Trypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI–TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.We present a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.Based on these findings we propose a simple typing assay for  Trypanosoma cruzi, the causative agent of Chagas Disease, infects approximately 8 million people in the Americas, with 200,000 new cases reported anually. The disease, in its chronic stage, has different manifestations: mega-colon, mega-esophagus, and cardiomyopathy, among others. The fact that infections by the same species cause these different clinical outcomes is believed to be determined, at least in part, by the genetic background of the parasite (infection by different strains). By analyzing a number of molecular markers, the population of the parasite has been divided into seven major evolutionary lineages, which evolve mostly independently, by clonal expansion with infrequent exchange of genetic material. Accurate classification of different strains and isolates into their corresponding evolutionary lineages is therefore essential to obtain a good map of biological, biochemical and ecoepidemiological features for the whole species. The current methods available to type T. cruzi stocks are either laborious and costly (requiring the amplification and sequencing of a variable number of genes or gene fragments), or limited in resolution. In this work we describe a number of key discriminant sites in a gene encoding a putative enzyme from the sterol pathway of the parasite, which were exploited to design a couple of alternative typing assays. Using these key discriminant sites, we can classify any T. cruzi stock into either six or seven evolutionary lineages using only one gene fragment, and in a matter of hours (depending on the assay used). To our knowledge, the proposed assays are the first typing assays that can discriminate T. cruzi stocks with such speed and low cost. Trypanosoma cruzi, a protozoan parasite of the order Kinetoplastida, is the causative agent of Chagas Disease, a neglected disease that is is endemic in South America, affecting 8 million people in Latin America T. cruzi species displays a considerable genetic and phenotypic diversity T. cruzi, CL Brener) have a very high degree of heterozygosity but otherwise very homogeneous population structures with low intra-lineage diversity Lineages TcV and TcVI  α-rRNA gene, and the 18S rRNA gene As mentioned, there are already a number of typing methods available, based on different methodologies. After a revision of all diagnostic molecular characters described, Lewis T. cruzi, bringing robust resolution of evolutionary lineages through sequencing of 4–10 diagnostic lociRecently, a number of multi-locus sequence typing (MLST) markers were introduced for T. cruzi we propose a simple typing strategy that relies on the amplification of a single locus, therefore providing a rapid assay that can be used routinely for typing T. cruzi strain collections.Based on the identification of a number of discriminating polymorphisms in the gene encoding a putative C-5 sterol desaturase in T. cruzi evolutionary lineages/DTUs, including T. cruzi reference strains, as well as isolates from bats that correspond to a new lineage/DTU T. cruzi marinkellei. Their names and corresponding lineage/DTU classification) are: Sylvio X10 cl1, Dm28c, TeDa2 cl4, PALV2-2cl5, CAI72, JR cl4, LL015, and Tul0 (TcI); MAS1 cl1, TU18 cl93, IVV cl4, and Y (TcII); M6241 cl6, M5631 cl5, Tc863, and X109/2 (TcIII); CanIII cl1, Dog Theis, Fuscicolis 15, and 92122102R (TcIV); Sc43 cl9, MN cl2, LL014, Teh53 cl4, and Bertha (TcV); Tulahuen cl2, CL-Brener, P63 cl1, and Tul2 (TcVI); TCC1994, and TCC1122 ; B3, and B7 (T. cruzi marinkellei). Genomic DNA from these strains was obtained from the T. cruzi collection at IIB-UNSAM, or were generous gifts from Dr. Patricio Diosque  and Dr. Marta M.G. Teixeira . The typing and lineage/DTU classification of these strains was carried out using the method proposed by S. Brisse, J Verhoef and M. Tibayrenc M loci .Thirty three parasite stocks were analyzed, covering all major TcSC5D gene  was amplified using the following oligonucleotide primers: TcSC5D-fwd 5′-GGACGTGGCGTTTGATTTAT-3′, TcSC5D-rev 5′-TCCCATCTTCTTCGTTGACT-3′. A fragment of 537 bp of the TcMK gene  was amplified using the following oligonucleotide primers: Tc-Mev-kinase26-Fw 5′-TTTTTGCATGTCATTTTGG-3′, Tc-Mev-kinase662-Rv 5′-AGCGGTCTTGTAATGAGCAC-3′. PCR amplification was performed using Taq polymerase (Invitrogen) in a Biometra T Professional Gradient 96 cycler. Amplification mixtures contained 10 pmol of each primer, PCR buffer (Invitrogen), 1.6 mM MgCl2, 100 ng of genomic DNA, 200 µM dNTPs, 2.5 U Taq polymerase (Invitrogen), and water to a final volume of 25 µl. After denaturing at 94 for 4.5 min, thermal cycling was performed for 35 cycles at 94 for 30 s, followed by 30 s at 58, followed by 72 for 30 s. Reactions were finished by a 5 min incubation at 72. Amplification products were checked in 1.2% agarose gels stained with ethidium bromide. A single amplification product was observed for the TcSC5D product in all T. cruzi stocks tested, including those from the Tcbat lineage (not shown). The same oligonucleotide primers were used to amplify the corresponding gene fragment from Trypanosoma rangeli and Trypanosoma cruzi marinkellei. In these cases, the yield of the amplification was lower, but a product of the expected size could be amplified in all cases, although in T. rangeli a couple of additional, non-specific bands of smaller size (∼300–250 bp) were also amplified. For the TcMK gene, a single amplification product was obtained in all strains tested  of the amplification reaction was treated with 1 U of Exonuclease I (Fermentas) and 10 U of Shrimp Alkaline Phosphatase (Fermentas) for 45 mins at 37 and then for 30 min at 80 to inactivate these enzymes. Subsequently two sequencing reactions were prepared , with the same primers used for the amplification of the product. Sequencing was carried out in an Applied Biosystems 3130 capillary sequencer using a Big-Dye terminator cycle sequencing kit, according to the instructions of the manufacturer. After sequencing, the length of each amplification product was such that, upon assembling a contig using the corresponding forward and reverse reads, there is a significant overlap, therefore providing sequence information from both DNA strands covering all key polymorphic sites.A fragment of 832 bp of the Base calling of chromatograms, assembly of sequences, detection of polymorphisms (including heterozygous peaks) and manual inspection of assembled sequences and polymorphisms was done using a software package composed of Phred (version 0.020425.c) TcSC5D amplification products where digested in a single incubation with 1 U of HpaI (NEB R0105) and 1 U SphI (NEB R0182) at 37 for 1 h. Digestion was performed in the same buffer used for PCR amplification (Invitrogen Taq PCR Buffer). The resulting restriction fragments were resolved by electrophoresis of in 2% TBE-agarose gels, applying 5,33 V/cm for 1 h. For TcMK fragment, an aliquot (20 µl) of the amplification products where digested in a single incubation with 1 U of XhoI (NEB R0146) at 37 for 1 h. The resulting restriction fragments were resolved by electrophoresis in 2.5% TBE-agarose gels, applying 5,33 V/cm for 1.5 h. The gels were stained with ethidium bromide for visualization under UV light.All restriction enzymes were purchased from New England Biolabs . An aliquot (20 µl) of the TcSC5D gene have been deposited in the GenBank database with the following accession numbers: HQ586986 (CL-Brener), and JN050564–JN050587 . Sequences of the TcMK gene have been deposited under the following accession numbers: HQ586987 (CL-Brener), and JN050693–JN050720. Heterozygous sequence polymorphisms have been submitted as ambiguities in the sequence using standard IUPAC notation .According to recent changes related to standardization in trypanosomatid T. cruzi, we turned our attention to the gene encoding a putative C-5 sterol desaturase (lathosterol/episterol oxidase). This gene, present as a single copy per haploid genome, and mapped to chromosome 22 TcSC5D gene, we observed a total of 81 fixed differences , the majority of which were bi-allelic (changing one specific nucleotide base for another). However, we also noticed the presence of some tri-allelic sites (a base is replaced by two different bases in the population), and 1 position where all possibilities  were observed in the population. This tetra-allelic SNP alone, at position 657 of the TcSC5D coding sequence, produced a separation of T. cruzi strains in five groups, that matched their classification in discrete typing units (DTUs), as obtained by other methods  step to discriminate all major evolutionary lineages/DTUs.TcSC5D gene, an initial simple typing assay can be proposed that is based on the direct sequencing of the amplification product. By analyzing the chromatogram peaks of only three positions of the TcSC5D sequence  it is possible to discriminate strains from all evolutionary lineages  nucleotide changes between the TcI and Tcbat lineages, a minimum of 4 changes between the TcII vs TcIV and TcIII vs Tcbat haplotypes, and a minimum of 5–8 changes for the remaining lineage comparisons , and HpaI . Three such sites were identified in our re-sequencing study, and are listed in T. cruzi sequences) was identified in T. cruzi marinkellei. These restriction enzyme sites are present alone and in homozygosity in three lineages/DTUs , while one DTU (TcIV) does not contain any of these restriction sites, and the Tcbat lineage contains two of these restriction sites in homozygosity. Therefore a unique restriction pattern was expected to arise from a double digestion of the TcSC5D amplification product in strains from these DTUs. The two hybrid lineages (TcV and TcVI) contain two of these sites . These patterns are unique for all DTUs, except for DTUs TcV and TcVI, that cannot be differentiated. In the