The Dutch government recently added universal Human Papilloma Virus (HPV) vaccination for 12-year-old girls to the existing national immunization program. The participation rate for the initial catch-up campaign for girls aged 13 to 16 years in 2009 was lower (47%) than expected (70%). To inform future HPV information campaigns, this paper examines the social and psychological determinants of the HPV vaccination intentions of girls aged 13 to 16 years and their mothers who were targeted by the Dutch catch-up campaign of 2009.A random sample of girls and their mothers was chosen from the Dutch vaccination register and received a letter inviting them to participate . In addition, a random sample was recruited via an online panel by a marketing research company . Both groups were asked to complete a web-based questionnaire with questions on social demographic characteristics, social-psychological factors and HPV vaccination intention. Backward linear regression analyses were conducted to examine which social-psychological factors were most dominantly associated with vaccination intention.2 = .01; p = .007), but not significant for daughters  after controlling for HPV vaccination uptake and the sample. In addition, social-psychological determinants largely contributed to the explained variance of HPV vaccination intention of mothers  and daughters . Attitudes, beliefs, subjective norms and habit strength were significantly associated with participants’ HPV vaccination intentions.Data from 952 mothers (14%) and 642 daughters (10%) were available for the intended analyses. The contribution of social demographic variables to the explained variance of HPV vaccination intention was small but significant for mothers (ΔRBecause of the large contribution of social-psychological variables to the explained variance of HPV vaccination intentions among the mothers and daughters, future communication strategies targeting HPV vaccination uptake should address attitudes, beliefs, subjective norms and habit strength. There is a need for longitudinal research to confirm the causality of the association between these determinants and HPV vaccination behavior indicated by this study. Despite a long-standing efficient national cervical cancer screening program for women aged 30 to 60 (uptake 66%) , 600 newIn 2009, a HPV vaccination catch-up campaign was organized for girls born between 1993 and 1996 . As with other NIP campaigns, this campaign was coordinated by the National Institute for Public Health and the Environment (RIVM). The accompanying information campaign consisted of an information pamphlet sent to the home addresses of the girls invited, a website with information about the HPV vaccination for girls and parents, and references to a help line. The Community Health Services, which is responsible for the local implementation of the HPV vaccination, organized local mass vaccination sites for girls.The Dutch NIP, initially implemented in 1999, is one of the most cost-effective public health programs with a consistently high and stable vaccine coverage . About 9In response to this rather low participation rate, research into the social-psychological determinants of the HPV vaccination decision was initiated to provide direction for the improvement of future HPV vaccine communication to girls and parents used by public health officials to improve vaccination uptake. Because dropout after the first HPV injection  was low in 2010 (5%), these insights into the decision to receive the vaccination can be used to improve the total vaccination uptake. This paper reports on the outcomes of our research on the social-psychological determinants of the HPV vaccination intentions of girls and parents who were targeted by the Dutch catch-up campaign in 2009.Because the HPV vaccine has only recently been introduced worldwide, most studies have focused on determinants of future acceptance of the HPV vaccination before implementation -15. ReviEven after implementation of the HPV vaccine, determinants of intention to take the HPV vaccination among girls and parents who do not have to make the decision in the immediate future may differ from those who have actually made the decision and were asked whether they would make it again. Focusing on participants in the latter group is important, because it may improve the explained variance of HPV vaccination intention.The present study is one of the first to examine determinants of the HPV vaccination intentions among participants who made a decision about the HPV vaccination. In addition, this study is one of the first to examine the extent to which determinants contribute to the HPV vaccination intention. The participants were Dutch girls and their mothers who received an invitation for the first catch-up campaign in 2009.Given the young age of the girls involved, most studies acknowledge that parents play a large role in their daughters’ HPV vaccination decision ,16-27. SThis cross-sectional study used a quantitative retrospective approach by means of a web-based questionnaire offered to girls born in 1995 and 1996  who were invited for the catch-up HPV vaccination campaign in 2009 and their mothers. We recruited study participants among this youngest cohort only, because they best resembled the girls who will be targeted by future campaigns. This research conformed to the Helsinki Declaration and to local legislation. The study was exempt from ethical review according to the Independent Review Board Amsterdam, a medical ethics committee acknowledged by the Central Committee on Research Involving Human Subjects (CCMO).The girls and their mothers received information about the study in the invitation letter. Those who chose to participate gave their informed consent by filling out the questionnaire. In the invitation letter and at the start of the questionnaire, participants were assured of their privacy, the confidentiality and security of responses, and informed that they could withdraw their participation at any time. Their responses were anonymized; the researchers were unable to verify participants’ names, addresses and e-mail addresses. Participants accessed the online questionnaire by using a log-in code, a unique number. This number was only used by a third party contact person from Praeventis for the Praeventis sample and an online panel bureau for the online panel sample to send reminders to those who did not respond to the first invitation; see “Participants” section for more information about the samples. Participants who completed the questionnaire were not able to participate for a second time.A flow diagram of the recruitment and response of participants is presented in Figure Participants were invited via Praeventis, the Dutch vaccination register hosted by RIVM. Mothers and daughters that were invited came from the same family unit. A computer program was used to draw a random sample of 6,000 addresses of girls born in 1995 or 1996 that was stratified on known geographical differences in vaccine uptake and ethnicity. Since the addresses of two participants were unknown, the study invitation was sent to 5,998 addresses. The invitation consisted of a single letter addressing mothers of girls born in 1995 or 1996 and the girls themselves. The invitation included a brief description of the study purpose, information about anonymity of participation, a log-in number and an explanation of the log-in procedure.The online questionnaire for the Praeventis sample was hosted on a private part of the TNO website, which was only accessible through use of the log-in number. Computer assisted self-interviewing was used for the online questionnaire and participants were not rewarded for filling out the questionnaire. The questionnaire was open for response from November 30, 2009, until January 10, 2010. Mothers and daughters  who had not responded within three weeks after the first invitation received a reminder. Of the 5,998 invited participants from Praeventis, 564 mothers (9%) and 537 daughters (9%) started the questionnaire, of which 445 mothers (79%) and 404 daughters (75%) fully completed the survey. A large percentage of mothers and daughters who started  failed to complete the questionnaire. Male participants (n = 5) and those with an invalid age (n = 4) were excluded from the analyses. The final response rate was 7% (N = 441) among the mothers, and 7% (N = 399) among the daughters.Due to the Praeventis sample’s limited response to the first invitation letter, we decided to recruit an extra sample by using an online panel of a private marketing research company. A computer program was used to draw a random sample of 650 mothers of girls born in 1995 or 1996, and 350 of their daughters that was stratified on age, education and geographical differences in vaccine uptake. Among the online panel sample, 350 invited mothers and daughters came from the same family unit. They were sent an e-mail that offered the opportunity to complete the questionnaire between December 18 and December 24, 2009. The online panel was responsible for collecting the data among the sample and TNO was owner of the data. Computer assisted self-interviewing was used for the online questionnaire, which was hosted on a private part of the online panel website that was only accessible through use of the log-in number. Participants from the online panel sample were rewarded for completing the questionnaire according to the standards of the online panel company: they received panel points that could be exchanged for gift coupons. The points that each participant received for completing the questionnaire about HPV vaccination were worth 4.35 euros. The response rate of people contacted by this recruitment strategy was 79% (N = 511) among the mothers, and 69% (N = 243) among the daughters.In total, data from 952 mothers (14%) and 642 of their daughters (10%) were available for the intended analyses. The total sample included 497 mother-daughter pairs, and 455 and 145 independent mothers and daughters, respectively.The online questionnaire was based on information derived from former empirical research on general vaccination intention, HPV vaccination intention  as well as the survey ). The percentage of agreement between objective and self-reported vaccination uptake for mothers and daughters from the Praeventis sample was determined by comparing the association between the Praeventis registry information with responses in the questionnaire. For participants recruited by the online panel, HPV vaccination uptake was available only via the survey (“How many HPV injections did your daughter/you receive?” (0 to 3 injections)).Vaccination uptake was assessed by a dichotomous variable representing complete vaccination Social-psychological factors included knowledge, risk perception, attitude, outcome expectations, anticipated regret, beliefs, perceived relative effectiveness of the HPV vaccination, ambivalence, confidence in responsible authorities, subjective norms, descriptive norms, self-efficacy expectations, information processing, habit, decisional conflict , opinions about alternative medicine and past cancer experience . Highest completed educational level for adults was classified as low , intermediate (secondary through pre-university education) or high . For the daughters, attained educational level was classified as intermediate  or low (other).Country of birth was also assessed for mothers and daughters. The variables were classified into four categories which represent the largest ethnic groups in the Netherlands and matched our recruitment strategy: Netherlands, Surinam/Antilles/Aruba, Turkey/Morocco and other countries.Religion was measured by asking participants about their religious convictions . Because a small minority of participants had another religion than Roman Catholic or Protestant, these participants were classified as “Roman Catholic” and people with “no religion” were put in the category “no religion”. This information was further dichotomized into “Protestant” or “not Protestant” because an ANOVA revealed that Protestant participants had a significantly lower vaccine intention than Roman Catholics or people with no religion. Finally, we asked participants if the parents had differences in opinion about the acceptability of the HPV vaccine.In all analyses, education level was treated as nominal, instead of ordinal, because we did not assume a monotonic increasing relationship of education level with the outcome variable “HPV-vaccination intention”. The association between objective and self-reported vaccination uptake  for mothers and daughters was determined by chi-square tests. Differences between participants recruited from Praeventis and the online panel were examined by t-tests or chi-square tests with Statistical Package for Social Sciences (SPSS) 17.0. A two-sided alpha of .05 was used as the criterion for significance.p ≥ .01 was used as the criterion for removal of a predictor in the backwards selection procedure. HPV vaccination uptake, sample, socio-demographic and social-psychological factors were included in the model by the forced entry method to determine their unique explained variance on vaccination intention.HPV vaccination intention was used as the criterion variable for examining the relationship between social-psychological factors and the HPV vaccination decision, as it is consistent with behavioral theories  that explain future behavior via intention, by social-psychological determinants measured at present. We first examined the univariate relationship between social-psychological factors and vaccination intention with