It is considered to be a great advance over smear microscopy and culture. However, there is very little information regarding the performance characteristics of Xpert MTB/RIF in Malawi.XpertWe aimed to evaluate the performance of Xpert MTB/RIF in a Malawian setting.® MTBDRplus assay. Rifampicin resistance was confirmed by DNA sequencing.Stored sputum pellets were processed on Xpert MTB/RIF between June 2012 and May 2014. Results were compared to mycobacteria growth indicator tube and Löwenstein-Jensen cultures, LED fluorescent microscopy and GenoTypeM. tuberculosis strains. Mutations were not detected by sequencing in one isolate which was rifampicin resistant on Xpert MTB/RIF but sensitive on MTBDRplus. Four non-tuberculous mycobacteria grew from four smear-negative specimens, namely, M. avium (n = 1) and M. intracellulare (n = 3). No cross-reactivity was observed with any of the non-tuberculous mycobacteria when using Xpert MTB/RIF.Of the 348 specimens with valid Xpert MTB/RIF results, 129/348 (37%) were smear-positive and 198/348 (57%) were culture-positive. Xpert MTB/RIF demonstrated a sensitivity of 93.8% (95% CI 89.4% – 96.8%) and specificity of 97.4% (95% CI 93.5% – 99.3%), with a positive predictive value of 97.8% (95% CI 94.6% – 99.4%) and a negative predictive value of 92.6% (95% CI 87.4% – 96.1%). Xpert MTB/RIF correctly identified 185/186 (99.5%) rifampicin-sensitive and 2/2 (100%) rifampicin-resistant When fully implemented, Xpert MTB/RIF may have an impact on patient care in Malawi. The increased diagnostic yield of Xpert MTB/RIF over smear microscopy can increase laboratory-confirmed tuberculosis detection and ensure that treatment is given to appropriate individuals or groups. Although more sensitive than smear microscopy, tuberculosis culture, when available, can take days to weeks before a result is available. Due to limitations of both smear and culture, pulmonary tuberculosis is often diagnosed late or presumptively. Low utilisation of laboratory confirmation and widespread use of empirical treatment can either lead to true tuberculosis cases being missed or to the initiation of tuberculosis treatment in people without the disease.1Tuberculosis remains a major health challenge that has worsened with the emergence of multi-drug resistant (MDR) tuberculosis strains that are resistant to rifampicin and isoniazid. Globally, the impact of tuberculosis is significant, with an annual estimate of 9.6 million tuberculosis cases and over 1.5 million deaths due to tuberculosis in 2014.3 Diagnosis of tuberculosis may be delayed or missed in HIV-positive, smear-negative tuberculosis patients. As such, it is imperative to rapidly diagnose and treat tuberculosis cases in people living with HIV.4 Introduction of simple and rapid diagnostic methods, such as the Xpert® MTB/RIF , could benefit smear-negative patients in countries like Malawi, where culture is limited to the National Tuberculosis Reference Laboratory. Currently, the Malawi National Tuberculosis Programme (NTP) is using Xpert MTB/RIF to increase tuberculosis case detection and to detect rifampicin resistance as a proxy for MDR tuberculosis followed by culture (liquid and solid) and drug-susceptibility testing (DST) as confirmatory tests.2Malawi has a high HIV burden, with an estimated one million HIV-positive people. The major cause of morbidity and mortality of people living with HIV is tuberculosis. It is estimated that of all tuberculosis cases, 41% are smear-negative and 64% are HIV co-infected.3 This can lead to the development of under-reporting of drug resistance, unnecessary suffering, additional costs for patients, and treatment with suboptimal regimens.5 Consequently, this might increase the spread of drug-resistant tuberculosis in the population.1DST is not routinely performed in Malawi, with the exception of retreatment cases and individuals at risk for MDR tuberculosis.7 and is reported as having excellent sensitivity and specificity in detecting Mycobacterium tuberculosis and rifampicin resistance.8 Some recent evaluations have demonstrated that Xpert MTB/RIF accurately detects 72.5% of smear-negative and 98.2% of smear-positive cases, and rifampicin resistance is detected with a specificity of 100% and sensitivity of 99.1%.9 The test has an overall pooled sensitivity of 90.4% (95% CI 89.2% – 91.4%), which is much higher than smear microscopy but is similar to that of solid culture in detecting tuberculosis.7 Among smear-positive tuberculosis samples, Xpert MTB/RIF has a pooled sensitivity of 98.7% (95% CI 98.0% – 99.2%), but has a substantially lower sensitivity of 75% (95% CI 72.0% – 77.8%) among smear-negative tuberculosis samples. The overall pooled sensitivity in diagnosing for rifampicin-resistant tuberculosis is 94.1% (95% CI 91.6% – 96.0%) and 97.0% (95% CI 96% – 97.7) pooled specificity.7 Furthermore, use of Xpert MTB/RIF has been linked to improved diagnosis, resulting in early appropriate treatment.10The Xpert MTB/RIF can both detect tuberculosis and identify rifampicin resistance in a single, rapid assay,® instruments distributed across 40 of the 315 public and private diagnostic centres (laboratories) with acid-fast bacilli testing capacity in the country.2 Xpert MTB/RIF’s performance is not known in the adult outpatient population in Malawi.The Malawi NTP recommended rolling out Xpert MTB/RIF in 2012 to increase detection of tuberculosis, especially in sputum smear-negative and HIV-positive individuals. While Xpert MTB/RIF has been approved for clinical use, its optimal use in the clinical algorithm for Malawi has not been established. There are currently over 50 GeneXpertM. tuberculosis complex and determining resistance to rifampicin among both HIV-positive and HIV-negative outpatients at Bwaila Hospital in Lilongwe, Malawi.In this study, we evaluated the performance of Xpert MTB/RIF in detecting Approvals for this study were granted by the University of the Witwatersrand Human Research Ethics Committee (M120256), National Health Sciences Research Committee (NHSRC) in Malawi (NHSRC # 999) and the University of North Carolina (Chapel Hill) Institutional Review Board (CID 1211).n = 351) obtained from an observational cohort study which collected 702 samples between April 2011 and July 2012 at Bwaila in Lilongwe, Malawi. The study was looking at the prevalence of drug-resistant tuberculosis among adult outpatients (≥ 18 years) with laboratory-confirmed or clinically-diagnosed tuberculosis registering for tuberculosis treatment at this HIV/tuberculosis clinic.11 Pellets were selected at random for use in this study without any special criteria to eliminate bias. Testing of these pellets using Xpert MTB/RIF started in June 2012 at the University of North Carolina (UNC) Project laboratory, Lilongwe, Malawi. Pellets were re-suspended in 1.5 mL phosphate buffer and an aliquot of 0.5 mL was processed on the Xpert MTB/RIF assay. In the primary study, all sample processes were performed at the UNC Project laboratory. In brief, sputum smears were prepared and stained with auramine-O stain. Smear microscopy was performed using LED fluorescent microscopy. Mycobacterial cultures (reference standard) were performed using both BACTEC mycobacteria growth indicator tube media (liquid) and Löwenstein-Jensen slants (solid). The reference standard was considered positive if there was growth of M. tuberculosis on either of the media and negative if both media were negative. Drug susceptibility was investigated using the GenoType® MTBDRplus assay (version 2) . Data collected from the above processes were included with data in this evaluation.We used frozen stored sputum pellets (12 Results were available within two hours of sample loading.Samples for Xpert MTB/RIF testing were prepared as per manufacturer’s instructions. Sample reagent was added to 500 µL of the re-suspended pellet in the ratio of 3:1 (sample reagent:specimen) as described previously.® kit  from mycobacteria growth indicator tube liquid media by transferring 1.0 mL to a Sarstedt micro-centrifuge tube. The tube was centrifuged for 15 minutes at 14 000 rpm. The supernatant was carefully removed and the pellet was re-suspended in 100 µL lysis buffer, vortexed thoroughly and incubated for five minutes at 95°C in a heating block. At the end of the five-minute incubation, tubes were briefly centrifuged to remove condensation. Neutralization buffer (100 µL) was added to each tube, vortexed for five seconds and then centrifuged for five minutes at 14 000 rpm. Supernatant was transferred to a new tube and the pellet discarded. Samples were stored at -80°C for use as DNA template.Genomic bacterial DNA was extracted using a GenoLyse® MTBDRplus (version 2)  was performed on positive culture isolates according to manufacturer’s instructions to identify M. tuberculosis complex and to determine drug susceptibility to rifampicin and isoniazid as described previously.13 Genotype® CM  was used to identify other common Mycobacterium species in samples which tested negative for M. tuberculosis complex on GenoType MTBDRplus.The GenoTypeM. tuberculosis strains were shipped to the HIV Genotyping Laboratory (University of the Witwatersrand) and were processed as described below.Determination of eligibility for DNA sequencing was based on detection of resistance to rifampicin by Xpert MTB/RIF. All eligible rpoB gene fragment which included the rifampicin resistance determining region was carried out by using forward primer rpoBF2 (5’-GAG GGT CAG ACC ACG ATG AC-3’) and reverse primer rpoBR2 (5’-GAG CCG ATC AGA CCG ATG T-3’) in a GeneAmp PCR system 9700 thermocycler . The total volume of the reaction mix was 50 µL, which contained 5 µL of 10X high fidelity buffer, 1 µL of 10 mM dNTP mix, 2 µL of 50 mM MgSO4, 1 µL of each primer, 0.2 µL of Taq HiFi , 36.8 µL of molecular grade water and 3 µL of genomic DNA. Amplification conditions were set as follows: 94°C for 2 minutes ; followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 68°C for 40 seconds; followed by a 10-minute final elongation at 68°C. Amplified products were visualised on a 1% agarose gel after staining with ethidium bromide.Amplification of the 450 bp rpoBS (5’-GCA GAC GTT GAT CAA CAT CC-3’) and reverse primer rpoBR2 (5’-GAG CCG ATC AGA CCG ATG T-3’). The resulting sequences were analysed using Sequencher software V4.8 .PCR products, after purification by GeneJet PCR purification kit , were subjected to DNA sequencing by automated DNA sequencer . PCR sequencing was carried out with a BigDye terminator V3.1 sequencing kit according to manufacturer’s instructions, using forward primer All data were double entered into a Microsoft Excel spreadsheet . Discrepancies were checked against source records for completeness and consistency. Data analyses were done in Stata Version 12 . The following statistics were calculated: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and 95% confidence intervals (CI). Mycobacterium culture was considered the reference standard.n = 1) and an invalid result (n = 2) on Xpert MTB/RIF. Of the 348 sputum pellets, 200 (57%) were from HIV-positive individuals, and 148 (43%) were HIV-negative. Among all samples, 219/348 (63%) were smear-negative and 129/348 (37%) were smear-positive. As shown in A total of 351 sputum pellets were processed both on culture and Xpert MTB/RIF. However, only 348 sputum pellets had valid Xpert MTB/RIF results. The remaining three samples were not included in the final analysis due to an error (M. tuberculosis in 58/219 (27%) of smear-negative and 127/129 (98.5%) smear-positive cases. Xpert MTB/RIF correctly detected M. tuberculosis in 181/198 (91.4%) of the culture-positive samples. Of the remaining 17 samples, 1/198 (0.5%) was positive while 16/198 (8.1%) were negative for acid-fast bacilli