This study aims to assess the diagnostic accuracy of digital breast tomosynthesis in combination with full field digital mammography (DBT + FFDM) in the charaterisation of Breast Imaging-reporting and Data System (BI-RADS) category 3, 4 and 5 lesions. Retrospective cross-sectional study of 390 patients with BI-RADS 3, 4 and 5 mammography with available histopathology examination results were recruited from in a single center of a multi-ethnic Asian population. 2 readers independently reported the FFDM and DBT images and classified lesions detected  based on American College of Radiology-BI-RADS lexicon. Of the 390 patients recruited, 182 malignancies were reported. Positive predictive value (PPV) of cancer was 46.7%. The PPV in BI-RADS 4a, 4b, 4c and 5 were 6.0%, 38.3%, 68.9%, and 93.1%, respectively. Among all the cancers, 76% presented as masses, 4% as calcifications and 20% as asymmetry. An additional of 4% of cancers were detected on ultrasound. The sensitivity, specificity, PPV and NPV of mass lesions detected on DBT + FFDM were 93.8%, 85.1%, 88.8% and 91.5%, respectively. The PPV for calcification is 61.6% and asymmetry is 60.7%. 81.6% of cancer detected were invasive and 13.3% were in-situ type. Our study showed that DBT is proven to be an effective tool in the diagnosis and characterization of breast lesions and supports the current body of literature that states that integrating DBT to FFDM allows good characterization of breast lesions and accurate diagnosis of cancer. From 2009 to 2015, the 5-year relative survival rate of women with breast cancer was 99%, 85% and 27% for local disease, regional disease and breast cancer with distant metastases respectively2.Breast cancer is the most common cancer in women worldwide4. Better visualization of lesions’ margin by DBT, particularly in dense breasts, has improved the sensitivity and specificity of lesion detection and allows for better categorization of suspicious and benign breast lesions6.Mammography is a proven screening modality to reduce breast cancer mortality and effective in detecting early stage breast cancer. Digital breast tomosynthesis (DBT) is a technique in breast imaging that overcomes the problems of overlapping breast tissues in full field digital mammography (FFDM) by producing multiple crosssectional images from reconstructed volume data. There has been a significant reduction in recall rates and an increase in breast cancer detection rates with the introduction of DBT into clinical practice7. Pathy et al. showed that 51% of Asian women were diagnosed with breast cancer before the age of 51 based on a hospital-based cancer registry from 1990 to 2007. This is in contrast to the Caucasian women, whereby only 23% of the total breast cancer incidence were younger than 50 year-old8. Asian women also presented with larger tumour size and presented at an advanced stage. 10% of patients in the Asian population has metastatic disease at initial presentation as opposed to 3–6% in the Western setting. The proportion of patients who were not treated, declined follow up or seeked traditional treatment were also not uncommon in Asian women8.As DBT is a relatively new technique, limited research has been done on the diagnostic accuracy in the characterisation of lesions, particularly in multi-ethnic Asian population. Studies done in the Caucasian populations may not be reflective of other populations due to the different variables, such as ethnic differences, lifestyle and psychosocial factorsTherefore, this study aims to assess the diagnostic accuracy of DBT in combination with FFDM (DBT + FFDM) in the charaterisation of mammographic abnormality in opportunistic screening, targeted screening and diagnostic groups in a multiethnic Asian population.This was a retrospective cross-sectional study involving consecutive 390 patients who had undergone DBT + FFDM imaging at the Department of Biomedical Imaging, University of Malaya Medical Centre (UMMC) between January 2015 and May 2017. The study was approved by Medical Ethics Committee of University Malaya Medical Centre (MECID No: 20154-1233).Patients who underwent DBT + FFDM with tissue diagnosis of the detected suspicious breast lesions were recruited. Inclusion criteria includes 1) patients presented for routine (opportunistic) screening (2) patients referred for triple diagnosis due to breast-related symptoms such as breast lumps, nipple discharge or breast pain, (3) symptomatic patients (diagnostic group), (4) women with high risk such as family history of breast cancer and on hormone replacement therapy (targeted screening group), (5) patients assigned to Breast Imaging Reporting and Data System (BI-RADS) 3, 4 or 5 with available histopathological diagnosis via core biopsy, excisional biopsy or mastectomy. Exclusion criteria were patients with known breast cancer, previous breast surgery or mastectomy, reconstructed breast and with breast implants. Flow chart of patients inclusion in the study with number of lesions detected and HPE results is summarized in Fig.  patientsAll patients underwent combined DBT + FFDM (Combo mode) using a 3D digital mammography system  in the same setting. A 2D synthesized images were automatically produced by summing and filtering the tomosynthesis images using an image processing software (C-View software by Hologic). Hence, each patient had three sets of images, i.e. the conventional 2D FFDM, DBT and 2D synthesized images (C-View). All sets consist of CC and MLO views. The processed image data were sent from the acquisition workstation to the reading workstation.All the patients were then subjected to supplementary breast and axillary ultrasound evaluation  with a high frequency (12.5 MHz) linear transducer.All the acquired image viewing and analysis were done in SecurView DX workstation . Data of FFDM, DBT and C-View were stored in the hospital Picture Archiving and Communication System (PACS) .The FFDM, DBT and C-View images were analysed consecutively by two breast radiologists (MR and FF) of 7 and 5 years breast imaging experience in a research setting. The reporting and grading of the BI-RADS score were determined as per American College of Radiology (ACR)-BI-RADS 5th edition (2013) mammogram lexicon and the assessment criteria included breast density assessment, calcifications, mass, asymmetric density, architectural distortiona dn other associated features. The final assessment category of BI-RADS 3 , BI-RADS 4  or BI-RADS 5  was determined after an adjunct ultrasound evaluation as per the ACR BI-RADS classification. The ultrasound images were not included in the image assessment if they were performed. The readers were blinded of the ultrasound findings, histopathology report and patient’s outcome.Biopsies for suspicious lesions were carried out either with stereotactic guided 14G core biopsy, 10G vacuum assisted stereotactic guided biopsy, ultrasound-guided core biopsy or freehand biopsy  by radiologists or clinicians who were not involved. n this study in a clinical setting. The biopsies were performed using 14G BARD Magnum instrument or 10G Mammotome Revolve Biopsy Device. HPE of breast specimens of patients who had undergone image-guided hook wire localization surgical excision and mastectomy were also included. Histopathology examinations were analysed and reported by histopathologists who were not involved in this study, in a clinical setting. Cancers were identified as invasive lobular, invasive ductal and ductal carcinoma in situ (DCIS). Cancers containing mixed invasive and in-situ components were classified as invasive.Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS version 23.0) software. Breast lesions were characterized using ACR-BI-RADS 5th edition (2013) mammogram lexicon. BI-RADS category 3 was considered as benign lesion and BI-RADS category 4 and 5 were considered as suspicious for malignancy. HPE results were referred as the gold standard. Correlations between the quantitative and qualitative characteristic of the lesions with the HPE results were studied. The quantitative assessment includes the size of malignant lesions, while qualitative parameters include mass, calcification, asymmetry, architectural distortion and associated features. The sensitivity, specificity, PPV and NPV were determined for mass. PPV was calculated for calcification, assymetric density and other associated findings. PPV is calculated as (number of true positives)/, and NPV is (number of true negative)/.Diagnostic accuracy of BI-RADS category were calculated and compared to the gold standard.Written consent was waived by the institution ethics committee due to retrospective nature of the study.n = 331). 43.1% (n = 168) patients were in BI-RADS density C and D category. BI-RADS density category was not assigned for the 3 male patients. Table n = 341) of the patients who did not have family history of breast cancer. Out of the 49 patients with family history of cancer (first or second degree relatives with breast cancer), 23 (46.9%) patients were positive for malignancy.A total of 387 women and 3 men were recruited in this study. Majority of the patients were Malay (39.7%), 33.1% were Chinese and 24.9% were Indian. The patients’ ages ranged between 28 and 86, with the majority in the 40–69 age group  lesions with ultrasound-guided core biopsy performed, 47 (11.1%) lesions had stereotactic-guided core biopsy performed and 3 (0.7%) lesions had vacuum-assisted stereotactic-guided biopsy (VAB). Free hand biopsy accounted for 26.2% (n = 111) of the lesions.Out of the 423 biopsied lesion, 196 (46.3%) were malignant and 227 (53.7%) were benign. The detailed HPE findings were outline in Table Table For ethnicity, the percentage of patients diagnosed with cancer in each ethnic group were almost similar, with 71 (45.8%) Malays, 59 (45.7%) Chinese and 49 (50.5%) Indians. As expected, invasive ductal carcinoma was the most common cancer across all ethnicity and age groups.There were 6.9% (n = 27 ) age < 40 years old, 79.7% (n = 311) age 40–69 and 13.3% (n = 52 )age ≥ 70 in the study population. Higher percentage of malignancy with increasing age group as depicted in Table From DBT + FFDM diagnosis, 253 lesions were categorized as BI-RADS 4 and 132 lesions as BI-RADS 5. There were 37 lesions in BI-RADS category 3 biopsied as requested by the primary team. Table Of the 423 lesions, 368 were identified on DBT + FFDM. 55 lesions were detected on ultrasound with normal or unremarkable DBT + FFDM findings. Of the total 196 malignant lesions, 189 were identified via DBT + FFDM, giving a PPV of 51.4%. Additional 7 (12.7%) cancers were detected via ultrasound in the patients with unremarkable DBT + FFDM findings. All the malignant lesions that were not visualized in the DBT + FFDM images were in BI-RADS density C and D.Of the 368 lesions identified on DBT + FFDM, there were 258 masses, 99 suspicious calcifications, and 61 asymmetry. These lesions were characterized as per BI-RADS lexicon for mammography and summarized in Table Features that were assessed for masses include shape, margin and density. Suspicious masses were those that showed irregular shape, high density and/or spiculated/indistinct margins. Some masses demonstrated benign appearance on DBT + FFDM . However, due to suspicious features on adjunct ultrasound, these masses were biopsied.Of the 258 masses, 152 (58.9%) appeared suspicious on DBT + FFDM. 135 (88.8%) were malignant and 17(11.2%) were benign. Of the 106 (41.1%) masses which appeared benign on DBT + FFDM, 9 (8.5%) were malignant.Out of the 144 malignant masses detected on DBT + FFDM, only 98 (68.1%) masses can be measured as the rest have ill-defined and obscured margins. The sizes of the malignant masses ranged between 0.5 and 8.9 cm with the mean size of 2.7 cm.Figure There were 99 suspicious calcification; 47 47.5%) were calcification with no associated mass and 52 (52.5%) were associated with a suspicious mass. There were 61 (61.1%) malignant lesions with PPV of 61.6%. Figure % were caThere were 61 asymmetries detected; 37 (60.7%) were malignant and the PPV was 60.7%. The detailed description in Table Associated features were seen in 189 lesions. The most frequently noted feature associated with malignancy is architectural distortion (92.8%). Presence of nipple retraction, skin thickening and lymphadenopathy were also predictive of malignancy Table .All architectural distortion detected in this cohort were associated with either a suspicious mass or calcifications.There were 69 cases of architectural distortion reported. 