simple regression analysis. Because of the large amount of univariate tests, we used a two-sided alpha of .01 as the criterion for significance. Secondly, we conducted a backward linear regression analysis to examine which social-psychological factors were most dominantly associated with vaccination intention. Only significant factors from the univariate tests were included in the regression analyses as predictors in a fourth step, after correcting for HPV vaccination uptake in the first step, sample in the second step and socio-demographic variables in a third step. HPV vaccination uptake was included in the regression analysis as a first step because past behavior best predicts future behavior . A two-sp ≥ .01) from the model, except for HPV vaccination uptake, sample and socio-demographic variables for which differences were found between the two samples. Because we wanted to adjust for the latter variables in the final model, we applied a manually backward selection, instead of an automatic procedure. There was no indication of multicollinearity between variables in the regression model . To further shape future recommendations, Pearson’s correlation coefficients were calculated for the relationship between vaccination intention and individual study measure items that were significantly related to vaccination intention according to the multiple regression model.To select the most important factors, the backward method was used to exclude non-significant variables (two-sided The univariate and multivariate tests were first performed on a random sample of 75% of the participants. Data from the other (25%) participants were used to check for stability and generalizability. The stability check examined which predictors from the final regression model among the 75% sample remained significant in the 25% sample; these were expected to be stable predictors. The generalizability check examined the predictive value of the final regression models for the vaccination intention of the population. This check was conducted to account for overestimation of the percentage of explained variance which mostly occurs in a regression analysis. The goodness-of-fit of the final regression model for the 75% sample was estimated for the 25% sample by keeping the estimated parameters (Betas) of the regression model for the 75% sample. This analysis was performed in R . SubsequIn total, we analyzed data from 952 mothers and 642 daughters. No data were available from participants who did not respond to the invitation. The sample description is depicted in Table The mean age of the mothers and daughters was 43 years (SD = 4.6) and 13 years (SD = 0.5), respectively. Most of the mothers (88%) and daughters (75%) were born in the Netherlands, and 24% and 18% were Protestant, respectively. One quarter of the mothers (26%) had a low educational level. More than half of the girls (58%) attained a low educational level. The sample appeared to represent the general population in the Netherlands with regard to educational level , countryr = .77; p < .001).Less than half of the mothers (45%) and daughters (39%) indicated that they would like or certainly like to receive the HPV vaccination if they were to decide in the future Table . Among tχ2 = 290.56; p < .001; daughters: n = 399; χ2 = 325.02; p < .001), and the percentage of agreement between both outcome variables appeared very large (91% for mothers and 94% for daughters). The correlation between HPV vaccination uptake and future vaccination intention was also significant for mothers  and daughters .Almost half of the mothers (45%) indicated that their daughter received all three HPV injections in 2009, and 51% of the daughters indicated that they had received them. The association between objective and self-reported data on the number of received HPV injections  in the Praeventis sample was high , fewer mothers were born in the Netherlands  and fewer mothers were Protestant . Among mothers, there were no differences between the samples with regard to HPV vaccination intention (p = .95) and vaccination uptake (p = .68).Compared to mothers recruited via the online panel, those recruited via Praeventis were older , were less likely to attain a lower educational level themselves , were less likely to have a mother who was born in the Netherlands , were less likely to have been born in the Netherlands themselves  and were less likely to be Protestant . Among daughters, there were no differences between the samples with regard to age (p = .73), HPV vaccination intention (p = .28) and vaccination uptake (p = .36).Compared to daughters recruited via the online panel, those recruited via Praeventis were more likely to have parents with a lower completed level of education . In addition, the samples (second step) did not significantly contribute to the explained variance of vaccination intention . The additional contribution of socio-demographic variables (third step) to the explained variance of vaccination intention was small but significant  for country of birth. The multivariate relationship between country of birth and intention was not found by the univariate test, which was most likely caused by the small percentage of mothers born in Turkey or Morocco (4%). The social-psychological determinants (fourth step) largely and significantly contributed to the explained variance of vaccination intention  after correcting for HPV vaccination uptake, sample and socio-demographic variables: attitude, anticipated regret when accepting or refraining from vaccination, beliefs, subjective norms, habit strength and decisional conflict (certainty). The relationship between decisional conflict and intention has to be interpreted with caution, because univariate analyses showed the opposite relationship, which indicates a possible suppressor-effect. This suppressor-effect was probably caused by the high correlation between decisional conflict and habit .The unique contribution of HPV vaccination uptake (first step) to the explained variance of vaccination intention was large and significant . The additional contribution of sample (second step) and socio-demographic variables (third step) to the explained variance of vaccination intention was not significant , whereas the additional contribution of social-psychological variables (fourth step) was large and significant . Attitude, beliefs, subjective norms and habit strength and risk perception  significantly and uniquely contributed to the explanation of HPV vaccination intentions.The unique contribution of HPV vaccination uptake (first step) to the explained variance of vaccination intention was large and significant (Rn = 220) and daughters (n = 160). Results have to be interpreted with caution for country of birth because of the small percentage of participants born outside the Netherlands (13% of mothers and 5% of daughters). The outcomes of these stability tests are also presented in Tables The stability of these models was tested on 25% of the total sample of mothers  appeared to be stable factors since they contributed to the explained variance in both the 25% and 75% samples.Beliefs, habit strength and risk perception no longer significantly contributed to the explained variance of HPV vaccination intention. Anticipated regret when refraining from vaccination significantly and positively contributed to HPV vaccination intention for the 25% sample, though it did not for the 75% sample. Attitude and subjective norms appeared stable since they significantly entered the equation in both samples.2 = .80 for mothers, R2 = .72 for daughters). This means that the final models found for the 75% samples appeared to predict to a large extent the vaccination intention of the mothers and daughters from the total population.The generalizability of determinants of HPV vaccination intention was explored by replicating the final regression model found for the 75% samples in the remaining 25% samples. The explained variance of the final models remained high for the 25% samples . Analyses for attitude were not conducted since the items on this scale were considered too general in nature to have practical relevance in this respect.p < .001 unless mentioned) correlated with the HPV vaccination intention of both mothers and daughters  and negative (−3 = completely agree to 3 = completely disagree) beliefs (see Table rMother (rM) = .65 and rDaughter (rD) = .57); “The HPV vaccination was only introduced because the pharmaceutical industry will earn a lot of money from it” (rM = .60 and rD = .40); “Our government is showing responsibility for the health of the Dutch population by introducing the HPV vaccination” (rM = .60 and Dr = .24); “There is too little known about whether the HPV vaccination effectively protects against cervical cancer” (rM = .59 and rD = .53); “There is too little known about the detrimental side effects of the HPV vaccination” (rM = .57 and rD = .42); “My daughter is too young to receive the HPV vaccination” (rM = .57 and rD = .40); and “My daughter does not need to get the HPV vaccination if she is already sexually active” . The belief that a daughter does not need the vaccination because she is not yet sexually active positively and significantly correlated with the mothers’ HPV vaccination intentions (r = .63), whereas this relationship was not significant among daughters . The belief that it is inappropriate that the daughter has to pay for the HPV vaccination if she decides to get the vaccination at a higher age was positively and significantly related to the HPV vaccination intention of daughters (r = .23), while it was not significantly related to the intention of mothers .The following beliefs significantly , general practitioners (rM = .52 and rD = .42), physicians or nurses from the municipal health service (rM = .46 and rD = .41) and the Ministry of Health . This also applied to mothers when they expected a positive influence from their partners (r = .80), daughters (r = .76) and parents (r = .66), and to daughters when they expected a positive influence from their mothers (r = .71) and fathers .Mothers and daughters were significantly more likely to vaccinate against HPV when they expected a positive influence from their friends (rM = .70 and rD = .61), without much thinking .Mothers and daughters were more inclined to accept the HPV vaccination when they perceived it as an automatic event . The item “As regards the HPV vaccination, I was clear about the best choice for my daughter” was borderline significant ; mothers seemed more likely to let their daughter receive the HPV vaccination if they agreed with this statement. The other item on this scale was not significantly associated with the mothers’ HPV vaccination intentions: “As regards the HPV vaccination, I felt sure about what to choose” .Decisional conflict (certainty) significantly contributed to the mothers’ HPV vaccination intention. Mothers were more likely to let their daughter receive the HPV vaccination if they perceived the decision as relatively easy to make . Another insight of this study is that we combined various predictors from social-psychological research used in previous studies in one comprehensive set. As such, we were able to demonstrate which predictors of the set proved to be a sufficient, stable and generalizable combination. This is also one of the first studies that examined the contribution of these determinants to the explained variance of the HPV vaccination decision. This study revealed that HPV vaccination uptake made the largest contribution to the explained variance of HPV vaccination intention. We also found that social-psychological factors were far more important than socio-demographic factors in explaining the HPV vaccination intention. Moreover, social-psychological factors appeared to predict to a considerable extent the HPV vaccination intention of mothers and daughters from the total population.2 = .83 excluding and R2 = .84 including HPV vaccination intake; daughters: R2 = .73 including and excluding HPV vaccination intake). In addition, secondary analyses showed that social-psychological variables explained more than two-thirds of the variance in HPV vaccination intention when HPV vaccination uptake was excluded from the model . These results indicate that our final regression model was sufficient and contained all important social-psychological determinants [HPV vaccination uptake made the largest contribution to the explained variance of HPV vaccination intention . It is a common finding that past behavior is the best predictor of future behavior . Secondarminants .The contribution of social demographic variables to the explained variance of the HPV vaccination intention appeared small (≤ 5%) and inconsistent. In contrast, the contribution of social-psychological determinants to the explained variance of the HPV vaccination intention was considerable . This was an important finding, because the implications for future HPV vaccination communication are best found through these ‘modifiable’ social-psychological determinants of the HPV vaccination decision. Social-psychological factors that were not related to the HPV vaccination intention were, for example past cancer experiences and knowledge. With regard to past cancer experience, results of the present study confirm outcomes from a systematic review on HPV vaccination intention  and a stAttitude, beliefs and subjective norms were the social-psychological factors that appeared to be most strongly and consistently related to HPV vaccination intentions. Attitude played the most prominent role in the explanation of the HPV vaccination