Human papillomavirus (HPV) testing is more sensitive than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We evaluated the performance of high-risk HPV (hrHPV) testing in routine screening.In all, 25 871 women (29–61) enrolled in our population-based cohort study were offered both cytology and hrHPV testing. High-risk HPV-positive women with normal cytology and an age-matched subcohort of hrHPV-negative women with normal cytology were invited for repeat testing after 1 and/or 2 years and were referred for colposcopy if they presented with abnormal cytology and/or a positive hrHPV test. The hrHPV-positive women with borderline or mild dyskaryosis (BMD) and all women with moderate dyskaryosis or worse (>BMD) were directly referred for colposcopy. Women with BMD and an hrHPV-negative test were advised to repeat cytology at 6 and 18 months and were referred for colposcopy if the repeat cytology test was abnormal. The main outcome measure was CIN grade 3 or worse (CIN3+). Results were adjusted for non-attendance at repeat testing.The hrHPV-positive women with abnormal cytology had a CIN3+ risk of 42.2% : 36.4–48.2), whereas the hrHPV-positive women with normal cytology had a much lower risk of 5.22% (95% CI: 3.72–7.91). In hrHPV-positive women with normal cytology, an additional cytology step after 1 year reduced the CIN3+ risk to only 1.6% (95% CI: 0.6–4.9) if the repeat test was normal. The CIN3+ risk in women with hrHPV-positive normal cytology was higher among women invited for the first time (29–33 years of age) (9.1% 95% CI: 5.6–14.3) than among older women (3.0% 95% CI: 1.5–5.5).Primary hrHPV screening with cytology triage in women aged ⩾30 years is an effective way to stratify women on CIN3+ risk and seems a feasible alternative to cytological screening. Repeat cytology after 1 year for hrHPV-positive women with normal cytology is however necessary before returning women to routine screening. Cytological screening has reduced the incidence and mortality of cervical cancer in countries with organised screening programmes .Although the data collected so far are in favour of implementing hrHPV testing in primary screening there is still debate about the management of hrHPV-positive women and about the screening ages at which hrHPV testing would be most beneficial. In young women, the prevalence of hrHPV is high and as a consequence the management of hrHPV-positive women may be complicated.The higher number of positive primary screening tests in this age group may lead to adverse effects of screening if more unnecessary follow-up tests and colposcopy referrals are made. This is of particular importance for these women of reproductive age, because it has been shown that the rate of serious obstetrical complications, such as preterm deliveries, low birth weight and premature rupture of the membranes, is increased after excisional treatments for precancerous lesions . The study was carried out within the setting of a routine cervical screening programme. We present the main results of this cohort study in which 3-year follow-up results were related to baseline hrHPV testing and cytology testing to find an optimal primary screening method. Special attention was given to the question whether hrHPV testing should be offered in combination with cytology or as a sole primary screening instrument. In addition, we study how hrHPV-positive women should be managed. Finally, we examine at what age (at 30 years or at older age) it would be most beneficial to start hrHPV testing.To evaluate for the Dutch cervical screening programme the effectiveness of implementing hrHPV testing and to assess future implementation issues, we set up the VUSA-Screen study . In the Netherlands, women are invited for cervical cancer screening at 5-year intervals starting in the year in which they reach the age of 30 and with the last invitation in the year in which they turn 60 . The study was carried out in the province of Utrecht in the Netherlands among women who were invited for the regular cervical screening programme between October 2003 and August 2005. The design of the study, including exclusion criteria, has been described previously . After preparation of the smear on a glass slide, the brush was placed in a vial containing 1 ml UCM  for hrHPV testing. Cervical cytology results were reported, blinded to the hrHPV testing results, according to the CISOE-A classification, which is routinely used in the Netherlands and can be easily converted into the 2001 Bethesda system (High-risk HPV testing was performed by HC2 high-risk HPV DNA test in an automated format on a rapid capture system according to manufacturer's instructions (Qiagen). This test uses a cocktail probe to detect 13 high-risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Samples with HC2 outcome of ⩾1 RLU/CO were considered as hrHPV positive. HC2-positive samples were tested with GP5+/6+-PCR-EIA  hrHPV-positive women as well as a subset of hrHPV-negative, cytologically normal women (n=3063) were included. To select the hrHPV-negative women, each hrHPV-positive woman was matched to three randomly chosen hrHPV-negative women of the same age. Women were not informed about the hrHPV test result. Women with normal cytology and hrHPV-positive test were offered cytology and a blinded hrHPV test at 12 months and combined hrHPV and cytology testing at 24 months. Women were referred at 12 months if cytology was abnormal and at 24 months if cytology was abnormal and/or the hrHPV test was positive.In the women with normal cytology at baseline, a subcohort was selected. In this subcohort, all , Excel , and Matlab version 7.9 .P=0.237). For women with normal cytology at baseline with follow-up, the histology follow-up at 24 months showed a higher referral rates after abnormal cytology than after an hrHPV-positive, cytologically normal test result . Among women who attended at repeat testing, the average time to the first follow-up test was 15.0 months with a standard deviation of 4.7 months. The follow-up time ranged from 1.3 to 28.6 months.Of the 25 871 women recruited for the VUSA-Screen study, 25 658 (99. 2%) had adequate baseline cytology and hrHPV HC2 test. The median age of participating women was 44.0 years (range 29–61 years). Among the women with adequate cytology, 97.4% had a normal result, 1.3% had BMD and 0.5% had >BMD. The proportion of women with hrHPV infection(s) was 4.1% in women with normal cytology, 49.6% in women with BMD and 92.0% in women with >BMD. Overall, 5.1% (1303 out of 25 658) of the women tested hrHPV positive by HC2. In women with BMD and negative hrHPV result, the overall compliance to repeat testing was 86.5%. In the subcohort of women with normal cytology, compliance to repeat testing was similar in the blinded hrHPV-positive and hrHPV-negative group . Among women with adequate cytology, 1.8% (95% CI: 1.6–2.0%) had an abnormal result (⩾BMD). The highest proportion of abnormal cytology was found in women aged 29–33 years  and the lowest proportion was found in women aged 59–61 years (0.6% 95% CI: 0.3–1.0%).We evaluated hrHPV prevalence in seven age groups corresponding to the screening rounds. We found the highest hrHPV prevalence among women between 29 and 33 years of age who were invited for the first time (10.5% 95% CI: 9.6–11.4%). As the age increased, hrHPV prevalence decreased until age 49 years. The hrHPV prevalence in women aged 59–61 years was 2.0%  tested hrHPV positive, 6 (6.5%) tested hrHPV negative and 10 (10.9%) women had an unknown hrHPV status. Of the women with normal cytology and a positive hrHPV test at baseline, 528 had normal cytology at 12 months follow-up of whom 224 (42.4%) tested hrHPV positive, 219 (41.5%) tested hrHPV negative and 84 (15.9%) had an unknown hrHPV status. The attendance at 24 months was comparable for women with normal cytology and hrHPV-positive test at 12 months  and women with normal cytology and hrHPV-negative test .vs 64.6%) at the cost of a lower specificity (95.6% vs 98.7%). The sensitivity of hrHPV testing for CIN2+ was 1.63-fold higher than cytology (82.0% vs 50.5%); however, the specificity was 0.97 fold lower (96.0% vs 98.9%).The absolute and relative sensitivity and specificity of cytology and hrHPV testing for detection of CIN3+ and CIN2+ are presented in The cumulative 3-year CIN3+ and CIN2+ risks, adjusted for non-attendance at repeat testing, are presented in The majority of the hrHPV-positive women had normal cytology and those women still had a CIN3+ risk of 5.22% (95% CI: 3.72–7.91). The HPV16/18 genotyping of hrHPV-positive women at baseline did not result in sufficient low risks for a screenings programme with 5 years interval. Women with hrHPV-positive normal cytology and HPV16 and/or HPV18-positive test had a CIN3+ risk of 13.0% (7.93–23.6), whereas women who tested hrHPV positive for other high-risk types had a much lower risk of 2.44% (95% CI: 1.61–5.25). We also evaluated a baseline triage and follow-up strategy for hrHPV-positive women. Baseline cytology triage followed by repeat cytology testing at 1 year showed that the CIN3+ risk reduced to only 1.6% (95% CI: 0.6–4.9) in women with normal cytology at the repeat test. In women with abnormal cytology at the repeat test, the CIN3+ risk was 25.0% (95% CI: 16.6–35.1). This CIN3+ risk is comparable to the CIN3+ risk of hrHPV-positive women with abnormal cytology at baseline.Analysis using CIN2+ as outcome measure found comparable results, albeit with higher absolute risks. The cumulative 3-year CIN2+ risk was 0.26% (95% CI: 0.14–0.69%) among hrHPV-negative women and 0.68% (95% CI: 0.54–1.13%) among women with negative cytology. The CIN2+ risk was similar for women with negative results on both tests (0.24% 95% CI: 0.12–0.64%) and women negative for hrHPV only (0.26% 95% CI: 0.14–0.69%). The hrHPV-positive women with normal cytology had a CIN2+ risk of 11.3% (95% CI: 8.90–15.2) and hrHPV-positive women with normal cytology and negative for HPV16/18 genotyping had a CIN2+ risk of 8.01% (95% CI: 5.53–12.6).When stratifying hrHPV-positive women into two age groups, there was a borderline non-significant difference in CIN3+ risk (adjusted for non-attendance at repeat testing) between women ⩾34 years of age and women invited for the first time (aged 29–33 years) (relative risk (RR) 0.78 95% CI: 0.52–1.15). The RR for CIN2+ was 0.87 (95% CI: 0.64–1.18). In hrHPV-positive women with normal cytology, the CIN3+ risk was significantly lower in women aged ⩾34 years  than in women aged 29–33 years (10.9% 95% CI: 5.9–19.2). The corresponding RR was 3.02 (95% CI: 1.39–7.07). The CIN2+ risk was 10.7 (95% CI: 7.3–15.3) in women with normal cytology and age ⩾34 years, 16.6% (95% CI: 10.7–25.3) in women with normal cytology and age 29–33 years, and the corresponding RR was 0.65 (95% CI: 0.37–1.14). When stratifying hrHPV-positive women with abnormal cytology into two age groups, no risk difference between the older and younger age group was observed for CIN3+ and CIN2+ .Implementation of hrHPV testing as a primary screening instrument in cervical screening is still under debate. The presented study enables us to examine three implementation issues in more detail. First, whether hrHPV testing should be offered in combination with cytology or as a sole primary screening instrument. Second, how hrHPV-positive women should be managed. Third, the screening ages at which hrHPV testing would be most beneficial.In our study, hrHPV testing has a 27.3% higher sensitivity but a 3.1% lower specificity than cytology for detection of CIN3+. For CIN2+, these figures were 31.5% and 2.9%, respectively. These results are in line with other screening studies . However, referring all hrHPV-positive women to colposcopy may result in overdiagnosis and overtreatment .In the present study, we have used cytology as a triage tool to identify women at high risk for CIN3+ among the hrHPV-positive women. Women with an hrHPV-positive test and abnormal cytology had a CIN3+ risk of 42.2% and obviously need immediate colposcopy. On the other hand, hrHPV-positive women with normal cytology have a low, but still non-negligible CIN3+ risk of 5.2%. This risk is too high to delay follow-up to the next screening round (in the Netherlands 5 years) but too low to refer them for immediate colposcopy. Therefore, hrHPV-positive women with normal cytology at baseline require further triage testing and/or follow-up. In our study, women were retested after 1 year