Sex estimation using orbital measurements in the Croatian population.

The aim of this study was to test the sexual dimorphism of orbital measurements in the Croatian population using multi-slice computed tomography (MSCT) images. We have retrospectively taken 414 head CT scans of adults from Croatian clinical hospitals in Split and Zagreb (214 males and 200 females) with slice thickness < 1 mm and no pathological or traumatic changes that could affect the measurements. DICOM files were imported into Stratovan Checkpoint Software and viewed in 2D and 3D using semi-transparent 3D volume rendering. Eight standard measurements were calculated based on twelve orbital landmarks (six paired). Principal component analysis (PCA) was used to explore sexual and regional differences, and linear discriminant analysis was used to develop sex classification models. The PCA showed separation based on sex and region, and additional analysis demonstrated that females and males in Split and Zagreb differed in four orbital measurements (P ≤ 0.001). Only those measurements that did not show regional differences were further analyzed, and all showed statistically significant sexual dimorphism. The accuracy of univariate functions for sex estimation ranged from 53.43 to 71.88%, and for multivariate function, the accuracy was 73.45%. The orbital measurements of the Croatian population showed restricted forensic significance for sex classification. On the other hand, we have shown that they can have a potential for exploring the inter- and intra-population differences.

The repeatability of standard cranial measurements on dry bones and MSCT images.

This study examined if the cranial measurements from Data Collection Procedures for Forensic Skeletal Material 2.0 are repeatable when measured in dry bones and MSCT images and if the virtual measurements correspond to the physical ones. The sample included 33 dry crania imaged by MSCT. Two observers measured dry bones, two placed landmarks on 2D and 3D MSCT reconstructions, and one conducted measurements/landmarking on both media. One of the observers for each media repeated the measurements. Technical and relative technical error of measurement (TEM and rTEM) and percentage differences were calculated to examine the repeatability of measurements and compare measuring modalities. Intraobserver rTEM was above 1.5% for six bone measurements: FOB, ZOB, OBB, NLH, DKB, MDH (1.51%-4.87%) and for seven MSCT measurements: OBH, FOB, OBB, MDH, NLB, ZOB, DKB (1.57%-5.55%). The interobserver rTEM was above the acceptable level (>2%) for 11 measurements: PAC, NLH, OBB, EKB, MAL, FOB, NLB, OBH, ZOB, DKB, and MDH (2.01%-9.34%). The percentage differences were not systematically larger for measurements taken by the same user on both modalities than those obtained by different users on the same modality. When physical and MSCT measurements were tested on sex classification standards, the proportion of crania classified as male or female did not significantly differ (p > 0.05). The study showed that physical and virtual cranial measurements could be interchangeable for developing or applying sex estimation standards. However, clarifications and adaptations are necessary for measurements of mastoid, nasal, and orbital regions that did not meet the standard criteria.

Efficiency of the Adjusted Binary Classification (ABC) Approach in Osteometric Sex Estimation: A Comparative Study of Different Linear Machine Learning Algorithms and Training Sample Sizes.

The adjusted binary classification (ABC) approach was proposed to assure that the binary classification model reaches a particular accuracy level. The present study evaluated the ABC for osteometric sex classification using multiple machine learning (ML) techniques: linear discriminant analysis (LDA), boosted generalized linear model (GLMB), support vector machine (SVM), and logistic regression (LR). We used 13 femoral measurements of 300 individuals from a modern Turkish population sample and split data into two sets: training (n = 240) and testing (n = 60). Then, the five best-performing measurements were selected for training univariate models, while pools of these variables were used for the multivariable models. ML classifier type did not affect the performance of unadjusted models. The accuracy of univariate models was 82−87%, while that of multivariate models was 89−90%. After applying ABC to the crossvalidation set, the accuracy and the positive and negative predictive values for uni- and multivariate models were ≥95%. Sex could be estimated for 28−75% of individuals using univariate models but with an obvious sexing bias, likely caused by different degrees of sexual dimorphism and between-group overlap. However, using multivariate models, we minimized the bias and properly classified 81−87% of individuals. A similar performance was also noted in the testing sample (except for FEB), with accuracies of 96−100%, and a proportion of classified individuals between 30% and 82% in univariate models, and between 90% and 91% in multivariate models. When considering different training sample sizes, we demonstrated that LR was the most sensitive with limited sample sizes (n < 150), while GLMB was the most stable classifier.

Croatian genetic heritage: an updated Y-chromosome story.