intention, confirming previous findings ,18,19,47With regard to subjective norms, the influence of important others on HPV vaccination intention was most dominant for close family members . These results are similar to other studies about HPV vaccination  or the iSocial-psychological factors that were also, but less consistently, related to HPV vaccination intention, were risk perception, anticipated regret, and habit strength. As for risk perception, daughters were more inclined to accept the vaccination if they felt a higher perceived susceptibility to cervical cancer without the HPV vaccination. This was also found by others ,16,17,49With regard to habit, mothers had higher intentions to let their daughters receive the HPV vaccination if they perceived getting the vaccination as something they did automatically or without thinking. This is probably caused by past confidence in the NIP. However, people who perceive the HPV vaccination as something one does automatically, without much thinking, may be more susceptible to counterarguments compared to deliberate decision makers . They shOne important strength of this study is the generalizability of the study results: the social-psychological factors appeared to predict to a large extent the vaccination intention of mothers and daughters from the total population. Another strength was that our final regression model contained all important social-psychological determinants . Also, tThe present study had some limitations that are worth mentioning. First, it was based on a cross-sectional design, which makes it impossible to draw definite conclusions about causality of the associations found. Second, the response rate among subjects from the Praeventis sample was very low (7%). A low response rate (25%) was also found in another study which recruited parents through Praeventis to examine their attitude towards universal vaccination against hepatitis B . The lowWe recommend a longitudinal research approach in order to confirm the causality of the associations between social-psychological factors and HPV vaccination behavior.We recommend future communication addressing social-psychological variables that appear to be related to HPV vaccination . For example, future communication could address important beliefs about the safety of the vaccine, could reduce unrealistic optimism about the expected monogamy of the daughter and could emphasize the importance of receiving the HPV vaccination before daughters become sexually active. Furthermore, future communication strategies about the HPV vaccination should be targeted at daughters and their parents. Both parents appeared to play the most important role in mothers’ and daughters’ vaccination decision making. In addition, we recommend maintaining or improving confidence in authorities involved in the HPV vaccination, for example, by preventing conflict of interest with the pharmaceutical industry, or by communicating about safety as well as risks of the HPV vaccination. Finally, a tailored communication approach should be considered, because it has already been successfully applied to a variety of health-related behaviors, especially in targeting social-psychological variables also found in the present study ,59.This is one of the first studies to examine determinants of the HPV vaccination intention among participants who had actually made the decision about the HPV vaccination in the preceding year. The study revealed that social-psychological factors were far more important than socio-demographic factors in explaining HPV vaccination intention. Attitude, beliefs, subjective norms and habit strength were significantly associated with both the mothers’ and the daughters’ HPV vaccination intentions. Moreover, the generalizability of the study results was adequate. Future communication strategies targeting HPV vaccination uptake should address these determinants.The authors declare that they have no competing interests.HMVK and MF developed and executed the study, with TWGMP, WO, RACR and JVS advising on its development. ED advised on the conceptions of data collection and executed parts of the data analyses. HMVK and TWGMP significantly contributed to the writing of the manuscript, while WO, RACR, MF, JVS and ED were involved in revising it. All authors have seen and approved of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/111/prepub


It is one of the important steps for metagenomic sequence data analysis. Although many methods exist, classification of high-throughput metagenomic sequence data in a limited time is still a challenge. We present here an ultra-fast metagenomic sequence classification system (MetaBinG) using graphic processing units (GPUs). The accuracy of MetaBinG is comparable to the best existing systems and it can classify a million of 454 reads within five minutes, which is more than 2 orders of magnitude faster than existing systems. MetaBinG is publicly available at  The culture-independent metagenomics methods try to sequence all genetic materials recovered directly from an environment. It has the potential to provide a global view of a microbial community There are two major types of computational methods for metagenomic sequence classification: alignment-based and composition-based. Alignment-based methods can determine that a sequence is from an organism only if the source genome or a genome with similar sequence has been sequenced. When the source genome is fully sequenced, alignment-based methods are accurate in general. However, it is difficult for alignment-based methods to do classification when the sequences of the source genomes or closely-related genomes are not available. Unfortunately, this is the case for many metagenomes. It is a significant limitation for alignment-based methods. Composition-based methods, on the other hand, are less accurate but are able to assign every read to a source bin, which can be one or more species, genera or other taxonomy ranks.Advantages of the next-generation sequencing (NGS) technologies such as the high throughput and low cost make them more and more attractive for metagenome sequencing. Among NGS platforms, the 454 sequencers provide the longest reads (up to 400 bps in average), and they can generate more than half a million reads in just one run There are many existing metagenomic sequence classification systems, such as Phymm and PhymmBL Graphic processing units (GPUs) were originally designed to accelerate graphic display only. In the past few years, GPUs have evolved to GPGPUs , which can do general purpose scientific and engineering computing. In many cases, programs implemented on GPUs can run significantly faster than on multi-core CPU-based systems since a GPU may have hundreds of cores. With the success of a parallel programming model called CUDA for GPUs, programming on GPUs for general scientific computing becomes much easier than before In metagenomics, “metagenomic sequence classification” is sometimes distinguished from “metagenomic sequence binning, ” which refers to the grouping of a dataset into subgroups but the subgroups remain unlabeled In this paper, we present a fast metagenomic sequence classification system (MetaBinG) using the power of GPUs. MetaBinG is able to classify accurately a single run of NGS-based metagenome shotgun sequence data in minutes instead of hours or days in a single desktop workstation.ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) on 14 December 2010. With NCBI taxonomy information, 390 genomes were removed to guarantee that every genus has at least two genomes. The remaining 822 genomes belong to 133 genera (534 species). The species in each genus were assigned to training and test groups. All genomes of one species were assigned into a same set, either the training set or the test set. In the end, we generated a training set of 468 genomes  and a test set of 354 genomes   for genomes in the reference database, which makes it very simple to parallelize the computing in a GPU. In practice, this computing is done by matrix multiplication. The matrix multiplication functions are from CUBLAS library, which is a Basic Linear Algebra Subprograms (BLAS) library ported to CUDA (Compute Unified Device Architecture) It may seem inefficient for sequences with lengths shorter than 4http://www.netlib.org, last updated on Jan. 20th, 2011). For the biogas dataset, the GPU version was about 25 times faster than the parallel CPU version (data not shown). Therefore, the speedup of MetaBinG is partially from BLAS library, and partially from GPUs. Meanwhile, the speedup of using GPUs is related to the size of inputs including the number of sequences and the size of reference genome database. The bigger input data sets, the higher speedup it can achieve.In order to check the impact of GPUs on the speed of MetaBinG, MetaBinG and its CPU version were compared. A naive single-threaded version of MetaBinG was implemented without using the BLAS library. K was set to its default value 5. For the same 6,640 input sequences as in In general, MetaBinG is an ultra-fast metagenomic sequence classification system for high-throughput sequence data. We demonstrated that MetaBinG could provide competitive results for sequences with long lengths in a speed 2 orders of magnitude faster. Due to the progress of sequencing technologies, the throughput gets higher and the reads get longer. The demand for a fast tool to analyze a huge amount of metagenomic sequences is constantly increasing. Therefore, MetaBinG can be a useful tool for the metagenomic classification.Latest version of PhymmBL can produce a confidence score for an input sequence at each taxonomy rank, which is very convenient for users to assess the reliability of the classification. MetaBinG has not implemented this though it will be a welcome feature to come in the near future.http://cbb.sjtu.edu.cn/~ccwei/pub/software/MetaBinG/MetaBinG.php. MetaBinG contains a pre-built 5th-order Markov Model for each of the existing 1212 genomes, so users do not need to train the models any more. The file size of current version of the full package is about 17 MB. MetaBinG has been tested on 64-bit Linux OS. One CUDA device is needed and it should be installed appropriately before running MetaBinG. In addition, users can add new Markov models (or genomes to the training set) using the addref.pl script in the package. In the near future, MetaBinG may become more accurate with more genomes available. This ultra-fast tool can be useful for a wide range of related research communities.MetaBinG is publicly available at th-order Markov models is used in MetaBinG. A state in the Markov model is defined as an oligonucleotide of length k, and each state connects to 4 other states. The previous state shares k-1 bases with the next state. Therefore, there are k+14 transitions in total. A genomic sequence under the kth-order Markov model can be viewed as a sequence of state-transitions. The transition probabilities can be calculated for each genome in the training data set according to its Markov model as following:mO and nO are oligonucleotides of length k, m | On)PF(O represents observed count of transitions from mO to nO in a genomic sequence i and m)FP. The high-order Markov models can be pre-computed from genomes in the training dataset. For each sequence, a genome in the database with the minimum score is selected as the source genome. At the end, each test sequence will be annotated with the taxonomy information of its source genome.A short sequence of length k represents the length of oligonucleotides and N′ stands for the number of genomes used for training.The algorithm complexity is determined by the number of genomes in the database and the order of Markov Models. It can be defined as followsiS in equation (2) is calculated by matrix multiplication. First, the transitions generated from each genome in the reference database are converted into a k+14diemension vector. Then, a matrix can be created from all vectors generated from genomes in the reference database. These can be prebuilt. For each short metagenomic sequence, the transitions generated from it are converted into a k+14dimension vector as well. Then, the scores are computed by matrix multiplication, which is done by calling the SGEMM function of CUBLAS library. At the end, the best score is picked and the associated genome is selected as its source genome. These are done by GPUs and the taxonomy information about the source genomes is printed out by CPUs.In practice, the score MetaBinG is implemented in C with CUBLAS library. The system design of MetaBinG is shown in Table S1The Complete list of training and test genomes. We downloaded 1212 fully sequenced bacterial genomes from the NCBI FTP site (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) on 14 Dec 2010. Using the NCBI taxonomy, 390 genomes were removed to guarantee that every genus has at least two genomes. The remaining 822 genomes were assigned to training and test groups. Genomes from a species were assigned to one and only one set, either the training set or the test set. In the end, we generated 468 training genomes and 354 test genomes.(DOC)Click here for additional data file.Table S2Accuracy of Phymm at different ranks. The accuracy of Phymm was reported at different ranks. The data sets, the software and parameters are all the same as in (DOC)Click here for additional data file.Table S3Accuracy of MetaBinG at different ranks. The accuracy of MetaBinG was reported at different ranks. The data sets, the software and parameters are all the same as in (DOC)Click here for additional data file.


Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes.Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates.A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215–221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established.This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner. Human papillomavirus (HPV), a small and nonenveloped double-stranded DNA virus, is established as the key etiological factor in cervical neoplasms HPV infection can be monitored by detection of thirteen high-risk oncogenic HPV types  using a commercially available HPV testing method such as the Hybrid Capture 2 assay , the only HPV assay approved by the US Food and Drug Administration Recently, a high-resolution melting (HRM) analysis method which incorporates double-stranded DNA saturating dye and specifically designed data collection and analyzing software has been developed. HRM analysis was first developed as a closed-tube technology for genotyping DNA variants and mutation screening with advantages over the other techniques such as high-throughput, rapid and non-destructive nature In this study, we reported a novel method based on the use of HRM analysis and unlabeled probes to rapidly identify and differentiate HPV genotypes in clinical specimens. Without multiplexing, HPV genotypes can be completed with one PCR within 3 h from the time of viral DNA isolation.All cervical samples were collected from the Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital with the approval by the Institutional Review Board (IRB 99-0112B) and with informed consent from the patients. The genomic DNAs of 140 consecutive HPV clinical specimens were obtained from the sample bank for this study. The individuals who performed the experiments do not know the genotype of these clinical specimens until completion of HRM analysis.The QIAamp DNA mini kit was purchased from Qiagen . The LightCycler 480 and the LightCycler 480 High Resolution Melting Master were purchased from Roche Applied Science . The T&A cloning vector was purchased from RBC Bioscience . The McTaq DNA polymerase was purchased from One-Star Biotechnology . The UniPOL-Long Range PCR enzyme mix containing Taq DNA polymerase and AccuPOL with 3′→5′ exonuclease activity was purchased from Ampliqon ApS . The EasyChip HPV genotyping system was purchased from KingCar .2), 2 µl of 10 mM dNTP, 0.5 µl of McTaq DNA polymerase (5 U/µl), 2 µl of 5 µM forward primer GAPDH-F, 2 µl of 5 µM reverse primer GAPDH-R  in a 50 µl of reaction containing 5 µl of 10× PCR buffer , 1 µl of 25 mM MgCl2, 2 µl of 10 mM dNTP, 0.5 µl of the UniPOL-Long Range PCR Enzyme mix (5 U/µl), 2 µl of forward primer FRG5 (5 µM), 2 µl of reverse primer FRG2 (5 µM) and 2 µl of template DNA. The PCR condition was 95°C for 5 min followed by 50 cycles of 95°C for 30 sec, 46°C for 40 sec, and 72°C for 30 sec. An additional 3 min of extension at 72°C was performed after the last PCR cycle to replenish PCR product followed by cooling at 4°C. The PCR product was then cloned into the T&A cloning vector as described by the manufacturer's instruction (RBC Bioscience) and was confirmed by DNA sequencing.Partial HPV L1 region was amplified by PCR using the clinical DNA samples of HPV16, 18, 39, 45, 52, 56, 58 and 68 and the primer pair FRG5/FRG2 . Briefly2, 0.8 µl of 5 µM forward primer FRG5, 0.8 µl of 5 µM reverse primer FRG2, and 2 µl of template DNA. The amplification condition was optimized for the use of FastStart Taq DNA polymerase by incubating the reaction mixtures at 95°C for 15 min, followed by 50 cycles of 95°C for 15 sec, 46°C for 20 sec, and 72°C for 30 sec with the ramp to 95°C at 4.8°C/s, to 46°C at 2.5°C/s, and to 72°C at 4.8°C/s. An additional 1 min at 72°C was added to replenish the PCR product.For symmetric amplification of the HPV genomic DNA, broad-range real-time PCR was performed in a 384-well format using LightCycler 480. Briefly, the PCR reaction (20 µl) was composed of 10 µl of 2× HRM master mix, 2 µl of 25 mM MgCl2, 0.4 µl of 2.5 µM forward primer FRG5, 2 µl of 5 µM reverse primer FRG2, 2 µl of 5 µM unlabeled probe for HPV16 and HPV18 when indicated, and 2 µl of template DNA. The unlabeled probe was modified by C6-amine or inverted dT at the 3′-end to prevent the probe from self-extension. The amplification condition was optimized for the use of FastStart Taq DNA polymerase by incubating the reaction mixtures at 95°C for 15 min, followed by 65 cycles of 95°C for 15 sec, 46°C for 20 sec, and 72°C for 30 sec with the ramp to 95°C at 4.8°C/s, to 46°C at 2.5°C/s, and to 72°C at 4.8°C/s. An additional 1 min at 72°C was added to replenish PCR product.For asymmetric amplification of HPV genomic DNA, the PCR reaction (20 µl) was composed of 10 µl of 2× HRM master mix, 2 µl of 25 mM MgClFor HRM analysis, the PCR product was denatured by rising temperature to 95°C at 4.8°C/s and was then cool down to 55°C at 2.5°C/s for hybridization. The melting curve was acquired by increasing the temperature from 55°C to 95°C at a ramp rate of 4.8°C/s with 25 acquisitions per degree of temperature.To assess the combined use of real-time PCR and HRM analysis for rapid detection and differentiation of HPV genotypes, genomic DNA sequences for 8 high-risk HPV genotypes  were subject to bioinformatic analysis. These genotypes represent 98% and 75% of the clinical isolates in Southeast Asia and Europe/US, respectively We determined whether symmetric PCR of the L1 fragment followed by HRM analysis provides distinguishable melting profiles for HPV genotyping. To facilitate the assay, the PCR products corresponding to L1 fragment of various HPV genotypes were subcloned into the T&A cloning vector. These plasmids were used as the template for PCR amplification using the primers FRG5 and FRG2. Despite that most of the HPV genotypes displayed unique HRM profiles, the melting profiles for some HPV genotypes were not distinguishable. For example, HPV18 and HPV45 exhibited almost identical derivative plots and the genotypes were not likely to be determined accordingly .To overcome this problem, we determined whether appropriate use of type-specific unlabeled probe(s) generates HRM profiles sufficient for differentiating HPV genotypes. To facilitate the binding of unlabeled probe to the PCR product, asymmetric PCR was performed to selectively amplify the DNA strain complementary to the unlabeled probe. An unlabeled probe specific to HPV18 was designed  and was The detection limit of this method was evaluated by asymmetric PCR amplification of 10-fold serially diluted plasmid DNA (from 1 pg to 10 ag) that contained 221-bp of HPV18 L1 fragment. The amplification reaction was performed in the presence of 40 ng of human genomic DNA to mimic the co-presence of HPV and human genomic DNA in clinical specimens. As revealed by amplification plots, as little as 10 ag plasmid DNA equivalent to 3 copies of HPV DNA was detectable in this assay condition . The staIt is known that the last 6–7 nucleotides of PCR primers are critical for amplification specificity and efficiency The HRM profiles for 8 high-risk HPV genotypes were then generated by asymmetric broad-range real-time PCR in the presence of the unlabeled probes specific for HPV16 and HPV18. The melting temperatures and melting profiles provide molecular signatures for the 8 high-risk genotypes that can be unambiguously identified through high-resolution derivative plots . When clWe further assessed our method in HPV genotyping of clinical specimens retrospectively. A total of 140 clinical samples from patients who were suspected of having precancerous lesion were blind tested. Due to the nature of sample storage, only 119 of the 140 DNA samples were informative and generated PCR products that were suited for HRM analysis . As reveDuring our analysis of clinical specimens, several low-risk HPV genotypes including HPV42, 62, 70, CP8304, CP8061 and MM8 were found to display their unique high-resolution derivative plots . Of the HPV is well recognized as a major cause of cervical cancer. The development of high grade pre-cancer and invasive cervical cancer could be due to persistent high-risk HPV infection. A rapid, accurate and sensitive method to detect and differentiate HPV genotypes is essential to identify high risk patients who are otherwise found to have normal cytological results and women with cervical cancer potential from the screened population. HPV genotyping provides a reference point for HPV vaccination and HPV prevalence in natural history studies during clinical practice. Unlike hepatitis B vaccine, there is no acceptable antibody test for HPV vaccines. If it is affordable, HPV genotyping assay should be the first choice for HPV testing. In this study, a novel approach for HPV genotyping is developed. This method is based on the combined use of asymmetric broad-range real-time PCR and HRM analysis. Accordingly, 8 high-risk and 6 low-risk HPV subtypes can be identified that offers a novel approach for HPV genotyping.A number of HPV DNA genotyping methods including real-time multiplex PCR, Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR, Roche Amplicor MWP HPV test and Digene HC2 assay have been reported in the literature Multi-infection of HPV strains with various genotypes accounts for approximate 8–22% of the HPV-infected patients In conclusion, the molecular signatures from HRM analysis of the broad-range real-time PCR products are useful for detection and genotyping of HPV infection. This approach can be extended further to cover all the 13 high-risk HPV genotypes. With an appropriate HRM molecular signatures database, this method should allow rapid and cost-effective differentiation of HPV genotypes.Figure S1Effects of human genomic DNA on the amplification of HPV DNA. A–C. Serial dilution of the plasmid harboring the L1 fragment of HPV18 (panel A and C) or HPV68 (panel B and C) corresponding to the indicated copy number of template DNA were subject to amplification by asymmetric broad-range real-time PCR in the presence or absence of 20 ng of human genomic DNA. The copy numbers of the template DNA were plotted against Ct values.(DOC)Click here for additional data file.Table S1The genotypes for the samples with multiple infection.(DOC)Click here for additional data file.


