Routine immunization is recommended for adolescents aged 11–12 years by the Advisory Committee on Immunization Practices (ACIP) for protection against diseases including pertussis, meningococcal disease, and human papillomavirus (HPV)–associated cancers .NIS-Teen monitors vaccination coverage among adolescents aged 13–17 years in the 50 states, District of Columbia (DC), selected local areas, and territoriesRevised methods for defining adequate provider data were implemented in 2014 and were retrospectively applied to 2013 NIS-Teen data for purposes of comparing these two most recent survey years. As a result, revised 2013 coverage estimates presented in this report differ from those previously published, and 2014 and revised 2013 NIS-Teen coverage estimates are not directly comparable to those published for the 2006–2013 survey years. This definition change will decrease some vaccination coverage estimates, particularly for some states and local areas. Details regarding this methodologic change and the assessment of its impact on vaccination coverage estimates are described elsewhere.Compared with revised 2013 estimates, coverage among adolescents aged 13–17 years significantly increased during 2014 for Tdap, MenACWY and for each HPV dose among females and males . PercentIn 2014, HPV coverage and series completion were higher among older females compared with females aged 13 years; these findings were observed less consistently among males . VaccinaCoverage estimates for each HPV dose and for ≥1 MenACWY were higher among Hispanic adolescents compared with non-Hispanic white adolescents, and estimates for each HPV dose were higher among adolescents living below the poverty level compared with those at or above the poverty levelIn 2014, vaccination coverage varied among the 50 states and DC  and 3. CFrom 2013 to 2014, vaccination coverage among adolescents aged 13–17 years increased for all vaccines routinely recommended for adolescents. Achieving high HPV vaccination coverage in early adolescence is important to optimize protection before HPV exposure. In 2014, the President’s Cancer Panel Report called for coordinated efforts to improve HPV vaccination coverage, including reducing missed opportunities to recommend and administer HPV vaccine at every clinical opportunity, increasing parents’ and adolescents’ acceptance of HPV vaccine, and maximizing access to HPV vaccination services . In late 2014, DC expanded its existing school requirement for HPV vaccination to include males and females through 12th grade, with a requirement for submitting exemption forms annually (http://www.dcregs.dc.gov/Gateway/NoticeHome.aspx?NoticeID=5225019).In six of the seven jurisdictions with increases in ≥1- or ≥3-dose HPV coverage among females, combinations of strategies were important. Immunization programs highlighted incorporating HPV vaccination in cancer control plans, joint initiatives with cancer prevention and immunization stakeholders, public communication campaigns, immunization information system–based reminder/recall, assessment and feedback activities , practice-focused strategies to educate staff and provide input on how to improve routine HPV vaccination within the practice, and using all opportunities to educate clinicians and parents about the importance of on-time HPV vaccination. These experiences are informing development of best practices for improving HPV vaccination coverage. At the start of 2014, only two jurisdictions had school requirements for HPV vaccination, both with broad exemption provisions  might still be low. In addition, because NIS-Teen includes adolescents aged 13–17 years, receipt of MenACWY at age ≥18 years is not captured in these coverage estimates.Estimated coverage with ≥1 MenACWY dose continues to increase among adolescents, but geographic disparities are evident and vaccination coverage estimates are still lower than for Tdap. Although 78.8% of adolescents aged 17 years received ≥1 dose of MenACWY, only 28.5% received the complete the 2-dose series. Further evaluation might identify factors that could lead to improved MenACWY series coverage, although older adolescents have fewer preventive health visits, and awareness of the 2-dose recommendation (http://www.cdc.gov/measles/cases-outbreaks.html). High MMR coverage is needed to sustain elimination and protect those who cannot be directly vaccinated. Health care providers of adolescents should assess their patients’ vaccination status at each clinical opportunity, take advantage of immunization information systems, which should reflect vaccines delivered in any setting, and offer all vaccines for which adolescents are eligible, including missing doses of MMR, varicella, and hepatitis B vaccines.MMR vaccine is routinely recommended at ages 12–15 months and 4–6 years –associated cancers. During 2006–2013, national coverage with ≥1 dose of tetanus-diphtheria-acellular pertussis (Tdap) vaccine and ≥1 dose of meningococcal conjugate (MenACWY) vaccine increased annually. Although ≥1-dose HPV coverage among females increased during 2007–2011, no change was observed during 2011–2012. However, during 2012–2013 and 2011–2013, ≥1-dose HPV coverage among females and males, respectively, increased.What is added by this report?During 2013–2014, vaccination coverage among adolescents aged 13–17 years increased for ≥1 dose of Tdap, ≥1 dose of MenACWY, and each HPV dose among females and males, with considerable variation in coverage by state. Although HPV vaccination coverage among females increased nationally for the second consecutive year, HPV coverage lags behind Tdap and MenACWY coverage. Seven jurisdictions achieved significant increases in ≥1- or ≥3-dose HPV vaccination coverage among females during 2013–2014, demonstrating that substantial improvement in HPV vaccination coverage is feasible.What are the implications for public health practice?Despite similar percentage point increases in coverage with Tdap and MenACWY vaccines, and ≥1 HPV dose among females in 2014, national HPV coverage estimates remain low for adolescents. Differences in coverage estimates by vaccine indicate missed opportunities for administering HPV vaccine at visits when Tdap or MenACWY vaccines are given. Routinely recommending HPV vaccine at ages 11–12 years, during the same visit and with the same emphasis used for other vaccines, is critical. Multifaceted interventions that engage clinicians and other immunization stakeholders and increase community awareness might improve HPV vaccination coverage.


PIK3CA (29.4%), KRAS (26.5%), TP53 (26.5%) MET (8.8%), SMAD4 (8.8%) and STK11 (8.8%). Multiple gene mutations were found in 14/21 (67%) patients with good prognosis, and 3/13 (23%) in the poor prognosis group. A single gene alteration was found in 5/21 (24%) good and 6/13 (46%) poor prognosis patients. No mutation was found in 2/21 (9.5%) and 4/13 (31%) of these groups, respectively. In the overall series, ß-catenin expression was the highest (82.4%), followed by E-Cadherin (76.5%) and FHIT (52.9%). The good prognosis group was characterized by a high mutation rate and microsatellite instability. Our proof-of-principle study demonstrates the feasibility of a molecular profiling approach with the aim to identify potentially druggable pathways and drive the development of customized therapies for RGC.In this study, we evaluated whether the presence of genetic alterations detected by next generation sequencing may define outcome in a prognostically-selected and histology-restricted population of resected gastric cancer (RGC). Intestinal type RGC samples from 34 patients, including 21 best and 13 worst prognostic performers, were studied. Mutations in 50 cancer-associated genes were evaluated. A significant difference between good and poor prognosis was found according to clinico-pathologic factors. The most commonly mutated genes in the whole population were  Gastric cancer (GC) is the fourth most common cancer type and the second cause of cancer deaths24Forty two patients made up the three-class model, including good (26 patients) and poor (16 patients) prognostic performers. These 42 patients had a 2-year CSS of 82.1% and 5.3%, and a 2-year OS of 79.3% and 5.3%, respectively . Good anPIK3CA (29.4%), KRAS (26.5%), TP53 (26.5%) MET (8.8%), SMAD4 (8.8%) and STK11 (8.8%). Seventeen (Good/Poor: 14/3), 11 (Good/Poor: 5/6) and six (Good/Poor: 2/4) RGCs contained multiple, single or no gene alteration, respectively. ATM, CDKN2A, EGFR, ERBB2, FBXW7 and SMARCB1 were mutated in two cases, and FGFR3, IDH1, JAK3, KIT, NOTCH1, NRAS and PTEN in one. Distribution of patients’ molecular characteristics in the NGS analysis according to prognosis is shown in KRAS and PIK3CA represented the most frequently altered genes in the good prognosis group (38.1%). TP53 gene was the second most frequently altered gene in the group (28.6%). Of interest was the fact that the good prognosis patients showed alterations in several genes involved in targetable pathways. In this regard, genes belonging to the mTOR pathway  were altered in 10 cases (47.6%). Tyrosine kinase receptors  were altered in seven cases (33.3%). Patients in the good prognosis group were characterized by microsatellite instability (MSI) in 42.9% of cases. The poor prognosis group was characterized by a relative low mutation rate compared with good prognosis patients. Four (30.8%) cases did not present any alteration in the 50 genes assayed, six (46.2%) cases showed only one mutation and two concurrent mutations were observed only in three (23.1%) cases. TP53 was the most frequently altered gene (23.1%). PIK3CA, SMAD4 and STK11 were altered in two samples each (15.4%). A KRAS alteration was observed only in one case (7.7%). The low mutation rate and the small number of samples made it more difficult to identify a targetable pathway, as was the case in the good prognosis group. The mTOR pathway was altered in two cases (15.4%) and one case showed alteration in the tyrosine kinase receptor MET (7.7%). Microsatellite instability was observed only in one patient with poor prognosis (7.7%).At least one mutation was observed in 28/34 cases (82.4%). Seventeen (50.0%) cases showed concurrent mutations in different genes and six (17.6%) showed no alterations in the 50 genes assayed. The most commonly mutated genes in the whole population were The results of IHC and FISH analyses are summarized in In this study we analyzed genetic alterations using NGS and protein expression by IHC and FISH according to the different prognoses of intestinal-type RGC. Although no specific genes associated with prognosis were identified, a specific molecular profile emerged as a tool to predict and define clinical outcomes.The concept to molecularly characterized different prognostic groups in order to identify the potential existence of differential driver molecular alterations is innovative and promising. In this regard, the tumor genetic analysis of the LUX-Lung 8, a phase III trial of afatinib versus erlotinib in squamous cell carcinoma of the lung, was performed in 2 subgroups of patients distinct according to treatment responseIn recent years, efforts have been made to apply NGS technologies to clinical practice, developing innovative predictive models for GC83PIK3CA (29.4%), KRAS (26.5%) and TP53 (26.5%). PIK3CA mutations were highly represented in our good prognostic subgroup (38.1%) and this evidence is coherent with the available data, supporting the stronger impact of the PIK pathway in MSI positive tumors characterized by intestinal histologyKRAS mutant patients was relatively high in the good prognosis population (38.1% compared to 7.7% in the poor prognosis group) suggesting that hyperactivation of receptor tyrosine kinase signaling positively impacts on prognosis. In this regard, the good prognosis subgroup had more alterations in the mTOR pathway (47.6%) and tyrosine kinase receptors (33.3%) compared to the poor prognosis group . TP53 mutations are equally stratified in the two prognostic populations according to that described by Cristescu et al. where TP53 status reflects intermediate prognosisThe most commonly mutated genes in our population were As suggested in our preliminary studyRecently, a comprehensive