on smear microscopy. A total of 150 samples were culture-negative regardless of their smear status. Among the 150 culture-negative samples, M. tuberculosis was detected by Xpert MTB/RIF in four (2.7%) samples which were smear-negative on microscopy. Xpert MTB/RIF did not detect M. tuberculosis in 12/70 (17%) of smear-negative, culture-positive samples. We further observed that 145/219 smear-negatives were culture negative and Xpert MTB/RIF negative. Out of these, 93/145 (64%) were from HIV-positive individuals. Four non-tuberculous mycobacteria grew from a total of four smear-negative specimens: M. avium (n = 1) and M. intracellulare (n = 3). No cross reactivity was observed with any of the non-tuberculous mycobacteria when using Xpert MTB/RIF  and specificity was 97.4% (95% CI 93.5% – 99.3%), with PPV of 97.8% (95% CI 94.6% – 99.4%) and NPV of 92.6% (95% CI 87.4% – 96.1%). Among smear-negative individuals, the overall sensitivity of Xpert MTB/RIF was 81.8% (95% CI 70.4% – 90.2%) and specificity was 97.4% (95% CI 93.4% – 99.3%), with PPV of 93.1% (95% CI 83.3% – 98.1%) and NPV of 92.5% (95% CI 87.3% – 96.1%) .Stratified by HIV status, sensitivity for smear microscopy was 78.9% (95% CI 69.4% – 86.6%) among HIV-negative individuals, with a specificity of 100% (95% CI 93.3% – 100%). Among HIV-positive individuals, sensitivity was 51.5% (95% CI 41.4% – 61.4%) and specificity was 99.0% (95% CI 94.4% – 100%). The sensitivity  and specificity  of Xpert MTB/RIF were lower in HIV-positive individuals, with PPV of 95.7% (95% CI 89.5% – 98.8%) and NPV of 90.6% (95% CI 83.3% – 95.4%). This was compared to HIV-negative participants, which showed a sensitivity of 97.9% (95% CI 92.5% – 99.7%) and specificity of 100% (95% CI 93.4% – 100%), with PPV of 100% (95% CI 96.1% – 100%) and NPV of 96.4% (95% CI 87.7% – 99.6%). Among smear-negative, HIV-positive individuals, the sensitivity of Xpert MTB/RIF was 78.7% (95% CI 64.3% – 89.3%) with a specificity of 96% (95% CI 90% – 98.9%) .plus was performed on 188/348 tuberculosis strains. When compared to Genotype MTBDRplus, Xpert MTB/RIF correctly identified 185 of 186 (99.5%) rifampicin-sensitive M. tuberculosis and 2/2 (100%) rifampicin-resistant M. tuberculosis. A single point mutation was detected in each of the two rifampicin-resistant strains (S531L and D516V). It was observed that for both strains, the corresponding Xpert MTB/RIF probes gave cycle threshold (Ct) value = 0 and were also detected as resistant to rifampicin by GenoType MTBDRplus line probe assay. One (0.5%) smear-negative, culture-positive tuberculosis strain was resistant on Xpert MTB/RIF but sensitive on GenoType MTBDRplus  observed in this study is in contrast to Dorman et al. and Geleta et al., who reported 62.6 % in South Africa and 65.5% in Ethiopia when compared with culture.6 and sensitivities of Xpert MTB/RIF ranging from 78% to 100% for smear-positive/culture-positive samples.22 Since Xpert MTB/RIF identified 58/219 (26.5%) of smear-negative samples, it can therefore assist in rapid detection of tuberculosis cases among smear-negatives and has the potential to impact patient care, although a small percentage  of the smear-negative/culture-positive cases was missed by Xpert MTB/RIF. In Malawi, smear-negative adult tuberculosis patients have poor treatment outcomes with high death rates, largely due to concurrent HIV infection.23 It is of particular interest to note that 64% of smear-negative samples which did not grow on culture and where M. tuberculosis was not detected by Xpert MTB/RIF, were from HIV-positive individuals. This may be interpreted as significant incorrect diagnosis of tuberculosis with a likelihood of treating people for tuberculosis who do not have the disease. It is therefore of paramount importance that steps should be taken to improve tuberculosis diagnosis to avoid exposing non-tuberculosis patients to the adverse effects of the tuberculosis medication. Misdiagnosis of tuberculosis can be a contributing factor to the high death rate observed in HIV-positive individuals globally.1 Due to the increased diagnostic yield and short turn-around time of Xpert MTB/RIF as compared to culture, Xpert MTB/RIF offers an opportunity to improve the diagnosis of tuberculosis, both in HIV-positive and HIV-negative individuals, if used as a first diagnostic for all presumptive tuberculosis cases in Malawi, considering the high proportion of misdiagnoses observed in this study.Prior studies have shown greater accuracy of Xpert MTB/RIF as compared to smear microscopy20 Another study demonstrated that 89% of rifampicin-resistant tuberculosis patients started second-line treatment when Xpert MTB/RIF was introduced and that the average time to start second-line treatment was reduced from 1 – 1.5 months by culture to one week with Xpert MTB/RIF.23 The current study does not highlight this for Malawi since Xpert MTB/RIF testing was performed retrospectively and the initial study did not have a long-term follow-up on patients, as it was only concentrated on diagnostics.Our results are comparable to other studies which have shown that Xpert MTB/RIF increased tuberculosis case detection by almost 31%, despite its low sensitivity with smear-negative samples.rpoB gene confirmed two mutations (S531L and D516V) and further helped to resolve the discordant result, since no mutation was detected. Mixed infection with multiple M. tuberculosis strains was excluded in this strain, given the wild-type rpoB gene sequence and no observed underlying peaks on the DNA sequence chromatogram. Prior studies demonstrated that DNA sequencing resolves discrepancies in favour of Xpert MTB/RIF,6 but this was not the case in our study; a false rifampicin resistance was most likely. In Malawi, the Xpert MTB/RIF is widely used to identify MDR tuberculosis patients and to initiate MDR tuberculosis treatment until culture and DST results are obtained. Malawi has a low MDR tuberculosis prevalence; as such the expected low PPV of Xpert MTB/RIF could contribute to false-positive results and would require a second Xpert MTB/RIF test as per current international recommendations.2Results in this study show that Xpert MTB/RIF detected primary rifampicin resistance in 3/188 (1.6%) specimens. DNA sequencing of the rifampicin resistance determining region of the 25 which was also the case in our study. We performed DNA sequencing on isolates from culture, whereas Xpert MTB/RIF was performed on sputum pellets. Theron et al.22 detected mutations in 5/6 cases which were rifampicin resistant on Xpert MTB/RIF but were susceptible on phenotypic DST. Marlowe et al.26 further investigated a sample which was susceptible on phenotypic DST and no mutation was detected in DNA sequencing but was repeatedly resistant to rifampicin on Xpert MTB/RIF. Investigations carried out in these studies show how difficult it is to distinguish true-positive rifampicin resistance from false positives in clinical practice. Also, the samples run on Xpert MTB/RIF might have included both dead and live bacteria since the specimens were taken directly from pellets, whereas for DNA sequencing, DNA extraction was performed after observed growth on culture (live bacteria). It was demonstrated previously that Xpert MTB/RIF has a pooled sensitivity of 94.1% (95% CI 91.6% – 96.0%) and pooled specificity of 97.0% (95% CI 96.0% – 97.7%) in diagnosing rifampicin resistance.7 In the present study, the sensitivity, specificity, PPV and NPV were not calculated due to the small number of rifampicin-resistant isolates identified.False resistance to rifampicin on Xpert MTB/RIF has been reported previously, although in some of the studies hetero-resistance could not be excluded, because neither cartridge amplicons nor unprocessed sputum were sequenced,27 Low rifampicin resistance detection among incident cases observed in this study reflects well on the prevalence of rifampicin-resistant tuberculosis strains in communities surrounding Bwaila. However, it is debatable whether Xpert MTB/RIF results alone can be used for making a decision to start on second-line treatment in outpatients detected with resistance to rifampicin in Malawi, considering the number of isoniazid mono-resistances detected in previous studies.11 It is therefore crucial to test tuberculosis strains for resistance to isoniazid as well, and to confirm rifampicin resistance detected by Xpert MTB/RIF before switching to second-line treatment. The success of the tuberculosis-control programme could be measured by the level of anti-tuberculosis drug resistance in a community. Consequently, the level of drug resistance gives future indications of suitable drug regimens.28The low rifampicin resistance detected in this study is similar to results obtained by Glynn et al., which identified 3/373 samples as resistant to rifampicin in Karonga, in the northern region of Malawi.2 As part of the scale-up plan for the GeneXpert technology, the Malawi NTP plans to assess the concordance between phenotypic and genotypic technologies in order to inform interpretation of single rifampicin-resistance results. Systematic confirmation of MDR tuberculosis status of rifampicin-resistant patients diagnosed with Xpert MTB/RIF is lacking in the country.2 Findings from the current study form a bank of data which can be beneficial to the NTP in its assessment of the Xpert MTB/RIF assay.The Malawi NTP has phased in plans within the context of national strategic plan for tuberculosis to expand and maintain Xpert MTB/RIF as a primary test for active case finding at high-volume antiretroviral therapy services and among high risk and vulnerable populations. This will consequently lead to improved tuberculosis diagnosis and initiation of appropriate treatment to patients, thereby reducing the spread of tuberculosis and/or transmission of resistant strains in the country. Additionally, Xpert MTB/RIF offers to help in reducing the time lost in the diagnostic pathway and potential loss of patients at each step of the pathway.29With our limited sample size from a single outpatient location, the observed results may not apply across the country due to differences in the prevalence of HIV in rural districts. The relatively small number of patients with drug-resistant tuberculosis limits the ability to evaluate comparative performance of Xpert MTB/RIF. We recommend that future studies extend to other sites with an overall sample size and/or focus on evaluating Xpert MTB/RIF on raw sputum, since is it known that testing frozen stored specimens may influence results.3In the current algorithm for Malawi, a residual specimen from the second sample of presumptive tuberculosis patients is tested on Xpert MTB/RIF if both samples are smear-negative. Xpert MTB/RIF is also performed on samples from all patients suspected of having MDR tuberculosis and retreatment cases, but not on new smear-positive cases.Our results demonstrate that Xpert MTB/RIF has the potential to increase the number of patients initiated early on appropriate treatment, given that additional tuberculosis cases were identified among smear-negative/culture-confirmed cases, one of which was resistant to rifampicin. It is expected that the use of Xpert MTB/RIF will offer a greater opportunity in increasing tuberculosis case detection among high-risk and vulnerable groups in Malawi where sputum smear microscopy is still widely used. We believe that results obtained by Xpert MTB/RIF may facilitate a proactive approach to tuberculosis by enhancing decisions towards treatment and ensuring that the tuberculosis programme is not treating people who do not have tuberculosis but are obviously sick. When this is achieved, it is anticipated that there will be a reduction in the burden of tuberculosis among people living with HIV.


