Very few efforts have been undertaken to scale-up low-cost approaches to cervical cancer prevention in low-resource countries.In a public sector cervical cancer prevention program in Zambia, nurses provided visual-inspection with acetic acid (VIA) and cryotherapy in clinics co-housed with HIV/AIDS programs, and referred women with complex lesions for histopathologic evaluation. Low-cost technological adaptations were deployed for improving VIA detection, facilitating expert physician opinion, and ensuring quality assurance. Key process and outcome indicators were derived by analyzing electronic medical records to evaluate program expansion efforts.Between 2006-2013, screening services were expanded from 2 to 12 clinics in Lusaka, the most-populous province in Zambia, through which 102,942 women were screened. The majority (71.7%) were in the target age-range of 25–49 years; 28% were HIV-positive. Out of 101,867 with evaluable data, 20,419 (20%) were VIA positive, of whom 11,508 (56.4%) were treated with cryotherapy, and 8,911 (43.6%) were referred for histopathologic evaluation. Most women  received same-day services (including 5% undergoing same-visit cryotherapy and 82% screening VIA-negative). The proportion of women with cervical intraepithelial neoplasia grade 2 and worse (CIN2+) among those referred for histopathologic evaluation was 44.1% . Detection rates for CIN2+ and invasive cervical cancer were 17 and 7 per 1,000 women screened, respectively. Women with HIV were more likely to screen positive, to be referred for histopathologic evaluation, and to have cervical precancer and cancer than HIV-negative women.We creatively disrupted the 'no screening' status quo prevailing in Zambia and addressed the heavy burden of cervical disease among previously unscreened women by establishing and scaling-up public-sector screening and treatment services at a population level. Key determinants for successful expansion included leveraging HIV/AIDS program investments, and context-specific information technology applications for quality assurance and filling human resource gaps. A high incidence of invasive cervical cancer (ICC) is observed in low- and middle-income countries (LMICs) where high quality prevention services are unavailable or inaccessible, awareness about cancer risk is low, healthcare infrastructures are fragmented and dysfunctional, lack of appropriate public health policies and competing priorities detract from making substantive improvements to the status quo –4. WhileIn 2013, the World Health Organization (WHO) endorsed the use of VIA and cryotherapy-based ‘screen-and-treat’ programs in countries without existing cervical cancer services . Yet, seWe sought to answer these implementation research questions by conducting an evaluation of the Cervical Cancer Prevention Program in Zambia (CCPPZ), a public-sector program initiated in 2006 that has provided services (as of March 2015) to over 200,000 women using a locally contextualized intervention with simultaneous health system strengthening. This effort represents the largest such national-level initiative and offers a model for scaling-up of cervical cancer prevention services in sub-Saharan Africa and globally.Zambia has some of the world’s highest rates of ICC incidence  and mortality  rates. ICC is the most commonly diagnosed malignancy among the country’s adult population (both sexes combined), and accounts for more than one-third of annual new cancer cases among women. Zambia is also experiencing a generalized HIV epidemic, with HIV sero-prevalence rates of over 14% in the general adult population. The HIV and cervical cancer epidemics intersect significantly, with HIV increasing the risk for cervical cancer and worsening its prognosis. Several national and international efforts, including those funded through the U.S. President’s Emergency Plan for AIDS Relief (PEPFAR), have invested significant resources in upgrading Zambia’s capacity for prevention, care, and treatment of HIV/AIDS over the past decade ,17.Fig 2)., peer review, quality assurance, continuing medical education, objective record of screening test results, and access to expert opinion through immediate distance consultation, if needed . Fig 4)( Cryotherapy was provided by trained nurses using a double freeze technique [To address the problem of patient adherence to treatment recommendations we tightly linked screening to treatment in the form of a single-visit ‘screen-and-treat (freeze)’ approach for cryotherapy-eligible lesions, and a two-visit ‘screen-referral-loop electrosurgical excision procedure (LEEP)’ approach (with no intervening punch biopsy except when the lesion is suspicious for ICC) for cryotherapy-ineligible lesions. An acetowhite lesion was considered cryotherapy-eligible if all the following criteria were fulfilled: the lesion involves three-fourths or less of the cervix, does not extend into the endocervical canal, can be fully covered by the cryoprobe, and has no clinical suspicion/evidence of invasive cancer. echnique  operatinVIA/cryotherapy-based ‘screen and treat’ service platforms were initially expanded into the 12 largest primary public health clinics within Lusaka, the country’s most heavily populated province, with one referral clinic for LEEP and biopsy located at the University of Zambia ,23. An iFrom the inception of the program, community health workers and community mobilization actions were central components of our health promotion and adherence efforts. Volunteer women with an aptitude for voicing health concerns were selected from communities surrounding their respective screening clinics and trained to conduct cervical health promotion talks to women, to inform them of CCPPZ’s free, walk-in/same-day services using simple, but persuasive, messaging. Women requesting more information or expressing a desire to be screened were ‘navigated’ by these ‘peer educators’ to the CCPPZ clinics.To evaluate the program operations and scale-up of CCPPZ’s services from 2006 to 2013, we analyzed data from the CCPPZ electronic medical records database. We derived key process and outcome metrics that reflected the success of CCPPZ’s prevention services and integration and interaction with and beyond the HIV/AIDS program infrastructure. These metrics included: (i) annual program uptake rate between 2006–2013 and comparison of year-on-year change and differences by age categories and HIV status; these were derived from comparisons of relative proportions and changes in age and HIV-strata over time relative to total women screened, (ii) rates and trends in screening positivity and cryotherapy: comparison of changes over time of the proportion of women screening positive  and those receiving cryotherapy  and differences by age category groups and HIV status, (iii) proportion of women receiving treatment on the same day of screening (the ‘screen-and-treat’ approach) among screen-positive women that received cryotherapy treatment, (iv) differences in rates of histopathologic detection of cervical precancer  and ICC by HIV status and age categories, and programmatic yield by histopathologic diagnoses, (v) trends in provision of ‘same-day services’  and rates of confirmed CIN2+ (CIN2/3 and ICC) diagnoses among women referred for histopathologic evaluation, (vi) differences in screening positivity rates and rates of treatment of cervical precancer by HIV status.This outcomes evaluation study of anonymized patient records of a public sector implementation program was deemed exempt from human subjects review or the need for informed consent by the Research Ethics Committee of the University of Zambia.Table 1. The vast majority (72%) of women attending the screening were between the ages of 25–49 years, i.e., the target age for screening. The relative composition of age-groups of the screened populations remained relatively stable across the screening period of 2006–2013. Almost half (45%) of the women reported their monthly household income being less than 500,000 Zambian Kwacha , and almost two-thirds (67%) had never completed a high-school education. Just under half reported their first sexual intercourse at age 17 or less, and almost three-quarters (74%) reported having multiple (2+) lifetime sexual partners. A large majority (80%) had multiple (2 or more) pregnancies, and almost one-third (30%) had 5 or more pregnancies. A very tiny minority (1.8%) had ever been screened with Pap smears for cervical cancer.A total of 108,330 women were offered screening in 12 public sector clinics in the Lusaka province in Zambia between January 2006 and December 2013, of whom 102,942 (95%) underwent screening. The absolute number of women undergoing screening increased significantly with every calendar year (p-trend<0.001). The socio-demographic and sexual-reproductive characteristics of these women are shown in Fig 5-Panel A), about half were HIV-negative  and the HIV status was unknown in about a quarter of the women . The proportion of HIV-positive women among the total screened population declined from 55% in 2006 to 26% in 2013, while the proportion of women who were HIV-negative increased during the same period from 23% to 56% . Of the remaining 101,867 with conclusive screening results, 20,419 (20%) were screen-positive. The screen positivity rate differed by age and HIV-status and was highest (23%) among women between 30–39 years and lowest (12%) among women >50 years Fig 5-Panel B) Fig 5-Panel C)( and the decline was consistent across HIV-status (data not shown).Among the 102,942 women screened, 1,075 (1%) had uncertain VIA results . The rate of cryotherapy  declined with age (p<0.001) Fig 5-Panel B) Fig 5-Panel C)(. The rate of cryotherapy as a proportion of VIA positive women was highest (65%) among women <30 years, and proportionally decreased with age  (p-trend <0.001).A total of 11,508  women received cryotherapy, and 8,911 (44%) were referred for further evaluation for cryotherapy-ineligible lesions to the referral clinic at the University of Zambia . These proportions were similar across different HIV