analysis of NGS and IHC in advanced GC demonstrated the existence of a complicated arrangement of protein expression and gene alterations, supporting the overall results of our study in RGCRegarding the future perspective for the presented analysis, an external validation of the identified molecular differences between the two prognostic subgroups will be performed. Moreover, considering the emerging clinical application of liquid biopsy as a feasible, non-invasive method for detecting and monitoring genomic alterations, this approach might represent a future step of the project, in the context of a prospective cohort of patients.The clinical application of molecular tumor profiling assays, such as NGS technologies, able to codify complicated factors such as tumor heterogeneity should be investigated in larger samples in order to complement clinical parameters to accurately predict individual patient risk and possibility of response in the context of clinical trials. In this context, our analysis represents a proof-of-principle study that demonstrates the feasibility of a comprehensive molecular profiling approach for cancer with the aim to identify potentially druggable pathways. This preliminary step is essential in paving the way for ideation of innovative and biologically relevant clinical trials (such as basket and umbrella studies) that rigorously test the efficacy of molecular targeted agents assigned on the basis of a specific genomic profile.Based on the results of a previously published three-class model , 34 tumor blocks from an original sample (208 patients) were included from those patients who had the best and the worst prognosis, both in terms of cancer-specific survival (CSS), and overall survival (OS) ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, JAK2, JAK3, IDH1, IDH2, KDR/VEGFR2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL. Emulsion PCR was performed with the OneTouch OT2 system (Life Technologies). The quality of the obtained library was evaluated by High Sensitivity Assay on the Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies). Sequencing was run on the Ion Torrent PGM (Life Technologies) loaded with a 316 chip. Data analysis, including alignment to the hg19 human reference genome and variant calling, utilized the Torrent Suite Software ver. 3.6 (Life Technologies). Filtered variants were annotated using the SnpEff software ver. 3.1 .Four 10-μm paraffin sections were manually microdissected to ensure that each tumor sample contained at least 70% of neoplastic cells. DNA was purified and its quality assessed as describedTo validate NGS results, KRAS , and TP53  specific PCR fragments were analyzed by Sanger sequencing. PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter), labeled with Big Dye terminator v3.1 (Applied Biosystems). Agencourt CleanSEQ magnetic beads (Beckman Coulter) were used for post-labeling purification. Sequence analysis was performed on an Applied Biosystems 3130xl Genetic Analyser.FFPE tissues of cases of GC with intestinal histology were used to construct tissue microarrays (TMAs) as previously described17Descriptive statistics was used to summarize pertinent study information. Follow-up was analysed and reported according to ShusterHow to cite this article: Bria, E. et al. Comprehensive molecular portrait using next generation sequencing of resected intestinal-type gastric cancer patients dichotomized according to prognosis. Sci. Rep.6, 22982; doi: 10.1038/srep22982 (2016).


In Romania, a country with no organized national surveillance program regarding cervical cancer, the early diagnosis of HPV (Human Papilloma Virus) infections is a major requirement, especially in HIV-infected women. The objective of this study was to determine the HPV prevalence and type distribution in young HIV-positive women and to assess the difference in the risk factors for developing cervical cancer compared to those of HIV-negative women.Linear Array HPV Genotyping test .We conducted one cross-sectional cohort study from June 2013–September 2014, including 1,032 women: 992 HIV- women who were 36.5 years old (limits: 17 ÷ 84) and 40 HIV + women who were 22.9 years old (limits: 17 ÷ 30) with iatrogenic HIV infected. We detected HPV types with the DNA/HPV was detected in 18/40 (45%) of the HIV+ patients and in 350/992 (35.2%) of the HIV- patients . After age adjustment, the overall HPV prevalence was 51.6% in HIV+ versus 63.2% in HIV- women aged under 25, and 22.2% in HPV+ versus 47.2% in HIV- women aged 25–34. We detect HIV being a risk factor for acquiring multiple HPV type infections . The eight most common HPV types  for women below age 30, HIV+ / - were: HPV 16, 18, 31, 51, 58, 68, and 6 and 82 respectively. To assess the risk factors of HIV-positive women for acquiring HPV infection, we analyzed the CD4/μL, ARN/HIV copies/μL, the age group, the number of sexual partners, smoking, and the type of HPV infection (single versus multiple infections). We found that the number of sexual partners and smoking are statistically significant risk factors.versus HIV – patients, multiple infections were more frequent in the first group. In our study group young HIV-infected patients under HAART therapy, high number of sexual partners (more than 3) and smoking were detected to be risk factors. Future organized screening for HPV infection using sensitive and specific methods are necessary at the national level in Romania.Even though there are no significant differences regarding the prevalence of HPV infection in HIV +  The primary cause of cervical cancer is persistent infection with high-risk human papillomavirus (HR HPV) types . HPV is In 1989, in Romania it was an important nosocomial HIV epidemic, when many institutionalized children contracted HIV through blood transfusions and/or infected needles . On30 JuCervical Cancer Action states that in Romania, there is a national program for HPV vaccination [In Romania 2012, the national government attempted to implement organized and free cervical screening by cytology but there was no centralized quality control or specific training and audits. Even under these conditions, it was possible to detect approximately 120 new cases of cervical cancer annually (data not published). The program has been stopped because of the lack of financial and political support, and screening by Pap test is now possible by request. The HPV vaccination program also failed, even though cination .In settings where HIV is endemic, screening for cervical cancer is particularly important. Although Romania is not included in the endemic area, HIV-positive patients represent a high-risk group for many infections, including HPV. The aim of this study was to evaluate the HPV type prevalence and distribution in HIV-positive women and to assess the difference in the risk factors for developing cervical cancer compared to those of HIV-negative women.We conducted one cross-sectional cohort study from June 2013- September 2014, including 1032HIV-negative women from “Cuza-Vodă” Gynecology Clinical University Hospital Iaşi and 40 HIV-positive/AIDS women from Infectious Disease “St. Parascheva” Clinical University Hospital Iaşi. The average age for 992HIV- women was 36.5 years old (limits: 17 ÷ 84) and 22.8 year old (limits: 17 ÷ 30) for40 HIV +patients. Half of the first study group (514 HIV-negative women respectively)was included in a previous study that was published in 2011 [Thestudy was approved by the Bioethical Research Committee of the “Gr. T. Popa” University of Medicine and Pharmacy Iaşi. All participants in the study signed informed consent form after they were shown/presented the benefits of inclusion in this study and filled out a questionnaire that was administered by an interviewer.The study participants completed a questionnaire concerning the known cofactors for cervical cancer  and provided cytological results of previous Pap tests. HPV genotyping was performed in the Virology Laboratory of “Gr. T. Popa” University of Medicine and Pharmacy, Iași, using the Linear Array (LA)HPV genotyping method as previously described (18). The LA method identified 37 HPV types: 13 high risk (HR) and 24 low or intermediate risk (LR) .p value of <0.05 was considered statistically significant.The age was categorized into the following groups: <25; 25–34; 35–44; 45–54; and 55+.years. Descriptive statistics were prepared for demographic variables: age, births, pregnancies, abortions, DNA/HPV presence, multiple HPV infections, and HR and LR HPV types. According to the presence or absence of HIV infection, after age adjustment, we used chi-2 test for OR, CI 95%, to determine statistically significant differences between the proportions for each variable. If OR was > 1, we considered the tested variable being a risk factors for acquiring HPV infections. The CD4+ cell count and ARN/HIV viral load were both divided into three groups , respectively ARN/HIV copies/mL: < 10,000, 10,000–1,000,000, >100,000), based on the median value for each respective variable. To assess the risk factors for HIV-positive women for developing cervical cancer, we used the Kruskal-Wallis test. Statistical analysis was performed using SPSS version 20.0 software. A versus 2.3% HPV 6 and 0.19% HPV 11 in HIV-negative women. We also detected that some LR HPV types  were found only in HIV + women. The prevalence of multiple HPV infections was 32.5% (13/40) for the HIV + women versus 20% (119 / 992) in HIV-negative women. Multiple HPV infections in HIV-positive women included the following types:16, 18, 31, 39, 51, 52, 58, 59, and 68; in one case, infection involved the following types:53, 59, 61, 68 and 84.For HIV-positive women, we detected 18/40 (45%) positive for DNA/HPV; the HPV prevalence for HIV-negative women was 35.2% (350/992).Among the HIV+ women, the most prevalent HR HPV were HPV 52 (12.5%), HPV 31 and 68 (each 10%), HPV 16 and 51 (7.5%) and HPV 18 (5%). For HIV-negative women, the descending order of HR HPV types was HPV 16 (11%), HPV 52 (4%), HPV 18 (3.9%), HPV 31 and 51 (3.7% each), and HPV 58 (2.75%). . There wThis is a pilot study assessing for the first time in Romania the HPV types distribution in HIV-positive women, and their risk for developing precancerous lesions and cervical cancer, was compared to that of HIV-negative women. The HPV type distribution in HIV-negative women in the present study does not bring any new significant information than those previous published :the sameIn We acknowledge that the group of HIV+ women who were infected iatrogenically is very small mainly because in Romania all HIV positive patients are treated and monitorized in 8 regional centers; in Iaşi Center from “Sf Parascheva” Hospital there are around 1000 patients treated with HAART therapy since 2010; therefore, many differences compared to HIV-uninfected women will not be statistically significant even if they are rather strong. The demographic data regarding the tested women are shown in HIV-positive women are more susceptible to HPV infection associated with multiple types and with loss of detection , 12. The3), and by a relatively high ARN/HIV median value (8575 copies⁄mL). For the patients with CD4 > 200 cells/mm3 and ARN/HIV < 20,000, the HPV prevalence was 33.33%, while for the patients having CD4 < 200 and ARN/HIV > 20,000,the HPV prevalence was doubled to 66.66%.A similar tendency was detected by Dames DN et al (in 2014), who reported that HR HPV infection increased 7 times in the case of CD4+ ≤ 200 cells/mm3versus> 200 in a study group of 167 non-pregnant HIV-positive women [The high HPV prevalence in this study group HIV-positive women can be explained by at least two main factors: the level of immunity, as demonstrated by a low medium level of CD4 cells  refused second sampling for the cytological examination because these women had a bloody cervix. We respected the patient’s decision in accordance with their informed consent; this possibility could have been avoided if LBC (liquid-based cytology) were available. Regarding the Pap smear examination, only two smears were detected with cytological abnormalities: one had LSIL  and infection with HPV 55 and 73, and one had ASC-US and AGC  with HPV 31, 61, and 84; the rest of the