92.8% (n = 64) were seen in association with a malignant lesion and 7.2% (n = 5) were in benign lesion. The PPV was 92.8%.The PPV, NPV, sensitivity and specificity for characteristics of masses detected on DBT + FFDM are tabulated in Table The lesions that were deemed to be benign were followed up for 2 years and no interval cancers detected from this cohort.The average glandular dose (AGD) for FFDM + DBT and DBT was 4.1 mGy (95% CI 3.98–4.25) and 2.0 mGy (95% CI 1.91–2.02) respectively.11.A number of studies have compared the clinical performance of DBT and FFDM, and have concluded that DBT showed superior image quality compared to FFDM in characterizing abnormalities and thereby more efficient in cancer detection and reduction in recall rates12. However, waiting until symptoms developed may lead to advanced cancer at presentation.There were 62.3% (n = 243) cases from the diagnostic group, 28.2% (n = 110) were from the opportunistic screening group and only 9.5% (n = 37) from the targeted screening group. From the 46.7% (n = 182) patients diagnosed with cancer in this study cohort, 84.6% (n = 154) were in the diagnostic group. Invasive cancers were detected in 37.8% (n = 160/423) cases and in-situ cancers were in 6.4% (n = 27/423) of cases. This is because there is no population-based breast screening programme in Malaysia, a middle-income country. Hence, most mammography cases were referrals for investigation of a symptom. Previous publication supported opportunistic screening in such setting and reported comparable performance indicators with international standardsIn the 187 malignant lesions, PPV in BI-RADS 4 category was 32.2%, with 4a, 4b and 4c accounted for 6.0%, 38.3% and 68.9% respectively. These are concordance with the percentage of likelihood of cancer as per ACR BI-RADS 2013 guideline; 2 to 10% for BI-RADS 4a, 10 to 50% for BI-RADS 4b and 50 to 95% for BI-RADS 4c. The PPV for BI-RADS category 5 was 93.1%. There were 9 patients with false positives in BI-RADS 5 category and this is justified as the tissue diagnosis were sclerosing adenosis, fat necrosis, and inflammation, all of which can mimic malignancies.There were 8.7% (n = 37) in BI-RADS category 3 and of these, 78.4% (n = 29) were biopsied and proven to be non-malignant. These lesions typically demonstrate benign appearances on imaging and are routinely followed up closely on ultrasound or mammography. However, biopsy were performed in these cases to concerning risk factors or upon patient/surgeon’s request. As per the ACR BI-RADS 2013 guidelines, the likelihood of cancer of BI-RADS category 3 is 0–2%, which is in agreement with our finding.14. Corsetti et al.13 found that ultrasonography screening in mammography-negative patients with dense breast contributed additional 20% cancer detection rate compared to mammography alone, predominantly in women younger than 50 year-old. McCavert et al.14 has shown that adding ultrasonography to mammography significantly increase sensitivity in cancer detection compared to mammography alone (80.8% vs 56.6%) with no significant difference in specificity.In our study, 87.2% (n = 340) patients had mammographic abnormalities and 12.8% (n = 50) had unremarkable mammogram, but with sonographic abnormalities. There were 14.0% (n = 7/50) malignant lesions which were detected on ultrasound, but not visualized on DBT + FFDM. They were all in BI-RADS density C and D category. Therefore, supplementary ultrasound has increased our cancer detection rate by 4%. Several studies have also shown improved detection of breast cancer when ultrasound was used an adjust tool to mammography15 on comparison of diagnostic accuracy of DBT and FFDM which showed similar results, with PPV of 48.1% for DBT.From the 189 malignancy cases that were identified on DBT + FFDM, 76% presented as masses, 4% as calcifications, and 20% as asymmetric density. Architectural distortion when present were all associated with either a mass, suspicious calcification or asymmetric density. The PPV for screen detected cancer in out study was 51.4%. This is in concordance with a study by Svahn et al.In tomosynthesis, margins of the mass lesions are delineated clearly and it was especially true for suspicious features such as indistinct, obscured and spiculated margins. In the present study, out of 149 patients with lesions that had margins with suspicious feature, 88.6% (n = 132) patients had cancer. Out of the 144 malignant masses which were mammographically detected, 89 (PPV = 96.7%) lesions were with spiculated margin, 32 (PPV = 72.7%) with indistinct margin, and 11 (PPV = 84.5%) with microlobulated margins. Only 9 (6.25%) lesions with well circumscribed margins were diagnosed to be malignant lesions.7. Our results were also comparable to Gilbert et al.16 which showed that, in cases where the dominant radiologic feature was a mass, sensitivity of DBT + FFDM was 92%. Out of the 144 masses which were malignant, 23.1% of cancers were detected in BI-RADS density A, 45.5% in BI-RADS density B, 25.9% in BI-RADS density C and 5.6% in BI-RADS density Bernardi et al.17 have shown that a greater advantage of tomosynthesis was observed in younger women who had dense breasts.There were 90.9% (n = 120/132) irregular shaped lesions and 87.1% (n = 128/147) high density lesions resulted positive for malignancy. 88.8% (n = 135/152) of masses that were described as suspicious were HPE proven carcinomas. One of the studies done in assessment of non-calcified breast lesions on tomosynthesis had concluded that radiologists were able to evaluate lesions more thoroughly by partially removing the tissues that are present above and below the plane of the lesion, which are overlying structures with conventional mammography, so that the shape and margin of lesions can be more readily assessed10 had also concluded similar findings to ours, whereby superior image quality were noted in the tomosynthesis images, especially in the characterization of masses and asymmetry.We described asymmetric density as asymmetry , focal asymmetry , global and developing asymmetry as per the BI-RADS lexicon for mammography. Cumulatively, 61 breast asymmetric densities were identified and biopsied and 37 (60.7%) lesions were proven to be malignant. The percentage of cancer diagnosed as asymmetry (1 view), focal asymmetry and global asymmetry were 51.1% and 58.8% respectively. Poplack et al.17. The Oslo study as reported by Skaane et al.18 had concluded that application of C-view 2D to replace FFDM 2D in a tomosynthesis screening exam offered comparable clinical performance to the FFDM 2D + DBT imaging. However, a meta-analysis by Gilbert et al.16 has shown mixed reports in previous studies regarding detection of calcifications on DBT. The TOMMY trial has demonstrated lower specificity for calcifications than for soft tissue masses which was 88% versus 92%, respectively19. Detection of suspicious calcifications on mammography is important in identifying the presence of breast lesions, especially in cases of non-palpable lesions. Calcifications were clearly seen in the C-view images which were noted in previous study from our centre and also resulted in reduced radiation dose20. This may be due to the manufacturers’ intention to highlight calcifications and avoid the ‘thin slice’ effect of thin tomosynthesis slices17.Calcifications are also the commonly appearing abnormalities in mammography. In this study, C-view images were also reviewed in the characterization of suspicious breast lesions. In detection of suspicious calcifications, the PPV was 61.6%. STORM II study showed higher cancer detection rate using C-View + DBT21. This may be because the readers in our study reviewed FFDM and DBT images together. More studies are recommended to assess this.From this study, 210 lesions were found to have other associated features. Architectural distortion were commonly seen in association to malignant lesions 64 (92.8%). Other associated features, such as nipple retraction , lymphadenopathy  and skin thickening  were also strongly associated with cancers. In this study cohort, no architectural distortion were noted without a suspicious mass lesion. This is in contrast to other studies which noted more architectural distortion without associated mass in DBT compared to FFDM, although they were less likely to represent malignancy23. The patients included in this study were representative only of the cohort that attended the breast imaging unit in a tertiary referral centre in a semi-urban to urban population.The majority of our patient population were from the Malay ethnic group. This is likely because this is the largest ethnic group in Malaysia. The cancer detection rate was similar in all other ethnic groups, which were 45.8% in Malays, 45.7% in Chinese and 50.5% in Indians. This may not be an accurate representation of the prevalence of cancer in Malaysian population. Previous study had reported higher breast cancer cases in Chinese ethnicity in Malaysia24.Invasive ductal carcinoma is the most common cancer across all ethnicity and age groups. The number of patients who are of less than 50 years old detected with cancer were 24% (n = 44). This is discordant with previous study on breast cancer in Asian women, which reported 40% of breast cancers diagnosed were in women aged below 50 yearsThe limitation of this study is it was performed in a single institution of a tertiary breast centre involving a small cohort of patients. Studies from other centres are recommended to validate our findings.We concluded that DBT is an effective tool in the diagnosis of breast cancer from a local Asian population perspective and supports the current body of literature that states that integrating DBT to FFDM allows good characterization of breast lesions and accurate diagnosis of cancer. This is supported by the diagnostic accuracy of suspicious and highly suspicious lesions which had given PPV for BI-RADS category 4 and 5 of 32.6% and 93.1%, respectively. DBT has proven to be a good diagnostic tool in identifying focal asymmetry with PPV of 100%. In addition, ultrasound has proved to be an effective adjunct tool to DBT + FFDM which yielded an additional 4% of malignancy.