status categories.Of the 7,527 screen-positive women treated with cryotherapy who had complete information on screening and treatment dates, over two-thirds  had treatment on the same day of screening and almost 80%  within one week of screening Table 2). Among those who complied with referral, histopathology results were available for 3,938 (81.7%) women , of which ICC was present in 710 (18%), CIN2/3 lesions were present in 1,025 (26%), while 2,203 (56%) women had ≤CIN1 lesions. Overall, among women with ICC, 53% were HIV-positive, 27% were HIV-negative and 20% were of unknown-HIV status, while among women with CIN2/3 lesions, the corresponding prevalence of HIV-positive, HIV-negative, and HIV-unknown status was 71%, 18% and 11%, respectively Table 3) of the 710 cases of ICC were diagnosed in screen-positive women whose lesions were evaluated with LEEP, as they were cryotherapy-ineligible but not grossly suspicions for invasion, the latter which were diagnosed with punch biopsy .Among 8,911 screen-positive women referred for physician evaluation, 4,819 (54%) complied with the referral to the University of Zambia (Table 3) women screened, or one case of CIN2+ (precancer or cancer) among every 59.3 (95%CI: 51.7–77.0) women screened Table 3), which is more than twice than the present IARC/WHO-reported ICC rate of 58.4/100,000 in Zambia.Over the eight-year period (2006–2013), among every 1,000 women who underwent screening, 10 (95%CI: 9.4–10.6) were detected with histologically-confirmed CIN2/3 lesions, and 6.9 (95%CI: 6.5–7.5) were detected with histologically-confirmed ICC Fig 5-Panel D). In effect, only one in ten women undergoing screening is required to undertake a second visit . Among women who underwent histopathologic evaluation, the overall rate of CIN2+ lesions was 44.1% . This rate improved significantly from 41% in 2006 to 48% in 2013 (p-trend<0.001) .Overall, among the 98,961  women with complete screening data, the program provided ‘same-day services’ to a total of 86,301 (87%) women, which included 4,853 (5%) screen-positives who underwent same-visit cryotherapy and 81,448 (82%) who were VIA-negative and were counselled about their risk and advised follow-up after 3–5 years (if HIV-negative) and annual follow-up (if HIV-positive). The rate of ‘same day services’ increased between 2006 and 2013 from 88% to 94% (p<0.001) . Among VIA screen-positive women, as compared to HIV-negative women, women with HIV were less likely to be treated by cryotherapy , more likely to be referred to UTH for histologic evaluation of cryotherapy-ineligible lesions , and more likely to have histologically-confirmed CIN2+  lesions .The results of this programmatic evaluation demonstrate that VIA-based screening services can be scaled-up at a population-level when adopted as part of the routine health care system in a low-resource, high disease burden country. Cervical cancer prevention services can benefit by efficient linkages with HIV/AIDS care and treatment services and, with time, such services can be offered to all women at need, regardless of HIV status. Our evaluation shows that the operational efficiency of the program as measured by rates of ‘same-day services’ and histopathologically-confirmed precancer and cancer improves with experience and sustained efforts in quality improvement. Setting-specific challenges in implementation can be overcome with locally-developed solutions, particularly with sufficient political will and stakeholder support. The findings emanating from this implementation research study could provide stimulus for replicating such national-level program expansions in sub-Saharan Africa, as well as in other resource-constrained settings globally. Although the cervical screening program in Zambia heavily relied on the HIV care infrastructure for implementation and scaling-up, the broader lesson for settings with a lesser HIV burden is that cervical screening services can be successfully developed and scaled-up in routine public health services by innovative integration with other vertical health initiatives.Our study has highlighted the substantially high burden of histologically-confirmed precancerous and cancerous (CIN2+) lesions that is uncovered by the establishment of a population-based screening program. It exemplifies the critical need for investments in oncologic surgery and radiation therapy alongside screening platforms, in order to avoid unnecessary deaths, suffering, and social consequences of detected but untreated disease. It also underscores the importance of continued global efforts to expand HPV vaccination.A retrospective critique and evaluation suggests six distinguishing facets of this program that contributed to the success of the scale-up of the implementation.Fig 5-Panel A) allowed the development of capacity for the prevention of a non-communicable disease . Focusing on the provision of services for high-risk HIV-infected women initially allowed a firm rooting of our program and provided a foundation for sustainability, but did not hamper or preclude eventual wider expansion of services to the general (HIV-uninfected) women in the community, as evidenced by the increasing proportion of HIV-negative women over the program expansion period Second, we adapted the screening intervention to local circumstances instead of rigorously retrofitting approaches that are successful, but in radically different environments. While our approach of screening promoted task shifting (doctors to nurses), we harnessed the power of mobile technology to address human resource gaps by allowing distance consultation to improve clinical decision making. Embedding the basic principle of cervical cancer prevention with visual-based screening in a public health context and linking the initial encounter within a matrix of an affordable and locally available telecommunications technology  allowed us to roll-out standardized, quality-assured, cervical cancer screening and real-time distance consultation at remote sites across Zambia where it would never have been possible before .Third, we heavily invested in a surgical excision (LEEP) infrastructure and expanded diagnostic services to facilitate management of complex cervical lesions that exceeded the therapeutic limitations of cryotherapy. The diagnosis and treatment of complex cervical lesions not amenable to local ablation by cryotherapy is as critical as, if not more than, providing same-visit screen-and-treat services for cryotherapy-eligible lesions. This was particularly critical for HIV-positive women who were more likely to require referral for histologic evaluation than HIV-negative women. Screening without linkages to treatment is unethical, hence both ‘single-visit screen-and-treat’ and ‘screen-referral-LEEP’ were frontline components of our comprehensive cervical cancer prevention program. The surgical excision component was a part of the initial programmatic vision itself and not an after-thought. This ensured that treatment of precancerous lesions was feasible for both low- and high-grade precancerous lesions, as reflected by the fact 70% of screen-positive women who were eligible for treatment received it. It also provided the basis for evaluating the most critical histologic outcomes  for monitoring programmatic impact. LEEP also served the dual purpose of diagnosis and treatment for the vast majority of women diagnosed with CIN3 or more severe lesions such as preclinical, occult invasive cancer.Fourth, we constantly assessed all phases of the program through a rigorous process of monitoring and evaluation of interim outcomes. Our use of routine programmatic indicators  provided ongoing evidence on the role of improvements in the quality of screening and treatment services over time ,25. For Table 4) and public health program staff, consisting of Zambian nationals and an expatriate U.S. board-certified gynecologic oncologist (GPP) living within the country, and represent vital and intermediate steps in the development and sustenance of efficient healthcare delivery systems in such settings.Fifth, we strengthened the existing healthcare delivery system as we gathered evidence and assessed needs, on-the-go, as an innovative approach to sustainability and long-term success. Whether to immediately implement cervical cancer prevention services with meagre resources, or working first to develop well-functioning health systems, was no longer an issue for debate. We arrived at innovative solutions Sixth, women detected with invasive cervical cancer are treated with radical surgery or megavoltage radiotherapy/chemoradiotherapy at the national cancer center in Lusaka . This matrix type of cancer center has been organized by the Zambian government at the University of Zambia Teaching Hospital in close proximity to and interaction with pathology, diagnostic imaging and other tertiary care services in the teaching hospital that permits comprehensive cancer diagnosis and treatment services for cancer patients within the country.Fig 2), CCPPZ presently (September 2014) supports 33 ‘screen and treat’ clinics and 18 LEEP clinics located throughout the country’s 10 provinces We have thus embarked upon a phase of screening which is intended to facilitate a radical and exponential uptake of services. The Zambian government has (as of March 2015) officially adopted CCPPZ as its national cervical cancer prevention program and it will be administered out of the Ministry of Health. Building on our present clinic infrastructure and telecommunications matrix it will focus on the provision of services in an outreach context and will include screening camps and village-based screening in collaboration with Ministry of Chiefs and Traditional Affairs, with local chiefs and chieftainesses, traditional marriage counsellors and traditional healers as primary advocates and facilitators. This new approach continues our use of the philosophy of adaptive innovation which, in principle, incorporates respect for local beliefs and culture while integrating new ideas and technology that fit the local circumstances.


