Objectives. In current study, the relationships between serum vitamin D3 levels and serum UA concentrations as well as their interaction with severity of PD were evaluated in a sample of Iranian PD patients. Method. In a cross sectional study at the one of the main referral hospitals in central region of Iran, during September to November 2011, 112 patients were recruited. Severity of PD was evaluated sing H&R stages and UPDRS. Results. The Spearman rank correlation coefficient suggests the negative significant association between serum vitamin D3 and UPDRS in patients aged >62 . No statistically significant association was observed between the UA levels and severity of PD (represented by H&Y categories) in different levels of serum vitamin D3 not only in total sample but also in separate age and sex groups. The linear regression coefficients suggested positive association between UA and serum vitamin D3 with UPDRSIII scores while negative relationship between UA and serum vitamin D3 interaction with UPDRSIII; however it was only statistically significant in age group ≤62 (P < 0.05). Conclusion. Our study revealed a negative correlation between interaction of serum vitamin D3 and UA with severity of PD; other studies are required to confirm our findings. Parkinson disease (PD) is a common neurodegenerative disorder, characterized by motor dysfunctions including rest tremor, rigidity, and bradykinesia , 2 whichPD was considered to be largely unknown and its pathogenic mechanisms remained uncertain. However, several genetic factors have been reported in the last decade , 5. AlthAccumulating evidence confirms that oxidative stress contributes to the loss of dopaminergic neurons in the substantia nigra of PD patients , 19. UriThe relationships between UA levels as well as serum vitamin D3 with PD have been demonstrated separately; however, to our knowledge a possible link between these factors and particularly their interaction with PD severity has not been explored yet. Therefore, in the current study, the relationships between serum vitamin D3 levels and serum UA concentrations as well as their interaction with severity of PD were evaluated in a sample of Iranian PD patients.Our cross-sectional, observational study was established from September to November 2011 at the Al-Zahra Hospital , one of the main referral hospitals in central region of Iran. One hundred and twelve patients diagnosed with clinically definite PD according to the clinical criteria of the UKPD Society Brain Bank  were recA diagnosis of PD was made clinically and disease severity was judged by experienced clinical neurologist. Two major clinical scales were used: the Unified Parkinson's Disease Rating Scale (UPDRS) and Hoehn and Yahr Scale (H&Y) , 30. DatInformed written consent was obtained from each study participant and research ethic committee of Isfahan University of Medical Sciences reviewed and approved the study protocol.For each participant, fasting blood samples were obtained on the day that H&Y stage and UPDRS were evaluated. Serum was immediately separated and stored at −20C. 25-Hydroxyvitamin D levels were measured by enzyme immunoassay   and seruIn order for analyses' proposes, we considered the following categories: uric acid level in two categories (the first category as ≤5.4 (lower than median) and the second category as >5.4 (higher than median)), age (≤62 (lower than median) and >62 (higher than median)), and H&Y .t-test and Mann-Whitney U test were used for comparing the continuous variables with normal and nonnormal distribution between considered groups, respectively. Chi-square test was used for evaluating the association between categorical variables. Spearman rank correlation coefficient was used for assessing the bivariate association between continuous variables. Linear and logistic regression models were fitted in different models for studying the association between UPDRS scores and H&Y stages as dependent variables, respectively, and UA and serum vitamin D3 levels as well as their interaction as independent variables. In crude model, the associations between two main independent variables as well as their interaction and dependent variables were evaluated, while in other models adjustments were made for different confounding variables including age, sex, and disease duration. All mentioned models were fitted in all samples as well as in age and sex groups separately. All statistical analyses were conducted using SPSS software version 15 .The continuous data were presented as mean ± SD and while categorical variables as frequencies (percentage). Normality of continuous data was evaluated using Kolmogorov-Smirnov test and P-P plot. Nonnormal data were subjected to logarithmic transformation. Independent samples One hundred and twelve patients agreed to participate in our study. Mean ± SD age and disease duration were 62 ± 11.95 years and 48 ± 49.65 months, respectively. Majority of participants were male (75 (66.9%)) and the remaining were female (37 (33.1%)). More than one-third of the patients (38.4%) showed deficiency levels (<20 ng/mL) and a majority of subjects (72.8%) had insufficient levels of serum vitamin D3 (<30 ng/mL). Mean ± SD of severity values of PD, measured by UPRDSIII and H&Y stages, were 18 ± 18.21 and 2 ± 0.90, respectively. The complete feature of bivariate association based on the Spearman rank correlation coefficient, between duration of the disease, serum UA, vitamin D3, and severity of the disease in total sample, as well as in different age and sex groups, has been presented in P < 0.05). It should be noted that when adjustment was made for sex and disease duration (Models 2 and 3), we did not find any significant association.In this cross-sectional correlational study, the associations of serum vitamin D3, UA levels, and their interaction with severity of PD in a sample of Iranian patients were investigated. Positive associations were observed between UA and serum vitamin D3 with severity of disease when measured by UPDRS and H&Y in total study's sample and in different age and gender groups based on simple correlation analysis; however, negative significant correlation was observed between serum vitamin D3 and UPDRS in patients aged above 62 years. We did not find any significant association between serum vitamin D3, serum UA concentrations, and their interaction with UPDRS and H&Y based on multiple linear and logistic regression analysis when adjustments were made for potential confounding factors such as gender, age, and disease duration.Continuously inadequate vitamin D status plays a major role in PD progression. In a longitudinal study in Finland, risk of PD obviously reduced with higher serum vitamin D level  and the On the other hand, although there are well-known evidences on the association of higher serum UA levels with a significantly reduced risk of PD, along with evidence for a dose-effect relationship, implying that reduced UA concentrations may have a causal role in PD , 22, 33,This inconsistency between our results and other previous studies could be explained by disproportion of the H&Y scores among our study samples. Only four patients with severe scores are included and the mean H&Y stage in current research was 1.8, which is lower compared with other studies.U-shaped effect, so that low UA level is accompanied by a higher PD risk, but, at higher concentrations, no further effect is observed. It could be seen that UA acts with dual antioxidant properties and it may also designate a prooxidative state under certain conditions. Regulation of this switch could be determined by the microenvironment in different compartment of human organism [In addition, it is known that lifestyle factors such as dietary habits and physical activity can affect serum UA . Hence, organism .α-hydroxylase and lead to decreasing vitamin D concentration in PD [More recently, a growing body of literature has focused on considerable influence of parathyroid hormone on UA. Vitamin D deficiency or insufficiency leads to releasing of parathyroid hormone . In the on in PD .Considering this point that UA was influenced by different conditions in body compartment, in the present study, we evaluated UA and serum vitamin D3 as important factors in PD and looked to the body as a whole. To the best of our knowledge, this is the first study on investigating the joint association of vitamin D and UA on disease progression in Iranian population. The observed controversial correlations in previous studies encouraged us to also investigate the interaction effect of serum vitamin D3 and UA on severity of PD. Although our study revealed a nonsignificant negative correlation between vitamin D3 and UA interaction with severity of PD, such findings need to be more investigated in larger samples of PD patients.The present study has some potential limitations. Serum UA and vitamin D3 levels are variable and were measured just once during the study. Also, our sample was small in a cross-sectional observational study's framework; hence our findings should be interpreted cautiously. Unavailability of data on dietary intake of vitamin D and protein as well as important factors such as body mass index (BMI), medications, and genetics can be considered as another important limitation.