Apical periodontitis (AP) is an inflammatory disease around the apex of a tooth root. Reported prevalence of AP ranges from 27% to 83% while the data about most post-Soviet countries are still missing. Knowing the prevalence of AP within a certain population helps to plan the treatment need and evaluate the success of endodontic interventions. We aimed to collect data about prevalence and determining factors of AP for the first time in Estonia.The cross-sectional study included 6552 patients (age 35.5±19.2 years). Radiographic examination was applied to investigate the prevalence of AP and quality of endodontic treatment.AP was diagnosed in 54.7% of subjects. Endodontically treated teeth were present in 58.2% of subjects. Periapical finding was present in 44.6% of endodontically treated and in 30.8% of untreated teeth.Out of 181,495 teeth, 52.7% were intact. AP was diagnosed in 6.3% of teeth, 6.9% of teeth were endodontically treated.Risk factors for AP included caries (OR = 2.30), male gender (OR = 1.44), too short (OR = 1.76) or too long root canal filling (OR = 2.51), root canal filling of low density (OR = 1.61) while not orthodontic appliance. Lower AP risk was associated with restoration of the tooth – filling (OR = 0.45), crown (OR = 0.34) and bridge (OR = 0.33).AP is highly prevalent in Southern Estonian population. Most cases are associated with root canal-treated teeth. The overall quality of root canal fillings tends to be low, consistent with the mediocre outcome of treatment. Considerable efforts are required to improve the standards of endodontic treatment.The online version of this article (10.1186/s12903-017-0429-7) contains supplementary material, which is available to authorized users. Apical periodontitis (AP) is an inflammatory disease around the apex of a tooth root that is caused by infection in root canal system , 2. Chro X-rays were taken for the first time at the radiology department at the Clinic of Dentistry, Tartu University Hospital, Estonia between November 2010 and May 2012 only for the consultation and/or treatment purposes.The study was conducted in compliance with the "Ethical principles for medical research involving human subjects" of Helsinki Declaration and approved by Ethics Review Committee (ERC) on Human Research of the University of Tartu (protocol no: 246/T-19). All of the collected data were coded and isolated; personal data and measurements data were kept separately.Planmeca Oy, Helsinki, Finland) with imaging values between 54 and 96 kV and 1–14 mA, depending on the subject’s size. For assessment of the radiographs, a HP 22uh 21.5-in. LED Backlit Monitor 1920 × 1080 @ 60 Hz using Romexis Imaging Software  was used. For better assessment the observers were able to use all software image enhancement functions whenever they felt the necessity for it.All radiographs were carried out by the same experienced dental radiologist using a panoramic digital radiography device Planmeca ProMax 3D Plus  in the Department of Oral and Dental Diseases examined all radiographs. All teeth were recorded according to FDI nomenclature, in all teeth the variables and findings listed in Additional file 1: Table S6 were assessed. To identify teeth with AP only the presence or absence of periapical radiolucency was used . The rooCalibration of observers was carried out on a selected set of 104 orthopantomographs from an unpublished pilot study from the same investigation group. The interobserver agreement scores gave Kappa-value of 0.51 for presence of periradicular radiolucency, 0.55 for length of root canal filling (RCF), 0.44 for quality of lateral seal, 0.56 for quality of coronal fillings, 0.63 for quality of crowns. Because the interobserver agreement was moderate (0.44–0.63), a highly experienced dentist served as the third observer (M.S.) for cases where disagreement occurred.In this study, 6552 subjects ranging in age from 3 to 93 years (mean 35.5 ± 19.2 years) were assessed, of them 2563 (39.1%) were male and 3989 (60.9%) were female. Majority of the subjects (62.6%) were 10 to 44 years old and the biggest age-group (11.3%) included the patients in the age of 15–19 years  had AP, and 58.2% (3815 patients) had root canal treatment (RCT) while 2737 patients (41.8%) had no RCT visible on X-rays  was present in 2920 subjects , whereas 2021 patients  had AP in non-endodontically treated teeth   and least often in the mandibular canines (0.4%) and mandibular central and lateral incisors (0.8%).p < 0.001), this shift was more prominent between the ages of twenties and thirties  and 49,303 teeth that had any type of restoration . Overview of the status of the teeth is presented in Table Among 181,495 teeth, AP was diagnosed in 11,438 teeth (4898 pAP and 6540 sAP), while 12,605 teeth  were endodontically treated.The highest prevalence of pAP was seen in lower molars (6.6%), followed by upper molars (3.0%) and lower premolars (2.5%). The highest prevalence of sAP was seen also in lower molars, followed by lower incisors and lower premolars . At the same time the teeth with orthodontic appliance did not have higher risk for AP  were treated using pulpectomy and 649 teeth (5.2%) were treated using the pulpotomy method Table . Of 11,9Root canal filling (RCF) length and homogeneity were measured only in pulpectomy group while coronal restoration was assessed in all endodontically treated teeth. Shorter RCF than adequate was associated with 1.76 times higher risk for AP while overfilled root canals were associated with 2.51 times higher risk for AP Table . There wThe authors undertook a cross-sectional observational study involving 6552 patients and focusing on the epidemiology and comorbid factors of periapical radiolucencies in a Southern Estonian population. The study revealed high prevalence of apical periodontitis (AP), observed in 54.7% of the subjects: 10.2% displayed AP without previous RCT or primary apical periodontitis (pAP), 23.9% had only post-treatment AP or secondary apical periodontitis (sAP) and 20.7% of the subjects had both teeth with and without previous RCT (pAP and sAP). A fifth of the 181,495 teeth studied had visible caries and 27.2% had undergone some type of restoration. AP was diagnosed in 6.3% of teeth while 6.9% of teeth were endodontically treated. Only 31% of teeth had received RCF’s of adequate length, 26% were filled homogenously, and only 19% of teeth had RCF’s acceptable in all terms. High quality root canal fillings and presence of coronal restoration were significantly associated with lower risk for AP and therefore better treatment outcome.Previous endodontic epidemiology studies have displayed numerous variations in study design, investigation methods, sample selection and evaluation criteria. As a result, a direct comparison is clearly difficult, although most of these studies are based on radiographic evaluation. Decades ago, PA radiography was the only imaging method available to diagnose AP . More reThe mean age of our study group, 35.5 years, was similar to some other studies , 25, 26 Our sample contained more females (60.9%) than males (39.1%). Preceding analogous reports have explained emerged gender disparity with women’s higher eagerness in receiving dental care, making them less predisposed to AP , 30. ThiThe prevalence of AP in our study (54.7%) was higher than generally outlined in other European studies. Higher figs. (63–80%) have only been reported in Belgium, Lithuania, Latvia, Austria, and Belarus in Europe , 32, 33 Out of all teeth evaluated in our study, 6.3% had signs of periradicular radiolucensies. This figure is lower than reported in studies carried out in some other Eastern and Southeast European countries that showed results ranging from 7% in Latvia to 12.3% in Kosovo , 39, 40;Some recent studies have shown molars and premolars to be the most common RCF teeth , 28 withDental caries is considered the main cause for pulpal and therefore periodontial disease . AlthougRadiographic evidence of lower quality RCF’s contribute to an increase in AP . MoreoveAt the same time, 40% of the teeth with technically adequate RCF still showed periradicular radiolucencies. Even though some of these lesions might have been actually healing at the time of evaluation, this result still indicates that the quality of RCF is not the only determinant for periradicular status. The presence of microorganisms in the root canal at the time of RCF or later contamination has been shown to increase the risk of failed treatment –65. The Alike Moreno et al.  we foundLong-term excessive orthodontic forces have been asserted to cause a predisposition for pulp inflammation and subsequent development of irreversible pulpitis and necrosis . PotentiAP tends to be a common sequela of pulp infection  and the Our study possesses several strengths. It included 6552 patients, making it one of the most extensive in the field of endodontic epidemiology. The sample size comprised about 0.5% of the total population in Estonia and about 7% of the population of Tartu area. The only recent study to involve a sample of comparable size was carried out in Finland by Huumonen et al.  forming Apical periodontitis is highly prevalent in the Southern Estonian population. Most periradicular radiolucencies are associated with root canal-treated teeth, and a half of all root canal-treated teeth show radiological signs of apical periodontitis. The overall quality of root canal fillings tends to be low, consistent with the mediocre outcome of treatment. Considerable efforts are required to improve the standards of endodontic treatment.


