Two herds of dromedary camels were longitudinally sampled with nasal and rectal swabs and serum, between September 2014 and May 2015, and the samples were tested for Middle East Respiratory Syndrome (MERS) coronavirus RNA and antibodies. Evidence of MERS-CoV infection was confirmed in one herd on the basis of detection of virus RNA in nasal swabs from three camels and significant increases in the antibody titers from three others. The three viruses were genetically identical, thus indicating introduction of a single virus into this herd. There was evidence of reinfection of camels that were previously seropositive, thus suggesting that prior infection does not provide complete immunity from reinfection, a finding that is relevant to camel vaccination strategies as a means to prevent zoonotic transmission. We therefore carried out a longitudinal study of two camel herds in the Kingdom of Saudi Arabia to elucidate MERS-CoV infection and transmission.Vaccination of dromedary camels has been proposed as a means to reduce the threat of zoonotic MERS.Nasal and rectal swabs and serum samples were collected from dromedary camels in two herds in the Eastern and Central regions of Saudi Arabia between September 2014 and May 2015. The same animals were resampled whenever possible. The ages of the animals were assessed on the basis of farm records and, when records were not available, by examination of dentation. Swab samples were collected in viral transport medium and stored at −80 °C.10This group was a closed camel herd of ~80 animals in the Eastern Province. The camels were housed in one compound and provided with feed in barns. There was no contact with nomadic camel herds. Occasionally, animals purchased from outside  may be introduced into the herd. A previous study of this camel herd has been published.This group was a camel herd of ~100 dromedaries in the Central Province. The animals were held in one barn and were separated into several subgroups . The different animal groups were separated within the same compound by only a fence. Although this herd was largely closed, animals purchased from local markets are occasionally introduced to the herd. Nomadic camel herds of Bedouins graze in the surrounding area, especially in the fall and winter. Other animals, such as sheep and goats, are also sometimes present in the same area. Stray dogs, foxes, rodents and birds such as doves and crows are also frequently seen in proximity to this herd.11 A 7675 nucleotide (nt) region of the genome from the spike gene to the N gene (22 140–29 814 nt in the reference MERS-EMC strain sequence) was RT-PCR amplified as overlapping PCR amplicons and sequenced by Sanger sequencing from any RT-PCR-positive samples.The total nucleic acid extracted from the swabs was tested for MERS-CoV RNA by using reverse transcription PCR (RT-PCR). An RT-PCR assay targeting the region upstream of the envelope protein gene (upE) was used for screening, and the confirmation of any screen-positive samples was done using RT-PCR that targeted open reading frame 1a (ORF-1a).12Evidence of other coronaviruses was sought by testing the swab samples using a pan-coronavirus-nested PCR that was targeted at the conserved RNA-dependent RNA polymerase (RdRp) gene of coronaviruses that we have previously developed and reported.13MERS-CoV antibody was tested using a validated MERS-CoV spike pseudoparticle neutralization test (ppNT) as previously described.In herd 1, 29 camels, including 2 calves, were sampled; 8 were sampled in October 2014, 9 in November 2014, 15 in January 2015 and 13 in February 2015. Animals had both swabs and serum collected, with the exception of one animal in November, one in January and one in February. The MERS-CoV RNA was not detected in any of the camels sampled from herd 1 . All theIn herd 2, 70 dromedaries were sampled, including 16 calves ⩽2 years of age, 5 animals 3–5 years of age, 35 animals ⩾6 years of age and 14 animals of undetermined age. Ten camels were sampled in September 2014, 43 in October 2014, 28 in November 2014, 45 in January 2015, 24 in February/March 2015 and 52 in May 2015. The number of swabs and sera collected at each sampling occasion is detailed in 3 copies per mL (specimen F2-3); 0.7 × 103 copies per mL (specimen F2-33) and 1.4 × 103 copies per mL (specimen F2-51)  . These tn F2-51)  and 4. Nn F2-51)  and 4. TA 7675 nt region of the genome from the spike gene to the N gene was sequenced from the three positive samples (GenBank accession numbers KY706245–KY706247). These three sets of sequences were found to be identical to each other, thus suggesting that a single virus had been introduced into this herd. The sequences were closely related but not genetically identical to the MERS-CoV strain Riyadh/Riyadh179/2015 (GenBank accession no.: KT368875), which has been previously reported in dromedaries in Saudi Arabia . In addiPan-coronavirus RT-PCR detecting most known (and likely unknown) coronaviruses was