Use of transcriptomics for diagnosis of infections and sepsis in children: A narrative review.

AIM: The aim of this review was to summarise the most recent evidence about the use of omics-based techniques as an instrument for a more rapid and accurate characterisation of respiratory tract infections, neurological infections and sepsis in paediatrics. METHODS: We performed a narrative review using PubMed and a set of inclusion criteria: English language articles, clinical trials, meta-analysis and reviews including only paediatric population inherited to this topic in the last 15 years. RESULTS: The examined studies suggest that host gene expression signatures are an effective method to characterise the different types of infections, to distinguish infection from colonisation and, in some cases, to assess the severity of the disease in children. CONCLUSIONS: 'Omics-based techniques' may help to define the aetiology of infections in paediatrics, representing a useful tool to choose the most appropriate therapies and limit antibiotic resistance.

Pertussis outbreak in neonates and young infants across Italy, January to May 2024: implications for vaccination strategies.

Since January 2024, Italy experiences a pertussis outbreak, primarily affecting neonates and unvaccinated infants at high risk of severe complications and mortality; 11 major paediatric centres noted 108 hospitalisations and three deaths by 10 May. The outbreak reflects increased circulation of Bordetella pertussis and non-adherence to immunisation recommendations during pregnancy. Public health interventions, including maternal immunisation, vaccination of infants as early as possible and post-exposure prophylaxis, are critical for reducing the burden of pertussis and preventing further mortality.

Sofosbuvir-velpatasvir-voxilaprevir in adolescents 12 to 17 years old with HCV infection.

BACKGROUND AND AIMS: Sofosbuvir-velpatasvir-voxilaprevir is a pangenotypic regimen for chronic HCV infection. In the USA and Europe, sofosbuvir-velpatasvir-voxilaprevir once daily for 12 weeks is indicated for adults who previously received an HCV NS5A inhibitor. In Europe, sofosbuvir-velpatasvir-voxilaprevir is also indicated in the absence of prior HCV direct-acting antiviral (DAA) therapy as an 8-week or 12-week regimen. In an open-label study, we evaluated the safety, efficacy, and pharmacokinetics of sofosbuvir-velpatasvir-voxilaprevir in adolescents 12 to 17 years with chronic HCV of any genotype. METHODS: In this Phase 2, multicenter study, sofosbuvir-velpatasvir-voxilaprevir 400/100/100 mg daily was administered to adolescents for 8 weeks if DAA-naïve or for 12 weeks for cirrhosis or prior DAA failure. The key efficacy endpoint was sustained virologic response 12 weeks after therapy (SVR12). Intensive pharmacokinetic sampling was done in 14 patients at week 2 or 4, and samples for population pharmacokinetics were collected in all patients. RESULTS: All patients (n = 21) were naïve to HCV DAAs, and none had cirrhosis. HCV genotype 3a infection was most common, occurring in 43% of patients. Overall, 100% of patients (21 of 21) reached SVR12. The most common adverse events were abdominal pain and headache (24% each) and nausea (19%); no adverse events led to discontinuation. The only serious adverse event, hypotension, was considered related to study drug and resolved the same day without interruption of treatment. Sofosbuvir-velpatasvir-voxilaprevir exposures were similar to those observed in adults. CONCLUSIONS: The pangenotypic regimen of sofosbuvir-velpatasvir-voxilaprevir is highly efficacious and well-tolerated in treating chronic HCV infection in adolescents.

The Immunological Profile of SARS-CoV-2 Infection in Children Is Linked to Clinical Severity and Age.