The aim of this study is to evaluate the new bone and connective tissue formation and the biomaterial remaining after maxillary sinus bone augmentation using 5 different bone substitutes. The osteocalcin immunolabeling was performed to demonstrate their calcification and the possibility of receiving dental implants. 40 patients underwent maxillary sinus bone augmentation and were divided in 5 groups: Group 1 with 8 maxillary sinuses were grafted with autogenous bone graft (AB); Group 2 with 8 maxillary sinuses grafted with bioactive glass (BG); Group 3 with 8 maxillary sinuses grafted with bioactive glass added to autogenous bone graft (BG + AB) 1:1; Group 4 with 8 maxillary sinuses grafted with Bio-Oss (BO) and Group 5 with 8 maxillary sinuses grafted with Bio-Oss added to autogenous bone graft (BO + AB) 1:1. In group AB, 37.8% of bone was formed in the pristine bone region, 38.1% in the intermediate and 44.5% in the apical region. In group BG, 43.6% was formed in the pristine bone, 37% in the intermediate and 49.3% in the apical region. In group BG + AB 1:1, 39.0% was formed in the pristine bone region, 34.8% in the intermediate and 36.8% in apical region. In group BO, 33.4% was formed in the pristine bone, 32.5% in the intermediate and 34.3% in the apical region. In group BO + AB 1:1, 32.8% was formed in the pristine bone, 36.1% in intermediate and 27.8% in the apical regions. The immunolabeling for osteocalcin showed an intensive staining for all groups, which could demonstrate the calcification of the bone formed. This study showed that the groups evaluated formed a suitable lamellar bone in the maxillary sinus reconstruction after six months of bone healing, thus being indicated to receive dental implants. Since its creation by Boyne and James, different bone substitutes have been used as autogenous bone grafts, xenografts and allografts, among others.5Different surgical techniques that depend on the residual bone on the maxillary sinus floor have been developed.6 Among the numerous biomaterials created and researched over the years, the autogenous bone graft (AB) is considered the “gold standard” due to its specific properties, namely osteoconduction, osteoinduction, and osteogenesis.However, an ideal bone substitute is yet to be described in the literature.® (BO)  is the most used bone substitute, consisting of a deproteinized bovine bone with osteoconductive properties that has shown promising results due to its morphology similar to the human bone.8 Bioactive glass (BG)  is another biomaterial with promising results, consisting of an osteoconductive ceramic with peculiar properties of graft resorption due to the chemical dissolution.10Bio-OssThe aim of this study was to compare the new bone formation in human maxillary sinuses grafted with these two bone substitutes (Biogran & Bio-Oss), which have different resorption pathways, using histomorphometric and immunohistochemical analysis, and to determine the effects of their addition to autogenous bone graft (1:1) after 6 months of bone healing.0 – There will be no difference in the new bone formation rates using biomaterials of different resorption pathways.HAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee approved with the number 47711015.4.0000.5420 by Plataforma Brasil/CONEP; with the 1964 Helsinki Declaration and its later amendments.11  to evaluate the maxillary sinuses, as well as the proximity of mandibular canal and inferior dental roots where the autogenous bone graft were harvested. The inclusion criteria were: patients with maxillary sinuses with less than 5 mm of bone remaining; those who decided to be rehabilitated with dental implants; and those with bone in the mandibular symphysis or in the retromolar region. Exclusion criteria were: patients that reported uncontrolled systemic disease, untreated periodontal disease, or sinus pathologies; smokers; patients without enough bone at the mandibular regions to be harvested for the autogenous bone graft; patients who reported medication-related osteonecrosis, or patients treated for head and neck cancer.Based on the aforementioned parameters, 40 patients were selected for unilateral maxillary sinus bone augmentation and divided into 5 groups:Group 1: eight maxillary sinuses grafted with AB as control group;Group 2: eight maxillary sinuses grafted with BG;Group 3: eight maxillary sinuses grafted with BG + AB 1:1;Group 4: eight maxillary sinuses grafted with BO,Group 5: eight maxillary sinuses grafted with BO + AB 1:1.13 The bone blocks were grounded with a bone crusher . The maxillary sinuses were treated according to Boyne and James3 (1980) using a posterior maxillary approach to expose the lateral wall of maxillary bone followed by a osteotomy using a nº 6 sphere drill to access the Schneiderian membrane and its elevation. The bone grafts were mixed using a syringe in cc to allow the 1:1 proportion. Post-operative pain was treated with 500 mg paracetamol , prescribed four times per day and 500 mg amoxicillin  was prescribed three times per day to reduce the chances of infection.Surgical procedures were performed under local anesthesia using lidocaine 2% with 1:100 epinephrine . The autogenous bone graft was harvested from retromolar region by vestibular approach similar to that of mandibular sagittal osteotomy and osteotomized using 701 drill. The harvesting from symphysis were performed by an intraoral anterior vestibular approach, 5 mm below the gingival line extending from canine tooth. The bone was also osteotomized using a 701 drill and removed using a chisel.12 (2017) in three regions using a light microscopy with a digital camera attached to capture the images at ×12.5 magnification:After 6 months of bone repair, bone biopsies were harvested using a 3.0 mm × 15 mm trephine bur  at the time of dental implants placement and stored in a 10% formalin solution (pH 7) for 48 h. The samples were washed in running water for 24 hours, decalcified for 4 weeks in an EDTA solution changed weekly. Subsequently, they were embedded in paraffin; sectioned and stained with hematoxylin and eosin. Samples were identified and stored following the apical orientation in all sequence during laboratory procedures. The biopsies were evaluated according to Pereira, et al.pristine bone (2 mm above the maxillary sinus floor),intermediate,apical (2 mm below the membrane)14 (1968).To exclude the influence of maxillary sinus floor and Schneiderian membrane during the evaluation, a 2 mm distance from upper and lower regions was respected. The height of pristine bone region was measured in the CBCT previously performed to exclude it during the microscope visualization. New bone formation, amount of connective tissue and the remaining biomaterial were analyzed by histometry using a grid of Merz15 (2017). Primary polyclonal goat antibodies against human Osteocalcin  were used in immunohistochemical assays to identify calcified tissue.16 A biotinylated donkey anti-goat secondary antibody  coupled to avidin  was used for signal amplification. The binding reaction was detected with diaminobenzidine . Data analyses were performed using a single-evaluator semi-quantitative approach, with score “0” indicating the absence of staining and scores “1,” “2,” or “3” indicating low, moderate, or intense staining, respectively.The immunohistochemical analysis was performed according to Pereira, et al.The Kolmogorov-Smirnov test was performed to indicate the parametric or non-parametric distribution of the samples. In case of normal distribution, a comparison among the groups was made using the ANOVA test followed by Tukey’s multiple comparison test. In case of non-parametric distribution, a Kruskal-Wallis test was performed. A priori, p-value<0.05 was considered significant for all tests.Forty patients (22 men and 18 women) aged between 32 and 65 years old were subjected to unilateral maxillary sinus bone augmentation using the 5 types of bone grafts purposed in our study.The group AB presented predominant lamellar bone formation in all three regions evaluated, with the presence of osteocytes in the bone matrix. The mean for the new bone formed was 37.8%±16.9 in the pristine bone region; 38.1%±21.4 in the intermediate region and 44.5%±18.6 in the apical region. The connective tissue formed presented a mean of 57.6%±16.3 in pristine bone; 58.8%±20.9 in intermediate and 54.0%±16.5 in apical region. The median for the remaining biomaterial was 1.5 in the pristine bone region, 0.5 in the intermediate region and 2.5 in the apical region.The group BG showed new bone formation, which were in the process of lamellar organization in all three regions. However, in most cases, the presence of lamellar bone organization was observed. We also observed the presence of cellularized connective tissue with remaining bioactive glass particles and osteoblasts in the periphery of the bone matrix. The mean of new bone formed was 43.6%±4.7; 37%±10.9 and 49.3%±13.2 in the pristine bone, intermediate and apical region, respectively. The connective tissue presented a 56.6%±6.5 mean for pristine bone; 63.4%±11.0 for intermediate and 49.3%±13.2 for apical region. The median for the 3 regions evaluated for the present group was 0.In group BG + AB 1:1, a new bone formation with lamellar organization were observed, mostly showing more woven bone formation presented in the apical region. The connective tissue had cells and osteoblasts in the periphery of the bone matrix. The mean for new bone formed was 39.0%±15.8 in the pristine bone region; 34.8%±14.5 in the intermediate and 36.8%±14.5 in apical region. For the connective tissue, a 60.3%±11.9 mean was observed in the pristine bone; 62.1%±14.5 in the intermediate and 58.5%±13.6 in the apical region. The median for the remaining biomaterial was 1.5 in the pristine bone region; 1 in the intermediate region and 2.5 in the apical region.The group BO presented lamellar new bone formation in the three regions evaluated in a cellularized connective tissue stroma and much remaining biomaterial. The mean for the new bone formed was 33.4%±12.6 in the pristine bone; 32.5%±10.8 in the intermediate and 34.3%±12.7 in the apical region. The amount of connective tissue formed in the pristine bone region was 38.3%±9.5; in the intermediate region was 40.9%±7.1 and 41.0%±7.2 in the apical region. The median for the remaining biomaterial in this group was 36.0 in pristine bone; 26.5 in intermediate and 25.5 in apical region.The group BO + AB 1:1 had lamellar bone formation with less remaining biomaterial due to autogenous bone graft particle resorption and less use of xenograft. The mean of the new bone formed in group 5 in the pristine bone, intermediate and apical regions was 32.8%±11.5; 36.1%±16.0 and 27.8%±19.8 respectively. For connective tissue, the mean in the pristine bone region was 46.9%±14.5; in the intermediate region was 40.9%±21.2 and in the apical region was 43.4%±15.9. The remaining biomaterial median was 22.5 for pristine bone; 24.5 for the intermediate and 36.0 for the apical region. Osteoclasts were absent around the bioactive glass or xenografts of specimens. .post hoc test detected statistically significant differences between groups AB and BG + AB 1:1 (p=0.001); groups AB and BO + AB 1:1 (p=0.012); groups BG and BO (p=0.001); groups BG and BO + AB 1:1 (p=0.023); groups BG + AB 1:1 and BO (p=0.0001) as well as groups BG + AB 1:1 and BO + AB 1:1 (p=0.001). The comparison among groups for remaining biomaterial also had a statistically significant difference . The test showed difference between groups AB and BO (p=0.0001); groups AB and BO + AB 1:1 (p=0.0001); groups BG and BO (p=0.0001); groups BG and BO + AB 1:1 (p=0.0001); groups BG + AB 1:1 and BO (p=0.0001) and groups BG + AB 1:1 and BO + AB 1:1 (p=0.001). Besides this, statistically significant differences were observed among regions for remaining biomaterial by the post hoc test : pristine bone region of groups AB and BO (p=0.048), pristine bone of group AB and apical region of group BO (p=0.025); pristine bone region of group AB and apical region of group BO + AB 1:1 (p=0.004). The differences were also observed among the intermediate region of group BG and the pristine bone (p=0.020), intermediate (p=0.022), and apical region (p=0.011) of group BO. The apical region of group BO + AB 1:1 showed difference among pristine bone region of group AB (p=0.004) and the intermediate region (p=0.002) and apical region (p=0.011) of group BG. . For connective tissue, the difference was statistically significant among groups (p=0.0001). Tukey’s roup BG.  