tested HIV-positive women had a negative Pap smear for intraepithelial lesions or malignity or an inflammatory result despite the high frequency of HPV type infection. Other studies reported an increased prevalence of HPV in association with high-grade lesions in HIV-positive women , 13; in versus multiple infections). The Kruskal Wallis score was significant for the number of sexual partners (> 3) and smoking condition , ARN/HIV copies/mL , age group, number of sexual partners and smoking in relation to the type of HPV infection (single With the exception of one single case (who refused the therapy), all of the HIV + cases received HAART therapy, which was given for the last 5 years. Antiretroviral therapy was associated with lower prevalence of HPV infections, similar to other authors . Recent versus persistent, considering that persistent HR HPV infections is a major risks for cervical cancer development.The most important strength of our study is the characteristics of HIV-positive women who were infected at birth or soon after, and this is the first study, to our knowledge, to assess the HPV type distribution in this category of patients from Romania. Additionally, the genotyping method has good strength and was demonstrated to be proficient by a WHO panel. The weaknesses of this study include the low number of HIV-positive women, explained by the real number of HIV—positive registered in our geographic area, and the lack of biopsies for them, due to the low medium age (22.9 years), which did not allow the gynecologist to see visible lesions in the colposcopic examination. Additionally, the cross-sectional feature of our pilot study did not enabled for investigating the effects of the CD4 count and HAART on HPV infection longitudinally. To study the natural history of HPV infection, it would be necessary to perform one longitudinal study to assess the type of HPV infection, transitory It is well known that only a small proportion of women infected with pathogenic HPV will develop a cancer. Therefore detecting such infections will unnecessarily alarm women and may lead to aggressive intervention from physicians, with a decrease in quality of life and fertility. Screening for pathogenic HPVs is costly. In addition, cervical cancer will develop  in HPV- women. The method of choice for a national surveillance program could remain the cytological examination of Pap smears, in the countries in which Pap smears is organized and HPV infection prevalence is low. For our country, in which in 2015 aproximatively 40% of family physicians did not registered themselves in the national cervical screening program (data not published), we can conclude that women registered to those doctors will not be not included in the future screening. In countries where cervical screening is well organized (e.g. Netherlands) it was observed that even with a very well organized cytology based cervical screening programm, the incidence and mortality did not decreased in the last 10 years. For this reason Pap test will be replaced with clinically validated test, more sensitive and specific, being also known that a HR HPV negative woman is having low risk for developing precancerous lesions.The HAART therapy and young age seems to be protective against developing precancerous cervical lesions in our study group, even the HPV prevalence and the multiple HPV type distribution were higher than those in the HIV-negative study group. The HPV prevalence in HIV-positive women increased (from 33.3% to 66.6%) in parallel with decreasing immunity. In particular, we noticed that serotypes 54, 81 and 83 were isolated only from HIV-positive patients. All women, regardless of their HIV status, should participate in an organized cervical cancer screening using clinical validated methods at affordable prices.In our region, it is necessary to improve medical knowledge of general population for a better understanding of the impact of HPV vaccination on the future cervical cancer incidence. Additionally, it would be cost efficient to use an HR HPV clinically validated test for the primary screening of cervical cancer.S1 Table(DOCX)Click here for additional data file.


These preliminary data suggest that a DUS-based assay could bypass sociocultural barriers and sampling issues and therefore could be a suitable, effective tool for epidemiological surveillance and screening programs, especially in low-income countries.Nowadays, several screening strategies are available to prevent cervical cancer, but inadequate resources, sociocultural barriers, and sampling issues impede their success in low-income countries. To overcome these issues, this study aimed to evaluate the performance of human papillomavirus (HPV) testing from dried urine spots (DUS). Eighty-eight urine samples (including 56 HPV DNA positive specimens) were spotted on filter paper, dried, and stored in paper-bags. HPV DNA was detected from the DUS after 1 week and 4 weeks of storage using a polymerase chain reaction (PCR) assay. The sensitivity, specificity, and concordance of the DUS-based HPV test were evaluated by comparing the results with those of HPV testing on fresh urine samples as the gold standard. The sensitivity of the test was 98.21% (95% CI: 90.56–99.68) for DUS stored for 1 week and 96.42% (95% CI: 87.88–99.01) for DUS stored for 4 weeks. The specificity was 100% (95% CI: 89.28–100) at both time points. The concordance between DUS and fresh urine HPV testing was “almost perfect” using the  Cervical cancer is a relevant public health problem for women worldwide, being the third most frequent cancer and the fourth most common cause of death from cancer in women , 2. OverHPVs are DNA viruses that are grouped into cutaneous and mucosal types according to their infection site and further subdivided into high-risk (HR) and low-risk (LR) types, depending on their association with disease malignancy. The International Agency for Research on Cancer (IARC) has included 25 types of HPV in the high-risk clade (HR-clade) by subdividing them into three groups . HPV typDifferent types of screening for cervical cancer are now available, such as the conventional cytology test (Pap test) and liquid based cytology (LBC), visual inspection with acetic acid (VIA), and HPV testing for HR-HPV from cervical brush. These types of screening require adequate financial resources, a developed infrastructure, trained labour, and surveillance mechanisms for screening, investigating, treating, and following up on targeted women , 6. MoreAlternative tools that can overcome these problems could improve screening coverage and reach the female population at risk in developing countries.The use of HPV testing on urine, a noninvasive and easy-to-collect sample, could be more attractive to women because it bypasses medical examination, as well as sociocultural and religious implications. The correlation between the detection of HPV in urine and cervical samples has been reported in several studies in the literature –12. In aHowever, urine samples require restrictive conditions for storage and transportation, especially when samples must be transported over large distances in a warm climate. This factor is particularly relevant in low-income countries where analysis laboratories can be located far away from the rural areas where women live. An alternative method of sample collection is the use of dried urine spots (DUS), that is, a urine sample spotted on blotting paper, which allows for stabilization by drying. This approach has logistical benefits because DUS are small and easily transported and the specimens can be stored at room temperature.DUS sample collection solves several problems associated with the sampling and storage of fresh urine samples. Therefore, the DUS approach is particularly interesting, especially in developing countries, even considering that dried spots on filter paper can be successfully used for the detection of various infectious agents, as well as metabolic and genetic diseases –16.Few studies have evaluated the use of dried samples on filter paper for the detection of HPV infection, and these investigations examined exclusively cervical brush samples –19. No dThus, the aim of this study was to evaluate the performance of HPV testing from DUS and to compare the results obtained with those from paired fresh urine samples. Chlamydia trachomatis infections [Urine samples were obtained from 88 immigrant women : 28–43 years) who attended NAGA Onlus in Milan, Italy, between June 2012 and December 2013 and were included in a large epidemiological study on HPV andOf the urine samples, 56/88 (63.6%) were HPV DNA positive and 32 (36.3%) were HPV DNA negative. Of the HPV DNA positive samples, 40/56 (71.4%) were sustained by single infections (24 belonging to HR-clade genotypes and 16 to LR genotypes) and 16/56 (28.6%) were caused by multiple infections (4 were caused by LR genotypes and 12 by at least 1 genotype of the HR-clade).μL urine sample was subdivided into eight 50 μL aliquots that were each spotted on preprinted circles on a piece of filter paper . The DUS filter papers were dried for 3 h and then stored in paper bags in a dry location at room temperature  for either 1 week or 4 weeks until the analyses took place. The analyses were carried out at the Laboratories of the Department of Biomedical Sciences for Health, University of Milan, Italy.Each 400 μL, was extracted using the commercial NucliSENS EasyMAG method , according to the manufacturer's instructions. The nucleic acids were eluted in 100 μL of the NucliSENS elution buffer.Four preprinted circles were punched or cut out from each piece of DUS filter paper using a sterile single-cut paper-punching machine or a new sterile scalpel blade. The circles were transferred into a 1.5 mL tube containing 1 mL of NucliSENS Lysis Buffer  and incubated on a roller mixer for 30 minutes at RT. Then the tube was centrifuged for 15 s at 1500 ×g. The lysate, with a volume of approximately 750 The concentration of the extracted DNA was evaluated using a spectrophotometer . The quality of DNA was validated by detection of a 268 bp fragment of the housekeeping beta-globin gene using an in-house polymerase chain reaction (PCR) assay .HPV DNA was detected using an in-house nested-PCR assay based on the amplification of a 150 bp open reading frame late gene 1 (ORF L1) fragment. The nested-PCR assay was performed using a two-step amplification to either increase sensitivity or mitigate the inhibitory effect of substances potentially present in the sample. Every PCR reaction included positive  and negative (water) controls. Strict laboratory precautions and quality assurance/quality control measures were followed to avoid cross contamination and carry over PCR. ELSI_F/ELSI_R primers were used for the first cycle of amplification and GP5+/GP6+ primers were used for the nested reaction, as previously described , 22.The amplification products were visualized by means of electrophoresis analysis on 2% agarose gels containing ethidium bromide (0.5 mg/mL). The amplified products were compared with molecular weight standards .HPV DNA positive DUS isolates were genotyped using INNO-LiPA HPV Genotyping Extra , a line probe assay based on the principle of reverse hybridization, according to the manufacturer's instructions. The resulting line patterns allowed for identification of 28 different HPV genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74, and 82.The sensitivity and specificity of HPV testing from DUS were evaluated in comparison to HPV testing from fresh urine samples as the gold standard. These results were presented as percentages with 95% confidence intervals (95% CIs). Proportions (95% CI) were calculated using the Wilson score model by the OpenEpi statistical program (version 3.01), which is available online .