Coronavirus disease 19 (COVID-19) is clinically less severe in children, even if the wide variety and degree of severity of symptoms reported in children pose a still-unresolved challenge for clinicians. We performed an in-depth analysis of the immunological profiles of 18 hospitalized SARS-CoV-2-infected children, whose results were compared to those obtained from 13 age- and sex-matched healthy controls (HC). The patients were categorized as paucisymptomatic/moderate (55.6%) or severe/critical (44.5%) according to established diagnostic criteria and further stratified into the categories of infants (1-12 months), children (1-12 years), and adolescents (>12 years). We assessed SARS-CoV-2-specific RBD antibodies (Ab), neutralizing antibodies (nAb), and circulating cytokines/chemokines in the plasma, and the SARS-CoV-2-specific immune response was measured in PBMCs by gene expression and secretome analyses. Our results showed peculiar circulating cytokine/chemokine profiles among patients sharing a similar clinical phenotype. A cluster of patients consisting of infants with severe symptoms presented hyperinflammatory profiles, together with extremely polarized antibody profiles. In a second cluster consisting of paucisymptomatic patients, a less pronounced increase in the level of inflammatory cytokines, together with an association between the selected cytokines and humoral responses, was observed. A third cluster, again consisting of paucisymptomatic patients, showed a circulating cytokine/chemokine profile which overlapped with that of the HC. The SARS-CoV-2-stimulated production of pro-inflammatory proteins, T lymphocyte activation, and migration-specific proteins, were significantly increased in SARS-CoV-2-infected children compared to the HC. Our findings suggest that immune response activation in the course of SARS-CoV-2 infection in children is directly correlated with clinical severity and, to a lesser extent, age.

Off-label use of combined antiretroviral therapy, analysis of data collected by the Italian Register for HIV-1 infection in paediatrics in a large cohort of children.

BACKGROUND: Early start of highly active antiretroviral therapy (HAART) in perinatally HIV-1 infected children is the optimal strategy to prevent immunological and clinical deterioration. To date, according to EMA, only 35% of antiretroviral drugs are licenced in children < 2 years of age and 60% in those aged 2-12 years, due to the lack of adequate paediatric clinical studies on pharmacokinetics, pharmacodynamics and drug safety in children. METHODS: An observational retrospective study investigating the rate and the outcomes of off-label prescription of HAART was conducted on 225 perinatally HIV-1 infected children enrolled in the Italian Register for HIV Infection in Children and followed-up from 2001 to 2018. RESULTS: 22.2% (50/225) of included children were receiving an off-label HAART regimen at last check. Only 26% (13/50) of off-label children had an undetectable viral load (VL) before the commencing of the regimen and the 52.0% (26/50) had a CD4 + T lymphocyte percentage > 25%. At last check, during the off label regimen, the 80% (40/50) of patients had an undetectable VL, and 90% (45/50) of them displayed CD4 + T lymphocyte percentage > 25%. The most widely used off-label drugs were: dolutegravir/abacavir/lamivudine (16%; 8/50), emtricitbine/tenofovir disoproxil (22%; 11/50), lopinavir/ritonavir (20%; 10/50) and elvitegravir/cobicistat/emtricitabine/ tenofovir alafenamide (10%; 10/50). At logistic regression analysis, detectable VL before starting the current HAART regimen was a risk factor for receiving an off-label therapy (OR: 2.41; 95% CI 1.13-5.19; p = 0.024). Moreover, children < 2 years of age were at increased risk for receiving off-label HAART with respect to older children (OR: 3.24; 95% CI 1063-7.3; p = 0.001). Even if our safety data regarding off-label regimens where poor, no adverse event was reported. CONCLUSION: The prescription of an off-label HAART regimen in perinatally HIV-1 infected children was common, in particular in children with detectable VL despite previous HAART and in younger children, especially those receiving their first regimen. Our data suggest similar proportions of virological and immunological successes at last check among children receiving off-label or on-label HAART. Larger studies are needed to better clarify efficacy and safety of off-label HAART regimens in children, in order to allow the enlargement of on-label prescription in children.

Efficacy of Sofosbuvir/Ledipasvir in Adolescents With Chronic Hepatitis C Genotypes 1, 3, and 4: A Real-world Study.

OBJECTIVES: Sofosbuvir/Ledipasvir (SOF/LDV) has been approved by the European Medicine Agency (EMA) for the treatment of children and adolescents (at least 3 years of age) with chronic hepatitis C (CHC) genotype 1, 3, and 4 infection. The aim of this study was to evaluate the efficacy and safety of SOF/LDV in adolescents (12 to <18 years old) with CHC in the real-world setting. METHODS: Prospective, open-label, multicentre study involving 12 Italian centres. Patients received the fixed-dose combination of SOF/LDV (400/90 mg) once daily ± ribavirin as per EMA approval and recommendations. The key efficacy endpoint was sustained virological response 12 weeks after the end of treatment (SVR12) as per intention-to-treat analysis. Safety was assessed by adverse events and clinical/laboratory data. RESULTS: Seventy-eight consecutive adolescents (median age 15.2 years, range 12-17.9; girls 53.8%) were enrolled and treated between June 2018 and December 2019. Genotype distribution was as follows: genotype 1 (82.1%), 3 (2.5%), and 4 (15.4%). Seventy-six (97.4%) patients completed treatment and follow-up. Overall, SVR12 was 98.7%. One patient was lost to follow-up after 4 weeks of treatment; 1 patient completed treatment and missed the follow-up visit. No virological breakthrough or relapse were observed. No patient experienced grade 3 to 4 adverse event or serious adverse event. CONCLUSIONS: The results of this real-world study confirmed the high efficacy and the optimal safety profile of SOF/LDV for treatment of CHC in adolescents.