by means of cytology and after 2 years by both cytology and hrHPV. An analysis of the repeat testing results showed that the decision either referral to colposcopy or return to routine screening can be made after 1 year on the basis of one repeat cytological test. The CIN3+ risk after 1-year normal cytology was only 1.6%. This risk is similar to the CIN3+ risk of women with BMD at baseline and normal cytology at 6 and 18 months follow-up (1.2%), which is presently accepted in the Netherlands. Furthermore, the CIN3+ risk of 1.6% is also below the CIN3+ risk threshold proposed by post hoc analysis on data of the present study is beyond the scope of this paper but has been presented in a separate paper  and for older women (⩾34 years). The same accounts for hrHPV-positive women with abnormal cytology. However, the CIN3+ detection rate in hrHPV-positive women with normal cytology was significantly higher among younger women (29–33) than among older women (⩾34). First, in the POBASCAM study the CIN2+/CIN3+ baseline detection in women over 30 years of age was higher in the hrHPV testing arm than in the cytology arm but over two screening rounds , the CIN2+/CIN3+ detection rates were similar in both arms  and in women ⩾34 years. Moreover, in hrHPV-positive women with normal cytology, the CIN3+ risk was higher in women invited for the first time than in older women. A possible explanation is that hrHPV infections detected in women invited for the first time may have persisted for many years before being identified by screening and therefore more likely to have developed into high-grade lesions. These results are in line with published data from the Guanacaste cohort (A limitation of our study is that women with normal cytology were not informed about the hrHPV status at baseline. This concealment was necessary to maximise attendance at repeat testing among hrHPV-negative women with normal cytology. The repeat testing attendance rate in women with normal cytology was 61.8% in hrHPV-negative and 59.7% in hrHPV-positive women. The attendance at repeat testing has been shown to be particularly poor after a cytologically normal test (We observed a higher percentage of histology reports after referral on the basis of abnormal cytology than after an hrHPV-positive, cytologically normal test result. This difference may be related to anticipated association between biopsy rate and colposcopic image of the cervix. If adjusted for, the effect of hrHPV testing on CIN3+ will be somewhat higher than the effect reported in this study. In this regard, several studies have indicated that the effect of hrHPV testing will be higher when a blind biopsy is carried out in women with a normal colposcopic impression (Another limitation of our study may be the use of a subjective test such as cytology as a triage test for hrHPV-positive women. However, A strong point of our study is that this study is population based and is integrated in the regular screening programme of eligible women aged 29–61 years. The differences in sensitivity between hrHPV screening and cytological screening are slightly overestimated because in the practice of screening some women will not attend repeat testing after an hrHPV-positive test. Nevertheless, a higher attendance at repeat testing is to be expected once the implication of a positive hrHPV test is well communicated to the women and hrHPV screening becomes routine .Although cytology adds little to the reassurance from a negative hrHPV test against high-grade lesions, it is a very useful risk stratifier in hrHPV-positive women and results in a feasible screening algorithm. We showed that repeat cytology testing after 1 year for hrHPV-positive women with normal cytology at baseline is critical for maximising the benefits of primary hrHPV testing in routine cervical cancer screening.


Cryptosporidium hominis, the predominant species responsible for human cryptosporidiosis. We conducted sequence analyses of 32 genetic loci of 53 C. hominis specimens isolated from a longitudinally followed cohort of children living in a small community. We identified by linkage disequilibrium and recombination analyses only limited genetic recombination, which occurred exclusively within the 60-kDa glycoprotein gene subtype IbA10G2, a predominant subtype for outbreaks in industrialized nations and a virulent subtype in the study community. Intensive transmission of virulent subtype IbA10G2 in the study area might have resulted in genetic recombination with other subtypes. Moreover, we identified selection for IbA10G2 at a 129-kb region around the 60-kDa glycoprotein gene in chromosome 6. These findings improve our understanding of the origin and evolution of C. hominis subtypes and the spread of virulent subtypes.Little is known about the emergence and spread of virulent subtypes of  Cryptosporidium spp. are emerging pathogens of humans and a variety of vertebrates, and cause severe diarrhea in immunocompetent and immunocompromised persons  identified by sequence analysis of the 60-kDa glycoprotein (gp60) gene, the Ib subtype is the major subtype responsible for waterborne and foodborne outbreaks of cryptosporidiosis in many countries. Subtype IbA10G2 has been found in ≈50% of C. hominis–associated outbreaks in the United States, including the massive outbreak in Milwaukee, Wisconsin, USA, in 1993  of 32 genetic markers. Data obtained should be useful in understanding emergence and spread of virulent C. hominis subtypes.We conducted a comparative population genetic analysis of virulent Cryptosporidium spp. were collected during a longitudinal cohort study of enteric diseases in children living in a periurban shantytown in Lima, Peru, during 2004–2006. The field study was conducted in the same community used in a previous longitudinal cohort study of enteric pathogens and had a similar study design  belonging to 4 gp60 subtype groups were selected for MLST analysis: Ia (9 specimens of IaA13R8 and 1 specimen of IaA13R7), Ib (26 specimens of IbA10G2), Id (6 specimens of IdA10 and 5 specimens of IdA20), and Ie (6 specimens of IeA11G3T3). No specimens used in the study had mixed C. hominis subtypes.DNA was extracted from 200 μL of microscopy-positive specimens by using the FastDNA SPIN Kit for Soil . Cryptosporidium spp., chromosome 6 was fully sequenced for 2 C. parvum specimens and 1 C. hominis specimen, which facilitated search for additional microsatellite and minisatellite markers for population genetics analysis. The 8 polymorphic markers on chromosome 6  used in several MLST and population genetic analyses of C. hominis and C. parvum (http://tandem.bu.edu/trf/trf.html). Of 325 short tandem repeat sequences identified, 46 loci were initially selected at spaced intervals that covered the entire chromosome 6. A nested PCR approach was used for amplification of all potential targets. After an initial evaluation of primers by PCR and DNA sequencing of IaA15R3, IaA20R3, IbA10G2, IdA10, and IeA11G3T3 subtypes of C. hominis from the United States and Peru, 24 polymorphic loci (Among the 8 chromosomes of C. parvum IIaA16G2R1 subtype) were used in each PCR. PCR products of the expected size were sequenced in both directions by using an ABI 3130 Genetic Analyzer . Sequences obtained from each locus were edited and aligned by using BioEdit 7.04 version (www.mbio.ncsu.edu/BioEdit/bioedit.html) and ClustalX version 1.81 , respectively. Nucleotide sequences of all haplotypes of polymorphic loci were deposited in GenBank under accession nos. JX088398–JX088417 and JX088427–JX088501.Conditions for nested PCR analysis of each marker were similar to those described , number of haplotypes, number of polymorphic and segregating sites, intragenic linkage disequilibrium (LD), and intragenic recombination rates by using DnaSP version 5.10.00 (www.ub.es/dnasp/). The ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site was calculated for gp60 and several nearby loci  by using DnaSP. Thereafter, sequences of polymorphic loci were concatenated as a single multilocus contig to calculate gene diversity, interlocus LD, and recombination rates by using DnaSP. Pairwise intergenic LD was also evaluated among polymorphic loci by using the Fisher exact test and Markov chain parameters in Arlequin version 3.1 (http://cmpg.unibe.ch/software/arlequin3/).For each marker, sequences of 53 http://pritch.bsd.uchicago.edu/structure.html). Conversion of microsatellite and minisatellite sequences to allelic data was performed by using the Excel Microsatellite Tool Kit 3.1.1  and Genepop version 4.0 (http://genepop.curtin.edu.au/). Several analyses of allelic data were performed by using K (likely populations) ranging from 2 to 8 and 50,000 iterations after a burn-in of 50,000 iterations. Output at K = 2–5 provided the best fit to MLST data and were used in further analyses. To provide an alternative view of substructuring, a median-joining algorithm analysis (www.fluxus-ngineering.com/sharenet.htm). Each gp60 subtype was further analyzed for LD and recombination by using DnaSP and Arlequin. Population differentiation (FST) between IbA10G2 and non-IbA10G2 subtypes was also assessed at each genetic locus by using Arlequin.Population substructures were analyzed by using the Bayesian analysis tool STRUCTURE version 2.2 , an Hd of 0.9898, and a nucleotide diversity of 0.339. The frequency of MLGs ranged from 7.5% (1 MLG with 4 specimens), 5.7% (1 MLG with 3 specimens), and 3.8% (5 MLGs each with 2 specimens) to 1.9% (36 MLGs each with 1 specimen). Because 431/968 polymorphic sites and 297/362 segregating sites occurred within the gp60 locus, a second analysis was performed by using concatenated sequences excluding gp60  with 537 polymorphic sites, including 65 segregating sites and 472 indel sites. A lower nucleotide diversity of 0.289 was observed. However, the number of MLGs (43) and haplotype diversity (0.9898) remained the same.Intragenic LD between pairs of segregating sites was assessed for each polymorphic locus. The analysis was possible only for 7 loci  that had at ≥3 segregating sites. Incomplete intragenic LD (|D′| Y = 0.9729 + 0.0130X), where Y is the LD value and X is the nucleotide distance in kb, was observed at the gp60 locus, and complete intragenic LD (|D′| Y = 1.0000 + 0.0000X) was found at each of the 6 remaining loci . An intrns) value of 0.2711 . Of 47,895 pairwise comparisons, 25,904 were significant by Fisher exact test, and 8,714 were significant after Bonferroni correction . Of 2,080 pairwise comparisons, 921 were significant by Fisher exact test, and 333 were significant after Bonferroni correction. Incomplete LD (|D′| Y = 0.7886 + 0.0051X) was also observed in LD analysis per site  was observed in subtype IbA10G2, suggesting recombination within this subtype. In contrast, all other subtypes showed complete LD (|D′| Y = 1.0000 + 0.0000X) among all sites, indicative of no genetic recombination within these subtypes . In addiTo validate these observations, recombination tests were conducted for all gp60 subtypes. Using full multilocus sequences, we found that among 33 potential Rms observed in overall recombination analysis, 4 Rms were detected in subtype IbA10G2 but no Rm was found within each of the remaining subtypes . When anFST analysis showed highly significant differentiation between the IbA10G2 subtype and non-IbA10G2 subtypes at 4 loci around gp60, C6–1000′ (p<0.00001), gp60 (p<0.00001), CP47 (p<0.00001), and C6–2600 (p<0.001). The only other locus that showed such a level of differentiation between the 2 groups was C6–3520 (The genetic determinant for differences between IbA10G2 and non-IbA10G2 subtypes was assessed by comparison of haplotype diversity of the 2 groups at all 25 polymorphic loci in chromosome 6. At most of the genetic loci, the haplotype diversity of the 2 groups was similar, and there was no clear population differentiation at these loci , Table 4Cryptosporidium spp. has a sexual phase, during which sexual recombination can occur between genetically distinct strains (C. parvum, the species that infects humans and some animals, undergoes meiotic recombination between different lineages in genetic-crossing experiments (C. parvum populations have also shown genetic recombination in most study areas (C. hominis is primarily infectious to humans, and previous MLST studies showed a clonal population structure. Genetic recombination was believed to be rare or nonexistent in C. hominis (C. hominis. In