case of these DTUs, the banding pattern is essentially the sum of the patterns of DTUs TcII and TcIII, which agrees with the currently favored hybridization hypothesis As can be seen in the agarose gel electrophoresis, the digestion with a mixture of these enzymes generates 7 unique restriction patterns , and also from the co-endemic trypanosome Trypanosoma rangeli. As shown, the RFLP analysis of the T. cruzi marinkellei genes produced a unique pattern of digestion that was different from those observed for T. cruzi DTUs .For comparison purposes, we analyzed DNA from the related bat trypanosome, DTUs see . For T. ands see , the comTcSC5D amplification product, in some cases a PCR-RFLP assay is desirable (e.g. to perform strain typing assays in the field or in remote settings where access to Sanger sequencing technology is limited). By including a second locus in the assay, it is possible to discriminate DTUs TcV from TcVI using a fast PCR-RFLP assay. In our analysis of the sterol biosynthesis pathway , we noticed another discriminating region in the mevalonate kinase gene , overlapping a XhoI site . Within the 5′-UTR of this gene , we identified a conserved XhoI site in strains from the TcV DTU (in some cases in heterozygosity), that is absent altogether in strains from the TcVI DTU  by sequencing a single amplification product and focusing the analysis on just 8 discriminant polymorphic sites. A faster PCR-RFLP variant of the typing assay can generate seven unique restriction patterns, effectively classifying strains in six major T. cruzi groups, and is also able to discriminate isolates from a related species, such as T. cruzi marinkellei. The majority of these patterns have only 2 bands, and all are nicely resolved in 2% agarose gels, greatly facilitating interpretation of the typing assay.In this work we propose a simple and fast typing assay for T. cruzi DNA samples. A schematic view of the proposed typing strategy is shown in loci, if resolution of lineages TcV and TcVI is required. In the figure, this marker is the polymorphic XhoI site in the TcMK gene  from all other DTUs, and can therefore be used to rapidly screen a large collection of gene see . But thiWith some caveats, the PCR-RFLP assay can also be applied to detect mixed stocks (isolates with different parasite populations). We have applied the PCR-RFLP typing assay to artificial DNA mixtures and have found that the method behaves as theoretically expected. For this we have prepared artificial mixtures for each DTU, in which we contaminated the DNA with increasing quantities of DNA from another DTU. In all cases amplification of the TcSC5D gene fragment was not affected, and the restriction pattern obtained was the sum of the expected banding pattern of the DTUs present in each mixture, with the intensities of the bands serving as indicators of the relative level of contamination (not shown). This essentially means that when dealing with isolates containing parasites from different lineages, the assay can help identify a mixed isolate (with one exception). The exception is a theoretical isolate that is a mixture of parasites from lineages TcII and TcIII. If the isolate has a 50∶50 mixture of strains, then this mixture will be indistinguishable from a TcV/TcVI hybrid strain. Therefore we believe the method has also great potential to rapidly screen field isolates, and detect candidate cases for cloning of parasite stocks.T. cruzi, and so it may seem redundant to propose yet another molecular marker for this purpose. However, there is still an unmet need for a simple and low cost typing analysis that can be employed in clinical and/or eco-epidemiological samples. Also, as recognized recently by a number of experts, there is an increasing need for clearly typing and reporting the classification of T. cruzi strains in scientific communications T. cruzi strains and isolates maintained in culture or as cryopreserved material. Because the proposed PCR-RFLP typing strategy is simple, and can produce results in hours, it can be also used as a preamble to more complex classification schemes.Many strain typing assays have been devised for T. cruzi marinkellei and T. rangeli  are both single copy genes, that are apparently under purifying selection, with a ratio of non-synonymous to synonymous changes of 0.16 and 0.39, respectively (data not shown for the TcMK gene). The fact that the same oligonucleotide primers are able to amplify the expected fragment from all evolutionary lineages, and from other related species such as locus, its main advantage is the ability to rapidly screen a collection of DNA samples from different isolates. When using the highly streamlined RFLP assay, results can be obtained in one day in a matter of hours. To our knowledge this is the only currently available typing assay that can discriminate strains with such speed and low cost. This fast typing method can classify T. cruzi strains in 6 groups, corresponding to DTUs TcI, TcII, TcIII, TcIV, Tcbat, and DTUs TcV and TcVI (as a group)  see . AlternaT. cruzi strains and isolates. Because it does not depend on sequencing of amplification products, the typing strategy proposed by Brisse et al.loci for each strain/isolate, the assessment of small differences in size (∼15 bp) between amplification products, and the comparison of band intensities. Furthermore, all this information has to be integrated into a complex decision tree to classify each sample  it was possible to obtain unique restriction profiles for DTUs TcI-TcIV, still failing to discriminate TcV from TcVI More recently, robust typing strategies have been proposed for use in eco-epidemiological and evolutionary studies The proposed assay strategy therefore, provides a key advantage over other currently available strategies, and that is its speed, based mostly on the fact that only one PCR reaction is needed for each sample, independently of the DTU/lineage. Furthermore, the post-amplification processing of samples is minimal, and is reduced to either an enzymatic cleanup incubation for Sanger sequencing, or an enzymatic digestion for the RFLP assay see . In eithlocus there are a number of homoplasic characters. Most notably, the key SNP at position 618 (selected because of its discrimination of DTUs TcV/TcVI) has one such trait in strains of lineage TcII. Also, when including additional strains in our panel, we uncovered an additional homoplasic trait in the tri-allelic SNP at position 138, in the case of the TcIV strain Fuscicolis 15. It has been shown already that the use of single molecular markers can lead to misclassification In spite of these advantages, it is important to emphasize a number of potential issues with this assay. First of all, the same feature that makes this strategy simple, cheap and rapid (e.g. being a single-locus assay) could easily turn into a major weakness, for example, if novel homoplasic traits are uncovered at any of the key discriminating sites when sampling additional strains. In the TcSC5D loci  to gain discriminatory power Secondly, when using the Sanger based sequencing assay, the analysis must focus on key discriminant positions in the sequence. As mentioned, there are a number of putative homoplasies in the dataset that would affect the results if all polymorphic sites are considered in the analysis without further inspection. These putative homoplasic traits correspond to those polymorphic sites in the alignment where a strain deviates from the expected character state for its current lineage/DTU classification see . That meloci, these ‘loss of heterozygosity’ events in TcVI were observed only in some stocks, and only for some lociAs shown in the results, the discrimination of stocks from DTUs TcV and TcVI relies on a single discriminant site in the case of the TcSC5D gene. In the case of the TcMK gene, there are another 6 discriminant sites that separate strains from these DTUs, all of which represent cases of loss of heterozygosity (not shown), that can be exposed by Sanger-based sequencing of the amplification product. However, only one of these allows a PCR-RFLP assay. In this regard it is also important to mention that in recent works analyzing a number of The observation of so few discriminant sites between DTUs TcV and TcVI is not surprising, given the relatively recent origin of these DTUs TcSC5D) (or throughout the TcMK gene fragment) to increase the confidence in the base call at these sites.Finally, although the RFLP assay is rapid and simple, the sequencing-based assay suffers from the same issues as the diploid sequence typing strategies recently published T. cruzi that can rapidly classify strains into six or seven discrete typing units (DTUs). The method is easy to implement, cheap, and can produce results in a matter of hours or days, therefore lowering the barrier to routine typing of strains in the field and in research laboratories working with T. cruzi. To our knowledge this is the only currently available typing assay that can discriminate strains with such speed and low cost.Based on the identification of key discriminant SNPs, and a number of polymorphic restriction sites, we propose two alternative simple typing strategies for Figure S1TcSC5D sequences. Thirty three (33) sequences were obtained by re-sequencing of the TcSC5D locus in T. cruzi (different DTUs) and T. cruzi marinkellei. The actual sequence is shown only for the first strain (Sylvio X10). For the rest of the sequences, only nucleotides differing from the top sequence are shown. Putative homoplasic traits (enclosed in red boxes), correspond to those sites where a strain deviates from the expected character state for its current DTU/lineage classification. Key informative polymorphic sites Click here for additional data file.Figure S2T. cruzi Discrete Typing Units. The table shows the total number of nucleotide changes observed between different DTUs. These include the 8 key informative changes shown in Nucleotide changes observed between (PDF)Click here for additional data file.