κ) statistic was calculated by dividing the difference between the observed proportion of agreement and the expected proportion of agreement by 1 minus the expected proportion of agreement. The concordance was defined as “poor” (k = 0), “slight” (0.01 ≤ k ≤ 0.20), “fair” (0.21 ≤ k ≤ 0.40), “moderate” (0.41 ≤ k ≤ 0.60), “substantial” (0.61 ≤ k ≤ 0.80), “almost perfect” (0.81 ≤ k < 1.00), or “perfect” (k = 1.00).To determine the proportion of agreement between DUS and fresh urine testing, Cohen's unweighted kappa  tested HPV DNA positive after 1 week. In contrast, HPV DNA was detected in 54/56  of the DUS samples after storage for 4 weeks.The DUS sample that tested HPV DNA negative after both 1 and 4 weeks of storage was prepared from an HPV-72 (LR HPV) infected urine sample. The other DUS that tested HPV DNA negative only after 4 weeks of storage was prepared from an HPV-56 (HR HPV) infected urine sample.All DUS prepared using HPV DNA negative urine samples tested negative for HPV DNA.κ statistic ≥ 0.81) (The sensitivity of the HPV DNA test in DUS at 1 and 4 weeks was 98.21% (95% CI: 90.56–99.68) and 96.42% (95% CI: 87.88–99.01), respectively. The specificity for the test was 100% (95% CI: 89.28–100) after both 1 week and 4 weeks of storage. The proportion of agreement between the DUS and fresh urine tests was “almost perfect” ( ≥ 0.81) .Of the 40 DUS prepared from fresh urine samples of women infected by a single HPV genotype, 38 (95%) tested HPV DNA positive after four weeks of storage. All amplified fragments from the DUS were properly genotyped, and the distribution of HPV genotypes matched across the two sample types . The disTo our knowledge, this is the first report of HPV detection from urine samples stored on filter paper. The quantity and the quality of DNA extracted from DUS were comparable to those obtained by standard collection from fresh urine, as estimated by the spectrophotometric readings and the detection of the housekeeping beta-globin gene using an in-house PCR assay. Accordingly, the high molecular weight DNA extracted from DUS samples is sufficient to perform molecular assays, either traditional or high throughput.The data obtained showed an elevated concordance between HPV DNA detection in DUS and fresh urine samples. Cohen's unweighted kappa values were very high, indicating an “almost perfect” agreement.These preliminary data support the use of DUS as simple, rapid, and safe sampling for HPV DNA detection and genotyping by using molecular tests, such as PCR and line probe assay based on the principle of reverse hybridization. Furthermore, the use of DUS strengthens the already recognized advantages of urine samples. Collection of these noninvasive specimens is more acceptable and can bypass the ethical, social, and religious barriers of speculum exams for the collection of conventional cervical brush , 8, 11.Moreover, drying urine samples on filter paper allows the DNA to be protected from degradation for a long period, as shown by the high percentage (96.4%) of HPV DNA positive samples detected after four weeks of storage at room temperature.Due to the introduction and increasing availability of novel and powerful high-throughput molecular technologies, the optimization of methods for biological samples collection and storage has become a critical issue. These results highlight the potential use of DUS samples as an alternative means of biobanking, avoiding the high costs and logistical problems associated with the storage and transportation, especially where a cold chain is absent , 24. FinHPV testing from DUS showed an elevated sensitivity and specificity and a high concordance rate compared to HPV testing from fresh urine samples. These preliminary data suggest that a DUS-based assay could bypass sociocultural barriers and sampling issues. This approach could be a suitable and effective tool for epidemiological surveillance and screening programs, especially in low-income countries.


Cervical cancer is the primary cause of cancer-related deaths in women living in Botswana.Paraffin-embedded blocks of formalin-fixed invasive cervical cancer specimens were identified from women living in the U.S. (n = 50) or Botswana (n = 171) from which DNA was extracted. Thin-section PCR was performed on each sample for HPV types and HIV. Comparisons were made between HPV types and groups of types identified in cancers.HPV DNA was identified in 92.0% of specimens from the U.S. containing amplifiable human DNA, and 79.5% of specimens from Botswana. HPV 16 was detected in 40 of 46 HPV-positive specimens (87.0%) from the U.S. vs. 58 of 136 (42.7%) from Botswana (p < 0.001). In contrast, non-HPV 16/18 types, all A9 species , non-HPV 16 A9 , HPV 18, all A7 types  types were detected significantly more often in specimens from Botswana. The prevalence of non-HPV 18 A7 types did not differ significantly between the two groups. For specimens from Botswana, 31.6% contained PCR-amplifiable HIV sequences, compared to 3.9% in U.S. specimens. Stratifying the samples from Botswana by HIV status, HPV 31 was detected significantly more often in HIV-positive specimens. Other HPV types and groups of types were not significantly different between HIV-positive and HIV-negative specimens from Botswana.This study demonstrates that there may be important HPV type differences in invasive cervical cancers occurring in women living in the United States or Botswana. Factors in addition to HIV may be driving these differences. Worldwide, cervical cancer is the second most common cancer in women and the most frequent cancer in women living in Sub-Sahara Africa, where the estimated incidence is 26.8 per 100,000 compared to 8.1 per 100,000 in the United States. PreviouArchived, paraffin-embedded blocks of formalin-fixed cervical cancer specimens were acquired from women residing in Indianapolis, Indiana (U.S.) or from throughout the country of Botswana. Cervical cancer specimens from the U.S. were collected during 2008 through 2011; those from Botswana between 2009 and 2010. Specimens from Botswana were re-embeded due to apparent dehydration of paraffin. Approval for this study was obtained through the Botswana Ministry of Health, Gaborone, Botwana and the Institutional Review Board at Indiana University School of Medicine, Indianapolis, IN. Sections (5 microns) were cut using a new blade for each block. The first and last sections were stained with hematoxylin and eosin for histopathologic classification.Specimens with no epithelium were eliminated from the study. For specimens containing less than an estimated 5% of the total specimen, macrodissection was performed using a microscope to identify cancerous tissue, which was marked under the undersurface of the slide, then removed using a sterile scalpel.Four to six tissue sections were scraped from slides with a sterile blade. Tissue fragments were placed in 2 mL sterile tubes containing 1 mL octane. Tubes were rocked at room temperature for 10 minutes (min) and centrifuged at 19,000 × g for 10 min in a microcentrifuge. The octane supernatant was removed and fresh octane (500 uL) was added. Tubes were rocked for an additional 5 min to dissolve remaining paraffin. Samples were centrifuged at 19,000 × g for 5 min. The octane supernatant was removed and 1 mL of absolute ethanol was added to the pellet, and tubes were capped and inverted to mix, followed by centrifugation for 10 min at 19,000 × g. The supernatant was removed, 500 uL absolute ethanol was added to the pellet, then centrifugation was performed for 10 minutes at 19,000 × g. The ethanol was removed and pellets were dried in a Savant Speed Vacuum  for 20 min.Dried pellets were suspended in 300 uL Specimen Transport Medium  and 10 μL proteinase K solution (20 μg/μL) was added (Qiagen). Samples were vortexed for 10 seconds and incubated at 55˚C overnight. DNA was extracted using the QIAamp MinElute Media Kit according to the manufacturer’s instructions (Qiagen). DNA concentration was determined by spectrophotometry  and 200 ng was used for each PCR.HPV detection and genotyping was performed using the Linear Array HPV Genotyping Test (LA-HPV)  as described previously-12. The Serum samples were not available, nor were medical records available for all subjects. Therefore, HIV was detected by thin-section PCR using DNA extracted from paraffin-embedded cervical cancer specimens. A 142 bp segment of the HIV-1 gag gene (nucleotides 1360-1507) was amplified using primers modified from An, SF et al[PCR products, including controls were analyzed on 1.8% agarose gels. To confirm the amplification of desired products, two reactions from U.S. and Botswana cancers with an amplicon of expected size were extracted from gels using the QIAquick Gel Extraction Kit (Qiagen), then sequenced bi-directionally. Homology of the sequenced samples with known HIV gag sequences was determined using the Basic Local Sequencing Alignment Search Tool (BLAST) search (NCBI).Adequacy of extracted DNA based on β-globin-positivity among cancer specimens, and HPV-positivity among β-globin-positive samples was compared between U.S. and Botswana specimens. Type-specific HPV prevalence was then compared between U.S. and Botswana HPV-positive samples. HPV types were divided by their respective classification into Alpha 9 (A9 or HPV 16-related) and Alpha 7 (A7 or HPV 18-related). The A9 types included HPV 16, 31, 33, 35, 52, and 58, and the A7 types included HPV 18, 39, 45, 59, and 68. Prevalence of A9 and A7 types between U.S. and Botswana were compared. The difference in the distribution of cancers with multiple HPV types was compared between the sites of collection the U.S. and using a dichotomized approach (1 HPV type vs. more than 1 HPV type detected in a cancer). Additionally, HPV prevalence was compared between HIV-positive and HIV-negative specimens among Botswana samples. Comparisons were made using Fisher’s exact tests or Chi-square tests. Analyses were performed using SAS .Paraffin-embedded tissue blocks were obtained from 51 women from the U.S.  cervical cancer specimens were adenocarcinomas and 166 of the 171 (97.1%) were squamous cell carcinomas.HPV (any type) was detected in 46 of the 50 92.0%) β-globin-positive cancers from the U.S. and 136 of the 171 (79.5%) β-globin-positive cancers from Botswana (p = 0.042)  HPV-positive cervical cancers from the U.S. and 49 of 136 (36.0%) HPV-positive cancers from Botswana (p = 0.008)  were identified in 61.8% of cancers from Botswana and 93.5% of cancers from the U.S. A “second generation” vaccine is currently under development that may also protect against HPV types 31, 33, 45, 52, and 58 in addition to these four types. One or more of the nine types in the “second generation” vaccine were identified in 77.9% of cancers from Botswana and 100% of cancers from the U.S.The HIV sequences were amplified in 54 of 171 (31.6%) of cancer specimens with amplifiable DNA from Botswana and 2 of 50 (3.9%) of cancer specimens with amplifiable DNA from the U.S. (p < 0.001). A statistical analysis of HPV types by HIV status was performed on cervical cancers from Botswana, but not on cancers from the U.S. In the case of the two HIV positive cancers from the U.S., HPV 16 was detected in one and no HPV types were detected in the other.The distribution of HPV types among HIV positive cancers and HIV negative cancers in Botswana is shown in Table All HPV-positive cancers were compared between countries for detection of multiple HPV types. A single HPV type was detected in 43 of 46 (93.5%) HPV-positive U.S. cervical cancers and 113 of 136 (83.1%) HPV-positive cervical cancers from Botswana (p < 0.082). The distribution of HPV types among cancers containing single vs. multiple HPV types by country is shown in Table For the 3 U.S. specimens that contained more than one type, all contained a total of 2 types. For the 23 specimens from Botswana that contained more than one type, 19 contained 2 types, 2 contained 3 types, 1 contained 4 types, and 1 contained 7 types. The HPV-positive cervical cancer specimens from Botswana were then stratified by HIV status. More than 1 HPV type was detected in 5 of 44 (11.4%) of cervical cancers testing HIV-positive and 18 of 92 (19.6%) of cervical cancers from lesions testing HIV-negative; a non-significant difference (p = 0.233).Cervical cancer is the most frequent cancer in women living in Sub-Sahara Africa. High quality data on HPV types in invasive cervical cancers from African women is lacking, because most studies utilized exfoliated cells from cervical swabs for type determination rather than actual cervical cancer tissue. Though useful in understanding the