GRS for AO-AD from COGA explained a modest but significant proportion of the variance in all alcohol-related phenotypes in SAGE. Despite including effect sizes associated with large numbers of single nucleotide polymorphisms , GRS explained, at most, 0.7% of the variance in these alcohol measures in this independent sample. In OZ-ALC, significant but even more modest associations were noted with variance estimates ranging from 0.03 to 0.16%. In conclusion, there is modest evidence that genetic variation in AO-AD is associated with liability to other aspects of alcohol involvement.Age at onset of alcohol dependence (AO-AD) is a defining feature of multiple drinking typologies. AO-AD is heritable and likely shares genetic liability with other aspects of alcohol consumption. We examine whether polygenic variation in AO-AD, based on a genome-wide association study (GWAS), was associated with AO-AD and other aspects of alcohol consumption in two independent samples. Genetic risk scores (GRS) were created based on AO-AD GWAS results from a discovery sample of 1788 regular drinkers from extended pedigrees from the Collaborative Study of the Genetics of Alcoholism (COGA). GRS were used to predict AO-AD, AD and Alcohol dependence symptom count (AD-SX), age at onset of intoxication (AO-I), as well as maxdrinks in regular drinking participants from two independent samples—the Study of Addictions: Genes and Environment (SAGE;  Similarly, Babor et al.2 defined Type A and B alcoholics—the latter were distinguished by early onset of alcohol problems. Across these typologies, early-onset problematic use was consistently associated with a more severe form of the disorder, which was often accompanied by polysubstance use and other psychiatric comorbidity, particularly externalizing disorders.Multiple epidemiological and genetically informed studies have documented the importance of age at onset of alcohol dependence (AO-AD) as a key feature of sub-types of alcoholism that vary in etiology and severity.2 and this relationship may be influenced by shared genetic liability. Cloninger et al.7 also posited that Type II/early-onset alcoholism may represent a more heritable form of the disorder. While there have not been studies that examine the heritability of AO-AD, numerous studies have robustly documented the role of additive genetic influences on AD itself9 and on both age at first drink12 and the speed of transitioning from first use to the development of alcohol problems.14 In support of these findings, a recent genome-wide association study (GWAS) in extended pedigrees from the Collaborative Study of the Genetics of Alcoholism (COGA) found several single nucleotide polymorphisms (SNPs) that were significantly associated with AO-AD (P<5E−8).15 For rs2168784, the most significant SNP, 30% of those homozygous for the minor allele met criteria for alcohol dependence while only 19% of those homozygous for the major allele did. This SNP was also associated with AD diagnosis in the COGA dataset.AO-AD is also correlated with other features of drinking. For instance, earlier AO-AD is associated with more alcohol dependence symptoms,et al.15 have yet been replicated. However, the significance associated with individual top SNPs might be subject to sample-specific characteristics . Recently, investigators have begun to use genome-wide risk scores (GRS) that reflect the polygenic and aggregate nature of genotypic effects. Effect sizes generated for one phenotype in a given sample can be used to generate GRS in additional samples; the association between these GRS and a similar phenotype may be seen as evidence for replication while correlations between the GRS and other related phenotypes provide support for shared genetic underpinnings.16To our knowledge, none of the top SNP effects identified in the previous study by Kapoor et al.15 and applies these scores to two independent samples, the Study of Addictions Genes and Environment 17 and a sample of Australian subjects (OZ-ALC).18 In contrast to COGA, which is comprised of extended pedigrees with a high rates of alcohol dependence, SAGE consisted of alcohol-dependent cases and alcohol exposed but non-dependent controls. Even though SAGE subjects were drawn from studies that used family history of alcohol and drug dependence to ascertain cases, including COGA, all overlapping subjects were removed and all subjects were unrelated to each other. OZ-ALC, on the other hand, consisted of pedigrees that were derived from various sources, including family studies ascertained for heavy drinking and heavy smoking and a sample consisting of large sibships. Despite being similar to COGA for sibship size, the density of alcohol-related problems in the OZ-ALC pedigrees is substantially lower. The variability in SAGE and OZ-ALC allowed us to investigate the generalizability of the COGA findings.The present study utilizes GRS generated from the analysis of AO-AD in COGA (discovery sample) conducted by Kapoor 15 is associated with AO-AD in the independent portion of SAGE and in OZ-ALC. Second, we examine whether GRS for AO-AD is associated with other features of alcohol involvement, including age at first intoxication, lifetime maximum drinks in a 24-h period and number of symptoms and diagnosis of alcohol dependence in the SAGE and OZ-ALC datasets.Overall, we were interested in replicating and generalizing our prior findings and extending them to other alcohol-related phenotypes. Thus, our goals were twofold: first, we examine whether GRS created from the GWAS of AO-AD in the COGA discovery sampleData were drawn from the three sources described below. The institutional review board at each contributing institution reviewed and approved the protocols.15 subjects from that study who were not regular drinkers were excluded. Ascertainment was based on a proband in treatment for alcohol dependence who had at least two first-degree relatives affected by alcohol dependence. Of these subjects, 685 met criteria for DSM-IV alcohol dependence (mean age of onset 22.5 years). A genome-wide Cox proportional hazards regression model was used to test the association between age at onset of AD and 4 058 415 imputed SNPs with minor allele frequency ⩾5%.15 A robust sandwich variance estimators approach was used to account for the familial correlation among observations .The discovery sample was genome-wide SNP data on 1788 regular drinkers (defined below) from 118 large European-American families densely affected with alcoholism;19 Family Study of Cocaine Dependence (FSCD)20 and Collaborative Genetic Study of Nicotine Dependence (COGEND).21 Further details of the SAGE sample are available elsewhere.17 One hundred and twenty nine individuals who were both in SAGE and the COGA discovery sample were excluded. The sample consisted of alcohol-dependent cases  and alcohol-exposed controls (N=1169).SAGE consisted of 2593 unrelated European-American subjects. Of these, 2336 individuals who reported regular drinking were included in these analyses. Subjects were selected from three large, complementary studies: COGA,et al.22); (iii) the OZ-ALC-BIGSIB study, which ascertained large sibships (4–14 full siblings), regardless of sibling phenotypes. Further details regarding recruitment may be found in the study by Heath et al.18 OZ-ALC was not ascertained for alcohol dependence  and includes 1714 alcohol-dependent individuals (mean age of onset 26.3 years).The OZ-ALC sample consisted of 6169 individuals  from 2356 families ascertained from 3 coordinated studies derived from a larger Australian twin registry: (i) the Nicotine Addiction Genetics (NAG) Study which ascertained heavy smoking index cases; (ii) the OZ-ALC-EDAC study, which ascertained index cases with a history of alcohol dependence or scoring above the 85th centile for heaviness of drinking factor score  were included. As is the norm for Cox survival modeling, those who did not meet criteria for DSM-IV AD were censored at their age at interview.The discovery and replication samples utilized versions of the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA)For the present analyses, in addition to AO-AD, which was coded identically as in the discovery sample, the following measures were drawn from SAGE and OZ-ALC:AO-I, defined as the age at which the respondent reported first getting drunk .AD diagnosis (binary) was based on DSM-IV; individuals who endorsed three or more criteria that clustered within a single 12-month period were diagnosed with AD.Alcohol dependence symptom count (AD-SX) was defined as the sum of the seven DSM-IV dependence criteria.Maxdrinks, defined as the maximum number of drinks consumed in a single 24-h period during their lifetime. The measure was Winsorized at the 95th percentile (>100 drinks) and log (10) transformed for analyses.Height, via self-report, was used as a negative control.et al.;15 data are available at dbGaP phs000763.Genotyping was conducted using the Illumina OmniExpress array . A total of 4 058 415 SNPs that were imputed in BEAGLE (https://faculty.washington.edu/browning/beagle/beagle.html) were analyzed. Further details are available in the manuscript by Kapoor 17 The software package EIGENSTRAT25 was used to calculate principal components reflecting ancestral differences. Only genotyped SNPs were selected from SAGE, resulting in 669 984 overlapping SNPs which were further pruned for linkage disequilibrium (maximum pairwise r2=0.25 within sliding windows of 100 SNPs), resulting in 90 365 SNPs that were used for all subsequent analyses.For SAGE, DNAs were genotyped on the Illumina Human 1 M beadchip (Illumina) by the Center for Inherited Diseases Research (CIDR) at the Johns Hopkins University; data are available at dbGaP phs000092. A total of 948 658 SNPs passed data-cleaning procedures and further within sample filtering for autosomal and X-chromosome markers yielded 948 142 markers. HapMap genotyping controls, duplicates, related subjects and outliers were removed from the sample set.N=4601) were genotyped on the Illumina CNV370-Quadv3 (Illumina); genotyping on a small number of additional individuals was conducted on the Illumina 317 K (N=20) and 610 Quad v1 (N=517) platforms; data are available at dbGaP phs000181 . To account for the lower density of genotyped SNPs and variation in platform contents, imputation to HapMap (http://hapmap.ncbi.nlm.nih.gov) CEU I+II data  was conducted