the present study, we examined population substructure of C. hominis in a cohort of children living in a small study area by using 32 genetic markers.Like other apicomplexan parasites, the life cycle of C. hominis. A recombination test showed only limited genetic recombination at the gp60 locus. Thus, the high level of LD and limited recombination found in the overall population could be explained by an essential clonal population structure of C. hominis in the field site in Peru, which is consistent with information on C. hominis population genetics in other countries (On the basis of multilocus sequence data and allelic profiles for 53 specimens, analysis showed strong LD among 25 polymorphic loci, suggesting an overall nonpanmictic population structure of C. hominis occurred mostly at gp60 or loci around gp60. Thus far, the gp60 gene is the most polymorphic marker identified in the Cryptosporidium genome. Because of its high sequence heterogeneity, gp60 has become the most widely used gene in Cryptosporidium spp. subtyping, which categorizes C. hominis and C. parvum into several subtype groups and various subtypes within each subtype group (Results of LD and recombination analyses suggest that limited recombination in the study population of C. hominis subtype IbA10G2 was shown to be more virulent than other subtypes in the study community (Recent studies have suggested that telomeric/subtelomeric regions are highly polymorphic and might encode putative virulence factors (FST analysis of 25 polymorphic loci in chromosome 6 between the virulent IbA10G2 subtype and non-IbA10G2 subtypes showed population differentiation at 4 loci around gp60. When a locus shows extraordinary levels of genetic population differentiation compared with other loci, this finding might be interpreted as evidence for positive selection (Pairwise Plasmodium falciparum chloroquine-resistance transporter gene in chromosome 7 as the result of selection for chloroquine resistance. Nair et al. (P. falciparum in association with pyrimethamine resistance. Consistent with these findings, we detected reduced sequence variation around gp60 in virulent subtype IbA10G2, suggesting that the 129-kb region surrounding gp60 in chromosome 6, perhaps gp60 itself, might be involved in selection for virulent gp60 subtype IbA10G2 in C. hominis. In a comparison of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site analysis of loci around gp60, we confirmed the presence of positive section at least at the CP47 locus.One explanation for this pattern of genetic diversity is that the region surrounding gp60 was probably affected by selective sweep or genetic hitchhiking caused by selection of the virulence subtype. A hallmark of a selective sweep is a chromosomal region with reduced diversity associated with a specific phenotype. In previous studies of other apicomplexan parasites, similar patterns of reduced genetic variations were observed in chromosomal regions surrounding sites under selection pressure. Wootton et al. (C. hominis responsible for numerous waterborne and foodborne outbreaks of cryptosporidiosis in many countries (C. hominis infections are caused mostly by IbA10G2 and imported cases by other subtypes (C. hominis subtypes and subsequent spread of the recombinant parasite with the fitness gene. Recently, genetic recombination was shown to be a key strategy for selection of virulent clones of Toxoplasma gondii, an apicomplexan parasite with a largely clonal population structure in North America and Europe (The reason for exclusive occurrence of genetic recombination in IbA10G2 is not clear. Because IbA10G2 is the major subtype of C. hominis population in a cohort of children living in a small community in Peru. Although C. hominis from the community has an overall clonal population structure, genetic recombination occurs within subtype IbA10G2 around the gp60 locus, which might be involved in pathogenicity. Common occurrence of its parental subtypes and biologic fitness of the recombinant subtype with the IbA10G2 trait have probably facilitated genetic exchange and spread of the virulent subtype. In addition, we were able to localize selection for the virulent subtype IbA10G2 to a 129-kb region surrounding gp60 in chromosome 6. These observations could improve our understanding of emergence and spread of virulent C. hominis subtypes.In conclusion, we have shown complex substructures in a natural


The stapled hemorrhoidopexy (SH) is a procedure for prolapse and hemorrhoids . At first SH seemed to be a good alternative for the Milligan Morgan (MM) hemorrhoidectomy and preliminary results in early 2000 confirmed it. However, further studies and evaluation of long-term results showed poorer outcomes.This study aimed to evaluate and compare the results of these 2 surgical procedures in terms of recovery, improvement of symptoms and incidence of complications.This study was conducted from April 2008 to August 2010. A total of 80 patients were divided into 2 groups of 40 each. In the SH group, there were 24 males (60%) and 16 females (40%) with a mean age of 48 ± 12.5 yrs. In the MM group, there were 30 males (75%) and 10 females (25%) with a mean age of 50.6 ± 17.3 yrs. Patients with grade 3 and 4 prolapsed hemorrhoids were entered in the study. Data were entered using SPSS software and analyzed using t-test and Chi-square test.The two groups had no significant difference in terms of age or sex. Duration of surgery was 35 ± 7 minutes in the SH and 23.6 ± 13.5 minutes in the MM group. This difference was statistically significant (P = 0.000). Post-operative pain and complete pain relief was slightly lower in the MM group (not significant). Hospital stay was significantly longer in the MM group (P = 0.003). Return to work was similar in both groups. Three patients in the SH group (7.5%) and 2 in the MM group (5%) had hemorrhoid recurrence.Both techniques are efficient treatment methods for grade III and IV hemorrhoids and are associated with greater than 95% recovery rate. Overall, outcomes are the same in both techniques. Lower postoperative pain was the only advantage of SH over MM technique. Stapled hemorrhoidopexy  was first introduced by Longo in 1998. At first it seemed to be a good alternative for the Milligan Morgan hemorrhoidectomy and preliminary results in early 2000 confirmed this , 4. The This study aimed to evaluate and compare the results of these 2 surgical procedures in terms of recovery, improvement of symptoms and incidence of complications.Positive history of anorectal surgeryHaving rectal prolapsePatients with fissure or fistulaThis study was conducted from April 2008 to August 2010. A total of 80 patients were divided into 2 groups of 40 each. In the SH group, there were 24 males (60%) and 16 females (40%) with a mean age of 48 ± 12.5 yrs. In the MM group, there were 30 males (75%) and 10 females (25%) with a mean age of 50.6±17.3 yrs. Patients with grade 3 and 4 prolapsed hemorrhoids who were candidates for surgery in the colorectal clinic of our hospital entered the study. The exclusion criteria were as follows:The study protocol was approved by the ethics committee of our university.First, type and method of surgical procedure (SH) was thoroughly explained to the patients and then if they were willing to participate in the study they were placed in the SH group . If they were not interested to undergo this type of procedure, they were placed in the conventional surgery group (MM). Afterwards, patients entered the research project and their data were recorded in specific questionnaires. All surgical procedures in both techniques were performed by experienced surgeons under spinal anesthesia with the patient in lithotomy position. For patients whom were selected for stapled hemorrhoidopexy (SH), 1 gram ceftriaxone injection and 50 mg intravenous metronidazole were administered preoperatively. No antibiotic was administered for patients in the MM group.Patients were visited and examined on the first postoperative day and one week after surgery in the colorectal surgery clinic. Follow ups were performed 1, 6 and 12 months later via phone. Patients were also visited by the surgeon at the clinic. Post-operative pain was measured according to Visual Analog Scale. For pain scale 1-3 oral acetaminophen, for scale 4-5 acetaminophen codeine and for scale 6-10 intravenous pethidine was administered. Psyllium powder and magnesium hydroxide syrup 8% were administered daily postoperatively. Data were entered using SPSS software and analyzed using t-test and Chi-square test.A total of 80 patients were divided into 2 groups of 40 each according to patient preference. In the SH group, there were 24 males (60%) and 16 females (40%) with a mean age of 48 ± 12.5 yrs. In the MM group, there were 30 males (75%) and 10 females (25%) with a mean age of 50.6 ± 17.3 yrs. The two groups had no significant difference in terms of age or sex. Ninety percent of patients had grade III and 10% of patients had grade IV hemorrhoids. The most important indications for surgery were prolapse in 18 patients (22.5%) and bleeding and prolapse in 62 cases (77.5%). Severity of post-operative pain according to VAS was 4 ± 1.7 in the SH and 4.6 ± 2.3 in the MM group. Although the severity of pain  was lower in the SH group, this difference was not statistically significant (P = 0.166).Duration of hospital stay after the surgery was 1.7 ± 0.6 days in the SH and 1.27 ± 0.6 days in the MM group. This difference was statistically significant (P = 0.003). Time required to achieve complete pain relief was 11.6 ± 10 days in the SH and 13 ± 12.2 days in the MM group. This difference was not statistically significant (P = 0.583).Time to return to work was almost similar in both groups (6.95 ± 3.6 days vs 6.13 ± 4.2 days). The mean follow up period in both groups was 13 ± 5.7 months. At the end of the follow up period, 3 patients in the SH group (7.5%) and 2 in the MM group (5%) had hemorrhoid recurrence. The difference in this regard was not statistically significant between the 2 groups. Full recovery was achieved in 92.5% of the cases in SH and 95% of the cases in the MM group. This difference was not significant either. Other minor complications observed post operatively included transient incontinence, 1 case of perianal abscess, and 1 case of urinary retention. Anal stenosis, or permanent incontinence, did not occur in any of the cases.In the MM technique hemorrhoidal tissue is completely excised but in SH hemorrhoid is not actually removed and in fact hemorrhoidopexy is performed. This explains the higher rate of recurrence in the SH group 7.5% vs 5%). Considering the obtained results, it seems that stapled hemorrhoidopexy has no advantage over MM in terms of final outcome. Duration of SH surgery is longer but patients are somehow more comfortable after surgery. Our study results in terms of severity of postoperative pain and time to return to work were in agreement with those of Mattana .5% vs 5% and TjanIn terms of general recovery our SH surgery results were in accord with those of Gravie  and Tjan


Usefulness of adalimumab (ADA) therapy in patient with rheumatoid arthritis has already established. But the efficacy of ADA in patient with juvenile idiopathic arthritis (JIA) and joint ultrasonography (US) findings of them are still unknown.To evaluate correlation of joint US findings and other clinical indices in JIA patient newly introduced ADA therapy.4 polyarticular JIA (p-JIA) children diagnosed as JIA according to ILAR criteria were enrolled. All were female. The median age was 12 years , and median disease duration was 5.6 years . Two patients were administered prednisolone and methotolexate, One prednisolone only, and one drug free, as preceeding therapy. ADA was administered biweekly at a dose of 40mg per body.Demographic data were collected. Joint US examination was demonstrated at 0 (prior to administration of ADA), 4, and 24 weeks.All of 4 patient had successfully continued ADA therapy for 24 weeks. There were no severe adverse events in this term. At 24 weeks after introduction of ADA, ACR pedi 50 were achieved by 3 patients. The average of MMP-3 at 0 weeks is 441.8 (56.1 – 690.8) ,and fell to 120.4 (45.5 – 185) after 24 weeks. R value of DAS28 and US grade of power doppler (grade 0 to 3) is 0.73.Efficacy of ADA therapy in children with JIA is demonstrated, and joint US is a good tool to evaluate disease activity of JIA.


Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome.MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis.The methylation status of MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p < 0.05), while CASP8 with older (p < 0.01). MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p < 0.05), while methylation of CASP8 was more frequent in patients who survived shorter than 36 months (p < 0.05). Cox regression analysis showed patient age, treatment, MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p < 0.05). MGMT and GATA6 were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy.The overwhelming majority (97.3%) of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome.High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of  In the first part of the study we have reported promoter methylation of MGMTGATA6CD81DR4, and CASP8 genes. In the second part we have analyzed gene methylation correlations with patient variables. And in the third part we have analyzed whether the methylation status of five genes separately or in clusters are independent predictors for patient outcome.Glioblastoma is the most common and aggressive astrocytic tumor of the central nervous system in adults. It is characterized by various genetic alterations, affecting genes that control cell growth, apoptosis, angiogenesis, and invasion. Epigenetic alterations also affect the expression of cancer genes alone or in combination with genetic mechanisms. Cytosine methylation of CpG dinucleotides in gene promoters is a common cause of DNA silencing and transcriptional repression that can modulate clinical features of globlastoma. The best known is Osurvival . Howeversurvival -4. The lferation  could bepurposes . To furtMGMTGATA6CD81DR4, and CASP8 genes in 76 patients with WHO grade IV glioblastomas was investigated. All glioblastomas were graded and classified according to the 2007 WHO classification criteria [The methylation profile of the criteria . Tissue MGMTCD81GATA6DR4, and CASP8 gene promoters was determined by bisulfite treatment of DNA. An amount of 400 ng of DNA was used for bisulfite modification. DNA modification was done using the EZ DNA Methylation Kit , and all the procedures were carried out according to the manufacturer’s protocol. Bisulfite-treated DNA was eluted in 40 μL distilled water and stored in –80 °C until PCR. Normal human blood lymphocyte DNA treated with bisulfite served as a negative control. As a positive control, standard Bisulfite Converted Universal Methylated Human DNA Standard  was used. Promoter methylation was detected by methylation-specific PCR (MSP). Each MSP reaction incorporated approximately 20 ng of bisulfite-treated DNA as a template. Specific for methylated and unmethylated DNA sequence primers are listed in Table MGMT[CD81[GATA6[DR4[CASP8[μL, using 10 μL Maxima® Hot Start PCR Master Mix with Hot Start Taq DNA polymerase  and 10 μM of each primer . MSP was performed for 38-40 cycles with start of 95 °C for 1 min, annealing for 1 min at temperature appropriate for an individual gene, and extension at 72 °C for 1 min. Amplification products were loaded on 2% agarose gels with ethidium bromide and after electrophoresis documented under UV. In a case of both, methylated and unmethylated signals appearance in a gel, methylation of the gene was considered.Brain tumor tissue specimens after dissection were snap-frozen and stored in liquid nitrogen until analysis. Tumor DNA was extracted using ZR Genomic DNA™-Tissue MiniPrep  from 25-40 mg of frozen tissue according to the manufacturer’s instructions. The methylation status of MGMT[CD81, GATA6[8GATA6[DR4, and CASDR4[CASP8). PCR ret test was used. For testing statistical hypothesis about the independence of two variables, the chi-square test was used.Statistical analysis was carried out with the software of IBM SPSS Statistics 19 . Quantitative data presented as mean and standard deviation (SD). To show the reliability of the estimate, the confidence interval (CI) with 95% confidence level was presented. For testing the statistical hypothesis the significance level of 0.05 was selected. The Kaplan-Meier method was used to estimate survival functions. For comparison of survival between two groups, the log-rank and generalized Wilcoxon tests were used. The Cox proportional hazard regression model was applied to determine independent variables and prognosis relative hazard. For comparing means of two groups the independent samples The mean age at diagnosis was 61.2 years . The male-to-female ratio was 1:1.5. The mean age of male (n = 30) and female (n = 46) patients was 59.9 years (SD: 13.3 years) and 62 years (SD: 11.6 years), respectively.The mean survival of patients with glioblastoma (n = 76) was 14.6 months (range: 0.26-52.60 months), while the median survival was 7.7 months (95% CI: 4.81-10.6 months). The survival rate at 12 and 24 months was 34.2% (95% CI: 23.7%-46.0%) and 22.4% (95% CI: 13.6%-33.4%), respectively.P = 0.001). The median survival of patients aged <60 years and ≥60 years was 15.6 and 4.5 months, respectively. Radiotherapy (RT) alone and RT plus concomitant chemotherapy (temozolomide) (RT + TMZ) were used for the postoperative treatment of glioblastomas in our study. RT alone was administered in 85.3% (n = 58) of cases and RT + TMZ was used in 14.7% (n = 10) of cases. Eight patients did not receive postoperative treatment due to their critical condition. The median survival for patients treated with RT + TMZ versus RT alone was 42.2 months (95% CI: 13.19-71.18 months) and 7.7 months (95% CI: 4.34-11.04 months).As expected, it was observed glioblastoma patient survival association with age and adjuvant treatment  for GATA6, 46.1% (n = 76) for CD81, 41.3% (n = 75) for DR4, and 56.0% (n = 75) for CASP8. Representative methylation-specific PCR for the mentioned genes is illustrated in Figure The overwhelming majority (97.3%) of tumors were methylated in at least one of five genes tested. Only 2.7% of glioblastomas had no detectable gene methylation, and 4.1% had concomitant methylation of all five genes tested. Methylation frequencies were 51.3% (n = 76) for P > 0.05).Despite the frequent co-methylation of two genes in the same tissue varying from about 15% to 37%, no significant associations in the frequencies of gene methylation were documented , while methylation of CASP8 more often was observed in older patients  , while CASP8 and DR4 gene methylation was more frequent event in patient group who survived up to 36 months  . Of the patients older than 60 years who lacked MGMT promoter methylation, 45.5% and 44.2% showed concomitant aberrant GATA6 and CASP8 methylation respectively. GATA6 methylation in older patients was significantly associated with co-methylated CASP8 and DR4 , as well as CASP8 methylation with co-methylated DR4 (34.9%) .Methylation of MGMT promoter was associated with longer survival   and 33.3% (95% CI: 19.1%-50.2%) versus 21.6% (95% CI: 9.8%-38.2%) and 10.8% (95% CI: 3.0%-25.4%), respectively.Figure ) Figure . The medly Table . The 12-GATA6, CD81, and DR4 and patient survival were identified .No significant associations between the methylation of  Figures . HoweverMGMT, the methylation of the CASP8 promoter was associated with shorter survival   as compared with 6.2 months (95% CI: 3.3-9.2 months) among their counterparts with the methylated CASP8 promoter.Contrary to the methylation profile of ) Figure . The medMGMT in a case of methylated GATA6 and CASP8 were significantly associated with worse outcome  as compared to that of the patients whose tumors had methylated MGMT with unmethylated GATA6 and CASP8 , as well as tumors with no methylation of both genes (MGMT and GATA6) , and tumors with double methylation  , and the difference was significant as compared with patients whose tumors had methylated GATA6 and unmethylated CASP8   Figure . Concomi) Figure .MGMT and CASP8 promoters as independent predictive factors significantly associated with patient survival. Patients older than 60 years were more likely to survive shorter than their younger counterparts . Moreover, patients who underwent treatment with RT + TMZ had a 76% reduced risk of death  as compared with patients treated with RT alone.Univariate Cox analysis identified patient age, treatment modality, and methylation profile of the MGMT, CASP8, and GATA6 was of prognostic value for survival in a multivariate Cox model, when patient age and treatment were excluded . Contrary, the methylated GATA6 and CASP8 promoters were found to be associated with shorter survival .The methylation status of ed Table . The metMGMT  and GATA6  were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy (data not shown).When the patient sample was divided into two groups by age (<60 years and ≥60 years), multivariate Cox regression analysis showed that MGMT, GATA6, and CASP8. To our knowledge, only MGMT has been previously widely described in glioblastomas, while the data on the epigenetic regulation of GATA6, CD81, DR4, and CASP8 in patient glioblastoma are scarce.Our study demonstrated that epigenetic alterations affecting multiple genes, which are important in several cell functions, such as DNA repair, cell migration and apoptosis, are a frequent event in glioblastoma. At least one of the five genes tested was methylated even in 97.3% of patients with glioblastoma, while only 2.7% of patients had no detectable methylation. Analysis identified 3 genes epigenetically deregulated in more than 50% of glioblastomas: GATA6, a transcription factor, with a methylation frequency of 68.4%. GATA6 is one of 6 members of the mammalian GATA family of transcription factors that regulates cell proliferation and differentiation and inhibits apoptosis [GATA6 expression was observed in colon and ovarian carcinomas as well [This study showed that the most frequently methylated gene was poptosis . Recentlpoptosis ,12-14. I as well ,16.CD81 gene showed promoter methylation in 46.1% of tested glioblastomas in the current study. This gene is a member of the membrane-embedded tetraspanin superfamily, which was found to be silenced by methylation in multiple myeloma cell lines [CD81 participates in several functions in a cell like adhesion and signal transduction [CD81 methylation rate of 54% [The ll lines . CD81 pasduction . Recent e of 54% ,3.CASP8 and DR4, in our study revealed that these genes were methylated in 56% and 41% of glioblastomas, respectively. The inactivation