As commercial human immunodeficiency virus type 1 drug resistance assays are expensive, they are not commonly used in resource-limited settings. Hence, a more affordable in-house procedure was set up taking into account the specific epidemiological and economic circumstances of Cuba. The performance characteristics of the in-house assay were evaluated using clinical samples with various subtypes and resistance patterns. The lower limit of amplification was determined on dilutions series of 20 clinical isolates and ranged from 84 to 529 RNA copies/mL. For the assessment of trueness, 14 clinical samples were analyzed and the ViroSeq HIV-1 Genotyping System v2.0 was used as the reference standard. The mean nucleotide sequence identity between the two assays was 98.7% ± 1.0. Additionally, 99.0% of the amino acids at drug resistance positions were identical. The sensitivity and specificity in detecting drug resistance mutations was respectively 94.1% and 99.5%. Only few discordances in drug resistance interpretation patterns were observed. The repeatability and reproducibility were evaluated using 10 clinical samples with 3 replicates per sample. The in-house test was very precise as nucleotide sequence identity among paired nucleotide sequences ranged from 98.7% to 99.9%. The acceptance criteria were met by the in-house test for all performance characteristics, demonstrating a high degree of accuracy. Subsequently, the applicability in routine clinical practice was evaluated on 380 plasma samples. The amplification success rate was 91% and good quality consensus sequences encoding the entire protease and the first 335 codons in reverse transcriptase could be obtained for 99% of the successful amplicons. The reagent cost per sample using the in-house procedure was around € 80 per genotyping attempt. Overall, the in-house assay provided good results, was feasible with equipment and reagents available in Cuba and was half as expensive as commercial assays. Access to antiretroviral therapy (ART) has been scaled up rapidly since 2001 in Cuba and this has markedly reduced human immunodeficiency virus (HIV)/Acquired Immune Deficiency Syndrome (AIDS) related morbidity and mortality –5. ThereThe aim of this study was to design an accurate and affordable HIV-1 genotyping system enabling the assessment of drug resistance against protease (PR) and reverse transcriptase (RT) inhibitors in the Cuban setting. The Cuban HIV-1 epidemic is characterized with the presence of almost all subtypes and particular circulating recombinant forms (CRFs). In 2003, the highest prevailing subtype was subtype B (41%), followed by CRF19_cpx (18%), BG recombinants (12%), CRF18_cpx (7%), subtype C 6%), subtype G (4%), subtype H (2%) and other subtypes and unique recombinant forms (URFs) (10%)[%, subtypA total of 424 clinical plasma samples was used in the validation of the in-house assay.A selection of 20 plasma samples from HIV-1 patients attending the Institute of Tropical Medicine “Pedro Kourí” (IPK) (Cuba) was used to determine the lower limit of amplification. The panel included the predominant subtypes in Cuba and comprised subtypes A1 (2 samples), B (4), C (4), CRF18_cpx (4), CRF19_cpx (4), and BG-recombinants (2). Viral loads were determined using Nuclisens Easy Q HIV-1 Kit version 2.0  or COBAS TaqMan HIV-1 Test For Use With The High Pure System .To assess the trueness (including sensitivity and specificity), 14 plasma samples from HIV-1 patients attending the University Hospitals in Leuven (Belgium) were selected based upon sequences previously obtained at the University Hospitals of Leuven (Belgium) using the ViroSeq HIV-1 genotyping system v2.0 . The panel included samples with subtypes A (2 samples), B (4), C (1), D (1), G (1), H (1), CRF02_AG (1) and URF (3), representing multiple resistance patterns including mixtures and viral loads ranging from 692 to 263,027 RNA copies/mL. Viral loads were determined using the Abbott RealTime HIV-1 assay .To evaluate the precision of the assay, repeatability and reproducibility were determined on 2 panels of 5 plasma samples from HIV-1 patients attending the IPK and displaying multiple drug resistance patterns and various subtypes (subtypes B (2), C (2), CRF18_cpx (2), CRF19_cpx (1), CRF20_BG (1), CRF24_BG (2)). The selection of patients was based upon sequences previously obtained at the IPK using the in-house drug resistance test described in this study.For the evaluation of the applicability of the test in a clinical setting, the genotyping results were evaluated for 380 plasma samples from 352 HIV-1 patients attending the IPK for clinical care between September 2009 and September 2011. This dataset included 92 plasma samples obtained from therapy naïve patients and 220 samples from therapy experienced patients. For the remaining 68 plasma samples, the therapy history of the patients was unknown.1. RNA extractionFor HIV-1 viral RNA extraction, 1 mL of plasma or serial dilutions of plasma in phosphate buffered saline (PBS) was ultra-centrifuged at 20,000 x g for 1 h at 4⁰C to pellet the virus. The supernatant was removed, retaining 140 μL to suspend the pellet again. Subsequently, the viral RNA extraction was performed manually or automatically on the QIAcube  using the QIAamp Viral RNA Mini Kit , as described by the manufacturer’s protocol. Isolated RNA was eluted in 60 μL elution buffer and stored at -80°C until use.2. Primer designgag-pol gene alignment of several HIV-1 group M strains available at the Los Alamos HIV Database (www.hiv.lanl.gov), with the inclusion of all available strains from CRFs in Cuba  [The PCR and sequencing primers were designed using the RF24_BG) . The pri3. Amplification4), 0.5 μL 20 μM primer AV159  and H2O until a final volume of 40 μL. Reverse transcription was performed at 55°C for 30 min. The amplification started with an initial denaturation at 94°C for 2 min, followed by 40 cycles of: denaturation at 94°C for 15 sec, annealing at 50°C for 30 sec and elongation at 68°C for 2 min, with a final elongation at 68°C for 5 min. A 2,060-bp amplicon, encompassing HIV-1 PR and the first part of RT, was thus obtained.Ten microliters of the extracted RNA was reverse transcribed and amplified in a one-step RT-PCR using the SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity kit . The reaction mixture was composed by: 25 μL of 2X Reaction Mix . Amplification products were separated on a 1% agarose gel and visualized by ethidium bromide staining and UV light. Only positive reactions were subjected to sequencing analysis.The inner 1,770-bp PCR fragment was generated using the Expand High Fidelity PCR System . To prevent degradation of primers and template, the PCR mixes were prepared in two separate tubes. Thus, a first tube was prepared containing 0.4 μL 25 mM of each dNTP, 1.25 μL 20 μM primer AV190 , 1.25 μL4. Sequencing analysis2O to complete the reaction to 20 μL. Cycling conditions on a Thermocycler Doppio were 50 cycles of 96°C for 30 sec, 50°C for 20 sec and 60°C for 4 min. The sequence products were purified according to the protocol described in the DTCS Quick Start Kit. The sequencing fragments were run on a CEQ 8800 Genetic Analysis System . Finally, the electropherograms were displayed and assembled, and sequences were manually edited using Sequencher version 4.10.1  and HIV-1 subtype B strain HXB2 as a reference (GenBank accession number K03455).The PCR products were purified using the QIAquick PCR Purification Kit  following the protocol indicated by the manufacturers. Purified PCR products were subjected to population-based bi-directional sequencing using the Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit . Separate reactions with 7 different primers were performed, in order to enable the coverage of a 1,302-bp fragment that encodes the 99 amino acids of HIV-1 PR and the first 335 amino acids of HIV-1 RT. Each mixture consisted of 1 μL 5 μM primer  , 8 μL QuThe FDA-cleared and CE-marked ViroSeq HIV-1 Genotyping System version 2.0  was used as the standard test for the validation of the in-house assay. ViroSeq is designed to detect drug resistance mutations (DRM) in the entire PR (amino acids 1 to 99) and the first part of the RT gene fragment (amino acids 1 to 335) from HIV-1 subtype B plasma samples with a viral load ranging from 2,000 to 750,000 RNA copies/mL (ViroSeq specifications). At a viral load of 2,000 RNA copies/mL and with 40% mutant viruses present, the sensitivity and specificity in detecting HIV-1 DRM was 98.1% and 99.9%, respectively. These values decreased to respectively 92.7% and 99.9% for samples with a viral load of 1,000 RNA copies/mL and 40% mutant viruses (ViroSeq specifications). For clinical samples with a viral load ranging from 1,800 to 10,500 RNA copies/mL, the sensitivity and specificity at a threshold of 40% mutants was 99.65% and 99.95%, respectively [In this study, ViroSeq testing was performed at the AIDS Reference Laboratory in Leuven (Belgium), where this system has been used since 2000 for monitoring drug resistance in HIV-1 infected patients . All steIn order to assess the performance of the in-house genotyping assay, the lower limit of amplification and the accuracy (including trueness and precision) in detecting NT and AA sequences as well as in detecting mutations at DRM positions were evaluated. The general definitions of these performance characteristics were adopted from Jennings et al. . AcceptaThe lower limit of amplification was defined as the lowest viral load at which samples could be amplified. Briefly, plasma samples were 10-fold serially diluted in PBS until a theoretical viral load below 1,000 RNA copies/mL was achieved. The serial dilutions were extracted and amplified with the in-house test. The procedure was considered successful if the amplicons could be visualized with ethidium bromide staining and UV light on an agarose gel. Subsequently, the last serial dilution that was considered positive, was subjected to 2-fold serial dilutions until a theoretical viral load below 100 RNA copies/mL was achieved, extracted, amplified and analyzed on an agarose gel. The lower limit of amplification should be ≤1,000 RNA copies/mL as this is the cut-off for which drug resistance testing is recommended [Trueness was assessed using 14 clinical samples and one negative control, and by comparing genotyping results of the in-house assay with the results of the ViroSeq HIV-1 Genotyping System version 2.0. The acceptance criterion for trueness was established as the pairwise agreement of ≥95% at NT level. At AA level, the threshold was defined as ≥99% identity at known DRM positions if no mixtures are present and >50% if mixtures are present in the reference standard –14.Precision (including repeatability and reproducibility) was assessed using 10 clinical samples with 3 replicates per sample. More specifically, the repeatability was determined using 5 samples for which the 3 replicates were generated by a single technician in a single run. The reproducibility was determined using 5 samples, of which one sample was diluted in PBS until theoretical viral loads of 2,000; 1,000; 500 and 250 RNA copies/mL. The latter experiment was performed to assess the reproducibility at low viral loads. For each aliquot, 3 replicates were generated in 3 separate runs and by 2 technicians. The acceptance criterion for repeatability and reproducibility was defined as ≥98% NT identities in ≥90% of the pairwise comparisons ,14,15.http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html) with default gap-penalty values. Amino acid sequence identity scores were defined as the percentage AA identity of a pairwise comparison of AA sequences. AA identity scores were also determined at DRM positions and at surveillance drug resistance mutation (SDRM) positions only. Calculations were performed using SeqScape version 2.6. Mixed versus unmixed AA were counted as discordant. DRM positions were defined as all positions for which AA changes are included in the resistance interpretation algorithms Rega v9.1.0, ANRS 2013.09 and/or HIVDB v7.0 (89 DRM positions). SDRM positions were defined according to Bennett et al. [Nucleotide sequence identity scores were defined as the percentage NT identity of a pairwise comparison of NT sequences. Mixed versus unmixed bases were counted as discordant. The NT sequence identity scores were determined using the EMBOSS pairwise alignment tool .Drug resistance interpretations were conducted using the Stanford website and resistance interpretation algorithms Rega v9.1.0, ANRS 2013.09 and/or HIVDB v7.0  and phylogenetic reconstructions were performed on all generated sequences. The 2008 subtypes and CRF reference dataset from Los Alamos database  was included. The sequences were aligned with Muscle in MEGA version 5. Surveillance drug resistance mutation (SDRM) positions [A negative control was included in each run to monitor for cross-contamination. Additionally, to confirm the absence of any sample mix-up and/or cross-contamination, sequence artifacts were investigated using the Calibrated Population Resistance tool version 6.0 , and complies with the principles laid down in the Declaration of Helsinki. All subjects included in the study, gave written