overall epidemiology of HPV in Africa, such studies do not provide an accurate representation of HPV types in cervical cancers. In the current study, a direct approach (thin-section PCR) was used to isolate DNA from formalin-fixed invasive cancer specimens.HPV 16 was the most frequently detected type in invasive cervical cancers from women living in the U.S. . HPV 16 was also the most frequently detected HPV type in cancers from women living in Botswana, but was significantly less often detected in this group  compared to the U.S. HPV 18 was less frequently detected in cancers from women living in the U.S. , compared to 23.5% in cancers from women living in Botswana. The combined frequency of HPV 16/18 detection in cancers from the U.S. was 93.5%, compared to a combined frequency of HPV 16/18 of 61.8% in cancers from Botswana. Overall, non-HPV 16 A9 family members were significantly more frequently detected in cancers from women living in Botswana, but the prevalence of non-HPV 18 A7 family members did not differ significantly between the U.S. and Botswana.only factor driving differences in HPV types in cervical cancers from women living in Botswana. However, factors related to HIV infection  were not analyzed in our study and may influence the HPV type distribution. Other factors to consider in future studies include geographical variation, nutritional factors and co-infections with other pathogens.HIV is a factor that likely contributes to differences in HPV types in cancers-16. Straet al., examined invasive cervical carcinoma specimens from women living in Kenya or South Africa, and found that the combined percentage of HPV 16 plus HPV 18 was approximately 68%, a percentage close to that presented in this report for women living in Botswana[10 DEIA-LiPA25 system. They found that HPV 16 was detected in 50.7% of cancers and HPV 18 in 19.2%. Similar to our study, multiple HPV types were detected in only 11.1% of cancers. HIV status was not known for most of the women in this study.What have other studies shown regarding HPV types in cervical cancers in African women? One study, utilizing cervical swabs from HIV-positive women living in Botswana with cervical intraepithelial neoplasia (CIN) grades 2 or 3 showed that a range of HPV types could be detected by PCR. Overall, 56% of these women were infected with multiple HR-HPV types. The most common HPV types found in women with CIN2 and CIN3 were HPV 16 and 18 accounting for 51% of their subjects. In anotBotswana. This stIn the current study, there were cervical cancers in which more than a single HPV type was detected. Cervical cancer is a clonal process, and no studies have demonstrated more than one HPV type in a single cell. However, cervical lesions have been demonstrated that contain separate dysplastic foci, each containing different HR-HPV types. One expThere are several limitations to this study. First, only one U.S. site and one African site were included, so these results cannot be generalized. Second, the study used paraffin-embedded specimens, and as a result, the sensitivity for HPV detection may be less than that if fresh cancer tissues had been available. It is possible that the techniques used to detect and type HPV have different performance capabilities for certain types, but the Roche Linear Array has become the standard typing method in epidemiologic studies. Certain conditions of fixation affect the recovery of DNA from paraffin-embedded tissues,22. BecaIn summary, amplifiable DNA was extracted from archived invasive cervical cancers from women living in the U.S. or Botswana. The contribution of HPV types 16 and 18 was significantly higher in cancers from the U.S. compared to cancers from Botswana. Conversely, non-HPV 16/18 oncogenic types were more frequently detected in cervical cancers from Botswana. Multiple HPV types were infrequently identified in cancers from either country. HIV status had a modest effect on HPV type distribution in cervical cancers from women living in Botswana. In larger future studies, a more pronounced effect of HIV on HPV types detected in invasive cervical cancers may be evident. Because current vaccines against HPV do not provide reliable or lasting protection against oncogenic types other than HPV 16 and HPV 18, such studies are important to conduct in other African countries.Darron R. Brown, MD received lecture fees, advisory board fees, and intellectual property fees from Merck and Co.AE- manuscript preparation, laboratory methods, data analysis and submission. DRM- study design, procurement of samples, manuscript preparation. NZ- data analysis and manuscript preparation. YT- data and statistical analysis and manuscript preparation. BQ- laboratory methods, testing of samples, and manuscript preparation. MA- testing of samples, data analysis and manuscript preparation. DB- principal investigator, study design, data analysis, and manuscript preparation. All authors read and approved the final manuscript.


The burden of HPV-related Head and Neck Cancers (HNC) has been rising in the U.S. and other developed countries but this trend has not been reported in Africa. Objective of study was to evaluate the prevalence of HPV infection in HNC cancer cases seen between 1990 and 2011 at the tertiary health care institutions in Nigeria.We retrieved 149 head and neck cancer formalin fixed, paraffin embedded tumor specimens diagnosed between 1990 and 2011 from four teaching hospitals in Nigeria. One hundred and twenty-three blocks (83%) contained appropriate HNC for analysis while DNA extraction was successful in 60% (90/149). PCR amplification was successful in 33% (49/149) and Linear Array genotyping for HPV was successful in 11% (17/149) of these cases. These were in tumors from the larynx (6), cervical lymph nodes (3), nasal cavity (2), parotid (1), palate (1), maxillary sinus (1) and mandible (1). Two cases were non-specific and none were from the oropharynx. Histologically, 41% (7/17) of the successfully genotyped blocks were squamous cell carcinomas .We were unable to detect HPV in any of the HNC samples in our study. Our result may suggest that there is a low prevalence of HPV-related HNC among the adult population in Nigeria. Our results provide a benchmark to compare future incidence of HPV -related HNC in this community in future. We had significant analytical challenges from possible poor tissue processing and urge that future studies should prospectively collect samples and ensure high quality sample processing. Head and Neck Cancers (HNC) are a significant public health concern globally with worldwide Age Standardized Incidence Rate (ASR) of 9.1 per 100,000 population according to GLOBOCAN 2012 data, 10.7 per 100,000 in the United States and 9.0 per 100,000 population on the African continent.Risk factors for HNC include consumption of alcohol and smoking and their joint effect can be synergistic –7. OtherAs much as 25% of all cases of HNC globally are related to high risk HPV (hrHPV). Persistent infection with hrHPV is now accepted to be a major risk factor for the development of HNC, almost exclusively in the oropharynx. According to the International Agency for Research on Cancer (IARC), HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 are carcinogenic, and are responsible for all cancers of the cervix and a varying proportion of HNCs. HPV-relThe incidence of HNC is rising throughout the world and most of this rise is attributed to increasing prevalence of hrHPV infection , 20. hrHStudies in the United States have shown that the burden of hrHPV-related Head and Neck Cancers (HNC) is lower in African Americans compared to white Americans–28, and ®. We tested for HPV types using Roche Linear Array® HPV Genotyping Test according to manufacturer’s instructions. Ethical approval for study was obtained from the National Health Research Ethics Committee of Nigeria (NHREC). The study used anonymized data from which patient identifiers had been removed prior to analysis.In this multi-center cross-sectional study in Nigeria, HNC tissue samples fixed using 10% Neutral Buffered Formalin (NBF) and embedded in paraffin blocks were used. We retrieved clinical data and Formalin Fixed, Paraffin-Embedded (FFPE) blocks of malignant head and neck tumors diagnosed between 1990 and 2011, from the Pathology Departments of 4 tertiary health institutions in Nigeria: University of Benin Teaching Hospital (UBTH) and University of Calabar Teaching Hospital (UCTH) in Southern Nigeria; including University of Abuja Teaching Hospital (UATH) and University of Ilorin Teaching Hospital (UITH)–both in North-central Nigeria. The tumor blocks were shipped to the United States where pathologists at the University of Maryland Greenebaum Cancer Center reviewed them to confirm the diagnosis. After confirmation of the histological diagnosis, we took sections from the tumor blocks and extracted DNA from these sections using QIAamp DNA FFPE Tissue KitOf the 149 tumor blocks retrieved see , most, 1In this retrospective study of HNC tumor blocks from 4 referral hospitals in Nigeria, we found that out of the 149 FFPE blocks available for studies, we did not identify any Oropharyngeal cancers; HPV genotyping was successful in 17 (11%) cases and none of these were positive for HPV DNA. We also did not find any tumor that specifically originated from the oropharynx in this sample. Despite studies from other parts of the world that indicate that the Oropharyngeal cancers account for a huge proportion of all HPV-related HNC, 4, our The low prevalence of hrHPV-related HNC among Nigerians may be due to several factors. hrHPV -related HNC has been associated with practice of oral sex and the prevalence of this sexual behavior has been rising among youths and young adults with a recent study showing that oral sex practice, though widely common, is more prevalent in younger age groups–37. In tFurthermore, the practice of oral sex appears to be less common among Africans than in Americans of any race. Studies on adolescents for example, shows that prevalence of having ever had oral sex for African adolescents ranges from 5.4%, to 13.3% compared with > 75% in American adolescents, population-wide studies of oral sex behavior in Africans shows a prevalence of less than 12% compared with at least 70% in Americans of all ethnicities, 40–43. et al[The difference in incidence of hrHPV -related HNC may also be related to genetic differences between Africans and White Americans. Ndiaye et al showed tet al. Consequet al. There iet al.There was a high prevalence of inadequate tumor samples for molecular biology analysis and HPV genotyping in this study, which may reflect poor infrastructure for tumor pathology in Nigeria. A study of HNC FFPE tumor blocks in South African blacks with oropharyngeal cancer detected HPV DNA in 94.1% of cases . This rein situ hybridization, making it even more complicated to pool results from several studies[There has been significant variation in prevalence of hrHPV-related HNC between studies that utilized different HPV detection techniques. Higher prevalence of HPV has been reported in HNC when PCR methods were used instead of l studies. Differel studies, 53.Our study suffers from several limitations. The quality of fixation was suboptimal in a significant proportion of the tumor blocks and DNA detection  was not successful for many samples. Attempts to repair formaldehyde-induced DNA damage did not yield meaningful success. Future studies of HNC and hrHPV -related HNC in Nigeria should be done prospectively and processed in good quality laboratories with standard QA/QC procedures in order to ensure that the resulting tumor blocks are amenable to molecular biology studies. Our sample size was small and this may be a reflection of the low incidence of HNC in our population. Many patients may also present late or do not present at all to hospital and die before a definitive diagnosis of HNC is made.In conclusion, we did not find any case of hrHPV-related HNC in our sample. While this is probably a result of low prevalence of HPV-related HNC, our results should be interpreted cautiously because of the small effective sample size and low DNA yield.S1 Table(XLSX)Click here for additional data file.