negative.17 One of these herds, namely, herd 2, had evidence of an active MERS-CoV infection. The RT-PCR detection of MERS-CoV confirmed the infection of three camels in November 2014, and viral molecular epidemiology confirmed that these three infections most probably arose from introduction of one virus into the herd. The three animals that were RT-PCR positive for MERS-CoV in November 2014 were seropositive in the previous serum sampling in October but were reinfected in spite of prior moderate (titer 1:320) or high (1:40 960) antibody titers. Because these animals were 2 years of age, the serum antibody that was detected prior to infection would have resulted from infection after birth rather than being antibody passively acquired from the dam. Given the camels’ age, such natural infections had occurred within the past 2 years.Our longitudinal study on two dromedary herds demonstrated high levels of seropositivity in both herds, a result in agreement with findings from many other studies reporting high seroprevalence in adult animals in the Arabian Peninsula and Africa.10 Because adult animals (which are very likely to be seropositive) and a 2-week-old calf  were found to be infected, it was presumed that neither prior natural infection nor passive antibodies would produce sterilizing immunity.10 However, there was no conclusive evidence of antibody detected prior to infection. The lack of correlation between the viral RNA loads and the levels of neutralizing antibody in camels slaughtered in an abattoir in Qatar has also been taken to indicate that prior antibodies may not provide sterilizing immunity.17 However, again, conclusive evidence of the presence of antibodies before infection was not available. In contrast, a study of infection within camel herds whose dam and calf pairs were studied, has reported that infection occurred in the calves but not in the seropositive dams, thus suggesting that prior immunity in older animals may prevent reinfection.16 In another study in which 11 calves were prospectively followed up during the first year of life, five calves were found to be infected within the first month of life even though they had evidence of passively acquired maternal antibodies, thus suggesting that the maternal antibodies do not provide complete protection from reinfection.18 Evidence of reinfection of adult seropositive camels was also reported in a longitudinal study of camels in Egypt.19A previous study of another infected camel herd has shown infection of both adults and calves.The present study provides conclusive evidence that reinfection of previously seropositive camels can occur. This observation has important implications for the feasibility of using vaccination of camels as a means to control MERS-CoV transmission within camel herds with the aim of reducing zoonotic transmission. Reinfection in previously seropositive animals may occur because MERS-CoV infection in camels is a mucosal infection and the serum antibody might not be an accurate predictor of the effective mucosal antiviral immunities that can provide sterilizing immunity. None of the studies to date, including our own, have tested for evidence of mucosal IgA immunity in the oral or nasopharyngeal cavity, and this deficiency remains a crucial gap in the understanding of protection from reinfection.7 TCID50 MERS-CoV inoculated intra-nasally showed a decrease, but not a complete elimination of detectable virus RNA or infectious virus.9 However, this test was an experimental challenge with what may be a non-physiological dose of virus and after a very short post-vaccination interval. These conditions may not reflect the situation in the field. Experimentally infected alpacas have been found to be protected against MERS-CoV reinfection; again, this study involved a challenge that occurred at a short interval after the previous infection.20The lack of protection against natural reinfection in the field thus raises questions about the potential duration of protection conferred by MERS-CoV infection or vaccines. Previous experimental studies of camels vaccinated with a vaccinia-vectored MERS-CoV vaccine that were challenged 3 weeks later with a dose of 103 to 2.2 × 103 copies per mL. In contrast, the viral loads in camels in our previously reported study of transmission within a camel herd ranged from 3.3 × 103 to 1.78 × 108 copies per mL. It is not clear whether the low viral load observed in the reinfected animals in herd 2 would be sufficient to result in onward transmission to other animals.In contrast to data from the experimental challenge of vaccinating animals, our studies have merit in that the infecting dose leading to infection was physiologically relevant to the field conditions and that the challenge probably occurred many months or years after the initial infection. The viral load in nasal swabs in the three RT-PCR-positive specimens was low and ranged from 0.7 × 1010 Data from this study showed that