The major problem with ventilation distribution calculations using DIR and 4DCT is the motion artifacts in 4DCT. Quite often not all phases would exhibit mushroom motion artifacts. If the ventilation series similarity is sufficiently robust, the ventilation distribution can be calculated using only the artifact-free phases. This study investigated the ventilation similarity among the data derived from different respiration phases. Fifteen lung cancer cases were analyzed. In each case, DIR was performed between the end-expiration phase and all other phases. Ventilation distributions were then calculated using the deformation matrices. The similarity was compared between the series ventilation distributions. The correlation between the majority phases was reasonably good, with average SCC values between 0.28 and 0.70 for the original data and 0.30 and 0.75 after smoothing. The better correlation between the neighboring phases, with average SCC values between 0.55 and 0.70 for the original data, revealed the nonlinear property of the dynamic ventilation. DSC analysis showed the same trend. To reduce the errors if motion artifacts are present, the phases without serious mushroom artifacts may be used. To minimize the effect of the nonlinearity in dynamic ventilation, the calculation phase should be chosen as close to the end-inspiration as possible. Lung function is often characterized by perfusion and ventilation. Clinically, the major imaging modalities for ventilation are SPECT  and PET However, the 4DCT-DIR technique is susceptible to noise and artifacts in the 4DCT data , 16. TheA set of 4DCT data consists of a number of respiratory phases, typically 10. By convention, phase 0% is the end-inspiration phase while phase 50% is the end-expiration one. Ventilation distribution is usually derived from the deformation matrix between phases 0% and 50%, which represent the largest respiration difference. Even when mushroom artifacts  are seenPart of the results of this study was presented at The International Congress on Clinical Trials in Oncoloty and Hemato-Oncology, March 16-17, 2017, London, UK.2 and 3 mm slice thickness.Fifteen lung cancer patients' 4DCT datasets were used in this study following a protocol approved by the institutional review board. No dataset used in the study had pronounced mushroom artifacts, to streamline the ventilation similarity comparison between the different phases. No other inclusion/exclusion criteria were applied. The case shown in Based on a previous DIR selection study , the DifV, method was used. It directly calculates the local volume change using the deformation matrix resulting from the DIR [V is the volume change between the involved respiration phases and Vex is the initial volume at expiration [The geometric, or Δ the DIR . In the piration .V50%0%, and is considered a standard. In this study, ventilation distributions using other phases were calculated and compared to this standard. To streamline comparisons, the base phase was always the end-expiration one (50%). A ventilation distribution calculated using an arbitrary phase x and the end-expiration phase can be further denoted as V50%x, where x = 10%, 20%, 30%, and so forth. A series of ventilation distribution data in a respiration cycle can also be considered a dynamic ventilation dataset.Each ventilation distribution calculation involves two respiration phases. The full ventilation is calculated using the end-inspiration (0%) and the end-expiration (50%) phases, which is denoted here as 3 (or 9 × 9 × 3 voxels) average filter [Errors in DIR caused by the image artifacts and noise will be reflected in the calculated ventilation distributions . To redue filter .V50%0% and V50%x, x = 10%, 20%, 30%,…, and also between neighboring phases, V50%x−10% and V50%x, were calculated.The voxel-wise nonparametric Spearman correlation coefficients (SCC) between the ventilation datasets, A and B are the two involved volumes, was applied for the upper/lower 10%, 20%, 30% 40%, and 50% ventilation regions between pairs of ventilation datasets, including the original and smoothed data.Dice similarity coefficient (DSC) , definedV50%40%, which is calculated using the phase closest to end-expiration (50%) phase, V50%30%, V50%20%, and V50%0%, which corresponds to the full ventilation distribution.V50%0% and other phases V50%x, x = 10%, 20%, 30%,… in the dynamic ventilation datasets. All SCC comparisons were statistically similar (p < 0.0001). Note how the similarity was high when the second phase was close to the end-inspiration phase  and decreased for the phases closer to end-expiration (base) phase. The average SCC value for the original datasets was 0.68 ± 0.10 between V50%0% and V50%10%, while it was 0.74 ± 0.09 after smoothing. For all the phases, the average SCC values were between 0.28 and 0.70 for the original datasets and between 0.30 and 0.75 after smoothing.V50%x and the previous phase V50%x−10%. For example, data point at 20% is the SCC between phases V50%20% and V50%10%. There are two special data points that need further explanation. The point at 0% is the SCC between V50%0% and V50%90%; the point at 60% is the correlation between V50%60% and V50%40%. There is no data point at 50% because it is the base phase. Compared to the plot of the SCC values between full ventilation V50%0% and other phases V50%x,  x = 10%, 20%, 30%,…  . The DSC versus phase trends for all the ventilation regions were similar to that shown in V50%0%–V50%10% and V50%0%–V50%90% were 0.58 and 0.61, respectively, lower than the average values 0.68 and 0.69 , indicating that the mushroom artifacts in the 0% phase introduced significant registration errors which caused low SCC of V50%0%–V50%x,  x = 10%, 20%, 30%,…. Using phases without mushroom artifacts can improve accuracy, resulting in higher similarity between calculations using different phases. Smoothed data (not presented) showed the same trend.For the case with mushroom artifacts in the end-expiration (0%) phase shown in and 0.69 . On the V50%0% and V50%x, where x ≠ 0%. SCC compares the voxel-wise order of ventilation magnitude and DSC compares relative ventilation volumes between ventilation datasets, both with nothing to do with the absolute magnitude difference between the datasets. Clinically, the parameter of interest is the relative ventilation distribution; that is why different imaging modalities can be applied for the same purpose although what is measured in different imaging modality is different.The correlation and similarity comparison used in this study are only useful for relative ventilation distribution comparisons and not for absolute ventilation (or magnitude of the ventilation) differences. The magnitude of ventilation is certainly different between V50%0%) and those based on the ones close to end-expiration phase is likely due to the ventilation distribution being nonlinear with respect to phase, or the ventilation change rate for the same location being not consistent between the phases. As a result, ventilation distributions differ somewhat depending on the respiration phase used. This is supported by the data shown in Figures V50%0%, consistent with ventilation distribution slowly shifting with phase. Both the overall SCC values and subvolumes' DSC values showed the same trends. The subvolumes' DSC analysis demonstrated that all ventilation levels, from 10% through 90% , had the similar nonlinearity property.The poor similarity between the standard calculation V0%0% and V50%60% and V50%40% was the lowest with the largest standard deviation. To avoid the effect of motion artifacts in ventilation calculations, if there were artifacts present in the phases close to end-inspiration, one should use the 30% or 40% instead of the 60% or 70% phases. Slightly better correlation of V50%40%–V50%0% than V50%60%–V50%0% and of V50%30%–V50%0% than V50%70%–V50%0% can be also observed in Based on Figures V50%0%–V50%40% and V50%60%–V50%70%.Smoothing of the ventilation distributions makes the correlation better in general, although occasionally, especially for the low correlation phases, smoothing may make the correlation worse. The reason for this phenomenon is most likely that smoothing can reduce the errors induced by image noise but not by other factors, for example, nonlinear change in ventilation. This can explain why, in Figures The results presented in This study attempts to improve ventilation calculations when obvious motion artifacts are present in 4DCT images. However the best approach would be to reduce or eliminate those artifacts in the first place. Amplitude-based binning, as opposed to the traditional phase-based binning, could reduce, but not completely eliminate, motion artifacts in 4DCT , 23. TheMotion artifacts are inevitably present in realistic 4DCT datasets. To reduce errors in the ventilation distributions caused by these artifacts, the respiratory phases that do not show serious mushroom artifacts should be used, as the correlation between the ventilation distributions obtained with different phases is reasonably good. To minimize the effect of the nonlinearity in dynamic ventilation, when the mushroom artifacts preclude the use of the end-inspiration phase, the phase used in the calculation should be as close to the end-inspiration as possible.