informed consent to the work.The lower limit of amplification ranged between 84 and 529 RNA copies/mL, and the median limit was 275 RNA copies/mL (IQR 225–363) . For allTrueness was assessed in each specimen of a set of 15 samples, by comparing genotyping results obtained with the reference method ViroSeq and the in-house assay . One negThe mean NT sequence identity score between both assays was 98.7% ± SD 1.0 (trueness). Each individual NT sequence identity score was well above the predefined acceptance criterion of ≥95%. Among the 14 samples, 235 NT differences were observed between both assays and were in 98% of the instances caused by a difference in the detection of mixtures (230/235). The other discordances (5/235) resulted from scoring a different nucleotide. In total, 486 NT mixtures were identified by the ViroSeq system and/or the in-house assay. From these mixtures, 256 (53%) were detected by both assays, whereas 150 (31%) were only detected by ViroSeq and 80 (16%) only by the in-house assay.The overall AA identity score at DRM positions was 99.0% (trueness) as 1,231 of the 1,244 evaluated DRM positions were identical for both methods . FurtherThe specificity and sensitivity of the in-house assay were respectively 99.5% and 94.1% in detecting mutations at DRM positions and 99.7% and 95.5% in detecting mutations at SDRM positions . In addiOnly 3 DRM  observed in the ViroSeq results were not detected by the in-house assay. The other differences observed at DRM positions were related to amino acid changes that are not included in the drug resistance interpretation rules. By not detecting 221Y in RT, drug resistance was missed for rilpivirine according to ANRS and Rega (from respectively resistant and intermediate resistant in the ViroSeq result towards susceptible in the in-house result). By not detecting 67N in RT, intermediate resistance was not detected for zidovudine and stavudine according to HIVDB. The differences for PR mutation 43R did not alter the drug resistance interpretations. In addition, 2 DRM (69N and 77I in PR) were detected by the in-house method, but not by ViroSeq. Only PR mutation 69N altered the ANRS drug resistance interpretation for tipranavir/r (intermediate resistance in the in-house result and susceptible in the ViroSeq result). Finally, one SDRM (67N in RT) was detected by ViroSeq, but missed by the in-house method.The in-house assay showed good results for both repeatability  and reprThe NT sequence identity ranged from 98.8% to 99.9% and from 98.7% to 99.7% in the repeatability and reproducibility assessment, respectively. For both analyses, 100% of the pairwise comparisons (15/15 and 20/20) were ≥98% identical on NT level, well above the predefined threshold of ≥98% nucleotide identities in ≥90% of the pairwise comparisons. At AA level, similarly high sequence identity scores were reached at all positions, DRM positions and SDRM positions, as shown in Tables Although in 2 samples not all mutations at DRM positions were detected in every replicate during the assessment of repeatability, this had only a minor impact on drug resistance interpretation, because the particular amino acid changes were not included in the drug resistance interpretation rules or they did not trigger a change in the interpretation. In total, only 2 DRM were not detected in the 3 replicates. In sample 17, the minor DRM 10F (in PR) was not detected in 2 replicates, resulting in an altered REGA and ANRS drug resistance interpretation for atazanavir/r (from resistant in the ViroSeq result towards respectively intermediate resistant and susceptible in the in-house result), an altered ANRS interpretation for fosamprenavir/r (from resistant towards susceptible) and a different ANRS resistance interpretation for saquinavir/r (from resistant towards intermediate resistant). In sample 20, the minor DRM 71V (in PR) was missed in 2 replicates, altering the REGA resistance interpretation for saquinavir/r (from intermediate resistant towards susceptible).In the assessment of reproducibility, only two DRM were not detected in the 3 replicates. 189I in RT was missed in the replicate generated by the second technician for sample 23. For sample 25–2000, 36V in PR was detected by only one of the replicates. However, the absence of 189I and 36V did not change the drug resistance interpretations for these samples.Finally, the sequences generated from 25–2000, 25–1000 and 25–500 were compared with the sequence generated from the original non-diluted plasma sample and the respective mean pairwise NT identity scores were 98.9% ± 0.6, 99.1% ± 0.2 and 99.0% ± 0.3. Mean pairwise AA identity scores were respectively 99.4% ± 0.5, 99.5% ± 0.0 and 99.8% ± 0.3 at all positions, 99.3% ± 1.3, 99.3 ± 1.3 and 100.0% ± 0.0 at DRM positions and 100.0% ± 0.0, 100.0% ± 0.0 and 100.0% ± 0.0 at SDRM positions. Drug resistance interpretations were concordant between all the replicates and the non-diluted plasma sample.To assess the amplification success rate, results obtained during routine clinical practice for 380 clinical samples from 352 different patients were evaluated. At first, the amplification was successful for 84% of the samples (321/380). For the 59 samples that could not be amplified in the first attempt, a second RNA aliquot from the same extraction was again subjected to the in-house amplification procedure and with this strategy another 19 samples could be amplified. Subsequently, for twenty of the samples that failed again in the amplification procedure and for which enough plasma was still available, a second extraction and amplification round was attempted. An additional 5 samples could be amplified, resulting into an overall amplification success rate of 91% (345/380).Sequencing results covering 1,302 nucleotides (amino acids 1 in PR to 335 in RT), could be obtained for all amplified reactions (345/345). However, 3 sequences were rejected as quality control analysis suggested APOBEC editing, resulting in an overall sequencing success rate of 99% (342/345).Phylogenetic analysis revealed that the subtype distribution among the 345 obtained sequences were 34.9% subtype B, 12.8% CRF19_cpx, 10.2% CRF20_BG, 9.0% CRF18_cpx, 9.0% URF, 7.8% subtype C, 7.0% CRF24_BG, 3.2% subtype G, 3.2% CRF23_BG, 0.9% subtype H, 0.6% subtype A, 0.6% subtype F, 0.6% CRF02_AG, 0.3% subtype J and 0.3% CRF31_BC.An estimate of the cost of reagents and disposables was made. The overall cost per sample using this procedure was around € 80 per single genotyping attempt and increased to € 100 per successful genotype in our local testing scheme after initial amplification failure, which is still only half as expensive as the ViroSeq system. The working time cost and necessary equipment, such as pipettes, centrifuges, thermal cyclers, electrophoresis unit and automatic sequencer were excluded from these calculations.The negative control added to each run was negative, suggesting the absence of cross-contamination and sample mix-up. In addition, none of the generated sequences revealed an unusual high number of mixed bases. Finally, a maximum likelihood tree was inferred using the sequences that passed the quality control assessment . SequencThe objective of this study was to design and validate an in-house system that could be implemented in Cuba, a resource limited setting with an HIV-1 epidemic with various subtypes and CRFs. To validate the here designed in-house system, the performance characteristics of the test were evaluated, including the determination of the lower limit of amplification and the assessment of accuracy (trueness and precision). Finally, the applicability of the test was evaluated in a clinical setting. Samples for validation experiments were carefully selected, so that the overall subtype distribution of the panels reflected the diversity in the Cuban epidemic. In addition, samples displaying a broad range of viral loads and complex drug resistance profiles were chosen when possible.For HIV-1 genotypic drug resistance testing, no gold standard is available. Therefore, trueness was assessed by comparing the in-house assay with the ViroSeq Genotyping System version 2.0. The use of this commercial test as a reference standard has been frequently described in previous studies ,22–32 anThe acceptance criteria defined in advance were met for all the evaluated performance characteristics, demonstrating a high degree of accuracy of the in-house assay. The in-house assay was able to amplify samples with low-level viremia, ranging from 84 to 529 RNA copies/mL. Although only a limited number of samples have been included in this analysis, it suggests that the lower limit of amplification is well below the threshold of 1,000 RNA copies/mL for the diverse strains circulating in Cuba. The in-house assay showed very good agreement with ViroSeq on NT level (98.7% ± 1.0) and minor differences were mainly caused by base mixtures. ViroSeq detected more base mixtures than the in-house assay, suggesting an increased sensitivity of ViroSeq in detecting NT mixtures, which has previously been reported for another in-house method by Saravanan et al. . Little A disadvantage of the in-house assay is that the risk for cross-contamination is higher than with ViroSeq, as it uses a nested PCR and no dUTP/UNG contamination control. This required more attention to avoid cross-contamination and the introduction of stringent precautions and quality control, such as the inclusion of a negative control in every run. The negative results of the negative controls in each run, the good quality of the sequences, the high specificity of the in-house assay as here assessed, and the phylogenetic results indicate that cross-contamination or sample mix-up had not happened using the implemented laboratory procedures.Furthermore, the intra- (repeatability) and inter-assay variability (reproducibility) were evaluated by generating different replicates per sample in the same run by the same technician and in different runs by different technicians respectively, both under similar assay conditions. Minimal differences were observed between resistance patterns of replicates of the same sample and NT sequence identities were excellent, even at viral loads below 1,000 RNA copies/mL. The good results for precision can be explained by the strict adherence to good laboratory practices and is an indication for the robustness of the assay.Subsequently, the in-house assay was evaluated in a clinical setting. Although the test was rather successful in routine practice and displayed a very good sensitivity for a broad range of HIV-1 subtypes, there is still a need for improvement as 16% of the samples could not be amplified in a single attempt. Although general guidelines for the pre-analytical steps and detailed standard operating procedures for the analytical steps do exist, deviations from these rules at some instances could not be excluded and could have contributed to the amplification failures. Furthermore, it is possible that low viral loads impacted the amplification success rate negatively. However, viral load tests are not systematically performed for all patients in Cuba, and if done, mostly not on the same isolation date as for genotyping, so viral load data were not available. Additionally, primers were designed in a way that they bind to the diverse strains, but strain specific mismatches at the primer binding sites could not be investigated for the amplification failures as these patients had not been tested yet with alternative methodologies and no sequences were yet available. Therefore, in future studies performance should be verified on a broad spectrum of samples with known viral load and quality indicators should be monitored regularly.Of the samples for which an amplicon could be generated, 99% were sequenced successfully using the in-house method. Generated sequences covered codons 1–99 of PR and 1–335 of RT, which is more than the minimal region for which sequence information must be collected according to the WHO guidelines [The in-house test provided a more affordable alternative to commercial assays as cost for reagents and disposables was less than half that of the ViroSeq HIV-1 Genotyping System . ExpensiIn conclusion, this study describes the design, evaluation and implementation of an accurate and affordable HIV-1 drug resistance assay that is applicable to various subtypes and CRFs in Cuba. The assay made the assessment of drug resistance against PR inhibitors and RT inhibitors possible for individual patient management as well as in surveillance studies. This is an important weapon against the accumulation of drug resistance in HIV-1 patients failing antiviral therapy and the subsequent spread to newly infected patients.S1 FigTrueness-IH and-VS sequences generated with respectively the in-house and ViroSeq assay to evaluate trueness (green); PC Positive Control; Reprod-A, -B and -C Replicates generated to evaluate reproducibility (red); Repeat-A, -B and -C Replicates generated to evaluate repeatability (blue); The subtypes and CRF reference dataset (black). Branches whithout taxa represent sequences for the evaluation of applicability. Only bootstrap values >70 are shown.(PDF)Click here for additional data file.