Chlamydia trachomatis interaction with HR-HPV types has highlighted a central role in cervical cancer development. The aim of this study was to investigate HPV prevalence and genotypes distribution in women at risk for C. trachomatis infection and negative for intraepithelial lesion or malignancy.C. trachomatis by Real Time PCR and genotyping by ompA gene sequencing. Additionally, a quantitative Real time-PCR was performed to assess the expression of the C. trachomatis Hsp60–encoding gene (Ct604 portion), linked to a persistent status of infection. HPV infection and genotypes was investigated in C. trachomatis positive women using Luminex technology.1071 cervical swabs were tested for C. trachomatis infection was detected in 53 out of 1071 (4.5%) samples, of which the 53% resulted positive for Hsp60 gene expression. The overall prevalence of HPV infection in C. trachomatis positive samples was of 60.4% (32/53): in 37.5% of samples was present a single genotype, while multiple genotypes infections were found in the 62.5% of them. Among women with a C. trachomatis chronic infection, 68% were HPV co-infected and the 79% showed multiple genotypes. Should be noted that levels of C. trachomatis Hsp60 expression in HPV co-infected women were significantly lower compared to women infected only with C. trachomatis. The C. trachomatis serotype F was found in the majority of samples, independently of HPV infection.C. trachomatis chronic infection. These observations suggested that the expression of CHSP60-1, interfering with both apoptotic and cellular senescence pathways, may promote a favourable local microenvironment for HPV infection.A high prevalence of HPV multiple infections have been found in young women affected with a  Chlamydia trachomatis (C. trachomatis), an intracellular bacteria characterized by a unique biphasic developmental cycle, is the most common sexually transmitted pathogens in women. Although C. trachomatis can cause pelvic inflammatory disease (PID), infertility, ectopic pregnancy, the clinical course is usually sub-acute and poorly symptomatic and the microorganism is rarely detected in subjects without clinical signs of infection [nfection .C. trachomatis to cause chronic persistent infection, characterized by the permanence of microorganisms in the host cells, can represent a common event [C. trachomatis produces a large quantity of Heat shock protein 60 (Hsp60) exhibited on the host cell surface and released into the extracellular space and in the bloodstream. This protein is considered a useful marker during clinical complications since its expression induce host chronic inflammation response [The ability of on event . During response ,3,4. Thuresponse .C. trachomatis in cervical cancer, the co-infection with Human papillomavirus (HPV), sharing the transmission route and the same risk factors, have recently highlighted [C. trachomatis as cofactor was suggested, since it seems to facilitate the penetration of HPV and the progress of cervical lesions interfering in the immunological response [C. trachomatis[C. trachomatis infection and its clinical persistence are poorly explored.Although epidemiological data have not yet provided consistent evidence about a real implication of hlighted -8. A rolresponse . Moreoveachomatis. NeverthC. trachomatis could be important in generating hypotheses regarding the possible synergism of these pathogens in cervical malignancy [HPVs are a family of DNA viruses that infect cutaneous epithelia, oral and genital mucosa. More than 100 different HPV types have been identified and characterized in two risk classes on the basis of their oncogenic potential: Low-risk (LR-HPV) types associated with benign genital warts and High-risk (HR-HPV) type considered the etiological agents of cervical cancer and other genital malignancies . Approxilignancy -16.C. trachomatis infection but negative for cervical lesions or malignancy. Furthermore, the role of C. trachomatis chronic infection in promoting HPV susceptibility has been evaluated.The aim of this study was to investigate HPV genotypes distribution and the frequency of infection in Italian women considered at risk for C. trachomatis infection were collected at the Virology laboratory of the IRCCS-Burlo Garofolo of Trieste, Italy, as part of C. trachomatis routine screening practices. Cervical samples were collected using a 200 mm polyethylene Cervex brush device  in 500 μl of TE buffer. The study was approved by the Institutional Scientific Board of the Institute for Maternal and Child Health - IRCCS “Burlo Garofolo”–Trieste, Italy and informed consent was obtained from each participant in accordance with the principles outlined in the Declaration of Helsinki.In 2013 year, cervical swabs (CS) specimen from 1071 women at risk for Genomic DNA was extracted, after samples centrifugation, using the QIAamp DNA Blood miniKit  as indicated by the supplier, and then stored at -80°C until analysis.C. trachomatis DNA was detected by Real Time PCR (RT-PCR), using a commercial kit , detection limit of the assay was 1 copies/μl, to amplify a conserved region of the cryptic plasmid element of C. trachomatis, following recommended protocol. The amplification and PCR product detection were performed with the ABI Prism 7900 Sequence Detection system .The presence of An aliquot, from each CS fresh specimen (1 ml), was centrifuged at 14.000 rpm for 15 min at 4°C and total RNA was extracted to the pellet obtained by RNeasy Mini kit  in according to the manufacturer’s instructions. The RNA final concentration (25 μg/ml) eluted in 50 μl of distilled water, was treated during sample processing with Dnase I , and subsequently stored at -80, in order to avoid DNA genomic contaminations.qPCR).cDNA synthesis was performed using kit SuperScript VILO™ cDNA Synthesis Kit , according to manufacturer’s instructions. Briefly, 14 μl of RNA were added to a mixture containing: 5X VILO™ Reaction Mix10, 10X Superscript Enzyme Mix and diethyl-pyro carbonate water (DEPC-treated) until a final volume of 20 μl and subsequently incubated at 42°C for 60 minutes and then at 85°C for 5 minutes. cDNA synthesized was employed for quantitative RT-PCR (C. trachomatis portion Hsp60 gene (Ct604) in specimen, a dedicated qPCR was used as previously described [-4 ng/ml corresponding to one genome copies/μl) and a standard curve equations were used to calculate the absolute copy number of gene mRNA.To quantify the transcript level for the escribed ,18. The C. trachomatis strain-TW-3). PCR reaction, in a final volume of 20 μl, contained: 5 μl of cDNA, 2 μl LC FastStart DNA Master SYBR Green I , 5 mM MgCl2 and 0.8 μM each primer. The thermocycling condition was: 95°C at 10 min followed by 40 cycles of 15 s at 95°C and 1 min at 63°C. The amplification was carried out in an ABI 7900HT Fast Real Time PCR System . The concentration of unknown samples based on their Ct values was determined with analytical software  [qPCR was further confirmed by agarose gel electrophoresis analysis, which showed the expected amplification product of 161 bp in length.In brief, the test included a serial standard curve, negative control with PCR-grade water and a positive control (systems) . The speC. trachomatis genotyping were performed by ompA gene primers; positive PCR amplification products were purified, sequenced and analysed with BLAST program (http://www.ncbi.nlm.nih.gov/BLAST) as previously described [escribed .HPV was detected in CS specimens using molecular assay supported by the Luminex technology . HPV genotyping was performed using the type specific E7 polymerase chain reaction bead-based multiplex assay  as recently described . The detChi Square Test was used to compare frequencies of discrete variables: Fisher Exact Test was applied when necessary. P value ≤ 0.05 was considered as the threshold of statistical significance for all tests.C. trachomatis infection  were analysed at the Virology laboratory of IRCCS-Burlo Garofolo, Trieste, Italy. Of these, 829 included Outpatients (mean age 35 ± 10 years), 161 patients from Sexually Transmitted Infection centre (STI) (mean age 30 ± 10 years) and 81 women from the Assisted Reproductive Technology (ART) (mean age 37 ± 10 years) clinic. All women were asymptomatic for C. trachomatis and other genital infections at the time of sampling, with the exception of STI women showing inflammatory symptoms. Moreover, all women were negative for cytological alterations, in accordance with Bethesda System 2001 diagnostic criteria [During 2013 year, 1071 cervical swabs from women at risk for criteria .C. trachomatis infection was 4.5% (53/1071) (mean age 35 years). As expected, C. trachomatis prevalence, stratified by the different clinical departments and by age, was found statistically significant higher (12.4%) in symptomatic women attending the STI center, than in asymptomatic women from the other groups (p < 0.001).In this study, as showed in Table C. trachomatis infection the overall prevalence of HPV was high, tested to 60.4% (32/53), as shown in Table In women with a o 60.4% 3/53, as so 60.4% 3/53, as sC. trachomatis were suffering from a chronic infection (Table C. trachomatis (±862 copies/μl). The evidence for viable organisms and not just residual DNA from a previous infection was supported to high correlation between DNA and RNA results (data not shown), considering that, the expressed gene should be linked to the C. trachomatis persistence.In order to characterize the chlamydial phase of infection, the mRNA expression of the Hsp60 gene showed that the 53% (28/53) of the women with C. trachomatis serotypes through the ompA gene amplification and subsequent sequencing, performed in available samples, had revealed a high frequency of serotype F, independently of chlamydial status or HPV infection.The classification of C. trachomatis and HPV-related cervical diseases. The co-presence of C. trachomatis and HPV was reported in cervical precancerous lesions, and high levels of specific IgG antibodies or DNA of C. trachomatis were recovered in HPV positive patients [C. trachomatis and HPV infection is still not completely understood.Several epidemiological studies have stated a positive association between patients ,14,15. HC. trachomatis prevention. For the first time, estimates are provided on the prevalence of HPV infections and about genotype distribution in women with a chronic C. trachomatis infection; this work has never been assessed before. Data from our study showed that the 60.4% of women with a diagnosis of C. trachomatis infection and in absence of cervical lesions, resulted co-infected with one or more HPV genotypes. To note, the 53% of them showed a chronic infection and, HPV was found more frequently associated (68%) to this specific chlamydial status. Moreover, a consistent portion of these women (79%) resulted to be infected with multiple HPV genotypes. To note, HPV multiple infections including specific genotypes such as HPV-6-51-59-66, was reported in the 31% of these women supporting evidence that the presence of one HPV type does not increase the likelihood of acquiring further infections, but that, HPV multiple infections might be the result of the local immune system impairment [In the present analysis, we evaluated the distributions of HPV DNA-positive infections analysing a large number of specimens collected as part of routine screening practices for ociated 6% to thispairment -31. In opairment ,33.In our series, HPV-31 and HPV-42 represented the genotypes more frequently detected, testing at 28% and 22%, respectively. The overall prevalence of these genotypes, with a smaller estimated oncogenic potentials than HPV-16, were usually lower in HPV screening Italian women  ,35. WhilC. trachomatis, a chronic infection is referred as a stop in development of chlamydial cycle, with aberrant bodies formation, and this state is characterized by high transcriptional activity [In the natural history of activity .C. trachomatis. Discussion on this finding can be merely speculative, suggesting that the maintenance of a steady-state level of transcription of Hsp60 gene could favour a balance between the Hsp60 induced pro-inflammatory microenvironment and HPV coexistence [In this study, the expression of Hsp60 gene, a marker of chronic infection, has been found significantly lower in HPV co-infected women compared to women infected only with xistence .C. trachomatis increases oxidative stress proteins that seem to trigger HPV cell entrance, and replication or enhance DNA breaks that may promote viral integration [C. trachomatis chronic status, independently of the genotypes risk [The chronic inflammation caused by egration ,14,16. Apes risk ,38.C. trachomatis chronic infection in young women without cervical lesions. In addition, specific HPV genotypes seem to be more frequently associated to C. trachomatis. This data may deserve further consideration, owing to accumulate evidence that the chlamydial chronic status could contribute to favour specific HPV genotypes representing possible implications for the prevention of cervical cancer.In conclusion, the results of the present investigation provide evidence for the notion that a high prevalence of multiple HPV infections has been associated with HPV: Human papilloma virus; LR-HPV: Low risk-HPV; HR-HPV: High risk-HPV; C. trachomatis: Chlamydia trachomatis; CIN: Cervical intraepithelial neoplasia; ICC: Invasive cervical carcinoma; PID: Pelvic inflammatory disease; Hsp60: Heat shock protein 60; CS: Cervical swabs.The authors declare that they have no competing interests.SS contributed to: study concept and design, acquisition of data (Chlamydia molecular and CT-Hsp60), drafting of the manuscript. FDS and CC contributed to: collection of clinical specimens and demographic data. RDS and VZ contributed to: technical assistance in molecular HPV analysis. GP contributed to: data analysis. PDA contributed to: data analysis. CC contributed to: drafting of the manuscript. MC contributed to: study concept and design, interpretation of data, and revising the manuscript. All authors read and approved the final manuscript.