A, B0.Therefore, our results were in accordance with hypothesis HGroup AB presented high level “3” immunolabeling results for osteocalcin, which points to a mature bone that is able to receive dental implants for group BG; for group BG + AB 1:1; for group BO and group BO + AB 1:1 (17(2010) evaluated Bio-Oss Collagen in human sockets, concluding that Bio-Oss particles were not reabsorbed and bone formation was delayed after 6 months. Hallman, Lundgren and Sennerby18 (2001) and Lindgren, et al.19 (2012) analyzed bone specimens from human maxillary sinuses augmented with Bio-Oss and found 12.4% and 24.0% of particles after 3 years, respectively. Our study showed a 36.0% median of Bio-Oss in the pristine bone region; 26.5% in the intermediate and 25.5% in the apical region. The presence of osteoclasts around the graft remaining were not observed. Our data are in line with previous studies, which outline that Bio-Oss tends to be “resistant to resorption”.19In our study, we evaluated the bone formation in human maxillary sinus bone augmentation using two different bone grafts. Previous literature reports that Biogran and Bio-Oss have different ways of graft resorption. Lindhe, et al.10 With simultaneous colonization of osteoblasts, the particle disintegrates. A rate of bioactive glass particles remaining from 0% to 4.2% was reported after 6 months of bone healing in maxillary sinus bone augmentation, similar to our study, in which we report a 0% median in all regions evaluated.20 Thus, we demonstrated that another crack formed after each particle divided, and the occurrence of a new osteoblast activity.Biogran has a peculiar way of resorption: body fluids react with these bone substitute due to its unstable structure. A silica gel and calcium phosphate layer are formed around the particle surface, which allow a combined process of chemical dissolution and macrophage action.21 (2017) reported a 27.5% bone formation using Bio-Oss and 16.6% of residual graft. Lee, et al.22 (2017) found 26.1% of new bone formed and 25.7% of residual Bio-Oss. Nizam, et al.23 (2018) demonstrated a 21.2% bone volume, similar to the outcomes reported in our study. Histometric studies using Biogran alone are scarce. Tadjoedin, et al.24 (2002) evaluated it; however, only 3 patients were operated upon and, therefore, their results cannot be considered reliable. Pereira, et al.20 (2017) researched the use of Biogran to reconstruct maxillary sinuses compared to autogenous bone graft. Their results showed a 42.0% mean of new bone formation in the pristine bone, 40.7% in the intermediate and 45.6% in the apical region, which is similar to the outcomes of our study.Previous studies show different rates of new bone formation in maxillary sinus bone augmentation using both biomaterials. Rodriguez y Baena et al.27 Recent studies reported post-surgical complaints immediately after the procedure, and that they tend to decrease in a long term.13 Besides this, osteoinductive properties and mesenchymal cells can be transferred with the autogenous bone graft and provide advantages when added to biomaterials to improve the bone quality.30Despite the autogenous bone graft being the most predictable for bone reconstructions, some authors showed that the requirement of another surgical site leads to post-operative morbidity.31 (2001) reported a 30.6% bone formation rate of Biogran and autogenous bone graft in a 4:1 ratio. Turunen, et al.32 (2004) grafted human maxillary sinuses with Biogran and autogenous bone graft from iliac crest in a 1:1 proportion with a 34.0% result, similar to the study of Menezes, et al.9(2018).In our study, the composite bone graft studied did not result in a statistically significant difference in the rate of new bone formation. Group BG + AB 1:1 showed a mean of 39.0%, 34.8%, and 36.8% in the pristine bone, intermediate region and apical region, respectively. Cordioli, et al.28 (2001) showed that 18.9% of bone is formed using this mixture, as well as Bonardi, et al.8(2018). When the Bio-Oss associated to A-PRF and i-PRF is used to reconstruct mandible, similar histological findings were identified by Lorenz, et al.33 (2018), in which the new bone formed was in direct contact with the biomaterial remaining with lamellar formation. In our study, we also found low rates of new bone formation; however, the histological findings presented lamellar bone and a connective tissue well cellularized. Thus, the H0 hypothesis was accepted.When Bio-Oss plus autogenous bone graft is used to reconstruct maxillary sinuses, lower rates of new bone is formed. Yildirim, et al.15The osteocalcin outcomes demonstrate that all bone substitutes evaluated in this study were calcified and able to receive dental implants, similar to previous studies.30 Despite the outcomes showed in this study, further studies are required to evaluate other periods and demonstrate if the biomaterial particle rates influence bone healing.The limitation of our study was the evaluation of dental implants in a long-term. According to literature, however, high rates of success are observed when Bio-Oss is used to reconstruct the bone height in maxillary sinuses.In conclusion, our study showed the formation of a suitable lamellar bone in the patients that underwent maxillary sinus reconstruction after six months of bone healing in all groups, thus being indicated to receive dental implants.


Measuring progress towards the Joint United Nations Programme on HIV/AIDS (UNAIDS) 90–90–90 treatment targets is key to assessing progress towards turning the HIV epidemic tide. In 2017, the UNAIDS model estimated that 75% of people living with HIV (PLHIV) globally knew their HIV positive status, 79% of those who knew their status were on antiretroviral therapy (ART), and 81% of those who knew their HIV status and were on ART had a suppressed viral load. The fifth South African national HIV sero-behavioural survey collected nationally representative data that enabled the empirical estimation of these 90–90–90 targets for the country stratified by a variety of key factors.To evaluate progress towards achievement of the 90–90–90 targets for South Africa, data obtained from a national, representative, cross-sectional population-based multi-stage stratified cluster random survey conducted in 2017 were analysed. The Fifth South African National HIV Prevalence, Incidence, Behaviour and Communication Survey (SABSSM V), collected behavioural and biomarker data from individuals residing in households from 1000 randomly selected Small Area Layers (SALs), across all nine provinces of the country. Structured questionnaires were used to collect socio-demographic data, knowledge and perceptions about HIV, and related risk behaviours. Blood samples were collected to test for HIV infection, antiretroviral use, and viral suppression (defined as < 1000 copies/ml). Weighted proportions of study participants aged 15 years and older who tested HIV positive were computed for those who reported awareness of their status (1st 90), and among these, those who were currently on ART (2nd 90) and of these, those who were virally suppressed (3rd 90).Among persons 15 years and older who were HIV positive, 84.8% were aware of their HIV positive status, of whom 70.7% were currently on ART, with 87.4% of these estimated to have suppressed viral load at the time of the survey. These estimates varied by sex, age, and geo-location type. Relatively higher percentages across all three indicators for women compared to men were observed: 88.7% versus 78.2% for those aware of their status, 72.3% versus 67.7% for on ART, and 89.8% versus 82.3% for viral suppression. Knowing one’s positive HIV status increased with age: 74.0, 85.8, and 88.1% for age groups 15–24 years old, 25–49 years old and 50–64 years old, although for those 65 years and older, 78.7% knew their HIV positive status. A similar pattern was observed for the 2nd 90, among those who knew their HIV positive status, 51.7% of 15 to 24 year olds, 70.5% of those aged 25–49 years old, 82.9% of those aged 50–64 years old and 82.4% of those aged 65 years or older were currently on ART. Viral suppression for the above mentioned aged groups, among those who were on ART was 85.2, 87.2, 89.5, and 84.6% respectively. The 90–90–90 indicators for urban areas were 87.7, 66.5, and 87.2%, for rural settings was 85.8, 79.8, and 88.4%, while in commercial farming communities it was 56.2, 67.6 and 81.4%.South Africa appears to be on track to achieve the first 90 indicator by 2020. However, it is behind on the second 90 indicator with ART coverage that was ~ 20-percentage points below the target among people who knew their HIV status, this indicates deficiencies around linkage to and retention on ART. Overall viral suppression among those on ART is approaching the target at 87.4%, but this must be interpreted in the context of low reported ART coverage as well as with variation by age and sex. Targeted diagnosis, awareness, and treatment programs for men, young people aged 15–24 years old, people who reside in farming communities, and in specific provinces are needed. More nuanced 90–90–90 estimates within provinces, specifically looking at more granular sub-national level (e.g. districts), are needed to identify gaps in specific regions and to inform provincial interventions. The 90–90–90 targets are that 90% of people living with HIV (PLHIV) know their HIV status, 90% of people who know their status be on antiretroviral (ARVs), and 90% of all patients receiving antiretroviral therapy (ART) are virally suppressed by 2020 . ModelliThe HIV epidemic in South Africa, with its estimated 7.1 million PLHIV, accounted for approximately 20% of the 36.9 million PLHIV in the world in 2017 . In thatIn 2015, the World Health Organisation (WHO) recommended that HIV ARV treatment should start as soon as possible after diagnosis of HIV infection regardless of CD4 count and/or WHO clinical stage . This reSeveral multi-country studies inclusive of South African sites have evaluated UTT using the 90–90–90 targets. Hayes et al. , estimatThe Human Sciences Research Council undertook the Fifth South African National HIV Prevalence, Incidence, Behaviour and Communication Survey (SABSSM V) in 2017/8 . This isDetails on the SABSSM survey methodology have been described elsewhere . In summThe study received ethical clearance from the Human Sciences Research Council Research Ethics Committee (REC 4/18/11/15) and The US Centers for Disease Control (CDC). In compliance to this ethical clearance, adults, 18 years or older provided written informed consent, while for children, younger than 18 years old, parental/guardian informed written consent and the minor’s assent were obtained before participants took part in the study. Household data for socio-demographic characteristics such as age, sex, race, education, and household assets were collected. Individual level data were also collected on health, sexual behavior, knowledge and perception of HIV and TB. Dried Blood Spots (DBS) were also collected to test for HIV status, recency of infection, exposure to antiretroviral (ARV) drugs, ART resistance, and levels of viral suppression. For this analysis, data for individuals aged 15 years and older were used.Dried Blood Spot (DBS) samples were used for national and sub-national estimates of HIV prevalence, incidence, ARV exposure, HIV drug resistance, and viral suppression. DBS samples were collected by finger prick from consenting individuals. To test for HIV infection, three enzyme immunoassays (EIA)  were used. Two assays were used to confirm a result, and in instances where there was a discordant result between the two tests, the third test was used as a tiebreaker. All samples testing HIV positive on two EIAs were subjected to a nucleic acid amplification test  for the final interpretation of the test result. High Performance Liquid Chromatography (HPLC) coupled with Tandem Mass Spectrometry was used to test for antiretroviral analytes in HIV-positive specimens.Viral load testing on confirmed HIV-positive