DFNB49 mutations in deaf Roma from 2 Central European countries (Slovakia and Hungary), and 2) to analyze a possible common origin of the c.1331+2T>C mutation among Roma and Pakistani mutation carriers identified in the present and previous studies.In the present study we aimed: 1) To establish the prevalence and clinical impact of MARVELD2 gene in a group of 143 unrelated hearing impaired Slovak Roma patients. Simultaneously, we used RFLP to detect the c.1331+2T>C mutation in 85 Hungarian deaf Roma patients, control groups of 702 normal hearing Romanies from both countries and 375 hearing impaired Slovak Caucasians. We analyzed the haplotype using 21 SNPs spanning a 5.34Mb around the mutation c.1331+2T>C.We sequenced 6 exons of the One pathogenic mutation (c.1331+2T>C) was identified in 12 homozygous hearing impaired Roma patients. Allele frequency of this mutation was higher in Hungarian (10%) than in Slovak (3.85%) Roma patients. The identified common haplotype in Roma patients was defined by 18 SNP markers (3.89 Mb). Fourteen common SNPs were also shared among Pakistani and Roma homozygotes. Biallelic mutation carriers suffered from prelingual bilateral moderate to profound sensorineural hearing loss.GJB2 negative Roma cases with early-onset sensorineural hearing loss.We demonstrate different frequencies of the c.1331+2T>C mutation in hearing impaired Romanies from 3 Central European countries. In addition, our results provide support for the hypothesis of a possible common ancestor of the Slovak, Hungarian and Czech Roma as well as Pakistani deaf patients. Testing for the c.1331+2T>C mutation may be recommended in  MARVELD2 gene (MAL and Related proteins for Vesicle trafficking and membrane Link Domain containing 2), also known as TRIC and included in the autosomal recessive group, is located on the chromosome 5q13.2 and linked to the DFNB49 locus , SIFT (http://sift.jcvi.org) [accessed 20 April 2014] and MutationTaster (mutationtaster.org) [accessed 20 April 2014] software were used to assess the potential pathogenicity of the novel variant.PolyPhen2  spanning 5.34 megabases around the c.1331+2T>C mutation were genotyped. Polymorphisms were selected from the dbSNP database with respect to their chromosomal position and minor allele frequency (MAF) value. Haplotype analysis was performed in seventeen c.1331+2T>C homozygous patients: 5 Slovak probands, 7 Hungarian probands, 5 Czech  and 4 Pakistani patients from 2 families (2 relatives homozygous for c.1331+2T>C mutation and 2 relatives with homozygous mutation c.1183-1G>A).A control group of 20 unrelated hearing impaired Roma patients and 36 unrelated normal hearing Roma individuals without the c.1331+2T>C mutation was genotyped along to determine genetic variability of the selected SNP markers. All markers were genotyped by Sanger sequencing using ABI BigDye v3.1 chemistry after standard PCR amplification  and ExoSAP  purification.Hearing thresholds were recorded by pure tone audiometry (PTA) or auditory steady state responses (ASSR), depending on the subject’s age and cooperation.MARVELD2 did not reveal any other pathogenic mutations. Additionally, five polymorphisms  were identified in homozygous state in five individuals and one variant of unknown pathogenicity (c.950G>A) (rs148416461) was found in heterozygous form. Bioinformatic tools predicted the c.950G>A variant to be probably pathogenic .Screening of 143 unrelated hearing impaired Roma individuals from Slovakia detected the c.1331+2T>C mutation in a homozygous state in five (3.5%) and in a heterozygous state in one affected individual. Subsequent sequencing of Screening of 200 unrelated normal hearing Romanies revealed nine individuals heterozygous for the c.1331+2T>C mutation. However, this mutation was not found in the group of 375 hearing impaired non-Roma (Caucasian) patients.Analysis of the c.1331+2T>C mutation in the group of 85 unrelated deaf Hungarian Romanies identified seven homozygous and three heterozygous patients. In this group, the prevalence of c.1331+2T>C homozygotes was 8.23%. In the control group of 502 normal hearing Hungarian Romanies, the c.1331+2T>C mutation was found in 5 heterozygous individuals.The observed frequency of the c.1331+2T>C allele in Slovak Roma hearing impaired patients was 3.85% (95% CI = 1.6–6.1%), while in unaffected Slovak Roma population the frequency was 2.25% (95% CI = 0.8–3.7%). The allele frequency of the mutation in the sample of Hungarian hearing impaired Roma patients was 10.0% (95% CI = 5.5–14.5%), which is nearly three times as much as in the Slovak deaf study group. On the other hand, the 0.5% (95% CI = 0.45–1.70%) allele frequency in the Hungarian healthy Roma population was approximately four times lower than in Slovak healthy Romanies. In the sample of 375 non-Roma Slovak patients with hearing loss, the c.1331+2T>C mutation was not detected.To confirm the common ancestry of the c.1331+2T>C mutation in all patients from our study, as well as among the Pakistani and Czech subjects where the mutation was first detected, we analyzed 21 SNPs located within approximately 5.34 megabase region around the mutation. The identified common haplotype defined by 18 SNP markers (approx. 3.89 megabases), was shared by all Czech, Slovak and Hungarian Roma patients in a homozygous state, suggesting a common ancestor for this mutation in Central European Roma patients . To deteFourteen common SNPs shared among two members of one Pakistani family and all Roma patients carrying the homozygous c.1331+2T>C mutation were found. In contrast, members of the second family of Pakistani origin harboring the c.1183-1G>A mutation possessed an identical homozygous region with the both the Pakistani and Roma patients of only nine SNPs wide .MARVELD2 gene.Relevant audiological data were only available for Slovak hearing impaired subjects (n = 6) from five families. The age of these patients ranged between 6–23 years. All of them suffered from prelingual non-progressive bilateral moderate to profound sensorineural hearing loss , with a In the Hungarian subgroup, all homozygous probands had bilateral severe to profound deafness with minimal residual hearing. The hearing loss onset was also in the prelingual period (diagnosed between 0–12 years).MARVELD2-associated deafness are known in the existing literature , but was detected in the NHLBI GO Exome Sequencing Project in one of 6502 individuals in heterozygous form (http://evs.gs.washington.edu/EVS/) [accessed 20 April 2014]. The structure-function relationship or clinical effect of the c.950G>A substitution are not yet known. The In silico programs PolyPhen2, SIFT and MutationTaster predicted c.950G>A to be probably pathogenic, with possible impact on protein features or splicing of mRNA. Further studies are required to evaluate the impact of this unknown variant.The other five pathogenic mutations of the akistani  and c.15By analyzing the c.1331+2T>C mutation in a Non-Roma (Caucasian) population (in a control group of 375 hearing impaired Slovak Caucasians), we have confirmed previous findings that occurrence of this mutation in Europe is probably restricted to the Roma population, and is still not known in Slavic Caucasian ethnicity .MARVELD2 homozygous patients in Europe have only been detected in Czech Republic [Until now, Republic , SlovakiWhen looking for the possible common ancestry, key historical events concerning the Central European Roma population must be taken into account. These 3 neighboring countries were inhabited by different Roma subpopulations. In the Czech Republic, nearly the entire original population of Bohemian Romanies was exterminated during the World War II holocaust in the Nazi concentration camps. Subsequently, according to the communist regime’s social engineering projects in former Czechoslovakia, Roma were moved from their original rural settlements in Slovakia (particularly from its eastern parts) to tenement blocks in Czech cities , 36. ConIn the present study, we tested whether patients with the c.1331+2T>C mutation share a common ancestral haplotype. In seventeen Roma patients , we genotyped 21 biallelic polymorphisms spanning approxximately 5.34 Mb around the mutation. Haplotype analysis revealed a common haplotype of 18 SNPs (approx. 3.9 Mb) which was present in the homozygous state in all Roma c.1331+2T>C patients but none of the 56 control Roma individuals. These data support the hypothesis of common ancestor for the c.1331+2T>C mutation in all analyzed Slovak, Czech and Hungarian Roma patients.MARVELD2 positive deafness cases are only known from Pakistan, Iran and Czech Republic  [MARVELD2 mutations, we only found 9 shared SNP markers  , 9, 10,  Europe) , 39. We  markers .MARVELD2 related deafness  may further illuminate the relative importance of MARVELD2 gene as a deafness cause in Roma. According to recent data and our opinion, its testing is not necessary in the majority (Caucasian) population.Our study provides the first record of


Soil salinity is a major constraint to rice production in large inland and coastal areas around the world. Modern high yielding rice varieties are particularly sensitive to high salt stress. There are salt tolerant landraces and traditional varieties of rice but with limited information on genomic regions (QTLs) and genes responsible for their tolerance. Here we describe a method for rapid identification of QTLs for reproductive stage salt tolerance in rice using bulked segregant analysis (BSA) of bi-parental recombinant inbred lines (RIL). The number of RILs required for the creation of two bulks with extreme phenotypes was optimized to be thirty each. The parents and bulks were genotyped using a 50K SNP chip to identify genomic regions showing homogeneity for contrasting alleles of polymorphic SNPs in the two bulks. The method was applied to ‘CSR11/MI48’ RILs segregating for reproductive stage salt tolerance. Genotyping of the parents and RIL bulks, made on the basis of salt sensitivity index for grain yield, revealed 6,068 polymorphic SNPs and 21 QTL regions showing homogeneity of contrasting alleles in the two bulks. The method was validated further with ‘CSR27/MI48’ RILs used earlier for mapping salt tolerance QTLs using low-density SSR markers. BSA with 50K SNP chip revealed 5,021 polymorphic loci and 34 QTL regions. This not only confirmed the location of previously mapped QTLs but also identified several new QTLs, and provided a rapid way to scan the whole genome for mapping QTLs for complex agronomic traits in rice. Rice is the most important cereal crop with maximum contribution to the global food requirements. The world cereal production is about 2500 mt with rice alone contributing 675 mt, accounting for 27 per cent of total . Importaet al. [Saccharomyces cerevisiae) also demonstrated the effectiveness of microarray-based BSA for QTLs mapping [et al. [S. cerevisiae). Magwene et al. [S. cerevisiae), but two recent studies have used NGS-assisted BSA for QTL mapping in rice; Takagi et al. [et al. [Wolyn et al.  first pr mapping , 17. Ehr [et al.  utilizine et al.  proposedi et al.  mapped s [et al.  mapped Q [et al. , 23. How [et al. . SSI is  [et al. . Analysi [et al. . Salt to [et al. , 28.Saltol, was mapped on the short arm of chromosome 1 between RM23 and RM140 (10.7–12.2 Mb), using recombinant inbred lines (RILs) developed from salt tolerant landrace Pokkali and salt sensitive variety IR29 [et al.