Despite significant efforts made for, most abstracts presented during a meeting do not proceed and publish as a manuscript in scientific journals.To investigate publication rates of national anatomy congresses.Descriptive study.All abstracts presented at two annual meetings in 2007 and 2008 were extracted. PubMed and Google Scholar database search used for publication history. Presentation and study types, publication rates and mean publishing times were evaluated. Inconsistency rates between meeting abstract and final published article were also considered.Among 342 abstracts, 195 (57%) were followed by a full-text article. Publication rates for oral and poster presentations were 75% and 52.2%, respectively. The mean publication time was 23.7±23 months. Overall, 89.2% of the articles were published within 5 years. There were no inconsistencies in 50 (25.6%) articles, while 145 (74.4%) had inconsistencies compared to the abstracts presented at the congress. Getting adequate information for 45 (23.1%) articles was not possible. There was no standard reporting format for the abstracts.Our study shows that, overall publication rates for abstracts presented at national anatomy meetings were higher than those presented at national meetings for clinical specialties. Congresses and symposia are necessary media for sharing scientific experiences, collaborating with colleagues and finding new ideas for future scientific research ,2. DespiThere are numerous studies that evaluate the publication rates of abstracts presented at scientific meetings and congresses in medical literature for various clinical specialties and subspecialties ,2. The pYalçınkaya and Bagatur  evaluateWe obtained the presented abstracts from both meetings via Anatomy - International Journal of Experimental and Clinical Anatomy ,14. AfteWe used the electronic search engines PubMed   and GoogWe identified the time of publication as the time lag between the congress and the final publication of articles in months. We included the studies that are published before the congresses and accepted their publication time as minus (-) months.®   of the journals, we calculated the average IF of each journal between 2010 and 2014 via Journal Citation ReportsWe compared the presented abstract and the final published article to evaluate any inconsistencies. We modified the methodology of Bhandari et al.  and YalçWe used statistical analysis software  to evaluate our results. Comparison of publication rates per year, presentation types and study topics was evaluated with chi-square analysis. We used the Mann-Whitney U test to compare the mean publication time and average IF for presentation types. We used the Kruskal-Wallis test to compare the mean publication time of the articles with/without inconsistencies.A total of 342 abstracts were presented at the 2007 (189 abstracts) and 2008 (153 abstracts) National Anatomy congresses. The total number of oral and poster presentations was 72 (21.1%) and 270 (78.9%), respectively.Three abstracts were followed by seven articles. The articles that were published earliest were accepted as final publications and the remaining four were excluded from the study to avoid iteration. As of August 2015, 195 abstracts were followed by a full-text article with a publication rate of 57%. Among these, 115 (33.6%) were published in MEDLINE-indexed journals. Twenty-one (6.1%) abstracts were published in journals indexed in Science Citation Index - Expanded but not in the MEDLINE database. Finally, 59 (17.3%) abstracts were published in national journals indexed in the TUBİTAK ULAKBİM database or international journals that are not indexed in Science Citation Index - Expanded. There was no statistically significant difference for publication rates between 2007 and 2008 meetings (p=0.342). The publication rates for oral and poster abstracts published in MEDLINE-indexed journals were 54.2% (39/72) and 28.1% (76/270), respectively. The publication rates for all oral and poster presentations were 75% (54/72) and 52.2% (141/270), respectively. Oral presentations had a statistically higher publication rate than poster presentations (p=0.001). The mean publication time for all abstracts was 23.7±23 months. Twenty (10.2%) abstracts were published prior to the meetings with a mean publication time of -9.1±-6.1  months. The number of published abstracts after congress per year was 52 (26.7%) for the first year, 48 (24.6%) for the second year, 24 (12.3%) for the third year, 21 (10.8%) for the fourth year, 9 (4.6%) for the fifth year and 21 (10.8%) for more than 5 years. Most of the abstracts (89.2%) were published within the first 5 years. One hundred and sixty-six (48.5%) abstracts were related to clinical anatomy, 76 (22.2%) were experimental, 57 (16.7%) were case reports, 22 (6.4%) were anthropometric, 8 (2.3%) were related to anatomy education, 6 (1.8%) to history, 4 (1.2%) concerned anatomical terminology and 3 (0.9%) were review studies. Final articles were published in 106 different journals. The six most widely preferred journals were: Surgical and Radiologic Anatomy (12-6.2%), Clinical Anatomy (9-4.6%), Journal of Craniofacial Surgery (9-4.6%), Turkish Journal of Medical Sciences (8-4.1%), Fırat Medical Journal (7-3.6%) and International Journal of Morphology (7-3.6%).The average IF of the journals indexed in the MEDLINE database for the 2010 to 2014 period for total, oral and poster presentations was 1.312±0.752, 1.581±0.770 and 1.173±0.709, respectively. The difference between the mean IF of the journals that published oral and poster presentations was statistically significant . Oral presentations tended to be published in journals with a higher mean IF.We could not find any inconsistencies in 50 (25.6%) articles. One hundred and forty-five (74.4%) articles had inconsistencies compared to the congress abstract. Among these, 78 (40%) had only minor, 6 (3.1%) had only major and 61 (31.3%) had both minor and major inconsistencies. Minor inconsistencies included changes to the title in 119 (61%) articles, the number of authors in 74 37.9 (%), the first author’s name in 30 (15.4%) and the number of other authors in 84 (43.1%) articles. Major inconsistencies included changes to the study design/hypothesis in 21 (10.8%) articles, the sample size in 52 (26.7%), the statistical method in 58 (29.7%) and the results in 49 (25.1%) articles. The mean publication times for articles with no, only minor, only major, and both minor and major inconsistencies were 16.9±24.3, 22.2±20.2, 30.8±27.1 and 31.3±23.5 months, respectively. Articles with no inconsistencies had a statistically significantly shorter mean publication time .We found that 45 abstracts (23.1%) did not provide sufficient data on the study. In 10 (5.1%) abstracts, the sample size was not mentioned. Forty-two (21.5%) abstracts did not define the statistical method(s) used. Eleven (5.6%) abstracts did not report results.Additionally, we detected author deletions between the meeting abstract and published article in 35 (18%) studies.Although there are numerous articles that evaluated the publication rates of abstracts presented at scientific meetings for different clinical specialties and subspecialties, we were unable to find a study that investigated this topic for basic medical sciences in international and national literature. Therefore, we compared our results with other national congresses.National meetings for clinical sciences had a publication rate of 29.5% for orthaopedics , 28.6% fThe publication time for abstracts presented at national meetings varied from 14.9 months to 30.72 months. The mean publication time (23.7 months) for abstracts presented at anatomy congresses was concordant with those presented at clinical meetings. Most of the congress abstracts 89.2%) were published as an article within the first 5 years of the presentation. This finding supports the results of Yalçınkaya and Bagatur 9.2% were,22. AddiWe found that the most widely preferred journals were Surgical and Radiologic Anatomy, Clinical Anatomy, Journal of Craniofacial Surgery, Fırat Medical Journal and Turkish Journal of Medical Sciences. After evaluating the published articles on human anatomy between 2000 and 2014, Tellioğlu et al.  reportedWe observed that the majority of the articles (74.4%) had changes from the congress abstract. This finding is similar to that from other national congresses . In 61% Frequent major inconsistencies included changes to the sample size (26.7%), statistical methods (29.7%) and results (25.1%). Our results are similar to those of previous studies . It shouWe found that 23.1% of presented abstracts did not report data including sample size, statistical methods and results. We accepted all abstracts with uncertain information as inconsistent. Providing inadequate data seems to be a common problem for meeting abstracts  and fullOur study has several limitations. We were unable to find any studies in international and national literature that provide publication rates for a basic medical science. Therefore, we compared our results with national congresses for different clinical specialties and subspecialties. Another limitation was that our cross-sectional study evaluated only two consecutive congresses. A longer time interval may have a more precise result for evaluating the publication rates of National Anatomy meetings. Finally, we were unable to explain why some of the studies were presented after their publication.In conclusion, we think that further sectional studies on basic medical sciences should be performed regarding national and international congresses for comparing results. Repeating these studies for selected time periods may be helpful in improving the scientific content of a given specialty. Finally, we observed that abstracts presented at anatomy congresses did not have a standard reporting format. In order to improve this inadequacy, we suggest implementing the STROBE  checklis


There is little scientific evidence for the influence of patient’s age, sex, and chemotherapy on the cell isolation efficiency and metabolic activity of the harvested hepatocytes. The purpose of this study was to investigate whether hepatocytes derived from different sources display differential viability and biosynthetic capacity. Liver cells were isolated from 41 different human tissue specimens. Hepatocytes were labeled using specific antibodies and analyzed using flow cytometry. Multiparametric analysis of the acquired data revealed statistically significant differences between some studied groups of patients. Generally, populations of cells isolated from the male specimens had greater percentage of biosynthetically active hepatocytes than those from the female ones regardless of age and previous chemotherapy of the patient. Based on the albumin staining  after donor liver exclusion (6 out of 41 samples), our results indicated that: 1. samples obtained from males gave a greater percentage of active hepatocytes than those from females ( According to data available from the Centers for Disease Control and Prevention chronic liver disease and cirrhosis are the 12th leading causes of death in the United States (1999–2014) . All theetc.) as well as preoperative blood tests and histopathology can also be a predictive factor for determining the likelihood of a successful isolation [Successful isolation of primary human hepatocytes remains a challenge and comparative studies need to be carried out in cells derived from different sources. The majority of published studies have focused on two aspects of the problem: cell yields and viability. Physiological activity or synthetic capacity of the isolated hepatocytes still pose a poorly recognized problem. It is commonly accepted that warm and cold ischemia time are one of the most important factors affecting hepatocytes isolation –7. Type solation –11. On tsolation –13.In this paper, we present a comparative study on the impact of the age, sex, and chemotherapy on the yield and viability of the metabolically active hepatocytes. We used a method based on immunostaining of cells and flow cytometry. To our best knowledge, the method developed to estimate quality, in particular ability to produce albumin and α-1-antitrypsin, of the isolated hepatocytes by the flow cytometric analysis is published for the first time. Our results enabled us to rank sources of hepatocytes by the synthetic capacity of the isolated cells.Liver tissue was obtained from surgical procedures carried out at the Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Poland (October 2012 to March 2016). Ethical approval for the study was granted by the Local Research Ethics Committee (reference number KB/182/2008). Study was performed according to the principles of the 1975 Declaration of Helsinki. None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given.Human liver cells were isolated from fragments of normal tissue obtained from hepatic resections carried out for metastatic disease (colon/ovaries/breasts), primary liver cancer or other reasons . Some of the isolations were performed using tissue fragments from livers not suitable for transplantation .Twenty six of 41 patients enrolled in the study had received perioperative chemotherapy (13 women and 13 men). Five different treatment regimens had been applied: FOLFOX4 (3 women and 3 men), FOLFOX6 (3 women and 2 men), FOLFIRI (2 women and 6 men), LF4 (3 women and 1 man), and XELODA (2 women and 1 man).In 10 patients who had received chemotherapy (10/26) 6 of their livers showed signs of steatohepatitis, 