specimens was performed using the recommended testing platform for HIV-1 Ribonucleic acid (RNA) testing in DBS samples .First 90/Diagnosed - weighted population percent of individuals who tested HIV positive and reported that they knew their HIV status or had antiretroviral analytes in their blood (numerator) out of all individuals who tested HIV positive (denominator).Second 90/on ART - weighted population percent of those testing positive for antiretroviral analytes (numerator) out of all those who knew their HIV status as defined numerator of 1 above.Third 90/Virally suppressed - weighted population percent of those who had viral loads of less than 1000 copies per ml (numerator) out of all those who had antiretroviral analytes (denominator) as defined numerator of 2 above.The ‘conditional’ 90–90–90 indicator is defined as:The results are reported by sex, age, self-reported race (black African and other), geographical type  and by each of the nine provinces in South Africa. Weighted population percentages of the first 90 vs the second 90, and the second 90 vs the third 90 were plotted by province and shown relative to each of the 90 targets to assess achievement of the targets provincially.Additionally, gaps to achieving the 90–90–90 targets were reported among all PLHIV to demonstrate the scale of the epidemic and inform programs. The number of individuals nationally who knew their status, those on ART and those with viral suppression among all PLHIV and the numbers needed to achieve the targets were estimated using population weights. Similar to the national presentation of gaps to achievement of the 90–90-90 targets, differences in percentages and numbers of individuals for those who knew their status, those on ART, and those with viral suppression among all PLHIV were estimated for each province.svyset” in Stata. Estimates are reported with 95% confidence intervals (CI) as appropriate. Stata Statistical Software Release 15.0  was used to conduct analyses of the data.Population weights based on province, sex, age, and race were used to account for the complex survey design and non-response, and weighted data analysis was implemented using “A total of 39,132 participants of all ages from 11,776 households were eligible to participate in the main study. Altogether, 93.6% agreed to be interviewed and, 61.1% agreed to provide specimens for HIV testing. Agreement to be interviewed and to provide a blood sample varied by sex (women - 64.3% vs men – 57.7%), age , race , and geographic area .Overall HIV prevalence among people aged 15 years or older was estimated at 18.8% (95% CI 17.5–20.1%), translating to 7,467,321 people infected with HIV in South Africa. The 90–90–90 indicator estimates for South Africa in 2017 were 84.8% (95% CI 81.6–87.6) of PLHIV knew their status, of those, 70.7% (95% CI 67.6–73.7) were on ART, and of those aware of their status and on ART, 87.4% (95% CI 84.9–89.6) had suppressed viral load (Table ). The gaps in the numbers of people along the treatment cascade in order to achieve these were: an additional 384,585 PLHIV to be diagnosed, an additional 1,777,710 individuals initiated on ART, and an additional 1,742,147 PLHIV to achieving viral suppression  aged 15 years and older need to be diagnosed, 6,048,530 initiated on ART, and 5,443,677 of PLHIV on ART must achieve viral suppression  versus 78.2% (95% CI: 72.4–83.1), respectively. Of those who knew their HIV status and who were on ART, women were more likely than men to be virally suppressed at 89.8%  versus 82.3%  (Table Point estimates for awareness of HIV status were higher in older age groups: 74.0% (95% CI: 63.6–82.2), 85.8% (95% CI: 82.2–88.8), and 88.1% (95% CI: 81.7–92.5) for 15–24 year olds, 25–49 year olds and 50–64 year olds respectively, with a slight dip, at 78.7% (95% CI: 63.1–88.9) for 65 year olds or older PLHIV. The second 90 target, (i.e. those on ART among those who knew their HIV status) was the lowest, 51.7% (95% CI: 43.2–60.0) for 15–24 year olds compared to the older age groups with the next closest age group, 25–49 year olds at 70.5% (95% CI: 67.0–73.9). For the third 90 estimate, viral suppression was near to the 90% target (range 84.6–89.5%) for all age groups.Among PLHIV, a higher proportion, 90.6% (95% CI: 87.0–93.4), of women aged 25–49 years old were aware of their HIV status (i.e. 1st 90) compared to their male peers, 77.6% (95% CI: 70.6–83.3), see Fig. For the second 90, those aged 15–24 years for men and women, had the lowest proportion of individuals on ART at 58.8% (95% CI: 41.1–74.5) and 49.1% (95% CI: 40.5–57.7),  in women aged 65 years or older and the highest in women aged 50–64 years at 92.7% (95% CI: 86.9–96.0).The 90–90–90-point estimates for Black Africans were, 85.3% knew their status, 70.8% were on ART and 87.3% were virally suppressed. This is in comparison with the respective estimates of 73.1, 68.2 and 92.7% for the other racial groups combined that include those that self-reported as White, Coloured, or Indian. Commercial farming communities were notable for much lower knowledge of status than the other geographic areas, with a 90–90–90 cascade of 56.2, 67.6, and 81.4% in commercial farming communities compared to cascades of 87.7, 66.5 and 87.2% in urban and 85.8, 79.8 and 88.4% in rural/traditional communities.Geographically, the Free State Province 91.1%) had achieved the first 90 target, and the Eastern Cape Province 89.3%) was close to achieving the same target as seen in Table .1% had a% was cloFigure ).The third 90 target for viral suppression among those knowledgeable of their HIV status and on ART was achieved or close to being achieved in Western Cape (89.0%), Eastern Cape 90.4%), North West (90.3) and the Free State (90.0%) provinces. Only Mpumalanga province (83.1%) had a third-90 indicator value below 85%  to achieve the 90–90–90 targets, are given in Table rd90) target level to be achieved. Thus, the North West Province , Limpopo Province , and Gauteng Province  had the largest gaps to reach the 73% target for viral suppression among all PLHIV, whereas the Eastern Cape Province again had the lowest gap .The gap on this second 90 for the cascade has a relational influence on the third 90 or the 73% (i.e. 1st 90*2nd 90 *3The highest need for diagnosis (1st 90) in terms of absolute numbers was in Limpopo Province , KwaZulu-Natal Province , North West Province , and Gauteng Province , while the highest needs for ART provision were in Gauteng Province , KwaZulu-Natal Province , Limpopo Province , and North West Province .In South Africa 84.8% of PLHIV were aware of their status, of those, 70.7% on ART, and of those knowledgeable of their status and on ART, 87.4% had suppressed viral load in 2017. The country had comparable figures on these 90–90–90 indicators with neighbouring countries for the first and third 90, while the second 90 was relatively lower. In nationally representative Population-Based HIV Impact Assessment (PHIA) surveys carried out in several countries in the Southern Africa region in 2016/2017, the 90–90–90 indicators were: 74, 87, and 87% for diagnosed, on ART and virally suppressed respectively for Zimbabwe, 73, 89, and 91% for Malawi, 85, 87 and 92% for Swaziland, and 81, 92, and 88% for Lesotho [The results demonstrate a significant gap in the 90–90–90 indicator for South Africa for, ART coverage among diagnosed PLHIV. Although the proportion of PLHIV aware of their status who are on treatment are higher than estimates in previous years, the current rate of increase is not yet sufficient to meet the 90–90–90 targets. Leigh Johnson et al. estimated that 56.9% of diagnosed HIV adults were on ART in 2015 . In 2012, 31.2% or 2.0 million PLHIV were on ART . EstimatThe percentage of PLHIV who knew their status was relatively high at close to 85% with some variation by sex, age and geographic location. This likely includes some individuals who were diagnosed before changes to the UTT guidelines, who at the time of diagnosis were not eligible for ART and have still not been initiated on ART. It is possible that a substantial number of people were not on treatment because they were unaware of the new eligibility criteria or were experiencing other barriers to re-engagement in care. It is important that targeted programmes rapidly identify and facilitate enrolment of these previously diagnosed individuals who were not on treatment.High proportions of women of reproductive age (25–49 years old) diagnosed and on ART likely reflected increased diagnosis and engagement in care due to pregnancy and Prevention of Mother-to-Child Transmission (PMTCT) programmes. Interestingly, higher proportions of men 50–64 years of age were on ART compared to their younger counterparts. Increased engagement with health facilities due to care-seeking for non-communicable diseases or psychosocial factors are possible explanations for further investigation. Low treatment coverage among young people aged 15–24 years old is of concern. Women in this age group have the highest rates of new HIV infections, (1.5% per year) , 19, homThere is a need to understand the relatively low levels of ART coverage given the changes in treatment guidelines to UTT in 2016. Previous studies found that being diagnosed with HIV does not necessarily translate to universal uptake of ART even though one is offered treatment , 11, 16.Fox at el reported retention in care of 63.3% after 6 years of treatment at the national level .There are noticeable variations in the first two 90s, diagnoses and ART coverage between provinces. For example, the Eastern Cape and Free State provinces achieved the first 90 (diagnosis), but they had low values on the second 90 indicator, while the Northern Cape and the North West provinces had low levels on both the first and second 90 indicators. The same can be said when one compares the first two 90s for geographical areas, commercial farms had very low rates of diagnosis and ART coverage compared to the other two geographic areas. Geographical differences in health systems such as waiting times for patients, drug availability, staff and system capacity to diagnose and treat more patients need to be better understood.It is encouraging to see that for those individuals who were on treatment as measured by bioavailability anti-retroviral analytes in their blood, viral suppression was high and close to the 90% target. However, as the gap analyses demonstrated, turning the HIV epidemic tide through controlled viral suppression can only be achieved if there are sufficiently large numbers of PLHIV who are diagnosed who are also enrolled onto the ART programme.Unlike other studies where self-reporting is used, the strength of our study is that the results presented in this study are calculated using population-based laboratory HIV, ART and viral load testing. The estimates for ART coverage may be lower than other studies and reflects “on ART”. Individuals on “treatment holidays” or defaulting for more than 14–28 days, the half-life for most ART drugs [There are several limitations associated with this study. Ninety four percent (94%) of survey participants agreed to answer the questionnaire, and of these, 61% agreed to provide blood samples for HIV testing, ART testing and viral suppression testing. If PLHIV who provided blood samples are different from PLHIV who were not tested, then the presented results may be biased. Extensive training of enumerators and undertaking multiple visits for those not at home, up to three visits were done to increase participation and minimise such biases.In conclusion, South Africa has made enormous strides to meet the 90–90–90 targets over the past 15 years, but it still has a significant way to go to reach them especially among some sub-population groups such as adolescents and young women.Critical in achieving 90–90–90 targets is the routine monitoring and analysis of the HIV treatment cascade framework that tracks the delivery of services from HIV diagnosis, linkage to care, initiation of ART, adherence and retention in care, and viral suppression . Targete


Genetics in Medicine; 10.1038/gim.2018.31; published online 15 March 2018Correction to: The PDF and HTML versions of the article have been updated to include the Creative Commons Attribution 4.0 International License information.