The goal of this study was to observe possible changes of the skin microvascular perfusion on the acupoints and related areas and to quantify influences of acupuncture stimulation on the volunteers' blood pressure, heart rate, and heart rate variability (HRV). During the measurement, the needling sensations of volunteers were enquired and recorded. Ten healthy volunteers with a mean age ± SD of 25.4 ± 2.6 years were enrolled, and acupuncture stimulation was performed on ST36 , in pure lifting-thrusting or twisting-rotating manipulation. During needling, we observed the changing of microvascular perfusion on ST36, 37, 38, and a control point using MOOR speckle laser blood flow scanning. Electrocardiogram and blood pressure were registered before, during, and after needling. Both lifting-thrusting and twisting-rotating needle manipulations could decrease blood pressure and heart rate while improving HRV significantly. There were significant differences in microvascular perfusion on acupoints ST36, 37, 38, and the control point following these two kinds of needle manipulation. The needling sensation caused by lifting-thrusting is stronger than that of twisting-rotating manipulation. Significant differences between lifting-thrusting and twisting-rotating acupuncture stimulation methods show that the mechanisms may be different and need to be researched thoroughly in the future. The former means that the acupuncturist inserts the needle into the skin to a certain depth to obtain the qi arrival and then moves the needle up and down perpendicularly to stimulate the tissues including skin, fascia, fat, and muscles [In clinical practice, the medical acupuncturist always stimulates the patients using compound manipulations including lifting, thrusting, twisting, and rotating. Lifting-thrusting , and heart rate variability (HRV) so as to investigate the relationship among acupoints, non-acupoints, and the local points on the same meridian. During the measurement, the needling sensations were inquired and recorded. Analysis of HR and HRV in this partially single-blind transcontinental study was performed in Europe.Ten healthy volunteers with a mean age ± SD of 25.4 ± 2.6 years , all from Beijing University of Traditional Chinese Medicine and Graduate School of China Academy of Chinese Medical Sciences, were enrolled in this study. The volunteers had no medical history whatsoever during the last year. All of them had had experience of acupuncture before, so they could accurately describe the needling sensation.Using single-use sterile needles , 0.30 (diameter) × 40 (length) mm, we punctured Zusanli , which is located in the lower limb, one finger's breadth from the anterior crest of the tibia, 3 cun below the knee joint . All acuThe needle was inserted to a certain depth till the acupuncturist felt qi arriving, asking the volunteer whether she/he felt any needling sensations at the same time. After 5 minutes scanning, the lifting-thrusting manipulation was done for 20 seconds, with a range of 10 mm, frequency 1–1.5 Hz see .The needle was inserted to a certain depth till the acupuncturist felt qi arriving, asking the volunteer whether she/he felt any needling sensations at the same time. After 5 minutes scanning, the twisting-rotating manipulation was done for 20 seconds, with an angle of 180–270°, frequency 2–2.5 Hz see .For visualization of microvascular changes a multipoint synchronization scanner Moor FLPI  was used. The scanning distance was about 11 cm. The acupoints ST36, 37, and 38 see  were marThe temperature of the laboratory was kept constant at 26°C, and the volunteers were asked to come into the room 5 minutes ahead of schedule to adapt to the room temperature. During the scanning procedure see , the volt-test, with P < 0.05 defined as the level of significance, were used. The HRV data were analyzed at the laboratory in Austria, and the analyzed results were sent back to Beijing. The researchers in Austria did not know the type of needle manipulation.The alterations of the skin microvascular perfusion were transferred into measurement data by the MoorVer2.0 software . All data including blood pressure, HR, and needling sensation (VAS) were analyzed using Friedman repeated measures ANOVA on ranks. In addition, Tukey test as post hoc analysis and the The results show that systolic blood pressure (SBP), diastolic blood pressure (DBP), and HR decreased significantly after lifting-thrusting manipulation, but only SBP and HR decreased significantly after twisting-rotating manipulation while DBP increased see .Among the 4 observed points, skin blood perfusion of the acupoint ST36 increased significantly after acupuncture needle insertion before lifting-thrusting needle manipulation, after manipulation and after removing the needle; the skin blood perfusion of the control point on the tibia bone decreased insignificantly. Skin blood perfusion of ST37 and ST38 changed inconspicuously .During the twisting-rotating needling manipulation, the skin blood perfusion of ST36 increased and reached its maximum, then decreased significantly step by step. Point ST37 showed similar changes. At ST38 and the control point, the skin blood perfusion decreased. But only the increase at ST37 after removing the needle and the decrease at ST38 after twisting-rotating manipulation was statistically significant .As mentioned in According to the questionnaires about the needling sensation, we found that the main feelings after inserting or removing the needle were tenderness and numbness , and theQuantifying acupuncture needle manipulation using modern techniques and measurement methods has been performed only by few research groups . There aNeedle manipulations and arrival of qi (needling sensation) are among the most important aspects in acupuncture. Needling manipulation, also known as needling transmission, refers to various manipulations of acupuncture to induce needling sensation after the needle is inserted. The arrival of qi, also known as needling sensation, refers to induction of channel qi after the needle is inserted. During this needling sensation, the patient feels numbness, soreness, or heaviness around the acupuncture point. Sometimes it is connected with coldness, warmness, itching, pain, electric shock feeling, ant crawling, feeling, and so on . The funBoth lifting-thrusting and twisting-rotating needling manipulations could effectively decrease BP and HR and improve HRV. However, the results were not completely uniform; they showed that the mechanisms are different and worth being studied deeply. In order to further elucidate these mechanisms, the results should also be compared with those of other modes of stimulation at ST 36, such as electroacupuncture, laser acupuncture, and moxibustion in future studies.In this experiment, the manipulation of twisting-rotating completely copied the clinical operation, the angle of rotation being smaller than 360°. Therefore, the needling sensation caused by lifting-thrusting manipulation was stronger than that of twisting-rotating.The control point showed a continuous decrease in skin blood perfusion, while the acupoints ST36, 37, and 38 all showed significant increases. This may indicate that there could be a specificity of channels and acupoints.


We aimed to evaluate the risk factors for recurrent wheezing in patients diagnosed with acute bronchiolitis.From 2009 to 2011, 500 patients from the pediatric clinics, with first attack of acute bronchiolitis were included in this prospective study. Each patient’s age, gender, birth weight, duration of breastfeeding, family history of atopy and asthma, smoking exposure, source of heating in the house, the presence of pets, any history of chronic disease have been questioned. The patients were followed for a duration of 12 - 24 months.In this study, 39% (n = 195) of the cases were female and 61% (n = 305) were male, with a median age of 3 months old. Male gender, low birth weight , low gestational age (< 37 weeks), breastfeeding of less than 6 months, congenital heart disease, family history of atopy, asthma, smoking exposure, stove warming, was found as significant risk factors for recurrent wheezing, however, presence of pets at home was found to be a protective factor.Informing parents about the risk factors such as exposure to cigarette smoke, heating mode, duration of breastfeeding can significantly decrease recurrent episodes of wheezing. Acute bronchiolitis is a lower respiratory tract disease characterized by inflammation of the bronchioles. It is often caused by viral agents in children under two years with the symptoms of wheezing, cough, rapid breathing, chest retractions and the incidence is 10-20% [Recurrent wheezing episodes can be seen after acute bronchiolitis attack. Acute bronchiolitis may change the immune response to facilitate the emergence of an acute asthma or acute bronchiolitis in children who already have asthma may result as repeated wheezing attacks. Risk factors for the development of recurrent wheezing episodes are as follows: family history of atopy and allergy in a child, narrow airways, lower socioeconomic status, crowded living conditions, absence of breastfeeding, passive smoking , 3. Pre-Knowing risk factors for acute bronchiolitis and recurrent wheezing attacks is of great importance in terms of child health and development. In this study, we aimed to determine the risk factors for recurrent wheezing in patients diagnosed with first attack of acute bronchiolitis.This hospital-based prospective study included infants with a diagnosis of acute bronchiolitis from the outpatient clinics of Zeynep Kamil Maternity and Children’s Disease Training and Research Hospital between June 2009 and June 2011. The diagnosis of acute bronchiolitis is made on clinical basis; infants with wheezing, retractions, tachypnea, increased respiratory effort with a history of upper respiratory tract infection symptoms such as runny nose, cough, fever were included in the study. Exclusion criterias were as follows: patients with a history of recurrent wheezing, age younger than 1 month and older than 24 months, families who did not agree to participate in the study.Each patient’s age, gender, gestational age at delivery, birth weight, duration of breastfeeding, family history of atopy and asthma, exposure to cigarette smoke, house warming mode , the number of people living at home, pets at home were recorded for all patients. The presence of chronic disease such as congenital heart disease, immune deficiency was also questioned.Patients participating in the study were followed up for a period of 12 - 24 months. For each episode of recurrent wheezing, patients were examined by the same doctor and the patients’ and his environments’ characteristics were recorded again. The average duration of each wheezing episode, number of wheezing attacks for each patient during the follow-up period, and the time spent during hospitalization, if any occurred, were recorded. The optimal duration of breastfeeding is defined as 6 months by the World Health Organization, premature birth is defined as birth before 37 weeks of pregnancy, low birth weight is defined as birth weight under 2,500 gr , 9. RecuThe study was approved by the local Ethics Committee of our hospital and an informed consent form was signed by parents who agreed to participate the study.Statistical Package for Social Sciences, 15.0 was used for the evaluation of data. For the comparison of qualitative data, chi-square test; for the comparison of two groups, Student’s t-test and Mann-Whitney U test; for the comparisons of the average of three groups, Kruskal-Wallis test was used. Logistic and linear regression analysis of the relationship between risk factors was made. Results with a P-value < 0.05 was considered significant.A total of 500 infant with acute bronchiolitis were included in this study . The medAttack duration of the patients was 4.8 ± 1.6 (median = 5) days, the mean number of episodes was 5.1 ± 2.9 (median = 6). A single episode was seen in 22% of the patients (n = 110), 5.4% (n = 27) had 2 episodes, 72.6% (n = 363) had ≥ 3 . Attack Recurrent bronchiolitis (≥ 3 episodes) was significantly higher in male infants, in infants who were born before 37 gestational week and with breastfeeding duration less than 6 months (P = 0.0001) .In this study, male gender, low birth weight, low gestational age, breastfeeding of less than 6 months, congenital heart disease, family history of atopy and/or asthma, cigarette smoke exposure, home heating with stove were found to be risk factors for recurrent wheezing.Male gender is an important risk factor for recurrent wheezing , 15-17. It is shown that duration of breastfeeding should be at least 6 months to be protective against respiratory tract infections . RecurreIn our study, in accordance with the literature, low birth weight and preterm delivery were found to be significant risk factors for recurrent wheezing , 24-27. Exposure to cigarette smoking is a significant risk factor for recurrent wheezing , 26, 28.Atopy and genetics can be predisposing factors in virus-induced wheezing. It is not clear whether reactive airways are genetically present or occur after acute bronchiolitis . Family Pets at home can be protective against recurrent wheezing episodes , 30, 31.In this study, stove warming was found to be an important risk factor for recurrent wheezing. Most of the infants with recurrent wheezing had stove heating (82.9%) at their homes. Infants whose house heated by a stove had their first bronchiolitis attack at younger ages; higher risk of hospitalization; significantly higher number and duration of attacks.In our study, patients’ younger age at the time of first acute bronchiolitis can be related to high incidence of premature and low birth-weigh deliveries at our hospital because of the presence of two neonatal intensive care units. Our outpatient clinics have patients characterized as families with low socio-economic conditions and high incidence of complications related to premature birth such as bronchopulmonary dysplasia, congenital heart disease.It is important to know risk factors for recurrent wheezing so as to predict and prevent recurrent episodes in terms of child health and development. For this reason, patients should be informed about potential risk factors such as tobacco smoke exposure, heating mode, and also the preventive role of breastfeeding. Larger-scaled prospective studies are needed to clarify the effect of pet to recurrent wheezing.