The objectives of this study were to summarize antiretroviral drug concentrations in breast milk (BM) and exposure of breast-fed infants.I2 statistic. PubMed Central, SCOPUS and LactMed databases were searched. No date or language restrictions were applied. Searches were conducted up to 10 November 2014. Clinical relevance was estimated by comparing ingested dose with the recommended therapeutic dose for each drug.This was a systematic review of pharmacokinetic studies of HIV-positive women taking antiretrovirals that measured drugs in BM. The quality of pharmacokinetic and laboratory methods was assessed using pre-defined criteria. Pooled ratios and 95% CIs were calculated using the generalized inverse variance method and heterogeneity was estimated by the Twenty-four studies were included. There was substantial variability in the clinical and laboratory methods used and in reported results. Relative to maternal plasma (MP), NRTIs accumulate in BM, with BM : MP ratios (95% CI estimates) from 0.89 to 1.21 . NNRTI estimates were from 0.71 to 0.94  and PI estimates were from 0.17 to 0.21 . Relative to the recommended paediatric doses, a breast-fed infant may ingest 8.4% (95% CI 1.9–15.0), 12.5% (95% CI 2.6–22.3) and 1.1% (95% CI 0–3.6) of lamivudine, nevirapine and efavirenz, respectively, via BM.Transfer to untreated infants appears quantitatively important for some NRTIs and NNRTIs. The pharmacokinetic methods varied widely and we propose standards for the design, analysis and reporting of future pharmacokinetic studies of drug transfer during breastfeeding. Pharmacokinetic (PK) knowledge of ARV transfer to BM and breast-fed infants is essential to understand the safety of prolonged exposure through breastfeeding and limit the development of drug resistance.In contrast to well-resourced settings,The objectives of this study were to summarize from existing studies of breastfeeding mothers taking ARVs: (i) ARV concentrations in BM; and (ii) ARV transfer to breast-fed infants.JAC Onlineavailable as Supplementary data at ), following PRISMA guidelines.9We wrote the protocol for this review before starting the analysis  administered to the mother and whether or not infant ARVs were administered were recorded.Data were extracted onto an ExcelFor clinical sampling, we assessed: (i) the PK sampling strategy in terms of number of BM samples obtained from each mother, timing of sampling relative to birth and to maternal dosing, or whether this was unclear; (ii) if the methods used to obtain BM were described sufficiently  for another investigator to be able to replicate the process; and (iii) whether the samples of maternal plasma (MP), infant plasma (IP) and BM were obtained at the same time, a different time or if this was unclear.We assessed the quality of the laboratory methods using seven criteria: (i) if the laboratory method was described in sufficient detail for another investigator to be able to repeat the procedure; (ii) whether the authors validated or referred to assay validation for the BM matrix (yes/no); (iii) if drug was measured in whole BM, if the lipid fraction was skimmed off or if both of these methods were used; (iv) the method of drug extraction; (v) the choice of detection method used; (vi) if the assay sensitivity was reported (yes/no); and (vii) if detail regarding the internal standard was provided (yes/no).10 where this was not specifically stated, it was calculated from median MP and BM concentrations where these were provided. BM : MP and IP : BM ratios were only determined when MP, BM and IP concentrations were detectable (above the assay limit of quantification). Hence not all studies provided sufficient data for inclusion in the statistical analysis or presentation in the figures.Medians and IQRs for ARV concentrations in human BM, MP and IP were extracted. The ratio of ARV in BM to MP (BM : MP ratio) is accepted as the key index of BM transfer of drug;11The individual studies identified in this review provided a median and IQR or single point estimates for drug concentrations and BM : MP ratios. Therefore, to provide a pooled estimate for the BM : MP and IP : BM ratios, it was necessary to assume that the ratios were normally distributed and that the median is approximately equal to the mean. Additionally, it is assumed that the IQR is four-thirds of the standard deviation, based on the fact that the standard normal has its 25th and 75th percentiles approximately two-thirds of a standard deviation away from zero.Having calculated an assumed mean and standard deviation for all relevant studies, pooled estimates of each outcome were obtained via the generalized inverse variance method. Studies without a spread measure were removed from the pooling exercise.12 that would be ingested by a fully breast-fed infant. If both treatment and prophylactic doses were available, we used the PMTCT dose reflecting that which a neonate might typically be prescribed. Whilst not included in WHO guidelines, the FDA has approved efavirenz use in infants aged >3 months and weighing >3.5 kg under exceptional circumstances. Genotyping for CYP2B6 metabolizer status is strongly recommended with appropriate dose adjustment, but if not available a dose of 100 mg would be used for a 3.5 kg infant;13 this was used in our calculations. The standard assumption of 150 mL/kg/day milk intake was made. As studies did not summarize infant weights, simulations were made for infants weighing 2, 3, 4 and 5 kg. CIs for the percentages were calculated using standard methodology and once again pooled estimates were obtained using the generalized inverse variance method with zero percentages being excluded from the pooling. CIs were capped at 0 and 100 as true percentages are being considered, not changes in percentages.To estimate clinical relevance, BM concentrations were interpreted as the percentage of the recommended infant doseI2 statistic. It was not possible to calculate an I2 statistic where there is only a single study or where only one of several studies for a drug has a measure of spread.Heterogeneity was estimated via the Twenty-four studies (19 full text and 5 conference proceedings) met the inclusion criteria and are summarized in Table The individual ARVs analysed in breastfeeding mothers were nevirapine (16 studies), zidovudine (12 studies), lamivudine (12 studies), stavudine (4 studies), lopinavir (4 studies), ritonavir (4 studies), nelfinavir (4 studies), tenofovir (2 studies), efavirenz (2 studies), atazanavir (1 study), indinavir (1 study), abacavir (1 study) and etravirine (1 study).Eight studies reported IP concentrations where the infants were not given the same drug as the mother. A further seven also reported IP concentrations where infants and mothers were receiving the same drug; here, MP and BM concentrations were retained and IP levels excluded, as drug exposures in the infant would reflect both orally administered drug and BM transfer, making it impossible to determine the contribution from the mother via BM. In two studies, infant blood sampling was performed at intervals over a period of several months; in this case, we included in the analysis only timepoints where the directly administered infant drug would have no longer been detectable based on the known ARV elimination half-life.Tables S1 and S2.The PK study design, types of matrices analysed  and the laboratory methods are described in 15 and two further studies performed truncated rich PK analysis from 0 to 6 h post-dose.17 In seven studies, the PK design was not clear. Fifteen studies sampled all three matrices , whereas 9 did not include infant concentrations. Twenty studies sampled the different matrices contemporaneously and 3 did not; in the final study, this was not clear (Table S1).For BM PK, 13 studies utilized a sparse PK sampling strategy , with sampling either within the first 6 weeks of life (6 studies) or at intervals up to 6 months of age (7 studies). Two studies undertook a rich PK profile on BM, sampling at 0, 2, 5, 8 and 24 h relative to dosing,Table S1). Regarding the fraction of milk analysed, three studies reported the measurement of drug in BM with the lipid layer skimmed off, six reported measurement in whole BM (one of which specified that this was homogenized prior to extraction)18 and three studies considered both whole and skimmed BM. In the remaining 12 studies, the milk fraction was not specified (Table S2).Nine studies provided detail on the clinical process of BM sampling .We evaluated whether matrix-specific assay validation for BM was described, in accordance with FDA bioanalysis guidelines.The ARV concentrations in MP and BM and the corresponding BM : MP ratios for each class of drug are summarized in Figures Seventeen studies reported on NNRTI levels in MP and BM, of which 14 measured nevirapine, 2 measured efavirenz and 1 measured etravirine.I2 = 95%), studies evaluating nevirapine transfer into BM consistently showed a BM : MP ratio of <1, with an overall pooled estimate of 0.73 (95% CI 0.71–0.76). Furthermore, three studies reported nevirapine levels in MP and BM at several timepoints post-delivery, demonstrating the BM : MP ratios to be relatively constant from 6 to 24 weeks post-partum, although numbers sampled at each timepoint were small.24 Eleven reporting IP nevirapine concentrations were confounded by administration of nevirapine to the infant. However, both Mirochnick et al.25 and Palombi et al.23 performed longitudinal analyses on MP, BM and IP; given the half-life of 30 h for nevirapine in infants from a similar population,26 by 3 months we presumed that detectable infant levels reflected BM transfer of drug. Both studies reported similar IP concentrations of ∼12% of MP at 14 weeks and <5% by 24 weeks.25Despite considerable heterogeneity . The remaining three studies45 found that lamivudine BM : MP ratios were <1. The overall pooled estimate was 0.93 (95% CI 0.89–0.98). The majority of studies25 demonstrated the BM : MP ratios were constant from delivery throughout post-partum. However, in studies that performed intensive PK sampling,16 there was evidence of differential PK within the MP and BM compartments; a slower elimination rate of lamivudine was observed in BM than in MP, which resulted in a gradual increase in the BM : MP ratio across the dosing interval. Breastfeeding infants also had measurable drug, with IP concentrations between 2% and 6% of MP.30 Infant lamivudine exposures and corresponding IP : BM ratios declined over the course of the post-partum period, in spite of a constant delivery of drug through BM, which potentially reflects maturation (or ontogeny) of the infant's metabolic clearance system. Two studies evaluated MP and BM levels of zidovudine and lamivudine at intervals up to 24 weeks post-partum,23 finding the BM : MP ratios of these drugs to remain constant over this time.Seven of 10 studies showed lamivudine accumulation in the breast, with a median BM : MP ratio of >1, although the variability both within and between studies was notable . Infant levels of zidovudine were determined in three studies.25 Zidovudine was measurable at the time of delivery , but was largely undetectable in IP after the neonatal period had passed.Regarding zidovudine, three studies reported lower concentrations in BM compared with MP,32 with a pooled estimate of 1.21 (1.07–1.36), which is significantly >1; however, infant concentrations approached the lower limit of quantification with a median value of zero.32 Two studies have measured tenofovir in BM. In one, tenofovir was measurable in the BM of only 4 out of 25 women sampled and neither study reported corresponding MP or IP concentrations.34 A single study measured emtricitabine in BM, but not MP. Abacavir had a reported BM:MP ratio of 0.85 in a single study, but BM, MP and IP concentrations were not reported.45Three studies report stavudine BM : MP ratios of >118 Of the five studies analysing infant concentrations of PIs, four reported levels below the limit of quantification. One study from Malawi was able to detect lopinavir and ritonavir in the plasma of breast-fed infants, at ∼2% of MP levels.23Eight studies reported on PI levels