[Saltol region between 11.0 Mb and 12.2 Mb and suggested that Saltol may involve the SKC1gene located at position 11.46 Mb, first identified in Indica landrace Nona Bokra [2:3 mapping population derived from Nona Bokra and susceptible Japonica variety Koshihikari identified several QTLs for salt tolerance on chromosomes 1, 4, 6, 7 and 9, including major QTLs for shoot K+ concentration on chromosome 1 (qSKC1) and shoot Na+ concentration on chromosome 7 (qSNC7) [A major QTL for salt tolerance at the seedling stage, namely 9 [et al. reportedna Bokra . A study (qSNC7) . There a (qSNC7) –35.QTLs for seedling stage salinity tolerance have been mapped on chromosomes 1, 5, 6 and 7 in earlier studies –39, but The stress intensity (SI) for grain yield under moderate sodic (pH ~9.5) and high sodic (pH ~ 9.9) environments as compared to control micro plots (pH ~7.5) was 0.51 and 0.85, respectively. Homogeneity of error variance across three years of data in 2009, 2010 and 2011 was evaluated by the F test. Combined analyses of variances for genotypes in three seasons were determined by comparing the genotype × season interaction for each trait. Significance levels were also determined for the combined analysis . The CSRAll the parameters showed transgressive segregation and near normal distribution, suggesting involvement of multiple genes with quantitative inheritance . AnalysiThe top and bottom 30 RILs with extreme phenotypes were selected based on consistent SSI for grain yield across the two levels of stress, moderate and high sodicity in three seasons , panicle length (-0.21and -0.25) and grain per panicle (-0.21 and -0.28), respectively.2 = 0.01).Array based SNP genotyping of pools of 10, 20, 30, 40 and 50 RILs was done to identify the optimum pool sizes for maximum heterogeneity  of alleles in the pool, so that the bulked tolerant and bulked sensitive pools did not differ for alleles other than those in the associated QTL regions. Out of 50,051 SNPs on the chip array, 41,327 were monomorphic in all the pools and 2,903 had missing calls in one or more pools. Hence, we analyzed the remaining 5,821 SNPs that showed heterogeneity in any of the five pools. As expected, pool of 10 RILs showed the minimum number of 4,115 (70%) heterogeneous SNPs, with successive increase in heterogeneity in higher pools of 20 (86.1%), 30 (90.13%), 40 (91.4%) and 50 (92%) RILs . Thus, oThe BSA method using high density SNP genotyping was applied to CSR11/MI48 RILs segregating for sodicity tolerance and several new QTLs were identified for SSI for grain yield. After optimization of the RIL pool size, SNP genotyping of CSR11, MI48 and the bulked-tolerant (BT) and bulked-susceptible (BS) pools of 30 RILs each were done using 50K rice SNP chip. Out of 50,051 SNPs on the chip we have analyzed 6,068 SNPs that were polymorphic between BT and BS pools . InteresqSSIGY2.1, qSSIGY2.2 and qSSIGY2.3 at positions 9.9, 23.1 and 34.8 Mb. Source of tolerance for QTLs qSSIGY2.1 and qSSIGY2.3 was CSR11 and at qSSIGY2.2 tolerant allele was coming from MI48. The QTL qSSIGY2.1, was marked by a homogeneous SNP in single copy rice gene Os02g17190 (MYB family transcription factor) know to be associated with stress tolerance. Five QTLs were mapped on rice chromosome 3 and tolerant alleles in four of these were from sensitive parent MI48 and one from the tolerant parent CSR11. QTL qSSIGY3.5 was marked by SNP in the gene Os03g55490 having kinase activity. Two QTLs were mapped on chromosome 5 at 23.5 and 26.1 Mb positions with tolerant allele contributed by CSR11. At 26.1 Mb, homogeneous SNPs were present in two consecutive genes , but it was considered one QTL because of their adjacent location. Five QTLs were identified on rice chromosome 6, out of which three had tolerant allele coming from CSR11 and remaining three from MI48. One QTL was mapped on rice chromosome 8, marked with SNP in gene Os08g06110, which is MYB transcription factor. One QTL was identified on rice chromosome 9 and source of tolerant allele for SSIGY was MI48. In the gene Os09g02790 that codes for a zinc binding protein. Another QTL was identified on rice chromosome 12 at position 23.1 Mb with tolerant allele coming from sensitive parent MI48. We found that out of 21 QTLs reported here, five were in the regions reported earlier in the CSR27/MI48 F2/F3 and RILs with one common parent [Three QTLs were identified on chromosome 1 at positions 32.3, 35.0 and 39.5 Mb with tolerant alleles contributed by CSR11. A homogeneous SNP was identified in single copy rice gene Os01g55530 at 32.3 Mb, which is reported to be involved in metabolism, signal transduction, and response to stress. Three QTLs were identified on chromosome 2, namely n parent , the remWe did bulked segregant analysis of BT and BS pools of 30 RILs each along with the parental lines CSR27 and MI48 using 50K SNP chip to validate the QTLs reported earlier in this population . Out of et al. [2/F3 populations have mapped 25 major QTLs on chromosomes 1, 2, 3 and 8 and Pandit et al. [et al. [et al. [Earlier, Ammar et al.  using CS [et al.  has repoet al. [qNa/KSV2.1/qNa/KSV2.2, qNa/KSV2.3, qClLR-2.1, qClSV2.2 at 4.4–8.98 Mb on chromosome 2. This QTL was not validated in the present study with the strict criteria of homogeneity in both the pools but at two positions  homogeneity of alleles in at least one bulk was present , qClLV-8.1a (9.2–10.29 Mb), qClLV-8.1b (10.29–19 Mb), and qSSISFH-8.1 (9.2–10.29 Mb). Here we found three QTL regions at 3.1, 7.9 and 14.8 Mb positions which are nearby the qNaSH-8.1, qClLV8.1b and qSSISFH8.1 QTLs. They reported most significant QTL for SSI for spikelet fertility at high salt concentration qSSISFH-8.1on chromosome 8 in the marker interval HvSSR08-25-RM3395 (9.2–10.29 Mb). This QTL was co-located with qClLV-8.1a for Cl- content in the leaves at vegetative stage. We found two QTLs, qSSIGY8.2 (7.9 Mb), with tolerant allele coming from CSR27 and qSSIGY8.3 (14.8 Mb), with tolerant allele coming from MI48. On chromosome 12, Pandit et al. [qNaSV-12.1 between 12.11–17.53 Mb. In our study we found two QTL regions at 9.4 and 23.5 Mb position, at 11.2–14.8 Mb position one QTL region was showing one homogeneous and one heterogeneous locus.Ammar et al.  has repoet al. [In our study out of 34 QTLs of CSR27/MI48 population five QTLs were reported earlier in the and found 29 novel QTL regions on rice chromosomes 1, 2, 3, 5, 6, 9, 11 and 12 due to dense SNP map of polymorphic locus covering all regions of the genome. Earlier highest 41 QTLs have been reported by Ghomi et al.  on all tet al. , 40–44 bs) and non-stress yield (Yp) was 0.46 for the moderate stress and 0.22 for severe stress conditions, which was almost similar to the present study for moderate sodicity (0.63) and high sodicity (0.17) stress.Analysis of variance revealed highly significant variation among the RILs for all the nine traits under the three stress environments, suggesting the presence of sufficient genetic variation and ample scope for the improvement of rice for reproductive stage sodicity tolerance. The stress intensity of phenotyping for grain yield indicates the moderate and high degree of sodic stresses. The degree of high sodic stress was almost two times more than that of moderate sodicity. Hence, the population expressed 70 per cent grain yield reduction under high stress over moderate sodic stress. Fernandez  has showet al. [et al. [Grain yield is a complex quantitative trait, greatly affected by environment. Hence, selection of superior genotypes based on yield per se is not effective. The association of plant characters and stress indices with yield thus, assumes special importance in formulation of selection criteria for yield. Stress susceptibility index (SSI) has been proposed for identifying genotypes with superior performance under stress as well as non-stress environments . It is wet al.  also repet al. , 50. Ali [et al.  have ind2 population using RFLP and RAPD markers [et al. [et al. [2 individuals and concluded that pooling larger numbers of individuals increases the probability that the two pools will not differ for alleles other than those linked with the trait. They have concluded that if the marker interval is larger, then the pooling size should be smaller. As the marker interval increases to 10 cM, fewer than 10 individuals can be pooled to maintain the same probability of double crossovers. Thus the conclusion is to use more number of individuals to create the bulks and simultaneously decrease the size of marker intervals examined. In the present study with high density genotyping we found empirically that a pool of 10 RILs was not enough (only 70% heterogeneity) and at least 30 RILs should be pooled to get heterogeneity for more than 90 percent of the loci in order to reduce the number of false positives. As expected the heterogeneity increased with the increasing pool size, but the maximum gain was between pool 10 to 30 where it increased from 70% to 90.2%, after this there was very little increment of heterogeneity; less than 2 percent by adding another 20 RILs to the pool. Hence, a pool of 30 RILs was considered optimal. Magwene et al. [The present method of QTL mapping by BSA using high-density SNP chip provides a rapid and efficient means for the identification of molecular markers linked to complex agronomic traits. BSA method was first applied to identify markers linked to specific genes in F markers , 6. Geno markers , 52–55.  markers , incorpoe et al.  based onet al [High density array chip however has the limitation of not being able to detect all the heterogeneous loci as we have observed in 1:1 ratio of two DNA samples which could detect only 86% loci as heterozygous, which is far below the expected 100%. Thus the high-density chip array has some inherent limitations of not being able to detect the allelic heterogeneity in the pool accurately. This is apparently because the Affymetrix allele-calling algorithm has been optimized for inbred individuals (homozygous) calls and this is presently being improved for the true heterozygote calls for pooled population samples with minor alleles. In our study of QTL mapping by BSA, this would lead to identification of false positive associations as QTLs by 1%. Earlier Takagi et al  describeet al. [At reproductive stage there are very few reports on QTL mapping for salt stress. Recently Hossain et al.  has repoet al. [et al. [For validation of 50K BSA approach it was compared with the SSR based QTL mapping described earlier by Ammar et al.  and Pand [et al.  in the s [et al. . Hence, A major disadvantage of QTL mapping by BSA is that we do not have an estimation of the proportion of variation explained by the QTL. For this at least we need to genotype sufficient number of individual RILs so that effect of different combination of genes can be examined. This individual RIL genotyping could be restricted to only those potential QTLs (SNPs) identified by BSA so that the entire procedure remains cost effective. The method of QTL mapping we have reported here is a fast method to make physical map of any particular trait using a reasonably high density array, which is cost effective and time saving.