AIM: To analyze an additional set of ˝Y-chromosome genetic markers to acquire a more detailed insight into the diversity of the Croatian population. METHODS: A total of 518 Yfiler Plus profiles were genotyped. Allele frequencies, haplotype frequencies, and haplotype diversity were calculated by using the STRAF software v. 2.0.4. Genetic distances were quantified by Rst with AMOVA online tool from the YHRD. The evolutionary history was inferred with the neighbor-joining method of phylogenetic tree construction in the MEGAX software. Whit Athey's Haplogroup Predictor v. 5 was used for additional comparison with regional and other European populations. RESULTS: A total of 507 haplotypes were used for genetic STR analysis. An interpopulation study on 17 Y-STR markers showed the lowest genetic diversity between the Croatian and Bosnian-Herzegovinian populations and the highest between the Croatian and Irish populations. Additional interpopulation comparison with the original 27 Y-STR markers (for the population with available data) was also performed. A total of 518 haplotypes were used in the determination of haplogroup diversity. Haplogroup I with its sublineage I2a expressed the highest prevalence. The second most prevalent haplogroup was R, with its major sublineage R1a, except for the subpopulation of Hvar, where E1b1b was the second most prevalent haplogroup. Rare haplogroups also confirmed in this study were L, T, and Q. G1 was detected for the first time in the Croatian population. CONCLUSION: We obtained a new insight into the differences between examined subpopulations of Croatia and their possible (dis)similarities with neighboring and distant populations.

ABO and Rh blood groups, demographics, and comorbidities in COVID-19 related deaths: A retrospective study in Split-Dalmatia County, Croatia.

BACKGROUND: Blood group phenotypes have been associated with COVID-19 susceptibility and severity. This study aimed to examine ABO/Rh blood group distribution in COVID-19-related deaths considering demographics and pathological conditions. METHODS: We conducted a retrospective study at the University Hospital Center Split, Croatia, that included 245 COVID-19 positive individuals that died from April 8, 2020, to January 25, 2021. We extracted data on their blood groups, demographics, and pre-existing comorbidities and compared findings with general population data from blood group donations (n = 101,357) and non-COVID-19 deaths from 2019 (n = 4968). RESULTS: The proportion of dead males was significantly higher than in non-COVID-19 cases (63.7% vs. 48.9%, P < 0.001), while the proportion of older individuals did not differ. The prevailing pre-existing diseases were hypertension (59.6%), diabetes (37.1%), heart failure (28.8%), digestive disorder (26.5%), and solid tumor (21.6%). The ABO distribution in the deceased and donors' group showed significant differences, with the higher prevalence of A/AB group and lower prevalence of 0, but with individual differences significant only for AB and non-AB groups. There was a reduced proportion of females within the deceased with group 0 (P = 0.014) and a higher proportion of AB individuals with coronary heart disease (P = 0.024). CONCLUSION: The study confirmed a higher risk of death in males. The lower proportion of type 0 in deceased individuals was greater in females, implying that group 0 is not necessarily an independent protective factor. Coronary heart disease was identified as a potential risk factor for AB individuals.

BUILDING THE COVID-19 TESTING CAPACITIES IN CROATIA: ESTABLISHING THE INTERDEPARTMENTAL COVID-19 UNIT AT THE SPLIT UNIVERSITY HOSPITAL CENTRE.

This paper presents the chronology, experiences, and challenges in introducing COVID-19 RT-PCR testing in Split, Croatia. We describe the processes from March 12, 2020 to May 26, 2020, starting from the initial knowledge transfer, expert team formation and management, testing implementation, and concluding with the standalone testing facilities, which used automated processes sufficient to meet testing requirements at that time. In the case presented, the COVID-19 unit was organized by joining human and laboratory resources from five clinical departments at the Split University Hospital Centre. Sample preparation procedures and analyses were launched within the restricted time frame while simultaneously training and organizing new laboratory staff and completing equipment requirements. As a result, the process that started with 30 tests per day was constantly improved over time and reached up to 160 tests per day when MagNA Pure was added to automatize RNA extraction at the end of April. At that pace, the cumulative number of samples soon exceeded the first thousand, and by the end of May it exceeded 4000. The case presented provides an example of good practice for crisis response and organization that successfully enabled sufficient COVID-19 testing capacities within the restricted time frame, human and technical resources. Despite limited understanding of COVID-19 at that time, appropriate management, transfer of knowledge, previous experiences in related laboratory and diagnostic work, as well as interdisciplinary and interdepartmental cooperation proved appropriate to overcome the above limitations and ensure adequate healthcare response.

Adjusted binary classification (ABC) model in forensic science: An example on sex classification from handprint dimensions.