in BM. One study measured atazanavir, one indinavir, four lopinavir used in combination with low-dose ritonavir and four nelfinavir. In all cases, the low-dose ritonavir was used as a boosting agent for lopinavir. Overall penetration of these PIs into BM was low (<40%) relative to MP. Only indinavir was found to accumulate in BM and this study must be interpreted with caution since the samples were drawn from a single mother on five consecutive days.2 understanding of the PK of transfer between mother and infant remains incomplete. Target concentrations have not been defined for any ARV in BM and many studies utilized an in vitro IC50 or IC95 with correction for protein binding. These approaches are not standardized and do not incorporate the active intracellular metabolites of NRTIs.36 Thus, we chose not to incorporate comparison against IC50 in the formal undertaking of this systematic review.Although increasing numbers of countries have adopted WHO option B+, meaning that all pregnant and breastfeeding women will receive lifelong ART,30 (exposure ∼5% of IC5037), more recent analysis of infants who acquired HIV through breastfeeding in the Kisumu Breastfeeding Study (KiBS)  found more than three-quarters to have resistance to both nevirapine and lamivudine.7 Further, amongst mothers who initiated triple therapy post-partum on the basis of clinical need in the PEPI-Malawi trial, almost one-third of HIV-positive infants were found to have multiclass drug resistance.8 The accumulation of zidovudine in BM was also highly variable between studies, but in most cases infant levels were undetectable, possibly attributed to the drug's rapid elimination half-life (∼1 h). Tenofovir and emtricitabine are measurable in BM, although the relationships with maternal and IP concentrations remain poorly defined; furthermore, tenofovir is not recommended for use in children aged <2 years. These findings have clinical and programmatic significance. Zidovudine and lamivudine remain components of first-line PMTCT and ARV regimens in low-resource settings and tenofovir and emtricitabine are increasingly used with implementation of WHO 2013 guidelines.38The NRTIs have higher and more variable BM penetration than the NNRTIs or PIs. Lamivudine has the highest accumulation in the BM and reaches detectable levels in the infant. Initially assumed to have unlikely clinical significance38 only two studies assessed efavirenz concentrations in BM. These showed infant levels approach the minimum levels considered necessary for effective treatment in adults, reaching 13%27 and 8%39 of MP concentrations. No data about effective efavirenz levels necessary for protection of neonates by PMTCT are published. In part, this is a consequence of efavirenz having been contraindicated in pregnancy until 2012 due to teratogenicity concerns, which have recently been refuted by meta-analysis of exposed pregnancies;40 additionally, the drug is not recommended for use in children aged <3 years or weighing <10 kg except under exceptional circumstances and ideally with pharmacogenomic testing.13 Even with the extension to FDA licensing for young infants, current guidelines state it should not be used in infants aged <3 months. The studies by Schneider et al.27 and Olagunju et al.28 indicate that the ‘dose’ received by the breast-fed infant may reach 3.5% of the paediatric dose. The consequences of this in the neonatal period and the influence of maternal and infant pharmacogenomics on the concentrations reached warrant further evaluation.Despite being a first-line ARV in PMTCT,24 differential accumulation of agents used within a triple-therapy regimen risks exposure to monotherapy within the compartment of the breast, potentially selecting resistant HIV strains. Furthermore, transfer of low levels of individual drugs to a breast-fed HIV-positive infant is associated with high rates of drug resistance as demonstrated in secondary analyses of both KiBS7 and PEPI-Malawi.8 Future studies should consider not only the levels of each individual drug, but also the optimal combination to be used to maximize benefits and reduce risks.The clinical relevance of ARV concentrations in BM is not fully understood. Whilst high levels have been correlated with reductions in the BM viral load, reducing the amount of virus to which the infant is exposed,The wide variability within and between studies may result from differences in sampling time relative to dose, differences in drug concentration assays and the statistical method used to report concentrations below the lower limit of quantification, in addition to biological differences between populations. However, it is noted that variability was particularly marked for the NRTIs. Intensive PK data suggest there is a possible lag in the elimination of lamivudine (and to a lesser extent zidovudine) from BM, as shown by a gradual increase in the BM : MP ratios over the course of the dosing interval. This may, in part, explain the extensive variation in NRTI BM : MP ratios across studies, as sparse samples are taken at different times relative to dosing.41 which explains lower levels of PIs penetrating BM compared with the other classes of drug (∼30% protein binding for zidovudine and lamivudine compared with >90% for the PIs).42 Maternal covariates influencing the variability in BM elimination of ARVs are poorly defined, but may include clinical states affecting circulating plasma proteins, such as intercurrent illness and poor nutrition.The accumulation of PIs in BM is low. Protein binding is the strongest predictor of drug transfer in BM,43 is an alternative approach).There are a number of limitations to the analysis. A normal distribution is assumed when converting medians and IQRs to means and standard deviations and comparison of pooled estimates must be made with caution. Significant heterogeneity was observed with CIs that frequently do not overlap Figures –5, necesWe have presented a pooled estimate for every drug, which has an associated measure of spread. However, in some cases this means we are presenting a pooled estimate for a single study, which some may consider as not being meaningful. This is related to the limited data available.The analysis of infant drug concentrations as a percentage of the recommended paediatric dose Figure  relied o44 published clinical PK studies of ARVs in breastfeeding mother/infant pairs rarely report detail. Descriptions of sample collection, fraction of milk analysed, extraction method, type of internal standard, stability, matrix effects, recovery, accuracy and precision are frequently lacking.Although validated methods are essential for accurate and reliable drug concentration data,et al.32 reported no significant difference in stavudine levels between whole and skimmed milk, Shapiro et al.45 noted significantly different recoveries of drug between skimmed and whole BM and consequently elected to report whole milk concentrations.45 Physicochemical principles influence the partitioning of drug into aqueous or lipid fractions of milk, but both are ingested by the infant and therefore whole milk is more likely to reflect the true clinical situation.The complexity of BM, particularly relating to variable lipid and protein content, requires that extraction methods are carefully validated. Among the studies reported here, most authors did not specify the milk fraction analysed or the extraction method used. This information is essential. Whereas Fogel The majority of BM PK studies included in this review followed the stringent eligibility criteria of the PMTCT trial within which they were nested. This selection process, rather than recruitment under operational conditions, may have excluded women with covariates influencing BM elimination of ARVs.Studies report BM concentrations of only 13 of the currently available ARVs. At the point of licensing, new drugs will not have been assessed in pregnant and breastfeeding populations. Whereas in well-resourced settings formula feeding is an option for mothers on these drugs, validated methodology is essential to enable investigation of BM elimination of drugs intended for widespread use in low-resource populations where prolonged breastfeeding is the norm.To yield precise, high-quality data in an efficient manner, the proposed study of a novel ARV in breastfeeding mother/infant pairs can be informed by the findings of this review as summarized in Table This systematic review reveals that the NRTI and NNRTI classes of ARVs are eliminated in the BM of HIV-positive women and transferred to breastfeeding infants, which may explain the development of drug-resistant HIV in infants where maternal ARVs have failed to prevent transmission. Available data are drawn from a small number of diverse studies employing laboratory methodology, which is frequently incompletely validated. Adoption of recent WHO guidelines will dramatically increase the use of ARVs in women of childbearing age in low-resource settings. Evaluation of the PK of both existing and novel drugs in breastfeeding mother/infant pairs is a matter of priority in order that best practice and safety can be ensured as novel regimens are rolled out.Academy of Medical Sciences Starter Grant for Clinical Lecturers investigating ARV drugs in breastfeeding Ugandan mother/infant pairs. C. J. W. commenced a Wellcome Trust Clinical Postdoctoral Fellowship (104422/Z/14/Z) to investigate PK of ARVs in breastfeeding mother/infant pairs on 1 February 2015.C. J. W. held an None to declare.C. J. W.: concept for review, study design and protocol development, data collection, data analysis and interpretation, preparation of figures and manuscript writing.P. G.: input into all areas listed for C. J. W., with particular input into protocol development and methods.L. J. B.: statistical analysis of drug levels.S. H. K.: discussion of all elements of study design, data collection and interpretation, particularly where this was challenging. Input into manuscript.L. J. E.: protocol development, literature searches, data collection and analysis and manuscript writing.JAC Online .The protocol and Tables S1 and S2 are available as Supplementary data at


Autosomal recessive cerebellar ataxias (ARCA) are a complex group of neurodegenerative disorders with great genetic and phenotypic heterogeneity, over 30 genes/loci have been associated with more than 20 different clinical forms of ARCA. Genetic heterogeneity combined with highly variable clinical expression of the cerebellar symptoms and overlapping features complicate furthermore the etiological diagnosis of ARCA. The determination of the most frequent mutations and corresponding ataxias, as well as particular features specific to a population, are mandatory to facilitate and speed up the diagnosis process, especially when an appropriate treatment is available.We explored 166 patients (115 families) refered to the neurology units of Algiers central hospitals (Algeria) with a cerebellar ataxia phenotype segregating as an autosomal recessive pattern of inheritance. Genomic DNA was extracted from peripheral blood samples and mutational screening was performed by PCR and direct sequencing or by targeted genomic capture and massive parallel sequencing of 57 genes associated with inherited cerebellar ataxia phenotypes.In this work we report the clinical and molecular results obtained on a large cohort of Algerian patients (110 patients/76 families) with genetically determined autosomal recessive ataxia, representing 9 different types of ARCA and 23 different mutations, including 6 novel ones. The five most common ARCA in this cohort were Friedreich ataxia, ataxia with isolated vitamin E deficiency, ataxia with oculomotor apraxia type 2, autosomal recessive spastic ataxia of Charlevoix-Saguenay and ataxia with oculomotor apraxia type 1.We report here a large cohort of patients with genetically determined autosomal recessive ataxia and the first study of the genetic context of ARCA in Algeria. This study showed that in Algerian patients, the two most common types of ataxia (Friedreich ataxia and ataxia with isolated vitamin E deficiency) coexist with forms that may be less