of these genes by promoter methylation has been previously reported in osteosarcomas [DR4 methylation in glioblastomas varied from 25% to 70% in different studies [DR4 methylation was observed, and we hypothesize that the difference could be attributed to the heterogeneity of glioblastomas. In contrast to the findings of Elias et al. [CASP8 in glioblastomas, in this panel more than half of tumors were methylated (56%). The tendency of association between methylation of CASP8 and DR4 in glioblastomas was noted: co-methylation was observed in 28.4% (21/74) of tumors. A study carried out by Elias et al. [CASP8 and DR4 co-methylation in glioblastoma specimens (10%). It is known that DR4 and CASP8 are factors affecting the nonmitochondrial apoptotic pathway, and the loss of DR4 expression (which was shown to be mediated by promoter methylation) attenuates apoptosis and is associated with the resistance of glioma cells to proapoptotic ligand therapy (known as TRAIL resistance) [CASP8 in TRAIL resistance in gliomas has been reported as well [CASP8-independent and DR4-specific [Analysis of the methylation status of two proapoptotic genes, sarcomas , melanomsarcomas , medullosarcomas , gastricsarcomas , and glisarcomas ,13,21,22 as well , while tMGMT gene, encoding a DNA repair enzyme, has been recently found to be of predictive value in glioblastoma. In agreement with numerous studies where the methylation of MGMT was detected in approximately 40%-48% of primary glioblastomas [MGMT and GATA6 (P > 0.05) despite rather high comethylation of these genes (37%).Epigenetic silencing of the lastomas ,2,24, oulastomas , this stMGMT and CASP8 gene methylation associations with patient age. Glioblastoma patients with methylated MGMT were significantly younger than those whose tumor lacked methylation (P < 0.05) and an opposite was observed for CASP8 (P < 0.01). Contrary, glioblastoma study of Cecener et al. [MGMT methylation association with older patient age (≥ 50 years), while in the study of Weller et al. [MGMT methylation status was not associated with clinical parameters .Correlations with patient parameters showed r et al.  showed Mr et al. MGMT methSeveral associations between survival and patients’ characteristics were observed. Survival was strongly correlated with patients’ age and treatment modality. In agreement with previous studies ,25, it wMGMTGATA6, and CASP8, but not CD81 and DR4, to be an independent factor for patient survival. Kaplan-Meier and Cox analysis showed the methylation status of MGMT to be significantly related to patient outcome. In agreement with previous studies [MGMT methylation status and patient survival, our study showed that patients with methylated MGMT were more likely to survive longer and were at lower risk of death than those with the unmethylated gene. After dividing the patient sample into the groups by age (<60 years vs. ≥60 years), methylated MGMT remained a significant independent predictor of survival among patients aged less than 60 years. Furthermore, we confirmed significant association between the methylation profile of MGMT and treatment modality: in the group of patients treated with RT + TMZ, patients with methylated MGMT survived significantly longer than those with unmethylated gene promoter  (data not shown).Cox regression analysis confirmed the methylation status of  studies ,26 reporCASP8 and survival in our study showed gene methylation being an important factor for worse outcome of patients. Patients with unmethylated CASP8 as a favorable genotype had prolonged survival as compared with patients having tumors with methylated CASP8. Although CASP8 may play an essential role in apoptosis induced by chemotherapeutic agents and radiation therapy [CASP8 and survival of the patients treated with RT, and no associations in the group of the patients treated with RT + TMZ (data not shown).In line with findings of the study by Martinez et al. , associa therapy ,28, our GATA6 gene differed significantly at early postoperative period . Cox regression analysis demonstrated CASP8 and GATA6 as independent predictors for patient survival. Furthermore, GATA6 became independent predictor for survival in younger patient group. Contrary to our results, recently it have been reported no relationship of GATA6[CASP8[GATA6 methylation in patient survival has been shown for the first time, our results suggest that the methylation of GATA6 is a frequent event and highly important for survival of patients with glioblastoma.Survival among patients with the methylated vs. unmethylated  of GATA6 and CASPTA6[CASP8 methylatMGMT, GATA6 and CASP8 promoter methylation in glioblastoma. GATA6 methylation occurred at a highest rate. Patient age, treatment regimen, MGMT, GATA6 and CASP8 methylation status were significantly associated with patient survival. MGMT and GATA6 were independent predictors for patient survival in younger patients’ group when adjusted for therapy. To further evaluate validation of the methylation profile of these genes is necessary for understanding their role in gliomagenesis and potential as GBM markers.The current study reveals new and important information on The authors are not aware of any biases that might be perceived as affecting the objectivity of this article.DR4, MGMT and GATA6 MSP analysis, statistical analysis and wrote a paper. PV did CD81 and CASP8 methylation analysis. VS did all statistical analysis. AT, KS, PVD generated an idea, gathered postoperative glioblastoma tissue and patient data.DS, PV, and VA generated an idea. DS did a part of The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/218/prepub


P. falciparum to anti-malarial drugs. To determine whether parasite susceptibility has been affected by the new anti-malarial policies, an ex vivo susceptibility and drug resistance molecular marker study was conducted on local isolates obtained from the Centre de santé Elizabeth Diouf .In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. Since the introduction of ACT, there have been very few reports on the level of resistance of pfcrt, pfdhfr, pfdhps and pfmdr1, were evaluated for a panel of 165 isolates collected from patients recruited from 17 August 2010 to 6 January 2011. The malaria isolates were assessed for susceptibility to chloroquine (CQ); quinine (QN); monodesethylamodiaquine (MDAQ), the active metabolite of amodiaquine; mefloquine (MQ); lumefantrine (LMF); dihydroartemisinin (DHA), the active metabolite of artemisinin derivatives; and doxycycline (DOX) using the Plasmodium lactate dehydrogenase (pLDH) ELISA.The prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance, i.e., in vitro resistant isolates, or isolates with reduced susceptibility, was 62.1% for MQ, 24.2% for CQ, 10.3% for DOX, 11.8% MDAQ, 9.7% for QN, 2.9% for LMF and 0% for DHA. The Pfcrt 76T mutation was identified in 43.6% of the samples. The pfmdr1 86Y, 184F and 1246Y mutations were found in 16.2%, 50.0% and 1.6% of the samples, respectively. The pfdhfr 108N, 51I and 59R mutations were identified in 81.9%, 77.4% and 79.4% of the samples, respectively. The double mutant (108N and 51I) was detected in 75.5% of the isolates, and the triple mutant  was detected in 73.6% of the isolates. The pfdhps 437G, 436A and 613S mutations were found in 54.4%, 38.6% and 1.2% of the samples, respectively. There was only one double mutant, 437G and 540E, and one quintuple mutant, pfdhfr 108N, 51I and 59R and pfdhps 437G and 540E. The prevalence of the quadruple mutant  was 36.7%.The prevalence of the in vitro P. falciparum susceptibility to anti-malarial drugs must be conducted in Senegal.The results of this study indicate that an intensive surveillance of the  Plasmodium falciparum have become resistant to chloroquine and other anti-malarial drugs [P. falciparum histidine-rich protein 2 (PfHRP2)-based rapid diagnostic test (RDT). Since this time, ACT use has been restricted to confirmed malaria cases to reduce drug pressure. In 2009, 184,170 doses of ACT were dispensed in Senegal [During the past 20 years, many strains of al drugs . One stral drugs . In 2006P. falciparum prevalence varied from 0.9% to 7.4% in asymptomatic women and children in Dakar [Malaria is transmitted in Dakar and its surrounding suburbs with a spatial heterogeneity of the human biting rate, which ranged from 0.1 to 250 bites per person per night during the rainy season from 2007 to 2010 . In 2008P. falciparum to anti-malarial drugs. To determine whether parasite susceptibility has been affected by the new anti-malarial policies, an ex vivo susceptibility study was conducted on local isolates from Dakar obtained from the Centre de santé Elizabeth Diouf . The malaria isolates were assessed for susceptibility to chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), the active metabolite of amodiaquine, mefloquine (MQ), lumefantrine (LMF), dihydroartemisinin (DHA), the active metabolite of artemisinin derivatives and doxycycline (DOX).Since the introduction of ACT, there have been very few reports on the level of resistance of P. falciparum CQ resistance transporter (pfcrt) for CQ [P. falciparum dihydrofolate reductase (pfdhfr) for pyrimethamine [P. falciparum dihydropteroate synthase (pfdhps) for sulphadoxine [P. falciparum multidrug resistance 1 (pfmdr1) for mefloquine resistance [In addition, the prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance was evaluated. The genes of interest included ) for CQ , P. falcethamine , P. falchadoxine  and P. fsistance  and potesistance ,14.pfcrt gene was first identified in 2000 [pfcrt gene, whose role is not yet defined. The odds ratio (OR) for CQ failure associated with the 76T mutation was 2.1  for a 14-day follow-up and 7.2  for a 28-day follow-up [The  in 2000 . To date in 2000 ,15,16, bollow-up . Howeverpfdhfr gene is associated with resistance to anti-folate drugs [in vitro resistance to anti-folate drugs and sulphadoxine-pyrimethamine. The OR values for codon 51 and 59 single mutants were 1.7 (95%CI: 1.0-3.0) and 1.9 (95%CI: 1.4-2.6), respectively [in vivo resistance to sulphadoxine-pyrimethamine by 4.3  [The S108N mutation in the te drugs . The OR te drugs . The addectively . The tristudies) .P. falciparum DHPS [in vivo resistance to sulphadoxine-pyrimethamine by 1.5  and 3.9 , respectively [Sulphones (dapsone) and sulphonamides (sulphadoxine) are inhibitors of the rum DHPS . The mutrum DHPS . The sinectively .pfdhfr (codons 51 + 59 + 108) plus pfdhps (codons 437 + 540) increases the risk of in vivo resistance to sulphadoxine-pyrimethamine by 5.2  [Pfmdr1, which encodes a 162 kDa protein named P. falciparum homologue of the P-glycoprotein (Pgh1), is located on chromosome 5. Field work has shown that the predictive value for CQ resistance and point mutations in the pfmdr1 sequence resulting in amino acid changes varies depending on the geographic area [pfmdr1 in P. falciparum[pfmdr1 86Y and pfcrt 76T increases the risk of in vivo resistance to CQ by 3.9  [The quintuple mutant of studies) . Pfmdr1,hic area ,21. Fivehic area . Howeverhic area . Using palciparum. Howeveralciparum. The comstudies) .In addition, the risk of therapeutic failure with amodiaquine is greater for patients harbouring the 86Y mutation with an OR of 5.4  . This mupfmdr1 mutations at codons 1034 and 1042 abolish or reduce the level of resistance to mefloquine [pfmdr1 allele at codons 1034, 1042 and 1246 confers mefloquine resistance to susceptible parasites [Pfmdr1 in P. falciparum isolates are not sufficient to explain variations in mefloquine susceptibility [P. falciparum isolates showed an association between mutation at codon 86 and an increase in susceptibility to mefloquine, halofantrine or artemisinin derivatives [It has been shown through heterologous expression that floquine . Moreovearasites . Howevertibility . Analyseivatives ,30.P. falciparum susceptibility to anti-malarial drugs was realised with the same venous blood sample used for diagnostic purposes. The study was reviewed and approved by the ethical committee of the Pasteur Institute and the Hôpital Principal de Dakar. Patients were treated by artemether-lumefantrine or artesunate-amodiaquine (depending on availability).In total, 165 patients  with malaria were recruited from 17 August 2010 to 6 January 2011 at the Centre de santé Elizabeth Diouf. Venous blood samples were collected in Vacutainer® ACD tubes  prior to patient treatment and transferred to the Hôpital Principal de Dakar within six hours. Parasitaemia ranged from 0.001% to 20% in the male group and from 0.001% to 10.6% in the female group. Informed verbal consent was obtained from patients and/or their parents before blood collection. Assessment of P. falciparum density and to confirm monoinfection. Parasitized erythrocytes were washed three times in RPMI 1640 medium  buffered with 25 mM HEPES and 25 mM NaHCO3. If parasitaemia exceeded 0.5%, infected erythrocytes were diluted to 0.5% with uninfected erythrocytes (human blood type A+) and re-suspended in RPMI 1640 medium supplemented with 10% human serum , for a final haematocrit of 1.5%.Thin blood smears were stained using a RAL kit  and were examined to determine the CQ, QN, DHA and DOX were purchased from Sigma . MDAQ was obtained from the World Health Organization , MQ was purchased from Roche , and LMF was purchased from Novartis Pharma . QN, MDAQ, MQ, DHA and DOX were first dissolved in methanol and then diluted in water to final concentrations ranging from 5 nM to 3200 nM for QN, 1.56 nM to 1000 nM for MDAQ, 3.2 nM to 400 nM for MQ, 0.1 nM to 100 nM for DHA and 0.1 μM to 502 μM for DOX. CQ was re-suspended and diluted in water to final concentrations ranging from 5 nM to 3200 nM. LMF was re-suspended and diluted in ethanol to obtain final concentrations ranging from 0.5 nM to 310 nM.msp1 and msp2 and microsatellite loci [The batches of plates were tested and validated on the CQ-susceptible 3D7 strain (West-Africa) and the CQ-resistant W2 strain (Indochina)  in 3 to 6 independent experiments using the same conditions described in the paragraph below. The two strains were synchronised twice with sorbitol before use , and cloite loci ,33 and ein vitro isotopic microtests, 200 μl of synchronous parasitized red blood cells  was aliquoted into 96-well plates pre-dosed with anti-malarial drugs. The plates were incubated in a sealed bag for 42 h at 37°C with the atmospheric generators for capnophilic bacteria, Genbag CO2® at 5% CO2 and 15% O2  [For the  France) . After t France) . The opt50) was calculated with the inhibitory sigmoid Emax model, with estimation of the IC50 through non-linear regression using a standard function of the R software (ICEstimator version 1.2) [50 values were validated only if the OD ratio (OD at concentration 0 / OD at concentration max) was greater than 1.8 and the confidence interval ratio  was less than 2.0 [The concentration at which the drugs were able to inhibit 50% of parasite growth (ICion 1.2) . IC50 vathan 2.0 .The total genomic DNA of each strain was isolated using the QIAamp® DNA Mini kit according to the manufacturer’s recommendations .pfcrt gene (containing codon 76) was amplified by PCR using CRTP1-sense 5′-CCG TTA ATA ATA AAT ACA CGC AG-3′ and CRTP1-antisense 5′-CGG ATG TTA CAA AAC TAT AGT TAC C-3′ primers [2 and 1 unit of RedGoldStar® DNA polymerase (Eurogentec) in a final volume of 25 μl. The thermal cycler  was programmed as follows: an initial 94°C incubation for 5 min; 40 cycles of 94°C for 20 sec, 56°C for 20 sec and 60°C for 40 sec; and a final 5-min extension step at 60°C. The PCR products were loaded on a 1.5% agarose gel containing 0.5 μg/mL ethidium bromide. The PCR products were diluted 1:100 in distilled water, and 2.5 μl of the final dilution was used for the second PCR. This PCR amplified a 275 bp segment around the mutation using a common inner primer CRTP3-sense 5′-TGA CGA GCG TTA TAG AG-3′ coupled with either CRTP4m-antisense 5′-GTT CTT TTA GCA AAA ATT G-3′ (detects the 76T codon), or CRTP4w-antisense 5′-GTT CTT TTA GCA AAA ATT T-3′ (detects the 76K codon) [2 and 0.75 U of RedGoldStar® DNA polymerase (Eurogentec) in a final volume of 25 μl.A 546-nucleotide fragment of the  primers . The reaK codon) . The reaP. falciparum clones 3D7 (chloroquine sensitive) and W2 (chloroquine resistant) were used as positive controls, and water and human DNA were used as negative controls. The PCR products from the amplification reactions were evaluated by electrophoresis on 2% agarose gels.The PCR conditions were as follows: initiation at 94°C for 5 min; 15 cycles at 94°C for 20 sec, 48.5°C for 20 sec and 64°C for 40 sec; and a final 5 min extension step at 64°C. Purified genomic DNA from pfmdr1 fragments carrying the five key codons [′-AGA GAA AAA AGA TGG TAA CCT CAG-3′ and antisense 5′-ACC ACA AAC ATA AAT TAA CGG-3′ to determine the sequences of codons 86 and 184 (MDR1-1), and a second fragment (968 base pairs) was amplified with the primer pair sense 5′-CAG GAA GCA TTT TAT AAT ATG CAT-3′ and antisense 5′-CGT TTA ACA TCT TCC AAT GTT GCA-3′ to determine the sequences of codons 1034, 1042, and 1246 (MDR1-2) [2, 200 μM deoxynucleoside triphosphate mixture   and 1 U of RedGoldStar® DNA polymerase (Eurogentec) in a final volume of 25 μl. The thermal cycler (T3 Biometra) was programmed for MDR1-1 as follows: an initial step at 94°C for 5 min; 40 cycles of 94°C for 30 sec, 52°C for 30 sec and 72°C for 1 min; and a final 10-min extension step at 72°C. For MDR1-2 the parameters were as follows: an initial step at 94°C for 5 min; 40 cycles of 94°C for 30 sec, 56°C for 1 min and 72°C for 1 min 30 sec; and a final 10-min extension step at 72°C. The PCR products were loaded on a 1.5% agarose gel containing 0.5 μg/mL ethidium bromide. Amplicons were purified using the QIAquick 96 PCR BioRobot Kit and an automated protocol on the BioRobot 8000 workstation . The purified fragments were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using the primers described above. The sequencing reaction products were purified using the BigDye XTerminator® Purification Kit (Applied Biosystems) in accordance with the manufacturer’s instructions. The purified products were sequenced using an ABI Prism 3100 analyser (Applied Biosystems). The sequences were analysed using Vector NTI advance (TM) software .Two primer pairs were used to amplify y codons . A 590-b(MDR1-2) . The reapfdhfr was amplified using the following primers: sense 5′-ACG TTT TCG ATA TTT ATG C-3′ and antisense 5′ TCA CAT TCA TAT GTA CTA TTT ATT C-3′[2, 200 μM deoxynucleoside triphosphate mixture   and 1 U of RedGoldStar® DNA polymerase (Eurogentec) in a final volume of 25 μl. The PCR conditions were as described in [A 562-bp fragment corresponding to the coding region of  ATT C-3′. The rearibed in . The amppfdhps was amplified using the following primers: sense 5′-GTT GAA CCT AAA CGT GCT GT-3′ and antisense 5′-TTC ATC ATG TAA TTT TTG TTG TG-3′[pfdhfr, and the PCR conditions were as described in [A 672-bp fragment corresponding to the coding region of TTG TG-3′. The fraribed in . The amp50 values were analysed after logarithmic transformation and expressed as the geometric mean of the IC50 and a confidence interval of 95% (CI95%). Using the Plasmodium lactate dehydrogenase (pLDH) ELISA under Genbag conditions, the cut-off values for in vitro resistance, or reduced susceptibility, were 77 nM, 61 nM, 115 nM, 12 nM, 611 nM, 30 nM and 37 μM for CQ, MDAQ, LMF, DHA, QN, MQ and DOX, respectively [ICectively .ex vivo, and 34 isolates were successfully cultured. The average parameter estimates for the seven anti-malarial drugs utilised against the P. falciparum isolates are given in Table P. falciparum isolates with decreased susceptibility to MQ in vitro reached 62.1%.Out of the 165 patients recruited at the Centre de santé Elizabeth Diouf, 63 were tested Pfcrt was examined in 156 of the samples. Within the pfcrt gene, the 76T mutation was identified in 43.6% of the samples. Eighteen samples (10.3%) were mixed, yielding both K76 and 76T.pfmdr1 polymorphisms are shown in Table pfmdr1 gene.The results for pfdhfr polymorphisms are presented in Table The results for pfdhps polymorphisms are presented in Table pfdhfr 108N, 51I and 59R and pfdhps 437G and 540E), and 58 isolates (36.7%) were quadruple mutants .The results for the ex vivo susceptibility of P. falciparum isolates, taken from patients in Dakar, to seven standard anti-malarial drugs and the prevalence of several molecular markers involved in anti-malarial drug resistance. The majority of the patients, recruited at the Centre de santé Elizabeth Diouf  from August 2010 to January 2011, said that they did not leave Dakar and its surrounding suburbs during the month preceding their malaria attack.This report describes the evaluation of the in vitro resistance to MQ has increased since previous studies conducted in Senegal. In Dakar, the percent of isolates with decreased