Defining the clinical phenotype of COVID-19 in children.

On January 7, 2020, a novel coronavirus (SARS-CoV-2) was identified as the causative agent of a cluster of pneumonia of unknown origin detected in Wuhan City by Chinese authorities. Since SARS-CoV-2 discovery, the corresponding disease (COVID-19) has rapidly expanded throughout the globe, making as a consequence the World Health Organization (WHO) declaring a pandemic. As of May 19, 2020, over 4.806.299 cases of COVID-19 had been confirmed worldwide, with more than 318.599 deaths.

Pregnancy and neonatal outcomes in women with HIV-1 exposed to integrase inhibitors, protease inhibitors and non-nucleoside reverse transcriptase inhibitors: an observational study.

PURPOSE: Recommended regimens for pregnant women with HIV-1 are composed of two nucleoside reverse transcriptase inhibitors (NRTI) plus either a ritonavir-boosted protease inhibitor (PI) or an integrase strand transfer inhibitor (ISTI), with non-nucleoside reverse transcriptase inhibitors (NNRTI) representing an alternative drug class. The study's purpose was to compare these three options in terms of pregnancy outcomes. METHODS: Data from a national observational study of pregnant women with HIV-1 were used. The analysis included all pregnancies reported between 2008 and 2018, ending in live births and exposed within 32 weeks of gestation to three-drug regimens composed of a NRTI backbone plus a PI, a NNRTI or a ISTI, without class switching during pregnancy. Clinical and laboratory outcomes were evaluated in univariate and multivariable analyses. RESULTS: Overall, 794 exposed pregnancies were analyzed (PI 78.4%, NNRTI 15.4%, ISTI 6.2%). Almost all outcomes had similar rates in the three groups. Women who received PI in pregnancy were less likely to be virologically suppressed at third trimester. PI use was associated with higher bilirubin and triglyceride levels, and ISTI use with a lower rate of low birthweight. The differences in viral suppression at third trimester and in low birthweight were not maintained in multivariable analyses that were adjusted for confounders. DISCUSSION: We found no major differences in a wide range of outcomes relevant for pregnant women with HIV. Such results are reassuring, and this information may be helpful in a context of preconception counseling when therapeutic choices for pregnancy are discussed between women and care providers.

Longitudinal Immune Phenotype Assessment and Serological Outcome in Foreign-born Children With Chronic Hepatitis B.

OBJECTIVES: The aim of the study was to assess changes in clinical phenotype, and identify determinants of outcome in children with chronic hepatitis B virus (HBV) infection born in HBV-endemic countries followed in 2 Italian tertiary care centers after immigration or adoption. METHODS: A prospective observational study on hepatitis B e-antibodies-negative chronic hepatitis B children started on 2002. Patients with liver fibrosis, or those needing antiviral treatment were excluded. Immune active patients were defined those with raised transaminases (alanine aminotransferase > 40 IU/L), immune tolerants those having normal alanine aminotransferase, both exhibiting substantial viral replication (HBV DNA >2000 IU/mL). RESULTS: Sixty-nine patients (44 boys, median age 4.7 years) had a median follow-up of 53 months. At entry, 18 (26%) children were immune tolerant, 47 (68%) immune active, and 4 had indeterminant immune status. At last follow-up, 14 (78%) of the immune-tolerant patients remained so, whereas only 23 (49%) of the immune active children maintained their initial immune phenotype. Seroconversion to hepatitis B e antibodies (SCHBe) occurred in only 2 (11%) immune tolerants, whereas 13 (28%) immune active patients achieved SCHBe.Ethnicity was the only feature independently correlated to SCHBe: Asian origin reduced by 4.1 times the probability of SCHBe (Asian vs other; odds ratio = 0.24 [95% confidence interval = 0.07-0.76]; P = 0.016) compared to other ethnicities, whereas viral genotype did not influence the outcome. CONCLUSIONS: Ethnicity and immune status phenotype against HBV, rather than HBV genotype, are the main determinants of SCHBe in foreign-born children with chronic HBV infection.