camels in herd 2 that were RT-PCR positive did not demonstrate a fourfold increase in antibody titer, although two camels showed a non-significant twofold increase in titers. Thus, we cannot exclude infections in other sampled animals whose antibody titers were not observed to rise by fourfold, the usual criterion for a significant antibody response. Because of these observations, and because all animals in herd 2 were not sampled, our detection of six infected camels is likely to be an underestimate of the extent of transmission within this herd.In addition to the three RT-PCR-positive camels, the serology data confirmed evidence of infection in three other camels from as early as September–October 2014 and confirmed that the infection was active until the November 2014–January 2015 period. Whether these infections were also a result of the same virus introduction cannot be confirmed, but this possibility is likely. If this were the case, a single introduction of MERS-CoV would probably have led to virus circulation in herd 2 from September 2014 until at least November 2014, a period of 2–3 months. In a previous study of herd 1, we have demonstrated virus circulation for at least 1 month, November–December 2013.P=0.0056). This result suggested that younger camels are more likely to acquire infection within an infected herd, as has been previously reported.16Age data were available for 56 of the sampled camels in herd 2. Among those animals with a known age, 5 (83.3%) of the 6 camels with evidence of MERS-CoV infection, either by RT-PCR or serological tests, were aged 2 years or younger, whereas only 11 (22.8%) of the 50 MERS-CoV-negative animals were of similar age  against viruses are very difficult to accurately assay because of the presence of many non-antibody-mediated mechanisms for virus neutralization in saliva.21 Thus, virus antigenic variation was unlikely to confound our serological tests or lead to antigenic escape from prior antibody immunity.Another limitation in our study was that our neutralization tests were done with MERS from the EMC strain rather than a contemporary MERS-CoV strain. However, we and others have shown that genetically diverse MERS-CoV are antigenically homogenous in cross-neutralization tests carried out by conventional microneutralization or pseudotype neutralization tests.In conclusion, we demonstrate the reinfection of camels that were previously seropositive with very high serum antibody titers. It may be possible that such reinfections are associated with lower viral loads and may be less transmissible to other animals, but this hypothesis remains to be confirmed. Well-designed field studies of vaccinated animals followed up over a period of longer than 1 year would be required to assess the effects of vaccination on decreasing virus transmission between camels. Longitudinal studies of dromedary herds such as those reported here provides useful data for the design of such systematic studies.


P = 0.911) and 6  months. The intervention decreased the time-to-recovery , improved the recovery rate , and the absolute weight gain . No statistical differences were noticed for the diarrhea longitudinal prevalence  and the weight gain velocity . Our results showed that adding a household WASH package did not decrease post-recovery relapse rates but increased the recovery rate among children admitted in OTP. We recommend further robust trials in other settings to confirm our results.Water, sanitation and hygiene (WASH) interventions have a small but measurable benefit on stunting, but not on wasting. Our objective was to assess the effectiveness of a household WASH package on the performance of an Outpatient Therapeutic feeding Program (OTP) for severe acute malnutrition (SAM). We conducted a cluster-randomized controlled trial embedded in a routine OTP. The study population included 20 health centers (clusters) from Mao and Mondo districts in Chad. Both arms received the OTP. The intervention arm received an additional household WASH package . The primary objective measures were the relapse rates to SAM at 2 and 6 months post-recovery. The secondary objectives included the recovery rate from SAM, the time-to-recovery, the weight gain, and the diarrhea longitudinal prevalence in OTP. The study lasted from April 2015 to May 2016. Among the 1,603 recruited children, 845 were in the intervention arm and 758 in the control arm. No differences in the relapse rates were noticed at 2 (−0.4%;  Interventions in HCs include the provision of a household WASH package designed to protect children against new episodes of diarrhea and aiming at improving nutritional outcomes.To increase synergies in the fight against acute malnutrition, WASH and nutrition actors led by United Nations International Children’s Fund (UNICEF) and European Civil Protection and Humanitarian Aid Operations, agreed in 2012 on a “WASH in Nut” strategy for the Sahel region.10 In 2013, the Kanem nutrition