Glossina palpalis palpalis  is one of the principal vectors of sleeping sickness and nagana in Africa with a geographical range stretching from Liberia in West Africa to Angola in Central Africa. It inhabits tropical rain forest but has also adapted to urban settlements. We set out to standardize a long-lasting, practical and cost-effective visually attractive device that would induce the strongest landing response by G. p. palpalis for future use as an insecticide-impregnated tool in area-wide population suppression of this fly across its range.2 targets covered with adhesive film proved to be as good as traps in phthalogen blue or turquoise blue for capturing G. p. palpalis. Trap efficiency varied (8–51%). There was no difference between the performance of blue-black and blue-black-blue 1 m2 targets. Baiting with chemicals augmented the overall performance of targets relative to traps. Landings on smaller phthalogen blue-black 0.25 m2 square targets were not significantly different from either 1 m2 blue-black-blue or blue-black square targets. Three times more flies were captured per unit area on the smaller device.Trials were conducted in wet and dry seasons in the Ivory Coast, Cameroon, the Democratic Republic of Congo and Angola to measure the performance of traps  and targets of different sizes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used as a practical enumerator at these remote locations to compare landing efficiencies of devices. Independent of season and country, both phthalogen blue-black and blue-black-blue 1 m2 cloth targets show promise as simple cost effective devices for management of G. p. palpalis as they can be used for both control when impregnated with insecticide and for population sampling when covered with adhesive film.Blue-black 0.25 m G. p. palpalis is one of the principal tsetse fly vectors of African Trypanosomiasis. Its range stretches from Liberia in West Africa to Angola in Central Africa. G. p. palpalis inhabits tropical rain forest but has also adapted to urban settlements. Reduction of tsetse populations remains one of the most effective methods to control disease transmission to man and animals, and development of visually-attractive insecticide-impregnated traps and targets for palpalis group tsetse dates from half a century ago. Here we describe field experiments made in wet and dry seasons in the Ivory Coast, Cameroon, Democratic Republic of Congo and Angola to establish the most efficient, long-lasting and practical object that induces the strongest landing response in G. p. palpalis. Independent of season and country, both phthalogen blue-black 1 m2 cloth targets covered with adhesive film proved as good as traps in phthalogen blue or turquoise blue cloth when employed as capturing and landing devices for G. p. palpalis. Pyramidal trap efficiency was inconsistent. As landings on 0.25 m2 square phthalogen blue-black targets were not significantly different from landings on the 1 m2 targets, these smaller targets show promise as simple cost effective devices for the management of G. p. palpalis populations. Human and Animal African Trypanosomiasis (sleeping sickness and nagana) are still a major constraint on the social and economic development of sub-Saharan Africa, G. p. palpalis is one of the principal vectors of sleeping sickness and nagana across large areas of central and West Africa. Its geographical range corresponds to the coastal belt of tropical rain forest stretching from Liberia in West Africa to Angola in Central Africa G. palpalis gambiensis in Burkina Faso palpalis and fusca group tsetse. Based on this model, simpler traps, the pyramidal G. p. palpalisG. p. palpalis are obtained on targets made of phthalogen blue cloth with its exceptionally high reflectivity in the blue part of the light spectrum G. p. palpalis than single-colour ones In the face of the continuing difficulties to treat human and animal trypanosomiasis, the reduction and eradication of the tsetse fly vector remains one of the most effective methods to control both diseases. Amongst the different control methods that have been employed, the deployment of visually attractive traps and targets impregnated with insecticide is the most widely used as it is one of the most accessible and efficient methods of control. Historically, the first trapping devices for controlling tsetse were black overalls worn by workers, coated in glue and hung up in the plantations of Sao Tome and Principe in 1910 G. p. palpalis, and to see if their efficiency and cost-effectiveness could be improved. The trials were based on existing trap/target/bait technology used at each location following a similar experimental approach throughout Africa G. p. palpalis for future use in area-wide population suppression of this fly with insecticide-impregnated devices.Within the Africa-wide WHO-TDR initiative to develop innovative control strategies for tsetse, we set out to standardize long-lasting, visually-attractive devices for Studies were conducted in four countries: three in central Africa  and one in West Africa . The site supports intact gallery woodland, surrounded by savannah grassland and bush; there are no domestic animals and the human population density is low but wild animals are still relatively abundant. A first set of field trials took place in 2010 in the wet season (January) and was repeated at the same site in the dry season (June). A second series of trials was conducted in 2010 in the wet season (November) and a third series in 2012 in the dry season (May).One set of field trials was conducted around Bechati near Fontem, in the South-West Cameroon , a hilly region with numerous streams with fragmented indigenous forest and plantations . The local human population density is high and there are many domestic animals. The trials took place in 2009 in the wet season (May) and were repeated at the same location in the dry season (December), but catches in the dry season were too low to be analysed.Hyparrhenia spp. and Panicum maximum grasses with riverside gallery forest, palm oil and coconut plantations. It is an area of intense human activity with numerous free-roaming goats and pigs and is an endemic focus for sleeping sickness. The trials were carried out in 2010 in the wet season; the first set in February and the second set in November.Two sets of field trials were conducted along the Ndongwa and Kamba watercourses near Malanga about 200 km south-west of Kinshasa . The site is in an area of wooded savannah of Two sets of field trials were conducted near Markouguié, Azaguié, 65 km north west of Abidjan  in a hilly region with numerous wet hollows and streams. The area is vegetated by a mosaic of relict indigenous forest and agricultural plantations of bananas, papaya and commercial flowers with livestock rearing  and fish-farming. The first set of trials took place in 2009 in the dry season (December) and was repeated again in 2010, in the wet season (April). A second set of trials was conducted in 2010 in the wet season (November).2 regular square cloth target  on either side of a single blue panel 2 target of equal vertical rectangles of black-blue-black cloth and a reduced regular square target of 0.25 m2 with vertical rectangles of blue and black cloth.Five catching devices were tested: standard biconical (1) C180 phthalogen blue 100% cotton, 180 g/m2, TDV, Laval, France  and referred to here as the standard fabric; (2) S250 phthalogen blue 65% cotton/35% polyester, 250 g/m2, TDV France (peak at 450 nm); (3) turquoise blue Q10067 65% polyester/35% viscose, 234 g/m2, Sunflag, Nairobi, Kenya (peak at 480 nm) and (4) Top Notch 6660-563 blue 100% polyester, 410 g/m2, Rochford Supply, USA (peak at 470 nm). One black fabric  was used for all devices.Four different blue fabrics were tested: G. p. palpalis are caught with traditional e-targets set up on their own.To monitor the numbers of tsetse landing on targets, one-sided sticky adhesive film  was attached to both sides of the targets. This film was also attached to the cloth component of traps in some experiments to enumerate flies that land on traps but may not be captured. To assess the influence of adhesive film, particularly its shininess, on landing responses, the number of flies attracted to non-sticky targets was compared to targets covered with adhesive film by using an electric grid of fine electrocuting copper wires (spaced 8 mm apart) mounted in front and behind the targets A 1∶4∶8 mixture of 3-n-propylphenol (P), 1-octen-3-ol (O), and p-cresol (C) was used as an attractant for experiments comparing baited devices based on general efficacy for several tsetse. The mixture was prepared at origin by the supplier  with a global purity of 98%. Sachets made of 500 gauge/0.125 mm polyethylene containing 3 g of the mixture were placed below the catching devices, 10 cm above the ground, alongside a 250 ml bottle buried up to the shoulders containing acetone (A) with a 2 mm aperture in the stopper. This combination, termed the POCA bait, was made up according to the method described by Torr et al. To assess which was the best catching device and the most attractive blue material, experiments were carried out to compare between four to six devices in a Latin square design of days×sites×treatments, with three simultaneous replicates. Trap positions were always >100 m apart and flies from each device were counted after 24 hours at each position. The various devices and blue materials tested were: biconical traps ; monoconical traps , pyramidal traps (in standard blue cotton or turquoise blue polyester/cellulose or Top Notch blue polyester) and a regular target in standard blue cotton and an Ivorian target in standard blue cotton or S250 blue cotton/polyester. The four to six device experiment (depending on location) was repeated using the POCA bait after the unbaited trial was completed in the same general area, with trapping positions >200 m apart. The objective was to determine whether baiting changed the performance ranking of the devices/fabrics .2 of adhesive film for the pyramidal trap and regular target and 0.9 m2 for the monoconical trap. All devices used to