Binary classification techniques are commonly used in forensic examination to test if a specimen belongs to a particular group and base the expert opinion on the questioned evidence. However, most of the currently used methods do not achieve sufficient accuracy due to the ignoring of the specimens classified in the overlapping area. To address the issue, we proposed a novel Adjusted binary classification (ABC) algorithm that automatically adjusts posterior probabilities to reach classification accuracy and positive/negative predicted values (PPV, NPV) of 95 %. In the presented example, we used three handprint measurements from 160 participants (80 males and 80 females) to develop models that would classify sex from their dimensions. The sample was split into the training/cross-validated (70 %) and testing sample (30 %). We developed four classification models using linear discriminant analysis (LDA) by employing traditional single cut-off values and ABC approach that for each group provides a specific posterior probability cut-off threshold. In the cross-validated sample, the accuracy of traditional models was 78.7-92.5 %, while PPVs/NPVs ranged between 78.2 and 93 %. ABC models provided 95 % accuracy, PPV, and NPV, and could classify 35.5-88.1 % of specimens. In the testing sample, ABC models achieved accuracy of 97.3-100 %, PPV/NPV 95.4-100 %, and could be applied to 29.1-87.5 % of specimens. The study demonstrated that the ABC approach could adjust classification models to reach predefined values of accuracy, PPV, and NPV. Therefore, it could be an efficient tool for conducting a binary classification in forensic settings and minimizing the possibilities of incorrect classifications.

SARS-CoV-2 Antibody Seroprevalence in Industry Workers in Split-Dalmatia and Sibenik-Knin County, Croatia.

OBJECTIVES: To examine seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in industry workers population sample. METHODS: From 23 to April 28, 2020, we conducted serological testing for antibodies (Immunoglobulin G (IgG) and Immunoglobulin M (IgM)) on 1494 factory employees living in the Split-Dalmatia and Sibenik-Knin County (Croatia). RESULTS: We detected antibodies in 1.27% of participants (95% confidence interval [CI] 0.77-1.98%). In Split facility 13/1316 (0.99%, 95% CI 0.53-1.68%) of participants were tested positive, of which 13/1079 (1.20%, 95% CI 0.64-2.05%) of those living outside the facility and 0/237 (0%, 95% CI 0-1.26%) of those living inside the facility. In Knin facility, 6/178 (3.37%, 95% CI 1.25-7.19%) participants were tested positive for antibodies. CONCLUSIONS: The study showed relatively small SARS-CoV-2 antibody seroprevalence in the DIV Group population sample.

A Forensic Genomics Approach for the Identification of Sister Marija Crucifiksa Kozulic.

Sister Marija Krucifiksa Kozulic (1852-1922) was a Croatian nun who is in consideration for beatification by the Vatican, which is facilitated by the identification of her 20th-century remains. Sister Marija was buried in a tomb in Rijeka, Croatia, along with other nuns including her biological sister, Tereza Kozulic (1861-1933). When the remains were exhumed in 2011, they were found in a deteriorated state and commingled with several other sets of remains. Thus, mitochondrial genome sequencing of the long bones was performed to sort the remains by mitochondrial haplotype. Two similar but unique haplotypes belonging to haplogroup H1bu were identified, and samples from these bones were subjected to autosomal short tandem repeat (STR) and single nucleotide polymorphism (SNP) sequencing. Although only partial profiles were obtained, the data were sufficient for kinship analysis with the profile of a paternal niece of Sister Marija (Fides Kozulic). The data indicate that it is 574,195-fold more likely that the two sets of skeletal remains represent 2nd-degree relatives of Fides than sisters who are unrelated to Fides. Although it is impossible to discern which set of remains belongs to Marija and which belongs to Tereza, forensic genomics methods have enabled identification of the sisters.

Adjusting posterior probabilities to meet predefined accuracy criteria: A proposal for a novel approach to osteometric sex estimation.