rehabilitation program admitted around 11,000 children for noncomplicated SAM . Besides acute malnutrition, diarrheal diseases represented the largest notified disease among children (32% of the HC admissions). The prevalence of intestinal parasitic infection is reported to be 60% , and worm infestation has been attributed to water quality.11In Chad, undernutrition remains a public health concern. In the Kanem region (northwest of the country), the prevalence of acute malnutrition was reported beyond emergency thresholds in 2014 .Against this backdrop, we hypothesized that nutrition rehabilitation of SAM could benefit from a complementary WASH intervention at the household level. Improving water quality and hygiene-related care practices at household level would decrease the incidence of diarrhea during the Outpatient Therapeutic feeding Program (OTP), and consequently, improve recovery, daily weight gain, and shorten the children’s time-to-recovery. Furthermore, it was expected that in the months after discharge from OTP, the improvement of WASH-related practices would decrease the risk of WASH-related infections. This would possibly improve the child’s immune recovery and reduce the risk of relapsing after successful discharge.12 or after discharge when immune recovery is still incomplete.13 Therefore, we investigated the additional benefits of a WASH intervention among 6- to 59-month-old children admitted to an OTP in the Kanem region in Chad. The primary outcome measures were the relapse rates at 2 and 6 months post-recovery. Secondary outcomes included recovery rate, time-to-recovery, and weight gain and diarrhea longitudinal prevalence at OTP discharge.To our knowledge, no WASH intervention has been assessed so far, either as a complement to a nutrition rehabilitation program,Z (WHZ) score of < −3 and/or a mid-upper-arm-circumference (MUAC) of < 115 mm, and/or the presence of mild or moderate bilateral edema. Children with signs of medical complications requiring inpatient management or severe bilateral edema were not included in the study and referred to the nearest hospital for inpatient management after agreement of the caretaker.This study was conducted in Mondo and Mao districts, Kanem region, Chad. The design was a cluster-randomized controlled trial embedded in a routine nutritional program for outpatient SAM management supported by the international non-governmental organization Action Contre la Faim (ACF). Health centers were the unit of randomization. Twenty of the 35 existing HC in the study area were included in the study based on their location (they were not contiguous from each other to avoid contamination among participants) and their performance . All new admissions without complications were eligible to the study. Children transferred from other HC, where their treatment had already started, were excluded. Routine criteria for OTP admission included children aged between 6 and 59 months with a weight-for-height 14 and basic hygiene education and care practice sessions during HC visits. Intervention clusters also received the household WASH kit at admission. This contained a safe drinking water storage container with a lid, water disinfection consumables (180 chlorine tablets), 12 bars of soap for hand washing, a plastic cup with handle (to be reserved for the child to facilitate safe drinking water practice), and a laminated leaflet with pictures representing the main hygiene messages. They also participated to a promotion session on the kit use at each weekly visit to the HC and two extra home visits for assessing and reinforcing adherence. Specifically, promotion at HC included key messages on 1) a protected space for children to play; 2) washing the child with soap; 3) cleaning and rapid burial of children’s stools; 4) hand washing at key times; 5) safe storage of water; 6) exclusive breastfeeding of children before 6 months; and 7) water treatment and food hygiene. The household WASH kit was designed to last for 3 months (2 months during treatment in the OTP and 1 month after the end of the treatment).Both groups received OTP services as routinely implemented ®; Hydrachem Ltd, Billlingshurst, United Kingdom) given was based on the need to purify around 40 L of water per family per day (two tablets). The number of soap bars corresponded to 250 grams per person per month for the mother and her child plus 250 grams per month for the rest of the family sufficient to cover a 3-month period. As compensation, the control group received a loincloth at the end of the OTP phase.The amount of chlorine tablets  during home visits at 2 or 6 months after a successful discharge. Relapsing at 2 months was considered as a censoring event and these children were not followed up at the 6-month visit.Secondary outcome measures included the recovery rate from SAM, the time-to-recovery, the weight gain and the diarrhea longitudinal prevalence at OTP discharge. Recovery rate was assessed based on