+ excluder and has tolerance to sodic (pH 9.6–9.9) soils [A mapping population of 216 recombinant inbred lines (RILs) was developed from a cross between rice varieties CSR11 and MI48 using single seed descent method. CSR11 is a salt-tolerant, Indica rice variety bred at Central Soil Salinity Research Institute (CSSRI), Karnal, India. It is a Na9) soils . In cont10 plants of 216 RILs along with two parents. The phenotyping was performed in 2009 (F10 plants), 2010 (F11 plants) and 2011 (F12 plants) under precisely controlled micro plots having two stress levels, moderate sodicity (pH ~ 9.5) and high sodicity (pH ~ 9.9), and non-stress (pH ~ 7.5) control plots at CSSRI, Karnal. Thirty day-old seedlings were transplanted in concrete microplots in simple lattice design for evaluation under normal, moderate and high sodicity stresses. At each stress level, genotypes were replicated twice with row×plant spacing of 20×15 cm. This micro plot facility has been created using brick mortar concrete materials having dimension of 6 m (length), 3 m (width) and 1 m (depth) under rainout shelter. The lysimeters were filled with soil which is uniform throughout the depth. The desired levels of moderate sodicity (pH ~ 9.5) and high sodicity (pH ~ 9.9) simulating the natural field conditions but without the field soil heterogeneity were created by adding required amount of NaHCO3 in the soil. The CSR27/MI48 RIL population was phenotyped for salt stress susceptibility index and other salt tolerance parameters as described earlier [Phenotyping of CSR11/MI48 RILs for salt tolerance parameters was commenced on the stabilized Fet al.[Homogeneity of error variance across the three seasons was tested by the F test  and combet al. using SA® 2.0 Assay Manual Target Prep Protocol was followed for DNA amplification, fragmentation, chip hybridization, single-base extension through DNA ligation and signal amplification. After Staining and scanning on the GeneTitan® Multi-Channel Instrument CEL file was extracted using the Affymetrix Genotyping Console™ v4.1 software package. SNP annotation library was used as standard reference set and development quality check was calculated. Threshold DQC (>0.85) and higher SNP call rate (>95%) was used for further analysis. To see the technical difficulties and sensitivity of array different proportion of two pure DNA samples of CSR11 and MI48 were used in ratio of 1:1, 1:2, 1:3, 1:4 and 1:5.For optimization of RIL pool size for BSA, genomic DNA was isolated from 50 random RILs of the CSR11/MI48 mapping population using CTAB method with minor modifications . Purifie2) used here was not the exact match with the MI48 used in the original cross to develop the mapping population, so if there was polymorphism between BT and BS and with monomorphic parental lines P1 and P2, then we considered the data on P1 (CSR11 and CSR27) as true result and assigned the alternate alleles to P2, as ignoring these loci may result in loss of important QTL regions. A QTL was called if homogeneous alleles were present in both tolerant and sensitive, either contributed by tolerant or susceptible parent. Sorting of polymorphic locus was done on Microsoft Excel sheet on the basis of chromosome number and then by their physical position in ascending order. Physical map of QTL regions was prepared in MapChart2.2, polymorphic homogeneous SNPs were color coded as red or green .SNP genotyping of parental lines CSR27, CSR11 and MI48 and the bulks of extreme tolerant and extreme susceptible RILs was done using the 50K chip (OsSNPnks). It was noticed that the sensitive parent MI48  and salt-sensitive MI48 (P2) were evaluated during 2009, 2010 and 2011 under moderate and high sodicity. The RILs showed significant variability for the nine salt tolerance parameters evaluated. All the parameters showed transgressive segregation and near normal distribution, suggesting involvement of multiple genes with quantitative inheritance. Tolerant CSR11 showed the least SSI in moderate (0.44) and high sodicity (0.75) as compared to the sensitive MI48 with SSI of 1.24 and 1.02, respectively across the three seasons.Parental lines and RILs derived from the cross between salt tolerant CSR11 (P(TIF)Click here for additional data file.S2 FigThe extreme top and bottom 30 RILs of CSR11/MI48 population were selected on the basis of their consistence SSI for grain yield undermoderate and high soidicity in three seasons . Red Bar, Top 30 RILs; Blue bar, Bottom 30 RILs(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableGreen background shows slected extreme tolerant RILs; Red background shows selected extreme susceptible RILs; RIL, recombinant Inbred Lines; SSIMS, stress susceptibility index at moderate stress; SSIHS, stress susceptibility index at high stress(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableGreen and red shaded homogeneous SNPs represent QTL regions with contribution of tolerant allele from the tolerant and sensitive parent, respectively, where as plain text with heterogeneous SNPs shows lack of association with the trait.(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 TableGreen and red shaded homogeneous SNPs represent QTL regions with contribution of tolerant allele from the tolerant and sensitive parent, respectively, where as plain text with heterogeneous SNPs shows lack of association with the trait.(XLSX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


To report on 4-year follow-up of corneal higher-order aberrations and daily visual functions of myopic patients after laser in situ keratomileusis (LASIK).One hundred thirty four eyes of 67 patients who underwent LASIK guided by aspherical ablation were included in this study. The vision, corneal spherical aberration (SphA) and Coma were recorded before LASIK and at 6 month and 4 year after LASIK. The evaluation of the questionnaire about daily visual functions was performed by the same physician after LASIK.p=0.040; r=-0.688, p=0.040 at 6 month and 4 year post-LASIK respectively).No eye decreased the BCVA during 4 year follow-up. The effect index and safety index were 1.08±0.16, 1.11±0.17 and 1.12±0.16, 1.13±0.14 respectively at 6 month and 4 year post-LASIK. After LASIK the corneal SphA and Coma were significantly increased, however the difference between 6 month and 4 year post-LASIK was no statistical significance. Most patients (94.3%-92.4%) felt satisfaction or high satisfaction about the ability to perform each daily visual function after LASIK. Meanwhile there was still about 7.4%-9.2% patients who complained that they could not drive at night. Further analysis showed that the score of driving at night was negative correlation with corneal SphA (r=-0.645, Our four-year follow-up outcomes indicated that the myopic patients after LASIK had the long-term stable corneal aberration and satisfaction of daily visual functions. Myopia is world problem, and the Laser in Situ Keratomileusis (LASIK) as a main therapy for myopia had been used for over twenty years, which brought good therapeutic effect for myopic patients. However some patients even had acuity vision, they often complain that they had poor vision at night, especially the difficulty of driving at night.Sixty seven patients  who underwent LASIK guided by aspherical ablation at the Guizhou Ophthalmic Hospital of the Affiliated Hospital of Zunyi Medical College from March 2010 to March 2011 with 4 years follow-up after surgery were included in this study. The patient age at the time of surgery was (24.56±5.57)  years. The preoperative manifest spherical equivalent (SE) was (-5.10±1.88) D . Patients were excluded if they had corneal surgery, keratoconus, flap complications, eccentric ablation, epithelial in growt.The routine examination about refractive surgery was performed. We used the Optikon-2000 and KeratronScout  to get the corneal topography. Every eye was checked 4 times, and then the best topography was selected to analyze the corneal higher-order aberrations. All these examinations were performed by one doctor. The LASIK procedure included mechanical flap preparation using Moria II microkeratome  with an intended 110-µm corneal flap. Excimer ablation guided by aspherical ablation for all eyes was performed using SCHWIND ORK Esiris excimer laser platform. An ablation zone of 6.5mm has been selected.The refraction, uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), anterior corneal spherical aberration (SphA) and Coma were recorded before LASIK and at 6 month and 4 year after LASIK. The evaluation of the questionnaireThe results are expressed as mean ± standard deviation (SD). All statistical analyses were performed using SPSS for Windows . The difference of corneal aberrations between pre-operation and post-operation were analyzed using analysis of variance. The score of visual functions between 6 month and 4 years after surgery was used for independent-sample t test to compare; and used Pearson correlation to analyze the correlation of the corneal aberration and the score of daily visual function after operation. A value of P<0.05 was considered statistically significance.All eyes had 0.8 or more UCVA, and no eye lost 1 line or more in BCVA during 48 month period after treatment. The effect index and safety index were 1.08±0.16, 1.11±0.17 and 1.12±0.16, 1.13±0.14 respectively at 6 month and 4 year after LASIK. No significant difference between 6 month and 4 years post-LASIK about the effect index and safety index .After LASIK the anterior corneal SphA and Coma were significantly increased, however the difference between 6 months and 4 years post-LASIK had no statistical significance .p=0.040; r=-0.688, p=0.040 at 6 months and 4 years post-LASIK respectively,  felt satisfaction or high satisfaction about the ability to perform each visual function in daily activities after LASIK. However the reduction of night vision was the main complaint in our follow-up. Early follow-up, there had six patients (9.2%) felt negative about the driving at night. At 4 year after LASIK, 5 patients (7.4%) still had the same complaint. The difference of the score of visual function between 6 month and 4 year after operation had no statistical significance . And thectively, .In the present study, we observed the long-term changes of anterior corneal SphA and Coma, and then evaluated the patients’ satisfaction about the daily visual functions after LASIK through questionnaire.It was well known that the BCVA was the most important index to assess the safety after refractive operations.Previous study showed that the third-order and fourth-order aberrations were the main component in human cornea,Though there are good long-term effect and safety, and we also found that most people (94.3% to 92.4%) felt satisfaction or high satisfaction about the ability to perform each visual function in daily activities after LASIK, there still some people complained the visual disturbance at night, especially driving at night. Early stage in our follow-up, 9.2% patients felt negative about the driving at night. Unfortunately, this complaint could not attenuate with time. Even at 4 year after LASIK, still 7.4% patients had the same complaint. However in our 4-year follow-up, no new patient had the same complaint. At six month and 4 year after LASIK, patients had the similar score about the daily visual function assessment. These indicated that at six month after surgery the visual function could satisfy with patients’ daily activities. Even with time, there had yet the stable evaluation about the ability to perform each function. We further analyzed the correlation of the score of visual function and the corneal aberrations. We found that the score of driving at night either at six months or 4 years post-LASIK was negative correlation with corneal SphA. And it was speculated that this phenomenon would not be improved for a long time. So it should be prudently selected to perform for myopic patients who work under high risk environment. However, the correlation of the higher-order aberrations of whole eye and the daily visual function and the longer follow-up should be further explored.