The osteometric methods are the most reliable way to estimate the sex of skeletons when DNA analysis is not used. However, as osteometric studies usually ignore the overlap in male and female skeletal dimensions, they rarely achieve accuracy sufficient for forensic application. To resolve this issue, recent studies suggest sex estimation only when posterior probability (pp) is greater than 0.95, but that approach does not always provide sufficient accuracy and creates a large proportion of unsexed skeleton. Thus, our study aimed to explore whether it is possible to adjust pp on skeletal measurements with pronounced sexual dimorphism to meet 95% accuracy and to enable sex estimation on a reasonable proportion of individuals. From 207 skeletons, we included 65 postcranial measurements and selected 10% of variables with the highest sexual dimorphism. We computed univariate and bivariate discriminant functions using pp threshold of 0.5, 0.95, and the threshold required to achieve accuracy of ≥ 95%. Discriminant functions with pp=0.5 obtained accuracy of 85%-93%, while those with pp≥0.95 and adjusted posterior probabilities obtained 94%-99%. However, we showed that by selecting a particular threshold, sex could be estimated on a greater proportion of individuals than for pp≥0.95: 42%-86% vs. 24%-62% for univariate and 69%-95% vs. 49%-78% for bivariate functions. Therefore, when developing sex estimation models, we suggest not to use fixed pp level, but to adjust pp to achieve 95% accuracy and to minimize the percentage of unsexed skeletons.

Impact of DNA degradation on massively parallel sequencing-based autosomal STR, iiSNP, and mitochondrial DNA typing systems.

Biological samples, including skeletal remains exposed to environmental insults for extended periods of time, exhibit increasing levels of DNA damage and fragmentation. Human forensic identification methods typically use a combination of mitochondrial (mt) DNA sequencing and short tandem repeat (STR) analysis, which target segments of DNA ranging from 80 to 500 base pairs (bps). Larger templates are often unavailable as skeletal samples age and the associated DNA degrades. Single-nucleotide polymorphism (SNP) loci target shorter templates and may serve as a solution to the problem. Recently developed assays for STR and SNP analysis using a massively parallel sequencing approach, such as the ForenSeq kit (Verogen, San Diego, CA), offer a means for generating results from degraded samples as they target templates down to 60 to 170 bps. We performed a modeling study that demonstrates that SNPs can increase the significance of an identification when analyzing DNA down to an average size of 100 bps for input amounts between 0.375 and 1 ng of nuclear DNA. Observations from this study were then compared with human skeletal material results (n = 14, ninth to eighteenth centuries), which further demonstrated the utility of the ForenSeq kit for degraded samples. The robustness of the Promega PowerSeq™ Mito System was also tested with human skeletal remains (n = 70, ninth to eighteenth centuries), resulting in successful coverage of 99.29% of the mtDNA control region at 50× coverage or more. This was accompanied by modifications to a mainstream DNA extraction technique for skeletal remains that improved recovery of shorter templates.

Sex estimation of the sternum by automatic image processing of multi-slice computed tomography images in a Croatian population sample: a retrospective study.

AIM: To determine the sexual dimorphism of the sternum with standard measurements in a contemporary Croatian population sample using multi-slice computed tomography (MSCT) and to compare the data obtained by an automatic with those obtained by a manual approach. METHODS: Five sternal measurements were obtained from MSCT images of 73 men and 55 women and three sternal indices were calculated. Custom image analysis software was developed for automatic segmentation and calculation of sternal measurements. Measurements of sexual dimorphism were automatically calculated and compared with manual measurements. RESULTS: All of the sternal measurements exhibited significant differences between men and women. The discrepancies between manual and automatic measurements ranged from 2.8% to 3.6% of the mean average values obtained with the automatic approach. The most accurate single-variable discriminant function was sternal body length (82.8%), the most accurate index was sternal area (89.1%), and the discriminant function using three variables was manubrium width, sternal body length, and sternal body width (90.6%). CONCLUSION: Sternal measurements are a reliable sex indicator and can be used in forensic casework. Computer-aided measurement methods can accelerate sex estimation and improve its precision and accuracy.

Attitudes and Knowledge About Plagiarism Among University Students: Cross-Sectional Survey at the University of Split, Croatia.

Plagiarism is one of the most severe academic integrity issues. This study examined students' knowledge of and attitudes towards plagiarism, tested their ability to recognize plagiarism, and explored the association of study levels and attendance in courses dealing with referencing rules and plagiarism with students' attitudes and knowledge. A cross-sectional online survey was conducted at the University of Split, comprising the students of all schools and study levels (n = 388). Overall, results indicate the students were not very familiar with referencing rules and did not perform well on either theoretical questions or practical examples. However, they demonstrated positive attitudes towards plagiarism avoidance as well as towards compliance with academic integrity with respect to the correct use of research publications. Students' self-reported attendance in courses dealing with referencing rules and plagiarism avoidance was not associated with their knowledge of and attitudes toward plagiarism. These findings are important for a general understanding of students' attitudes, and the relation of practical and theoretical knowledge of plagiarism. Furthermore, the academic community addresses plagiarism not only as an ethical and regulatory violation but also as a direct consequence of a lack of knowledge, and of academic illiteracy. Study programs should be adjusted and long-term policies developed at all academic levels to promote a positive climate among students towards responsible academic writing.