measure landing responses were made of standard phthalogen blue cotton. Flies caught in the cage of the traps with adhesive film on the cloth component were not included in the total for this comparison. Pyramidal and monoconical traps not treated with the adhesive film were included as controls to estimate trap efficiency (percentage flies caught in the control compared to those caught in the cage and on the cloth by the trap with adhesive film). In the DRC and Angola, a three-day experiment was conducted to compare three devices in four replicates. In the Ivory Coast, three devices were compared in four replicates in a six-day experiment  or monoconical (Ivory Coast) traps with sticky adhesive film on the cloth component were compared to targets covered with adhesive film. This gave a surface area of 2 m2 target ; a square 1 m2 Ivorian type target of equal vertical rectangles of black-blue-black cloth and a reduced regular square target  of 0.25 m2 , with no film applied and targets covered on both sides by the adhesive film with the sticky side inwards. The two types of targets were placed within electric grids (above), orientated E-W, and the experiments were conducted following a 2×2 Latin square design of days×sites×treatments, with two replicates, over eight days. The experiments were carried out simultaneously from 10:00 am to 02:00pm each day and trapping positions were always >100 m from one another.To assess whether the addition of the adhesive film could affect the attraction of tsetse to a catching device, a comparison was made in the Ivory Coast between catches of tsetse attracted to a 1 magricolaeIn all trials randomization was set up using design.lsd in the package In the Ivory Coast the target with adhesive film consistently captured significantly more flies than the traps. The better performance of the target was less consistent in the other three countries, where on at least one occasion, the traps performed equally as well as the targets and actually outperformed the target in Cameroon .There was no difference between the performance of the same trapping device made from different blue cloths P>0.05;  with theThe relative rankings of POCA-baited devices were very similar to those in the unbaited trials, but the capture rate on the target covered in adhesive film increased relative to the number of flies caught in the cages of the traps in all countries, most noticeably in the Ivory Coast and Angola . The POCVery similar numbers of flies landed on the traps and targets in Angola and the Ivory Coast and the slight differences recorded are not significant P>0.05; . In cont2 targets in blue-black (regular) and black-blue-black (Ivory Coast style) equal sized vertical stripes covered with adhesive film caught very similar numbers of flies .The daily landing rate of flies on the smaller 0.25 mTrap efficiency, defined here as the proportion of flies caught in the cage of the unaltered trap relative to those caught in the cage and on the cloth by the same trap with adhesive film, has been estimated by dividing the mean daily catch of the unaltered pyramidal and monoconical traps by the mean daily catch of the matching traps with adhesive film on the cloth , reduced by over half the total number of G. p. palpalis that apparently attempted to land on the device. The detransformed catch index compared to the unmodified target is 0.45  G. p. palpalis than black-blue-black and blue-black targets. However, our results imply that G. p. palpalis attraction to targets is underestimated in the presence of adhesive film by up to 50% which would mean that the targets systematically surpass traps as landing devices. It is the landing response that underlies the principle of using insecticide-impregnated targets as control devices for tsetse. To determine whether traps impregnated with insecticide . This confirms observations made by Rayaisse et al. (2010) G. p. palpalis in the Ivory Coast. However, considering the efficacy of smaller targets for G. p. palpalis (see below), one could ask how much effort should one invest in deploying and maintaining chemical baits in control campaigns  when it may be possible to compensate adequately by simply deploying more targets.As the baited and unbaited trials were sequential at each location they cannot be compared directly. Baits were used to see whether they increased trap efficiency as has been shown for other tsetse species G. p. palpalis. These results agree with findings for the same fabrics tested in similar devices for several riverine and savannah tsetse species in East and West Africa ad hoc use of several alternative blue fabrics in tsetse control, some of which are less than optimal for attracting tsetse The blue fabrics chosen for these experiments  were manufactured with differences in fabric texture and with clear differences in blue-green colour, yet with only one exception  all performed equally well in capturing G p. palpalis by half on the 1 m2 blue-black target, accounted for in the main by reduced landings on the black portion of the target. We assume that landings on panels of monoconical and pyramidal traps are affected to the same extent by the presence of the adhesive film. In any case, the surface area of blue and black parts of pyramidal traps and targets covered with adhesive film were the same in these field trials. The two trap types performed equally as well as the target as a landing device in both Angola and the Ivory Coast. In contrast to this, over twice as many flies landed on the target as on the cloth portion of the pyramidal trap in the DRC. This may be partially explained by the behavioural responses of G. p. palpalis as a relatively high proportion of flies were captured in the cage of the adhesive traps in the DRC (62%) as well as in the similarly treated monoconical and pyramidal traps in the Ivory Coast and Angola . This is in contrast to the results of identical experiments conducted on other tsetse species where very few flies flew directly into the cage  and Mramba et al. (2013) G. p. palpalis was the same on the standard blue-black and Ivory Coast type black-blue-black 1 m2 targets covered with adhesive film, with a noticeable preference for landing on the black portion on both targets . These results would suggest that there is little difference between the two target designs to induce landing by G. p. palpalis. In contrast, landing was equally divided between the blue and black panels on the pyramidal trap. However, the trials using electric grids in the Ivory Coast show that numbers of G. p. palpalis landing on the black portion of the targets would be three times higher on unmodified targets and similar results were recorded using the same experimental approach for the closely related G. p. gambiensis in Burkina Faso G. p. palpalis.The 2012 field trial in Angola shows that alighting by G. p. palpalis as has recently been demonstrated for this species in West Africa G. p. palpalis on the 0.25 m2 blue-black target in Angola were not significantly different to those on either the blue-black or blue-black-blue 1 m2 targets covered with adhesive film. In fact, fly catches normalised by unit area were three times higher on the smaller device. This confirms the three-fold higher attraction per unit area recorded for G. p. palpalis to 0.25 m2 black cloth targets over 1 m2 targets of the same colour by Tirados et al. in the Ivory Coast G. p. palpalis and that 0.25 m2 is near the optimum target size. Such devices are also less prone to wind damage and theft because of their smaller size.The 2012 Angolan trial also included a 0.5×0.5 m blue-black target to test if smaller devices could prove effective for G. p. palpalisG. p. palpalis cannot be ascribed to known population structuring in this species across its West and Central African range It is a well-established fact that traps used for tsetse capture only a proportion of the flies that are attracted to their vicinity or that may even land on them G. p. palpalis across the diverse range of natural and man-made habitats this species occupies from West Africa to Central Africa. Targets that attract flies to land on insecticide-impregnated surfaces are most suitable for population suppression of this vector. We have found no significant difference between the performance of regular blue-black and traditional blue-black-blue 1 m2 targets in experiments performed in West and Central Africa. Furthermore, our results show that landings by G. p. palpalis on 0.25 m2 blue-black targets are not significantly different from those on either blue-black or blue-black-blue 1 m2 targets, with three times more flies per unit area on the smaller device. It is thus possible that a number of smaller insecticide-impregnated targets in blue and black could achieve the same result as larger targets in G. p. palpalis control campaigns across its geographical range. However, the most effective size of devices for controlling G. p. palpalis in terms of the costs of fabrication, deployment and maintenance of large targets versus a higher number of smaller targets needs to be determined through field trials. Either phthalogen or turquoise blue cloth would be suitable for these visual control devices.There is a need for reliable and inexpensive devices for population suppression and monitoring of G. p. palpalis population visiting the device. However, for long-term eradication goals, the detection of very low-density residual pockets is also critical and 0.25 m2 targets covered with adhesive film would be a more effective tool, as already been proven in the eradication programme against G. p. gambiensis in the Loos islands (Guinea) .Effective control requires adaptive management