P = 0.001), insulin, and HOMA-IR (P < 0.001) were observed in the G2 adolescents. In multivariate linear regression analysis, the % BF from equation (5) was associated with TG, diastolic blood pressure, and insulin in G1. Among the G2 adolescents, the % BF estimated by (5) and (9) was associated with LDL, TG, insulin, and the HOMA-IR. Body fat anthropometric equations were associated with cardiovascular risk factors and should be used to assess the nutritional status of adolescents. In this study, equation (5) was associated with a higher number of cardiovascular risk factors independent of the nutritional status of adolescents.The aim of this study was to analyze body fat anthropometric equations and electrical bioimpedance analysis (BIA) in the prediction of cardiovascular risk factors in eutrophic and overweight adolescents. 210 adolescents were divided into eutrophic group (G1) and overweight group (G2). The percentage of body fat (% BF) was estimated using 10 body fat anthropometric equations and 2 BIA. We measured lipid profiles, uric acid, insulin, fasting glucose, homeostasis model assessment-insulin resistance (HOMA-IR), and blood pressure. We found that 76.7% of the adolescents exhibited inadequacy of at least one biochemical parameter or clinical cardiovascular risk. Higher values of triglycerides (TG) ( Obesity is a proinflammatory state involving the hypertrophy and hyperplasia of adipocytes related to metabolic and cardiovascular diseases. In recent years, it has become evident that adipose tissue is not a passive receptacle of lipids, but a dynamic organ involved in the process of obesity, type 2 diabetes mellitus, hypertension, atherosclerosis, dyslipidemia, acute and chronic inflammatory processes, and metabolic syndrome, among others .Obesity is a chronic, complex, multifactorial disease resulting from the interaction of genetics, environment, and lifestyle, and it is considered a public health problem worldwide due to its increasing prevalence in recent decades in developed and developing countries .Obesity in adolescence tends, in 80% of cases, to perpetuate in adulthood and it is associated with high mortality . Excess On the basis of their body mass index (BMI), normal individuals may carry a high percentage of body fat because the BMI cannot discriminate excess body fat from the fat-free mass (FFM) . HoweverIdentification of body fat evaluation methods that associate to higher number of cardiovascular risk factors is necessary on obesity prevention and treatment, specially during adolescence, to avoid that harmful health effect persists in future life. The objective of this study was to analyze the body fat values obtained by anthropometric equations and electrical bioimpedance analysis (BIA) in the prediction of cardiovascular risk factors in eutrophic and overweight adolescents of both sexes in Viçosa, Minas Gerais, Brazil. This was a cross-sectional study conducted between March and October 2010 involving adolescents of both sexes aged 15–18 years from the urban area of Viçosa, Minas Gerais, Brazil.The sample size was calculated using software Epi Info version 6.04 based on a specific formula for cross-sectional studies. We considered the population of 5010 adolescents at the age studied, in Viçosa-Minas Gerais , prevaleThe participants were selected by simple random sampling according to the following inclusion criteria: to be postpubescent, for so, it was considered as criterion presence of menarche of at least 1 year for girls and axillary hair for boys; not having participated in studies/nutrition consultations in the last 6 months; to be normal weight presenting BMI/age ≥25 percentile and BMI/age ≤75 percentile or to be overweight, BMI/age ≥85 percentile . Those cThe exclusion criteria included reports of infections or diagnosis of acute and chronic noncommunicable diseases; use of medications or supplements that might interfere with the metabolism of carbohydrates, lipids, and blood pressure; regular use of diuretics/laxatives or contraceptives for less than 2 months; use of a pacemaker or prosthesis; and pregnancy.n = 140): normal weight, BMI/age ≥25 percentile, and BMI/age ≤75 percentile.Group 1 (G1) (n = 70): overweight, BMI/age ≥85 percentile.Group 2 (G2) , and all volunteers and their guardians signed an informed consent form before the study commenced.For anthropometric evaluation, weight and height were measured using the techniques proposed by the World Health Organization (WHO) . Weight The brachial perimeter was measured on the right side, avoiding compression of the soft tissues, with an inextensible, 1.5 m long measuring tape divided into centimeters and subdivided into millimeters . MeasureThe biceps, triceps, suprailiac, subscapular, midaxillary, and calf skinfold thicknesses were measured on the right or left, with the subject in a standing or seated position, according to protocol , 26, 27.The percentage of body fat (% BF) was estimated by tetra-polar horizontal BIA  (BIA1), and tetrapolar vertical BIA using 8-point tactile electrode  (BIA2), being all participants fasted for at least 12 hours and undergoing the proposed evaluation protocol ; and by The resistance value provided by BIA1 was used in the predictive equation of FFM. Body fat (kg) was determined by the difference between the body weight (kg) and FFM (kg), and consequently, the value of the % BF was derived from the ratio between body fat (kg) and body weight, multiplied by 100. The % BF was classified according to Lohman . AdolescWe measured total cholesterol (TC), high-density lipoprotein (HDL), and triglycerides (TG) by enzymatic calorimetric method and low-density lipoprotein (LDL) calculated by Friedewald's formula, once no TG value was higher than 400 mg/dL. We measured fasting glucose by glucose oxidase enzymatic method and insulin by electrochemiluminescence method.Lipid profiles and hyperinsulinemia were classified according to the I Guideline for Prevention of Atherosclerosis in Childhood and Adolescence . FastingInsulin resistance was calculated by homeostasis model assessment-insulin resistance (HOMA-IR). Presence of insulin resistance was defined as HOMA-IR ≥ 3.16 .Uric acid was measured by enzymatic calorimetric method classified according to sex, considering adequate values of 2.0–7.0 mg/dL for boys and 1.5–6.0 mg/dL for girls.Blood pressure was measured and analyzed according to the recommendations of the VI Brazilian Guidelines on Hypertension  using an t-test or the Mann-Whitney test, Chi-square Pearson test, and linear regression to assess the relationship between the cardiovascular risk factors (dependent variable) and the % BF estimated by body fat anthropometric equations and BIA (independent variable), according to the study groups. The variables TG, insulin, HOMA-IR, LDL, TC and the ratios LDL/HDL and TC/HDL were submitted to logarithmic transformation, for not presenting normal distribution. The values of body fat anthropometric equations and BIA that presented coefficient β exhibiting significance of P < 0.20 in the bivariate analysis were used in multiple regression models adjusted for sex, and in G1, also for normal individuals with excess body fat. The level of physical activity, considered a confounding factor, was not included in the model because it was not associated with risk factors and % BF in the bivariate analysis. We used the Kolmogorov-Smirnov normality test to assess the type of distribution of variables, and Student'sP > 0.05. The presence of heteroskedasticity was verified by the White test; when necessary, the consistent variance matrix modified for small samples was used to correct the estimated error pattern [To analyze the fit of the models, we first evaluated the normality of the distribution of residuals, which should be normally distributed, in order to examine the prerequisites of the classical linear regression model. For this, we used the Shapiro-Wilk statistic level of significance  pattern . For mulP < 0.05.The data were double-entered in Microsoft Office Excel 2007, and statistical analyses were performed in SPSS for Windows version 13.0 and STATA version 11.0, with a significance level of n = 110) were women, 61.4% (n = 129) were non-whites, 51% (n = 107) were actives, 76.7% (n = 161) exhibited inadequacy of at least one biochemical parameter or clinical cardiovascular risk, 44.8% (n = 94) had TC ≥150 mg/dL, 24.3% (n = 51) had LDL ≥100 mg/dL, 43.3% (n = 91) had HDL ≤45 mg/dL, 15.2% (n = 32) had TG ≥100 mg/dL, 1% (n = 2) had uric acid >7 mg/dL, and 3.9% (n = 8) had systolic or diastolic blood pressure ≥120/80 mmHg. Higher values of TG (P = 0.001), TC/HDL (P = 0.001), LDL/HDL (P = 0.005), insulin, and HOMA-IR (P < 0.001) were observed in the G2 adolescents (P < 0.05).The study evaluated 210 adolescents, mean age was 16.8 (±1.0) years, 52.4%  of these adolescents had excess adiposity according to least one method of body composition assessment, being more frequent in feminine sex (P < 0.001).After analyzing only the G1 adolescents, we found that they had excess body fat of 10.7–82.1% according to the body fat anthropometric equations or BIA, and 92.1% , (8), and (10) for TG and from (4) for LDL . HoweverP < 0.05); however, when adjusting the model by sex, this association did not remain significant   .Considering the number of risk factors associated with the % BF, we found that the best equation for the G1 adolescents was equation (5), and (9) was the best suited for the G2 adolescents.The results of this study are broadly in agreement with the literature –36 that As expected, the frequency of excess adiposity varied according to the body fat anthropometric equations and BIA, possibly due to the peculiarities of each method, which involves characteristics related to the population, age, race, and nutritional status in addition to anthropometric measurements used as independent variables. Excess body fat was more frequent in girls, which may in part be explained by the differences in body composition between the sexes. Although weight gain is also a result of increased muscle mass and adipose tissue in both sexes during puberty, the gain in muscle mass is higher in boys and that for adipose tissue is higher in girls . AnotherAs in other studies –36, overConversely, there was no difference in blood pressure when comparing G1 and G2 individuals, which was contrary to the findings of Falaschetti et al. , who assThe body fat anthropometric equations and BIA evaluated in this study as a method of estimating the % BF are generally associated with cardiovascular risk factors and are good predictors of changes in TG, insulin, and HOMA-IR, mainly in overweight individuals.These results are relevant and are consistent with the literature, which has reported that metabolic and hemodynamic changes are more frequent in obese subjects , 38 and According to the present study results, the % BF obtained by body fat anthropometric equations was a better predictor of cardiovascular risk than that estimated by BIA for both study groups, especially G1, as multiple regression analysis determined that did not exist associations between the % BF estimated by BIA and the cardiovascular risk parameters.The multiple linear regression analysis performed in the G1 group revealed that the association of the % BF with TG, insulin, HOMA-IR, and