Sweet orange ranks as one of the most acceptable fruit juices across the globe as it offers a healthy choice and pleasant taste to a wide spectrum of consumers. This makes it a promising vehicle for conveying functional ingredients into the human body. The present study was designed to produce functional orange juice by incorporating Kersting's groundnut proteins (isolate and hydrolysates) at different proportions  into freshly produced sweet orange juice. The sample formulations were further analysed for changes in their physicochemical , sensory , antioxidant  and antidiabetic (inhibition of α-amylase and α-glucosidase enzyme activities) properties as well as their storage stability over a 90-day storage period. The functional orange juice exhibited an improved physicochemical, antioxidant, antidiabetic and shelf life properties based on the respective protein concentrations used over the 90-day study period while the sample with the lowest proportion (0.6%) of functional ingredient had the highest sensory acceptability. Hence, the study concluded that Kersting's groundnut proteins could find useful applications in the formulation of functional orange juice. Ascorbic acid; ferric ion-reducing; metal chelating; proteins isolate; radical scavenging; total antioxidant This is not only as a cheap alternative to animal protein offering nutritional benefits, but also for the numerous health-promoting functions with guaranteed safety unlike those of synthetic origin . Hence, ). Although, functional foods range cut across different food products and classes including infant formula, beverages, confections, dairy, etc.  fruit (Orange juice is the liquid extracted from the endocarp of the sweet orange (s) fruit . It is hs) fruit . GalaverMacrotyloma geocarpum [Harms] Kerstingiella geocarpa [Harms] , Ground beans or Hausa groundnut is a subterranean legume belonging to the family fabaceae and with seeds resembling cowpea (M. geocarpum is utilized as edible seeds which are rich in protein (25%) and contain 42% of essential amino acids  and 60–70% of carbohydrates with comparatively low level of antinutrients  fruits were purchased within 4 h of harvest (for immediate use in the laboratory) from a local farm in Ile-Ife, south-western Nigeria. All chemicals and reagents used were of analytical grade and were purchased from Sigma and Aldrich Chemical company, USA.Dried, fully matured Kersting's groundnut seeds were bought from a local market in Abakaliki, Ebonyi state, Nigeria where it was stored in woven sandbags (after about 6–8 months from harvest and drying). Fully-ripe orange (2.22.2.1Kersting's groundnut defatted flour was prepared from the whole full fat flour Kersting's groundnut using the method of 2.2.2Kersting's groundnut protein isolate was prepared from the defatted flour by a method described by 2.2.3-protein hydrolysate (PcKPH) and trypsin-Kersting's groundnut-protein hydrolysate (TpKPH) but at pH of 7.5 and temperature of 40 °C and 50 °C respectively.Kersting's groundnut protein hydrolysate was prepared by using pepsin, pancreatin and trypsin - proteolytic enzymes according to the method reported by The digestion was carried out for 4 h keeping the temperature constant and pH maintained by adding 1 M NaOH or HCl when necessary. The reaction was terminated/stopped by adjusting the pH to 4.5 (with 1 M NaOH or HCl), then placing the mixture in boiling water for 20 min then placed on ice-bath for 1 h. The mixture was then centrifuged . These were stored at -4 °C until needed.2.2.4The fruits were sorted, cleaned and washed under running water. The cleaned fruits were peeled to remove the pericarp and seeds with a sharp stainless-steel knife. Extraction of juice from orange fruit was carried out as described by 2.2.52.2.5.1This was determined using a digital pH meter  according to 2.2.5.2This was determined using the hand-held sugar refractometer according to 2.2.5.3This was determined according to the AOAC 942.15 (2000) method.Ten millilitres of the juice was pipetted into a conical flask and 25 ml of distilled water was added. Three drops of phenolphthalein were added (as an indicator) and the mixture was titrated against 0.1N KOH. Titration continued until a pink colouration was observed and the corresponding burette reading taken. Blank titration was carried out by replacing the sample with distilled water. TTA was calculated in 2.2.5.4This was estimated by titrimetric method described by Exactly 5 ml of standard ascorbic acid (100 μg/ml) was measured into a conical flask containing 10 ml 4% oxalic acid. The mixture was titrated against the 0.0005M of 2, 6-dichlorophenol indophenols dye. Distilled water was added to the final volume of 1000 ml). The appearance and persistence of pink colour for 30 s was taken as the end point. The quantity of dye consumed (Va ml) was equivalent to the amount of ascorbic acid. Exactly 5 ml of sample  was measured inside a conical flask containing 10 ml 4% oxalic acid and titrated against the dye (Vb ml). The amount of ascorbic acid was calculated using the x (mg) = quantity of ascorbic acid dissolved in a known volume of oxalic acid.Va = volume of dye consumed by the sample.Vb = volume of dye consumed by the sample.15 ml = total volume of sample and oxalic titrated.100 ml = volume of oxalic acid solution used in dissolving the sample.2.2.5.5w/v) Na2CO3 solution. After 2 h of incubation at room temperature, the absorbance was measured at 750 nm. A standard curve was created with a linear range of 0.0–0.1 mg/ml using Gallic acid as the standard. The results were expressed as milligram Gallic acid equivalent (mgGAE/ml) of juice by extrapolation from the standard curve. Distilled water was used as blank.This was estimated following the procedure described by 2.2.62.2.6.1The method described by 2.2.6.2control is the absorbance of the control reaction , and Asample is the absorbance of the sample extract. Sample concentration was calculated from the graph by plotting inhibition percentage against sample concentration.This was determined using the stable radical DPPH  method as reported by 2.2.6.33 solution and FRAP reagent was added to each of the tubes to make it up to 5 ml. The tubes were incubated for 10 min and the absorbance of the mixture was read at 593 nm against the blank. Acetate buffer was used as a blank control sample.This was determined using colorimetric method described by 2.2.6.42 (0.05 ml). The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml) and total volume was adjusted to 4 ml of ethanol. Then, the mixture was shaken vigorously and left at room temperature for ten minutes. Absorbance of the solution was then measured spectrophotometrically at 562 nm. The percentage inhibition of ferrozine–Fe+2 complex formations was calculated using control = absorbance of control sample  and Abs sample = absorbance of sample.This was carried out according to the method of 2.2.7Linoleic acid oxidation was measured using a slight modification of the method described by 2.3(i)Inhibition of α-glucosidase activity2O).). In order to get entire enzyme inhibition, phosphate buffer was used to prepare multiple solutions. Phosphate buffer (75 μl), 20 μl of the enzyme solution in phosphate buffer  and sample (5 μl). This was followed by initial incubation at 35 °C for 10 min prior to the addition of P-NPG to initiate the reaction. P-NPG (20 μl) was added in phosphate buffer  and then incubated at 35 °C. Acarbose compound was used as positive control. The absorbances were spectrophotometrically measured at 405 nm. One unit of α-glycosidase equals amount of enzyme that catalysed the hydrolysis of 1.0 mol substrate per minute at pH 7.4.(ii)Inhibition of α-amylase activityThe method described by 2O). Phosphate buffer was used to prepare multiple solutions in order to get entire enzyme inhibition. Substrate (35 μl), phosphate buffer , and sample (5 μl) solutions were mixed and preincubated at 35 °C for 30 min. Afterwards, 20 μl of a 50 μg/ml enzyme solution was added and the solution incubated for another 30 min. The reaction was terminated by adding 50 μl of 0.1MHCl. The absorbance was measured spectrophotometrically at 580 nm. One unit of α-amylase enzyme equals the amount of enzyme that released 1.0 mg of maltose from starch in 3 min (pH 6.9 at 20 °C).The α-amylase inhibition assay was carried out according to the method of 2.3.1. During this period, the juice samples were evaluated every 2 weeks for quality characteristics  using some of the methods described earlier. The functional orange juice samples were stored at ambient temperature under cool and dry environments.The shelf-stability of the functional orange juice samples was evaluated for 90 days2.3.2Sensory characteristics of the fortified juice were evaluated based on colour, taste, flavour, mouthfeel and overall acceptability using a 9-point Hedonic scale. This was done on a scale of 1 (for dislike extremely) to 9 (for like extremely). The taste panel analysis was conducted using 20 semi-trained panelists who are familiar with orange juice. The evaluation was conducted at room temperature in a well-lit and properly ventilated environment. There was water to rinse mouth in between tasting protocols of the samples. The samples were randomly coded and presented as KCF, FKC, KFC and FCK .2.3.2.1The semi-trained panelists selected to participate in the study were made voluntary and confidential. Twenty (20) panelists  were selected from University community based on their knowledge about conduct of sensory evaluation exercise and familiarity with the type of product under evaluation.The panelists gave their consent and confirmed their willingness to participate before taken part in the evaluation exercise.2.3.2.2The protocol and the instruments (Questionnaire) used were approved by the Department of Food Science and Technology, Obafemi Awolowo University, Ile-Ife Research Committee in line with the approved ethics of the Institutional Research Committee.2.3.3Experiments were performed in triplicates, and data analyzed were mean subjected to analysis of variance (ANOVA). Means were separated by the Tukey's multiple range test (SPSS version 20) while results were taken to be significant at 5% level (p < 0.05).33.1Citrus sinensis juice for (.uice for .(p < 0.05) except those fortified with trypsin-hydrolysed protein . As expected, the TTA values showed a reverse order when compared with the pH values, thus a direct relationship with the proportion of isolate/hydrolysate. This study presented comparable range of values with 0.56% reported by Citrus reticulata juice  but much lower range compared with 1.01% reported for manually-expressed Citrus sinensis nectar (Also presented in s nectar . The obs< 0.05) from the control sample. A comparable value of 10.0 was reported for manually-extracted sweet orange juice by Citrus sinensis nectar was reported by Citrus sinensis and 11.78 reported for hand-squeezed Saudi Arabia Citrus sinensis (cv. Orlando) by Citrus sinensis (cv. Range and cv. Jincheng) by The refractive indices of the juice samples are also indicated in From citrus sinensis juice.The total phenol content (TPC) of the orange juice samples is shown in From the present result, there is an indication that the TPC of the orange juice samples increased with increasing proportions of protein isolate/hydrolysate. This could be as a result of higher proportions of basic and or hydrophobic amino acids with greater tendency to interact with the polyphenol compounds in the orange juice . Phenoli3.2The antioxidant and antidiabetic properties of freshly-prepared orange juice fortified with varying proportions of isolate/hydrolysate fractions were carried out.Citrus aurantifolia and persimmon as reported by Citrus sinensis and mechanically-squeezed Citrus aurantifolia by Also presented in (p < 0.05) from the control sample, an indication that fortification improved the FRAP of the orange juice. The FRAP values recorded in this study are comparable with the 0.41mMol for fresh orange juice and 0.15 to 0.46 mMol obtained for Citrus aurantium reported by The Ferric-reducing antioxidant power (FRAP) of fortified orange juice samples are presented in Citrus aurantium while 7.98 mg EDTA/L of juice was recorded for hand-squeezed, white Citrus maxima  ability of fortified orange juice samples is also shown in s maxima . The difAlso presented in From this study, the inhibition of α-amylase and α-glucosidase enzymes ranged between 28.88 and 42.11% and 27.32–40.97% respectively. For the two enzymes, the control samples had the lowest values of inhibition while PA-A recorded the highest values. Also, there were significant differences (p < 0.05) between the control samples and the fortified samples in both cases, an indication that incorporation of protein isolate/hydrolysate had a direct effect on inhibiting the activities of the enzymes. It was noted that the percentage inhibition increased with increasing level of functional ingredients. This agrees with earlier findings that the activities of α-amylase and α-glucosidase enzymes increased with increasing concentration of yellow pea hydrolysate  some selLuffa cylindrical) alcalase-hydrolysate and tryptic-hydrolysate respectively  of functional orange juice samples is presented in 3.3.4In other words, all the samples showed a decrease in vitamin C content with storage period with the decrease exhibited by the control sample being significantly lower than that of the fortified samples. 