Purpose. To quantify the impact of diagnostic confidence on radiological diagnosis with a fuzzy logic-based method. Materials and Methods. Twenty-two oncologic patients with 20 cysts and 30 metastases ≤1 cm in size found at 64-row computed tomography were included. Two readers (R1/R2) expressed diagnoses as a subjective level of confidence P(d) in malignancy within the interval  rather than on a “crisp” basis ; confidence in benignancy was 1 − p(d). When cross-tabulating data according to the standard of reference, 2 × 2 table cells resulted from the aggregation between p(d)/1 − p(d) and final diagnosis. We then assessed (i) readers diagnostic performance on a fuzzy and crisp basis; (ii) the “divergence” δ (%) as a measure of how confidence impacted on crisp diagnosis. Results. Diagnoses expressed with lower confidence increased fuzzy false positives compared to crisp ones . Crisp/fuzzy accuracy was 94.0%/93.6% (R1) and 94.0/91.6% (R2). δ (%) was larger in the case of the less experienced reader (R2) (up to +7.95% for specificity). According to simulations, δ (%) was negative/positive depending on the level of confidence in incorrect diagnoses. Conclusion. Fuzzy evaluation shows a measurable effect of uncertainty on radiological diagnoses. The confidence underlying medical diagnosis has a pivotal impact on clinical decisions . Based oQuantifying the effect that a diagnostic test has on DC serves as an assessment of the efficacy of that test , 5. To oTo our knowledge, no previous studies compared crisp and fuzzy diagnostic performances. In this study, we assumed that levels of DC in a given diagnosis are equivalent to the fuzzy membership degrees expressing how much that diagnosis belongs to the sets of correct diagnoses. We then adjusted the diagnostic performance of radiologists for the level of DC, based on the method used by Castanho et al. , to “fuzBecause of the retrospective design and the theoretical nature of the study , the approval by Institutional Review Board was not required, according to laws and regulations of our country. However, patients' data were managed according to the ethical principles for medical research as stated by the Declaration of Helsinki, and patients gave informed consent to undergo computed tomography (CT).We searched in our institutional database for all oncologic patients who performed a baseline abdominal or thoracoabdominal CT between March 2009 and March 2010. Of them, we included those presenting up to five focal livers lesions matching the following criteria: (i) a maximum diameter ≤1 cm on axial images; (ii) hypodense appearance on venous and/or equilibrium phases as compared to the surrounding liver; (iii) to have been assessed as metastases or cysts by a consensus panel of two experienced radiologists who reviewed images of baseline and follow-up CTs, magnetic resonance imaging (MRI), and/or ultrasonography (US) examinations. They assessed as metastases those lesions showing any modification in dimensions and attenuation characteristics at CT, whereas lesions remaining stable were assessed as cysts . Alternan = 5), colonic cancer (n = 16), cholangiocarcinoma (n = 3), pancreatic cancer (n = 3), lung cancer (n = 2), and melanoma (n = 1).Final population was represented by 22 patients , who presented 50 focal liver lesions . According to the standard of reference , 30 lesions were metastases and 20 cysts . Of lesions, 5/30 metastases and 2/20 cysts had additional confirmation at subsequent MRI and/or US examination. Primary site of metastases was breast cancer  with 64 sections at a detector collimation of 0.625 mm, a table feed speed of 39.37 mm per rotation, a pitch of 0.98, and a gantry rotation time of 0.5 s. Reconstructed images were displayed as 1.25 and 4 mm thick images. Both image sets were accessible to radiologists for image analysis.All patients received i.v. injection of 600 mg iodine/Kg of nonionic iodinated contrast material at the concentration of 400 mg iodine/mL , using a commercially available power injector . After the acquisition of unenhanced images, a bolus-tracking program  was used to determine the time to initiate diagnostic scanning, after placing a circular Region of Interest in the aorta just above the diaphragmatic dome and fixing a threshold of 70 Hounsfield Units. When the bolus-tracking threshold was reached, triple-phase contrast-enhanced diagnostic scans were acquired with acquisition delays of 35 s for the arterial, 110 s for the venous, and 180 s for the equilibrium phases.The notion of fuzzy set has been introduced by Zadeh  in orderμF(u), which measures the degree of membership of an element u in a fuzzy set F, defined on a referential U. Literature provides at least three different interpretations of the concept of membership function, which are degree of similarity, preference, and uncertainty  (0 ≤ P(d) ≤ 1). Values were limited to the first decimal position, except for 0.51 and 0.49 indicating a P(d) near to the perfect uncertainty (0.50). In accordance with Castanho et al.  operator, within the fuzzy subsets P and N (expressing the test results) and the subsets “test results associated with malignancy” (M) and “test results associated with benignancy” (B), that is,At the end of R1 and R2 readings, fifty couples of n et al. , the 2 ×PM, PB, NM, and NB corresponded, in the 2 × 2 table, to those of fuzzy true-positive (fTP), fuzzy false-positive (fFP), fuzzy true-negative (fTN), and fuzzy false-negative (fFN) cases, respectively. It has been demonstrated elsewhere [Tables lsewhere  that gloP(d) or N(d) was equal to or larger than 0.51, respectively. In the case of perfect uncertainty (P(d) = 0.5), we assumed a crisp diagnosis of metastasis.Second, we calculated per-lesion sensitivity, specificity, positive-predictive value (PPV), negative-predictive value (NPV), and accuracy for malignancy (together with 95% C.I.s) (i) on a crisp basis, that is, by assuming that 2 × 2 table cells were filled with mutual exclusive 1/0 values; (ii) on a fuzzy basis, that is, by using fTPs, fFNs, fFPs and fTNs resulting from the above method , 14. Criδ between fuzzy and crisp proportions, calculated as follows:Finally, we assessed the impact of DC on diagnosis as the “divergence” δ is here assumed to represent the “error” inherent to crisp values of diagnostic performance. In other words, δ measures how much crisp evaluation differs from “the real state” of uncertainty (unexpressed in crisp setting) with which radiologists achieve a diagnosis, that is, the “real” diagnostic performance as adjusted for DC levels.In the classical crisp approach for diagnosis, the reader is forced to press his own uncertainty  on a “flat” dichotomic basis 0-1. This leads to a skewed building of the 2 × 2 table based on the standard of reference. So the divergence P(d) of 0.9 for TPs. On the contrary, DC for incorrect diagnoses was simulated as follows: (i) scenario 1, in which R3 showed high DC levels, with P(d) of 0.9 for FPs; (ii) scenario 2, in which R3 showed lower DC levels, with P(d) of 0.7 for FPs.We supposed that one reader (named R3) had examined CT images, showing crisp sensitivity, specificity, PPV, and NPV 66.7% (20/30), 50.0% (10/20), 66.7% (20/30), and 50.0% (10/10), respectively . DC for correct diagnoses was assumed to be high, corresponding to δ between crisp and fuzzy proportions.Based on simulated membership degrees, we estimated fuzzy sensitivity, specificity, PPV, NPV, and accuracy (on a per-lesion basis) for both scenarios, together with the divergence P(d) and N(d) ranged from 0.8 to 1 in 27 and 2 patients, respectively (P(d) = 1) and one incorrect diagnosis (N(d) = 1). However, since P(d) for the one FN diagnosis was 0.2  = 0.8), fTPs (28.2) and fFNs (1.8) results slightly increased and decreased compared to crisp ones, that is, 28.2 and 1.8 versus 28 and 2, respectively  = 0  = 1) to 19 of 20 of them, but (s)he was less confident in one single diagnosis of benignancy (P(d) = 0.2). Compared to the crisp scenario, the effect was to slightly increase the FPs (from 0 to 0.2) and decrease the TNs (from 20 to 19.8) (P(d) = 0, i.e., N(d) = 1). Concerning the remaining 7 diagnoses, P(d) ranged between 0.1 and 0.3, leading to an increase in FPs (from 1 to 2.4) and a decrease in TN (from 19 to 17.6) compared to the crisp setting  . R2 made setting .δ (%) values are reported as well. In the case of R1 crisp diagnostic performance was slightly “overestimated” as compared to fuzzy ones in terms of specificity, PPV, NPV, and accuracy. In other words, positive δ (%) values indicate that crisp performance was higher than that adjusted for the level of DC, that is, when R1 was not forced to express diagnoses dichotomically. The effect of DC level was more complex for R2, showing that the crisp performance was (i) slightly “underestimated” in the case of sensitivity and NPV and (ii) more largely overestimated in the case of the remaining estimates, especially specificity  = +7.95%).When estimating sensitivity, specificity, PPV, and NPV with the above fuzzy values, variations with respect to crisp proportions occurred as shown in Difference between crisp and fuzzy proportions was not statistically significant , as arguable from the overlap in 95% C.I.s.