DBP remained significant, indicating the presence of cardiovascular risk factors even in individuals with adequate nutritional status. These results are suggestive of the influence of other factors such as high-energy density diet, physical inactivity, and heredity, among others, that may be involved in the changes within these parameters .r = − 0.576, P < 0.0001). This result can be explained partly by the fact that, unlike the other authors whose equations are analyzed in this work, Weststrate and Deurenberg [In this study, equation (5) was associated with a greater number of cardiovascular risk factors in the G1. Similarly, in the study by Burrows et al. , equatiourenberg  developeurenberg .With regard to the G2 adolescents, equation (9) explained 5 of 7 risk factors, followed by (10) and (5). Equation (9) for the FFM differed from the others in that it had as independent variables height, weight, and resistance, which was determined by the BIA equipment and can be related with the best results. Another factor that may explain its association with risk factors is the fact that this equation be specific for adolescents . It is nAlthough BIA to be a fast method, of easy utilization, noninvasive, and in this study the body fat estimated by equation (9) was associated with expressive number of cardiovascular risk factors, it is worth noticing this method tends to underestimate % BF, specially in subjects with body fat excess , not preIn order to encourage the use of the body fat anthropometric equations in routine healthcare and research, we suggest the use of equations (10) and (5). The advantages of equation (10) are its simplicity and ease of collection of the variables weight, height, and age, and the measurements and information obtained are on an outpatient basis and available to most research efforts; however, this equation was better associated with cardiovascular risk factors only in overweight adolescents .Although involving more complex calculations and obtainment of measurements because it uses skinfolds thicknesses, equation (5) has the advantage of being the equation that was most associated with cardiovascular risk factors in both study groups, which means it can be used regardless of nutritional status.In this study, we found that none of the estimates of body fat was associated with HDL, demonstrating that the variation of this parameter is independent of adiposity, being regular physical activity, dietary habits, and family history of diseases being some of the factors associated with its modification , 44.Many studies have associated cardiovascular risk factors with the BMI, waist circumference, hip circumference, waist/hip and waist/height ratio, and the sum of skinfolds thicknesses as central and peripheral body fat measures, and other parameters , but lit The present study results are in agreement with the literature and demonstrate that excess adiposity is associated with cardiovascular risk factors, especially those related to metabolic syndrome. As expected, excess adiposity varied according to the anthropometric equations and BIA used in this study, being more prevalent in girls.Broadly, body fat anthropometric equations and BIA were capable of predicting cardiovascular risk factors in postpubertal adolescents, especially for overweight subjects (G2), once it was verified associations of all equations and BIA to at least one risk factor in this group.From the results obtained and considering that all body fat anthropometric equations are differentiated by sex, it can be inferred that, in this study, the best anthropometric equation was the one of Weststrate and Deurenberg (equation 5), as it was associated with a higher number of cardiovascular risk factors independently of the nutritional status of adolescents.Considering the increasing prevalence of overweight/obesity and cardiovascular risk factors in adolescents, the identification of a noninvasive and inexpensive method, as is the case with body fat anthropometric equations, permits the early detection of excess body fat, allowing for interventions in a period conducive to the reduction of such risk factors.


Despite epidemiological evidences of relationship between poor lung function and atherosclerosis, the relationship between poor lung function and microalbuminuria (MAU), an early surrogate marker of both kidney damage and atherosclerosis, is not well understood. Hence, we plan to investigate the relationship between poor lung function and MAU using multivariate models to adjust for other atherogenic risk factors.th Korean National Health and Nutrition Examination Survey. Poor lung function is determined by spirometric measurement, primarily through estimation of the forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1). Declines in the percent predicted FVC (<80%) and in the FEV1/FVC ratio (<0.7) are defined as restrictive and obstructive patterns, respectively. Urine albumin to urine creatinine levels ratio (UACR) were measured in spot urine samples. MAU was defined as UACR >30 mg/g.We used data from the 5P = 0.002), which remained significant after adjustment for SBP, FBG, triglyceride level, BMI, smoking history, and heavy alcohol consumption . When the restrictive pattern was compared to the normal pattern, the odds ratios (OR)  for MAU were 1.90 (1.32–2.72) in men, after adjustment for age, hypertension, diabetes mellitus, triglyceride level, obesity, smoking history, physical activity, and heavy alcohol consumption.Inverse relationship was observed between lung function and UACR. In an age-adjusted regression model, the regression coefficient (B) of 10% lower FVC was 11.09 in men (Our study, the first investigation in Asia, demonstrated that the restrictive pattern is related to MAU in men. Furthermore, there was linear relationship between lower FVC and UACR. Thus, our current study suggests that poor lung function, particularly the restrictive pattern, is related to kidney damage as well as atherosclerosis. Microalbuminuria (MAU), as indicated by the urine albumin-to-creatinine ratio (UACR), occurs when blood albumin leaks from the kidneys into the urine Multiple pathological steps such as retention, modification, and accumulation of lipids in the endothelium occur prior to vascular damage or atherosclerosis, all of which are negatively influenced by systemic inflammation 1/FVC ratio (<0.7) are key phenotypes of restrictive and obstructive patterns of poor lung function, respectively. Both poor lung function patterns worsen detoxification of systemic inflammation brought on by physical activity, eventually resulting in persistent inflammatory status even after rest 1Poor lung function is determined by spirometric measurement of dynamic lung capacity, primarily through estimation of the forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1). Clinically, a decline in the percent predicted FVC (<80%) and in the FEVDespite the epidemiological evidence of a relationship between poor lung function and the risk of atherosclerosis, the relationship between poor lung function and MAU, as an early surrogate marker of both kidney damage and atherosclerosis, has not been investigated. Tobacco smoking is a strong risk factor of both MAU and poor lung function The aim of our study was to investigate the relationship between poor lung function and MAU as an early surrogate marker of kidney damage as well as atherosclerosis and thus, to generate important information concerning the association between poor lung function and MAU in an Asian population. We used a multivariate model to adjust for obesity, hypertension, and diabetes mellitus as well as the lifestyle factors of smoking history, heavy alcohol consumption, and regular physical activity.th Korean National Health and Nutrition Examination Survey (KNHANES). Data from 2011 to 2012 were selected for analysis. All voluntarily engaged participants provided written informed consent prior to their enrollment. The participants’ records, except survey date and home region, were anonymized prior to analysis. This study was approved by the Institutional Review Board (IRB) of Korea Centers for Disease Control and Prevention .We used data from the 5th KNHANES from 2010 to 2012. A geographic region based-multistage probability sampling of household units, stratified according to sex and age, was undertaken throughout the entire Korea population. Using the above sampling method, 192 household units consisting of 3,800 individuals were selected each year, resulting in a total of 576 units with 11,400 individuals included in the 5th KNHANES from 2010 to 2012. The response rates in 2010, 2011, and 2012 were 81.9%, 80.4%, and 80.0%, respectively. The KNHANES comprises 3 major examinations. The first is a clinical health examination, including blood and urine biochemistry analyses, blood pressure assessment, pulmonary function tests, and roentgenography. The second is a comprehensive self-reported questionnaire of anthropometric and demographic characteristics such as health-related behaviors. The third is a questionnaire on 24-hour recall of nutritional information.The Korea Centers for Disease Control and Prevention conducted the 51normal predicted values of FVC and FEV1 in Korean general population were used to calculate the values of % predicted FVC and FEV1/FVC ratio <0.7.Spirometry tests to determine lung function patterns were performed by trained technician using dry rolling-seal spirometers  in participants older than 40 y Smoking status was categorized as non-smoker, former smoker, and current smoker using a self-reported questionnaire. “Non-smoker” was defined as a lifetime history of smoking fewer than 100 cigarettes. Regular physical activity was defined as more than 20 min of physical activity sufficient to cause sweating, 3 or more times per week. Heavy alcohol consumption was defined as drinking 7 or more glasses of alcohol on 2 or more occasions per a week in men, and 5 or more glasses on 2 or more occasions per a week in women.2), normal (18.5–25.0 kg/m2), and overweight (≥25.0 kg/m2) categorized as dummy variables.Hypertension was defined as blood pressure controlled using pharmacological treatment or systolic blood pressure (SBP) or diastolic blood pressure (DBP) exceeding 140 mmHg or 90 mmHg, respectively. Normal blood pressure was defined as SBP below 120 mmHg and DBP below 80 mmHg. Participants who fell below the hypertension threshold but above the threshold for normal blood pressure were defined as prehypertension. Diabetes mellitus was determined based on 8-h fasting blood glucose (FBG). FBG levels less than 100 mg/dl, 100–125 mg/dl, and 126 or above were defined as normal, glucose intolerance, and diabetes mellitus, respectively. Participant with diabetes mellitus controlled by pharmacological treatment were categorized as diabetes mellitus regardless of FBG level. Body mass index (BMI), the ratio of weight to squared height, was used for obesity parameters Spot urine samples were used to analyze MAU in KNHANES Because UACR was measured from 2011 to 2012, 6,053 participants older than 40 y who completed UACR measurement and spirometry testing were selected for analysis . We excluded 33 asthma patients receiving pharmacological treatment, because spirometry was not undertaken after bronchodilation. Ultimately, a