This investigation seeks to help fill this gap by quantitatively determining whether a random sample of underage (n = 158) and legal (n = 298) drinkers differed with regard to their alcohol-related behaviors, responsible drinking behaviors, and responsible drinking beliefs.To date, scholarly discourse over the Amethyst Initiative has primarily debated the relative effectiveness of the 21 year-old Minimum Legal Drinking Age (MLDA). Unfortunately, this discourse has failed to account for the Amethyst Initiative’s central tenet/mission: p < .003; d = -0.297], (b) significantly more perceived barriers to responsible drinking , and (c) significantly lower behavioral intentions to perform responsible drinking behaviors the next time they consumed alcohol . Each of these differences remained statistically significant, even after controlling for sex and race, in three separate multiple linear regression models.Compared to legal drinkers, underage drinkers reported: (a) significantly less confidence to perform responsible drinking behaviors during their next drinking episode [t(446) = -2.97, While college students both above and below the 21 year-old MLDA have similar beliefs regarding what constitutes responsible drinking, students below the current MLDA have less intention to drink responsibly regardless of their behavioral beliefs and/or motives. College/university administrators should consider the negative repercussions that are possible if underage students who are less confident in their ability to drink responsibly are given the legal right to drink on campus. Amethyst Initiative, this proposal supports a series of educational and policy level efforts to enable 18–20 year old adults to purchase, possess, and consume alcoholic beverages at their own discretion.Debate over the United States’ minimum legal drinking age (MLDA) has recently erupted -4. Despifacilitating responsible drinking among college students. There is a dearth of literature investigating differences in how underage and legal drinkers practice and/or conceptualize responsible drinking behavior(s) specifically, yet studies have explored other aspects of responsible drinking among college students , age , Greek (fraternity/sorority) status , full-time student status , binge drinking status , or the number of days in which alcohol was consumed in the past 30 days . Consequently, incomplete surveys were retained for analysis.Only a small percentage of respondents had missing data on any of the subscales (2.0% of d) were computed for all statistically significant mean differences. All between-group differences found to be statistically significant were further explored in multiple linear regression models, controlling for sex and race/ethnicity.Among underage and legal drinkers, independent sample t-tests were performed to compare mean scores on the continuous alcohol-related behaviors and 5 subscales of the CHORDS. Effect sizes  and legal  drinkers completed the web-based survey. Participants were primarily full-time students (93%) who were Caucasian (79.3%) and female (54.5%), with a mean age of 22 years (SD = 5.47 years). Approximately 11% of respondents were members of a fraternity or sorority, and the majority (78%) resided in off-campus housing or an on-campus residence hall (18%). There was an equal representation (~ 18% per each year in school) across all student classifications . Both gender and ethnic distributions of those surveyed were comparable to the institutional population from which the sample was drawn .459 self-identified underage  and legal drinkers  consumed similar amounts of alcohol the last time they were in a social situation . Likewise, underage drinkers  and legal drinkers  reported comparable rates of binge drinking . Compared to legal drinkers , however, underage drinkers  reported significantly less self-efficacy to perform responsible drinking behaviors during their next drinking episode. The magnitude of difference  in self-efficacy  was relatively small, however (Cohen’s d = -0.297). Underage drinkers  also reported significantly more perceived barriers to responsible drinking compared to their of-age counterparts . The magnitude of difference in barrier means between groups  was medium (Cohen’s d = .368). Moreover, compared to legal drinkers , underage drinkers  also reported significantly lower behavioral intentions to perform responsible drinking behaviors the next time they consumed alcohol . No statistically significant differences were reported between groups on responsible drinking behavioral beliefs or motivations.Table Table Amethyst Initiative continues to attract supporters and attention [Despite a lack of empirical evidence suggesting that lowering the MLDA will curb binge drinking among college students,  the Ametttention . Underagttention , and heattention . Future Amethyst Initiative, including an increased social availability of alcohol (campus wetness) that will likely overshadow any anticipated benefits stemming from allowing those 18 and older to consume alcohol legally [Current evidence suggests that age-based restrictions on access to alcohol have substantial impact on alcohol consumption, such that the MLDA clearly reduces alcohol consumption and its associated harms . Grantin legally . Therefo legally  warning The authors declare that they have no competing interests.AEB conceptualized the manuscript and prepared the first draft of the manuscript. MLS and CW contributed to the introduction and review of literature. MLS consulted on the statistical analysis. AEB, MLS and CW contributed to the conceptual development of the discussion and conclusion section. Both MLS and CW reviewed the study design, statistical approaches, and each successive draft of the manuscript. All authors contributed to the overall construction of this manuscript, read, edited, and approved the final draft of the manuscript. AEB takes responsibility for the paper as a whole.The findings reported herein build upon AEB’s previous work, which examined how college students interpret and practice “responsible drinking”. As readers will note, the responsible drinking dimensions reported herein, and elsewhere, echo those of previous investigations focusing on protective behavioral strategies (PBS) drinkers employ to stay safe while drinking. Thus, this manuscript contributes to both the scant literature base focused on responsible drinking, as well as the larger burgeoning literature base focused on harm reduction strategies.


Malnutrition is associated with adverse outcomes for hospital inpatients and is a significant economic burden on hospitals. Malnutrition is frequently under-recognised in this setting and valid screening and early diagnosis are important for timely nutritional management. Aboriginal Australian and/or Torres Strait Islander peoples  are likely to be at increased risk of malnutrition due to their disproportionate burden, pattern and age-distribution of chronic diseases. Despite this increased risk, the burden and impact of malnutrition in Indigenous Australians is poorly understood. Furthermore, a suitable screening tool has not been validated for this vulnerable patient group. The aim of this study is to determine the burden of malnutrition, understand its impact, and validate a malnutrition screening tool for Indigenous Australian inpatients.This project involves cross-sectional, prospective cohort and diagnostic validation methodologies to assess the burden and impact of malnutrition and to validate a malnutrition screening tool. A target of 752 adult Indigenous and non-Indigenous Australian inpatients will be recruited across three different public hospitals in the Northern Territory and far north Queensland of Australia. Cross-sectional data collection will be used to determine the prevalence of malnutrition using the Subjective Global Assessment and to stratify participants based on the International Consensus Guideline Committee malnutrition aetiology-diagnostic framework. Subjects will then be followed prospectively to measure short and long-term health outcomes such as length of hospital stay, in-hospital mortality, 30-day and 6-month readmission rates. Finally, the utility of a new screening tool, the Australian Nutrition Tool, will be assessed against an existing screening tool, the malnutrition screening tool, used in these settings and the malnutrition reference standard, the Subjective Global Assessment.Indigenous Australians continue to experience poorer levels of health than non-Indigenous Australians and issues such as food insecurity, poor diet, and a disproportionate burden of chronic disease play a key contributing role for malnutrition in Indigenous Australians. To improve the health and hospital outcomes of Indigenous and non-Indigenous Australians, it is important that patients are routinely screened using a validated screening tool. It is also imperative that the burden and impact of malnutrition is properly understood, and fully appreciated, so that early and appropriate nutritional management can be provided to this group of hospital patients. Under-nutrition is a term used synonymously with protein-energy deficiency, where there is a loss of body cell mass with signs of subcutaneous fat loss and skeletal muscle wasting and therefore  as any “any nutritional imbalance” , the prevalence of PEM in adult inpatients has been reported as ranging from 17 to 58 %  at 2015 valuations ; clinical assessment of weight history (i.e. loss of weight); changes to food intake ; gastrointestinal signs and symptoms ; functional capacity (i.e. ability to complete day-to-day activities); a physical assessment for signs of subcutaneous fat loss and skeletal muscle wasting; anthropometric measures such as BMI; hand-grip strength; and blood chemistry data  or obese (BMI ≥ 30.0 kg/m2) compared to non-Indigenous Australians  is a factor in the prevention and management of chronic disease for Indigenous Australians  using the MST and ANT. The ANT was developed with the involvement of dietitian experts from each of the three study sites, Indigenous and non-Indigenous health providers , and academic experts.Following screening, all participants, irrespective of MST and ANT scoring, will have a diagnostic assessment for malnutrition using the SGA  on length of hospital stay, in-hospital mortality, discharge destination, and 30 days and 6 months readmission and survival using hospital and jurisdictional death registry data.Comorbidities, the severity of the presenting illness and other contributors to health outcome will be controlled for using the Charlson Comorbidity Index  to identify suitable scoring cut-offs and to determine overall utility as a screening tool in this setting.The sample size required to detect a 10 % absolute difference in the prevalence of malnutrition in Indigenous as compared with non-Indigenous Australians . Descriptive analysis of participants will utilise standard univariate techniques and will be reported as percentages with 95 % confidence intervals (95 % CI), means with standard deviations (SD) or medians with interquartile range depending on the data format and distribution. Comparisons between Indigenous and non-Indigenous participants will be undertaken using χ2 for categorical data and Student’s t Test or Mann–Whitney U test for continuous normally distributed data or non-normally distributed data respectively. A p value <0.05 will be taken to indicate statistical significance and all tests will be two-sided. The utility of the MST and ANT to detect PEM as measured by the SGA will be investigated using diagnostic test analysis including determining sensitivity and specificity, and positive and negative predictive values. Optimal scoring cut-offs for the MST and ANT will be determined using receiver operator characteristic (ROC) analysis.Data will be analysed using StataThe impact of PEM on the outcomes listed above will be first assessed using bivariate analysis for length of hospital stay, in-hospital mortality, discharge destination, 30 days and 3 months readmission. Survival to 6 months post-discharge will be presented using Kaplan–Meir curves and analysed using the log rank test to compare survival in patients with and without PEM, and in Indigenous and non-Indigenous Australians.Multivariable linear, logistic and Cox proportional hazard models will be developed to identify independent factors associated with outcome measures. These will use a backwards stepwise approach including in the first model all factors associated with a particular outcome variable using bivariate analysis with a p value <0.1. Factors with a p value ≥0.05 will be progressively removed from the models starting with those variables with a regression co-efficient closest to 0 or an odds or hazard ratio closest to 1. Final models will be limited to predictive factors with significant coefficients (p < 0.05).Diagnostic validation analysis will first involve the assessment of concordance between the MST and ANT by the technique of Lin  that assThis project has been approved by the Central Australian, Menzies, Far North Queensland, and Monash University Human Research Ethics Committees.The study outlined in this paper aims to provide a new and detailed perspective on the burden, impact and diagnosis of PEM in a high income country setting and in an underserved population, in this instance, Indigenous Australians. The prevalence of PEM and those at-risk of malnutrition is significant in hospital inpatients (Allard et al.