susceptibility was 17% in 2001 [in vitro resistance to MQ was 22% in 1995 [in vitro decreased susceptibility to MQ and its clinical implications in Africa. It is important to monitor the evolution of P. falciparum susceptibility to MQ, to archive suspicious isolates and to correlate clinical outcomes with pharmacokinetic and phenotypic responses and with molecular markers.The prevalence of isolates with reduced susceptibility to MQ remains high (62.1%) in Dakar, but relatively stable compared with the previous year (55%) . The lev in 2001  and 13%  in 2001 . In Diel in 1995  and 15%  in 1995 ,44. Prop in 1995 , and MQ in vitro P. falciparum resistance to CQ was reported in Dakar, and reports of resistance in other regions of the country followed shortly [in vitro resistance to CQ increased from 1995 to 1999 in Dielmo with 32% resistance in 1995 [in vitro resistance to CQ in Dakar was low and stable in comparison with the previous year (24.2% versus 22%) [in vitro resistance in Thies in 2007 (23% of isolates exhibiting CQ resistance) [pfcrt have been shown to be correlated with CQ resistance in different parts of the world [pfcrt 76T mutation has decreased since 2004 in Dakar. From 2000 to 2001 in Guediawaye, a suburb of Dakar, a prevalence of 92% of was observed for the 76T mutation in pregnant women with malaria [pfcrt 76T mutation was 65% in patients hospitalised for malaria at the Hôpital Principal de Dakar [pfcrt 76T mutation was 75.8% in pregnant women taking chloroquine prophylaxis in Thiadiaye (84 km southeast of Dakar) [pfcrt 76T mutation was identified in 43.6% of the patients recruited from August 2010 to January 2011. These data are consistent with previous works on CQ molecular resistance in Dakar in 2009 (37.8%) [pfcrt 76T mutation ranged from 64% to 79% before CQ withdrawal (2000 to 2003) [As far back as 1988,  shortly . From 19 shortly . The in  in 1995  compared in 1995 , 44% res in 1995  and 55%  in 1995 . In 2010sus 22%) . These distance) . The evohe world . The pre malaria . In Piki malaria , 63.9% i malaria . In 2002de Dakar . From 20f Dakar) . In this (37.8%) , in cent (37.8%) . Howeverto 2003) ,55,60, tto 2003) ,60 whilein vivo CQ study in Malawi five years later, in which CQ was found to be 99% effective [This decrease in CQ resistance parallels the withdrawal of CQ treatment and the introduction of ACT in 2002 in Senegal. However, in 2003, CQ was still being administered to patients. The prevalence of CQ in the urine ranged from 14.5% to 47.5% in two- to nine-year-old children from northern Senegal and from 9.0% to 21.4% in children from southern Senegal . In 2006ffective . The rapffective . Despitein vitro reduced susceptibility to MDAQ slightly increased in 2010, 11.8% versus 6% in 2009 [The prevalence of isolates with  in 2009 . The pre in 2009 ,48, whil in 2009 .pfmdr1 mutations 86Y, 184F and 1246Y were 16.2%, 50.0% and 1.6%, respectively. In 2000 and 2001, prevalence rates of 31% and 30.6%, respectively, were observed for pfmdr1 86Y in Pikine [pfmdr1 is still debated. The pfmdr1 86Y mutation has been shown to be associated with in vivo resistance to amodiaquine in recrudescence after monotherapy with amodiaquine [pfmdr1 1246Y mutation has also been found to be associated with in vitro resistance to amodiaquine [pfmdr1 86Y mutation was found to be associated with amodiaquine failure, with an odds ratio of 5.4 [pfmdr1 of 86Y predicts that 16.2% of isolates would be resistant to amodiaquine in 2010 in Senegal. The resistance to amodiaquine has remained low even after the introduction of artesunate-amodiaquine in 2006 in Senegal. A study in Dakar and Mlomp from 1996 to 1998 showed that monotherapy with amodiaquine remained effective for treating uncomplicated malaria in areas where CQ resistance was prevalent [in vitro and in vivo surveillance programmes, such as those championed by the Worldwide Antimalarial Resistance Network [The prevalence of the n Pikine ,55, and n Pikine . Howeverdiaquine  or afterdiaquine . The pfmdiaquine  and withdiaquine ,72. In ao of 5.4 . Based orevalent . The artrevalent . The fixrevalent . Howeverrevalent ,77. In t Network ,79.in vitro susceptibility to DHA. This result is consistent with previous studies that did not show any parasites resistant to artesunate [et al. reported that 3% - 23% of isolates had IC50 values greater than 15 nM in Mlomp between 2000 and 2004 [50 values can also be found for artemisinin, with an IC50 > 30 nM in Dakar [50 > 45 nM [No isolate exhibited reduced tesunate ,43,44. Hand 2004 . High ICin Dakar  and arte > 45 nM .P. falciparum malaria in Senegal is the combination of artemether-lumefantrine. Only 2.9% of the isolates presented reduced susceptibility to LMF, and this prevalence did not rise in Senegal after the introduction of ACT. In 1996, 6% of isolates from Dielmo were resistant to LMF in vitro[The other ACT first-line treatment for uncomplicated  in vitro; and 1%  in vitro.50 to QN were already identified in 1984 [P. falciparum isolates to QN has varied widely. The IC50s for isolates collected in Senegal were 31 to 765 nM in 1984 (Thies and Kaolack) [In 2009, 7% of isolates showed low reduced susceptibility to QN, which is in accordance with previous studies in Dakar ,40,41 an in 1984 . Even inKaolack) , 5 to 93Kaolack)  and 6 toKaolack) ,83 or inin vitro reduced susceptibility to DOX was observed in Dakar in 2010, which is similar to the prevalence observed in 2009 (12%) [50 of DOX was similar to those estimated in Dielmo in 1998 [in vitro has a delayed effect upon growth and requires the prolonged incubation of parasites [in vitro susceptibility.A prevalence of 10.3% of isolates with 09 (12%) . The mea in 1998 ,86. The arasites ,87. Howepfdhfr 108N mutation has been shown to be correlated with in vitro and in vivo resistance to pyrimethamine [in vitro resistance to anti-folate drugs and sulphadoxine-pyrimethamine. The OR values for single mutants of codons 51 and 59 are 1.7 (95%CI: 1.0-3.0) and 1.9 (95%CI: 1.4-2.6), respectively [pfdhfr 108N was 81.9% in patients with malaria who were treated at the Centre de santé Elizabeth Diouf, which is similar to the results of those treated in 2009 at the Hôpital Principal de Dakar (82.4%) [in vivo resistance to sulphadoxine-pyrimethamine by 4.3 . Isolates carrying a combination of three mutations  were associated with high-level pyrimethamine resistance and represented 73.6% of isolates. In 2002, in Dakar, the prevalence of pfdhfr 108N was 65%, and triple mutants were identified in 50% of the isolates [pfdhfr codon 108 was 78% in Pikine, and the prevalence of the triple mutant was 61% [The ethamine . The OR ethamine . The addectively . In 2010 (82.4%) . The triisolates . In 2003 was 61% . Additio was 61% .pfdhps 437G mutation has been shown to be correlated with in vitro and in vivo resistance to sulphadoxine [in vivo resistance to sulphadoxine-pyrimethamine by 1.5  and 3.9 , respectively [pfdhps 437G mutation was 54.4% in patients with malaria in Dakar. Only one isolate (0.6%) carried the double mutation (437G and 540E) that is associated with high-level sulphadoxine resistance. The mutation of codon 613 (A613S) (1.2%) is very rare in Africa. In 2002, in Dakar, only 20% of isolates harboured the pfdhps 437G mutation [pfdhps codon 437G was 40% in Pikine [pfdhps 437G significantly increased after intermittent preventive treatment of infants with sulphadoxine-pyrimethamine [pfdhfr 108N, 51I and 59R triple mutant and 36.7% for the quadruple mutant pfdhfr 108N, 51I and 59R and pfdhps 437G), the use of sulphadoxine-pyrimethamine as an intermittent preventive treatment (IPT) must be monitored. Encouragingly, only one quintuple mutant, Pfdhfr 108N, 51I and 59R and Pfdhps 437G and 540E, which is associated with high-level sulphadoxine-pyrimethamine resistance has been identified to date.The hadoxine ,17. The mutation . In 2003n Pikine . Severalethamine ,90. GiveIPT with anti-malarial drugs given to all children and pregnant women once per month during the transmission season can provide a high degree of protection against malaria. Seasonal IPT with sulphadoxine-pyrimethamine and one dose of artesunate resulted in a 90% reduction in the incidence of clinical malaria in Senegal . The comP. falciparum susceptibility to individual drug components, such as DHA, MDAQ and LMF. However, the prevalence of P. falciparum isolates with reduced drug susceptibility to MQ increased, and clinical failures with QN have been reported in Senegal. Additionally, in Senegal, isolates with high IC50 values for artemisinin derivatives. Since 2004, the prevalence of chloroquine resistance has decreased, but the data argue against the re-introduction of chloroquine for mono-therapy in places where the resistant allele has dropped to very low levels following discontinuation of chloroquine treatment. The prevalence of isolates resistant to pyrimethamine is high (81.9%), with 73.6% of parasites exhibiting high-level pyrimethamine resistance. The prevalence of isolates resistant to sulphadoxine was 54.4%. Susceptibility to anti-malarial drugs remains stable between 2009 and 2010 in Dakar. Nevertheless, an intensive surveillance of the susceptibility of P. falciparum to anti-malarial drugs in vitro must be conducted in Senegal. In addition, maximising the efficacy and longevity of ACT as a tool to control malaria will critically depend on pursuing intensive research into identifying in vitro markers as well as implementing ex vivo and in vivo surveillance programmes. In this context, there is a need to identify molecular markers that predict ACT resistance, which can provide an active surveillance method to monitor temporal trends in parasite susceptibility.The introduction of ACT in 2002 in Senegal did not induce a decrease in The authors declare that they have no competing interests.in vitro testing of drug susceptibility. AP and NW carried out the molecular genetic studies. FDS, VR and AT supervised, carried out and coordinated the field collection of isolates from patients. RB, BW and BP conceived and coordinated the study. BP and SB analysed the data. BF, AP, NW, SB and BP drafted the manuscript. All authors read and approved the final manuscript.BF, EB, YD, RB and BP carried out the