total of 6,020 participants  were included in data analyses.1 values in multiple regression models. Odds ratios (ORs) with 95% confidence intervals (95%CI) were calculated in multiple logistic regression models according to poor lung function pattern .Frequency with percentage, mean with standard deviation (SD), or median with interquartile range was used to interpret the participant characteristics according to non-MAU vs. MAU status as appropriate. Furthermore, age adjusted comparisons were undertaken by using generalized linear model in P<0.001 in men; 57.7±10.6 y vs. 61.8±10.9 y, P<0.001 in women). The proportion of poor lung function patterns differed between the MAU and non-MAU groups. The proportions of restrictive and obstructive patterns were higher in the MAU group than the non-MAU group . The percent predicted value of FVC in the non-MAU group was higher than that in the MAU group for both sexes . The percent predicted value of FEV1 in the non-MAU group was also higher than that in the MAU group for men , but not for women.Participants with MAU of both sexes were older than those without MAU . Mean SBP level was higher in the MAU group than in the non-MAU group for both sexes . The proportion of participants with diabetes mellitus was greater in the MAU group than in the non-MAU group for both sexes . Mean FBG level was higher in the MAU group than the non-MAU group for both sexes . Median triglyceride level was higher in the MAU group than in the non-MAU group for both sexes . The proportion of overweight women was higher in the MAU group than in the non-MAU group . No differences in the lifestyle factors of smoking history, heavy alcohol consumption, and physical activity were observed between participants with MAU and without MAU of either sex.The proportion of participants with hypertension was higher in the MAU group than in the non-MAU group for both sexes . But age adjusted comparison did not alter those significant associations in the others variables.P = 0.002), which remained significant after adjustment for SBP, FBG, triglyceride level, BMI, smoking history, and heavy alcohol consumption . The relationship was not significant in women. No significant association between UACR and FEV1 was observed in either sex.An inverse relationship was observed between lung function and UACR. In an age-adjusted regression model, the regression coefficient (B) of 10% lower in predicted FVC was 11.09 in men  for MAU were 2.60 (1.87–3.62) in men and 1.55 (1.07–2.25) in women. That relationship remained significant after adjustment for age in men  but not in women. Further adjustment for hypertension, diabetes mellitus, triglyceride level, obesity, smoking history, physical activity, and heavy alcohol consumption did not attenuate that association in men . The obstructive pattern was related to MAU in the crude logistic regression model , but further adjustment for age attenuated that association in both sexes. Hypertension and diabetes mellitus were related to MAU in a multiple logistic regression model . In men, heavy alcohol consumption was related to the MAU in a multiple logistic regression model (OR [95%CI]: 1.49 [1.03–2.17]).Our current study shows that restrictive lung function pattern was associated with MAU, an early surrogate marker of kidney damage and atherosclerosis, in men. That relationship remained significant even after adjustment for age, hypertension, diabetes mellitus, triglyceride level, obesity (using body mass index), smoking history, physical activity, and heavy alcohol consumption. In a multivariate regression model, a linear relationship was revealed between the lower predicted FVC and UACR, which was not attenuated after controlling for conventional atherogenic risk factors such as age, SBP, FBG, triglyceride level, BMI, smoking history, and heavy alcohol consumption. To the best of our knowledge, this study is the first investigation in Asia to examine the relationship between poor lung function and MAU.MAU and increased UACR are regarded as early predictors of kidney damage as well as atherosclerosis The obstructive lung pattern manifests as COPD, and COPD has been reported to increase systemic inflammation and elevate the risk of atherogenic events In the current study, the restrictive pattern was strongly related to MAU as an early indicator of kidney damage and atherosclerosis. Although the pathological mechanism underlying this relationship is not clear, previous reports have shown that lower FVC is linked to atherogenic diseases Vascular damage and endothelial dysfunction are key mechanisms of both atherosclerosis and kidney damage. Poor lung function is also related to vascular damage and endothelial dysfunction P = 0.008, respectively, data not shown in any table). Additionally, the percent predicted FVC of the MAU group was 87.0% in men and 91.1% in women . Therefore, the lower proportion of women with MAU and relatively better lung function in women than in men may partially explain the lack of statistical significance in women. However, to elucidate the relationship between poor lung function and MAU in women, a large sample size including participants with poor lung function is needed.Sex differences were observed in the current study. The restrictive pattern was significantly related to MAU in men but not in women. We also observed a significant linear relationship between lower FVC and UACR in men but not in women. However, we did not find any suitable explanation for these sex differences in the literature. Among participants demonstrating the restrictive pattern , the proportion of cases with MAU was lower in women than in men (n [%]: 42 [13.3%] vs. 62 [21.5%], The current study has several limitations. Due to the cross-sectional study design, the direction of causality in our study is not clear. However, evidence such as the “spilling over” theory supports our current results. To summarize the “spilling over” theory, inhaled particles cause lung inflammation and oxidative stress. When compensations to reduce inflammation and oxidative stress are insufficient, inflammatory mediators spill over from the lung to systemic circulation, subsequently exacerbating pathological change in extrapulmonary organs and causing chronic disease such as ischemic heart disease, osteoporosis, anemia, and depression Our study, the first investigation in Asia to report a relationship between poor lung function and MAU, demonstrated that the restrictive pattern is related to MAU, an early indicator of kidney damage and atherosclerosis, in men. Thus, our current study suggests that poor lung function, particularly the restrictive pattern, is related to kidney damage as well as atherosclerosis. Furthermore, the association remained significant after controlling for conventional atherogenic risk factors such as age, hypertension, diabetes mellitus, lipid profile, obesity, smoking history, and heavy alcohol consumption.


Airway management, especially outside the operating room, needs meticulous observation in order to avoid certain risks, such as; endotracheal tube (ETT) dislocation, esophageal intubation, and unwanted extubation. ETT or tracheostomy dislocation is responsible for one-half of death or brain damage cases in the ICU. Despite appropriate fixation of an ETT, the previously mentioned complications can still occur . A broken ETT and consequent airway obstruction may lead to lethal complications.We report a case of death caused by tracheal tube aspiration, where it was located distal to the vocal cords, with a part of it entering the right bronchus and the mediastinum, after tearing the right bronchus.The vigilance and experience of medical personnel in the ICU, appropriate IV sedation, and using a bite block are the best ways to prevent mortality caused by aspiration of an ETT in all intubated patients. Airway management, especially outside the operating room, needs meticulous observation in order to avoid certain risks, such as; endotracheal tube (ETT) dislocation, esophageal intubation, and unwanted extubation.ETT or tracheostomy dislocation is responsible for one-half of death or brain damage cases in the ICU. These events may happen during; patient’s transportation, discontinuing sedative drugs infusion, abrupt wakening, coughing, or during suctioning of ETT. Despite appropriate fixation of an ETT, the previously mentioned complications can still occur . A brokeIn this report, we present a case of death caused by a broken ETT. Our patient  was a 32-year-old, with a known case of Hodgkin's lymphoma of eight years duration, prior to admission to our infectious disease department  with fever, cachexia, and dyspnea. Antibiotic treatment  was started with a diagnosis of pneumonia. After 48 hours from the admission, the patient was transferred to the ICU because of his decreased level of consciousness, and intubation was ordered.When visited by an anesthesiologist, the patient was confused, cachectic, sweating, and suffering from respiratory distress. Vital signs were recorded as follows: PR = 110 /min, RR = 40/min, axillary temperature = 38°C, and blood pressure = 90/50 mmHg. In lung auscultation, the breathing sounds were muffled in the left superior and middle lobes, and coarse crackles were heard in the lung bases.2, and saturation = 85%.Arterial blood gas analysis results were: pH = 7.18, HCO3 = 15 mmol/L, PCO2 = 23 mmHg, PO2 = 48 mmHg, BE = -8 mmol/L OConsidering the patient’s condition, cardiorespiratory monitoring and pulse oximetry were established. After suctioning the oral secretions, an intravenous injection of 150 µg fentanyl and 80 mg lidocaine 2%, plus four puffs of lidocaine 10%, were administered to anesthetize the glossopharyngeal nerve and a direct laryngoscopy was carried out. Then, the patient was intubated with a high volume low-pressure tracheal tube No 8, with an inflated cuff. After the correct position of tube was confirmed, the cuff was filled with 5cc air. 2O, pressure support = 15 cm H2O, flow trigger = 2 L/min, inspiratory time = 1.5 sec, inspiratory flow rate = 50 L/min, and FIO2 = 100%. Mechanical ventilation was established in the synchronized intermittent mandatory ventilation (SIMV) mode with pressure support parameters as follows: RR = 12/min, TV = 450 cc, PEEP = 3 cm H2 = 90-92%.Hemodynamic parameters after intubation were s follows: BP = 100/60, HR = 120/min, and SPOWe ordered an intravenous sedation with 1-2 mg midazolam and 50-100 mg fentanyl to be started if the patient's BP fell below 90 mmHg.After one hour of intubation, the ventilator began to alarm, and the ICU nurse informed us about the disappearance of a part of the tracheal tube.2 saturation to be 60%. It was impossible to open the patient’s mouth and although a jaw thrust maneuver was performed, ventilation with a mask was unsuccessful. Direct laryngoscopy did not show any traces of the aspirated part of the tracheal tube, and two consecutive attempts with Magill forceps to extract the tube were unsuccessful. The patient entered cardiorespiratory arrest; hence, reintubation of the patient was performed with tracheal tube number 7. Cardiopulmonary resuscitation was performed for 45 minutes with no success and finally, the patient expired.An anesthesiologist was dispatched immediately to the ICU and determined the disappearance of a part of tracheal tube from around number 22, with bite marks in the remaining portion. The patient was cyanotic and his hemodynamic parameters were unstable. Pulse oximetry identified the OA postmortem chest X-ray was requested to evaluate the position of the aspirated piece of tracheal tube. X-ray images showed that it was located distal to the vocal cords, with a part of it entering the right bronchus and the mediastinum, after tearing the right bronchus. The chest X-ray was not of acceptable quality, thus it was impossible to localize the exact location of the ETT. However, no further chest X-ray was performed due to a lack of consent from the patient’s relatives. A postmortem autopsy was performed and it confirmed that the ETT was located distal to the vocal cords, with a part of it entering the right bronchus and the mediastinum after tearing the right bronchus. This is the first case report of mortality and rupture of the bronchus after aspiration of an ETT. The aspirated ETT entered the right bronchus and mediastinum after tearing the right bronchus. This event caused airway obstruction and an inability to ventilate.In reviewing the reports of aspirated ETT, we found only a few case reports. In one report, a 10.5 mm ETT in a 40-year-old patient with a stab wound in the chest area was aspirated . The autIn our case, the ETT was cut at number 22, indicating aspiration of 22 cm of the tracheal tube . As we cIn most cases, the diagnosis is confirmed by chest radiography . ComputeIn one case report, authors were successful in reintubation, without extracting the aspirated tube, with a second tube, as it was a smaller size than the primary tube . Therefo