Swaziland is the first country to introduce national early infant male circumcision (EIMC) into voluntary medical male circumcision (VMMC) programming for HIV prevention. With more than 5,000 EIMCs performed between 2010 and 2014, Swaziland learned that EIMC requires inclusion of stakeholders within and outside of HIV prevention bodies; robust support at the facility, regional, and national levels; and informed demand. Expansion of EIMC and VMMC has the potential to avert more than 56,000 HIV infections in Swaziland over the next 20 years. Swaziland is the first country to introduce national early infant male circumcision (EIMC) into voluntary medical male circumcision (VMMC) programming for HIV prevention. With more than 5,000 EIMCs performed between 2010 and 2014, Swaziland learned that EIMC requires inclusion of stakeholders within and outside of HIV prevention bodies; robust support at the facility, regional, and national levels; and informed demand. Expansion of EIMC and VMMC has the potential to avert more than 56,000 HIV infections in Swaziland over the next 20 years. The government of the Kingdom of Swaziland recognizes that it must urgently scale up HIV prevention interventions, such as voluntary medical male circumcision (VMMC). Swaziland has adopted a 2-phase approach to male circumcision scale-up. The catch-up phase prioritizes VMMC services for adolescents and adults, while the sustainability phase involves the establishment of early infant male circumcision (EIMC). Swaziland does not have a modern-day tradition of circumcision, and the VMMC program has met with client demand challenges. However, since the launch of the EIMC program in 2010, Swaziland now leads the Eastern and Southern Africa region in the scale-up of EIMC. Here we review Swaziland’s program and its successes and challenges.From February to May 2014, we collected data while preparing Swaziland’s “Male Circumcision Strategic and Operational Plan for HIV Prevention 2014–2018.” We conducted structured stakeholder focus group discussions and in-depth interviews, and we collected EIMC service delivery data from an implementing partner responsible for VMMC and EIMC service delivery. Data were summarized in consolidated narratives.Between 2010 and 2014, trained providers performed more than 5,000 EIMCs in 11 health care facilities in Swaziland, and they reported no moderate or severe adverse events. According to a broad group of EIMC program stakeholders, an EIMC program needs robust support from facility, regional, and national leadership, both within and outside of HIV prevention coordination bodies, to promote institutionalization and ownership. Providers and health care managers in 3 of Swaziland’s 4 regional hospitals suggest that when EIMC is introduced into reproductive, maternal, newborn, and child health platforms, dedicated staff attention can help ensure that EIMC is performed amid competing priorities. Creating informed demand from communities also supports EIMC as a service delivery priority. Formative research shows that EIMC programs should address the fears and anxieties of parents so that they, especially fathers, understand the health benefits of EIMC before the birth of their babies.The vast majority of public-sector facilities in Swaziland are led by nurses, and nurses and midwives have borne the brunt of caring for patients with HIV/AIDS in Swaziland. Like prevention of mother-to-child transmission, EIMC provides an opportunity for nurses and midwives to stand at the forefront of HIV prevention efforts. Rapid scale-up of VMMC and EIMC in Swaziland has the potential to avert more than 56,000 HIV infections and save US$370 million in the next 20 years. The hospital introduced the pilot to strengthen delivery of maternal and newborn health services. Data from the RFM Hospital pilot demonstrated the feasibility of implementing EIMC as an integral part of maternal and newborn care services in Swaziland. Factors contributing to the successful adoption of EIMC at RFM Hospital included hospital ownership of the intervention, effective communication with stakeholders, no severe adverse events, and collaboration with development partners to meet the start-up costs of training medical staff, facility improvements, and supplies.In October 2009, while the pilot was under way at RFM Hospital, the Swaziland MOH initiated planning for an EIMC program by hosting an international expert consultation on EIMC. Hosting the first consultation of its kind gave Swaziland a voice in the global discussion around EIMC. Swaziland’s MOH immediately moved forward on clinical and programmatic recommendations that emerged from the consultation. The resulting EIMC surgical guidelines endorsed the Mogen clamp as the preferred EIMC method for Swaziland. Less than a year after the consultative meetings, the MOH incorporated these guidelines into the existing national male circumcision surgical protocol.The timeline in Between 2011 and 2013, Swaziland expanded integrated EIMC services to 17 facilities.The MOH and stakeholders established EIMC within its national VMMC and HIV prevention program and integrated it into existing health service delivery outlets, namely the maternal, newborn, and child health (MNCH) service delivery platforms. The Swaziland EIMC experience offers a useful template for other countries with generalized HIV epidemics and low circumcision prevalence that are embarking on EIMC programming.We collected data during a consultation process with both VMMC and EIMC stakeholders between March and May 2014 in preparation for the document, “Male Circumcision Strategic and Operational Plan for HIV Prevention 2014–2018.” The drafting of this important guiding document offered a timely opportunity to reflect on the first years of EIMC program implementation and consolidate lessons learned.We collected data through structured stakeholder focus group discussions and in-depth interviews with key informants. We conducted 30 in-depth interviews with:Individuals from key MOH departments, including Central Medical Stores, the Public Health Unit, the Strategic Information Department, the Legal Advisor, the Swaziland National AIDS Program, the Rural Health Motivator Program, the Sexual and Reproductive Health Unit (SRHU), and the Expanded Program on Immunization (13 interviews)NGOs involved in EIMC service delivery and programming, including PSI, the Family Life Association of Swaziland, the Elizabeth Glaser Pediatric AIDS Foundation, and mothers2mothers (5 interviews)Professional associations, including the Swaziland Democratic Nurses Union, the Swaziland Nursing Council, and the Private Providers Association (3 interviews)Donors supporting EIMC programming (2 interviews)A traditional organization, Khulisa Umntfwana (“Grow a Child”)Providers and clinical managers working at 2 regional hospitals, 3 health centers, and 1 private clinic that offer EIMC (6 interviews with 5 doctors and 5 nurses). These were among the most informative interviews.We collected data through structured stakeholder focus group discussions and in-depth interviews with key informants.We selected stakeholders for in-depth interviews based on their involvement with MNCH or HIV/AIDS health issues from a policy or practice perspective, those with expertise in the Swazi health care workforce, and those with direct clinical or program experience with EIMC. While it was not feasible to visit all 17 facilities conducting EIMC services, we involved facilities from all 4 geographic regions of Swaziland as well as the varying types of facilities. Each interview lasted between 30 and 60 minutes, and we tailored them to informants’ areas of expertise: EIMC technical approach and service delivery, coordination, human resources, or communication and education.We convened 2 focus group discussions. Traditional leaders from Manzini Region attended the first discussion, which addressed cultural values related to EIMC, and optimal approaches to building informed community demand for EIMC. The second discussion, an MNCH stakeholders’ forum with 27 participants, included 10 EIMC-trained nurses and facility matrons representing facilities across all 4 regions, as well as other NGO and national MOH representatives. Forum participants broke into small groups to discuss questions of (1) provider support and staffing for MNCH services, including EIMC, (2) EIMC entry points and service coordination and linkages between VMMC and EIMC, (3) facility-based client education and counseling, (4) EIMC demand creation and community awareness, and (5) EIMC expansion and scale-up. Note-takers documented the focus group discussions, and we analyzed the notes for consistent themes. An implementing partner responsible for VMMC and EIMC service delivery contributed all service delivery data presented here: numbers of EIMC procedures performed by month, year, and location, as well as the incidence of documented moderate and severe adverse events. All data collection and analysis were conducted according to international principles of maintaining privacy and confidentiality of personal information.With a total of 5,149 EIMCs performed between 2010 and the end of 2014, Swaziland now leads the Eastern and Southern Africa region in the scale-up of EIMC. By mid-2014, 123 health care workers (45 doctors and 78 nurses) had been trained through 11 clinical trainings. Five providers, trained as trainers, facilitated clinical trainings and conducted mentorship visits to newly trained providers. Approximately 80% of the infants were circumcised in the immediate postpartum period before discharge. The remaining approximately 20% of infants were circumcised later, and included referrals from other facilities or in tandem with immunization or routine postpartum visits. Notably, Swaziland’s EIMC program has not reported any moderate or severe adverse events to date at the time of publication of this article. Severe adverse events require extensive intervention with referral or specialist input, mild adverse events require minimal or no intervention, and moderate adverse events can be classified as neither severe nor mild, but do require intervention and are usually managed on-site. Because moderate adverse events are managed on-site, providers may have underreported these adverse events, but this did not emerge as an issue in any focus group discussions or in-depth interviews. In 2015, Swaziland became the first PEPFAR male circumcision priority country to draft a costed operational plan that includes EIMC.80% of circumcisions took place in the immediate postpartum period.In 2015, Swaziland became the first PEPFAR male circumcision priority country to draft a costed operational plan that includes EIMC.In Swaziland, EIMC integration starts during the pre-pregnancy period and continues through the postnatal period . AntenatSwaziland registers approximately 34,249 births per year.In 2011, the MOH developed the “Swaziland Task-Shifting Implementation Framework in Support of Quality Health Service Provision.” According to the framework, some minor procedures were to be shifted from doctors to nurses.According to a representative sample of both doctors and nurses at RFM Hospital, the facility with Swaziland’s most established EIMC program, when doctors and nurses are trained together, doctors feel more comfortable providing nurses the autonomy to lead the program. RFM Hospital started task shifting for EIMC in 2010 because doctors could not meet client demand for the service. Although hesitant in the beginning, hospital management embraced task shifting for EIMC because of the excellent clinical outcomes. The strong safety record to date at the time of publication of this article, as evidenced by the absence of documented severe or moderate adverse events, has further strengthened national confidence in EIMC-trained nurses. Global evidence in support of task shifting for male circumcision, particularly from Kenya, reinforces the argument in its favor.Development partners have primarily supported the procurement and distribution of EIMC instruments, including Mogen clamps, prepackaged EIMC kits, and consumables. Within the next 5 years, the MOH plans to assume responsibility for EIMC supply and equipment procurement within its supply chain systems for the MNCH platform.In 2011, the MOH established the position of