Three headache disorders – migraine, tension-type headache (TTH) and medication-overuse headache (MOH) – are major contributors to population ill-health. Policy-makers need local knowledge of these to guide priority-setting. Earlier we reported the prevalence of these disorders in Zambia; here we describe the burdens attributable to them.This was a cross-sectional population-based survey of adults aged 18-65 years, selected by cluster-randomized sampling in the mostly urban Lusaka Province and mostly rural Southern Province. Interviewers visiting households used a structured questionnaire. Diagnoses made algorithmically applied ICHD-II criteria. Burden enquiry focused on the previous 3 months and the day before interview. Disability was estimated by applying disability weights (DWs) from the Global Burden of Disease Survey 2010.From 1,134 households, 1,085 unrelated adults  were interviewed . The gender- and habitation-adjusted 1-year prevalence of migraine was 22.9%, of TTH 22.8%, of headache on ≥15 days/month 11.5%, of probable MOH (pMOH) 7.1%. Reported mean intensity of migraine attacks was 2.7, representing severe pain. People with migraine spent 10.0% of their time in the ictal state (DW: 0.433); they were therefore 4.3% disabled overall. Disability from TTH was much lower. People with pMOH  were 8.3% disabled overall. Average lost productive time in the preceding 3 months for migraine was 4.1 days from work (6.3% loss) and 4.2 days (4.7% loss) from household work. Losses for pMOH were 4.8 days (7.4% loss) from work and 4.5 days (5.0% loss) from household work. In the population aged 18-65 years (effectively the working population), estimated disability from migraine was 0.98%, with 1.4% of workdays lost, and from pMOH was 0.59%, with 0.53% of workdays lost. Headache yesterday was reported by 28.3% of participants, whose average productivity yesterday was 55.9% of expectation.Zambia loses 1.93% of GDP to headache, and action is required to mitigate this loss and the associated suffering. Structured headache services with their basis in primary care are the most efficient, effective, affordable and equitable solution. They could be implemented within the existing health-care infrastructure of Zambia. These matters require urgent political attention. Three headache disorders – migraine, tension-type headache (TTH) and medication-overuse headache (MOH) – are major contributors to population ill-health. Worldwide, migraine is the seventh leading specific cause of years of life lost to disability (YLDs), responsible for 2.9% of all YLDs, and more than half of all YLDs attributable to neurological disorders -3. This For health policy-makers, all of this is important knowledge. Not only do headache disorders affect very large numbers of people through their high prevalence ,2, imposlocal knowledge to guide decision-making and priority-setting. For headache disorders, this has been unavailable in many countries of the world . Thus the mean estimated disability attributed to MOH per adult with this disorder would be 8.3% (0.220*37.5%).We had data for lost productive time to relate to these estimates, deriving from the HALT questionnaire  Table 22. On aveP = 0.07) but those with pMOH had significantly lower income (2-tailed Student’s t test vs migraine: P < 0.0001). WTP perhaps reflected this: means ranged from USD 1.54 to USD 1.67 per month, or, as a percentage of income, from 1.4 to 1.8%. Differences between the diagnoses in absolute WTP were small and insignificant; in percentage terms, those with migraine were willing to pay less than all others, but this appears artefactual. A total of 39 participants claimed a WTP >5% of their income, 14 (5.6%) diagnosed with migraine , 15 (5.7%) with TTH  and 10 (7.3%) with pMOH .We also had data on WTP for effective health care for headache, if it were available, and on income to set this in context Table . Both quReturning to Tables Similar estimates were possible and informative for pMOH. Individual disability was 8.3%; prevalence 7.1%. The calculated population-level disability from pMOH was 0.59%%. Lost workdays were 7.4%, and therefore 0.53% among the population of 18-65 year-olds, only slightly less than the conservatively estimated underlying disability.In our earlier manuscript, we noted the high headache frequency overall of 10.3 days/month, creating, among the 72.0% with headache, a probability of headache on any particular day of 0.34 . The preJust as our first manuscript showed headache to be highly prevalent in Zambia – no less so than elsewhere in the world  – here wThese estimates were not exactly reflected in other measures of burden. People with migraine lost 6.3% of workdays and 4.7% of household workdays; more than might be expected in the case of the former, but the discrepancy might be explained by the very skewed distribution and a highly-disabled minority. What is at first surprising is that losses from people with pMOH were only 7.4% from workdays and 5.0% from household workdays – higher than for migraine but not greatly so, whereas the disability estimate was two-fold higher. The explanation almost certainly lies in the disruptive effect of episodic migraine, with its short sharp bursts of high disability, which makes coping difficult. People manage chronic pain and disability differently, with more emphasis on distraction and less on palliation . Nonethemore they would pay than they were already. The 39 respondents who would pay >5% of their income  were distributed reasonably equally between the three diagnostic groups, slightly more having pMOH.Perhaps this was why it was migraine, not pMOH, that generated the highest WTP in absolute terms; indeed, pMOH generated the lowest. Of course, other factors come into play with WTP, including affordability, and, set against income, people with migraine (who had the highest incomes) demonstrated the lowest relative WTP. It should be noted that none of these differences was significant. Respondents with pMOH must already be spending significant amounts on medication, presumably with perceived benefit, which would condition their responses to the enquiry. This does question their low WTP, unless they saw the enquiry as asking what Part of our purpose was to inform policy, which the population-level findings do very well. Individual disability of 4.3% attributable to migraine extrapolated to a disability of 0.98% among the entire population aged 18-65 years – effectively the working population. It is important to recognise this. Individual disability of 8.3% among those with pMOH extrapolated to 0.59% in the population. The total was 1.6%, not including TTH (which added little). Lost workdays from migraine (6.3%) and pMOH (7.4%) translated to 1.4% and 0.53% respectively, which together represented a loss of workforce capacity of 1.93%, and this huge economic burden would be reflected in national productivity and gross domestic product (GDP).recalled lost productivity per person over 3 months was much the same .We would like to make comparisons with other countries, but there is no point in doing so when methodological differences may be the explanation of any differences . Therefoversus Zambia’s USD 22.38 bn , slightly higher than that in Zambia, but with nowhere near the level of headache on ≥15 days/month, we have comparative lost workforce capacity data: for all headache, 1.1% . In ZambEnquiry into headache yesterday obviates the problem of faulty recall – highly likely over periods of 3 months . In factIt is a study limitation that we cannot be precise about this. In our earlier paper, we also recorded our inability to complete the diagnostic validation exercise satisfactorily owing to country-based factors beyond our control , but theHeadache disorders are not only common in Zambia but also highly burdensome. However, burden does not sit quite as might be expected: measures of disability and lost productivity show only loose relationships with headache type and frequency.At population level, Zambia effectively loses 1.93% of its GDP to headache – more than in Georgia  and almoUnderlying these losses are the high prevalences of headache disorders, especially pMOH. These are treatable disorders . Much covs 2.1%). In other words, a remediable problem will instead become worse.Inaction is not an option: medication overuse, and MOH, are predictable consequences of lack of health care for migraine and TTH , lack of public health education, and easy access to non-prescription analgesics (hence the urban-rural divide for pMOH: 14.5%


In Korea, cancer is the third leading cause of death among adolescents and young adults (AYAs). However, cancer incidence and survival trends among AYAs (15–29 years) have never been studied in Korea. Therefore, this study aimed to investigate the incidence and relative survival rates and their trends among AYAs in Korea.Cancer incidence data from 1999–2010 were obtained from the Korea Central Cancer Registry (KCCR). Each cancer was classified into subgroups according to the National Cancer Institute Surveillance, Epidemiology, and End Results (SEER) AYA site recode. Percent distributions, age-specific incidence rates, age-standardized incidence rates per million, and annual percent changes (APCs) were calculated for AYAs according to sex. Five-year relative survival rates were estimated for cases diagnosed between 1993 and 2010 and followed up to 2011.P<0.001). The five most common cancers among AYAs were thyroid carcinoma, non-Hodgkin lymphoma, stomach carcinoma, breast carcinoma, and acute myeloid leukemia. In males, the 5-year relative survival rate improved, from 46.5% in 1993–1995 to 75.9% in 2006–2010. In females, the 5-year relative survival rate also improved, from 66.7% in 1993–1995 to 89.1% in 2006–2010.The age-standardized incidence rates of all cancers combined were 196.4 and 367.8 per million for males and females, respectively  ratio: 0.5). The age-standardized incidence rates increased from 208.7 per million in 1999 to 396.4 per million in 2010, and the APC was 6.3% (Our study showed increases in cancer incidence and improvements in the 5-year relative survival rate among Korean AYAs. This study also provides additional data regarding temporal and geographic trends in cancer that may enhance future efforts to identify factors affecting cancer incidence and responses to treatment among AYAs. Cancers in adolescents and young adults  have distinctive characteristics compared with cancers in children and older cohorts.The incidence pattern of specific cancer types differ between AYAs and younger and older patients. In addition, the genetic and histologic patterns of cancers among AYAs differ from those of older patients Over 200,000 new cancer patients are diagnosed annually in Korea In 1980, the Korean Ministry of Health and Welfare started the Korea Central Cancer Registry (KCCR), a nationwide, hospital-based cancer registry Incidence data were collected for Korean AYAs aged 15–29 years who were newly diagnosed with cancer between 1999 and 2010. The incidence data were collected from the KNCIDB of the KCCR and included age, sex, diagnosis date, primary tumor site, morphology, the diagnostic method, and stage at diagnosis.Survival data for individuals aged 15–29 years who were newly diagnosed with cancer from 1993–2010 were obtained from the KNCIDB, and the patients’ vital status was followed until December 31, 2011. The survival analysis was based on the KNCIDB data and mortality data obtained from Statistics Korea.In accordance with the guidelines of the National Cancer Institute SEER Program Cancer sites were coded by primary site and morphology using the International Classification of Diseases for Oncology (third edition) β-1), where β is the slope calculated from a linear regression of log age-standardized incidence rates in a calendar year Age-specific incidence rates per million were analyzed in each diagnostic subgroup according to the age at diagnosis , and age-standardized rates (ASRs) according to sex were calculated using the world standard population defined by the World Health