common or underdiagnosed. To refine the genotype/phenotype correlation in rare and heteregeneous diseases as autosomal recessive ataxias, more extensive epidemiological investigations and reports are necessary as well as more accurate and detailed clinical characterizations. The use of standardized clinical and molecular protocols would thus enable a better knowledge of the different forms of ARCA. Inherited cerebellar ataxia is the consequence of an impairment of the cerebellum, the brainstem and/or spinocerebellar tracts. Phenotypic manifestations may include gait-limb ataxia, frequent falls, dysarthria, adiadochokinesia, ophthalmological abnormalities and may also involve other neurological and/or extraneurological symptoms. Progression of these features lead to the loss of ambulatory abilities and often to an early death , 2.Although all patterns of inheritance have been described in hereditary cerebellar ataxia , 3, we fARCA’s first symptoms usually appear before 25 years old, despite the fact that late-onset forms have been described , and areDespite the increasing knowledge in the molecular aspects of ARCA, a significant proportion of patients remain with unidentified ARCA. In addition to the genetic heterogeneity of this group of ataxias, there is an important phenotypic variability in the expression of the cerebellar impairment. The clinical manifestations are indeed as varied as the possible underlying mutations. The heterogeneous expression of these disorders occurs within unrelated patients as well as between siblings of the same family and with the same ataxia form. Furthermore, the existence of atypical phenotypes and overlapping characteristics bring more complexity to the etiological diagnosis of ARCA. The delineation of the specific clinical features to each ARCA is in fact under permanent discussion, thus complicating the molecular investigations, but also make it an absolute necessity.Despite ARCA’s high physiopathological diversity, some clinical entities are known to be the most frequent, such as Friedreich ataxia (FRDA) and ataxia telangiectasia (AT) in Europe –8 or FRDGiven the great clinical and genotypic heterogeneity, as well as overlapping features, identification of the most common mutations and corresponding ataxia forms, together with the specific clinical features observed in a particular population, is crucial to facilitate and speed up the diagnosis process, especially when an appropriate treatment could be provided.This work reports the molecular findings and phenotypic features of a large cohort of 110 Algerians patients with autosomal recessive cerebellar ataxia representing 9 different ataxia entities. We also report some novel mutations, additionaly to mutations already described so far.We studied 166 patients (115 families) refered to the neurology units of Algiers central hospitals (Algeria) and report a cohort of 110 Algerian patients, belonging to 76 families, with identified causal mutations.All patients studied had cerebellar ataxia phenotypes with an autosomal recessive pattern of inheritance. The existence of similar cases among siblings (and/or family members) were observed in 30 families and a consanguineous background were noted for about 80 % of the families. Whenever possible, affected siblings and other family members were also analyzed.Blood samples were obtained for all the patients who gave informed consent to take part in the study which was approved by the Ministry of Health, Health Ethic Committee, Algeria.Genomic DNA was extracted from peripheral blood samples using QIAamp DNA Mini Kit®.n expansion in the first intron of the frataxin gene causing Friedreich ataxia, and the c.744delA mutation in exon 5 of the TTPA gene which causes ataxia with isolated vitamin E deficiency.For all 166 patients, the approach consisted in performing a systematic screening, using PCR and direct sequencing, of the mutations responsible for the major forms of ataxia: the (GAA)When these targeted mutations were absent, patients’ DNA was then analyzed by PCR and sequencing of genes accountable for some other forms of ataxia  by taking into account the specific clinical indications, while not always strictly relying on them because of the encountered phenotypic heterogeneity, as well as on the basis of previous reports incriminating these genes in our population.et al. [Finally, taking advantage of the availability of targeted genomic capture of 57 genes (responsible for inherited cerebellar ataxia phenotypes as well as genes associated with pathologies presenting ataxia as a major and/or only sign) and massive parallel sequencing using the Agilent SureSelect kit®, we were able to select 16 of our patients, taking into account specific clinical indications as well as more heterogeneous phenotypes. The used strategy was similar to the one described by Vasli et al. .Out of 110 patients with identified molecular alterations, 68 had undergone a complete neurologic examination. Those patients belonged to 52 unrelated families among which 40 families (55 patients out of 68) were consanguineous. 57 patients were males and 53 females. The sex ratio was not statistically different (1.07). The mean age at onset for those 68 patients was 11.1 ± 5.97 SD years and ranged from birth to 35 years old.The most common onset sign was gait limb ataxia with unbalance and lack of coordination. Cerebellar syndrome was a constant feature, although with various degrees of disability. Most patients had ataxia  and experienced frequent falls along with different symptoms like dysmetria and adiadochokinesia. Different stages of disability were observed: most patients were capable of walking without help, some of them with unilateral or bilateral help, while 3 were wheelchair-bound at the time of clinical diagnosis. A few patients had a mild cerebellar syndrome. Table Otherwise, we noticed that the 56 patients whose diagnosis was not clarified, mostly issued from consanguineous families (39 patients), were represented by 30 males and 26 females (sex ratio of 1.15), a ratio close to the group of 110 patients with genetic diagnosis. The mean age at onset was 7.63 ± 8.17 SD years and ranged from birth to 31 years old, data not statistically different from the subgroup of patients with genetic diagnosis, while cerebellar syndrome, from severe to moderate, was always present. When available, MRI results and EMG examination showed the same heterogeneous features as the group of patients with genetic diagnosis, with no remarkable delineation of particular phenotypic characteristics or additional associated signs. Also, when available, deficiency of vitamin E, hypoalbuminia, or elevated AFP, cholesterol or cholestanol levels, could not be noticed and were in the normal range.Among the 110 patients issued from 76 Algerian families, consanguinity was present in 76.36 % of patients (84 patients/56 families) and absent in 18.18 % of patients (20 patients/15 families), whereas an undetermined inbreeding context could be noted for the remaining 5.45 % (6 patients/5 families).We report 23 different molecular alterations, including 6 not as yet described, in 9 different genes responsible for autosomal recessive cerebellar ataxia forms. In all but one patient, these mutations were found at the homozygous state.n expansion in the first intron of the FXN (frataxin) gene which is responsible for the Friedreich ataxia phenotype [The most common mutation was the (GAA)henotype , presentTTPA  gene, responsible for ataxia with isolated vitamin E deficiency [In our cohort, 19 patients representing 16 families were found to be homozygous for the c.744delA mutation in the fifth exon of the ficiency . 12/19 pSETX (senataxin) gene responsible for ataxia with oculomotor apraxia 2 (AOA2). 14/18 patients (9/12 families) were consanguineous.18 patients representing 12 families were homozygous for different mutations in the SACS (sacsin), APTX (aprataxin), ADCK3 (aarf domain-containing kinase 3), ABHD12 (abhydrolase domain-containing protein 12), SYNE1 (synaptic nuclear envelope protein 1), and SIL1 (SIL1 nucleotide exchange factor 1), respectively responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), ataxia with oculomotor apraxia type 1 (AOA1), spinocerebellar ataxia, autosomal recessive 9 (SCAR9), polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract syndrome (PHARC), spinocerebellar ataxia, autosomal recessive 8 (SCAR8) and Marinesco-Sjögren syndrome (MSS). These forms were found in less than 10 patients and some of them in a single patient:SACS gene.- 8 patients belonging to 6 families carried mutations in the APTX gene.- 6 patients belonging to 5 families had mutations in the ABHD12 gene.- 6 patients belonging to 3 families showed mutations in the SYNE1 gene and another patient was homozygous for a mutation in the SIL1 gene.- 1 patient carried a homozygous mutation in the We also report patients with mutations in the following genes: Table The present study is the first description of Algerian patients with autosomal recessive ataxia and represents the largest cohort of patients with genetically characterized ataxia, with 110 Algerian patients (76 families). Table Our cohort did not include any ataxia telangiectasia patients, although it is reported to be the second most common ARCA , 13. DesHowever, we were able to report 23 different molecular alterations, including 6 novel mutations, allowing us to identify 9 different forms of autosomal recessive cerebellar ataxias in our population Fig. . Five foFXN gene responsible for Friedreich ataxia (FRDA) [n expansion associated with a point mutation, were described in some patients, all our patients carried exclusively the homozygous unstable trinucleotide repeat (GAA)n in the first intron of the FXN gene, as reported in the majority of patients reported worldwide [We identified 49 patients (31 families) carrying mutations in the a (FRDA) , in accoa (FRDA) . Even iforldwide .In our cohort, 26 of the 49 patients had undergone neurological examination. 24 of them had gait-limb ataxia as a first sign. Head tremor was the first sign in one patient, while another patient displayed muscle weakness.The age at onset in our cohort ranged from 4 to 35 years old, with a mean onset age of 12.74 ± 7.15 SD years. Most patients (24) had a classical onset age, between 4 and 20 years old, while one patient had onset at 27 and another at 35 years, considering them affected by late onset forms.Dysarthria, which is considered as a clinical feature in Friedreich patients , was preThe upper limb tendon reflexes were abolished in 15 patients, were brisk in 3 and normal in 5. At the same time, lower limb tendon reflexes were abolished in 19 patients, brisk in 4 and normal in one patient. Babinski sign was noted in 12 patients, as pointed out by previous reports , 17, 18.In 22 patients, dysmorphic features was either scoliosis (8 patients), pes cavus (3 patients) or both scoliosis and pes cavus (11 patients), one patient presented with a thoracic deformation, according to the progression of the symptoms in Friedreich ataxia .MRI was mostly normal (10 patients), whereas 2 patients presented with cerebellar atrophy as can be observed in advanced stages of the disease , 16. TheFXN mutations had hypoacusia, as has already been reported in a similar proportion [In our cohort, 4 patients (15.4 %) with oportion , 20.Electromyography (EMG) was normal in 2 patients, while sensory motor polyneuropathy was found in 10, sensory neuropathy in 8 and axonal neuropathy detected in 2 other patients.Cardiac impairment, frequent and usually correlated to a premature death in Friedreich ataxia , 19, 21,None of the patients of our cohort had diabetes, although it has been reported in approximately 10 % of FRDA patients .TTPA gene was found at a homozygous state in all the patients, which further confirms its high frequency in the Mediterranen basin [et al. [et al., 2014 [The second most common ARCA diagnosed was the ataxia with isolated vitamin E deficiency (AVED), with 19 patients (16 families) genetically confirmed: a unique mutation (c.744delA) in the en basin  and its  [et al.  and suppl., 2014  or our pAmong our AVED