National Male Circumcision (MC) Coordinator under the Swaziland National AIDS Program. This position supports both the VMMC and EIMC programs. According to the National MC Coordinator, Swaziland’s MOH viewed the EIMC program as both a way to further its HIV prevention goals and as an opportunity to strengthen MNCH services. EIMC stands to improve health outcomes for mothers and their babies by increasing attention to improving services, including strengthening infection prevention measures, reinforcing close monitoring of newborn health, encouraging routine neonatal physical examinations, discouraging early discharge, and promoting postpartum follow-up for both mothers and babies.Swaziland has not yet completed a formal evaluation of EIMC, but EIMC integration into the MNCH platform appears to have been an effective approach to the delivery of services based on the number of EIMCs conducted as well as the following markers:EIMC indicators were integrated into national monitoring tools including the antenatal care (ANC) register, ANC card, child welfare card, child welfare register, and postnatal register.EIMC has been integrated into the comprehensive package of essential services for healthy baby boys.EIMC is part of the health education content discussed during routine antenatal care visits.EIMC consent forms for parents  are distHealth facility management has identified EIMC focal people.All public health facilities that offer labor and delivery services have allocated space to perform EIMC within their maternity units.Facility doctors oversee the EIMC service delivery as well as facility-level EIMC training activities.In the early stages of VMMC programming in Swaziland, a technical working group led by the MOH with participation from development and implementing partners provided oversight. Under the broad technical working group for VMMC, a subgroup focused on technical aspects of EIMC. This subgroup coordinated various aspects of the EIMC program, including macro-level program planning, clinical skills training, facility identification and assessment, and quality assurance. In February 2011, this technical team developed and piloted EIMC quality assurance supportive supervision tools, as well as a quality assurance implementation plan. As EIMC expanded to additional facilities, program planners recognized that it was critical to fully engage the MNCH platform for an integrated, robust, and sustained EIMC program. Efforts are currently under way to bring the SRHU, which is responsible for the MNCH platform, fully into the coordination framework.It is envisaged that the VMMC technical working group, under the leadership of the National MC Coordinator, will include the manager of the SRHU. EIMC issues will be addressed through ad hoc working groups under the VMMC technical working group.The majority of key informants stressed that the human resource needs of an EIMC program are different from those of the intensive, time-bound, VMMC catch-up program. While EIMC is the long-term sustainability plan for VMMC, it is linked to, but also distinct from, the VMMC program. For adolescent and adult VMMC services, dedicated nurses and doctors were hired to meet the ambitious VMMC catch-up targets. MOH interviewees stated that this approach would be neither practical nor desirable for an integrated, sustainable EIMC program.-To face the human resource challenges for EIMC scale-up, the MOH, in collaboration with its partners, first trained EIMC providers, doctors, midwives, and nurses, through competency-based clinical training beginning in February 2011. Swaziland was the first country to pilot the competency-based clinical skills training in EIMC using the WHO/Jhpiego “Manual for Early Infant Male Circumcision Under Local Anaesthesia” and the associated UNICEF/Jhpiego facilitators guide and learners workbook.With the introduction of EIMC services, MOH stakeholders anticipated concerns about the introduction of an “additional” task without providing additional compensation. This was a particular challenge because doctors and nurses involved in the VMMC program had been compensated for the extra hours worked in support of the program. The MOH also needed to ensure that routine and emergent demands of the MNCH platform were not compromised by the introduction of EIMC in MNCH units. For these reasons, according to representatives from the Swaziland National AIDS Program, the MOH scaled up EIMC slowly. A considered, unhurried scale-up allowed time for facility teams to develop creative and practical solutions to any problems that arose. While the issue of compensation is still a sensitive one, ongoing inclusive dialogue is helpful in clarifying decision-making rationale.A considered, unhurried scale-up allowed sufficient time for facility teams to develop creative and practical solutions to any problems that arose.To address human resource constraints, some higher-volume sites, such as the Mbabane Government Hospital and the RFM Hospital, allocated dedicated, trained midwives to EIMC. Facility management redistributed staff despite human resource shortages and without financial incentives, which demonstrates ownership of the EIMC program at the facility level. Assistance from partner-supported EIMC motivators—to educate clients, link clients to services, and help complete paperwork—also relieved providers of some supportive functions.,Formative research has emphasized that EIMC sensitization efforts must provide parents with the information to make informed decisions.factors and themes that contributed to the development of Swaziland’s fledgling EIMC program:In reviewing EIMC program data and feedback from key stakeholders, several critical program components emerge. Distilled findings across all results areas highlight the presence of the following Program backing from committed MOH leaders who have been termed early adopters in implementing an evidence-based EIMC intervention.Consistent and coordinated funding for EIMC across multiple funding sources during the early stages. UNICEF funded the first EIMC pilot at RFM Hospital in 2009. Since then, PEPFAR has provided consistent funding through USAID-supported EIMC expansion, which has helped the program establish a sound foundation.The role played by RFM Hospital, whose model EIMC pilot encouraged Swaziland to move quickly from EIMC adoption to program implementation. RFM Hospital’s initiative and leadership informed the later roll-out of the EIMC program, highlighting the need for dedicated EIMC staff and space as well as linkages and coordination across the MNCH platform.Targeted technical support from implementing partners. An example of technical assistance that supported program scale-up included formative research, supported by PSI and completed in 2013, that informed EIMC program decision making. This research found that there was a near-universal perception that consent for EIMC cannot be granted by one parent alone, and emphasized that EIMC sensitization efforts must provide parents with the information needed to make informed decisions. Another example of targeted technical support included the efforts of Jhpiego’s Maternal and Child Health Integrated Program to help providers achieve competency through a mix of classroom and clinical mentorship. Other organizations, such as the Elizabeth Glaser Pediatric AIDS Foundation and mothers2mothers, also played pivotal roles in informing potential clients through community and facility education sessions.Formative research that informed decision making. A study of knowledge, attitudes, and practices (KAP) conducted by RFM Hospital helped direct the EIMC pilot and, eventually, national implementation. Later, an MOH-led, PSI-supported KAP study informed community- and facility-based EIMC demand-creation efforts.A policy environment open to task shifting. To overcome the human resource challenge, in 2011 Swaziland’s MOH developed the “Swaziland Task Shifting Implementation Framework in Support of Quality Health Service Provision.” This framework allows nurses to perform minor procedures that were previously conducted only by doctors.challenges, listed by results area:While these components contributed to a growing national program, EIMC stakeholders also stated that scale-up and implementation of EIMC in Swaziland were not without the following Legislation is required to enact task shifting. Although the MOH approved the task shifting framework, fully operationalizing it remains a challenge, as does securing task shifting support from all relevant stakeholders.Early postpartum discharge of the mother/baby pair results in missed opportunities for EIMC. WHO recommends that EIMC should be performed at least 12 hours after birth to ensure that babies are healthy and stable before the procedure. However, due to high birth volumes and limited space, clients are often discharged before 12 hours postpartum, despite global guidance that mothers and babies should remain at facilities for 24 hours after birth, the period of greatest risk.EIMC requires broad coordination across multiple stakeholders at the national level. In Swaziland, because EIMC was conceived within the larger VMMC strategy and coordinated by the Swaziland National AIDS Program, involvement of key stakeholders from the SRHU was not as extensive as desired.Program ownership should be fostered at all levels to avoid the perception of a donor-driven process. Although the EIMC program has been implemented within public health facilities, it still depends on donor support for material and financial resources. Moving forward, Swaziland will need to assume greater financial ownership as donors limit funding for EIMC.Human resource constraints can pose challenges for service continuity. The EIMC program is constrained by regular staff rotations of EIMC-trained providers. EIMC services are further challenged by limited numbers of EIMC-trained providers inhigh-volume facilities and already-limited human resources for health care services.The EIMC program is constrained by staffing shortages as well as regular staff rotations of EIMC-trained staff.The consent process for EIMC is complex. Parents should be ready to make a decision about EIMC by the time they are admitted to facilities for deliveries. Parents and key decision makers should be sensitized before pregnancy or in early pregnancy. Early education allows couples and families the time to make considered decisions about EIMC.Client counseling needs dedicated educators. Especially in the early stages of program implementation and scale-up, dedicated educators can help establish and sustain EIMC services amid the multiple demands of busy and often understaffed MNCH units.EIMC has vocal global and regional opponents. Misinformation can jeopardize fledgling EIMC programs unless these programs continue to involve and educate communities and health care workers.Like PMTCT, EIMC provides an opportunity for nurses to help lead the way toward an HIV-free generation. Because there are approximately 12 times as many registered nurses as doctors in Swaziland, and because an overwhelming number of public sector facilities are led by nurses,As with all VMMC programming, program staff must establish a careful balance between service delivery and community awareness. Informed demand from parents and communities for the service may also support the establishment of EIMC as a service delivery priority. However, for countries with developed VMMC programs that are now introducing EIMC for sustainability, it is also important to recognize that EIMC requires a different approach—standards and guidelines for VMMC must be expanded and adapted to address service provision on a different platform.Swaziland has become the first country to implement EIMC programming quickly in response to the growing evidence supporting the health benefits of the procedure. In a country without a tradition of circumcision, where the sociocultural norms are not favorably predisposed to EIMC, and where VMMC scale-up faced challenges related to client demand, the EIMC program has expanded, which is particularly impressive because of the absence of a targeted national EIMC communication campaign. Continued expansion holds the promise of averting thousands of HIV infections and is a large step toward creating an HIV-free generation in Swaziland.