In vitro studies were conducted using a liposome model system to simulate biological cell membranes. Total antioxidant potential and polyphenol content of the herbal preparation was measured. For in vivo analysis, 10 healthy human subjects consumed either three or six teaspoons of greens+ per day for four weeks. Blood samples were analyzed at baseline and at the conclusion of the treatment period for total antioxidant capacity, polyphenol content, protein, lipid and LDL oxidation, and the level of glutathione peroxidase. Results showed that greens+ supplementation was well tolerated and increased serum antioxidant potential at higher levels of intake in a dose-dependent manner. HPLC analysis showed the presence of quercetin, apigenin, kaempferol and luteolin in the supplement. Plasma analysis indicated the presence of kaempferol only. A statistically significant (p < 0.05) reduction in protein and lipid oxidation was observed. Based on its antioxidant properties, the results suggest that greens+ might play a role in reducing the risk of chronic diseases involving a burden of oxidative damage.Dietary antioxidants play an important role against oxidation, an underlying mechanism in the incidence of chronic diseases. Greens+ is a commercially available preparation containing a variety of plant-derived ingredients. The aim of the current study was to evaluate the antioxidant potential of the methanolic extract of greens+ powder using  A clear understanding of the antioxidant properties of this herbal preparation will contribute towards evaluating its efficacy and make appropriate recommendation for its use.Oxidative damage to cellular biomolecules such as lipids, proteins and DNA is thought to play a crucial role in the incidence of several chronic diseases –5. Flavo2.2.1.In vitro experiments consisted of: (i) measuring the overall antioxidant potential in a methanolic extract of greens+ of various concentrations. The ABTS  decoloration assay [on assay  was used2.2.2.2.1.The overall antioxidant potential of the greens+ expressed as trolox equivalent was estimated using the ABTS  method . Results2.2.2.4 in the presence of 100 μL of greens+ solution at various concentrations . The final concentrations of liposome and FeSO4 in the incubation mixture were 5 mg/mL and 0.09 mM respectively. Lipid oxidation was measured by the thiobarbituric acid (TBA)-malondialdehyde (MDA) assay [Multilamellar liposome membranes were prepared using 100 mg egg phosphatidyl choline in 20 mL of 2 mM phosphate buffer using the method as described by Balachandran and Rao . They weA) assay .2.2.3.One gram of greens+ powder was extracted with 20 mL 50% acetone and hydrolyzed with 5 mL of 6 M HCl for 1 h. The hydrolysate was cooled and centrifuged at 3,000 rpm for 15 min at room temperature. The supernatant was then neutralized with 10 N NaOH to pH 6.6 and diluted with distilled water. The diluted extract was passed through Sep-Pak C18  cartridge column for solid phase extraction. The column was then eluted with 100% acetone.2.2.4.et al. [4H2PO4, pH 4.4 and solution B: 0.1 M NH4H2PO4, pH 4.4 (20%), acetonitrile (60%), methanol (10%) and water (10%) with gradient 0–15% B, 0–5 min; 60% B, 5–30 min; 100% B, 30–50 min. The flow rate of the mobile phase was 1 mL/min. Ten microlitre was injected onto the column. The external standards of flavonoids such as quercetin, kaempferol, myricitin, luteolin and apigenin  were used as reference standards.The analysis was carried out according to the method of Hollman et al. . Chromat2.3.2.3.1.2.3.1.1.in vivo study involved (i) measuring serum total antioxidant potential; (ii) measuring serum lipid, protein and LDL oxidation; (iii) measuring erythrocyte glutathione peroxidase activity; (iv) HPLC measurement of the levels of plasma polyphenols. Ten healthy human subjects (Five men and five women), nonsmokers who were not taking any medication or vitamin supplements, were recruited into the study. Average age (years), weight (kg), and body mass index of the subjects were 29.3 ± 2.3, 72.0 ± 3.37, and 24.1 ± 0.70 respectively. The average systolic and diastolic pressure were in the normal range of 104 ± 1.63 and 66.5 ± 1.30 respectively. Subjects maintained their body weight during the study period.The 2.3.1.2.Study protocol used in this study is shown in 2.3.2.For the analysis of polyphenols in plasma, the method as described previously for greens+ herbal preparation was used.2.3.3.2.3.3.1.+ • method [in vitro procedures.Trolox equivalent antioxidant capacity in serum was measured by the spectrophotometric ABTS• method  as in th2.3.3.2.−1.Protein oxidation was estimated by measuring the loss of reduced thiol (-SH) groups using 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) assay . Thiols 2.3.3.3.535 1.56 × 105 M−1.Malondialdehyde, as a measure of lipid peroxidation, was measured by the thiobarbituric acid-malondialdehyde (TBA-MDA) assay and reported as TBA reactive substances (TBARS) . Results2.3.3.4.535 1.56 × 105 M−1.For LDL oxidation analysis, serum LDL was isolated by isoelectric precipitation with buffered heparin . LDL cho2.3.3.5.et al. [Glutathione peroxidase activity in erythrocytes was measured using the method of Pleban et al. . The enz2.4.t test was used to compare the results of the RI and treatment groups. Values are reported as means ± SEM. A probability value less than 0.05 (p < 0.05) was considered statistically significant.All calculations were performed using Excel 5.0 (Microsoft Corporation). The paired Student’s 3.3.1.3.1.1.Greens+ is a commercially available herbal supplement containing several botanical products that are considered as good sources of the polyphenols having antioxidant properties. The herbal preparation itself was never tested for its antioxidant properties. Results obtained in this study are shown in Based on this observation, the antioxidant potential of greens+ was evaluated in a liposomal lipid peroxidation model. Liposomes are commonly used to simulate cell membranes. As shown in 3.1.2.HPLC procedure was used to detect the presence of polyphenols present in greens+ and to quantify them. Polyphenol composition of the herbal preparation is shown in 3.2.in vivo estimation of the antioxidant potential of greens+, ten healthy human subjects consumed either three or six teaspoons of the herbal preparation per day for four weeks. Blood samples were collected, processed and analyzed for the presence of polyphenols, total antioxidant potential, lipid, protein and LDL oxidation, and glutathione peroxidase activity.For the 3.2.1.No changes were observed in the body weights, BMI and blood pressure of the subjects after oral supplementation of greens+ at two different doses for four weeks. These observations suggest no adverse effects of ingesting greens+ even up to six teaspoons per day for four weeks.3.2.2.Results are shown in 3.2.3.3.2.4.Results are shown in 3.2.5.Results are shown in in vitro and in vivo antioxidant properties of the herbal preparation greens+. The liposome model was used in the in vitro studies to simulate a biological cell membrane. Greens+ showed a dose-dependent trolox equivalent antioxidant capacity under the in vitro conditions. A similar effect on the total antioxidant potential was also seen in the serum of human subjects consuming greens+ for a period of four weeks. HPLC analysis of greens+ showed the presence of four major polyphenols: quercetin, apigenin, kaempferol and luteolin. Since the powdered greens+ preparation contains several plant-derived products that are evidently significant sources of antioxidant polyphenols, the increase in the serum antioxidant capacity, following greens+ consumption, could be related to the absorption of the polyphenols. Kaempferol was found to be the only major polyphenol present in the human plasma at higher concentration (six teaspoons) after four weeks of consumption. Some of the other polyphenols found in greens+ were undetected in the plasma of subjects. This may be due to the accumulation of flavonoids mainly as glucoronide and sulfate conjugates in blood plasma [A study was undertaken to investigate the d plasma . Neverthd plasma  found thet al. [The liposome model system used in this study also showed a significant antioxidant capacity at higher concentrations of ingestion. Cao et al.  demonstrThe consumption of greens+ by healthy human subjects showed a significant increase in the levels of reduced thiols indicating a decrease in the level of oxidized proteins in the serum. The damage caused to enzymes via protein oxidation is significant in discussions regarding human health. The activity of metabolic enzymes that otherwise play a crucial role in the detoxification of carcinogens and repair the damaged DNA may be inhibited, the net result being a contribution to metabolic disorders and increased risk of chronic disease –29. AlsoSerum MDA, indication of lipid peroxidation, was reduced significantly with the consumption of greens+. This effect is likely related to the presence of polyphenols. Plant phenolic compounds trap chain-initiating radicals at the interface of the membrane, thus preventing the progression of the radical chain reaction. Additionally, the phytochemicals such as the polyphenols can chelate the transition metal ions responsible for the generation of reactive oxygen species and therefore inhibit the initiation of the lipoxygenase reaction .Greens+ administration did not change LDL oxidation as measured by LDL malondialdehyde. This could be ascribed to the presence of more water-soluble polyphenols that might have been absorbed by the protein-rich HDL fraction. Polyphenols have been shown to have high affinity for proteins . Other sResults obtained in the present study also showed an increase in the erythrocyte glutathione peroxidase activity during greens+ supplementation.4.in vitro testing. The results of the in vivo arm of the study suggest that one or more of these phytochemicals are well absorbed, retain their antioxidant properties in vivo and lower oxidative stress at an intake level of six teaspoons per day for four weeks. The trend observed with three teaspoons could be due to shorter period of intake and smaller number of subjects. A long-term study with larger number of subjects should be undertaken to confirm the effect of low level intake of greens+ on oxidative stress. Overall, the results suggest a potentially important role of greens+ in reducing oxidative stress and as such, it may play a long-term role in the prevention of chronic diseases.In summary, the greens+ formulation includes a variety of plant-derived ingredients with polyphenolic antioxidants that have been identified via