Organization Relative survival rates were estimated according to the time period: 1993–1995, 1996–2000, 2001–2005 and 2006–2010. Relative survival rates according to the diagnostic group were calculated by dividing observed survival by expected survival among comparable groups in the general population From 1999 to 2010, 39,639 cancer cases  were newly diagnosed in AYAs. Of these cases, 14,016 (35.4%) and 25,623 (64.6%) cases were diagnosed in males and females, respectively. According to the age at diagnosis, 6,396 (16.1%) cases were diagnosed at 15–19 years of age, 10,433 (26.3%) cases were diagnosed at 20–24 years, and 22,810 (26.3%) cases were diagnosed at 25–29 years.The number of cases per age group  by sex, age-specific incidence rates, and age-standardized incidence rates among both males and females and M/F ratios according to the diagnostic group are shown in P<0.05). The higher rate among females was largely due to a much higher incidence rate of thyroid carcinomas . Because the incidence rate of thyroid carcinoma was unusually high, the age-standardized incidence rates of all cancers combined were recalculated, excluding thyroid carcinoma (group 8.1). Removing thyroid carcinomas, the overall ASR of cancers was 192.2 per million   than in males (196.4 per million), and for all cancers combined, the male/female ratio was 0.5  .The incidence increased with age, from 150.9 in males and 160.4 in females per million at 15–19 years of age to 170.7 in males and 305.6 in females per million, respectively, at 20–24 years of age. The incidence further increased at 25–59 years of age, to 273.4 in males and 663.3 in females per million, respectively . The incP<0.05). Over the studied time period, there was also a steady increase in the incidence of cancer among AYAs for both males (APC = 3.9%) and females (APC = 7.8%) ( = 7.8%) .P<0.05). The annual percent change in all cancer combined, excluding thyroid carcinoma, was 1.8% (P<0.05) among AYAs (P<0.05) for males and 1.8% (P<0.05) for females (data not shown). Although the incidence of kidney carcinoma also exhibited a notable increase (APC = 9.1%), the number of cases was small.Most cancer sites showed a trend of increasing incidence, with the exception of unspecified malignant neoplasms (group 10) among AYAs (APC  =  −10.9%). For miscellaneous specified neoplasms, NOS (group 9), a rapid increase in the incidence rate was observed for both sexes (APC = 9.8%), but the number of cases was small. For carcinomas (group 10), a large increase in the incidence rate was also observed among AYAs (APC = 9.4%). In particular, among carcinomas, the incidence of thyroid carcinoma showed the most rapid increase . In particular, Hodgkin lymphoma rapidly increased in incidence  (The incidence of lymphomas (group 2) exhibited a large increase among AYAs  .P<0.05), followed by germ cell and trophoblastic neoplasms . Among females, the incidence of most cancer sites was also increased. Carcinomas  and miscellaneous specified neoplasms, NOS , showed the greatest increases. However, osseous/chondromatous neoplasms (APC  =  −0.5%) and germ cell and trophoblastic neoplasms (APC  =  −0.4%) slightly decreased in incidence (P<0.05) and breast  (data not shown).Cancer incidence trends within diagnostic groups were observed to differ by gender. Among males, the incidence of most cancer sites was increased. Miscellaneous specified neoplasms, NOS, showed the largest increase in incidence . AYAs with leukemia and lymphoma showed the most marked improvement in survival from 25.8% (95% CI: 22.9–28.7) and 55.4% (95% CI: 50.6–59.9) in 1993–1995 to 58.8% (95% CI: 55.5–61.8) and 83.6% (95% CI: 80.9–85.9) in 2006–2010, respectively. Conversely, decreases in survival were observed from 1993–1995 to 2006–2010 for other glioma (group 3.2), carcinoma of gonads (group 8.5.3), and carcinoma of pancreas (group 8.6.4)  .Survival rates for thyroid carcinoma (group 8.1) and skin carcinoma (group 7.2) were very high across all time periods. The five-year relative survival rate for thyroid carcinoma among males increased slightly, from 95.3% in 1993–1995 to 99.7% in 2006–2010, whereas the rate was unchanged among females from 1993–1995 (99.9%) to 2006–2010 (100.0%). The survival rates for germ cell and trophoblastic neoplasms (group 6), skin carcinoma (group 7.2), and carcinoma of the genitourinary tract (group 8.5) consistently exceeded 80–90% in all time periods. Conversely, the lowest survival rates were observed for rhabdomyosarcoma (group 5.2); carcinoma of the trachea, bronchus, and lung (group 8.3); and carcinoma of liver and intrahepatic bile ducts (group 8.6.3) .P<0.05) and from 66.7% to 89.1% in females (P<0.05). However, the 5-year relative survival rate for all cancers combined was slightly lower in males than in females, regardless of whether thyroid carcinoma was excluded  showed the greatest increase in survival in both males (32.5%) and females (33.7%). In particular, chronic myeloid leukemia had the largest and second-largest increases in survival in males  and females , respectively.P<0.05), from 1999 to 2010 and that age- and gender-related cancer incidence patterns differed according to the primary site. Moreover, five-year relative survival rates for most cancers improved from 1993–1995 (58.9%) to 2006–2010 (84.8%) among AYAs.This is the first study to investigate cancer incidence, survival and their trends among AYAs using the population-based national cancer registry in Korea. The major findings of this study were that cancer in AYAs showed a trend of increasing incidence, with an increase of 6.3% per year . An increased incidence rate of thyroid carcinoma has also been noted among AYAs in Western countries In this study, a notable trend of increasing incidence was also observed for cervical carcinoma (APC = 6.2%) among female AYAs in Korea. Although the incidence of cervical carcinoma in Korean females of all ages is decreasing (APC  =  −4.3%) In terms of survival, our data are consistent with that reported for other geographic regions. Although the time period in our study differed, the overall cancer survival rate among AYAs in Korea was similar to the rate and significantly improvement reported in the U.S. and Germany. Improvements in relative survival rates among AYAs can be partially explained by advances in cancer detection, more intensive treatments, and increased expertise in adolescent oncology However several important differences should be highlighted in lymphoma and leukemia. In the present study, the most significant improvements in survival were observed in leukemia and lymphoma patients, but the survival rates for leukemia and lymphoma were noticeably lower than in the U.S. and Germany Compared with the survival of patients aged 1 to 10 years, overall survival and disease-specific survival are clinically significantly poorer among AYA patients with acute lymphoblastic leukemia Among AYAs, breast cancer accounts for approximately 7% and 4.9% of all cancers diagnosed in the United States One limitation of our study is that the follow-up period began relatively soon after the diagnosis of cancer, in contrast to the protocols in other studies In conclusion, our study provides representative cancer statistics regarding temporal trends in the AYA population in Korea. In particular, the results showed an increasing trend in cancer incidence and an improving survival trend among AYAs in Korea. These results may support cancer control and prevention plans focusing on AYAs.In the future, further research will help to identify factors affecting cancer incidence and responses to treatment among AYAs. In particular, research on the etiological factors related to the rapid increase in thyroid carcinoma in AYAs is needed.


The influence of age on the performance of percent free prostate-specific antigen (%fPSA) in diagnosing prostate cancer (PCa) in East Asians is controversial. We tested the diagnostic performance of %fPSA in a multi-center biopsy cohort in China and identified the proper age-specific cutoff values to avoid unnecessary biopsies.Consecutive patients with a prostate-specific antigen (PSA) level of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml who underwent transrectal ultrasound-guided or transperineal prostate biopsy were enrolled from 22 Chinese medical centers from Jan 1, 2010 to Dec 31, 2013. The diagnostic accuracy of PSA and %fPSA was determined using the area under the receiver operating characteristic (ROC) curve (AUC). Age-specific cutoff values were calculated using ROC curve analysis.The median %fPSA was much lower in younger patients compared with older patients with a PSA level of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml. The AUC of %fPSA was higher than PSA only in older patients. In patients aged 50 to 59 years, %fPSA failed to improve the diagnosis compared with PSA in these two PSA ranges. Age-specific cutoff values were 24%, 27% and 32% for patients aged 60–69, 70–79 and ≥80 years, respectively, to reduce unnecessary biopsies in men with PSA levels of 4.0–10.0 ng/ml to detect 90% of all PCa.The effectiveness of %fPSA is correlated with age in the Chinese population. Age-specific cutoff values would help avoid unnecessary biopsies in the Chinese population. Prostate cancer (PCa) is the second most common diagnosed malignancy in males globally . AlthougOesterling et al.  establisThe diagnostic efficacy of %fPSA is weaker in Chinese and Korean populations than Western populations , 12. SomThe Institutional Review Board of Shanghai Changhai Hospital approved this study. Written informed consent was obtained from the participants. This retrospective study consisted of a total of 5915 consecutive patients who underwent initial transrectal ultrasound-guided (TRUS-guided) biopsy or transperineal prostate biopsy from Jan 1, 2010 to Dec 31, 2013 in the 22 participating hospitals. All patients were enrolled at the outpatient Department of Urology with/without urinary symptoms. Patients were recommended to undergo biopsy as long as abnormal PSA and/or abnormal digital rectal examination (DRE) results were obtained. A few patients (1.7%) who were not willing to undergo this procedure were completely informed of intensive follow-up, and they were not included in this study. Exclusion criteria were urinary tract infections, urinary retention, recent instrumentation or catheterization of the urethra, and finasteride or hormonal treatment.Peripheral blood samples were obtained within two weeks prior to DRE and prostate biopsy. Prostate volume (PV) was calculated using the equation D1*D2*D3*(π/6), and the three dimensions of the prostate were measured by TRUS. Three types of PSA/fPSA electro-chemiluminescence immunoassays were used in the participating hospitals . Recalibration was performed to WHO standards (PSA-WHO 96/670) using an appropriate correction factor.The Mann-Whitney U test was used to analyze PSA, %fPSA, PV, and age. Simple linear regression analyses were used to evaluate the correlation between clinical parameters and %fPSA. Univariate logistic regression analyses were used to assess correlations between clinical parameters and biopsy results. Clinical parameters that were significantly associated with biopsy results were further tested using multivariate logistic regression analyses. The demographic and clinical variables used in the model included age, PSA, %fPSA and prostate volume. Parameters were included and excluded in a stepwise manner (α<0.05 for inclusion and α<0.1 for exclusion). We further combined PSA and %fPSA using logistic regression to generate a new parameter (%fPSA + PSA) and estimate the influence of age on the performance of the combination of these two parameters. Receiver operating characteristic (ROC) curves were calculated for PSA and %fPSA by plotting the sensitivity versus (1 – specificity) for predicting PCa. The areas under the ROC curves (AUC) were used to measure the diagnostic accuracy of PSA and %fPSA. The statistical significance of any difference was calculated using the z test. Sensitivity, specificity, positive predicted value (PPV) and