Factors Affecting Outcome of Tuberculosis in Children in Italy: An Ecological Study.

INTRODUCTION: Tuberculosis is a major problem in children depending on their families for management and a re-emerging disease in low incidence countries, where foreign-born cases account for a large proportion of cases. METHODS: We investigated socioeconomic features of families and their impact on management and outcome of children with tuberculosis disease seen at a tertiary care centre for paediatric infectious diseases in Italy. RESULTS: Forty-nine Italian and 30 foreign-origin children were included. Children from foreign families had more complicated diseases (20 % vs 0 %; P = 0.002), harbored more drug resistant strains (20 % vs 2 %; P = 0.011), showed longer hospital stay (12 ± 13.1 vs 5.1 ± 6.5 days; P = 0.012) and higher proportion of missed medical visits (15.7 ± 16 vs 8.6 ± 9.6; P ≤ 0.042) than those from Italian families. Harboring drug resistant strains was an independent risk factor for complicated disease course (OR: 72.98; 95 %CI: 1.54-3468.58; P = 0.029), and this risk is higher in children from Eastern Europe (OR: 10.16; 95 %CI: 1.7-61.9; P = 0.012). CONCLUSIONS: Children from immigrant families showed an increased risk of complicated course of tuberculosis due to a higher rate of resistant strains and raise problems in clinical management. Specific protocols are needed to support these populations ensuring easy access to health services and monitoring.

Atazanavir and lopinavir profile in pregnant women with HIV: tolerability, activity and pregnancy outcomes in an observational national study.

BACKGROUND: Atazanavir and lopinavir represent the main HIV protease inhibitors recommended in pregnancy, but comparative data in pregnant women are limited. METHODS: Women from a national observational study, exposed in pregnancy to either atazanavir or lopinavir, were compared for glucose and lipid profiles, liver function tests, CD4 count, HIV RNA and main pregnancy outcomes. Statistical methods included univariate and multivariable analyses. RESULTS: The study population included 428 pregnancies (lopinavir, 322; atazanavir, 106). The lopinavir group was characterized by higher rates of HIV diagnosis in pregnancy and treatment indication for maternal health, lower CD4 counts, higher HIV RNA levels, less frequent antiretroviral treatment at conception and shorter duration of drug exposure during pregnancy. No differences in pregnancy outcomes, glucose metabolism and weight gain were observed. The two groups also showed in a multivariable analysis similar odds for detectable HIV RNA in the third trimester (adjusted OR 0.85, 95% CI 0.35-2.10, P = 0.730). Total lipid levels were significantly higher in the lopinavir group (median values in the third trimester 239 versus 221 mg/dL for total cholesterol and 226 versus 181 mg/dL for triglycerides; P < 0.001 for both comparisons) and bilirubin levels were significantly higher in the atazanavir group (1.53 versus 0.46 mg/dL, P < 0.001). CONCLUSIONS: In this observational study atazanavir and lopinavir showed similar safety and activity in pregnancy, with no differences in the main pregnancy outcomes. Atazanavir use was associated with a better lipid profile and with higher bilirubin levels. Overall, the study findings confirm that these two HIV protease inhibitors represent equally valid alternative options.

Areal bone mineral density in pediatric patients with chronic hepatitis B or chronic hepatitis C.