patients, 12 (10 families) had undergone neurological examination and 8 patients (6 families) were consanguineous.The mean age at onset was 11 ± 4.69 SD years, ranging from 4 to 17 years old, in accordance with the fact that most of the patients already described have onset before 20 years old , 23.All the patients but one (11) had gait-limb ataxia as a first sign. Head tremor was the initial sign for the remaining patient. Head titubation was noted in 7 out of the 12 patients, a sign that could in fact be considered significant to distinguish FRDA from AVED –25.The upper limbs reflexes were abolished in 10 patients and normal in 2, while the lower limb reflexes were abolished in all the patients. These observations were expected as most AVED patients reported in the literature have absent or weak tendon reflexes , 25.Nystagmus was present in 7 patients and one patient had a visual acuity decline. None of the Algerian patients presented other ocular features such as retinitis pigmentosa , 27 or oDysmorphic signs were either solely pes cavus (1 patient) or scoliosis, which was associated with either pes cavus (6 patients) or flat feet (3 patients).In this cohort, MRI was normal in 7 patients, showed the presence of cerebellar and vermian atrophy in one patient and was not available for 4 patients. EMG results were normal in 4 patients and abnormal in 8: two patients had axonal neuropathy, four patients presented with sensorimotor neuropathy and two patients displayed purely sensory neuropathy. Although AVED is considered to be an ataxia form presenting with a pure sensory neuropathy , 15, 28,TTPA gene showed significantly low levels of vitamin E in plasma, as initially stated by this biomarker for this disease [All the patients carrying the c.744delA mutation in the  disease .Although it has been reported that an early supplementation with vitamin E can slow down the progression of the disease and maintain the walking capability of the patients , 30, mosTTPA gene [One of our patients that had generalized tonico-clonic seizures presented with early cerebellar syndrome, dysarthria, abolished tendon reflexes, scoliosis and sensorimotor neuropathy at 4 years of age, while cerebral computed tomography was normal, as has been reported for a Norway patient with vitamin E deficiency and epileptic seizures but with compound heterozygous mutations in the TPA gene .SETX gene. This cohort included 18 patients (12 families) with AOA2, and this study backs up the hypothesis already stated by Tazir et al. [The third most frequent form of ataxia in our cohort was the ataxia with oculomotor apraxia type 2 with different mutations in the r et al.  setting SETX mutations. This patient was a compound heterozygote for a new mutation c.985C > T; p.Arg329* associated with a previously described mutation c.915G > T; Trp305Cys, both in exon eight of the gene [Most of the mutations in our cohort have been previously described Table . One mutSETX mutations, 10 patients (6 families) have been previously reported [Out of the 18 patients with reported , while tThe mean age at onset in our patients was 14.57 ± 3.87 SD years, ranging from 9 to 21 years old, in accordance with the data of other published series of AOA2 patients –35.The 8 newly diagnosed patients presented with cerebellar syndrome, dysarthria and nystagmus, as indicated by the AOA2 phenotype that includes such features in the great majority of the patients , 33, 34,SETX gene. Although pes cavus and scoliosis are considered to be occasional features [All patients had elevated alpha-fetoprotein levels which is the main biological marker of AOA2 , 33. Duefeatures , 33, 34,Our study suggests that ARSACS is the fourth most frequent form of ataxia in Algeria, with 8 patients and 6 families.SACS gene in 8 patients. One of the mutations  was novel, and the mutation , previously described in Tunisian families [We identified 4 different mutations in the families , was theSACS mutations had undergone neurological examination. The 8 patients (6 families) were consanguineous.All the patients with The mean age at onset for our patients was 7.75 ± 4.62 SD years, ranging from 2 to 14 years old. Two patients had onset at 12 years old and one at 14 years old, which can be considered as late onset for ARSACS phenotype , 37.Dysarthria was present in 7 of our patients and was acute for one of them, in accordance with one of the major manifestations of the disease during the first two decades of disease progression .Although hyper myelinated retinal fibers has been constantly described in Canadian ARSACS patients , 39, we Pyramidal signs were observed in our patients: Babinski sign in 6 of our 8 patients; spasticity, either severe or moderate, in 6 patients; upper and lower limbs reflexes, brisk or very brisk, in 4 patients; while tendon reflexes were normal in 3 patients, which could be explained by the progression of peripheral neuropathy that thus mask spasticity and pyramidal signs with the evolution of the disease .Cognitive impairment has been noted for two of the patients of our cohort.SACS mutations [Two patients had epileptic seizures, a feature which has been reported in a small proportion of patients harboring utations , 41, whiA larger study of ARSACS is being conducted in our population and will provide more insights on this ataxia in our population, as the patients from Algeria seems to share clinical phenotypes that can be rather rare and can mislead the diagnosis .SACS gene complicates the screening of patients presenting atypical clinical phenotype and indicates that this form of ataxia may be under-diagnosed in several populations, including ours. The screening of the gene is therefore worthwhile even when some features are absent.The huge size of the APTX mutations, although it seems to be a rare condition outside Japan or Portugal [The ataxia with oculomotor apraxia type 1 (AOA1) was the fifth most frequent ARCA in our cohort since we have identified 6 patients (3 families) carrying Portugal , 43.The nonsense mutation, c.837G > A; p.Trp279*, known as the most frequent mutation in the European population , 45, wasA consanguineous context was noted in 5 patients (4 families) and two of these patients (from two unrelated families) had undergone neurological examination.The age at onset in our patients was 3 and 7 years old respectively, with gait-limb ataxia as a first sign for both patients, accordingly with the early-onset status of this form of ataxia , 46, 47.Our patients had moderate cerebellar syndrome with dysarthria, but absence of nystagmus although this later feature accounts for one of the main clinical signs of AOA1 . HoweverMRI showed in our patients a severe cerebellar atrophy in one case and a severe vermian atrophy in the other, as has been reported in all affected AOA1 patients , 47, 48.The report of a new case of SCAR9 in Algeria in this study, after the identification of the causal gene in two Algerian families , brings ADCK3 gene, highlighting that all the mutations identified so far in Algeria are truncating.The patient we report here carried a novel frameshift mutation c.1334_1335delCA, p.Thr445Argfs*51 in the Although the delineation of the SCAR9 phenotype remains difficult because of the great clinical differences from one patient to another , the cliThe upper and lower limb reflexes were weak for 2 patients including the newly reported one but were either brisk or normal for the others. All patients had dysarthria and pes cavus but only the patient carrying the newly found mutation presented with mild scoliosis. The MRI showed no abnormalities for this patient, which can be explained by the early diagnosis and the short time of disease duration. All the other patients (with 14 to over 30 years of disease duration) showed cerebellar atrophy.Our patient should also be surveyed for signs of intelectual deficiency as mild forms have been observed in two of the Algerian patients and was reported as mild, moderate or severe in non-Algerian patients –52.All the Algerian patients with SCAR9 seems to have a mild, slowly progressive or even stable cerebellar ataxia, which was suggested as a main feature for SCAR9 phenotype, in contrast with most of childhood-onset ARCA –52. The ABHD12 gene for two Algerian patients who presented a phenotype somewhat similar to the Algerian patients identified in the report of Fiskerstrand et al. [ABHD12 mutations. Although our criteria were mainly the presence of ataxia and polyneuropathy associated with either cataract, a decline in visual acuity and/or hearing loss, it could not exclude the presence of atypical clinical presentations, as was the case for non-Algerian PHARC patients. Indeed, neither signs of polyneuropathy nor ataxia were observed in these siblings, while their hearing loss and cataract were wrongly attributed to their age or the progression of the retinitis pigmentosa, thereby expanding the phenotypical spectrum of PHARC [Our cohort of 6 patients included 2 of the siblings previously described in studies of PHARC and which allowed the identification of the gene, showing that all reported mutations so far results in premature stop codons presumed to cause a loss of function of the protein , 55. Alld et al. . The twoSYNE1 gene, thereby reporting the first Algerian patient with a genetically confirmed recessive ataxia SCAR8.We identified in the present cohort a patient carrying a novel mutation, c.3715G > T; p.Glu1239*, in the SYNE1 with its 145 coding exons and complicates considerably the mutational screening.The patient had consanguineous parents and 10 siblings who were all healthy. The age at onset was 7 years old, which can be considered as early-onset in contrast to the findings in the French-Canadian patients who showed a middle-aged onset , 58. TheSIL1 gene, c.1285 T > G; p.Tyr429Asp, at the homozygous state.We report in this study the first genetically confirmed case of Algerian patient with Marinesco-Sjögren syndrome (MSS) and the characterization of a new mutation in the This patient had consanguineous parents and had an initial psychomotor impairment that started at birth . The patient was 14 years old when diagnosed and had severe cerebellar syndrome , severe dysarthria, nystagmus and visual acuity decline. The upper and lower limbs reflexes were brisk and the patient presented a mild spasticity but no Babinski sign. Dysmorphic features consisted in pes cavus and craniofacial dysmorphy. MRI showed cerebellar atrophy and hypoplasia. The patient had congenital cataract, but has undergone surgery at 6 years of age. Once the molecular diagnosis established, the patient was also found to have hypergonadotropic hypogonadism, in accordance with the presentation of the syndrome , 61.Although MSS patients have been reported in many countries , 62, 63,We report with this large cohort of genetically determined autosomal recessive ataxia, the first study of the genetic context of ARCA in Algeria. Our study shows that, in addition to the most common mutations and ataxias, other rarer or underdiagnosed forms might be involved and could be underestimated. Taken together, FRDA and AVED patients thus represented 61.82 % of the patients in this cohort (68 patients) while combined, FRDA, AVED, AOA2, ARSACS and AOA1 accounted for 90.9 % of the cohort (100 patients) of the whole autosomal recessive forms of ataxia in our cohort. Besides these most frequent forms, we were able to identify other types of ataxia seemingly rare in our population.A precise assessment of the frequent forms and mutations specific to our population can strongly help the orientation of the investigations in the long term. However, for rare and heterogeneous pathologies as autosomal recessive ataxia, there is still a long way to go in order to refine genotype/phenotype correlations. More extensive molecular and epidemiological investigations will allow a better knowledge of the different ARCA and facilitate the genetic explorations. The power of innovative approaches, such as high-throughput sequencing, opens now new perspectives for diagnosis and research and will certainly provide mandatory answers for patients remaining without molecular diagnosis.