Water supply systems are mainly classified into branched and looped network systems. The main difference between these two systems is that, in a branched network system, the flow within each pipe is a known value, whereas in a looped network system, the flow in each pipe is considered an unknown value. Therefore, an analysis of a looped network system is a more complex task. This study aims to develop a technique for estimating the optimal pipe diameter for a looped agricultural irrigation water supply system using a harmony search algorithm, which is an optimization technique. This study mainly serves two purposes. The first is to develop an algorithm and a program for estimating a cost-effective pipe diameter for agricultural irrigation water supply systems using optimization techniques. The second is to validate the developed program by applying the proposed optimized cost-effective pipe diameter to an actual study region . The results suggest that the optimal design program, which applies an optimization theory and enhances user convenience, can be effectively applied for the real systems of a looped agricultural irrigation water supply. Water supply systems are mainly classified into branched and looped network systems. The main difference between the two is that, in a branched network system, the flow within each pipe is a known value, whereas in a looped network system, the flow within each pipe is considered an unknown value. Therefore, an analysis of a looped network system can be a more complex endeavor.Water supply systems form part of a larger social infrastructure of an industrial society; their objective is the effective supply of water from a water source to an area in demand. The analysis of a water supply system can be one of the more complex mathematical problems. A significant fraction of the entire set of equations consists of nonlinear equations, and a large number of these equations must be solved simultaneously.This process requires sufficient consideration of the law of conservation of energy and a continuity equation of mass. In this regard, over the past few decades, many methods have been developed to analyze water supply systems and perform hydraulic simulations of their steady state conditions. Commercial hydraulic analysis programs such as EPANET  and WateThe development of such models has played an important role in the design and operation of water supply systems. However, problems related to the selection of the pipe diameter for configuring low-cost water supply systems have emerged as important issues that need to be resolved.In recent years, many optimization methods have been used for the design of low-cost water supply systems. The process for obtaining an optimal water supply system and pipe diameter is considered important because it helps in determining the final operational costs. However, because the aforementioned problems are extremely complex, they are constrained by the types of methods selected for defining the problems, as well as by analysis methods; thus far, only a minimization of the construction costs has been experimentally applied –6.If the structure of a water supply system and its constraints (water pressure and velocity) are known, the optimal design of the water supply system can be expressed in terms of the selection of a pipe diameter that minimizes the total cost. The mathematical optimization methods described earlier can easily be used to find the optimal solutions in small systems within an ideal environment.However, if existing mathematical optimization methods are applied to actual civil engineering problems, the limitations of these methods are revealed. For example, in linear programming, because all functions applied to such problems are linear, simplified assumptions lower the accuracy of the final solution. On the other hand, in dynamic programming, too many combinations have to be considered to obtain the optimal solutions, thereby requiring a considerable amount of computational effort and storage space.In nonlinear programming, if the initial solution is not located at a good position within the solution zone, the global optimum is unobtainable, and the initial solution cannot escape from the local optima. To overcome these disadvantages, during the last 20 years, researchers have attempted to apply new approaches to optimization technologies that do not use an existing mathematical methodology.Rather than relying completely on conventional differential derivatives, the technologies currently being developed apply a natural evolution phenomenon, that is, the principle of the survival of the fittest, and artificial imitations of this phenomena in the optimal system design; these have yielded better results than those obtained using an existing optimal mathematical design.Such nature-inspired optimization algorithms are called metaheuristic algorithms. Some examples of such algorithms include a Genetic Algorithm (GA), Simulated Annealing (SA), Tabu Search (TS), Ant Colony Optimization (ACO), Harmony Search (HS), and Particle Swarm Optimization (PSO). Reca et al. , Monem aHowever, most of these studies have disadvantages in that they were applied to small benchmark problems and were not reflected in the actual plans . The preThe Saemangeum Project is a reclamation project intended to create land from a mud flat and sea waters along the western coast of South Korea by constructing a 33.9 km long seawall. Under the Saemangeum Project, which was started on November 16, 1991, the construction of a cofferdam was completed on April 21, 2006, and the reinforcement and embankment projects were completed on April 27, 2010.2, of which land and a fresh water lake account for 283 km2 and 118 km2, respectively.The Saemangeum seawall is listed in the Guinness Book of World Records as the longest seawall on record and is 1.4 km longer than the Zuider seawall (32.5 km) of the Netherlands, which was earlier regarded to be the longest. The construction of the seawall has resulted in the reclamation of a new region with an area of 401 kmUpon completion of the seawall construction, the South Korean Government newly formulated its Comprehensive Saemangeum Development Plan. According to this plan, the reclaimed land is to be developed as the central agricultural and economic sector of Northeast Asia, with the reclaimed land mainly divided into nine areas, as shown in Agricultural lands account for the largest share (30.3%) among the divided areas. These agricultural lands are used for ensuring national competitiveness, producing high-value-added agricultural products, and developing food-industry facilities through mixed environment-friendly agriculture and ecological crop cultivation. Traditionally, agricultural irrigation water supply systems have been designed as branched water supply networks, which incur less initial costs. However, such networks are disadvantageous in that they do not ensure the reliability of the water supply.In recent years, new cultivation methods such as greenhouse crop cultivation, high-value-added crop cultivation, and perennial cultivation have been adopted. Accordingly, agricultural irrigation water supply systems also need to provide a stable and reliable water supply, as achieved by urban water supply systems.The HS algorithm proposed by Geem et al.  is an opWhen sounds are produced by various sources, they together create a single harmony. Some of these created harmonies sound pleasant, whereas others sound dissonant. Eventually, the discordant harmonies disappear through practice, and among the more appropriate harmonies , those that are aesthetically the most beautiful  are achieved.In other words, the HS algorithm considers an optimal solution to be an optimal harmony found through practice. The principle of the HS algorithm can be explained in detail by first comparing how music improvisation and optimization calculations correspond to each other.χ1, χ2, and χ3 in Improvisation is the spontaneous creation of notes by performers without relying on sheet music (score). The ability of the performers improves the more they perform together, and ultimately a top-level harmony is created. In such an improvisation, each performer  by substituting the value of each variable.The musical range of each instrument  made by the corresponding performer can be referred to as the range of each variable , harmony memory considering rate (HMCR), and pitch adjustment rate (PAR) are important factors in the HS method for finding an optimal solution. First, each musical performer should have a memory space to preserve a good harmony; before starting the important process of the HS algorithm, a harmony memory space is created by consolidating existing memory spaces.This is called the HM, and the maximum number of harmonies that can be stored in this storage space is called the harmony memory size (HMS). Next, to produce better solutions from the harmony storage space, which is initially filled by as many random vectors as the HMS, the HS algorithm employs three types of operators.K is the total number of all possible variable values, one of them is randomly selected, and the probability of this technique being adopted is 1-HMCR.In the random selection technique, the value of a variable is randomly selected from all values of the playable note range. If The memory consideration technique picks the value of a variable from the existing high-quality notes. In other words, a single value is picked from all values possessed by a variable within the storage space. Its probability is HMCR, and although it can have a value between 0 and 1, a value between 0.7 and 0.95 is usually used; nevertheless, the value is changeable.For a pitch adjustment, a note obtained through a memory recall technique is considered a basic note, and its pitch is trimmed by adjusting the note based on the surrounding upper and lower notes. In an actual calculation, when a single value is obtained using a memory recall technique, it is adjusted by a one-step higher or lower value. The PAR is the probability of this technique actually being applied, and it can attain a value between 0 and 1. Generally, the PAR has a value of around 0.01 to 0.3, but this can vary. CC(Di) is cost function (construction cost) per unit length (m) for each pipe diameter, CM(Di) is cost function (maintenance cost) per unit length (m) for each pipe diameter, CP(Di) is cost function  per unit length (m) for each pipe diameter, Li is length of the pipe (m), Di is pipe diameter (mm), and N is total number of pipes.An objective function minimizes the design cost of an irrigation system. The algorithm developed in the present study was applied to an optimization; the