2—signs of fibrosis, and 2—signs of steatosis. Additionally, two individuals (2/41) had diabetes. None of the patients had cirrhosis.The type of liver tissue and the results of cells isolations are presented in 2+ and Mg2+, pH 7.2; Lab Empire, Rzeszow, Poland) until all remaining blood was removed (flow rate 10 mL/min). Next, the tissue was minced with a scalpel in pre-warmed (37°C) 0.05% collagenase I  solution in the William’s E Medium  and kept under constant agitation on a magnetic stirrer at 37°C for 1 hour. The obtained cell suspension was filtered through the cell strainer with 70 μm nylon mesh . After centrifugation at 366xg , the residual erythrocytes were destroyed by the incubation of the pellet in the red blood cell lysis buffer solution  at 37°C for 5 min. Next, the cells suspension was centrifuged over a Percoll  cushion (1.04 g/ml) at 366xg  to remove cell debris. Finally, after a washing step, the cell pellet was resuspended in the Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum . Viable and dead cells were discriminated and counted using the 0.4% Trypan blue  staining with a hemacytometer.The isolation of liver cells was carried out using a method based on enzymatic tissue disaggregation. Following resection, the fragments were immediately placed in a sterile sealed string bag containing ice-cold University of Wisconsin  solution and processed within 1 hour. In cases of donor liver fragments, the specimens have been kept in sterile UW solution on ice for several hours (less than 12 hours). The isolation started with the washing of liver fragments through the exposed vessels with ice-cold phosphate-buffered saline  were incubated with fluorophore-conjugated specific antibodies . The immunostaining of the cells was described elsewhere . Brieflytibodies  for 1 hotibodies  were useHuman hepatoma C3A (ATCC No. CRL-10741) and osteosarcoma HOS (ATCC No. CRL-1543) cell lines were used to verify the specificity of antibodies against hepatic markers. FACSDiva software  was used for data acquiring and analysis.M) of size of 18 patients, females (F) = 17, without chemotherapy (Ch-) = 9, with chemotherapy (Ch+) = 26, under 60 years old (A60-) = 18, 60 years old and older (A60+) = 17, and certain combinations of above-mentioned ones. The group of 4 patients was the smallest one used for comparison (F Ch-).Thirty five patients from whom the liver tissue samples were collected ) of viable cells (97.2% (91.8%–99.9%)) ) . FACS an–99.9%)) .The hepatocytes are present in the P2. Nonparenchymal cells  belong to the other populations. The identity of the hepatocytes was confirmed by labeling with fluorophore-conjugated albumin (Alb)- and α-1-antitrypsin (A1AT)-specific antibodies . The popThis staining is not sufficient to discriminate between different cell types populating liver but eventually allows for distinguishing parenchymal from nonparenchymal cells. As shown in 47.6. There were differences between groups observed in the entire patients’ population (see p = 0.002) was observed only between donors (n = 6) and non-donors (n = 35) groups (64.1% (55.2%–86.2%) and 38.5% (14.6%–81.2%), respectively). This above-average percentage of the P2 cells in the isolates from the donor livers is probably resulting from the different procedure applied for the pre-preparation of the whole donor organs. Nevertheless, the donor group was omitted from further statistical analysis, because it is not representative of the entire patients’ population  and we found this tissue not suitable for hepatocytes’ isolation (this issue is discussed in the following section).As expected, after the initial removal of most of the blood cells from the tissue fragment, hepatocytes represent a large fraction of the isolated cells. A median percentage of cells belonging to the P2 population in all samples (n = 41) was F = 17) gave greater percentage of the cells belonging to the P2 population then those from males (M = 18) (47.6 (22.0–81.2) and 32.9 (14.6–75.3), respectively; p = 0.162), from people with no chemotherapy (Ch- = 9) greater than from treated (Ch+ = 26) patients (40.6 (18.8–75.3) and 35.1 (14.6–81.2), respectively; p = 0.823), and from younger people (60- = 18) greater than from older ones (60+ = 17) (48.6 (18.7–81.2) and 32.3 (14.6–75.2), respectively; p = 0.186). The two-way ANOVA model detected no interactions between above variables. The broad range of the percentage of cells found in the P2 population (14.6–81.2) is notable and suggests that these outcomes can be affected by both the quality of the tissue and by the effectiveness of blood removal (variable size of the P3–P5 populations). Thus, the meaning of these results is not clear and is not further discussed.Generally, samples obtained from females (85.8 (56.5–98.7) and A1AT-possitive (data available for 29 samples: H9-13 – H44-16)– 88.9 (30.4–97.1).Since the production of Alb and A1AT is a commonly accepted measure of metabolic capacity and viability of hepatocytes we have focused on these two markers. When all tissue samples were taken into account (n = 35), a median percentage of Alb-positive cells in the P2 population was M = 18: 86.9 (59.9–98.7)) and females (F = 17: 80.3 (56.5–97.5)) was statistically significant (p = 0.038) (Ch- = 9: 84.0 (59.9–97.5) and Ch+ = 26: 86.1 (56.5–98.7), p = 0.902) (p = 0.04 after correction) between M Ch+ = 13 (87.3 (82.1–98.7)) and F Ch+ = 13 (80.3 (56.5–97.3)), but not significant (p = 0.61 after correction) between non-treated patients: M Ch- = 5 (84.0 (59.9–94.5)) and F Ch- = 4 (86.6 (73.4–97.5)) (M Ch+/M Ch-: p = 0.40 and F Ch+/F Ch-: p = 0.40).In the single variable comparison, the difference in the percentage of Alb-positive hepatocytes in the P2 population between males (4–97.5)) . Additiop-value for the interaction term “M/F” was significant (0.034), whereas for term “Sex/Chemotherapy” it was not (0.058). However, it is close to the significance level and the interaction plot ) and A60+ = 17 (84.7 (56.5–97.5)), p = 0.547 (M A60- = 7 (92.6 (81.2–98.7)) and M A60+ = 11 (85.8 (59.9–97.5)), yet this difference was not statistically significant (p = 0.42 after correction). Such a difference was not noticeable for females (F A60- = 11: 80.3 (59.9–97.5) and F A60+ = 6: 79.8 (56.5–95.9), p = 0.52 after correction), but was considerable  between M A60- and F A60-. In the group of A60+ patients (n = 17) the difference between males and females was smaller and not significant (p = 0.32 after correction) (p = 0.039 after Tukey HSD test) and no interaction between the sex and the age of the patients (p = 0.772)  . In the = 0.772) .M = 14: 89.4 (41.4–97.1) and F = 15: 83.5 (30.4–95.8), p = 0.79; Ch+ = 22: 84.6 (30.4–97.1) and Ch- = 7: 92.5 (61.7–96.5), p = 0.092; A60- = 16: 89.2 (30.4–97.1) and A60+ = 13: 81.5 (41.4–95.8), p = 0.20  isolated human hepatocytes from tissue after partial hepatectomy (n = 50). As it was shown in our report, the patient’s sex and previous chemotherapy had no influence. Contrary to our results, donor age significantly affected the isolation outcome, but it was correlated with the overrepresentation of the primary and secondary tumors in the group of older patients. Thus, the quality of the individual specimen rather than the age was important and this parameter was not found suitable for predicting cell yields [p = 0.669) or the viability (p = 0.303) between specimens from the younger and the older patients. However, in a study including liver pieces from as many as 1034 individuals, carried out in order to establish an algorithm that predicts the yield and viability of isolated hepatocytes, statistically significant differences between various groups have been found. After multiple regression analysis, apart from the tissue quality correlated with the disease, such variables as chemotherapy, sex, and age influenced the analyzed parameters. This study detected statistical significance in more variables than any other research due to the remarkable increase in the statistical power [We have observed only minor differences in both isolation efficiency and cells viability between analyzed groups. Neither age of the tissue donors, sex, and chemotherapy nor combinations of above-mentioned parameters gave l yields . Also prl yields –11. Neveal power . The aut6/g and 96.7% vs. 3.4 × 106/g and 97.3%, respectively. These results, although not statistically significant , are in line with others. Bhogal et al. (2011) in the study on isolation of the cells from 104 liver fragments reported lower viability and yield of hepatocytes from donor livers (n = 7) due to the longer cold ischemia time that donor livers have been exposed to when compared to normal livers [et al. (2006) cells isolated from fragments of the livers used for transplantation exhibited a significantly higher viability than those from livers rejected for transplantation (10 of 15 rejected livers were steatotic) [p = 0.458. Thus, it turned out that tissue quality played a major role in the successful isolation of healthy hepatocytes. Taking into account that donors group is not representative of the patients’ population  we did not include it into the further statistical analysis.In our report, median isolation efficiency and cell viability in the group of liver donors n = 6) were lower than those obtained in the rest of the population (n = 35): 2.7 × 10 were lowp = 0.038) when cells were labeled with anti-Alb antibody. To our best knowledge, this report is the first to indicate, using FACS, that human male hepatocytes may be more productive in terms of albumin synthesis. Generally, we have found the anti-A1AT antibody less useful for our purposes. The reason for this could be explained by the characteristic of the particular antibody we have used for hepatocytes’ staining or by the differential expression pattern of these two proteins. Nevertheless, albumin is the major blood protein product of the liver. Difficulties in obtaining statistically significant differences between studied groups result from the relatively low size of the groups in combination with the broad range of the acquired data. Such a situation is typical for biological objects and more data are required to draw far-reaching conclusions. Nevertheless, based on the Alb staining, our results certainly indicated  that: 1. samples obtained from males gave a greater percentage of active hepatocytes than those from females and 2. specimens from the males after chemotherapy greater than those from the treated females. In a large study on the cytochrome P450 enzyme activity in human liver microsomes (n = 142) and its inducibility in cultured human hepatocytes (n = 64) Parkinson et al. (2004) concluded that gender, age, ethnicity, and liver cirrhosis of the donor should not influence the selection of human liver samples for routine studies of drug metabolism and enzyme induction in vitro. The results indicated that the CYP enzyme activity varied considerably from one sample to the next and this variation was observed in human liver samples from males and females, young, middle-aged and elderly donors, and in livers from people of different ethnicity. A lack of statistical significance was apparent between analyzed groups (with some exceptions), yet the authors did not use combinations of these parameters for comparison [In order to find the optimal source of human hepatocytes we have focused on the method that would enable us to estimate biosynthetic capacity of the isolated cells. We have developed a fast and reliable technique, based on flow cytometry, to analyze production of hepatocytes’ proteins within few hours after isolation. The method to estimate quality of the hepatocytes by the FACS analysis of the percentage of the Alb-positive cells in the P2 population is presented here for the first time. The idea is based on the assumption that using the given antibody one can detect target protein present in the cell in amounts above some threshold and that only certain levels of the albumin synthesis, characteristic of active hepatocytes, allow their detection. As it was shown for the parameters of cells isolation, patients’ characteristics such as age and chemotherapy, analyzed in the entire population individually, did not reveal statistically significant differences in the number of active hepatocytes (percentage of the Alb- and A1AT-possitive cells in the P2 population) in studied groups. On the other hand, the sex variable gave us satisfying statistical significance . Some differences between groups were visible when certain combinations of these parameters were used. The most pronounced differences  were observed between: males and females, and males and females after chemotherapy. The low number of samples is a major limitation of our report. For this reason, we have applied very restrictive statistical analysis, including tests for the correction for the variations of the sample size (see: Patients and Methods section), that allowed us to draw careful conclusions. Moreover, due to the low accessibility of such specimens for a research, many other papers report similar sizes of the analyzed samples or sizes of the same order of magnitude ,6,11–13.Although more samples are needed to draw firm conclusions we ranked sources of active hepatocytes by the median percentage of Alb-positive cells in the P2 population as follows:92.6 (81.2–98.7)Men under 60 years old (n = 7)– 85.8 (59.9–97.5)Men over 60 years old (n = 11)– 80.3 (59.9–97.5)Women under 60 years old (n = 11)– 79.8 (56.5–95.9)Women over 60 years old (n = 6)– S1 Fig(PDF)Click here for additional data file.