whether a child was cured at discharge. Depending on the admission criteria, being cured was defined as either achieving a MUAC of 125 mm or a WHZ score of ≥ −2 taking length at admission, with no presence of bilateral edema in the previous 14 days. It was noticed during the analysis, that in the routine OTP practice, anthropometric criteria for discharge were not always strictly applied and discharge defined as “cured” was decided based on clinical appraisal before anthropometric normalization. Poor adherence to the protocol could have introduced a misclassification bias. To assess this potential bias on the recovery rate, we performed a sensitivity analysis by defining children as cured strictly according to their anthropometric measurements (and not to their discharge status).Daily weight gain velocity was calculated for cured children according to the following formula:In addition, absolute weight gain was calculated using weight at discharge minus weight at admission (in gram) as numerator and length of stay (days) as denominator. Length of stay (days) was calculated only for cured children. Diarrhea was defined as having loose or watery stools at least three times per day during the week before the visit at the HC during the OTP phase.Other possible outcomes at discharge were “defaulter” (when the child was absent during two consecutive visits), “died,” “transferred to a stabilization center,” or “nonresponder” . The number of days with vomiting, fever, and cough were based on recall of the mother during the week before the visit during the OTP phase.Hygiene knowledge and related care practices were evaluated through a knowledge, attitude and practice survey using standardized questions based on self-reporting. A WASH score (ranging from 0 to 8) was calculated by assessing eight behaviors of the child’s caretaker (one point for a correct and 0 for an incorrect behavior). The eight WASH behaviors included 1) child’s body hygiene; 2) fecal contamination–free child play areas; 3) safe disposal of child’s faces; 4) household drinking water treatment; 5) transport of water from the source to the point-of-use; 6) safe water storage; 7) knowledge of the key times for hand washing; and 8) food hygiene.Health centers were stratified in pairs (intervention and control) according to the monthly number of SAM admissions (historic data from the year 2013) to obtain a balanced number of enrolments in the two arms. We randomly extracted one letter of the alphabet and we assigned within each pair the intervention to the HC with the first letter of its name closest to this letter. Masking of participants was not possible because of the nature of the intervention. To limit judgment bias, the research and HC staff members were different. Whereas the HC staff collected nutritional and morbidity data in OTP, research staff was responsible of collecting WASH data. It was not possible to blind research staff, but they rotated so they covered different groups. Recruitment started 1 month after allocation of each HC to either group.15 we estimated a cumulative true relapse rate of 12% during the 6 months after successful discharge. Furthermore, we estimated a coefficient of variation of the true rate between clusters within each group (k) of 0.17 based on the number of admitted cases in the 20 clusters as reported in program data in 2013. Using the formula presented by Hayes et al.,16 an alpha error of 5% and a beta error of 20%, we needed 1,400 cured children from 20 clusters to detect a 5% difference between the two groups.Program data indicated that 20% of cases were relapse cases at admission but the relapse rate post-recovery remained unknown in the study area. Based on other studies,Z (HAZ) scores and WHZ scores were calculated based on the UNISEX Z scores table, as used by the Chad Ministry of Health.17 Water, sanitation and hygiene variables were collected by the field monitors at the HC through a standardized questionnaire, at admission and discharge. During the OTP phase, two home visits in the intervention group aimed to measure water quality , to collect information on the adherence to the WASH package, and to provide extra promotion session on the usage of the WASH kit. In the study area, chlorine was not available, and therefore, no data were collected in the control group. Turbidity was measured with a two-part turbidity tube to assess the suitability of the chlorine intervention. Free chlorine  was measured with a pooltester with a detection limit of 0.1 mg/L. According to ACF standards,18 correct turbidity and correct chlorine residual were defined between 0 and 20 nephelometric turbidity units and ≥ 0.5 mg/L, respectively. Acceptance of chlorinated water was self-reported by the mother and defined as “at least the child drinks the chlorinated water.” In addition, cured children from both groups received home visits 2 and 6 months after OTP discharge to assess whether the child relapsed and to collect