in MACH27 and best guess genotypes were selected based on Rsq⩾0.3 and imputation quality⩾0.9, resulting in 112 594 autosomal SNPs. Nuanced admixture was determined using EIGENSTRAT25 and outliers were removed, as outlined in the study by Heath et al.18For OZ-ALC, most subjects , 0.05 (GRS0.05), 0.10 (GRS0.1) and 0.50 (GRS0.5) were created in SAGE and OZ-ALC sample using PLINK28 and SAS . Briefly, SNPs in COGA that were significant at each P-value threshold  were selected. For each SNP, the effect size was calculated as the natural logarithm transformation of the hazard ratio from COGA. For every individual in SAGE and OZ-ALC, this effect size was multiplied by the number of copies of reference allele, and this product was summed across all SNPs.28 For the SAGE dataset, the number of SNPs for each score was: GRSall (110 797), GRS0.5 (58 374), GRS0.1 (12 254), GRS0.05 (6147) and GRS0.01 (1441), while for OZ-ALC number of SNPs for each score was: GPSall (112 594), GPS0.5 (57 053), GPS0.1 (12 161), GPS0.05 (6268) and GPS0.01 (1402). The resulting GRS was used to predict AO-AD, as well as other measures  in those datasets. Associations between age of onset measures and GRS were conducted using Cox proportional hazards analysis. Logistic and linear regression was used for dichotomous (AD) and continuously distributed  measures, respectively. Similar to the analysis in the discovery sample, a robust sandwich variance estimator approach was used to account for the familial correlation among OZ-ALC families. For both studies, sex, age at last interview and study source  were included as covariates.Based on effect sizes for the analysis of AO-AD generated in the discovery (COGA) sample, GRS at 29 that replication of polygenic scores is optimized when the size of the discovery and test samples is approximately equal; we performed 10 000 iterations in which we randomly resampled 1788 individuals from the pool of 2336 subjects in SAGE. Cox proportional hazard models were fit to each randomly drawn sample to examine whether the magnitude of the association was modified by selection of a comparatively sized test sample. Similar analyses were not conducted in OZ-ALC as random selection of subsets of individuals nested in pedigrees would not be representative of the sampling design nor would selection of subsets of whole pedigrees allow for adequate numbers of individuals with AO-AD.Based on recommendations by Dudbridge,NSAGE=1167; NOZ-ALC=1714) were more likely to report an earlier AO-I. They also reported higher Maxdrinks. In general, individuals from SAGE were heavier drinkers and have a greater number of AD-SX than those from OZ-ALC. This is unsurprising given the differences in ascertainment strategies. Modestly, earlier onset of drinking to intoxication and AD was noted in SAGE relative to OZ-ALC. No differences in height were noted across individuals with and without AD or across SAGE and OZ-ALC.Characteristics of the replication samples, SAGE and OZ-ALC, are presented in 0.05. Increasing P-value thresholds resulted in greater proportion of variance explained with the adjusted R2 ranging from 0.3% for GRS0.1 to 0.7% for GRS0.5. The variance explained was maximum when we included the SNPs with P⩽0.5. In addition to AO-AD, GRS explained a modest proportion of the variance in AO-I (≈0.3%). GRS from COGA were also related to AD-SX and Maxdrinks in SAGE, explaining 0.2 to 0.8% of the variance in these measures.As shown in P>0.05) with any GRS across SAGE and OZ-ALC.In contrast, AO-AD GRS from COGA explained only a very modest (but significant) proportion of variation in AO-AD (0.06–0.07%) and AD (0.03–-0.06%) in OZ-ALC . The GRSP<0.05) in the reduced SAGE dataset in all 10 000 permutations. However, the reduction in sample size influenced the magnitude of P-values and only about 12% of the time, the association P-values were equal to or more significant than the original P=6.70 × 10−06 observed with the full SAGE sample.Resampling 10 000 subsets of SAGE individuals to create a sample size that was equivalent to COGA resulted in similar results. GRS showed statistically significant association with the AO-AD  in two independent and differently structured and ascertained datasets, SAGE and OZ-ALC. GRS, especially when including SNPs associated with AO-AD at more liberal P-value thresholds, were significantly associated with a range of these alcohol-related measures in those two independent and distinctly ascertained samples. In contrast, there was no evidence for replication of the top 10 most significantly associated variants from COGA in either SAGE (6.6 × 10−1–8.1 × 10−1) or OZ-ALC (8.7 × 10−1–8.9 × 10−1). This strongly underscores the idea that multiple common genetic effects contribute to the etiology of complex disorders like addictions.Using effect sizes generated via a prior GWAS of AO-AD in the COGA family sample,32 For instance, Vink et al.32 used GRS constructed from a large meta-analysis of GWAS of tobacco smoking measures to predict variance in alcohol, tobacco and cannabis-related outcomes. In that study, polygenic scores that were associated with tobacco-related measures at P<10−70 explained, at most, 1.5% of the variance in any substance-related outcome. Similarly, Power et al.30 examined the relationship between cannabis involvement and GRS generated from a meta-analysis of schizophrenia  which included 13 genome-wide significant loci. Even though schizophrenia GRS were significantly associated with cannabis use, the scores, even at P<0.05 explained <1% of the variance in cannabis-related phenotypes. For alcohol-related measures, Salvatore et al.31 found that GRS generated for alcohol problems (N=4304) only predicted 0.6% of the variance in a similar measure in an independent sample. Despite relying on a smaller discovery sample , our findings are consistent with these estimates. Nonetheless, the small sample size of the discovery set limits the accuracy of predicted SNP effect sizes and likely influenced our ability to generate GRS that might be reliable predictors of alcohol involvement in independent samples.GRS comprised of >110 000 SNPs only captured very modest proportions of the variance in any alcohol-related measure (<1%). This observation is consistent with other studies.An additional consideration when viewing these results is the difference in ascertainment method across COGA, SAGE and OZ-ALC. The discovery sample (COGA) consisted of extended pedigrees ascertained for a dense family history of alcoholism and it was not expected that all variants associated with alcohol-related measures in such densely affected pedigrees would generalize to other cohorts. SAGE cases were selected for DSM-IV alcohol dependence from among several studies focused on alcohol, tobacco and cocaine and thus, as expected, we note a stronger degree of replication in this sample. In contrast, OZ-ALC comprises of samples ascertained for heavy smoking, discordance of heavy alcohol consumption measures and also for large sibship size (without any oversampling for substance-related phenotypes). Therefore, it is not surprising that replication in OZ-ALC, a less severely affected sample, is weaker for AO-AD, but occurs for ages of onset for earlier drinking milestones  and for measures that are quantitative indices of problem drinking . In OZ-ALC, AO-I and AO-R, as well as AD-SX and Maxdrinks may serve as proxies for genetic liability to problematic drinking, while in COGA and SAGE this liability may be appropriately captured by AO-AD itself. Even so, the statistical significance of the associations and the proportions of variance explained in OZ-ALC are markedly lower than those in SAGE. Nonetheless, as OZ-ALC is so markedly distinct from COGA and SAGE, any level of association between COGA GRS and alcohol-related measures in OZ-ALC may be considered as support for the generalizability of the COGA results.et al.29 has noted that there are two purposes for GRS: association testing  and prediction of phenotypic variance . Based on numerous simulations, he concluded that while most studies with approximately equally sized training  and testing  samples are well-powered for association testing, current training samples are underpowered for prediction. Consistent with this observation, we were interested in the former rather than the latter and even though we report R2 values, the emphasis of our analysis was association testing. We confirm that selecting a testing sample of approximately the same size as the training cohort, as was achieved via our bootstrapping approach, yields significant association. Dudbridge29 notes that 10-fold cross-validation might be a more efficient approach to maximizing prediction. As two of our samples are family-based (COGA and OZ-ALC), such cross-validation approaches, which may necessitate disaggregating members of pedigrees or selecting subsets of pedigrees that may or may not be informative for the etiology of genetically transmitted AD, may not be applicable. Hence, our findings should be observed in the context of association (and not prediction) testing alone.Dudbridge Finally, it is worth noting that we did not attempt to characterize the SNPs that comprised each GRS nor did we create biologically informed GRS by selecting variants that related to a specific neurotransmitter pathway . Given the high false discovery rates typically associated with ascribing a functional direction to such variants of purported biological importance  and our currently limited understanding of the etiology of AD, we employed a more conservative and agnostic approach of utilizing genome-wide data. Future studies that contrast such genome-wide PRS with biologically informed risk scores may be valuable in the construction of the architecture underlying the polygenicity associated with complex traits such as AO-AD. Nonetheless, our approach precludes any mechanistic interpretation of the polygenicity represented by each GRS.In conclusion, using GRS generated for AO-AD, we document that similar genetic factors might underpin the liability to alcohol dependence, drinking to intoxication and indices of problematic drinking in independent datasets. Future studies could strengthen these interpretations by conducting meta-analyses across multiple samples to produce larger discovery samples for generating GRS that could be applied to a wider range of alcohol and other substance phenotypes.