3.3.5A similar loss (66–58% at 28 °C and 51–22% at 4 °C) in TPC during storage was also reported by 3.3.6The pattern exhibited by total antioxidant activity (TAA) of fortified orange juice samples over a 90-day storage period is presented in 3.3.73.3.8in-vitro ability of isolate/hydrolysate-fortified orange juice samples to inhibit the activities of alpha-amylase and alpha-glucosidase enzymes over a 90-day storage period are shown in citrus spp  is presented in For the isolate-fortified juice samples, the values for the appearance ranged between 6.36 and 8.00 with KCF recording the lowest and KFC the highest preference. No significant differences (p > 0.05) were exhibited by samples KCF, FKC and FCK (the control) while KFC was significantly different (p < 0.05) from all others. The values for colour (6.55–7.55), flavour (6.55–7.73), mouthfeel (6.36–7.46) and overall acceptability (7.00–7.55) exhibited no significant difference (p > 0.05).The pepsin-hydrolysate fortified juice samples presented a range of 6.27–7.55 for appearance with only FKC and KFC being significantly different (p < 0.05) from each other. The colour presented a range of 6.55–7.55 with only FKC and KFC being significantly different (p < 0.05) from each other. The values for flavour ranged between 5.82 and 7.46 with samples KCF and KFC being significantly different (p < 0.05) from the control (FCK). The values for mouth feel and overall acceptability showed no significant difference (p > 0.05) among one another and the ranges are 6.36–6.55 and 6.55 to 7.00, respectively.The values for the pancreatin-hydrolysate fortified juice samples in terms of appearance and colour ranged from 5.63 to 7.55 and 5.27 to 7.46 respectively, with the two sensory parameters showing significant difference (p < 0.05) only between sample KCF and the control (FCK). The respective values for flavour and mouth feel ranged from 6.18 to 7.46 and 7.09 to 7.27 with no significant difference (p > 0.05) among all the samples. Overall acceptability recorded a range of 6.55–7.55 with significant difference existing between only samples KFC and FCK. Trypsin-hydrolysate fortified juice samples indicated no significant difference (p > 0.05) for all the parameters and the range of values recorded were 7.09–7.27 for appearance; 6.73 to 7.27 for colour; 6.18 to 6.77 for flavour; 6.45 to 6.55 for mouth feel and 6.36 to 7.00 for overall acceptability.Based on the evaluation, most of the judges perceived a very low difference between sample KFC  and sample FCK  while samples KCF and FKC recorded higher level of significant difference (p < 0.05) in comparison with the control. KCF similar report was presented for protein-fortified mango juice by The trypsin hydrolysate-fortified samples were more preferred by the panelists in terms of colour and appearance, the pepsin-hydrolysate fortified juice recorded the least preference in terms of flavour, while the isolate hydrolysate-fortified samples were least preferred for mouthfeel. For overall impression, no significant difference (p > 0.05) was recorded except in the pancreatin hydrolysate-fortified samples.From the responses in this study, it can be deduced that individually, the opinions of the respondents were diverse due to the randomized order of presentation. However, the outcome of the evaluation still pointed to a single direction, an indication that the responses were based wholly on the sensory experience of the panelists.4It was concluded from this study that production of functional orange juice by incorporating Kersting's groundnut protein isolate and hydrolysate  was able to improve the overall physicochemical, antioxidant and antidiabetic properties while the sample with the lowest proportion (0.6%) of functional ingredient had the highest sensory acceptability. Also, the shelf stability of orange juice was increased in terms of these properties over a 90-day period. Hence, Kersting's groundnut proteins could find bioactive roles in the production of functional beverages.Omolayo R. Osungbade: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Abiodun V. Ikujenlola: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Saka O. Gbadamosi: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


A prospective cohort study was conducted on a convenience sample of 1370 pregnantwomen with a gestational age of 20 to 25 weeks in the city of Ribeirão Preto.Data on obstetrical history, maternal age, parity, smoking habit, and a historyof preterm delivery was collected with the application of a sociodemographicquestionnaire. Cervical length was determined by endovaginal ultrasound, andurine and vaginal content samples were obtained to determine urinary tractinfection (UTI) and bacterial vaginosis (BV), respectively. The aim of thisstudy was to verify the association of cervical length and genitourinaryinfections with preterm birth (PTB). Ultrasound showed no association of UTI orBV with short cervical length. PTB rate was 9.63%. Among the women with PTB, 15showed UTI , 19 had BV ,and one had both UTI and BV . Nineteen (14.4%) PTBoccurred in women with a cervical length ≤2.5 cm .Among the 75 patients with PTB stratified as spontaneous, 10 showed UTI  and 14 had a diagnosis of BV . A short cervical length between 20 and 25 weeks of pregnancy wasassociated with PTB, whereas UTI and BV determined at this age were notassociated with short cervical length or with PTB, although UTI, even ifasymptomatic, was related to spontaneous PTB. Preterm birth (PTB) is an important public health problem due to its high incidenceand perinatal morbidity-mortality .BraziliStudies have demonstrated that infections such as bacterial vaginosis (BV) andurinary tract infections (UTI) may be associated with a higher risk of PTB and lowbirth weight ,6, sinceCervical effacement causing cervical shortening precedes by five to six weeks theclinically determined labor ,10. ThusOn this basis, our objective was to assess the association of genitourinaryinfections with cervical shortening and their relationship with previous PTB(PPTB).The study was approved by the Research Ethics Committee of the University Hospital,Faculty of Medicine of Ribeirão Preto, University of São Paulo (HC-FMRPUSP) in 2008(protocol No. 11157/2008), and all women gave written informed consent toparticipate.https://nesca.fmrp.usp.br/brisa-botoes) conducted in Ribeirão Preto(SP) and São Luís (MA). The Ribeirão Preto data were used in the present study.This case-control study was nested in a prospective convenience cohort investigatedin 2010 and 2011. The cohort has been described in the thematic project entitled"Ecological factors of preterm birth and consequences of perinatal factors onchildren’s health: birth cohorts in two Brazilian cities - BRISA"  and based on the gestationalultrasound performed before 20 weeks of pregnancy, thus minimizing determinationbias in the estimate of outcome .Lactobacillus,Gardnerella/Bacteroides, andMobiluncus morphotypes. Morphological types where scored as 1+to 4+ according to the number of microorganisms present. A score of seven or morewas considered to indicate positivity for BV , and atechnique systematized and validated by the Fetal Medicine Foundation . ControlAfter birth, newborn data regarding hospital, weight, and gestational age wereobtained and a standardized puerperium questionnaire was applied in order to obtainchildbirth information about the 95 participants, with the analysis of replies suchas presence of painful contractions preceding the outcome in order to determinewhether labor was spontaneous. Participants whose questionnaires did not containsufficient information were contacted by telephone and 7 of them were located. Thedifference between groups was analyzed by the Fisher exact test, and the relativerisk of the various variables was calculated by the adjustment of log-binomialmodels and confidence intervals (95%CI) using the SAS 9.2 software (USA).The variables were defined according to their relationship with risk of PTB: cervicallength (≤2.5 or >2.5 cm); presence or absence of urinary infection and/or BV; age; smoking (yes or no); previous preterm birth (yes or no) –25.The general characteristics of the study population are shown in The Fisher exact test was applied to determine the association between the presenceof genitourinary infections and a short cervix in an ultrasound exam. A cervicallength of 2.5 cm or less was observed in 7 (5.79%) of 121 patients with a positiveUTI exam and in 68 (5.46%) of the 1245 patients with no UTI (P=0.83). A cervicallength of 2.5 cm or less was observed in 13 (7.07%) of 184 patients with a positiveBV exam and in 61 (5.21%) of those with no BV, again without statisticalsignificance (P=0.30). Among the 13 patients with a positive exam for the twoinfections, no cervical length ≤2.5 was observed.Regarding the infections, patients with UTI had a crude RR of 2.02 with 95%CI:1.05–3.863 and an adjusted RR of 2.205 with 95%CI: 1.109–4.385, whereas patientswith BV had a crude RR of 1.72 with 95%CI: 0.97–3.04 and an adjusted RR of 1.2 with95%CI: 0.64–2.24. No patient had association of the two infections. Regarding thecervix (see details in Taking into account previous preterm births (PPTB), for patients with 1 PPTB, thecrude RR was 16.94 (95%CI: 10.46–27.43) and the adjusted RR was 15.83 (95%CI:9.37–26.74). For patients with 2 or more PPTB, the crude RR was 16.52 (95%CI:8.56–31.87) and the adjusted RR was 12.83 (95%CI: 5.97 to 27.56). Patients whoreported that they were smokers had a crude RR of 1.85 (95%CI: 1.17–2.93) and anadjusted RR of 1.31 (95%CI: 0.79–2.18). The reference for this calculation was thegroup of nonsmokers. Regarding parity, primiparous patients had a crude RR of 0.90(95%CI: 0.55–1.46); the adjusted RR could not be calculated since a history of PTBwas one of the variables that did not apply to this group. Patients in their secondor third pregnancy were the reference used for this calculation. The group ofpatients with 4 or more pregnancies had a crude RR of 1.34 (95%CI: 0.71–2.45).Regarding age, pregnant women younger than 19 years had a crude RR of 1.16 (95%CI:0.61–2.23) and the adjusted RR could not be calculated. The group aged 19 to 35years was used as reference for the calculations. Patients older than 35 years had acrude RR of 1.18 (95%CI: 0.52–2.67) and the adjusted RR could not be calculated.Details are presented in The present study revealed a 9.63% incidence of PTB, a value below that reported inthe literature. In Brazil, a 2010 study coordinated by the Postgraduate Program ofEpidemiology of the Federal University of Pelotas with the participation of 12universities, reported that 11.7% of the births occurring in Brazil are preterm. The 201Data analysis revealed that cervical shortening was not associated with theoccurrence of infections (UTI and BV) between 20 and 25 weeks of pregnancy. Thehypothesis of such association was raised because infections, as well cervicalshortening, are associated with PTB. We considered the immuno-inflammatory theory ofPTB and attempted to determine whether the cytokines and prostaglandins produced ininflammatory and infectious processes, which trigger uterine contractions anddepolymerization of the cervix, were linked to cervical shortening, as proposed byRomero et al. , and ChaA cervical length ≤2.5 cm determined between 20 and 25 weeks of gestation wasassociated with PTB, supporting data obtained by others. In 1966, Schaffner andSchanzer  who inveOf 199,093 patients studied by Scheiner et al. , 4890  being usedto assess the association of factors related to PTB, it was observed that the samplesize used had enough power for the estimate of associations of interest.A limitation of the present study was that the patients were not monitored during theprenatal period, being evaluated only once prenatally and once after childbirth.Thus, there was no control of the number of pregnant women who had symptomatic UTIand BV throughout gestation and it was not known whether or not they had beentreated. An additional limitation was that we did not know how the patients with ashort cervical length had been followed up.In the present study, there was no association of asymptomatic UTI or BV withcervical shortening determined between 20 and 25 weeks of pregnancy. UTI, butnot BV, was associated with spontaneous PTB. There was a relationship betweenearly cervical shortening and prematurity, with a lower incidence than thatreported in the literature.