Decreased bone mineral density (BMD) is a known complication of chronic liver disease in adults. Data on bone mass, an important factor for the development of osteoporosis in adult life, in young patients with chronic hepatitis B (HBV) and C virus (HCV) infections are scarce. We measured BMD at the lumbar spine and whole skeleton by dual-energy X-ray absorptiometry in 11 HBV- and 21 HCV-vertically infected untreated youths (3.9-21.1 years). BMD measurements were compared to those of 202 healthy subjects (3.0-21.9 years). The median BMD Z-score of the lumbar spine of HBV-infected patients was -0.3, ranging from -1.6 to 0.6, while the median whole skeleton BMD Z-score was 0.1 (-0.8 to 0.6). HBV-infected patients showed a median Z-score of the lumbar spine of 0.6 (-1.6 to 1.9), and a median whole skeleton BMD Z-score of 0.6, ranging from -1.5 to 1.4. Multivariate analyses have been performed to correct for differences in sex, age, and anthropometric measurements. Lumbar spine BMD values of HBV and HCV-infected patients were not significantly different from those of controls. Similarly, no differences were found between groups in total body BMD measurements. Our data suggest that, unlikely adult patients, untreated young patients with chronic HBV and HCV infection may not have impaired bone mass measurements.

Unexpected vertical transmission of HIV infection.

Mother-to-child transmission of HIV infection occurred in a child born from an HIV-infected mother with HIV-RNA undetectable during pregnancy. She was suffering from gastroenteritis in the last 3 weeks of gestation.

Immunophenotypes of Newborns From SARS-CoV-2-infected Mothers.

BACKGROUND: Little is known about the neonatal immunologic response to a maternal SARS-CoV-2 infection present during childbirth. Here we analyze a cohort of 75 neonates from SARS-CoV-2-infected mothers. METHODS: The SARS-CoV-2 infection status was laboratory assessed by real-time reverse transcription polymerase chain reaction on nasopharyngeal swabs (NPS) in both mothers during childbirth and neonates within 24 hours of life. Immunophenotypes of peripheral blood mononucleated cells and SARS-CoV-2 antispike IgA, IgM and IgG of the newborns were recorded. Ten (13.3%) of 75 neonates had positive NPS for SARS-CoV-2; 17 of 75 (23%) were SARS-CoV-2-IgG seropositive, of which one with positive NPS. All the newborns resulted seronegative for SARS-CoV-2 IgA and IgM and were asymptomatic. Our cohort of newborns was divided into groups according to IgG seropositivity (IgG+/-) and NPS results (NPS+/-). RESULTS: The count and proportion of lymphocyte subsets (evaluated measuring CD3, CD4, CD8 and CD19 markers) and of natural killer cells (evaluated by measuring the CD3-/CD16+/CD56+ subset) were all in the normal range, with no statistical differences among groups. We found a significant expansion of the T cell (CD3+) subset in the IgG+ group interpreted as the result of immune effects triggered by trained immunity in these newborns, but a decrease in CD4+ T cells for NPS+ neonates. It is therefore difficult to conclude that the decrease in CD4 can certainly be caused by an infection. CONCLUSIONS: A maternal SARS-CoV-2 infection resulted in an expansive effect of CD3+ T cells in IgG+ newborns; nonetheless, it seems not to affect structural and functional development of the newborn immune system.

Natural History and Management of Hepatitis C in Children: 25 Years Experience of a Reference Center in Northern Italy.

Hepatitis C virus (HCV) infection natural history and management in the pediatric population are still debated. We retrospectively evaluated the outcome of a HCV pediatric population managed at the Pediatric Infectious Disease Unit of Luigi Sacco Hospital (Milan, Italy) from January 1997 to January 2022 (median follow-up 10 years) and we focused on the role of new drugs and transient elastography. Fifty-seven patients were enrolled: 8 (14%) had a spontaneous clearance, 33 were treated (58%), 7 (12%) were not treated because they were under 12 years old and 9 were lost at follow-up. HCV RNA was undetectable in all treated patients at the end of therapy, after 12 weeks (SVR12) and for the rest of their follow-up. All patients treated underwent elastography before and 1 year after therapy. Median stiffness pretherapy was 5.6 kPa, and 9 patients (16%) had abnormal transient elastography (>7 kPa, median 8.7 kPa). Median stiffness after treatment in the abnormal group was 6.8 kPa. Direct-acting antiviral agents are a safe and effective therapy for HCV chronic infection in the pediatric population. Liver elastography is normal in many vertically infected children before 12 years, but, when abnormal, it shows a significant improvement after direct-acting antiviral agent treatment. Further studies are needed to evaluate the role of elastography at diagnosis and follow-up in children.

Pediatric Lyme Disease in Northern Italy: An 18-Year Single-Center Case Series.