ABO-incompatible (ABOi) renal transplantation (RTx) from living donors is an established procedure to expand the donor pool for patients with end stage renal disease. Immunoadsorption (IA) is a standard procedure for the removal of preformed antibodies against the allograft. In this study, antigen-specific and non-antigen-specific IA in ABOi RTx were compared.10 patients underwent antigen-specific IA (Glycosorb group) and 13 patients non-antigen-specific IA (Immunosorba group). The effects of both procedures regarding antibody reduction, number of treatments, complications, costs, as well as the allograft function and patient survival were compared between both groups.Although the IgG levels were reduced equally by both procedures (p=0.82), the reduction of the IgM level was more effective in the Glycosorb group (p=0.0172). Patients in both groups required a median number of 6 IA before ABOi RTx. Allograft function at one year after AB0i RTx was similar in both groups (estimated glomerular filtration rate: 66 vs. 64 ml/min/1.73m² respectively), with a death-censored graft survival of 90.0% and 92.3% respectively. Complication rates did not differ between procedures. Due to the reuse of non-antigen-specific Immunosorba columns, costs were considerably lower in this group; however, the use of the Immunosorba-based IA was less time-efficient.Considering upcoming alternatives as simultaneous performance of dialysis and IA or a possible reuse of Glycosorb columns, this might become less relevant in the future. As the shortage of deceased donor kidney transplants is a growing problem for patients suffering from end stage renal disease (ESRD), ABO-incompatible (ABOi) living renal transplantation (RTx) has become an established alternative procedure . ABOi RTs, the perioperative management has continuously improved ”. The Mann-Whitney U test was performed for comparison. Regarding boxplots, mild outliers are defined as values more than 1.5*interquartile range (IQR) from the rest of the scores and extreme outliers as values more than 3*IQR from the rest of the values.This study includes only a small number of patients. Therefore, statistical analysis has to be interpreted only as a trend for different characteristics.The 23 patients who underwent ABOi RTx were between 19 and 68 years of age (46.1±13.8) and 15 (65%) were male. 2 6 and 12 months after RTx, respectively.Although additional PPh before ABOi RTx were performed in 60% of the Glycosorb group and in 77% of the Immunosorba patients, the number of PPh treatments in each patient before ABOi RTx was low. Postoperative IA and PPh until the first discharge from our hospital were rarely required. The efficiency of IgG antibody reduction—related to the number of performed IA—was not different between the study groups . Costs fThe death-censored graft survival 12 months post RTx was similar in both groups (90.0% in the Glycosorb group vs. 92.3% in the Immunosorba group). One patient of the Glycosorb group experienced renal graft loss 107 days after ABOi RTx due to a severe ABMR.A patient of the Immunosorba group suffered from a haemolytic uremic syndrome (HUS) a few days after RTx, which was primarily thought to be tacrolimus-induced. Hence, PPh was started, but due to severe allergic reactions during the first procedure, it was switched to IA (10 sessions) before eculizumab was given. Furthermore, the immunosuppressive therapy was switched from tacrolimus to everolimus. 6.6 months after RTx this patient underwent allograft loss due to HUS, followed by several humoral and cellular allograft rejections and recurring pulmonary infections and UTI. The patient died from a bradycardia during hemodialysis 13.3 months after transplantation.Overall renal function did not differ between the two IA groups at the first discharge from our hospital after ABOi RTx  and in aIn 70% (Glycosorb group) and respective 77% (Immunosorba group) of the patients, at least one allograft biopsy was performed . In 3 paBoth groups showed similar incidence of biopsy-proven acute rejections. Two patients of the Immunosorba group who experienced an ABMR had HLA specific DSA (14 and 504 days after RTx). Nevertheless, all patients were successfully treated, either with PPh and prednisolone in case of ABMR or with prednisolone only in case of TCMR and borderline rejection. BK nephropathy and CNI nephrotoxicity occurred only in a small number of cases in the Immunosorba group.Typical surgical and infectious complications were observed in both groups . The mosDesensitization procedures using antigen-specific IA in ABOi kidney transplant recipients lead to considerably good graft and patient survival rates. However, non-antigen-specific IA with reusable Protein A columns has emerged as an attractive alternative . Morath Herein, for proof-of-concept, the non-antigen-specific Immunosorba column was compared to the antigen-specific Glycosorb column for desensitization of ABOi RTx recipients.We used a modified Stockholm protocol for desensitization of recipients. Patients with IgG isoagglutinin titers above 1:128  were included in our study. However, the median isoagglutinin titer in both groups was low (1:32) but comparable to data presented by Morath et al. . This isSuccessful desensitization in our cohort was independent of the IA column used. Morath et al. who failed to achieve a sufficient reduction in isoagglutinin titers by solely use of IA in one third of patients supposed that the observed treatment failure with IA, and the beneficial effects of additional PPh, might be explained by the presence of larger amounts of isoagglutinins of the IgG3 or IgM isotype, which can be removed more easilyby PPh than by IA . It has According to our data and to other studies, IgG titer reduction in Immunosorba-based protocols can be achieved as efficient as in Gylcosorb-based treatment, if the plasma volume per session is increased , 19. ThiNon-antigen-specific IA could be of advantage in cases when other, potentially harmful HLA alloantibodies might play a role. In the present study, both procedures depleted HLA-specific DSA in two patients. However, one patient of the Immunosorba group developed an ABMR and TCMR, due to the existence of strong DSA. The overall rate of observed acute rejections in our cohort 40 and 31%) appears to be high, but is comparable to that in other studies % appears, 12. RejSince protective antibodies might also be depleted by both IA procedures, we assessed the infection rate after RTx. It was hypothesized that the intensified (pre-) treatment of patients promotes infections and possibly infection-related cancer like Karposi sarcoma after RTx. Indeed, treatment with rituximab or splenectomy was reported to increase the risk for viral infections , 25; howDue to the depletion of coagulation factors, PPh is thought to cause more bleeding complications compared to IA. However, the development of hematomas or bleeding episodes after ABOi RTx is rarely reported by centers using PPh or double filtration plasmapheresis and prevalence seems to be low with <5% , 28, 30.This study is somehow limited by the mainly retrospective analysis and short follow-up period. As the entire study population consists of Western Europeans, the data is only representative for this ethnical group with similar genetic characteristics. Although the study includes only a relatively small number of patients, this is the largest report comparing these modalities to date.We were able to effectively reduce isoagglutinin titers in all ABOi RTx patients regardless of the column used. Allograft function and complication rate were comparable between the antigen-specific and non-antigen-specific IA group. While Immunosorba-based IA was more cost- effective than Glycosorb, it was more time-consuming. However, as IA could be performed combined with dialysis or a reuse of Glycosorb columns might be performed in the future, these aspects might become less important.