data on hygiene knowledge and related care practices. All collected forms were inserted in an open data kit database with integrated validity checks. Data were double entered by two different data managers and checked for consistency and accuracy by the project manager.The study recruitment phase lasted from April to December 2015 and the follow-up phase finished by May 2016. Ten field monitors were recruited locally and received a technical training in Mao on appropriate data collection, water quality tests, and how to promote the WASH kit. The HC staff members as part of their routine procedures collected weekly data on sociodemographic characteristics, nutritional status, morbidity, and treatment. Height was measured to the nearest 1 mm  and weight was measured to the nearest 100 grams  for children weighing less than 10 kg). Mid-upper-arm-circumference was measured to the nearest 1 mm using an arm circumference measuring tape. Height-for-age 19 We compared categorical and continuous variables at baseline with clustered χ2 tests and clustered t tests, respectively (clustering was at HC level). Kaplan–Meier multilevel survival analysis was used to display the length of stay, with time-to-recovery as censoring event and a maximum stay of 12 weeks. Multilevel mixed-effects linear regression models at individual level were used for all outcomes, with a random effect for clusters to account for the clustered design.20 All models were adjusted for the number of missed visits (no treatment because of ready-to-use therapeutic foods shortage), WHZ score at admission, gender, and age of the child. We ignored the pairing in our analysis as it has been shown to be valid and efficient when trials have small number of relatively large clusters.21 Outcomes were presented as means with their standard errors for continuous outcomes and as rates for binary outcomes estimated from the models. Intervention effects were presented as absolute differences with their 95% CI. Longitudinal prevalence for morbidity outcomes was only measured during the time that the child was in OTP and defined as proportion of time under observation with the disease.22 Total morbidity was calculated by summing up all symptom days.All analyses were conducted in STATA 13  on an intention-to-treat basis by using the full dataset. Statistical significance was set at < 5% with a two-sided test. For anthropometric values, World Health Organization standards with recommended flag limits were used . In the sensitivity analysis, despite a lower absolute recovery rate, the effect of the WASH intervention did not change .The recovery rate was 10.5% higher in the intervention group compared with the control group, with 92.4% and 81.9% of the cases, respectively . Survival functions show that time-to-recovery curves diverge . Absolute weight gain was 3 g/d significantly higher in the intervention group . Diarrhea longitudinal prevalence was low in both groups and not statistically different.Time-to-recovery was 4.4 days shorter in the intervention group (51.7 days) compared with the control group (56.0 days) ( diverge  over theP = 0.009). Compared with the control group, the longitudinal prevalence of reported symptoms was lower in the intervention group, although not statistically different, except for vomiting (P = 0.023).The rates of the other types of discharge did not differ between the groups, except for the nonresponder rate, which was lower in the intervention group (P = 0.6695). Each visit with a shortage in RUTF was associated with a 2.5% reduction in the recovery rate (P = 0.000) and with a 0.2% increase in the transfer rate (P = 0.039).During the OTP phase, shortages of RUTF affected similarly the two groups . Both groups improved their WASH-related practices from admission to discharge but the increase was statistically higher in the intervention group . After discharge, whereas the WASH score remained stable in the control group, it decreased in the intervention group and became not statistically different from the control group at 6-month follow-up.The WASH score was not significantly different at admission between the two groups, with a mean of around five points on a scale of 8  to one-third (flocculation and disinfection sachets).25 Still, other pathways in the fecal-oral transmission routes remain. Latrine use is limited in the study area and a large part of the population still practices open defecation. This can provide another source of contamination that offsets the gains of safe water provision and soap. The process indicators also showed that practices improved but without reaching a high level of compliance. The adoption of improved practices might just not have been enough to show a difference in longitudinal diarrhea prevalence. The relatively small number of reported diarrhea cases in the whole group was unexpected and may have diluted the intervention effect. Underreporting is classically seen for self-reported morbidity