Mycobacterium tuberculosis isolates from Northwest Ethiopia, and to assess the performance of the GenoType® MTBDRplus and GenoType® MTBDRsl assays as compared to the BacT/ALERT 3D system.The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany.Two hundred sixty katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType® MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType® MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance.Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR) was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene and codon 531 of the rpoB gene, respectively. The GenoType® MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH, RMP and MDR. However, the GenoType® MTBDRsl assay shows limitations in detecting resistance to EMB.The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the  According to the World Health Organization (WHO) report, the proportion of multidrug resistant tuberculosis (MDR-TB), resistant to at least isoniazid and rifampicin among new and previously treated TB cases globally ranges from 0% to 28.3% and from 0% to 61.6%, respectively . In most3 (RMP-INH)3), respectively [M. tuberculosis usually grows slowly, the identification and drug-resistance testing usually require several weeks. The gold-standard of TB diagnosis by culture takes weeks to become positive, and even with the up-to date automated fluid culture methods it takes an average of 14 days. Another 14 days for additional testing are required to get the information on drug susceptibility [In Ethiopia, the treatment regimens for category I and category II (retreatment regimen) tuberculosis cases are 2 (RMP-INH-EMB-PZA)/4(RMP-INH) and 2 STM (RMP-INH-EMB-PZA)/1(RMP-INH-EMB-PZA)/5. These assays are DNA strip assays that use PCR and hybridization. Mutations in katG gene and inhA gene were related to the high-level and low-level INH resistance, respectively [rpoB gene, coding RNA polymerase subunit ß and mutation in the embB gene was associated with EMB resistance [Molecular methods that have been developed to detect drug resistance include the GenoTypeectively . Nearly sistance ,15.M. tuberculosis are not performed routinely in clinical microbiology laboratories. Laboratory diagnosis of TB remains in a stage of acid-fast staining. Currently, five regional laboratories are being rebuilt and equipped to perform culture and drug susceptibility testing using methods including GenoType® MTBDRplus assay. The GenoType® MTBDRplus and GenoType® MTBDRsl assays have been studied in several laboratories of other countries, however, there is a wide variation in circulating M. tuberculosis strains worldwide [M. tuberculosis isolates from Ethiopia. In this study, we analyzed the frequency of gene mutations associated with resistance to INH, RMP and EMB among M. tuberculosis isolates from Northwest Ethiopia, and assessed the performance of the GenoType® MTBDRplus for detection of resistance to INH, RMP and MDR and GenoType® MTBDRsl assay for detection of EMB resistance compared to the automated, culture-based, BacT/ALERT 3D system drug susceptibility testing.In Ethiopia, culture and drug susceptibility testing (DST) for orldwide ,17, and orldwide -24, affeM. tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 at Gondar Hospital, Gondar Health Center, Metemma Hospital, Bahir Dar Hospital and Debre Markos Hospital in Northwest Ethiopia were included in this study. Diagnosis of smear-positive tuberculosis was based on the national guideline for microscopic examination of tuberculosis (6): direct smears were prepared from three sputum specimens and stained by Ziehl-Neelsen staining technique for microscopic examination. Drug susceptibility tests using GenoType® MTBDRplus, GenoType® MTBDRsl and BacT/ALERT 3D system were performed at the mycobacteriology laboratory in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany. Informed consent was obtained from the study subjects. Institutional ethical clearance was obtained from the research and publication committee of Gondar University, Ethiopia. Details of sputum storage, transportation, isolation and identification of the isolates have described previously [Two hundred sixty eviously .® MTBDRplus assay for detection of INH and RMP resistance, and GenoType® MTBDRsl assay for detection of ethambutol resistance were performed according to the manufacturer's instructions . Briefly, DNA was extracted from cultures by heating the bacteria in a heating block for 20 minutes at 95°C followed by sonication in ultrasonic water bath for 15 minutes. Amplification was performed using 2.5 μl (1 unit) Taq DNA polymerase . For the amplification profile the instructions of the manufacturer were followed. Hybridization of the single-stranded, biotin-labeled amplicons to membrane-bound probes on the strip followed by addition of conjugate, and substrate to detect visible band patterns on the strip was performed manually using a shaking water bath, Memmert-SV1422  at 45°C. Then strips were allowed to dry and interpreted according to the manufacturer's recommendation.GenoType® MTBDRplus has incorporated one wild type (WT-315) and two mutation-type probes specific for mutation Ser315Thr1 and Ser315Thr2 of the katG gene. For detection of low-level INH resistance this assay has two wild-type probes (WT-15/-16 and WT-8) and four mutation-type probes, covering mutations of C15T, A16G, T8C and T8A in the inhA gene. To detect rifampicin resistance, the Genotype MTBDRplus has incorporated eight wild-type probes for the rpoB gene, covering codons in the rpoB gene from 505 to 533, and four other probes specific for mutations Asp516Val, His526Tyr, His526Asp and Ser531Leu. For detection of ethambutol resistance, the GenoType® MTBDRsl employs one wild-type probe (WT-306) and two mutation probes specific for mutations Met306Ile and Met306Val in the embB gene.To detect high level INH resistance, the GenoTypeM. tuberculosis isolate was determined to be resistant to an antibiotic when the drug-containing bottle had a time to detection (TTD) that was less than or equal to the TTD of the 1% control.Drug susceptibility testing for isoniazid, rifampicin and ethambutol was performed by BacT/ALERT 3D system  according to the methods published previously ,27. The χ2) were used to assess statistical relationships between predisposing factors and drug-resistant TB. Sensitivity, specificity, positive predictive value and negative predictive value of the molecular methods were analyzed using crosstabulation after arranging the results of the molecular methods in the rows and gold standard, BacT/ALERT 3D system in columns. P values of less than 0.05 were considered statistically significant.All laboratory data were entered, cleared and analyzed using SPSS version 13 statistical package software . The standard chi-square tests (P = 0.001), RMP (P = 0.002) and MDR (P = 0.044). HIV co-infection, gender and age of the study subjects had no significant association with gene mutations conferring resistance to INH, RMP and EMB. A summary of patient demographic characteristics and associated drug susceptibility pattern according to the molecular methods is shown in Table Of the 260 patients included in this study, the majority of patients, 59% were males. The median age of the study subjects was 28.0 years . History of previous treatment for tuberculosis was significantly associated with gene mutations conferring resistance to INH P = 0.00, RMP  gene with amino acid change of Ser315Thr1, indicating high level resistance, while 6% of the strains had mutation in the inhA gene, C15T, indicating low level resistance. All katG gene mutations detected at wild type probes were also present at mutant probes, as was the case with the inhA gene mutations  gene with amino acid change of Met306Ile and one strain had mutation in the embB gene with amino acid change of Met306Val, whereas five strains had mutation that detected only at the wild type probes (embB WT) but not at the mutant probes . Suggesting that the set of the DNA probes used in the GenoType® MTBDRplus assay covers most of the mutations prevalent in Northwest Ethiopia. However, previously reported associations between the gene mutations and Beijing strains [In this study, the GenoTypee et al.  reportede et al.  showed t strains ,31 suggekatG gene mutations. Of which, 75-90% are recognized as mutations in the 315th codon of the katG gene, which mainly result in Ser315Thr1 and Ser315Thr2 mutation [katG mutations which confer high level resistance to INH. Of which, 100% were identified as Ser315Thr1 mutation. Studies have also shown that 8% to 43% of INH resistance are defined as the low-level drug resistance mainly caused by the mutations in the promoter region of inhA gene [Previous studies have shown that 40-95% of isoniazid resistance are defined as the high-level drug-resistance due to the mutation ,15,32. InhA gene . In thisrpoB gene mutations which are found to cluster mainly in the region of codon 507-533. In this study, the distribution of gene mutation among RMP resistant isolates was 73% at position Ser531Leu and 7% at His526Asp. In 20% of the resistant isolates, mutation was detected only at the wild type probes, which is different from the previously reported gene mutation distribution in China, 37% at Ser531Leu, 3% at His526Asp and in 60% of the isolates, mutation was detected only at the wild type probes [rpoB gene that can not be detected by the current version of the GenoType® MTBDRplus assay.In the previous studies ,15,34, ae probes , reflect® MTBDRsl assay was 25% at Met306Ile, 13% at Met306Val and 63% of the strains had mutation only at the wild type