Short-term impacts of high temperatures on the elderly are well known. Even though Italy has the highest proportion of elderly citizens in Europe, there is a lack of information on spatial heat-related elderly risks.Development of high-resolution, heat-related urban risk maps regarding the elderly population (≥65).A long time-series (2001–2013) of remote sensing MODIS data, averaged over the summer period for eleven major Italian cities, were downscaled to obtain high spatial resolution (100 m) daytime and night-time land surface temperatures (LST). LST was estimated pixel-wise by applying two statistical model approaches: 1) the Linear Regression Model (LRM); 2) the Generalized Additive Model (GAM). Total and elderly population density data were extracted from the Joint Research Centre population grid (100 m) from the 2001 census (Eurostat source), and processed together using “Crichton’s Risk Triangle” hazard-risk methodology for obtaining a Heat-related Elderly Risk Index (HERI).The GAM procedure allowed for improved daytime and night-time LST estimations compared to the LRM approach. High-resolution maps of daytime and night-time HERI levels were developed for inland and coastal cities. Urban areas with the hazardous HERI level (very high risk) were not necessarily characterized by the highest temperatures. The hazardous HERI level was generally localized to encompass the city-centre in inland cities and the inner area in coastal cities. The two most dangerous HERI levels were greater in the coastal rather than inland cities.This study shows the great potential of combining geospatial technologies and spatial demographic characteristics within a simple and flexible framework in order to provide high-resolution urban mapping of daytime and night-time HERI. In this way, potential areas for intervention are immediately identified with up-to-street level details. This information could support public health operators and facilitate coordination for heat-related emergencies. Short-term impacts of high air temperature on human health are well known , 2. FurtHigh temperature has the worst impact on the elderly . The higPeople living in heavily built-up areas, especially if they live in old buildings without insulation or air conditioning, are at a greatest risk  because It is well known that temperature has a great spatial variability in metropolitan areas because of the complex interaction of physical variables, such as surface and air temperature gradients, turbulent transport, and surface energy exchange.Temperatures typically rise in the most densely urbanized areas, creating a concomitance of adverse environmental conditions and high levels of exposure of the population that need to be assessed by integrated methodologies for accurately resolving spatial scales.As meteorological station networks are generally scattered and often located in suburban areas (i.e. airports or parks), they only provide partial representations of air-temperature variations in heterogeneous urban/suburban environments. For this reason, tools for intra-urban hazard risk assessments are required. In particular, the development of geoscience and the wide availability of remote sensing data allow for ongoing accurate estimations of the Land Surface Temperature (LST) and the Normalized Difference Vegetative Index (NDVI). LST-NDVI relationships have already been studied through linear regression analyses over a wide range of moisture and climatic/radiation regimes  and specRemote sensing data, together with spatial socio-demographic datasets describing the exposed vulnerable population, are valuable indicators of urban hazard health risk analyses. We hypothesize that these datasets, if available at high spatial resolution, might represent an essential contribution to prevent the heat impact on human health with detailed information at the urban small-area level.Several examples of heat risk assessment in urban areas exist in the USA , Canada 2 European population density on a 100-m grid by using the downscaling method described in Gallego [Spatial population density is the main socio-demographic indicator of heat exposure generally used in risk assessment studies. Population and housing censuses have a long tradition in the European Union. However, the way the census methods are developed in each country depends on the availability of data sources and technology, data protection requirements, the burden on the respondents, and the census operation costs. More recently, the Joint Research Centre (JRC) has produced a raster dataset representing the 1-km Gallego . To our The main aim of this study was to develop high-resolution daytime and night-time heat-related population risk maps, specifically referring to the elderly (people aged 65 or over) and to summer (May-September), for the major Italian cities with different geographical features . In order to achieve the main aim, a preliminary analysis was addressed to define a reliable downscaling procedure (100 m) in order to obtain a long time-series (2001–2013) of MODIS LST data. For each city, Heat-related Elderly Risk Index (HERI) maps were developed through the interaction of the natural hazard layer (LST) with spatial demographic data by using “Crichton’s Risk Triangle”  frameworThis study was carried out during the warmest period of the year (May-September) over a 13-year period (2001–2013). The city selection criterion was based on the number of residents. Eleven major Italian cities with more than 200,000 inhabitants were selected from all over the country: five in the North , two in the Centre (Florence and Rome), and four in the South  .These cities are located in different geographical  and climThe borders delimiting each city study-area were defined in order to include, besides the main city itself, an outer belt. The latter was represented by the municipalities of all satellite residential towns, suburbs or settlements around the city.The study design focuses on “Crichton’s Risk Triangle” hazard-risk assessment methodology  adopted The hazard layer is obtained for the period 2001–2013 using two remote sensing MODIS data products : LST at 8 days temporal and 1-km spatial resolution (MOD11A2) both for daytime and night-time conditions; NDVI at 16 days temporal and at 250-m spatial resolution (MYD13Q1).http://due.esrin.esa.int/globcover) was used based on 22 land cover classes as per the UN-FAO Land Cover Classification System at 300-m spatial resolution [The ESA (European Space Agency) Globcover land cover dataset (solution .2); statistical model significance; root-mean square error (RMSE). In addition, the linear regression coefficient was also calculated. The RMSE represents a good measure of model accuracy and is widely used to assess the accuracy of spatial analysis and remote sensing data [A nearest-neighbour resample procedure was applied using GDAL  operators working on raster-tiles to homogenize the different spatial resolution of data layers. In particular, the monthly average LST and land-cover layers were initially resampled at 250-m resolution. Then the mean daytime and night-time LST was estimated pixel-wise by the corresponding mean NDVI value, also considering the land cover stratification, by applying two nested statistical model procedures: 1) the Linear Regression Model (LRM); 2) the Generalized Additive Model (GAM) . LST moding data .http://cran.r-project.org/ (packages link). Demonstrative work-flow, data and code functions built-up for this work are available on github.com/meteosalute/mapheatrisk.The same nearest-neighbour resample procedure was also used by averaging the monthly LST outcomes in summer (May-September) to obtain a high-resolution (100 m) product spatially consistent with the population layers (100 m). Water surfaces were excluded from the procedure. Geostatistical and GIS procedures were written in R-language using specific R-packages, such as rgdal, raster and mgcv, available online on: http://www.eea.europa.eu/data-and-maps/data/population-density-disaggregated-with-corine-land-cover-2000-2) which entails the 1-km2 population density for 2001 (source Eurostat) on a 100-m grid for the European Union. Population by commune was disaggregated with the CORINE land cover 2000 using the downscaling method described in Gallego [http://epp.eurostat.ec.europa.eu). We represented the vulnerability layer by the aggregated age class of people aged 65 or over. For each city, the vulnerable population layer (people > 65 years) was calculated by multiplying the cumulative percentage of the vulnerable population for the pixel-wise total population layer. In this way, the consistency of the elderly was obtained numerically and spatially in compliance with the LAU2 European statistical data (Eurostat)  .The spatial methodology employed to provide heat-related health risk mapping envisaged a normalization procedure, followed by a weighted-layers combining procedure and development of the final mapping of the HERI . The norThe following step was the combination of the normalized layers through a weighting procedure. To avoid subjective manipulation, all weightings were kept equal. In particular, the exposure and vulnerability layers were combined in a single “exposed and vulnerable” layer (each weighted at 50%) which was then spatially combined with the hazard layer (weighted at 50%). This spatial methodology follows the one successfully applied in previous recent studies , 15. In The percentage of the urban coverage area for each HERI level was also assessed.2) such as Milan, Naples, Turin and Palermo , C130 (19.6%), C50 (18.1%), C14 (16.8%) and C190 (13.1%). The C190 land cover class is of great interest because it describes all the areas that have an artificial cover as a result of human activities such as construction , extraction (open mines and quarries) or waste disposal. The C190 showed the highest percentage of coverage area values in cities with the greatest total population density  LST-NDVI regression coefficients for every land cover class Tables  and 2. H2 values than those observed for LRM. The accuracy of the LST estimation was better when the GAM procedure was applied: the RMSE-related GAM procedure was always lower than the RMSE-related LRM approach in both daytime  in Rome and Turin  and almoThe two most dangerous HERI levels were characterized in all cities by the highest total and elderly population frequency and density. The population density generally showed a progressive increase from the very low to the very high risk level Tables  and 4. TThe results demonstrated a heterogeneous intra-urban variation of night-time HERI levels in both inland  and coasMost of cities showed a progressive rise in the mean LST from the lowest value at the very low level to the highest value at the very high risk level. In particular, the highest mean LST was observed in Bologna (18.6°C), followed by Florence (17.6°C) and Rome (17.5°C) . Among cThe low risk level had the highest coverage area in most of cities . This variable is commonly adopted to characterize the UHI effect , recentlThis study confirms the negative LST-NDVI relationship of land cover classes highlighted in previous studies, especially during the summer months , 10, 11.For the first time LST was combined with high-resolution (100-m grid) exposure and vulnerability layers assessed by using population density grids  spatially available over a wide geographical area .http://demo.istat.it/pop2014/index.html). Therefore, an increasing number of more vulnerable individuals is expected to become exposed to future heat events and detailed methodologies are indispensable for assessing the intra-city heat-related elderly risks.The decision to focus on urban areas stems from recent extreme-heat events causing serious health, economic and social problems worldwide  with strong heat-impact due to global warming predicted in cities . In addiThe highest heat-related vulnerability of the elderly is due to physiological, health and socio-economical status . ConsequIn this study, all areas characterized by the hazardous risk level also have the highest total and elderly population densities. For this reason, healthcare should immediately be provided in these zones in case of excessive heat events. Most cities showed the hazardous HERI level in inner-city areas, which is in agreement with previous studies showing that the heat effect was mainly evident among people living in inner-city neighbourhoods , 31, alsInterestingly, urban areas with the hazardous HERI level were not necessarily characterized by the highest daytime LST. Conversely, there was a night-time correspondence between the harmful HERI level and the highest LST, in agreement with the fact that UHI is a marked night-time phenomenon  when eneThe urban areas affected by the two most dangerous HERI levels were greater in coastal  than inland (8.1% and 3.3% for the high and very high risk levels respectively) cities. This