Sex estimation standards for medieval and contemporary Croats.

AIM: To develop discriminant functions for sex estimation on medieval Croatian population and test their application on contemporary Croatian population. METHODS: From a total of 519 skeletons, we chose 84 adult excellently preserved skeletons free of antemortem or postmortem changes and took all standard measurements. Sex was estimated/determined using standard anthropological procedures and ancient DNA (amelogenin analysis) where pelvis was insufficiently preserved or where sex morphological indicators were not consistent. We explored which measurements showed sexual dimorphism and used them for developing univariate and multivariate discriminant functions for sex estimation. We included only those functions that reached accuracy rate ≥80%. We tested the applicability of developed functions on modern Croatian sample (n=37). RESULTS: From 69 standard skeletal measurements used in this study, 56 of them showed statistically significant sexual dimorphism (74.7%). We developed five univariate discriminant functions with classification rate 80.6%-85.2% and seven multivariate discriminant functions with an accuracy rate of 81.8%-93.0%. When tested on the modern population functions showed classification rates 74.1%-100%, and ten of them reached aimed accuracy rate. Females showed higher classified in the mediaeval populations, whereas males were better classification rates in the modern populations. CONCLUSION: Developed discriminant functions are sufficiently accurate for reliable sex estimation in both medieval Croatian population and modern Croatian samples and may be used in forensic settings. The methodological issues that emerged regarding the importance of considering external factors in development and application of discriminant functions for sex estimation should be further explored.

From Biography to Osteobiography: An Example of Anthropological Historical Identification of the Remains of St. Paul.

In the identification process of historical figures, and especially in cases of Saint's bodies or mummified remains, any method that includes physical encroachment or sampling is often not allowed. In these cases, one of the few remaining possibilities is the application of nondestructive radiographical and anthropological methods. However, although there have been a few attempts of such analyses, no systematic standard methodology has been developed until now. In this study, we developed a methodological approach that was used to test the authenticity of the alleged body of Saint Paul the Confessor. Upon imaging the remains on MSCT and post-processing, the images were analyzed by an interdisciplinary team to explore the contents beneath the binding media (e.g., the remains) and to obtain osetobiographical data for comparison with historical biological data. Obtained results: ancestry, sex, age, occupation, and social status were consistent with historical data. Although the methodological approach proved to be appropriate in this case, due to the discrepancy in the amount of data, identity could not be fully confirmed. Nonetheless, the hypothesis that the remains do not belong to St. Paul was rejected, whilst positive identification receives support. Anat Rec, 300:1535-1546, 2017. © 2017 Wiley Periodicals, Inc.

Rich but poor: life in the Roman period with extreme rheumatoid arthritis.

In a Sidonian sarcophagus, from the Late Antique/early Christian period, skeletal remains of two persons were found. One of them, male, 30-50 years old, was found almost completely ankylosed, with highly osteoporotic bones and prominent erosion of joint surfaces. We diagnosed rheumatoid arthritis based on the eroded odontoid process, mandibular condyles, distal humerus, proximal and distal ulna, as well ankylosed hand and foot bones. Despite the fact that ankyloses of vertebrae and sacroiliac joint could point towards ankylosing spondylitis, the lack of typical vertebral ankyloses and new bone formation led to exclusion. In a practical sense, due to the advanced stage of the disease, the man was fixed in the supine position, on the left, with his head turned to the right. Apparently, he could not move and had problems with chewing and breathing. But, the high standard of provided healthcare probably enabled him to survive in advanced stages of the disease. This case shed light on the antiquity of the disease, its medical, and social context and provided the example of most extreme osteological changes reported in the paleopathological and medical literature.

Anthropological analysis of the Second World War skeletal remains from three karst sinkholes located in southern Croatia.

Although in the cases of war crimes the main effort goes to the identification of victims, it is crucial to consider the execution event as a whole. Thus, the goal of the research was to determine the trauma type and probable cause of death on skeletal remains of civilians executed by partisans from WWS found in the three karst sinkholes and to explain the context in which the injuries occurred. We determined biological profiles, pathological conditions, traumas, and assessed their lethality. Nineteen skeletons were found, 68.4% had, at least, one perimortem trauma, classified as lethal/lethal if untreated in 69.2% cases. The type of execution and administered violence showed to be age and health dependent: elderly and diseased were executed with the intention to kill, by the gunshot facing victims, whilst the more violent behavior expressed towards younger and healthy individuals was indicated by the higher frequency of blunt force trauma.