probes. Furthermore, 58% of the isolates showing resistance to ethambutol by the BacT/ALERT 3D system did not display a similar result by this molecular assay even on repeat assays. Consequently, in the present study, GenoType® MTBDRsl assay had sensitivity of 42% and specificity of 100% for ethambutol resistance. Similarly, other previous studies have shown that this assay has low sensitivity, only about 50% for detection of EMB resistance [M. tuberculosis is still insufficiently understood to allow detection of EMB resistance by molecular methods.In this study, the distribution of gene mutation among 8 isolates showing resistance to EMB by GenoTypesistance -38. The katG gene and codon 531 of the rpoB gene was observed. The GenoType® MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH, RMP and MDR. The short turnaround times and the potential for rapid screening of large numbers of isolates make it suitable as a first-line screening assay for TB drug resistance. Its application and popularization will help better solve the long-standing problem of laboratory diagnosis of drug resistance in Ethiopia. In the majority of phenotypically ethambutol resistant isolates, gene mutation associated with the resistance to ethambutol was not detected by this assay. This indicates that the present version of the GenoType® MTBDRsl assay shows limitations in detecting resistance to ethambutol. Further studies are required to understand the mechanism of resistance to ethambutol and to evaluate GenoType® MTBDRplus assay for the diagnosis of INH and RMP resistance from direct sputum specimens of tuberculosis patients in Ethiopia.In our study, the dominance of single gene mutations associated with the resistance to isoniazid and rifampicin in the codon 315 of the The authors declare that they have no competing interests.BT was the primary researcher, conceived the study, designed, participated in sample collection, performed laboratory experiments, conducted data analysis and drafted the manuscript for publication. JB participated in doing the laboratory experiments, interpreting the results, and reviewed the initial and final drafts of the manuscript. FE, US and AR reviewed the initial and final drafts of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/37/prepub


To examine the role of perceived stressfulness of trauma exposure and economic, social, occupational, educational, and familial adaptation after trauma in posttraumatic stress disorder (PTSD) and depression in displaced war survivors.A cross-sectional survey was conducted between March 2000 and July 2002 with a sample of 173 internally displaced persons or refugees and 167 matched controls in Croatia. Clinical measures included Structured Clinical Interview for DSM-IV and Clinician-Administered PTSD Scale.Displaced war survivors reported the exposure to a mean ± standard deviation of 13.1 ± 8.3 war stressors, including combat, torture, serious injury, death of close persons, and loss of property. Compared to controls, they reported higher rates of marked to severe impact of war on family (16.2% vs 51.6%), social (7.2% vs 43.5%), economic (12.6% vs 55.4%), occupational (1.8% vs 15.9%), and educational (2.4% vs 8.8%) adaptation. In two logistic regression analyses, the strongest predictor of PTSD and depression was high level of perceived distress during trauma exposure. PTSD but not depression was associated with economic, social, occupational, educational, and familial adaptation after trauma.Displaced survivors who experienced multiple war events perceived greater negative impact of war on their life domains compared to individuals who lived in a war setting but had no trauma exposure. The most important determinant of psychological outcomes was perceived stressfulness of war stressors. Although post-trauma adaptation in different life spheres had an impact, its effect was not robust and consistent across disorders. These findings suggest that it would be effective to use a trauma-focused approach in rehabilitation of war survivors. Armed conflicts, wars, and associated displacement affect large numbers of people in the world . AccordiThe most common mental health outcome of exposure to war-related traumatic stressors is posttraumatic stress disorder (PTSD) and depression . In a meSome studies identified older age ,21, femaPolitical violence and terror during the 1991-1995 war in Croatia and the 1992-1995 war in Bosnia and Herzegovina were categorized as the highest level on the Political Terror Scale . Four yeOur research was part of a large survey conducted between March 2000 and July 2002. Details of the study methodology were presented in the main report . BrieflyPsychiatric outcome was assessed with the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV)  . AssessmThe interviews were conducted by 5 trained professionals in Rijeka, Croatia. The interviews were evaluated for validity of diagnoses, rapport, adherence to the assessment protocol for each instrument, and overall quality of assessment. The concordance rates  were 0.81 for PTSD, 0.84 for depression, and ranged from 0.83 to 0.99 for SISOW items in the overall sample . Sample characteristics were examined with frequency and descriptive statistics. Displaced war survivors were compared to matched controls on study variables of interest to produce evidence in support of the validity of the economic, social, family, occupational, and educational adaptation after trauma using McNemar test for related samples and paired or a mix . As in mThe socio-demographic characteristics of the sample are given as web-extra material. There was a balanced distribution of male and female participants in the sample. The majority of the displaced war survivors were Croats  and Catholics . The mean ± standard deviation time since the most distressing traumatic event was 9.0 ± 1.6 years. Participants were exposed to a mean of 13.1 ± 8.3 war-related stressors. Some of these stressors included combat experience , prisoner of war experience , torture , serious injury , death of close persons , exposure to aerial bombardment , and sudden loss of property . About 70% of the participants reported severe/extremely severe distress, while 94% reported slight to extreme loss of control during exposure to such traumatic stressors. Sixty-one participants (35.5%) had lifetime PTSD, 39 (22.5%) had current PTSD, 22 (12.7%) had current depression, 20 (11.6%) had past depression, 16 (9.2%) had alcohol abuse, and 10 (5.8%) had dysthymic disorder. Rates of the other psychiatric diagnoses were lower than 3%.P < 0.001) and depression . Among displaced war survivors, 51.6%, 43.5%, and 55.4% reported marked to severe negative impact on their family, social, and economic adaptation, respectively (P’s significant at 0.001 level). The latter findings supported the validity of the scale.The matching procedure resulted in groups similar in sex, age, education, and marital status . There wectively . A compa2 = 72.0, df = 1, 11, P < 0.001) and explained 52.0% of the variance (Nagelkerke R2). PTSD significantly related to old age, male sex, high levels of perceived distress during exposure to traumatic stressors, and negative cumulative impact of trauma on life spheres. When the logistic regression analysis was repeated with current major depressive episode as the dependent variable  and explained 32.2% of the variance (Nagelkerke R2). Only high levels of distress during exposure to war stressors related to current depression. Interestingly, there was no significant association between the impact of trauma on economic, social, family, occupational, and educational adaptation and depression.Two logistic regression analyses (method enter) were conducted using the diagnoses of current PTSD and current depression as the dependent variables. The ratio of cases to independent variables was 15.6 to 1, which was satisfactory. In the analysis where current PTSD was used as the dependent variable , the ovevariable , the oveComparisons between displaced war survivors and control participants matched on age, sex, and education revealed significant differences in terms of impact of war stressors on family, occupational, social, economic, and educational adaptation. This finding supported our first hypothesis that refugees and internally displaced persons from war settings endure socioeconomic disadvantage, problems in family functioning, lack of access to educational and occupational opportunities, loss of social support, and perhaps marginalization and isolation. It needs to be noted, however, that it is not possible to attribute this negative impact on life domains to displacement only, because our study participants had also been exposed to multiple war-related traumatic events, which may have had independent effects on adaptation.The strongest predictor of PTSD and depression in internally displaced persons and refugees was the level of perceived distress during exposure to war-related stressors This finding supported our second study hypothesis and is consistent with previous studies which showed that perceived stressfulness of the stressors, rather than mere exposure to them, determines traumatic stress in war and torture survivors ,19,32,33The significant association between distress during trauma and major depressive episode is consistent with previous similar association in war , tortureGlobal impact of trauma on adaptation in different life domains showed independent but rather weak association with PTSD and it was not associated with depression, a finding that provided only partial support for our third study hypothesis. The mechanisms by which such secondary effects of trauma have an influence on PTSD symptoms are not entirely clear. On the one hand, psychological trauma could be conceptualized as involving exposure to primary traumatic events which then lead to secondary stressors, all contributing to an ongoing traumatization process ,15,40-42The findings need to be interpreted in the light of some methodological limitations. Although the sample size afforded sufficient power for the analyses it was still small and findings from such small samples may have limited generalizability. However, the fact that the predictors were consistent with previous research supports the validity of the findings. Furthermore, although measures were taken to minimize sampling bias, non-random selection of study participants may have resulted in a sample with limited representativeness of displaced war survivors in Croatia. Finally, the retrospective design of the study, which involved recall of events and associated reactions assessed on average nine years post-trauma may have affected the internal validity.In conclusion, our findings suggest that psychological processes during exposure to war stressors are the most important determinants of PTSD and depression in displaced war survivors. Although adaptation in different life spheres after trauma exposure may have an impact on mental health, its effect is not robust and consistent across disorders. These findings imply that a non-trauma-focused approach in rehabilitation of displaced war survivors is not justified. Trauma-focused cognitive-behavioral treatments and exposure-based interventions have been shown to be effective in a wide range of trauma survivors . Given t