Chordomas are rare, slow-growing sarcomas without any accepted prognostic biomarkers. Owing to their proximity to critical neurovascular structures, discovering predictive biomarkers in chordoma has been a significant research effort because it may potentially reduce risky therapies in patients with less aggressive tumors. In response, because cyclin E1 overexpression correlates with patient prognosis in several malignancies, we investigated its expression in chordoma and whether it informs patient prognosis.via immunohistochemical staining. Western blot was used to assess cyclin E1 expression in chordoma cell lines and fresh tissues. We then correlated cyclin E1 staining intensity in the TMA to clinicopathological features and chordoma patient outcomes.Seventy-five chordoma patient specimens were enrolled in a tissue microarray (TMA) to evaluate cyclin E1 expression Sixty-three percent of the chordoma patient specimens in the TMA, fifty-six percent of the fresh chordoma tissues, and all chordoma cell lines showed high cyclin E1 expression. In TMA analysis, cyclin E1 expression positively correlated to chordoma patient disease status. By survival analysis, high cyclin E1 expression was an independent prognostic risk factor for chordoma patients along with advanced disease status and positive surgical margin.Cyclin E1 is a promising biomarker predicting chordoma patient prognosis. In addition, even with aggressive surgery, recurrent rates remain high. There is, therefore, an urgent need for the identification of chordoma biomarkers to better ascertain patient prognosis and whether high-risk surgeries or adjuvant therapies are indicated.Chordomas are rare bone sarcomas that arise from the transformed remnants of notochord, with an incidence of 0.1/100,000 people per year . SurgeryCyclin E1 was originally described in 1991 as the prototype cyclin E. It is a 47 kDa protein localized within both the nucleus and cytoplasm . By compIn response, we sought to investigate the expression of cyclin E1 in chordoma and its correlation, if any, to clinicopathologic features or prognosis within chordoma patients. To our knowledge, this is the first and only work evaluating cyclin E1 expression in chordoma and its clinical significance.The study protocol and the consent of the informed patients were approved by the Partners Human Research Committee (number: 2007-P-002464/5) of Partners HealthCare as previously described . All patSeventy-five chordoma patient specimens in formalin fixed paraffin embedded (FFPE) blocks obtained during surgery were used to construct the tissue microarray (TMA) as previously described . The inc® Boost Detection Reagent  and SignalStain® DAB . Finally, all sections were counterstained with Hematoxylin QS , and the slide was mounted with VectaMount AQ  for long-term preservation.Immunohistochemistry (IHC) staining was performed to evaluate for cyclin E1 expression. In brief, the paraffin-embedded slide was baked for 1 h at 60°C before xylene deparaffinization. The slide was then rehydrated through graded ethanol (100% and 95%). We then used 3% hydrogen peroxide to quench endogenous peroxidase activity after heated epitope retrieval. After blocking for 1 h with normal goat serum, the slide was incubated with polyclonal rabbit antibody to human cyclin E1  overnight in a humidified chamber set at 4°C. Afterwards, the bound antibody was detected by SignalStainTwo independent pathologists blinded to patient clinical information and tumor characteristics assessed and scored the IHC-stained slide. Cyclin E1 expression was scored and categorized according to the staining intensity of chordoma tissues: 0, no staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining. The low cyclin E1 expression subset included group 0 and 1+, while the high cyclin E1 expression subset included group 2+ and 3+ (2 in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin .The human chordoma cell line UCH2 was established and kindly provided by Dr. Silke Brüderlein  . The celWe used 1× RIPA lysis buffer  combined with protease inhibitor cocktail tablets  to extract protein from cells and fresh tissues. The DC™ protein assay reagents  and a spectrophotometer SPECTRA max 340PC  were then used to determine the protein lysate concentrations. Briefly, our Western blot protocol began with an SDSPAGE gel to run the denatured proteins before transfer to nitrocellulose membranes. These membranes were incubated with monoclonal rabbit antibodies to human cyclin E1  and mouse monoclonal antibody for human β-actin  at 4°C overnight after they were blocked in Odyssey Blocking Buffer  for 1 h. Following incubation with the primary antibody, TBST was used as a membrane wash (4 times for 5 min each at room temperature). Next, goat anti-rabbit IRDye 800CW  and goat anti-mouse IRDye 680LT secondary antibody   were applied for 1 h at room temperature followed by another TBST membrane wash (four times for 5 min each at room temperature). Bands were detected using Odyssey Infrared Fluorescent Western Blot Imaging System from Li-COR Bioscience , and Odyssey software 3.0 was used to quantify the bands.P<0.05) in the univariate analysis were involved in the multivariate analysis. A P-value < 0.05 was considered significant.GraphPad Prism 7 software  and SPSS 19.0 software  were used for statistical analyses. Nonparametric testing (Mann-Whitney U test) was performed to compare the two groups and determine statistical significance. One-way analysis of variance and the least-significant difference test were performed for multiple comparisons. As for the survival analysis, overall survival (OS) was defined as the time from surgery (when the tissue was obtained) to death of the patient. Recurrence-free survival (RFS) was defined as the time from the surgery to the first local recurrence after this surgery. Metastasis-free survival (MFS) was defined as the time from surgery to the detection of new onset metastasis. The survival curves were produced by Kaplan-Meier methods. The relation between various key factors and OS, RFS, or MFS was evaluated by Cox regression both univariately and multivariately. Only those factors that were statistically significant  with the remaining 65 specimens having various staining intensities, including 1+ staining in 18 specimens (24.0%), 2+ staining in 23 specimens (30.7%), and 3+ staining in 24 specimens (32.0%)  and 4 with low expression (44.4%). In chordoma cell lines, Western blots showed cyclin E1 was expressed in both UCH2 and CH22, with CH22 having notably high expression. (P=0.23)  (P=0.002) , local recurrence was found in 37 patients (49.3%) and new onset metastasis was found in 17 patients (22.7%). Of the 75 chordoma patients, the mean OS in months was 102.3 (95% CI 83.7 to 121.0), RFS was 102.4 (95% CI 79.5 to 125.3) and MFS was 180.2 (95% CI 151.5 to 208.9). Five-year rates included OS of 63.1% (95% CI 51.7% to 74.5%), RFS was 52.4% (95% CI 40.2% to 64.6%), and MFS rate was 79.3% (95% CI 69.1% to 89.5%). Finally, the ten-year rates included OS of 38.2% (95% CI 25.3% to 51.1%), RFS of 47.0% (95% CI 33.9% to 60.1%), and MFS of 69.5% (95% CI 55.6% to 83.4%) , breast cancer (53%–61%), liver cancer (36%–68%) and some sarcomas 29%–61%) , 22–28. %–61% , 2Our TMA analysis showed cyclin E1 expression positively correlates with chordoma patient disease status. Of note, these results echo works in other malignancies where high cyclin E1 expression is associated with high-staging as well as tumor aggression and therapeutic-resistance , 33, 34.Cyclin E1 predicts poor prognosis in several malignancies , 28, 37.Finally, we assessed the predictive potential of other clinicopathological features for chordoma patients. As expected, an advanced disease status of chordomas engenders more aggressive future tumor behavior. It is an independent risk factor for OS and MFS in chordoma, which is similar to the results of previous studies , 5, 38. To our knowledge, ours is the first study evaluating the expression and prognostic role of cyclin E1 specifically in chordoma. While we have identified a significant correlation between cyclin E1 expression and chordoma patient prognosis, future works should focus on the mechanism of the overexpression of cyclin E1 in chordoma and how cyclin E1 overexpression causes chordoma cell proliferation and poor outcomes. This would inform future targeted therapies and clinical trials making use of our initial findings. Moreover, while molecular targeting therapies have been investigated in chordoma but have largely been underwhelming, the cyclin E1 should be investigated as a novel therapeutic strategy for chordoma patients –43.via orthopedic surgeons, who usually treated chordoma on mobile spine or sacrum. Third, the mechanisms of cyclin E1 overexpression in chordoma remains unknown and need be further investigated. Future investigations to validate and build on the prognostic value of Cyclin E1 in chordoma will be carried out on a larger sample size collaborating with neurosurgeons to include skull base chordoma, and will focus on the detail mechanism and regulation of cyclin E1 expression in chordoma.There are some limitations in our study. First, 75 chordoma cases is comparatively large sample size for this rare disease as compared to other previous studies on investigating prognostic biomarkers in chordoma , 42. HowIn summary, we show cyclin E1 is overexpressed in most chordomas, and its expression positively correlates to disease status and inversely correlates with OS, RFS, and MFS of chordoma patients. Our work suggests cyclin E1 as a prognostic biomarker for chordoma patients and supports future works investigating its mechanism and pathway as a potential drug target.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study protocol and the consent of the informed patients were approved by the Partners Human Research Committee (number: 2007-P-002464/5) of Partners HealthCare as previously described. All patients provided their written informed consent to participate in this study.Research design and data preparation: RW, PT, FH, WG, and ZD. Drafting and revising the paper: RW, DD, and ZD. Approval of the submitted and final versions: WG and ZD. All authors contributed to the article and approved the submitted version.This work was supported, in part, by the Musculoskeletal Tumor Center and Beijing Key Laboratory of Musculoskeletal Tumor at Peking University People’s Hospital, Beijing, China, and Department of Orthopaedic Surgery at UCLA, Los Angeles, California, USA. WG is supported, in part, through a grant from Sanming Project of Medicine in Shenzhen, China. ZD is supported, in part, through a grant from Sarcoma Foundation of America (SFA), 222433, a Grant from National Cancer Institute (NCI)/National Institutes of Health (NIH), U01, CA151452-01.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.