Salmonella serovars isolated from pork and Slaughterhouse environment.The aim was to detect virulence gene associated with the Salmonella isolates (n=37) used in this study were isolated from 270 pork and slaughter house environmental samples collected from the Ahmedabad Municipal Corporation Slaughter House, Ahmedabad, Gujarat, India. Salmonella serovars were isolated and identified as per BAM USFDA method and serotyped at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli . Polymerase chain reaction technique was used for detection of five genes, namely invA, spvR, spvC, fimA and stn among different serovars of Salmonella.Salmonella were isolated with two serovars, namely Enteritidis and Typhimurium. All Salmonella serovars produced 284 bp invA gene, 84 bp fimA and 260 bp amplicon for enterotoxin (stn) gene whereas 30 isolates possessed 310 bp spvR gene, but no isolate possessed spvC gene.Out of a total of 270 samples, 37 (13.70%) invA, fimA and stn gene in all isolates shows that they are the specific targets for Salmonella identification and are capable of producing gastroenteric illness to humans, whereas 20 Typhimurium serovars and 10 Enteritidis serovars can able to produce systemic infection.Presence of  Pork is one of the most widely eaten meats in the world, accounting for about 38% of meat production worldwide, although consumption varies widely from place to place . Most ofSalmonella remain a major problem for the food industry. Salmonellosis is an important health problem and a major challenge worldwide having greater significance in developing countries [Salmonella is an important cause of food-borne  health problems in humans [Salmonella might differ between the production systems, caused by components of the husbandry systems affecting disease development and pathogen shedding or differences in the level of resistance to the pathogen [Food safety hazards caused by food-borne pathogens such as ountries . Pork anountries . Salmonen humans . The rispathogen . The incSalmonella is linked to a combination of chromosomal and plasmid factors. Different genes such as inv, spv, fimA and stn have been identified as major virulence genes responsible for salmonellosis. Salmonella pathogenicity islands (SPIs) are large gene cassettes within the Salmonella chromosome that encode determinants responsible for establishing specific interactions with the host, and are required for bacterial virulence in a given animal like other pathogenicity islands. More than 20 SPIs have been described [invA codes for a protein in the inner membrane of bacteria that is necessary for invasion of epithelial cells [spvRABCD), containing five genes, is present on plasmids commonly associated with some serotypes. One main function of the spv operon is to potentiate the systemic spread of the pathogen [spvC is virulence-related gene on the plasmid required for survival within host cell [Salmonella induced diarrhea is a complex phenomen on involving several pathogenic mechanisms, including production of enterotoxin. This enterotoxin production is mediated by the stn thus it plays a significant role in causing gastroenteritis by producing enterotoxin [The virulence of escribed . The chral cells . Whereaspathogen . The spvost cell . Some stost cell . Salmoneerotoxin .Salmonella isolates from eggs and poultry house environment by detecting the presence of the invA, spvR, spvC, fimA and stn virulence genes using the polymerase chain reaction (PCR).The purpose of this study was to evaluate the potential virulence of Approximately, a total of 270 samples of pork and slaughterhouse environment will be collected from the Ahmedabad Municipal Corporation Slaughterhouse, Ahmedabad, Gujarat under aseptic precautions. The samples were collected in sterilized polyethylene bags and transported to the departmental P.G. Research Laboratory in an icebox for further processing and microbiological analysis. All the samples collected are shown in Salmonella isolates (n=37) recovered from pork and Slaughterhouse environmental samples collected from the Ahmedabad Municipal Corporation Slaughterhouse, Ahmedabad, (Gujarat), India. 13 Salmonellaenteritidis and 24 SalmonellatyphimuriumSalmonella serovars were isolated and identified as per BAM USFDA method [Salmonella and Escherichia Centre, Central Research Institute, Kasauli . The DNA of isolates of Salmonella was prepared by boiling method. Approximately, loop full of culture was taken in microcentrifuge in 100 µl of sterilized DNAse and RNAse-free milliQ water . Then, vortexed and samples were heated at 95°C for 10 min, cell debris was removed by centrifugation and 3 μl of the supernatant was used as a DNA template in PCR reaction mixture. PCR was performed with four sets of primer pairs specific for the invasion gene invA, spvR gene, spvC gene, fimA gene and stn gene as shown in Our study used A method  and seroinvA gene was carried out as described by Kumar et al. [spvR gene was carried out similarly by employing standardized annealing temperature. The fimA gene fragment was amplified at annealing temperature of 56°C and extension for 30 s. The spvC gene fragment was amplified at annealing temperature of 63°C and extension for 1 min. The amplification for stn gene was carried out employing same conditions as invA except annealing at 55°C. Amplification products were separated by electrophoresed on 2% agarose gel stained with 5 µg/ml of ethidium bromide with a 100 bp DNA ladder as molecular weight marker.PCR amplifications were performed in a final volume of 25 µl containing DNA template (3 µl), ×2 PCR Mastermix (MBI Fermentas) (12.5 µl), 10 pmol/µl of each primer  (1 µl) and 5.5 µl nuclease-free water. Amplification for r et al.  with minSalmonella isolates (13 of which belonged to serovar Enteritidis and 24 belonged to Typhimurium) contained the invasion gene invA, other studies having reported similar results [invA is an invasion gene conserved among Salmonella serotypes.All 37  results ,21-24, winvA gene all isolates produced 260 bp DNA fragment specific for stn gene which was in agreement with other authors [Salmonella isolates were found highly invasive and enterotoxigenic.Similar to  authors -27. ThusfimA gene was detected in all 37 isolate produced 85 bp DNA fragment. Which is similar to that of Naravaneni and Jamil [fimA gene has a high degree of sequence conservation among Salmonella serovars. This is very useful in the diagnosis of Salmonella organisms at the genus level.The nd Jamil ,19 and tspvR gene was detected in 30 isolates belonged to Typhimurium and Enteritidis, which is similar to that of Araque [spvC was not detected in any isolates, which is in contrast to that of Soto et al. [spvC in all the isolates  are not specific targets for the same.We can conclude that the JBN and MNB planned and designed the study. JHC collected and processed samples, the experiment was conducted, and laboratory work was done by JHC and PPM. All authors participated in the preparation of draft of the manuscript and read and approved the final manuscript.


Thus accurate identification of these disordered regions have significant implications in proper annotation of function, induced fold prediction and drug design to combat critical diseases. We introduce DisPredict, a disorder predictor that employs a single support vector machine with RBF kernel and novel features for reliable characterization of protein structure. DisPredict yields effective performance. In addition to 10-fold cross validation, training and testing of DisPredict was conducted with independent test datasets. The results were consistent with both the training and test error minimal. The use of multiple data sources, makes the predictor generic. The datasets used in developing the model include disordered regions of various length which are categorized as short and long having different compositions, different types of disorder, ranging from fully to partially disordered regions as well as completely ordered regions. Through comparison with other state of the art approaches and case studies, DisPredict is found to be a useful tool with competitive performance. DisPredict is available at  Many protein regions and some entire proteins do not adopt well-defined, stable three-dimensional 3D) structures in an isolated state and under different non native environments –3. TheseD structuFor the last two decades, many works have been presented in evidence that many proteins do not follow the well-known paradigm of sequence to stable structure to function. Rather these proteins adopt disordered state for complex and essential biological functions , 6, 8, 9In reality the IDPs are abundant. Approximately 70% of the structures released by Protein Data Bank (PDB)  contain Several computational methods have been developed to fulfill the fast annotation requirements for the rapidly growing known protein sequences. Machine learning based some of these well-known approaches are PONDR series , 29, 30,Disorder Predictor)”  before using.We included the information of neighboring residues within the features of each residue by using a sliding window, keeping the target residue at the center of the window. The motivation was to incorporate the native interactions and contacts of neighboring residues which are found to play essential roles in determining protein structures and protein folding dynamics , 72. We optimization-layer and classification-layer. The classification-layer is developed using a single support vector machine (SVM), namely LIBSVM [i.e. Hilbert space), which results in a linear separating hyperplane. On the other hand, in the optimization-layer of DisPredict, we selected two parameters, C and γ, where C is the cost of misclassification and γ is the parameter of fitting best mode of RBF. The optimal values for the parameters C and γ are determined by grid search using 5 fold cross validation. However, in our case the grid search turned out to be computationally very intensive. Thus, we used 5% of the training dataset to determine the optimal parameters instead. The DisPredict output classes such as disordered or ordered residue, in terms of probability, is optimized by another round of 5-fold cross validation. Using the threshold value 0.5, the probabilities are converted into binary decision variables, where probability ranges 0.5 ≤ ranged ≤ 1.0 is considered as disordered and 0.0 ≤ rangeo < 0.5 is considered as ordered. DisPredict is a two-layer disorder predictor that integrates y LIBSVM . Due to https://github.com/tamjidul/DisPredict_v1.0 with a user manual.We implemented our software in C++. The software is developed and tested on Linux platform. It is dependent on two external packages, namely PSI-BLAST  and NR dThe performance of DisPredict is evaluated using the criteria followed in the past Critical Assessment of protein Structure Prediction (CASP) competitions –76. The Binary value, defining whether a residue is disorder or not (“+1” for disorder and “−1” for order) andReal value, quantifying the probability of a residue being disorder (“≥ 0.5” for disorder and “< 0.5” for order).TP (True Positive) = number of correctly predicted disordered residues, TN (True Negative) = number of correctly predicted ordered residues, FP  = number of incorrectly predicted disordered residues and FN  = number of incorrectly predicted ordered residues. To determine the total number of correct prediction (both ordered and disordered), Ncorrect = TP + TN is calculated. Sensitivity (SENS) and specificity (SPEC) are two complementary statistical measures identifying the proportionate values of correct prediction of disordered (positive class) and ordered (negative class) residues, respectively.In binary (two-class) prediction of disorder, Nd and No are the total number of disordered and ordered residues, respectively. As increment of one of these measures (SENS and SPEC) usually leads towards the decrement of another measure, neither of these two measures is a suitable indicator of performance for an imbalanced dataset. On the contrary, the balanced accuracy (ACC), weighted score (Sw) and Mathews correlation coefficient (MCC) are the measures that take all four components of prediction quality  into account and thus can be regarded as more important indicators.wd is the weight for Nd = percentage of ordered residues wo is the weight for No = percentage of disordered residues Sw measure includes weight to address the imbalance in the ratio of ordered and disordered residues and rewards correct disorder classification over correct classification of ordered residues, which is later found to have a linear relationship with ACC (Sw = 2 × ACC − 1) [Sw) have been used in CASP assessment, we have also included both of them in our paper instead of just one. MCC score, another measure that accounts for all four parameters of the prediction quality, is