ING1b is a tumor suppressor that affects transcription, cell cycle control and apoptosis. ING1b is deregulated in disease, and its activity is closely linked to that of p53. In addition to regulating protein-coding genes, we found that ING1b also influences the expression of large intergenic non-coding RNAs (lincRNAs). In particular, lincRNA-p21 was significantly induced after DNA-damage stress or by ING1b overexpression. Furthermore, lincRNA-p21 expression in response to DNA damage was significantly attenuated in cells lacking ING1b. LincRNA-p21 is also a target of p53 and can trigger apoptosis in mouse cell models. We found that this function of lincRNA-p21 is conserved in human cell models. Moreover, ING1b and p53 could function independently to influence lincRNA-p21 expression. However, their effects become more additive under conditions of stress. In particular, ING1b regulates lincRNA-p21 levels by binding to its promoter and is required for induction of lincRNA-p21 by p53. The ability of ING1b to cause apoptosis is also impaired in the absence of lincRNA-p21. Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21. Our findings suggest that ING1b induces lincRNA-p21 expression independently of histone 3 lysine 4 trimethylation mark recognition and that lincRNA-p21 functions downstream of ING1b. Thus, regulation at the level of lincRNA-p21 may represent the point at which ING1b and p53 pathways converge to induce apoptosis under specific stress conditions. This effect requires domains in the amino terminus of ING1b but appears to be independent of the ING1b PHD or the presence of HuR. Moreover, lincRNA-p21 expression is crucial for ING1b-induced apoptosis in response to DNA-damage stress. This effect is significantly enhanced by p53. Thus, lincRNA-p21 may represent the point at which ING1b and p53 pathways converge to induce apoptosis in cells.In recent studies, large intergenic long non-coding RNA p21 (lincRNA-p21) has been shown to regulate apoptosis in response to p53 signaling and to facilitate suppression of target gene translation.15 Moreover, p53 was shown to require ING1 for induction of programmed cell death,14 suggesting the potential for a functional interdependency between ING1 and p53. Recently, lincRNA-p21 was shown to be a transcriptional target of p53 that mediates gene repression to promote apoptosis.16 Given this relationship between ING1 and p53, we asked if ING1b could influence lincRNA-p21 expression. ING1b was overexpressed using an adenoviral construct encoding GFP and ING1b expressed from different promoters, or knocked down with siRNAs in Hs68 human diploid fibroblasts. Levels of lincRNA-p21 was subsequently examined using quantitative real-time polymerase chain reactions (qRT-PCR). In this human diploid fibroblast strain, ING1b increased lincRNA-p21 levels by ~40-fold  to induce DNA damage. Under control conditions, lincRNA-p21 expression increased significantly after DNA damage (P<0.05), suggesting ING1b was transducing a major part of the ADR stress signal. This was not due to the effects of ING1b on p53 transcription , knockdown of ING1b might also interfere with the ability of ActD to block transcription, leading to apparent increased lincRNA-p21 stability.Previous studies have reported that ING1b-induced apoptosis can be either p53-dependent or p53-independent, with variations being contingent on cell type.~40-fold . In cont~40-fold , suggestA damage , but indA damage , P<0.05,cription . InsteadA damage . In thisIn human non-small cell lung carcinoma cells that are p53-null, ADR failed to induce expression of lincRNA-p21. In these cells, ING1b knockdown by ~80%  also dro2O2) or UV and tested for lincRNA-p21 expression. Results indicated that like ADR, H2O2 and UV increased lincRNA-p21 levels but knocking down ING1b had a less significant effect on lincRNA-p21 expression  or p53 knockout (p53−/−) mouse embryo fibroblasts (MEFs) with ADR and compared lincRNA-p21 expression in different treatment groups. As shown in 22 and ING1b,13 also had a similar pattern of expression as lincRNA-p21  showed that ING1b binds to a promoter region ~150–400 bp upstream of the first lincRNA-p21 exon (10 Binding efficiency was similar between untreated and ADR-treated p53-null cells but was reduced in p53-induced cells (17) into a luciferase reporter and transfected this plasmid into cells along with β-galactosidase to normalize for transfection efficiency. We also included control or ING1b plasmids to determine effects of ING1b on reporter expression. Results showed significant expression of firefly luciferase when ING1b was overexpressed  primary cells, whereas ING1a increases significantly at high-passage levels.pression . Interespression . Furtheression29 . As show15 Interestingly, p21, a known target of ING1b and p53 that is found directly downstream of lincRNA-p21 on the DNA, also required the same domains of ING1b to remain intact for proper induction  requires ING1 for maximal induction of lincRNA-p21. ADR is a DNA-intercalating agent that poisons toptisomerase II and induces double-stranded breaks. It can also evict histones including the DNA-damage-associated H2AX variant from chromatin, altering the transcriptome and epigenome,34 which might be expected to affect epigenetic regulators such as the ING proteins.Our experiments showed that lincRNA-p21 levels positively corresponded with ectopic ING1b expression and ING1b alone was sufficient to increase lincRNA-p21 in p53-null cells. Furthermore, ING1b was necessary for a robust rise in lincRNA-p21 upon exposure of cells to stress using ADR, but not other stresses such as UV and H32 but rather appears to protect p53 from degradation35 to enhance its activity. This represents another mechanism by which ING1b and p53 may interact to increase levels of lincRNA-p21, which might also explain their convergence of function in inducing apoptosis, an observation made independently in previous studies.12The observations noted above indicate that both ING1 and p53 contribute approximately equally to the induction of lincRNA-p21. We found that ING1b bound the lincRNA-p21 promoter and drove the expression of reporter constructs, whereas a previous study noted the presence of p53-binding sites in the same region, consistent with ING1 and p53 jointly inducing lincRNA-p21 transcription. A model highlighting this relationship is shown in 17 All plasmid transfections were carried out using Lipofectamine LTX . Cells infected with Ad-GFP or transfected with appropriate empty vectors were used as controls. For induction of p53, H1299 cells harboring an inducible p53 expression construct  were treated with 100 ng/ml of doxycycline for 24 h. For DNA damage, cells were treated with 400–500 nM ADR  for 16 h, 100 μM H2O2 for 24 h (Sigma), and exposed to 85 J/m2 of UV (CL-1000 UV Crosslinker) and harvested at 24 h.To overexpress ING1 wild-type proteins and deleted mutants, cultured cells were transfected with pCI-ING1bΔPHD/ pcDNA-ING1bΔLID/ pEGFP.C2-ING1bΔNLS/ pEGFP.C2-ING1bΔUBD/ PBR plasmids or treated with adenoviruses containing GFP-ING1 for 24–48 h at a multiplicity that yielded 95% infection efficiency. Overexpression of human lincRNA-p21 was accomplished using pMS2-lincRNA-p21 plasmids .17 human HuR (5′-CGUAAGUUAUUUCCUUUAAtt-3′),17 and mouse lincRNA-p21 .16 ING1 and non-targeting siRNAs were purchased from Dharmacon  . All transfections were carried out with 100 nM of siRNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.Knockdown experiments were performed using siRNA oligos that target the genes of interest. The custom siRNAs used were as follows: human p53 (5′-GACUCCAGUGGUAAUCUACtt-3′), human lincRNA-p21 (5′-UCAUCAUGCGGCCUUGCAGtt-3′)RNA was isolated from cells using Trizol (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen). The purity and concentration of the RNA were assessed using a Nanodrop ND-1000  as per the manufacturer's instructions.2, 10 mM dNTPs, 10 × SYBR Green (diluted 1:1000), ROX passive reference dye, nuclease-free water and platinum Taq DNA Polymerase (Invitrogen). For RNA expression measurements, both the standard curve and the ΔΔCt methods were performed using the 7900HT thermal cycler system from Applied Biosystems. The human primers used in this study are listed in Reverse transcription was conducted to create templates for qRT-PCR using the High-Capacity cDNA synthesis kit  or the iScript cDNA synthesis kit . Quantitative real-time PCR was performed using a SYBR green master mix containing 10 × PCR buffer, 50 mM MgCl36 In brief, cells were crosslinked with 1% formaldehyde, harvested, resuspended in lysis buffer that was supplemented with 1 mM PMSF, and sonicated. The chromatin was sheered to an average size of 500–1000 bp and immunoprecipitated using either ING137 or IgG  background control antibodies. After extensive washing steps, the formaldehyde crosslinks were reversed by treatment at 65 °C. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation and samples were used in semi-quantitative PCR  and left at −20 oC overnight. Cell extracts were assayed for luciferase and β-galactosidase activity the following day.The human lincRNA-p21 promoter region was cloned into the pGL3-basic vector . H1299 cells containing a doxycycline-inducible p53 promoter were transfected with either pCI or pCI-ING1b in conjunction with 100 ng of pGL3-lincRNA-p21 and 25 ng of pCMV-Viability of Hep3B cells was assessed using Trypan Blue exclusion assays. In brief, cells were collected by trypsinization after transfection with either control vector or pMS2-lincRNA-p21 plasmids and stained with 0.4% trypan blue reagent (Sigma). The number of live cells was determined using a hemocytometer. All experiments were performed in triplicate.In Situ Apoptosis Detection Kit  according to the manufacturer's recommendations. Post processing, the cells were visualized using a Zeiss Axiovert 200 microscope and images were captured using AxioVision software.Cells were seeded into eight-chamber culture slides  where they were transfected with either control or lincRNA-p21 siRNAs, and infected with adeno-GFP or adeno-ING1b viruses for a total of 48 h. Subsequently, TUNEL assays were performed using the ApopTag Red