geographical heterogeneity is particularly interesting because it is well known that tolerance to temperature extremes varies regionally  accordinThis study represents a starting point for identifying areas requiring adaptation-planning strategies  such as github.com/meteosalute/mapheatrisk, by using a well-referenced open source software . These strengths enable easily replicable and comparable studies. In this regard, one of the main strengths is the potential geographical reproducibility of the study. All grid-raster data used are spatially continuous, thus allowing for future investigations in marginal and developing urban areas. Since the same demographic grid-data and satellite data are also available in all European Union countries, the heat-risk analysis assessed nationally in this study could easily be extended to the rest of Europe in future studies.Other strengths of this study are linked to the fact that well-known, referenced and free public remote sensing (satellite MODIS data) and demographic (Eurostat source) datasets were used for the HERI assessment. Furthermore, all computerized procedures were coded, and made freely available on This study also has some limitations. Firstly, the LST was used as a hazard-layer measure for potential population exposure. However, the relationship between LST and ambient temperature is very complex  and stroAnother limitation is the fact that the exposed and vulnerable layer was only represented by the total and elderly population density. Conversely, no other socio-demographic and health variables  were considered as often spatially fractioned and when available, they only apply to very few cities. Furthermore, specific grid platforms for other high-resolution socio-demographic or health variables are still not available. However, as these variables become available as gridded datasets in the future, they could easily be implemented in the risk assessment methodology and provide an improved version of urban hazard maps.This study shows the great potential of combining geospatial technologies and spatial demographic characteristics within a simple and flexible conceptual framework in order to provide high-resolution urban mapping of daytime and night-time heat-related elderly risk index. This result was achieved thanks to an effective downscaling smoothing procedure (GAM approach) of thermal surface satellite MODIS data. The GAM procedure allowed for improved daytime and night-time LST estimations compared to the linear regression model approach normally used in previous studies.HERI maps in major Italian cities generally showed the hazardous HERI level localized to encompass the city-centre in inland cities and the inner areas of coastal cities. Furthermore, the two most dangerous HERI levels were greater in the coastal than the inland cities. This information is a potentially useful tool for delineating daytime and night-time high-risk intra-urban areas with high—not necessarily the highest—temperatures, as well as the greatest total and elderly population density. In this way, potential areas for intervention are immediately identified, thus supporting public health operators and facilitating coordination for heat-related emergencies.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


To assess HIV incidence and its associated risk factors among young men who have sex with men (YMSM) in urban areas, China.The study used a prospective cohort study design and standard diagnostic tests.A twelve-month prospective cohort study was conducted among YMSM (18–25 years old) in 8 large cities in China. The participants were recruited via snowball sampling. A total of 1102 HIV-negative YMSM completed baseline assessment, 878 YMSM participants completed 6-month follow-up, and 902 completed 12-month follow-up. HIV was screened by an enzyme-linked immunosorbent assay and confirmed with Western Blot. Syphilis was screened via rapid plasma reagent and confirmed by treponema pallidum particle agglutination assay.78 HIV seroconversions were identified within 1168.4 person-year observations yielding an incidence rate of 6.7 per 100 person-years. HIV seroconversion was associated with non-student status , low HIV transmission knowledge , and syphilis infection .Incidence of HIV among YMSM is high in urban areas of China. Interventions measures are required to contain the HIV epidemic within this population. The heavy burden of HIV infection among men who have sex with men (MSM) has been demonstrated around world. The HIV prevalence within this subpopulation appears to be rising rapidly and MSM continue to be disproportionately affected by HIV in many international settings A number of studies in China have revealed rapidly increasing rates of HIV infection among MSM Young MSM  have been shown to be a particularly high risk population in many international settings During August 2009 to December 2010, a cohort study was conducted in eight cities of China including: Beijing, Shanghai, Kunming, Guiyang, Chongqing, Chengdu, Urumqi and Nanning. Eligibility criteria of participants were men ages 18–25, who had anal or oral sex with men in the past 6 months, who were tested to be HIV negative at recruitment, and who were willing to participate in 6- and 12-month follow-up surveys as well as complete follow-up HIV testing. Because of the hard reach to the population, we used snowball sampling to recruit participants. In the baseline study, 10 initial seed subjects from each city were recruited. After training of recruitment eligibility, these subjects were given “recruitment coupons” to refer other YMSM in their networks to the study, who were then screened for eligibility and invited to participate if eligible. All subsequent participants were asked to refer other YMSM to this study using the recruitment coupons. Each recruitment coupon had an expiration date, information about the study testing site, and a contact telephone number. The baseline survey was conducted with 1242 YMSM. Of them, 140 (11.3%) were HIV positive at baseline and were excluded due to calculate the rate of new infections. The remaining 1102 YMSM were eligible for the cohort study.After completing written informed consent, structured questionnaire-based interviews were administered in a private room by health workers from the local Centers for Disease Control and Prevention (CDC). Blood samples were collected from participants for tests of HIV and syphilis infections by health workers. An enzyme-linked immunosorbent assay was used to screen for HIV antibodies  and Western Blot immunoassay  was used for HIV-1/2 confirmation if ELISA was positive. All participants received pre-and post-test HIV counseling by CDC workers. Rapid Plasma Reagent  was used for syphilis screening and Treponema pallidum particle agglutination assay  was used for confirming syphilis infection. Individuals who were HIV positive or syphilis infected were referred to HIV/STD care clinics for appropriate services. Participants provided contact information and were informed about the 6- and 12-month follow-up assessments.We conducted 6-month follow-up assessments with 878 YMSM and 12-month follow-up assessments with 902 YMSM. Follow-up assessments involved identical procedures including the behavioral risk questionnaire and HIV test.This study, including design, recruitment, consent, and assessment procedures, was reviewed and approved by the Institutional Review Board of the Chinese National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention. Written informed consent was obtained from all participants. Each participant received 50 RMB (8 USD) and 30 condoms as incentive for participation in baseline and follow-up assessments.Questionnaire-based interviews were administrated to assess socio-demographic characteristics , behavioral information , and any STD diagnosis. Knowledge related to HIV transmission was measured by a 8-question measure . Knowledge scores were created by counting the number of correct responses; “I don’t know” was coded as an incorrect response.EpiData 3.0  software was used to input the original data and SPSS 10.01  was used for data analysis. Descriptive analyses were conducted to describe socio-demographic characteristics of the sample. Chi-square tests were conducted to examine differences in socio-demographic characteristics between baseline study and follow-up studies. HIV incidence was calculated by using the number of seroconversions within the follow-up period as the numerator and the cohort’s total number of person-year (PY) exposure to the risk of HIV transmission as the denominator. Cox regression analysis was conducted to identify the risk factors for HIV seroconversion. Independent variables were entered into the multivariate Cox regression models in 3 blocks including socio-demographic characteristics (block 1), HIV/AIDS related knowledge and sexual behaviors (block 2), and syphilis and STD infection (block 3).Socio-demographic characteristics of the respondents are described in Of 1102 YMSM in the baseline survey, 878 (79.7%) participated in the 6-month follow-up. Nine hundred and two YMSM (81.9%) completed assessment at the 12-month follow-up. There were no significant differences on socio-demographic characteristics between participants who completed the baseline and 6- or 12-month follow-up visits .We used multivariate Cox regression models to examine associations of risk factors with HIV seroconversion in one year . In the This cohort study brings attention to YMSM in China as a sub-group of MSM at high risk for HIV infection. The present study reveals that the HIV incidence rate was 6.7 per 100 person-years among YMSM. In addition, the study shows that YMSM with non-students status, low HIV/AIDS transmission knowledge, and syphilis infection were more likely to become infected with HIV. These findings propose some measures for developing HIV intervention and prevention programs targeting YMSM. In particular, prevention and intervention efforts should target non-student YMSM populations in urban China to improve their HIV/AIDS transmission knowledge, and encourage them to seek testing and treatment for syphilis.From February 2008 to September 2009, a nationwide cross-sectional survey was conducted in 61 cities in China reported that the overall prevalence of HIV was 4.9% among MSM. In that survey, 43.3% of participants were 18–24 year-old MSM, and HIV prevalence in this subgroup was 3.8% Data in the current study indicates that YMSM who were non-students status were more likely to be infected HIV compared to student YMSM (7.9/100 PY vs.4.2/100 PY). These findings are similar to a study conducted in Chongqing province in 2009, which found that compared to students, non-students MSM have higher HIV infection prevalence (4.4% versus 20.9%) Higher HIV/AIDS-related knowledge was associated with more consistent use of condoms with primary and commercial sex partners There are several limitations to this research. Firstly, due to the hard-to-reach nature of the targeted population, the snowball sampling method might not have recruited a representative sample. Moreover, because many MSM in China do not choose to disclose their sexual orientation or might self-identify as heterosexual, this sample might be affected by selection bias. Secondly, non-attendance at follow-up assessments might have affected the HIV incidence rate estimates. However, we believe that this bias might be limited because of the reasonably high follow-up rate (81.9%) and the similarity between those who failed to attend follow-up and those who remained in the cohort. Third, due to the brief nature of the questionnaire, we were unable to examine other potentially important risk factors for HIV infection including alcohol and other drug use, sex with women, health and prevention services utilization, and specific contexts of risk behavior, which need further investigations. Finally, due to the different economic and cultural features in different cities, caution is need when grouping findings across cities to calculate the whole seroconversion rate.Study strengths included the novelty of addressing the subpopulation of YMSM and assessing HIV incidence in eight large cities in China. This study provides evidence for the high HIV incidence rate among YMSM in China and draws attention to younger MSM as a population in need of urgent public health intervention. Future research is needed to explore the underlying psychological, social, economic and structural factors related to these high HIV risk behaviors among YMSM. Efforts to reduce risk behavior and improve preventative strategies such as HIV/STI testing and treatment are important steps in order to reduce HIV transmission among and from this special group. Future efforts must be attentive to cultural factors, such as stigma and secrecy about same-sex behaviors, which may shape the implementation feasibility and acceptability of HIV interventions for YMSM in China.