the most reasonable and consistent measure for disorder prediction assessment because of not being favorable to over prediction of any class (order/disorder). MCC and Sw scores vary from −1 to 1, where −1 and 1 represent perfect misclassification and classification, respectively with a random classification scoring by 0. More recently, precision  . Since bn is the total number of protein chains, and ith protein chain, respectively. The lower value of MAE corresponds to better prediction.We calculated Mean Absolute Error ROC) curve, which depicts the correlation between the true positive rate  and false positive rate (FPR = 1—SPEC) for a probability threshold. The area under the ROC curve (AUC) quantifies the predictive quality of a classifier, where the AUC value equal to 1 indicates a perfect prediction and 0.5 corresponds to a random prediction. Moreover, 95% confidence interval (CI) for the AUC score is evaluated using DeLong’s [The SVM model of DisPredict generates a predicted probability value for each residue which signifies the disorder confidence of that residue. This probability value is then binarized using a threshold of 0.5 to generate class annotation. If the probability is greater than or equal to 0.5, the predicted class is ‘disorder’ and if the probability is less than 0.5, the predicted class is ‘order’. Assessment of the predicted probability by a DisPredict is performed by receiver operating characteristic  and total number of residues  in a dataset, we have a feature matrix of dimension, Residuetotal × (Wsize × 56). Therefore, the increase in window size leads towards the increase in the dimensions of the feature space, which in turn makes the time expensive grid search for parameters slower. To trade off between performance with optimization and time complexity of parameter selection along with model generation, we determined the optimal values of parameters with a 5% randomly selected subset of residues from training dataset for 3 window sizes . The optimal parameters (C and γ) found from grid search are reported in Note that, this preliminary extensive analysis of performance with multiple window sizes is done without selection of optimal parameters for SVM. For a specific window size .The improvement of performance with optimized parameters over non-optimized one was significant. To compare, for SL477 dataset (window size 21), FP and FN values are reduced to 1,002 and 1,083 from 1,125 and 1,152 due to optimization. In case of MxD dataset (window size 21), the FN value is increased by 133 residues. However, the FP value is also decreased by 1,812 residues which maintains the overall increase in the total number of correctly predicted residues from 16,691 to 18,370. The improvement of prediction, both in terms of increased correct classification and decreased misclassification, is also visible from both the sensitivity and specificity scores. For window size 21, the values of N sequences in the dataset and the expected length of the sequence is Wsize) is N = 477, approximate Wsize = 21, the probability of the overlap is 2.44 × 10−06, which is significantly low and thus can be safely ignored. Further, we reevaluated DisPredict’s 10 fold cross validation performance with sequence level sampling by modular operation for SL477 dataset to generate training and test subsets. To uniformly distribute the residues into ten subsets for cross validation, we applied modular arithmetic operation to split the dataset in residue level. As the residues are already included within the neighboring information based on the window, they are detachable from their original sequence. However, this inclusion of residue information within window may yield overlap of information between training and test sets in case of residue level splitting of dataset for cross validation. We analyzed the probability of this residual overlap between training and test sets. Let, there are Sw: 0.672, 0.667. These consistencies are also evident in case of training with MxD444 dataset while tested by different datasets and the evaluations are, ACC: 0.805, 0.789 and Sw: 0.611, 0.577. We calculated the Mean Absolute Error (MAE) which is also reported along with its corresponding STDEV from mean. The score indicates that the error does not increase from cross validation to independent test as the test-results were robust.With optimized parameters and balanced dataset, we carried out independent training on SL477 and MxD444 datasets followed by testing the resulting predictor model with MxD134 and SL171 dataset, respectively. Note that, these independent test datasets (MxD134 and SL171) were generated at low sequence identity (10%) with the corresponding training datasets (SL477 and MxD444). The consistent results of these two tests done through cross validation and independent test confirm the usage of robust technique and effective feature set in DisPredict as well as training efficacy avoiding possible over-fittings. To analyze the probability prediction, the ROC curves given by DisPredict are plotted in The performance of DisPredict is compared with the state-of-the-art disorder predictors, MFDp  and SPINSw score is also 19.6%. DisPredict showed lower sensitivity (7%) than MFDp while at the same time improved specificity by 20%, which in turn improved the balanced accuracy by 6.67%. Moreover, DisPredict outperformed MFDp in AUC score by 1.29% which is used to assess the probability based prediction.In particular, MFDp  is a metw score. On the contrary, DisPredict is a SVM based two state disorder predictor using a more meaningful threshold for two-class classification of value 0.5. DisPredict outperformed SPINE-D in terms of sensitivity as well as specificity by 5.19% and 1.18% respectively which leads to 3.7% improvement in overall accuracy. DisPredict also outperformed SPINE-D in terms of Sw, MCC and AUC by 8.06%, 6.34% and 10.34% respectively.The other state of the art predictor, SPINE-D  utilizesIn addition to the comparison on cross validation test, we evaluated DisPredict, SPINE-D  and MFDpMFDp and SPINE-D have been established as the best disorder predictor among 8 and 11 existing disorder predictors , 56, resProteins with disordered regions are found to contain several regions of interest, such as self-stabilizing folded regions, DNA or, nucleotide binding regions, short (up to 20 amino acids) conserved regions of biological significance (known as motif), mediating regions for protein interaction with different partners etc. These characteristic regions undergo various conformational changes, gain structure and affect many important biological functions. We selected three proteins as cases  with experimentally verified regions of interest to analyze per residue disorder confidence score assigned by DisPredict, SPINE-D and MFDp. In this article, we proposed a canonical support vector machine which uses a RBF kernel and includes useful and advanced features for predicting disordered residues, called DisPredict. DisPredict not only generates the binary class annotation for ordered and disordered residues but also provides order-disorder probabilities that can be treated as the confidence level of the prediction too. The DisPredict outperforms other existing top performing predictors both in predicting binary annotation and probability. The competitive performance of DisPredict is mainly due to the use of a novel methodology that incorporates firstly, radial basis kernel function (RBF) that can implicitly map the feature space in infinite dimension, secondly and most importantly the optimization of the parameters and thirdly, the novel features monogram (MG) and bigram (BG) assisted in determining an optimal as well as effective class separating hyperplane.This overall performance of DisPredict is also persuaded by the use of a comprehensive set of features that well captures the sequential (amino acid composition) and structural characterization of ordered and disordered residues or, proteins. We used SPINE X  to generThe robust performance of DisPredict is also justified by training and testing the predictor with multiple datasets: SL477, SL171 and MxD444, MxD134. The datasets used to train DisPredict encompass disorder annotation from several complementary sources (X-ray and NMR defined disorder from PDB and DisProt) as well as disorder region of various lengths. The SL dataset comprises of 81 full disordered proteins (IDPs) while the rest of the chains contain 928 disordered regions (IDRs). On the other hand, the MxD dataset is composed of 55 full disordered chains, 4 full ordered chains and 385 chains, sharing both structured and disordered regions, which include 730 disordered regions (IDRs). Furthermore, 70% of the IDRs included within partially disordered proteins are short (≤ 30 residues) and 30% of them are long (> 30 residues). This combination of several length disordered regions  includedIt is interesting to note that, regardless of cross validation or independent test, DisPredict’s performance is relatively better while it is trained on SL477 dataset than that of MxD444 . To furtPcoil) for each ordered or disordered region and computed the fraction of exposed residues with greater than 25% solvent accessibility (Fexposed) of that region. In this analysis, we discarded 5 residues from N and C-terminal regions of each protein sequence as they are mostly found on the surface of a protein chain (not buried in the core) and more likely to be affected by the interaction with nearby structured protein, yielding to a highly flexible and dynamic conformation. The plots for both datasets show that the ordered regions are mostly concentrated in the portion with relatively low coil probability, 0.3 ≤ Pcoil < 0.5  and low exposure, 0.2 ≤ Fexposed < 0.5. While on the contrary, the disorder regions are found abundant in the area of high coil probability, 0.5 ≤ Pcoil ≤ 0.9  and high exposure, 0.5 ≤ Fexposed ≤ 1.0. However, we found the intrinsic difference between these two datasets according to their annotation of residues as order and disorder. This difference is also evident from the top right location of the correlation plot, 0.6 ≤ Pcoil ≤ 0.8 and 0.4 ≤ Pcoil ≤ 0.9, designated for disordered regions. For SL477 dataset  and predicted ratio (rp) of each amino acid type out of total annotated and predicted disordered residues.We would like to highlight that the amino acid residue compositions may vary in different datasets as well as within short (≤ 30 residues) and long (> 30 residues) disordered regions , 29. Spera and rp) for each amino acid type. For MxD134 test dataset, we found RMSE of 0.0046, which was comparatively higher than the RMSE value computed for SL171 which equals to 0.0018. However, the correspondence between actual composition and predicted composition by DisPredict measured with PCC  was found equally positive, 0.9976 and 0.9897 for SL171 and MxD134 dataset, respectively. It is important to note that, this consistent result is corresponding to the independent test where the dataset used to train DisPredict shared significantly low sequence identity (at most 10%) with test dataset, which once again implicates the strength of the classification methodology of DisPredict.The composition profile demonstrates that SL171’s disordered residue set accommodates relatively higher ratio of amino acid type E (10%) and K (9%), which are long disorder prone residues. In contrast, MxD134’s disordered residue set is enriched with high ratio of amino acid type S (11%), G (10%) and D (9%), known as short disorder prone residues. Another significant difference between the intrinsic compositions of these two datasets is in the proportion of histidine (H). Disorder annotation from PDB includes higher ratio of H-tag , which is sometimes used for protein purification. The predicted proportion of all these amino acids given by DisPredict ensures its capability of detecting residues in disordered region of all length accurately with no significant over prediction. Moreover, DisPredict could also accurately predict methionine (M) at highly flexible N-terminal region. To further quantify DisPredict’s performance in detecting residue composition, we evaluated the Root Mean Square Difference (RMSE) and Pearson Correlation Coefficient (PCC) between actual and predicted ratio (Finally, accurate prediction of disorder has useful implication in proteomic studies due to its direct involvement in the proper function of a protein. Successful detection of disordered region of a protein is considered to be the first step in drug design to combat critical diseases. We have built DisPredict using the canonical SVM classifier with RBF kernel and established it as a successful fine predictor of disorder by utilizing the benchmark datasets. In addition to that, our case studies ensure biologically relevant performances of DisPredict.