To investigate the prevalence of mental disorders in (subgroups of) juvenile suspects who sexually offended (JSOs), and its relation with criminal re-referrals five to eight years later.A sample of 106 JSOs (mean age 15.0 ± 1.5 years) referred to the Dutch Child Protection Board between May 2003 and December 2006 was classified into JSOs with child victims (N = 19), solo JSOs with adolescents and/or adults victims (N = 29), and group JSOs with adolescents and/or adults victims (N = 58). Mental disorders were assessed at baseline by means of a semi-structured interview (K-SADS-PL), the level of functioning by means of the Children’s Global Assessment Scale (CGAS) and re-referrals was ascertained from official judicial registration systems.Three quarters of JSOs met criteria for at least one mental disorder. Comorbidity was found in more than half of the subjects and almost two thirds of the JSOs were functionally impaired. As compared to the other subgroups, JSOs with child victims showed higher rates of affective disorders and had a lower overall level of functioning. Furthermore, JSOs re-referred for another sexual offense were more often diagnosed with an affective disorder, were more often sexually abused and had a lower level of global functioning than JSOs who were not re-referred for another sex offense.JSOs should receive mental assistance, as more than two thirds are functionally impaired due to mental problems. This may not only be important to safeguard the development of the juvenile offender but might also reduce repeated sexual offending. Sexual offending behavior of juveniles is often a reason for public concern. It is estimated that minors commit about 20% of all rapes and 30-50% of all child molestations . A recenFurthermore, JSOs constitute a heterogeneous group. Previous research reported differences in mental health problems between subgroups of JSOs. While in rapists, for example, externalizing problems are more common , offendeSexual recidivism rates in JSOs were repeatedly found to be relatively low. A recent review and meta-analysis showed that only 7% of JSOs reoffended sexually . This waThe overall aim of this study was to examine the prevalence of mental disorders in JSOs and to assess its relationship with repeated sexual offending behavior. More specifically, the prevalence of mental disorders will be studied in the group of JSOs, as well as in specific subgroups. In line with previous research, a distinction will be made between JSOs with child victims, solo JSOs with adolescent/adult victims, and group JSOs with adolescent/adult victims ,24,25. IParticipants in this prospective longitudinal study were 106 male juvenile suspects (aged 12 to 18) of a sexual offense referred to the Child Protection Board (CPB) between May 2003 and December 2006. For the sake of readability, these juvenile suspects will be referred to as juvenile(s) (suspects) who sexually offended (JSOs). Due to the limited number of female JSOs, all girls were excluded from the sample. As the police in the Netherlands is obliged to refer all 12 to 18 year old suspects of a sex crime to the CPB, regional CPB offices were the primary sites of inclusion. Out of twenty-two regional offices, four were selected because of their location spread over rural and urban regions. Also JSOs who immediately were admitted to four (out of the thirteen) pretrial Juvenile Justice Institutions in The Netherlands were asked to participate as well.Exclusion criteria were an IQ below 70 and insufficient command of the Dutch language. The Ethics Committee of the VU University Medical Center Amsterdam, The Netherlands, approved the study. After explaining the study to the participants, we obtained written informed consent from them and their parents or legal representatives.χ2 = 0.00; p = 1.00), age of victim  or type of offending  . Responders were more often of non-Dutch ethnicity than non-responders .In total, 161 boys were eligible for participation, of which 106 agreed to participate (response rate = 66%) . Non-responders did not differ from responders with respect to age  or offense characteristics, such as gender of victim   retrieved from both official registration systems and CPB files, JSOs were classified into three subgroups: a) JSOs with child victims, suspected of having sexually abused children (below 12 years of age) who were at least four years younger than the offender himself (N = 19); b) solo JSOs with adolescent/adult victims, suspected of having raped or sexually assaulted adolescents (at least twelve years old) or adults (N = 29); c) group JSOs with adolescent/adult victims, suspected of having raped or sexually assaulted adolescents (at least twelve years old) or adults (N = 58). The three subgroups were mutually exclusive.Mental disorders were assessed by means of the K-SADS-PL (Schedule for Affective Disorders and Schizophrenia for school age children - Present and Lifetime -28), a sThe CGAS  providesBy means of the official judicial registration systems of the Dutch Ministry of Security and Justice (JDS: Justice Documentation System [Justitieel Documentatie Systeem]) both the referrals prior to the index referral and the re-referrals in the five to eight year follow-up period after the index referral were examined. Prior referrals were defined as referrals registered before the index referral. Re-referrals were defined as referrals registered after the index referral. With respect to offending behavior, a distinction was made between referrals for sexual offenses and non-sexual offenses. Sexual offenses were defined as sexual offenses according to the Dutch Criminal Code. Non-sexual offenses were all offenses according to the Dutch Criminal Code without sexual offenses. In the JDS records, violations that yield a more serious punishment when repeated were also included.The data were processed and analyzed with IBM SPSS 19 . For all calculations the level of statistical significance was set at .05. First, in order to determine the prevalence of mental disorders, the global assessment of functioning, and the prior referrals and re-referrals of the subjects, descriptive statistics were performed. Second, differences between subgroups of JSO  were analyzed using Chi-square tests (or Fisher Exact when expected cell counts less than 5) for categorical variables, and ANOVA’s  for continuous variables. It should be noted that using corrections as Bonferroni increases the risk for Type I errors.Prevalence rates of mental disorders and scores on the CGAS are shown in Table Although subgroups did not differ in presence of any mental disorder, comorbidity was least present in the group JSOs with adolescent/adult victims  was retrieved. The index referral was found in 98 cases. In 8 cases we could not retrieve information about the juvenile offender and/or the index referral from the official judicial registration system. Of the 98 JSOs, 41% were referred for a sexual offense preceding the index referral Table . WhereasJSOs re-referred for another sexual offense were diagnosed more often with an affective disorder than both JSOs re-referred for a non-sexual offense and JSOs who were not re-referred at all. JSOs re-referred for another sexual offense were also more often sexually abused than JSOs re-referred for a non-sexual offense, and had a significantly lower level of global functioning than JSOs who were not re-referred at all Table .Table 3In the current study, we examined mental disorders in juvenile suspects of a sexual offense, as well as subgroups of these suspects. For the sake of readability, these juvenile suspects will be referred to as juvenile(s) (suspects) who sexually offended (JSOs). Furthermore we examined differences in mental disorders between JSOs re-referred for another sexual offense, re-referred for a non-sexual offense, and without a re-referral. In total, over three-quarters were found to meet criteria for at least one mental disorder, with over half being diagnosed with comorbid conditions, and two thirds being functionally impaired. Rates differed between subgroups of JSOs, with JSOs with child victims showing the most mental disorders. With regard to re-referrals, 7% of JSOs was re-referred for another sexual offense, whereas 80% was re-referred for a non-sexual offense. JSOs re-referred for a sexual offense were more often diagnosed with an affective disorder, were sexually abused more often and had a lower level of global functioning than JSOs who were not. This study indicates that the mental health needs of JSOs deserve special attention.The current study suggests that JSOs have relatively low prevalence rates of CD and SUD and high rates of ADHD and internalizing disorders as compared to findings on general offenders -5. FirstAs was found in previous research with regard to sexual re-offending e.g., ,22), onl, onl22])In addition, a larger group of JSOs was re-referred for a non-sexual offense. Our results are in line with the higher prevalence rate for general re-offending compared to sexual reoffending found in the studies of Caldwell . At . At 22])Caldwell  and FortCaldwell  study anCaldwell ).With regard to the relationship between mental disorders and reoffending, our results showed that, whereas non-sexual re-referral in JSOs was characterized by the presence of CD, JSOs re-referred for another sexual offense were characterized by the presence of an affective disorder and a history of own sexual abuse. Hence, especially non-sexually reoffending JSOs may be the subgroup of JSOs who commit their sexual offense in the context of a problematic antisocial behavior pattern. In line with expectations, JSOs without any re-referrals had the least mental disorders. With regards to the higher rate of an affective disorder in the group of JSOs referred for another sexual offense compared to those who were not, previous research in general offending juveniles found that affective disorders decreased the risk for reoffending . An explFindings of this study must be interpreted in the light of some limitations. First, because of the relatively low prevalence of sexual offenses committed by juveniles, it is difficult to include large groups of sex offenders. For that reason, specific subgroups, such as JSOs with child victims (N = 19) or JSOs referred for another sexual offense (N = 7), were small. This also accounts for the prevalence of some mental disorders, such as affective disorders. This may well explain the wide range of the Odds Ratios (ORs), and, therefore, findings need to be replicated in larger samples. Second, at follow up re-referral was determined from an official registration system. Offenses unknown to police and the justice system, the so-called dark number, could, therefore, not be included. In addition, as we examined (re-)referral, not all juveniles had to be convicted for their (sex) offenses. However, acquittal of an offense does not necessarily mean that the  behavior did not take place. For treatment purposes it is important to examine the prevalence of sexual misbehavior: do JSOs persist in sexual and general delinquent behavior and do they continue to victimize other people? Third, as information regarding mental disorders and the index referrals was cross-sectional, no conclusions can be drawn with regard to causal or time-related paths. Fourth, due to the absence of a general offending control group, it is unknown to what extend the mental disorders are related to sexual deviant behavior or offending behavior in general. Finally, a possible sample bias limits the generalizability of our results. Although responders and non-responders did not differ in age or offense characteristics, responders were more often of non-Dutch ethnicity. As it is suggested that minority and non-minority adolescent offenders differ in prevalence of mental disorders , more reIn conclusion, mental disorders are highly prevalent in JSOs, especially in those with child victims. Although most JSOs were re-referred for a non-sexual offense, 7% persisted in their sexual offending behavior. The presence of an affective disorder, a history of sexual abuse, and a lower level of global functioning were more prevalent in JSOs re-referred for another sexual offense than those who did not. The differences in mental disorders between subgroups of JSOs as well as between JSOs re-referred for a sexual offense and JSOs who were re-referred for another  offense are important because of their consequences for treatment as well as for the course of offending behavior. It is clear that this relationship is complex, but of importance for both early intervention and treatment to prevent recidivism, especially sexual recidivism.