The World Health Organization (WHO) 2010 guidelines for intensified tuberculosis (TB) case finding (ICF) among people living with HIV (PLHIV) includes a recommendation that PLHIV receive routine TB screening. Since 2005, the Rwandan Ministry of Health has been using a five-question screening tool. Our study objective was to assess the operating characteristics of the tool designed to identify PLHIV with presumptive TB as measured against a composite reference standard, including bacteriologically confirmed TB.In a cross-sectional study, the TB screening tool was routinely administered at enrolment in outpatient HIV care and treatment services at seven public health facilities. From March to September 2011, study enrollees were examined for TB disease irrespective of TB screening outcome. The examination consisted of a chest radiograph (CXR), three sputum smears (SS), sputum culture (SC) and polymerase chain reaction line-probe assay (Hain test). PLHIV were classified as having “laboratory-confirmed TB” with positive results on SS for acid-fast bacilli, SC on Lowenstein-Jensen medium, or a Hain test.+ cell count was 385  cells/mm3. Of the patients screened, 138 (7.8%) were diagnosed with TB of which; 125 (90.5%) were laboratory-confirmed pulmonary TB. Of 404 (22.9%) patients who screened positive and 1,363 (77.1%) who screened negative, 79 (19.5%) and 59 (4.3%), respectively, were diagnosed with TB. For laboratory-confirmed TB, the tool had a sensitivity of 54.4% (95% CI 45.3–63.3), specificity of 79.5% (95% CI 77.5–81.5), PPV of 16.8% and NPV of 95.8%.Overall, 1,767 patients were enrolled and screened of which; 1,017 (57.6%) were female, median age was 33 , and median CD4TB prevalence among PLHIV newly enrolling into HIV care and treatment was 65 times greater than the overall population prevalence. However, the performance of the tool was poorer than the predicted performance of the WHO recommended TB screening questions. Tuberculosis (TB) alongside HIV remains a leading cause of morbidity and mortality worldwide. In 2014, there were an estimated 9.6 million new cases of TB and 1.5 million deaths from TB globally. Overall, 12% of TB cases were among people living with Human Immunodeficiency Virus (HIV). There has been important progress worldwide in implementing TB/HIV collaborative activities and the numbers of people in HIV care and treatment services screened for TB increased by 27% from 5.5 million to 7.0 million from 2013 to 2014. 8]Data analysis was conducted using STATA Version 12 (Copyright 1984–2007 StataCorp TX USA). Baseline characteristics are presented by means and medians as appropriate for parametric and non-parametric data; ANOVA/ Student’s T-test and Kruskil-Wallis / Mann Whitney U test were used to compare groups . Comparisons of the statistical significance of differences between categorical data were done using the chi-square test.Operating characteristics, including sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) with 95% CI and receiver operating curves (ROC), using a logistical model, were calculated for the TB screening tool (five screening questions together) and each question separately. Operating characteristics of the screening tool were determined based on the composite reference standard for confirmed TB cases and then confirmed and probable TB cases combined (which included those diagnosed by radiography).Subgroup analysis on the added diagnostic value of the chest radiographs was completed using sensitivity and specificity compared to the composite reference standard for TB confirmed cases only .3).Analyses were stratified by factors affecting operating characteristics  because of the additional health facility visits required to undergo diagnostic evaluation per the protocol.This study was approved by the National AIDS Commission and Rwanda National Ethics Commission in Rwanda, and the Columbia University Institutional Review Board at Columbia University Medical Center. The study was determined to be non-research by CDC Atlanta based on the role of CDC investigators.A total of 2,928 PLHIV sought HIV care and treatment at the seven study sites from March 1st, 2010 to September 16, 2011. Of these, 2,416 (82.5%) patients were referred to study staff.  Of thoseAmong the 1,767 participants, seven were transferred-in from other HIV care and treatment sites and were currently receiving ART; 937 (53.0%) were from Kigali, with the remainder from outside Kigali.  The mediTwenty-three percent (n = 404) of the participants screened positive based on answering “yes” to at least one of the five questions on the screening tool. Of the 404 participants screening positive, 203 (50.2%) had cough for two weeks or more, 225 (55.7%) had experienced weight loss of three kg or more in the last four weeks, 81 (20.0%) had fever for three weeks or more, 112 (27.7%) presented with night sweats and 111 (27.5%) had been in contact with someone known to have TB disease. Among all participants screened, 410 (23.3%) were found to have any cough, regardless of the duration.M. tuberculosis. Anterior-posterior and lateral chest radiographs were taken for 349 (86.4%) participants screening positive, of which 89 (25.5%) were classified as abnormal.  were smear-positive cases. Of those who screened positive, 358 (88.6%) had a sputum culture done at the NRL of which 52 (14.5%) were positive for bnormal. .M. tuberculosis. Of the 1,150 (84.4%) patients who underwent a CXR, 100 (8.7%) were interpreted as abnormal.Of the 1,363 participants who answered “no” to all five screening questions, 100% had at least one sputum smear, of whom 24 (1.8%) were sputum smear-positive. Eighty-seven percent  had cultures performed with 44 (3.7%) positive for M. tuberculosis. Of the 13 participants defined as probable cases of TB, 10 (7.2%) were pulmonary and three were extra-pulmonary . These participants with probable cases of TB had negative sputum smears and culture (Hain test was not done). All 13 participants diagnosed with probable TB cases presented with clinical symptoms and a chest radiograph determined to be suggestive of TB.Overall, 138 (7.8%) of 1,767 participants enrolled were diagnosed with TB, of which 125 (90.6%) were laboratory-confirmed pulmonary TB cases. Of the lab-confirmed cases, 74 (59%) participants had a positive Hain and/or Capilia test, of which 13 (10.4%) were diagnosed only with Hain/Capilia and the remaining cases were diagnosed with a combination of sputum smear examination and or culture. Forty (29%) participants had negative sputum smear results but a positive culture for Of the 404 participants who screened positive, 68 (16.8%) had laboratory-confirmed TB. Of the 1,363 participants who screened negative, 57 (4.2%) had laboratory-confirmed TB, including 27 with a positive culture but negative smears. The sensitivity of the national screening tool for laboratory confirmed TB cases was 54.4% (95% CI: 45.3%–63.3%) and increased to 67.8% (95% CI: 57.9%–76.3%) by replacing “cough for two weeks” with “any cough” and adding a CXR to the screening tool. The specificity of the tool was 79.5% (95% CI: 77.5%–81.5%) and decreased to 65.3% (95% CI: 62.7%–67.8%) after replacing the “cough for two weeks” with “any cough” and adding a CXR .Of the 13 participants diagnosed with probable TB, 11 (84.6%) screened positive and two were missed using the screening tool. When combining the confirmed and probable TB cases, the sensitivity of the national screening tool was 57.2% (95% CI: 48.5%–65.6%) and the specificity was 80.0% (95% CI: 78.0%–82.0%).3, the tool has sensitivity and specificity of 71.9% (95% CI: 58.5%–83.0%) and 68.8% (95% CI 63.5%–73.8%) and for patients with CD4 cell count ≥200 46.3% (95% CI: 35.0%–57.8%) and 82.8% (95% CI: 80.6%–84.8%) respectively. Similarly, when used for patients at WHO stage III or IV the tool had a specificity of 62.6% (95% CI: 55.0%–69.7%) compared to 82.1% (95% CI: 80.0%–84.1%) for patients with WHO stage I and II. The highest sensitivity of 78.3% (56.3–92.5) was found among participants with WHO clinical stage III & IV and those with a CD4+ count <200 cells/mm3 71.9% (95% CI: 58.5–83.0).Significant differences in the performance of the tool among participant subgroups were observed . When stWhen analyzing the performance of each of the screening questions for the confirmed cases only or when the confirmed and probable TB cases were combined, no single indicator achieved a sensitivity of greater than 55% . HistoryIn This study aimed to understand the performance of the Rwandan national TB screening tool among PLHIV enrolling in out-patient HIV care and treatment settings. During the study enrollment period in 2011, WHO published updated guidelines for intensified TB case finding among PLHIV, which recommend a four-element symptom-based screening tool consisting of any current cough, fever, night sweats, or weight loss. The 54.4TB screening tools are developed to have a high sensitivity and negative predictive value to identify people suspected of having TB who require additional diagnostic testing and avoid false negatives. ScreeninThe overall prevalence of TB in PLHIV newly enrolled in HIV care and treatment in this study was higher (7.8%) than expected based on routine monitoring and evaluation data from Rwanda. Routine programmatic data from Rwandan HIV care and treatment clinics since 2006 suggest a prevalence of TB disease of about 2% among PLHIV. The high+ cell count decline, the proportion of smear negative pulmonary TB and extra-pulmonary TB cases increases. In this sample, patients initially had relatively high median CD4+ cell counts (385 cells/mm3). For participants with confirmed TB, the proportion with CD4+ cell count ≥200 cells/mm3 was 61.3%, while in smear negative and extra-pulmonary (probable) TB the proportion was 30.8%. The low proportion of smear-negative pulmonary tuberculosis and extra-pulmonary tuberculosis cases observed in this study may be a function of participants registering for HIV care when CD4+ cell counts were relatively high and that nearly 90% of the study population had a WHO clinical staging of I or II. Also of note, the HIV program in Rwanda has matured as evidenced by the national trend of increasing median baseline CD4+ cell counts in patients enrolling in HIV care.[The proportion of probable pulmonary TB and extra-pulmonary cases was lower than expected in a population newly enrolled in HIV care.,15 It haHIV care.Of the different components of the screening tool, cough for two weeks or more was the strongest predictor of TB (AUC 0.67). This is mainly due to the fact that most of the cases were pulmonary TB (98%). Cough for two weeks or more as a single symptom was poorly sensitive consistent with earlier studies conducted in Ethiopia, Zimbabwe and South East Asia.,18,19 UsChanging cough of two weeks or more to any cough and adding the results of the chest X-ray improved sensitivity of the tool from 54.4% to 67.8%. However, the specificity dropped to 65.3% and PPV to 13.3% while the negative predictive value remained high at 96.2%. These results are similar to other published reports, although there were variations in screening tool components and populations screened within the studies. The addeThe NPV of the national tool was high which made the tool appropriate for excluding active TB prior to initiation of IPT. This was also observed in other studies in Ethiopia, Zimbabwe and South East Asia.,18,19 Ho7 Unfortunately because of stock-outs at the NRL, study staff were unable to perform Hain and/or Capilia on all samples. In addition, cultures were done on solid media in accordance with national guidelines which is known to be less sensitive than culture performed using liquid media.[This study had several limitations. There was initial training and periodic supervision of study nurses however variability in use of the screening tool within and across sites was not systematically evaluated. The screening tool may not have been applied consistently and uniformly across sites. Also, although temporality of cough was removed for the analysis of the additional yield for screening, this was not recorded for other screening components  as recommended by the WHO guidelines for intensified TB case finding that were issued after our evaluation protocol was implemented.id media.Overall, the Rwandan TB screening tool that included temporal parameters performed poorly compared with the predicted performance of the WHO-recommended screening questions. This finding reinforces the current international recommendations—and current, revised Rwandan national guidelines—to assess for the presence and not the duration of symptoms when screening for TB among PLHIV. Also, our study demonstrates the importance of routine TB screening among PLHIV. Using a screening tool with relatively poor operating characteristics we were still able to document a TB prevalence among PLHIV that was 65 times greater than what is observed in the general population in Rwanda.