Prestige oil spill, in 2002. Individuals who participated in the clean-up tasks showed increased chromosome damage two years after exposure. Long-term clinical implications of chromosome damage are still unknown.The north-west coast of Spain was heavily contaminated by the To realize a follow-up genotoxic study to detect whether the chromosome damage persisted six years after exposure to the oil.Follow-up study.Fishermen cooperatives in coastal villages.Local fishermen who were highly exposed (n = 52) and non-exposed (n = 23) to oil seven years after the spill.Chromosome damage in circulating lymphocytes.Prestige spill vs. those detected two years after the exposure.Chromosome damage in exposed individuals persists six years after oil exposure, with a similar incidence than those previously detected four years before. A surprising increase in chromosome damage in non-exposed individual was found six years after The sample size and the possibility of some kind of selection bias should be considered. Genotoxic results cannot be extrapolated to the approximately 300,000 individuals who participated occasionally in clean-up tasks.The persistence of chromosome damage detected in exposed individuals six years after oil exposure seems to indicate that the cells of the bone marrow are affected. A surprising increase in chromosome damage in non-exposed individuals detected in the follow-up study suggests an indirect exposition of these individuals to some oil compounds or to other toxic agents during the last four years. More long-term studies are needed to confirm the presence of chromosome damage in exposed and non-exposed fishermen due to the association between increased chromosomal damage and increased risk of cancer. Understanding and detecting chromosome damage is important for detecting cancer in its early stages. The present work is the first follow-up cytogenetic study carried out in lymphocytes to determine genotoxic damage evolution between two and six years after oil exposure in same individuals. Prestige wrecked offshore of the Spanish North-West  and thousand tons of crude oil were spilled contaminating more than 1,000 km of coast [In 2002, the oil tanker of coast . Most oiof coast ,5 and reof coast –16.Prestige oil spill which have been previously described [The effects of oil spills on human health have mainly focused on the acute phase. Most studies described respiratory problems; headache; irritation of the skin, eyes and mucous membranes; nausea; dizziness and psychological symptoms ,5. Moreoescribed –18.Prestige oil spill [Prestige accident, no genotoxic analyses have been conducted on the three other large oil spills, despite its proximity to the coast: Tasman Spirit , Hebei Spirit  and Deepwater Horizon .Most of the genotoxic studies have been done on the effect caused by the il spill –16. AftePrestige, using different biomarkers, with controversial results [Prestige oil spill (P-2y study) [Among the oil genotoxic studies, the most common are performed at moment to exposure or few months later. These studies, using different type of biomarkers, have demonstrated an association between exposure to oil and genotoxic damage –13. Howe results –16. In oy study) ,15. Longy study) –24.Leishman stain/G-banding technique.The purpose of the present work was evaluate whether chromosome damage  of circulating lymphocytes detected in the P-2y study in exposed individuals persists six years later of the accident (P-6y study). The follow-up genotoxic study was performed in 52 exposed and 23 non-exposed individuals previously studied, using the same experimental protocol. More than 7,700 metaphases from standard lymphocytes culture were analyzed using a sequential Prestige oil spill. Only fishermen were included in our study in order to minimize other occupational sources of exposure that could act as confounders. A questionnaire survey among 6,780 fishermen one year after exposure was performed. Very strict selection criteria for exposed and non-exposed individuals to oil were applied as detailed in a previous report [In November 2002, an important number of local fishermen participated in the clean-up of the s report . In bries report ,26. In as report . A totals report ,15. The In the genotoxic follow-up study (P-6y) only 75 fishermen who had been included in the P-2y study accepted the offer to participate (52 exposed and 23 non-exposed). The exposed group was 15 men and 37 women and the average age was 49.3 years (ranging from 29.2 to 64.4).The non-exposed group was 2 men and 21 women and the average age was 52.2 years (ranging from 31.1 to 63.9). This Project has been approved by the Ethics Committee of Clinical Research of Galicia, Spain, and all participants signed the participating consent.Prestige accident. The individuals were six men and four women with ages ranging between 21 and 42 years, and all individuals had never smoked, nor had they been exposed to Prestige oil or any other known genotoxic factor.The control group used in this work, which was described previously , includePeripheral blood was extracted and circulating lymphocytes cultured for 72 hours in RPMI-1640 medium  and then harvested according to standard procedures ,15. AsseFor chromosome lesions analysis, preparations were uniformly stained with Leishman. For each participant, at least 100 metaphases were investigated. The same preparations were stained and banded using Wright staining for detecting structural chromosome alterations. At least 25 G-banded karyotyped for each individual were realized.All slides were coded before cytogenetic analysis so that information about whether individuals were exposed or non-exposed was not available until the completion of data collection. Aberrant metaphases from each individual were determined by two independent observers.All chromosome lesions and chromosome structural alterations have been classified according to the International System for Human Cytogenetic Nomenclature .Differences in cytogenetic results  between the exposed and non-exposed groups adjusted by age were evaluated using a generalized estimating equation, GEE method ,30. The All individual had a normal constitutional karyotype , except one exposed individual with a polymorphic inversion of chromosome 9 in her karyotype, 46,XX,inv(9)(p11q12).The follow-up cytogenetic study (P-6y) was performed in 52 exposed and 23 non-exposed individuals previously studied (P-2y). The analysis of genotoxic oil effect was performed using two biomarkers: chromosome lesions and chromosome structural alterations.In present P-6y study, a total of 5,346 and 2,385 metaphases were analyzed from exposed and non-exposed individuals respectively. No significant statistical differences were observed between exposed and non-exposed individuals for chromosome lesions (P = 0.2879) or structural chromosome alterations (P = 0.9263).Chromosome damage in exposed and non-exposed to oil individuals obtained in the two studies, P-2y and P-6y, is showed in Cytogenetic results for each individual are shown for exposed and non-exposed individuals in supplementary dataPrestige accident using chromosome lesions and structural chromosome alterations as biomarkers.Genotoxic oil exposure studies are limited; most of them carried at the moment of exposure or a few months later and long-term clinical implications are still unknown. The present work is the first genotoxic follow-up study carried out in the same individuals, exposed and non-exposed, four years after of the previous study which itself had been done two years after the The frequency of chromosome lesions detected in the P-6y study is similar in exposed and non-exposed individuals, as observed in previous the P-2y study . Up to nPrestige exposure, using biomarkers other than those employed in our study  [In the P6y study the frequency of the structural chromosome alterations in exposed individuals (6.7%) was similar to that observed in non-exposed individuals (6.6%). These findings suggest that oil toxicity disappears after seven years. In a recent study performed seven years after oil  assays)  no diffe assays) ,40.Prestige accident  decreases in relation to the previous study (P2y-study), but no statistical significant differences were found between the two studies see . Moreoveaccident . This peUnexpectedly, an increase in chromosome damage in non-exposed individuals was detected four years after of initial study (6.6% in P6y vs. 2.1 in P2y). Furthermore, when comparing the chromosomal damage non-exposed and control individuals in both genotoxic studies, a significant increase in the follow-up study (at least two times higher) compared to the level observed in controls was observed, whereas in the previous study the non-exposed and control groups had similar values. This finding is consistent with the increase in respiratory symptoms and markers of oxidative stress observed in these same non-exposed individuals .Prestige had been stored within 50 km of the fishermen’s cooperatives, from which the non-exposed individuals were selected [We do not know the reason for the increase in chromosome damage in non-exposed individuals found in this study, but there is the possibility of indirect exposure to some compounds of oil or other toxic agents during the last four years. One candidate to explain this indirect exposure would be improper storage of oil collected during the clean-up tasks. Once having finished the P6y study we discovered that around 60,000 tons of oil from the selected ,15,17. Aselected –49. Whenselected . MoreoveFor the same reasons as those mentioned above, it is possible that the persistence of chromosome damage observed in exposed individuals could be the result of oil exposure both during the clean-up and from contaminated food ingestion.The present work is the first follow-up genotoxic study carried out in circulating lymphocytes to determine genotoxic damage evolution between two and six years after oil exposure in a set group of individuals. Structural chromosome alterations in exposed individuals persist six years after exposure to oil, suggesting that the cells of the bone marrow could be affected. In addition, the unexpected increase in chromosome alterations in non-exposed individuals suggests that they have also been indirectly exposed to some oil compounds. Some of chromosomal damage detected in exposed individuals, may be due to indirect oil exposure or other genotoxic agents, as suggested in the non-exposed ones. More long-term studies are needed to confirm the presence of chromosome damage in exposed and non-exposed fishermen due to the association between increased chromosome damage and an increased risk of cancer in order to detect cancer in early stages.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.