We have developed a highly versatile platform for the systematic retrieval of T-cell receptors (TCRs) from single-antigen-reactive T cells and for characterization of their function and specificity. This approach enables rapid extraction of multiple TCRs from repertoires in individuals and not only broadens the diversity of TCRs suitable for clinical use, but also sets the stage for actively personalized immunotherapeutic strategies. The tailored reprogramming of immune cells from cancer patients to recognize and attack their tumors has potential as a disruptive medical innovation.The pool of antigens potentially suitable as immunotherapeutic targets for TCR-engineered T cells has grown considerably. However, for the vast majority of these antigens, neither TCRs nor T-cell clones have been defined as yet.As a consequence, there is an ample need for a technology enabling the systematic recovery of sets of functional TCRs against defined antigens from individual repertoires as well as swift definition of their specificity.in vitro-transcribed synthetic RNA encoding a defined antigen of interest.+ and CD8+ T cells are identified by assessing activation markers and then isolated by cell sorting. The TCRα and -β chains are amplified from these single T lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primer sets designed to cover all TCR gene families. For functional characterization, T cells are transfected with synthetic RNA to express the identified TCRα/β pairs and are assessed by using target cells that are reconstituted for the expression of the respective antigen in conjunction with the donor's HLA alleles.Recently, we presented a platform capable of fulfilling this need  phosphoprotein pp65 as a model antigen. We obtained 13 different CMV–pp65-specific TCRs recognizing HLA class I and II restricted epitopes, both known and novel ones.+ and CD8+ T-cell responses in patients with cancer, the specificities of which have been mapped over the last few years. Sixteen NY-ESO-1–specific TCRs were isolated from 3 seropositive patients with non–small cell lung cancer (NSCLC). These were directed against 11 different HLA class-I and -II restricted epitopes, again known and novel ones, clustering in known immunogenic regions of the NY-ESO-1 protein.For further proof of concept, we resorted to NY-ESO-1, one among the immunologically best-characterized members of the cancer/germline antigen family. NY-ESO-1 is known to frequently elicit spontaneous CD4Finally, we applied this TCR identification approach to the transmembrane phosphatase with tensin homology (TPTE), which belongs to a large group of antigens with high cancer cell selectivity, for which spontaneous T-cell responses have not yet been reported in cancer patients. TPTE-specific T cells were isolated from 3 seropositive patients with NSCLC, and a total of 27 TPTE-specific TCRs were cloned. Thus, multiple HLA class-I and -II restricted epitopes distributed over the whole protein sequence were identified, none of which had been identified earlier.+ T cells are of particular interest, as coadministration of CD4+ T cells to TCR-engineered adoptively administered T cells has been shown to enhance tumor infiltration of CD8+ T cells and to prevent their exhaustion.In summary, we identified 398 TCRs representing 189 different clonotypes. Among these, 56 TCRs were shown to be antigen specific corresponding to approximately one third of the uniquely identified TCRs. Notably, the majority of specificities have not been described earlier, although 2 of the antigens have been extensively studied as T-cell antigens for decades now. Moreover, the majority of TCRs were shown to recognize HLA class-II restricted epitopes, whereas the currently available and clinically explored TCRs are all HLA class-I restricted epitopes. The CD4in vitro-sensitized lymphocytes or in vivo-primed T cells obtained from cancer patients. T cells from peripheral blood or from infiltrates in tumor lesions may also be used as a TCR source.The single-cell TCR isolation technology is versatile and can be adapted to clone human TCRs from different sources such as It can be applied to identify single TCRs of interest against defined antigen–HLA combinations to be developed for stratified approaches.As the discovery process is efficient and the output is high, the technology allows setting up a warehouse of TCRs against multiple antigens and various HLA restrictions to accommodate personalized approaches. The TCR matching each individual patient's tumor and HLA haplotype would be chosen “off the shelf.” As studies suggest a benefit of targeting 2 or more antigens and simultaneously addressing HLA class I and II epitopes to circumvent tumor escape and achieve optimal T-cell effector functions, combinations of TCRs could be customized from such TCR warehouses.The rapidity of obtaining TCRs confers the power to enter the new dimension of actively individualized approaches.This technology can be envisioned to foster 3 clinical treatment concepts:in vivo, they should have a sufficient affinity conferring sensitive recognition of the respective mutation. Their mutation specificity excludes on-target toxicity. Moreover, the negative selection in the autologous host minimizes the risk of off-target toxicity.Cloning and functional immunological characterization of TCRs from 1 individual is accomplished in less than 2 weeks, and the platform is operated by liquid handling stations and a barcode-assisted sample tracking and documentation system. TCRs of individual patients can be defined and manufactured on-demand for “just-in-time” customized, engineered T cells for adoptive transfer. This is particularly attractive in conjunction with cancer-specific mutations as immunotherapy targets. The first evidence of the spontaneous T-cell responses directed against mutated gene products in cancer patients was generated in the 1990s.In summary, the single-cell TCR isolation approach presented here has the capability to broaden the range of patients and malignancies eligible for TCR-engineered immunotherapies and opens up avenues for personalized TCR-engineered treatment concepts.