Tracing the profile of pediatric Lyme borreliosis (LB) in Europe is difficult due to the interregional variation in its incidence and lack in notifications. Moreover, the identification of LB can be challenging. This study is an 18-year case series of 130 children and adolescents aged under 18 years referred to the Pediatric Infectious Diseases Unit at L. Sacco Hospital, Milan, with suspicion of LB, between January 2005 and July 2023. The routine serological workup consisted of a two-step process: an initial screening test followed by Western blot (WB). Forty-four (34%) patients were diagnosed with LB. The median age was six years, and 45% were females. Of the children with erythema migrans (EM), 33 (57%) were confirmed as having true EM, and, of these, 4 (12%) were atypical. Ten (23%) patients had early disseminated/late diseases, including facial nerve palsy (n = 3), early neuroborreliosis (n = 1), arthritis (n = 3), relapsing fever (n = 2), and borrelial lymphocytoma (n = 1). No asymptomatic infections were documented. Over seventy percent of confirmed LB cases (n = 31/44) recalled a history of tick bites; in this latter group, 19 (61%) were from the area of the Po River valley in Lombardy. Almost half of the children evaluated for LB complained of non-specific symptoms (fatigue, musculoskeletal pain, skin lesions/rash, and persistent headache), but these symptoms were observed in only two patients with confirmed LB. Most LB cases in our study were associated with EM; two-tier testing specificity was high, but we found frequent non-adherence to international recommendations with regard to the timing of serology, application of the two-step algorithm, and antibiotic over-prescription. Most children were initially assessed for a tick bite or a skin lesion suggestive of EM by a family pediatrician, highlighting the importance of improving awareness and knowledge around LB management at the primary healthcare level. Finally, the strengthening of LB surveillance at the national and European levels is necessary.

Children with perinatally acquired HIV exhibit distinct immune responses to 4CMenB vaccine.

Children with perinatally acquired HIV (PHIV) have special vaccination needs, as they make suboptimal immune responses. Here, we evaluated safety and immunogenicity of 2 doses of 4-component group B meningococcal vaccine in antiretroviral therapy-treated children with PHIV and healthy controls (HCs). Assessments included the standard human serum bactericidal antibody (hSBA) assay and measurement of IgG titers against capsular group B Neisseria meningitidis antigens (fHbp, NHBA, NadA). The B cell compartment and vaccine-induced antigen-specific (fHbp+) B cells were investigated by flow cytometry, and gene expression was investigated by multiplexed real-time PCR. A good safety and immunogenicity profile was shown in both groups; however, PHIV demonstrated a reduced immunogenicity compared with HCs. Additionally, PHIV showed a reduced frequency of fHbp+ and an altered B cell subset distribution, with higher fHbp+ frequency in activated memory and tissue-like memory B cells. Gene expression analyses on these cells revealed distinct mechanisms between PHIV and HC seroconverters. Overall, these data suggest that PHIV presents a diverse immune signature following vaccination. The impact of such perturbation on long-term maintenance of vaccine-induced immunity should be further evaluated in vulnerable populations, such as people with PHIV.

Antiretroviral therapy and pregnancy: effect on cortical bone status of human immunodeficiency virus-infected Caucasian women as assessed by quantitative ultrasonography.

Treatment with antiretroviral agents (ARVs) during pregnancy is important to prevent mother-to-child transmission of the human immunodeficiency virus (HIV), but their use has been associated with low bone mineral density in adult patients. Currently, there are no data regarding the bone status of HIV-infected women who received ARV during pregnancy. The aim of this study was to evaluate cortical bone status at delivery in a group of HIV-infected women who received ARV during pregnancy and to monitor the changes occurring during the first year postpartum. We studied 33 HIV-infected and 116 HIV-uninfected healthy Caucasian women within 4 days from delivery. Follow-up measurements were performed at 4 and 12 months postpartum in 17 HIV-infected and 55 healthy women. Cortical bone status was evaluated by quantitative ultrasonography at the mid-tibia, and bone measurements were expressed as the speed of sound (SOS). HIV-infected women after delivery had a median SOS of 3,985 (3,567-4,242) m/s, while the median SOS of healthy women was 4,025 (3,643-4,250) m/s. The difference was not significant (t = 0.39, P = 0.69). No significant differences were observed between ARV-exposed and control subjects at 4 and 12 months. Our data suggest that ARV during pregnancy and the first year after delivery does not affect negatively cortical bone status.