negative predicted value (NPV) were calculated at all possible cutoff values in the application of %fPSA to diagnose PCa. All of the statistical analyses were performed using the Statistical Package for Social Science (SPSS), v.17.0  and MedCalc v.10.4.7.0 . All of the p-values were two-sided, and p<0.05 was considered statistically significant.Detection rates of patients with PSA levels of 4.0–10.0 ng/ml or 10.0 to 20.0 ng/ml were 25.3% and 36.5%, respectively. The median age of PCa patients was 71 and 70 years in the PSA 4.0–10.0 ng/ml and 10.1–20.0 ng/ml groups, respectively, which were higher than men with negative biopsies  . Total P2 = 0.067, p<0.001 and R2 = 0.039, p<0.001, respectively). The median %fPSA ranged from 12.0% in patients aged 50–59 years to 17.2% in patients aged 70–79 years and 15.7% in patients aged over 80 years in the PSA range of 4.0–10.0 ng/ml  and PSA 10.1–20.0 ng/ml . Results of stratified analysis by age, prostate volume, biopsy scheme and biopsy path are summarized in The diagnostic performances of %fPSA and PSA in different age decades are also illustrated in the ROC curves in The diagnostic accuracy of %fPSA in patients who underwent extended biopsy (10- or 12-core-based biopsy) was not higher than PSA in patients aged 50–59 years with a PSA levels of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml . The results of other age groups were similar to patients who underwent all four types of biopsy protocols .The ability of %fPSA to predict HGPCa was similar to its ability to predict any prostate cancer in each age group. %fPSA significantly outperformed PSA in men aged 70–79 years with a PSA level of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml. The AUC of %fPSA also outperformed PSA in men aged 60–69 years with a PSA level of 10.1–20.0 ng/ml. The AUC of %fPSA was higher than that of PSA in other age groups, but the benefit failed to reach statistical significance.The diagnostic accuracy improvement was minor when PSA and %fPSA were both incorporated in the PCa prediction . NeitherWe categorized patients into six age groups with intervals of five years. The AUC of %fPSA in the PSA range 4.0–10.0 ng/ml was significantly better than PSA in patients aged 75–79 years (p = 0.024), and 70-74-year-olds were borderline significant (p = 0.085). However, %fPSA improved the AUC of PSA in all age ranges except men aged 50–59 years with a PSA range of 10.1–20.0 ng/ml. The highest AUC was 0.673 in patients aged 65–69 years.Different %fPSA cutoff values were evaluated in patients with PSA levels of 4.0–10.0 ng/ml. Sensitivity, specificity, positive predicted value (PPV) and negative predicted value (NPV) were calculated at all possible cutoff values. The highest NPV were 86.6%, 79.5% and 76.1%, respectively for patients aged 60–69, 70–79 and ≥80 years, respectively. We failed to identify a %fPSA value above which the likelihood of detecting PCa is minimal (less than 10%). Nevertheless, if our aim was to detect the dominant majority of PCa (90%), while reduce unnecessary biopsies, we identified age-specific cutoff values to reduce unnecessary biopsies on the basis of maintaining the detection of 90% of all PCa patients in that age group. The cutoff values were 24%, 27% and 32% for patients aged 60–69, 70–79 and ≥80 years, respectively . The NPVThe fixed cutoff of 25% for the PSA range 10.1–20.0 ng/ml yielded a detection rate of 90.2% to 93.4% in patients aged 60–69, 70–79 and ≥80 years. This cutoff would reduce unnecessary biopsies by 17.2% in patients aged 70–79 years while compromising sensitivity by only 9.8%. A cutoff of 25% would avoid 29.0% of unnecessary biopsies in patients aged 80–89 years while maintaining a PCa detection rate of 90.4%. A cutoff value for each age group was not necessary in this circumstance.%fPSA is an important predictor of PCa in patients with a PSA level of 4.0–10.0 ng/ml However, there are huge unexplained differences between races. For instance, the European Association of Urology Guidelines indicated that %fPSA was not applicable for patients with a PSA level >10 ng/ml. However, we demonstrated that %fPSA was not more effective than PSA in younger patients (<60 years), and %fPSA also improved the diagnosis of PCa in PSA ranges of 10.1–20.0 ng/ml and 4.0–10.0 ng/ml in Chinese men in our previous studies. To translate the results in the ROC curve analysis to a binary and clinical applicable test, we tried to identify the specific cutoff values to reduces unnecessary biopsies using the same data set. The underlying reason for all of these findings may be the lower PCa detection rate at the same PSA level in Chinese compared with Western patients. For instance, the PCa detection rate of patients with PSA levels of 4.0–10.0 ng/ml was 25.1% in this Chinese cohort, but it was 40.3% and 43.4% in consecutive clinical patients in the Cleveland Clinic and Durham Veteran’s Affair Hospital, respectively, and 34.8% and 41.4% in the Tarn and SABOR screening programs, respectively , 16. TheChinese, Japanese, Korean and other Asian populations share a similar incidence rate and genetic and social environments. Therefore, the findings of this study may offer a hint for further studies in these countries . HoweverThere is great ethno-geographical variation in the prevalence of prostate cancer. The differences in PSA reference range and detection rate of the same PSA level are significantly different between different races. Our findings illustrate that age-specific cutoff values are helpful to reduce unnecessary biopsies, but the influence of age should also be considered. Multiple age-specific cutoff values, instead of a single cutoff value, should be considered for men with a PSA level of 4.0–10.0 ng/ml to maintain the same level of sensitivity. Increasing numbers of urologists realize that a lower %fPSA poses a continuous risk for PCa similarly to rising PSA. Therefore, we put particular emphasis on the impact of age on the diagnostic accuracy of %fPSA.Previous studies in Western populations identified age-specific cutoffs for %fPSA, but these values were not strongly recommended. Catalona et al.  demonstrWe did not emphasize the application of age-specific cutoffs for patients with a PSA level of 10.1–20.0 ng/ml, although these cutoff values were also identified. First, the AUC of %fPSA was higher in patients with a PSA level of 10.1–20.0 ng/ml than patients with a PSA level of 4.0–10.0 ng/ml. The single cutoff of 25% would maintain a sensitivity of over 90% in all age decades. Second, the detection rate of Chinese patients with a PSA level of 10.1–20.0 ng/ml (36.4%) was similar to consecutive Western patients with PSA levels of 4.0–10.0 ng/ml from outpatient departments , 16. TheSeveral circumstances must be considered when drawing conclusions from this data set. First, there is an inherent limitation of multicenter studies because the biopsies were performed by different physicians and examined by different pathologists. Second, 22 institutes and three different assays were involved in PSA testing. However, the variability of the total and fPSA results between the commercial assays was diminished by calibration. Therefore, these findings should be validated in prospective multicenter studies. Despite this caveat, our data depict the effectiveness of %fPSA in a practical setting for Chinese men. These findings should be validated in prospective multi-center studies. Finally, the age-specific cutoff values were proposed on the basis of maintaining the detection of maximal prostate cancer patients , however, we failed to propose these cutoff values to ensure that all the patients were excluded from biopsy would have a minimal chance of PCa (more than 90% of the patients who were determined to not to undergo biopsy were negative). This drawback should be fully acknowledged in applying this strategy.Our results indicate that age has a strong impact on %fPSA and its diagnostic performance in predicting prostate biopsy outcomes in Chinese men with a PSA level of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml. We do not recommend the use of a fixed cutoff for all clinical patients, but we emphasize the importance of taking age into consideration when using %fPSA to reduce unnecessary biopsies.S1 Table(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Preoperative therapy with angiotensin-converting enzyme inhibitors (ACEI) is common in patients undergoing cardiac surgery. The aim of this study was to evaluate the still-debated impact of preoperative use of ACEI on postoperative renal function in cardiac surgery patients ,2]..2].A total of 624 consecutive patients, who underwent cardiac surgery from July 2012 to October 2013, were evaluated. Data were prospectively collected in our clinic's electronic database and were retrospectively analyzed as to preoperative ACEI therapy. The chi- square test was used for correlations. Endpoints of the study were the development of postoperative acute kidney injury (AKI) and the difference between hospital admission and discharge glomerular filtration rate (GFR). The AKI definition was based on modified RIFLE classification. GFR values were estimated by the MDRD formula.P = 0.981). Mean GFR values on admission day were 65.23 ± 16.89 for ACEI users, and 65.06 ± 19.62 for the rest of the cohort. Mean GFR values on discharge day were 66.77 ± 21.25 and 67.35 ± 25.64 respectively. The difference in GFR at the time of hospital admission and on discharge day had no statistical difference, P = 0.511  were treated with ACEI preoperatively. Overall, 95 patients 15.3%) developed postoperative AKI. Preoperative use of ACEI was not associated with the development of postoperative AKI (5.3% deveThe impact of preoperative ACEI treatment on postoperative renal function after cardiac surgery is still debated. We found no association, neither protective nor harmful, between preoperative ACEI therapy and renal function impairment.


Identifying patients who may deteriorate on the ward and need admission to an intensive care unit (ICU) is imprecise. Numerous variables, alone or in combination, have been used to identify patients who may benefit from ICU admission. A predictive test should be accurate, reliable, easy to measure and cheap. 15 years ago an observational study from our unit showed that both base excess (BE) and lactate, or combination of both, can be used to predict outcome in patients admitted to ITU . We replAs in the 2001 study we examined the records of 148 unselected, consecutive general medical and surgical admissions to St George's Hospital General ITU in September 2013. We recorded blood BE and lactate levels at admission and at 24 hours and calculated corresponding Sequential Organ Failure Assessment (SOFA) scores. The data was taken from the patients' ITU charts and our computer laboratory result system EPR. We compared this data to that presented in 2001. The chi square test was used to determine the statistical difference between groups.44 patients were admitted with acute medical problems, 68 after elective surgery and 36 after emergency surgery . As in 2001 we divided the patients into three groups: those with admission serum lactate ≥ 1.5 or < 1.5 mmol/l, those with serum BE > -4.0 or ≤ -4.0 mmol/l and those with admission BE ≤ -4.0 mmol/l and a lactate ≥1.5mmol/l. Calculated admission SOFA scores were not significantly different between and of the 2001 and 2014 groups.Patients with a deranged admission lactate had a mortality of 10%, versus 4.4% in the normal lactate group. If the lactate failed to clear at 24 hours, mortality was 32% versus 2% if it had cleared. Similarly, patients admitted with a BE ≤ -4.0 mmol/l had a mortality of 15% compared to 4% in patients with a BE >-4mmol/l. If BE was still ≤ -2.5 mmol/l at 24 hours, mortality was 25% versus 0% if the base deficit had improved. A combination of deranged lactate and BE ≤ -4.0 mmol/l showed a mortality of 17% and 33% if it had failed to clear at 24 hours. (Table 1)Base excess and lactate are still indicators of severity of disease and can be used to monitor treatment and response.