regression analysis only female sex (p = 0.005), multifocal involvement (p = 0.000001) and absence of foot bones involvement (p = 0.000001) were independent risk factors of spinal involvement (p = 0.000001). The response rate on bisphosphonates and tumor necrosis factor-a inhibitors was 90.9% and 66.7%, consequently. Only 4/29 (13.8%) SpCNO patients underwent surgery due to severe spinal instability or deformities. The spinal involvement is frequent in CNO and could be crucial for choosing a treatment strategy. Bisphosphonates and TNFa-inhibitors could be effective treatment options for severe SpCNO.

Comparison of different treatment approaches of pediatric chronic non-bacterial osteomyelitis.

Chronic non-bacterial osteomyelitis (CNO) is a chronic inflammatory bone disease which usually manifests in children and adolescents. There are a few data about pathogenesis and treatment. The aim of the study to compare the efficacy of different treatment approaches in pediatric CNO cohort patient. Fifty two children (25 boys and 27 girls) with CNO with average age at the onset of the disease 8.4 years (5.4; 11.0), number of foci - 3.0 (2.0; 6.0, incl. multifocal cases in 80.8%). Non-steroid anti-inflammatory drugs (NSAID) was the first-line treatment for non-vertebral cases, as well as pamidronate (PAM) for vertebral involvement. Second-line treatment includes sulfasalazine (SSZ), methotrexate (MTX), PAM and tumor necrosis factor-α inhibitors (TNFα-inh). We evaluated the dynamics of pain, patient's and physician's (MDVAS) assessment with visual-analog scale (VAS) and ability to each medication to achieve remission of CNO activity. According to the NSAID, MTX, SSZ, PAM and TNFα-inh groups the following data were registered: patient's VAS: - 14.2% (p = 0.05), - 50.0% (p = 0.04), - 23.1 (p = 0.89), - 83.3% (p = 0.0001), - 73.6% (p = 0.0007); painVAS: - 21.9% (p = 0.01), - 18.6% (p = 0.13), + 36.4 (p = 0.89), - 79.7% (p = 0.00016), - 74.1%, (p = 0.0015); MDVAS: - 13.8% (p = 0.13); - 56.4% (p = 0.09), + 30.8% (p = 0.89), - 74.7%, (p = 0.0001), - 82.1 (p = 0.0015) respectively. The ability of each treatment strategy to achieve the CNO remission was 52.6%, 44.4%, 57,1%, 88.8% and 73.3%, respectively (log-rank test, p = 0.001). The efficacy of treatment approaches for CNO depended on the severity of the disease. NSAID, methotrexate, and sulfasalazine were effective in forms without spine involvement, but pamidronate and TNF-a inhibitors were useful in vertebral forms of CNO. Pamidronate and TNF-a inhibitors more extensively suppressed CNO activity. The randomized controlled trials for assessment of the efficacy and safety of these medications is mandatory to confirm these results.

Multilevel spinal reconstruction in pediatric patients under 4 years old with non-congenital pathology (10-year single-center cohort study).

PURPOSE: To evaluate the influence of anterior fusion option on the short- and long-time outcomes on multilevel spinal reconstructions in young children. METHODS: Forty-five patients aged under 4 years old (2 years 2 months ± 11 months in average) underwent spinal reconstruction due to tuberculosis spondylitis (35), pyogenic spondylitis (9) and spinal tumors (1) complicated by angular kyphosis exceeded 20° (49.2° ± 14.3° in average). All lesions involved two or more spinal motion segments. Clinical and radiographic data were compared in two groups depended on the types of anterior fusion: titanium mesh cage with bone graft (TMC + BG) (19 patients) and cortical BG only (26). The average follow-up was 5 years 10 months ± 2 years 8 months (min = 3 years; max = 12 years). RESULTS: The deformity correction was similar in groups. The operation time and blood loss were less in TMC + BG group (p = 0.001) as a times for anterior bone block formation (p < 0.001) and posterior instrumentation removal (p = 0.003). Ten late post-op complications registered include disease's recurrence (1), pseudoarthrosis (6), deformity progression (1) and graft resorption (1). The complication rate was less in TMC + BG than in BG group: two and eight cases consequently, p = 0.024. CONCLUSIONS: Multilevel spinal reconstruction in early aged patients is safe and effective procedure. The anterior fusion by TMC with bone autograft has advantages of reducing blood loss, operation time, time for anterior block formation and complications rate compared with bone autograft only. These slides can be retrieved under Electronic Supplementary Material.

Tuberculous spondylitis in Russia and prominent role of multidrug-resistant clone Mycobacterium tuberculosis Beijing B0/W148.

Extrapulmonary and, in particular, spinal tuberculosis (TB) constitutes a minor but significant part of the total TB incidence. In spite of this, almost no studies on the genetic diversity and drug resistance of Mycobacterium tuberculosis isolates from spinal TB patients have been published to date. Here, we report results of the first Russian and globally largest molecular study of M. tuberculosis isolates recovered from patients with tuberculous spondylitis (TBS). The majority of 107 isolates were assigned to the Beijing genotype (n = 80); the other main families were T (n = 11), Ural (n = 7), and LAM (n = 4). Multidrug resistance (MDR) was more frequently found among Beijing (90.5%) and, intriguingly, Ural (71.4%) isolates than other genotypes (5%; P < 0.001). The extremely drug-resistant (XDR) phenotype was exclusively found in the Beijing isolates (n = 7). A notable prevalence of the rpoB531 and katG315 mutations in Beijing strains that were similarly high in both TBS (this study) and published pulmonary TB (PTB) samples from Russia shows that TBS and PTB Beijing strains follow the same paradigm of acquisition of rifampin (RIF) and isoniazid (INH) resistance. The 24-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) subtyping of 80 Beijing isolates further discriminated them into 24 types (Hunter Gaston index [HGI] = 0.83); types 100-32 and 94-32 represented the largest groups. A genotype of Russian successful clone B0/W148 was identified in 30 of 80 Beijing isolates. In conclusion, this study highlighted a crucial impact of the Beijing genotype and the especially prominent role of its MDR-associated successful clone B0/W148 cluster in the development of spinal MDR-TB in Russian patients.