and might have affected all participants in our study.29 Indeed, both groups received basic hygiene education and care practice sessions. Furthermore, study staff who provided these sessions was different from the HC staff who collected information about child morbidity. This low longitudinal prevalence could also explain the nonsignificant increase in weight gain velocity, which has been reported to be strongly associated with diarrhea longitudinal prevalence in children.22Our results reported an increased recovery rate in the intervention group. This result was based on clinical judgment but was confirmed in the sensitive analysis based on anthropometric data, excluding any potential misclassification bias. Difference in the total recovery rates between the two analyses highlighted some difficulties for field staff to adhere strictly to the SAM protocol in routine practice. The causal mechanism leading to increased recovery rate in the intervention group is not straightforward to identify. One possibility is through a reduced duration of infectious disease episodes;30 This reduction would reduce the costs, both for the program and the beneficiaries. However, this result should be taken with caution, as it is not possible to exclude a misclassification bias due to the staff judgment. The sensitive analysis reported a nonsignificant difference, which could also be due to a lack of power.The observed reduction in the time-to-recovery is in line with a study conducted in the Democratic Republic of Congo, where the use of flocculent-disinfectant for water was linked to a 4-day reduction in the time-to-recovery.31Finally, our results showed that although the household WASH package was well accepted and used, there is still potential for improvement and therefore potential in its effect. Indeed, self-reported practices often overestimate true practices because of social desirability bias. Although it was reported that more than 80% used the correct chlorination procedure at the second visit, the correct level of residual chlorine in the water was only reported in 50% of the households. We cannot exclude that other non-WASH–related behaviors  may have accounted for the improved recovery. This particularly indicates the need to improve the promotion component when delivering the kit. The storage container was correctly used in only 50% of the households. Containers with a tap would be a solution to avoid contamination of water when chlorine residual is < 0.5 mg/L. Alternatively, an increased chlorine dosage (without compromising acceptance) could maintain an adequate concentration for a longer duration.Giardia33 or environmental enteric dysfunctions (EED).34 We cannot exclude that these may have affected recovery rates. Easy-to-use diagnostic tools for EED are necessary if we want to better interpret WASH intervention pathways. Unfortunately, logistical and organizational constraints in our setting did not allow us to conduct more advanced etiological diagnostics on diarrhea. Further studies should include this component. It is also to be mentioned that the observed positive results were obtained after two home visits in the intervention group, which is not done in standard “WASH in Nut” interventions.The shortage in RUTF during the study period was a potential bias. However, our results showed that both groups were equally affected. Furthermore, all models were adjusted for the number of visits with shortage. The way diarrhea was defined excludes more chronic forms of enteropathies with less frequent passages of stools, such as In the Sahel context, the provision of a household WASH package increased the recovery rate among children treated for noncomplicated SAM and decreased their time-to-recovery. This study shows that the integrated “WASH in Nut” approach has the potential to increase program performance. We recommend further robust trials in other settings to confirm our results. In the meantime, our study can inform health authorities in settings where the WASH component is not yet integrated into the nutritional programs.


Despite investigation of many methods, such transmutation remains technologically difficult. To establish an effective and efficient transmutation system, we propose a novel neutron moderator material, yttrium deuteride (YD2), to soften the neutron spectrum leaking from the reactor core. Neutron energy spectra and effective half-lives of LLFPs, transmutation rates, and support ratios were evaluated with the continuous-energy Monte Carlo code MVP-II/MVP-BURN and the JENDL–4.0 cross section library. With the YD2 moderator in the radial blanket and shield regions, effective half-lives drastically decreased from 106 to 102 years and the support ratios reached 1.0 for all six LLFPs. This successful development and implementation of a transmutation system for LLFPs without isotope separation contributes to a the ability of fast spectrum reactors to reduce radioactive waste by consuming