Type 2 diabetes and prediabetes are established risk factors for atherosclerosis. The aim of this study was to evaluate the atherosclerotic plaque burden in the carotid arteries of patients with acute coronary syndrome according to their glycemic status.Patients with acute coronary syndrome and no previous history of type 2 diabetes were consecutively included in the study. Glucose metabolism was evaluated with fasting glucose in plasma, HbA1c and a standard two-hour oral glucose tolerance test. Atherosclerotic plaque in the carotid arteries was evaluated with a standardized ultrasound examination where total plaque area was measured and patients classified as having no plaque or a significant plaque formation.A total of 245 acute coronary syndrome patients ) were included. The proportion diagnosed with normal glucose metabolism, prediabetes and type 2 diabetes was 28.6%, 64.1% and 7.3%, respectively. A significant atherosclerotic plaque was found in 48.5%, 66.9% and 72.2% of patients with normal glucose metabolism, prediabetes and type 2 diabetes, respectively. An incremental increase in total plaque area was found from normal glucose metabolism to prediabetes (25.5%) and from normal glucose metabolism to type 2 diabetes (35.9%) (p = 0.04). When adjusted for conventional cardiovascular risk factors the OR of having significant atherosclerotic plaque in the carotid arteries was 2.17 (95% CI 1.15–4.15) for patients with newly diagnosed dysglycemia compared to patients with normal glucose metabolism. When additionally adjusted for the 2-hour plasma glucose after glucose loading (2hPG) the OR attenuated to 1.77 (95% CI 0.83–3.84).Newly detected dysglycemia is an independent predictor of significant atherosclerotic plaque in the carotid arteries with oral glucose tolerance test as a major determinant of carotid plaque burden in this group of individuals with acute coronary syndrome. Type 2 diabetes mellitus (T2DM) and prediabetes are established risk factors for atherosclerosis and cardiovascular disease (CVD) and associated with increased mortality compared to those with normal glucose metabolism (NGM) . PatientAtherosclerotic disease in the carotid arteries has been associated with coronary artery disease and is an indicator of an increased atherosclerotic burden . MeasuriWe hypothesized that patients with ACS have a high prevalence of atherosclerotic plaque in the carotid arteries and that ACS patients with newly diagnosed T2DM and prediabetes have increased atherosclerotic burden compared to ACS patients with NGM. Therefore, we set out to evaluate the atherosclerotic plaque burden in the carotid arteries of patients with ACS and related the plaque burden to newly detected metabolic derangements.Patients admitted to the coronary care unit of Landspitali, the University Hospital of Iceland with the diagnosis of ACS were consecutively included in the study between June 2013 to October 2014. ACS was defined according to the joint European Society of Cardiology (ESC) and American College of Cardiology recommendations . Patientst degree family members with coronary artery disease (CAD) were defined as having family history of CAD.Prior to any study related procedure informed written consent was obtained for each participant. The study protocol adhered to the principles laid out in the Declaration of Helsinki  and was Glucose metabolism was evaluated after an overnight fast of at least ten hours with fasting plasma glucose (FPG), glycated hemoglobin (HbA1c) and a standard oral glucose tolerance test (OGTT). Two hours after ingesting a solution containing 75 g of glucose measurements of plasma glucose (2hPG) were made. Measurements of glucose metabolism were made during hospitalization, generally on third to fifth day after admission, and repeated at least three months later. All samples collected for venous plasma glucose measurements were centrifuged and analyzed immediately after blood collection. Glucose levels were determined using reagents, calibrators and Vitros 250/950 analyzers from Ortho Clinical Diagnostics, Rochester, USA and HbA1c levels were determined using reagents, calibrators and Cobas c311 analyzer from Roche, Mannheim, Germany.The classification of glucose metabolism was based on the American Diabetes Association (ADA) criteria, where prediabetes was defined as HbA1c 5.7–6.4% (39–47 mmol/mol), FPG 5.6–6.9 mmol/l (100–125 mg/dL) or 2hPG 7.8–11.0 mmol/l (140–199 mg/dL) and T2DM as HbA1c ≥6.5% (48 mmol/mol), FPG ≥7.0 mmol/l (126 mg/dL) or 2hPG ≥11.1 mmol/l (200 mg/dL) . PatientThree months after discharge an ultrasound imaging of the carotids was performed according to a standardized protocol . A Toshi2) for each plaque automatically computed by the program. The TPA was calculated by summing the area of all individual plaques. Acquisition and interpretations were made by trained sonographers [Atherosclerotic plaques in the carotid artery segments (BIF and ICA) extending 10 mm proximal and distal from the tip of the flow divider, were categorized as a significant or not. The most severe lesion per segment was assessed . Patientgraphers .All patients underwent coronary angiography during admission. Coronary arteries with lumen reduction of more than 70% were considered to have a significant stenosis and patients classified of having 0-, 1-, 2- or 3-vessel disease. Gensini score was applied to evaluate the coronary artery disease by the degree of lumen reduction and the importance of the lesion´s location .p<0.05. All statistical analyses were performed using the R software version 3.2.2. [Categorical variables were presented as percentage. Normally distributed continuous variables were presented as means , otherwise as median with interquartile range (IQR). Between group comparison of NGM, prediabetes and T2DM was made using the chi-square test and analysis of variance or Kruskal Wallis test for categorical and continuous variables, respectively. The odds ratio (OR) of having significant plaque in the carotid arteries was estimated with a multivariable logistic regression model. The predictors used were HbA1c, FPG and 2hPG. The association between TPA and risk factors for atherosclerosis was evaluated with general linear regression models, both unadjusted and adjusted for glucometabolic factors and conventional atherosclerotic risk factors: age, gender, hypertension, hypercholesterolemia, smoking status and BMI. The level of statistical significance was set at n 3.2.2. A total of 435 patients admitted to the coronary care unit with the diagnosis of ACS were considered for inclusion in the study. Among these, 90 were discharged before the OGTT could be performed, 60 refused participation and two died shortly after admission. Therefore, 283 patients were included in the study shortly after admission. Before the follow-up visit at 3 months, 21 patients were lost to follow-up, 13 patients withdrew consent, three died and one had a stroke. The remaining 245 patients comprised the study population.2 (SD 64.7) and 0.90 mm (SD 0.15), respectively.The baseline characteristics of the study population are presented in 2 (SD 57.2)) prediabetes (108.9 mm2 (SD 66.1)) and T2DM (118.0 mm2 (SD 70.7)) (p<0.04). The incremental increase in TPA was 25.5% and 35.9% among patients diagnosed with prediabetes and T2DM, respectively, compared to patients with NGM. A significant difference in BMI, waist-to-hip ratio, smoking status, high-sensitivity C-reactive protein (hS-CRP), insulin resistance (HOMA-IR), number of diseased coronary arteries and Gensini score was found between different glucose metabolism categories  and 2.25 (1.21–4.35), respectively. For HbA1c and FPG classification of dysglycemia the unadjusted OR for the detection of atherosclerotic plaque in the carotids did not reach statistical significance .When adjusted for conventional atherosclerotic risk factors, the OR for patients diagnosed with dysglycemia according to the 2hPG during admission of having significant plaque in the carotid arteries was 1.92 (95% CI 1.05–3.55) and 1.51 (0.74–3.18) at follow-up. However, the ORs for HbA1c and FPG remained statistically non significant.2  and 24.0 mm2  greater TPA, respectively, compared to those with normal glucose level.The relationship between the diagnosis of dysglycemia by the three different methods and TPA is shown in The unadjusted OR of patients diagnosed with prediabetes, T2DM and dysglycemia of having significant atherosclerotic plaque in their carotid arteries compared to patients with NGM was 2.14 , 2.75  and 2.19 , respectively , model 1From the multiple logistic regression analysis after adjusting for conventional atherosclerotic risk factors the ORs of having significant atherosclerotic plaque for patients diagnosed with prediabetes, T2DM and dysglycemia  compared to NGM was 2.14 (95% CI 1.13–4.12), 2.50 (0.75–9.51) and 2.17 (95% CI 1.15–4.15), respectively , model 22 , 31.2 mm2  and 23.0 mm2 , respectively, compared to patients with NGM  and 10.0 mm2 , respectively, compared to patients with NGM Click here for additional data file.