construction costs, pipe material costs, and maintenance costs are considered as the design costs according to the pipe diameter. Therefore, the equation for the objective function is as follows:hj is pressure head of each node (m), hmin is minimum pressure head (m), hmax is maximum pressure head (m), vi is velocity of each pipe (m/s), vmin is minimum pipe velocity (m/s), vmax is maximum pipe velocity (m/s), Pj, Pi are penalty functions with regard to the pressure and pipe velocity, and M is total number of nodes.Hydraulic constraint equations are considered in optimization problems. Therefore, a penalty function method is introduced to convert the optimization problem subject to constraint conditions into an optimization that is free from the constraint conditions. The final objective function, which is applied using a penalty function, can be defined as follows:α, β are penalty constants.The above penalty function is applied only when the pressure of each node and the velocity of the pipe exceed either the minimum or maximum value; the equation below represents the penalty function equation applied to the present model. In the target water supply system, the minimum and maximum nodal pressures were set to 10 and 35 m, respectively, and the minimum and maximum pipe velocities were set to 0.01 and 2.5 m/s, respectively:α so that the solution will not be selected. To prepare for a case in which the pressure head and pipe velocity fall short of the design conditions by a small margin, a model that largely satisfies all of the design conditions was implemented by assigning a large value to β. A trial-and-error analysis was conducted using the α and β values for Saemangeum, which is the target area of the present project. The results indicate that an effective optimal design is possible when α and β are assigned values of 10,000,000 and 100,000,000, respectively. But, detailed studies about constraint handling techniques and determination of their parameters should be tackled to improve model efficiency and reliability in future.When running an optimization model, if the pressure head of each node and the velocity of the pipe do not satisfy the minimum and maximum values, which are the design conditions, the penalty cost is increased by assigning a significantly greater value to In the present study, proposal data on the loop-type design of the six zones of Saemangeum were obtained and applied to one of the zones. A diagram of the corresponding water supply network is shown in The data on the cost incurred per unit of pipe length for the different diameter pipes used in this study are listed in The number of decision variables, which should be determined through optimization, is 356 because there are 356 pipelines in the target water supply network. As indicated in The parameters applied in the present program for the Saemangeum target water supply network are listed in These values, which correspond to the optimum results, are adjusted; therefore, the convergence time and efficiency of the optimal solution vary. However, when there are many decision variables, and in such a case, if large HMCR and small PAR values are used, the efficiency of the optimization generally increases.To compare and evaluate the optimization results of the pipe diameters for the initial design, the cost results and hydraulic analysis results of the initial design were first reviewed according to The minimum nodal pressure head is mostly stable at a value greater than 10 m. In the present initial design, a looped network irrigation system is implemented by installing an additional pipeline to a branched network system. In this case, the supply path up to the demand node is determined to be a branched network, that is, only a single type.However, in the initial design, because various supply paths are possible, the head loss is slight, and a water supply is possible through the hydraulically satisfied supply paths, a system that is more hydraulically stable than a branched network system that can be implemented. Thus, because various supply paths are possible in a looped irrigation water supply system, a looped system provides a better water supply than a branched system during abnormal operating conditions such as during an irrigation path failure or closure.The pipe diameter was optimized by considering the pressure and pipe velocity constraint conditions and the HS parameters, which were explained earlier in this study. The optimization results from a cost-effective pipe diameter are shown in The statistical values of the nodal pressure head and pipeline velocities, which are the results of a hydraulic analysis based on cost-effective pipe diameter and the optimal cost results, are shown in Tables The application results indicate that the cost reduction rate of the optimal design was considerably greater (9.08%) than that of the initial design. These results were further analyzed from the viewpoint of current practices that do not employ optimization techniques; this analysis indicates that even without using any optimization technique, branched network systems that do not significantly differ from the optimal designs can be created using the current techniques.However, in the case of a looped network system, such as the water supply network applied in this study, the differences in the results were significant; therefore, it is necessary to determine an cost-effective pipe diameter for the optimization technique based on the results obtained when employing current practices. The hydraulic analysis results indicate that the minimum pressure head (more than 10 m) was mostly satisfied, as observed in the initial design. Furthermore, the statistical values of the nodal pressure head and pipe velocity indicate that the minimum pressure head, allowable pipe velocity, and average pipe velocity all satisfy the economical pipe velocity requirements.In the present study, optimal design reviews of two other design plans in addition to the proposed looped network design plan were conducted. These two design plans are of a branch type and a pump type, as shown in Figures The branch-type water supply network comprises 335 pipelines, with a total length of 37.88 km. The pump-type water supply network comprises 345 pipelines; for the water supplied by the pumping of this irrigation network, the entire area encompassing the six zones was reclassified into four new areas. The total length of the pipelines is approximately 41.39 km.To compare and evaluate the estimation results for the optimal pipe diameter of the three water supply network systems, that is, the loop type (plan 1), branch type (plan 2), and pump type (plan 3), the cost results according to the final optimum pipe diameter and the pipe diameters of the initial plan of each of the three networks are listed in The results indicate that the cost of applying the optimal design was at a minimum for plan 2 and at a maximum for plan 3. This is similar to the trends found in the initial plan. However, an examination of the varying cost rate shows that the cost reduction of the optimal design for plan 2 was 4.19% less than that of the initial plan. On the other hand, the cost increased by 0.72% for plan 3, whereas in the case of plan 1, the cost reduction rate was very high (9.08%). The results for plan 1 show that the reduction rate between the optimal cost and the total length of the pipes is inversely proportional when the pressure head and velocity conditions remain constant. Moreover, a looped irrigation system has many nodes and pipes, which vary hydraulically because pipes of different diameters are used in a pipe system; this proves that it is difficult to design a looped irrigation system economically without using an optimization technique.These results are attributed to the fact that the self-nodal pressure head of the initial version of plan 1 is relatively greater than that of the initial version of plan 2. However, from the viewpoint of current practices, which do not employ optimization techniques, branch-type systems such as plans 2 and 3, which do not differ greatly from optimal systems, can be designed by applying current techniques. In the case of a looped network system such as plan 1, the differences between the results corresponding to the initial and optimal designs were considerable. Therefore, based on the results from current practices, it is necessary to determine a cost-effective pipe diameter using an optimization technique.The results of a hydraulic analysis in which the optimal pipe diameters for plans 1, 2, and 3 were considered are shown in A comparison of the three optimal design types shows that plan 2 (branch type) is the most economic optimal design based only on the criterion of minimum costs. However, because plan 2 does not differ greatly from plan 1 in terms of costs, it is necessary to derive the final design results by considering the hydraulic and maintenance aspects. Plan 1 is a case in which a looped network irrigation system is implemented by installing additional pipelines to plan 2, which is a branched system.If the pipelines supplied up to the demand node correspond to plan 2 (branch network type), the supply path is determined to be of only one type. However, in the case of plan 1, many supply paths are present; the water supply is made possible through the supply paths, which are hydraulically satisfactory. Therefore, plan 1, which is a more hydraulically stable system than plan 2, can be implemented.Thus, the supply of a looped irrigation water supply system during abnormal situations such as an irrigation path failure or closure is better than that of a branched irrigation water supply system because the former has various supply paths. Unlike plans 1 and 2, plan 3 was designed by reclassifying the target pipeline system into four hydraulically independent sections, and water was supplied to each section through pumping heads. By dividing the target pipeline system into four hydraulically independent sections, the fluctuations in the water quantities by each area can be more effectively and reasonably handled, and plan 3 can respond to future pipeline maintenance and expansion plans. However, the increased use of pumps can cause maintenance difficulties and an increase in maintenance costs.In the present study, the HS algorithm, which is one of the latest optimization techniques, was introduced in the design of an agricultural irrigation system, and a corresponding program was developed. The developed program was applied to the actual target area , and the results were presented in this paper. Currently used methods have disadvantages in that the pipe diameter has to be adjusted through a hydraulic calculation of the given water supply network, and this process has to be repeated until satisfactory results are obtained. Unlike this calculation method, the model presented herein yields results that automatically meet the hydraulic conditions through the combined use of the HS algorithm and a hydraulic analysis. Hence, a comparative analysis is simple and effective. The results obtained by applying this method to an actual large-scale water supply network are better than those obtained using existing mathematical algorithms even after considering the nonlinearity, which is inevitable during the analysis. The calculation results of the optimal construction costs and the pipe diameter when applying the proposed model to the actual target region (Saemangeum business area zone 6) indicate that the optimal design results obtained using HS yield much better results (9%) in terms of cost than those of the presently utilized economic pipe diameter calculation techniques. In particular, the optimization technique was found to be more necessary in the optimal design of a looped network irrigation system than for a branched network irrigation system. Furthermore, an examination of the hydrological factors of a pipeline system in which cost-effective pipe diameters were applied showed that based on the statistical values of the head and pipe velocity, the minimum pressure head, the allowable pipe velocity, and the average pipe velocity all satisfy the requirements of an economical pipe velocity. Therefore, if the benefits of the proposed model are proven through application in future systems, it will show the model to be a useful decision-making tool for designing looped network water supply systems.