Atrial fibrillation (AF) can be treated with percutaneous catheter ablation procedures into the left atrium. Pulmonary veins stenosis (PV) stenosis is a severe complication of this procedure.we report a case of late hemoptysis secondary to severe PV stenosis in a man who underwent AF ablation 9 months before onset of symptoms. He presented four episodes of bleeding and developed an acute respiratory failure (ARF). Parameters of respiratory mechanics and medical investigation did not show any abnormalities. Only computed tomography (CT) angiography showed stenosis of 3 out of 4 native PVs. PV balloon dilatation in all affected PVs and a stent was implanted in 1 of the 3 PVs with full restoration of respiratory function during 1 year follow-up.PV stenosis may be the underlying cause of recurrent haemoptysis after AF ablation in the presence of normal respiratory parameters. This diagnosis can be confirmed by means of CT angiography and magnetic resonance imaging can provide accurate localization of stenosis. Massive haemoptysis is characterized by a relevant bronchial hemorrhage, usually quantified in 100–1000 ml in about 24 hours, and it is potentially life-threatening, representing a medical emergency , 4.Usually, the first approach to AF is medical therapy or electric cardioversion. Catheter ablation strategies should be reserved for patients with AF, which remains symptomatic despite optimal medical therapy, or for paroxysmal AF in young patients with severe symptoms .Percutaneous catheter ablation procedures involve the application of radiofrequency energy into the left atrium. Specifically, the technique consists of the electrical isolation of pulmonary veins (PV) from left atrium by encircling their ostium , 5. InitThe clinical presentation, investigation, management, and outcome of this disease have not been completely explained.This report describes a case of severe pulmonary vein stenosis associated with major haemoptysis and ARF requiring intensive treatment.A 40-year-old male, heavy smoker, occupational exposure to inhaling substances (his work consisted in floor tiling), was admitted to our intensive care unit on December 2013 because of major haemoptysis thus causing respiratory failure.The patient’s medical history included hypertension and AF which began on December 2011, with no response to antiarrhythmic medications. In September 2012, the patient underwent catheter ablation for AF in another hospital. The procedure was conducted as conventionally with irrigated tip radiofrequency electrode. In particular, circumferential ablation about 1 cm away from PV orifice for all PVs was performed with maximum delivered power 35 watts at pre-set maximum temperature setting 38 °C. Since July 2013 he referred the onset of dyspnea and asthenia with several episodes of spontaneous haemoptysis. In October he was admitted to a medical ward. Complete blood count, coagulation study, arterial blood gases, bacterial and fungal cultures, virologic test, autoimmunity study, electrocardiogram, chest radiography and Positron Emission Tomography were carried out, but did not show any abnormalities. An echocardiogram performed on September 2013, showed a normal dimension and normal biventricular systolic function (estimated Ejection Fraction >70 %) and pulmonary artery pressure was estimated about 25–30 mmHg. Reumatological and Otolaryngological examinations were normal. He had no fever, chills, night sweats or weight loss. A thoraco-abdominal Computed Tomography study, performed in October 2013, documented the presence of mediastinal lymphoadenomegalia.At the beginning of the flexible bronchial endoscopy, which was aimed at perform transbronchial fine needle aspiration of the node station 4R, 4 L and 7 (in order to exclude a proliferative pathology), the patient had hemorrhaged (about 400 ml in 30 minutes). The bleeding started immediately spreading from tracheobronchial mucosa. However, the bronchial tree anatomy was normal, and no evident hemorrhage source was found. Bleeding was copious and persistent, thus making the collection of a sample impossible and orotracheal intubation was required. Then, an angiography of the bronchial arteries was performed, but it did not reveal any active source of bleeding. However, a selective embolization was performed because of convoluted aspect of the bronchial arteries. Following the procedure, the patient was transferred to our intensive care unit in order to treat the respiratory failure.aO2/FIO2 = 118 with a positive end expiratory pressure (PEEP) of 10 cmH2O. The pulmonary shunt fraction, calculated with the Automatic Lung Parameter Estimator system  was about 16 %. A protective lung ventilation was established. The patient was paralyzed and ventilated in Volume controlled mode with a VT of 5.7 ml/Kg and a PEEP of 10 cmH2O. Chest X-ray showing patchy bilateral infiltrates , equal to 52 ml/cmH2O, did not change by rising PEEP and by placing the patient in prone position. Cst, rs was calculated as VT/(plateau pressure – PEEPtot), where Plateau pressure and PEEPtot are the pressure registered after an end inspiratory/end expiratory occlusion of at least 5 sec, respectively. After 24 h of prone position, without further benefit in terms of oxygenation, the patient returned in supine position and the curarization was stopped. The shunt fraction was about 14 % with a PEEP of 10 cmH2O.Subsequently, he presented four more episodes of sudden major bleeding with acute desaturation. At the beginning, the oxygenation improved few hours after the bleeding events. Then the patient became severely hypoxic since the PThe evident relationship between bleeding events and hypoxia imposed the re-analysis of the medical history of the patient. This helped us to notice the temporal correlation with the cardiac ablation. Indeed the latter could be responsible for severe complications, such as the PVS. Hence, to clarify this aspect, another Chest CT angiography was performed, at the seventh day of hospitalization. This showed the concentric severe stenosis of the right superior pulmonary vein Fig.  and the PVS is a relatively rare condition, which can be both congenital or acquired. The congenital form is linked almost completely to congenital heart diseases and inadequate embryological cardiovascular connections, and evidences in childhood . In adulThe most common causes of acquired PVS in adult patients are radiofrequency ablation procedures for AF treatment. However, the underlying molecular mechanisms remain poorly defined, probably involving scarring, contraction of the venous wall and peri-adventitial inflammation or collagen deposition as a result of thermal injury, which may compromise or even occlude the lumen of PV , 5.PVS is asymptomatic in most cases, especially when only one vein is involved. When clinically evident, the presentation varies depending several factors including: 1) the number of PV involved; 2) the severity of the lesions; 3) the response of the entire pulmonary vasculature to the lesion; 4) the time course of stenosis; 5) the clinical setting; and 6) the presence and extent of collateral vessels [Patients with more extensive and severe involvement may present respiratory symptoms like dyspnea, orthopnea, cough, chest pain, recurrent pulmonary infections. Rarely, does an X-ray show bronchitis or pneumonia , 5.Haemoptysis, the main symptom described in our case report, is relatively infrequent , 7. Its 2O) and even for patients ventilated for other diseases (44 ml/cmH2O) [Interestingly, our patient presented a severe hypoxemia requiring prone positioning . This acl/cmH2O) . Hence, The diagnosis of PVS may not be simple to identify, given the variability of clinical presentation and the atypical findings. Presence of dyspnea or cough after catheter ablation in the region of the PV should raise suspicions of PVS , 5. Our PVS can be treated acutely with balloon dilatation of the PV, although the long-term outcome is uncertain . TreatmePrevention of PVS is mainly related to placing the ablation site from inside to outside the orifice of the PV and reducing the ablation temperature and energy. This strategy should decrease the risk of PVS down to less than 1 % , 19.Clinicians should be aware of the possibility of PVS in patients who underwent ablation procedure for AF and those which present haemoptysis and acute hypoxemic respiratory failure. Hence pulmonary venous hypertension should be considered among the causes of ARF. Presence of dyspnea, cough, haemoptysis and acute respiratory failure after catheter ablation in the region of the PV should raise a high suspicion of PVS and a CT angiography should be performed since it can provide accurate location and evaluation of the extent of stenosis.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.