δ  (%) = 0).Results of simulations are shown in δ (%) as low as −16.7% in the case of specificity). It is easy to demonstrate that δ might have assumed a positive sign by entering less incorrect diagnoses in the model.On the contrary, lower level of DC in incorrect diagnoses led to a decrease of crisp performance in the second scenario , as arguable from the overlap in 95% C.I.s.Fuzzy logic has been used in medicine to define complex epidemiological and clinical models for health decisions or risk prediction . ApplicaP(d) ranging from 0 to 1, based on the degree of DC at a single-time reading. A simplified clinical model was set for this purpose, providing one complementary alternative only (N(d) = 1 − P(d)) to main diagnosis . Both radiologists were shown to be highly confident in making diagnoses, regardless of their correctness: they used P(d)/N(d) values of 1 or 0 in the majority of cases . The degree of DC was relatively high even in more uncertain cases, corresponding to P(d)/N(d) no less than 0.8 for R1 and 0.7 for R2. Moreover, R2 showed average lower DC compared to R1, as expected on the basis of differential effects of experience  [Given the above premises, we asked two radiologists to quantify the diagnosis of liver metastasis as a whichever value , resp.) . In summδ between fuzzy and crisp sensitivity, specificity, PPV, and NPV. In other words, we measured how much crisp performance is affected by levels of DC underlying radiological diagnoses, that is, how much information do diagnoses carry in the light of their subjective origin. Positive δ values indicate that crisp diagnoses are overestimated as compared to fuzzy ones, because of lower levels of DC. In the case-study, R2 showed larger positive δ (up to +7.95% in the case of specificity) compared to R1. One can suppose that a clinician aware of δ values would probably find R1 diagnoses more informative than R2 ones and would probably change (or not) patients management based on the level of DC underlying radiological diagnoses, for example, by adding (or not) further diagnostic procedures before any treatment. It should be pointed that the estimate of δ refers to a set of readings given the “truth” established by a standard of reference, that is, to the body of evidence provided by one radiologist on a large series of readings, as for example, occurs in an experimental study. The effect on the referring physician of expressing each single diagnosis as a fuzzy value in clinical practice, where the “truth” about the patient just comes at the end of the work-up process, should be a matter of a specific study, and its estimate was beyond the purpose of our work. In the case of negative values, δ expresses how much crisp diagnosis is underestimated as compared to fuzzy one. This occurs when incorrect diagnoses are expressed with lower levels of DC, as shown in the case of R2 in the clinical setting. Since N(d) for one FN negative diagnosis was 0.8, the number of fNs slightly decreased compared to crisp ones . Accordingly, δ for sensitivity and NPV was −0.71 and −0.27, respectively. This effect was emphasized by supposing more incorrect diagnoses with low DC, as we did in the second simulation. The diagnostic performance of the simulated reader (R3) was systematically lower on a crisp rather than a fuzzy basis, that is, when accounting for DC levels (up to −16.67% in the case of specificity). Thus, in a context of low accuracy readers should be unconfident in incorrect diagnoses in order to be more efficacious. On the other hand, high DC in incorrect diagnoses in the first simulation had the main effect to increase diagnostic accuracy (up to 5.26% in the case of sensitivity). In other words, DC level and δ are able to communicate how confidently wrong diagnoses impact readers performance, leading to potential consequences in terms of clinical practice or evaluation of study results. Finally, δ, equal to zero when the algebraic sum of fuzzy and crisp elements in the 2 × 2 table is equivalent, as occurred for TP/fTP and TN/fTN cases of R1 in the clinical scenario (crisp and fuzzy sensitivities of 90.0% each) and FP/fFP and TN/fTN cases of R3 in the first simulation (crisp and fuzzy sensitivity of 50.0%). δ might be zero also in the case readers would express all correct and incorrect diagnoses with P(d) = 1 and N(d) = 0, that is, in the case fuzzy membership degrees would correspond to crisp ones. We did not observe this result in our clinical series, confirming that readings of an experimental set are unavoidably affected by uncertainty, even if low. Accordingly, fuzzy diagnosis is expected to be more informative than crisp one.DC has been advocated to impact on diagnostic efficacy . It is rδ potentially related to our series. Of note, we did not aim to test diagnostic accuracy of fuzzy evaluations per se but rather to verify whether effects of uncertainty are measurable in terms of δ. For this purpose, we used a simple, dichotomic model, regardless of its clinical importance. It is reasonable to infer that differences between crisp and fuzzy sensitivity, specificity, predictive values, and accuracy would be larger and statically significant in the case of more complex clinical scenario . Nonetheless, the absence of statistical significance does not imply a lack of clinical significance, as arguable from large δ values in R3 simulations. Second, one might argue that two readers only might have been insufficient to achieve adequate statistical power in our analysis. However, we did not mean to estimate a somewhat variant of inter- or intrareader agreement and to establish how precise the estimate was. Divergence consists in the algebraic difference between crisp and fuzzy diagnostic performance as determined by readers' DC (see above); thus statistical power has no influence in its determination. Third, we gave emphasis to the concept of δ in this study. One might argue that area under the curve (AUC) at receiver operating characteristic (ROC) analysis is more helpful in assessing uncertainty than δ. However, the role for fuzzy ROC curves in refining crisp evaluations was investigated elsewhere [Our work has some limitations. First, although differential diagnosis between small cysts and metastasis is relatively easy during imaging and clinical followup of oncologic patients, no histological examination was available to establish final diagnosis. However, general assumptions and applicability of our model do not depend on the presence of incorrect final diagnoses and subsequent variations in lsewhere . Our aimlsewhere  is appliδ between fuzzy and crisp diagnostic accuracy as a measure of the effect of DC levels on the nominal, crisp diagnostic performance . Potential applications of our method are in the context of clinical practice and/or experimental studies, and should be a matter for targeted investigation: first, by adjusting test results for readers' subjective interpretation; second, by testing the efficacy of readers involved in a study; third, by showing whether confidently incorrect diagnoses significantly affect readers' interpretation and fourth, by evaluating how training or educational programs improve the knowledge of less experienced radiologists. We expect that major application for this method would be—at least initially—in the setting of experimental studies. From the practical point of view, the estimate of δ can be easily performed using a software program (see our website http://trm.twilightsubnet.com/fuzzy.php/). According to our results, ideal radiologists  should be accurate, highly confident in correct diagnoses, and highly unconfident in incorrect diagnoses, leading to a δ close to zero.In conclusion, we tested a fuzzy logic-based method accounting for readers' uncertainty (DC levels) on a real, simplified clinical scenario and two simulations using CT. According to this approach, a diagnosis can belong to positive or negative test results at the same time, differently from what occurs when considering diagnosis with binary  logic. By operating with the 2 × 2 table cells as fuzzy subsets, we calculated the divergence


To the editor:The original research article by Mills et al  providesUnpublished data from our group suggest that lack of knowledge about HPV may play a crucial role in shaping Latinas’ use of cervical cancer preventive health service. Using a convenience sample of Latina women recruited from 2 clinics serving indigent populations in 2009, we performed descriptive analyses of an HPV knowledge survey. The number of correct responses to 5 questions acted as a measure of knowledge.A total of 86 Latina women participated: 80% were of Mexican origin. Among the participants, 54% correctly identified HPV as the most common sexually transmitted infection (STI), 54% responded incorrectly that herpes was the most common STI, 74% correctly responded that HPV causes cancer, 29% correctly answered that HPV can cause penile cancer, and only 12% responded correctly that HPV may not have symptoms.P = .005). In addition, scores were higher for those who feel they speak English better than Spanish (P = .06), those who read or speak primarily in English (P = .03), those who think primarily in English (P = .04), and those who speak in English with friends (P = .05).Chi-square analyses revealed that those who already had received the vaccine scored higher on the knowledge questions (P = .005), those who think in English (P = .04), those who feel they speak English best (P = .008), and those that speak in English with friends (P = .03) were more likely to have received the vaccine.Further examination found that those who speak or read primarily in English (Most of this sample of underserved Latinas correctly responded that HPV is associated with cervical cancer. The more knowledgeable were the more vaccinated. However, the highest level of knowledge was associated with those who are comfortable with the English language.Our findings echo what was found with young Appalachian women: there is a knowledge gap among HPV-unvaccinated women. We agree that educational interventions for at-risk populations are needed. However, many distinct populations are vulnerable to HPV and cervical cancer, and the complexity and cost of customizing a campaign for each group may prove too expensive. The question is how to effectively reach these populations in a cost-effective manner, transcending the differences among them. Lay health workers, peers, and others who are trusted within the community could offer education about cervical cancer, HPV, and prevention. Pharmacies in neighborhoods with high proportions of vulnerable women could act as nontraditional HPV vaccination delivery sites, overcoming barriers such as limited free time and difficulty with transportation. Pediatricians’ offices could be universally equipped to offer HPV vaccines to mothers. Beyond applying some of the population-specific strategies mentioned by Mills et al, we recommend universal implementation of aggressive, unconventional methods to decrease disparities in vaccine uptake and the disproportionately large burden of cervical cancer faced by underserved women nationwide.Anna M. Acosta, MDEmory University School of Medicine49 Jesse Hill Jr Dr SE Atlanta, Georgia 30333Loida E. Bonney, MDEmory University School of Medicine49 Jesse Hill Jr Dr SEAtlanta, Georgia 30333Michael Fost, MScEmory University School of Medicine49 Jesse Hill Jr Dr SEAtlanta, Georgia 30333Victoria L. Green, MDEmory University School of Medicine49 Jesse Hill Jr Dr SEAtlanta, Georgia 30333Carlos del Rio, MDEmory University School of Medicine and Rollins School of Public Health of Emory University49 Jesse Hill Jr Dr SEAtlanta, Georgia 30333