In many developing countries nutritional, and epidemiological transitions are emerging into continuing undernutrition and escalating overnutrition, giving rise to the double burden of the malnutrition phenomenon.This study aims to determine the prevalence of the dual form of malnutrition (overweight mother/underweight child) in the same household and its associated factors in the Gaza Strip.2. Anthropometric indices were measured for children aged two to five years to classify the underweight children Z-score <-1.A total of 357 mother-child pairs from the same households were surveyed from three different geographical locations in the Gaza Strip, namely, El Remal urban area, Jabalia refugee camp, and Al Qarara rural area. The height and weight of mothers aged 18–50 years were measured, and their body mass index (BMI) was computed. The mothers were categorized according to the criterion of the World Health Organization (WHO) for BMI as overweight if they have a BMI ≥ 25 kg/madj, 1.50, 95% CL, 1.22, 1.82; p = <0.001), father’s educational (low or medium) levels , or , high scores of mothers' nutrition knowledge , and low monthly income .The results showed the prevalence of the dual form of malnutrition in the Gaza Strip was 15.7%, and its associated factors were child’s birth order (ORThe results from this study showed the dual form of malnutrition in the same household was prevalent in the Gaza Strip. This is a public health issue that must be understood and addressed and policy makers must implement an appropriate nutrition action plan to control dual form of malnutrition based on the underlying specific risk factors in the study population. In addition, interventions are needed to help individuals to translate their nutrition knowledge into healthy dietary behaviors. In developing countries nutrition transition is a continuous process that occurs in all societies as patterns of food consumption and physical activity change over time . In low Findings of increasing prevalence of obesity and overweight among adults and underweight among preschool children lead to the hypothesis that overweight and underweight might cluster within the same household. Previous studies supported the presence of the dual form of malnutrition in the Palestinian community . The duaThe aim of this study was to determine the dual form of malnutrition and its associated factors in the Gaza Strip.This cross-sectional study was conducted in three different areas in the Gaza Strip, namely, El Remal urban area, Jabalia refugee cam and Al Qarara rural area. It was carried out from April to October 2012.The sample size was calculated using the single proportion formula , as follP) was 32.0%, which was for obesity prevalence among women in the Gaza Strip  was 15.7%, the prevalence of normal weight mother/normal weight child (NWM/NWC) was 25.2%, while the prevalence of normal weight mother/underweight child (<-1 SD) was 8.1%. In Tables / underweight child (<-1 SD)] to the normal weight mothers/normal weight children. The majority of the participants in the two groups OWM/UWC and NWM/NWC were more likely to be found in the Jabalia refugee camp (64.3% and 60.0% respectively). No significant difference was observed in the geographical locations between the two groups. Our results showed a high proportion of participants lived in poor households and no significant difference was found. This piece of data indicated that most mothers and children were from a deprived socioeconomic background. Similarly, no significant differences were observed between OWM/UWC and NWM/NWC pairs in terms of household characteristics such as: father’s job, mother’s job, and household assistance. But results revealed that comparing the mean difference (MD) of mothers’ age between OWM/UWC 31.55 (6.62) year and NWM/NWC 27.93 (5.27) year was statistically significant (p<0.001). Additionally, the mean difference (MD) of child’s birth order between OWM/UWC 4.43 (2.35) and NWM/NWC 2.94 (1.88) was statistically significant (p = 0. 028). The mean difference (MD) of the number of children in the household between OWM/UWC 6.96 (1.90) and NWM/NWC 5.60 (1.67) was statistically significant (p <0.001). Moreover, an association was found between the mother’s educational level in the two groups OWM/UWC and NWM/NWC . Results revealed that there was an association between child’s appetite and groups of OWM/UWC and NWM/NWC . p = 0.044). Results revealed that when comparing the energy and macronutrients intake  between the two categories of overweight mothers lived in the households with dual form of malnutrition, and normal weight mothers, the difference between proportions wasn’t statistically significant, but overweight mothers lived in the households with dual form of malnutrition consumed higher energy and macronutrients than the others. The underweight children lived in the households with dual form of malnutrition consumed lower energy and macronutrients than normal weight children, and there was no statistically difference. A binary regression analysis was done with the outcome and explanatory variables described in adj, 1.50, 95% CL, 1.22, 1.82; p = <0.001). Households with father’s education level low or medium were more likely to have the dual form of malnutrition , or , respectively, than households with father with high education level. Poor households were less likely to have the dual form of malnutrition ; p = 0.030). The finding of the study revealed that the dual form of malnutrition increased with increasing scores of woman nutrition knowledge .This study presents evidence of a dual-pronged problem of malnutrition, which consists of underweight children and overweight/obese mothers living in the same household. The Palestinian community, like other countries, is experiencing rapid changes in diet and activity. The prevalence of hypertension, diabetes mellitus, and tobacco smoking are now higher and similar to those in neighbouring countries . Often tThe study design was cross-sectional to identify associated factors with the dual form of malnutrition, which could not determine the cause and effect relationship. Case control studies should be conducted in the future to address the risk factors of the dual form of malnutrition in the Gaza Strip. For 24-hour recall analysis, the results were based on estimates; therefore, they may give rise to recall bias.The coexistence of maternal overweight with child undernutrition in the same household is a health concern in the Gaza Strip. The socioeconomic situation of the household and birth order of the child appears to be important predictors of the dual burden of malnutrition. That highlights the importance of having effective strategies within families, focusing on nutrition education and the effective guidance to help parents promote caring and proper eating patterns among their children. In addition, maternal nutrition knowledge is also an important indicator for underweight/overweight households. Thus, interventions are needed to help individuals to translate their nutrition knowledge into healthy dietary practices. This is the first study conducted in the Gaza Strip, thus the results shed light on the public health concern of the dual form of malnutrition in the same household in the Gaza Strip. The sample of this study was selected randomly from three different sociodemograhic areas in the Gaza Strip. Thus, the results of this study can be generalized to the whole population, however, we are aware our sample is small. Further studies with larger sample sizes are needed to develop programs that could effectively address the existence of the dual form of malnutrition on a population’s health and nutrition. It is important to investigate these determinants of the underweight-overweight pairs at the same household for planning successful nutrition intervention programs that consider the coexistence of two individuals representing the opposite types of malnutrition (underweight and overweight) while sharing the same household environment.


Plasmodium malariae is widely distributed across the tropics, causing symptomatic malaria in humans with a 72-hour fever periodicity, and may present after latency periods lasting up to many decades. Delayed occurrence of symptoms is observed in humans using chemoprophylaxis, or patients having received therapies targeting P. falciparum intraerythrocytic asexual stages, but few investigators have addressed the biological basis of the ability of P. malariae to persist in the human host. To investigate these interesting features of P. malariae epidemiology, we assembled, here, an extensive case series of P. malariae malaria patients presenting in non-endemic China, Sweden, and the UK who returned from travel in endemic countries, mainly in Africa. Out of 378 evaluable P. malariae cases, 100 (26.2%) reported using at least partial chemoprophylaxis, resembling the pattern seen with the relapsing parasites P. ovale spp. and P. vivax. In contrast, for only 7.5% of imported UK cases of non-relapsing P. falciparum was any chemoprophylaxis use reported. Genotyping of parasites from six patients reporting use of atovaquone-proguanil chemoprophylaxis did not reveal mutations at codon 268 of the cytb locus of the P. malariae mitochondrial genome. While travellers with P. malariae malaria are significantly more likely to report prophylaxis use during endemic country travel than are those with P. falciparum infections, atovaquone-proguanil prophylaxis breakthrough was not associated with pmcytb mutations. These preliminary studies, together with consistent observations of the remarkable longevity of P. malariae, lead us to propose re-examination of the dogma that this species is not a relapsing parasite. Further studies are needed to investigate our favoured hypothesis, namely that P. malariae can initiate a latent hypnozoite developmental programme in the human hepatocyte: if validated this will explain the consistent observations of remarkable longevity of parasitism, even in the presence of antimalarial prophylaxis or treatment. Plasmodium malariae is a widely distributed parasite infecting humans across the tropics, and causing symptomatic malaria with a 72-hour fever periodicity. It is rarely studied. This parasite has the notable ability to recur after many decades of apparent absence. Further, people who have received drug treatment for other malaria infections, or to protect themselves when travelling, sometimes develop malaria illness due to P. malariae resurgence months or years later. We set out to understand the ability of this largely ignored parasite to persist in the human host. To do this we studied 359 P. malariae malaria patients in non-endemic China, Sweden, and the UK following endemic country travel, mainly in Africa. These patients were much more likely to report protective use of malaria drugs while travelling than were patients with P. falciparum infection. We tested for drug resistance, but did not find any evidence that this played a part in parasite survival in these travelers. We propose that P. malariae may have a relapsing form in the liver, which permits emergence of parasites into the blood months, years or decades after initial infection from a mosquito bite. Plasmodium falciparum, P. knowlesi, P. malariae, P. ovale curtisi, P. ovale wallikeri and P. vivax [P. knowlesi (1 case), and P. malariae, responsible for 688 cases, 1.8% of the total overall, although in both 2013 and 2014 P. malariae infections accounted for 2.6% of all imported malaria [P. malariae, the general assumption being that infection with this species will be prevented by any prescribed regimen that is effective against the more common P. falciparum or P. vivax. Despite being widely distributed and present in all malaria endemic zones, only a single case of P. malariae infection, in Costa Rica, is mentioned in the most recent World Malaria Report [Human malaria is caused by six species of protozoan parasite: P. vivax . AccordiP. vivax , it is eP. vivax ,4. The lP. malariae. Whereas an acceptable level of efficacy of most antimalarial drugs against P. malariae has been reported in both field and clinic [P. malariae infections after ACT treatment is not uncommon [P. malariae parasitaemia in travellers treated weeks or months prior for imported P. falciparum malaria [P. malariae malaria in individuals with no reported exposure to infected Anopheles mosquitoes for several decades [P. malariae does not have a latent liver-stage hypnozoite analogous to that well-described for P. vivax and the monkey parasite P. cynomolgi [The existing literature presents a conflicting picture of the drug responses of d clinic –7, recenuncommon ,9. Furth malaria ,11, and  decades –14. Thesynomolgi –16.P. falciparum is prevalent [P. vivax, P. ovale spp., and P. malariae [P. ovale curtisi and P. ovale wallikeri, can occur despite patients reporting good adherence to AP chemoprophylaxis during endemic country travel [P. falciparum three or more weeks after treatment does occur in a small proportion of AP-treated acute malaria patients [pfcytb locus of these recrudescent parasites has confirmed that a single nucleotide polymorphism at codon 268  is associated with treatment failure in vivo [P. falciparum infection documented in the literature, and this regimen has an estimated prophylactic efficacy against falciparum malaria of greater than 99.9% [P. vivax liver stages [P. vivax-infected patient who experienced both chemoprophylaxis breakthrough and treatment failure with AP, the relapse occurring five months after the initial episode in the absence of pvcytb mutations [P. malariae surviving treatment and chemoprophylaxis with a number of regimens including AP [cytb mutations have not previously been evaluated in P. malariae, so it is unclear whether this phenomenon represents an acquired resistance mechanism, or an intrinsic biological characteristic of this parasite.Among the more effective regimens for chemoprophylaxis in travellers, atovaquone as a fixed dose combination with proguanil (AP) is particularly recommended in areas where revalent . Althougmalariae ,19, we hy travel . Recrudepatients –23. Sequ in vivo –23. Despan 99.9% . Althougutations . Curiousuding AP , 24, 26.P. malariae infections were derived from data compiled by specialist centres in China and Sweden and from the UK MRL database. Cases of P. malariae infection in travelers reporting use of AP for chemoprophylaxis, and for whom frozen blood samples were available for DNA analysis, were identified in the UK MRL database between 2007 and 2010, and sequences at codon 268 of pmcytb elucidated in each of these putative breakthrough infections. Our findings are discussed with reference to published examples of long-term persistence of infections with this parasite species, and propose reconsideration of the possibility that latent liver-stage hypnozoites do occur in P. malariae.In this study, estimates of the latency period of imported P. malariae malaria reported to the PHE MRL, London, from 1991 to 2010, cases of P. malariae malaria seen at the Karolinska University Hospital, Stockholm, Sweden, between 1997 and 2013 and cases of P. malariae infection diagnosed in Jiangsu Province and reported to the Institute for Parasitic Diseases, Wuxi, China, were available. For a subset of the London series, DNA samples were provided for pmcytb gene sequencing. All cases in each site were identified by expert microscopists working at Reference Laboratory standard. PCR confirmation was also carried out for all cases from China, five of those from Sweden and all six UK cases subjected to pmcytb sequencing.Anonymised clinical and epidemiological data on cases of pmcytb analysis was identified among entries in the database collected by the PHE MRL at the London School of Hygiene and Tropical Medicine (LSHTM). Patients were selected who satisfied all three of the following criteria:The sample set for reported use of AP chemoprophylaxis whilst travelling abroad;P. malariae infection confirmed by the MRL;diagnosis of slide-positive availability of a blood sample or purified DNA in the MRL sample archive.Sixteen samples were identified which fitted criteria 1 and 2, for six of which a blood sample was available. Drug resistance testing was performed under the surveillance remit of the MRL. All patient identifiers have been removed.P. malariae cytochrome b . Primer sequences were as follows:Parasite DNA was extracted from patient blood samples using the QIAamp DNA Blood Mini Kit  according to the manufacturer’s instructions. Primers were designed specifically for this study, based on Pmcytb1 5’-TGTTTTACCATGGGGACAAAT-3’Pmcytb2 5’-CACCAATACAAATTGTTCCAGA-3’Pmcytb3 5’-TGTGGTAATTGACATCCAATCC-3’pmcytb locus. The presence of mutations at codon 268 was investigated by direct sequencing of PCR amplicons.Sequencing templates were amplified using a hemi-nested protocol which generated a 635bp nest 1 product (Pmcytb1 + Pmcytb3) and a 253bp nest 2 product (Pmcytb2 + Pmcytb3) encompassing codons 255 to 337 of the For the primary amplification reaction, a 25μL mix that contained 1X KCl PCR buffer , 15mM MgCl2, 0.2mM deoxyribonucleotide triphosphates , 0.25nM each of primers Pmcytb1 and Pmcytb3 , 0.2μL Taq polymerase , and 2μL of extracted parasite DNA was initially heated at 92°C for 3 minutes, then cycled at 92°C for 30 seconds, 55°C for 50 seconds, and 68°C for 90 seconds for 35 cycles, with a final extension at 68°C for 5 minutes. 1μL of PCR product was then added to 19.3μL of nuclease-free water, 0.5μL Pmcytb2 and Pmcytb3 each, and identical volumes of dNTPs, Taq polymerase, and PCR buffer as described in the first amplification reaction to make a total mix of 25μL. 30 further cycles of PCR were applied, as above. PCR products were sequenced using the ABI Prism BigDye Terminator kit  according to the manufacturer’s instructions, primed by Pmcytb2 and Pmcytb3. Sequencing data was visualized and analyzed using Chromas software .2 distribution. Statistical analysis was performed in STATA 12 . All data were de-linked from personal identifiers.Continuous data (number of days delay to appearance of symptoms) were not normally distributed, and comparisons between categories were therefore performed using the non-parametric Wilcoxon’s rank-sum test. Categorical data were analysed in 2 x 2 tables to provide odds ratios (OR), and statistical significance was estimated from the χP. malariae infection. These comprised:Two parameter, time elapsed from arrival in a non-endemic setting to symptomatic presentation for diagnosis, and the delay to onset of symptoms, were explored among 638 cases of -P. malariae malaria in various hospitals, and confirmed by the Jiangsu Institute of Parasitic Diseases (JIPD), in Jiangsu province, China between 2012 and 20136 travellers diagnosed as imported cases of -P. malariae malaria identified at Karolinska University Hospital, Stockholm, Sweden, between 1997 and 201321 cases of imported -P. malariae malaria presenting to UK hospitals between 1991 and 2010 and reported to the UK MRL.611 cases of imported For many individuals, insufficient data were available to estimate either or both parameters of interest. P. malariae malaria was tested as a likely modulator of delay to onset of symptoms. Patients reporting no chemoprophylaxis while travelling in endemic countries experienced a geometric mean delay of 20.0 days  between arrival in the non-endemic country and onset of symptoms, compared to a geometric mean delay of 36.1 days  in those reporting any chemoprophylaxis use . Reported use of chemoprophylaxis prior to diagnosis with P. malariae malaria cases for which data were available reported chemoprophylaxis use, an estimate very similar to that for 4,133 cases of the relapsing parasite P. vivax  [Among 77 patients reporting use of a particular chemoprophylaxis regimen, and for whom date of onset of symptoms was also available, the three most frequently used drugs were mefloquine (N = 29), chloroquine  and AP (N = 17). Of these, mefloquine generated the greatest increase in geometric mean delay to symptoms , followed by AP . In total, 26.5% of the 378 P. malariae cytochrome b sequence (NCBI accession number: AB354570.1) isolated from a human case  [DNA sequence data were successfully obtained for isolates from six patients with recorded use of AP chemoprophylaxis prior to onset of malaria. These data were aligned with published man case . No muta68607.1) .P. malariae malaria evaluated the time elapsed between post-travel arrival in China, Sweden or the UK and clinical presentation and diagnosis of malaria as a proxy measure of latency. A significant relationship between use of chemoprophylaxis and delayed presentation was observed, although some late presentations with P. malariae malaria were also seen in patients with no history of chemoprophylaxis use during travel. Out of 359 evaluable P. malariae cases in this study, 26.5% reported using at least partial chemoprophylaxis, closely resembling the patterns seen with the relapsing parasites P. ovale curtisi, P. ovale wallikeri and P. vivax, whereas for 2,814 imported cases infected with the non-relapsing parasite P. falciparum, only 7.5% reported chemoprophylaxis use , or mutations at other positions of pmcytb, as were reported in experimentally drug-pressured atovaquone-resistant P. yoelii parasite lines, where codons other than position 268 of the pycytb locus were found to be substituted with novel amino acids [In our series of 6 patients with no acids . Furtherpmcytb gene only previously reported from a wild-caught chimpanzee in Sierra Leone. This provides some support for the hypothesis that African malaria parasites of all species shuttle between great ape and human hosts [P. falciparum and P. vivax suggest [pmcytb could be of human origin, existing as a minor variant across P. malariae populations among Homo sapiens in Africa, which happened to be transferred to Pan troglodytes verus in Cote d’Ivoire. Alternatively, there may be genetically diverse P. malariae across the wild ape populations of central and western Africa, which provide a reservoir of new genetic variants for human-focussed transmission cycles [An unexpected finding among our sample set was an isolate from Uganda that encoded an amino acid change in the an hosts , as rece suggest ,31. Thisn cycles . FurtherP. malariae is a truly relapsing parasite species, and has a quiescent liver-stage form analogous to the well-described hypnozoites of P. vivax and the simian parasite P. cynomolgi. This hypothesis could be tested experimentally, as Dembele et al [Macaca fascicularis with sporozoites of Plasmodium cynomolgi and directly demonstrate persistence and later activation of hypnozoites. Infection of human hepatocytes with P. malariae sporozoites should be technically feasible. Our conclusion is in contradiction to the findings of Ciuca et al. [P. malariae to three volunteers by sporozoite inoculation together with a single dose of 50mg pyrimethamine. Over 8 to 18 months’ follow-up, none of the volunteers developed a blood-stage P. malariae infection. Similarly, six volunteers with experimentally-induced blood-stage parasitaemia with the same Romanian P. malariae isolate were successfully treated with various antimalarial regimens. No relapses occurred in any of these volunteers over 15 months’ follow-up. These authors conclude the following: “Ces constatations imposent la conclusion logique que P. malariae n’aurait qu’une phase tissulaire préérythrocytaires unique << la schizogonie exoérythrocytaire primaire>>.” (There is only a single hepatic shizogony cycle in each infection.) [et al comment on these findings of Ciuca et al thus: “In this connection, we also have observed no relapse activity after adequate blood schizonticidal therapy in our volunteers exposed to infection with a Nigerian strain of P. malariae. It seems safe to say that, in this respect, P. malariae resembles P. falciparum more than it does the human tertian malarias which do relapse.” [We find support in our data for the notion that le et al  have reca et al. , who admelapse.” P. malariae does not relapse. Firstly, the initiation of a secondary hepatic cycle may be a rare event in P. malariae and thus a handful of volunteers may not have been a large enough sample to detect relapse events. Secondly, follow-up of less than 2 years may have missed late events in these individuals, as P. malariae is without doubt the human parasite with the greatest within-host longevity. Thirdly, the putative hypnozoite stage of P. malariae may not be identical to that of P. vivax in its mechanism of arrest; if metabolism halts at a later stage of intra-hepatocytic development, these forms may not be completely insensitive to full therapeutic doses of pyrimethamine or other antimalarials . Finally, some “strains” of P. malariae may have lost the ability to form hypnozoite-like stages in the host liver, and thus the single Romanian parasite line of Ciuca et al. [et al. [There are several tentative explanations that might help us to reconcile our findings with the earlier evidence from experimentally induced infections that a et al. , and the [et al.  could ha [et al. .pmcytb sequences of P. malariae isolates from a subset of users of atovaquone-proguanil chemoprophylaxis, lead us to conclude that P. malariae may be able to form hypnozites, or an analogous liver-stage form. As Garnham has observed: “… true long term relapses of sporozoite induced infections seem more likely to be due to a secondary exoerythrocytic cycle in the liver, known to occur in other species, than to a persistent low-grade parasitaemia.” (P. 269) [P. malariae is a relapsing parasite.Analysis of delay to onset of symptoms in a large series of imported quartan malaria cases in three non-endemic countries, and elucidation of the (P. 269) . Whereas(P. 269) , our dat