their own LLFPs.Transmutation of long-lived fission products (LLFPs:  Although nuclear energy does not release greenhouse gases, it does produce highly radioactive spent nuclear fuel (SNF)2. After recovering U and Pu from SNF by conventional reprocessing, such as the PUREX process, the disposal of high-level radioactive wastes is a major concern in many countries. Most of the radioactive hazard remaining in high-level radioactive wastes after thousands of years comes from minor actinides  and some long-lived fission products 3. The disposal of such SNF in stable deep geological repository has been considered, however, it has not been fully implemented.Nuclear energy provides 16% of the world’s electricity production14. The P&T strategy consists of two major techniques, elemental separation of MAs and LLFPs from high-level radioactive wastes by a chemical process (partitioning) and nuclear reactions of these nuclides into short-lived or non-radioactive nuclides (trasmutation) in nuclear reactors or accelerator-driven transmutation systems(ADS). The P&T strategy has been well studied for MAs20, while little attention has been paid to LLFPs especially for transmutation of LLFPs. After the amount of MAs is reduced by transmutation, LLFPs become the main source of the hazard. It is because they are associated with radioactive release from geological repositories owing to their high solubility in water and mobility in the geosphere22.The partitioning and transmutation (P&T) strategy has been proposed and research and development have begun in several countries to reduce the inventory of radioactive wastes to be stored and the long-term hazard for future generations25. The transmutation of LLFPs is a neutron-consuming process and it requires high neutron flux and excess of neutrons in principle. After partitioning, the isotopic compositions of the LLFPs are not monoisotopic, but are a mixture of stable and radioactive isotopes including LLFP. The consumption of neutrons to transmute the LLFPs is much larger than when isotope separation is applied because isotopes other than the LLFPs also capture neutrons. Only 129I and 99Tc have been considered that can be transmuted because of their relatively large neutron capture cross sections and large isotopic abundance27. Therefore, in most studies, the transmutation of LLFPs involves isotope separation30. However, no isotope separation system for elements related to LLFPs is technologically and economically feasible at an industrial scale32.There are two technical difficulties that make transmutation of LLFPs less efficient: (1) the isotopic compositions and (2) small neutron capture cross sections of LLFPs33. The transmutations of LLFPs in common light water reactors has been known to be difficult mainly because of lack of excess neutrons35. The subcritical ADS or also called hybrid systems, which produce energy and to transmute radioactive wastes, appear to be a possible alternative to critical reactors36. Although numerous programs have conducted to evaluate the ADS availability, the high-power accelerator technology is a complex system and any ADS system had not yet been demonstrated.There are some studies on transmutations of LLFPs by reactor systems to address these two technical difficulties. The feasibility of reactor systems depends on the neutron balance. All LLFPs and the fuel are neutron consumers. Even with isotopic separation, it needs at least 0.3 neutron/fission for LLFP-transmutation39. Since most LLFPs have large neutron capture cross sections in the 1 to 103 eV region, the neutron spectrum must be softened to use the fast reactor system for transmuting LLFPs via resonance absorption. Metal hydrides  have been frequently proposed as neutron moderator materials for fast reactor systems because of the good neutron moderating ability of 1H and the high density of the materials41. However, a fission of fissile nuclide (e.g. 235U 239Pu) and the neutron capture reaction of stable 92Zr both produce 93Zr, which is one of the LLFPs. Moreover, metal hydrides sometimes induce a thermal spike by elastic collisions due to over-moderated neutrons near the core, causing the dissociation of hydrogen at high temperatures. Therefore, alternative moderator materials are required to develop a more effective transmutation system for LLFPs41.In this context, fast neutron spectrum reactor is the most attractive solution in the existing system with respect to the neutron balance, because of their high neutron flux and excess of neutrons2) as a moderator material for the transmutation of LLFPs in a fast spectrum reactor. Yttrium is a possible alternative to Zr because Y has a small neutron capture cross section over a wide neutron energy range and produces no radioactive isotopes with long half-lives. The bulk density of YH2 is smaller than that of ZrH2 and the dissociation temperature of YH2 is 1200 °C, which is 300 °C higher than that of ZrH2. These physical and chemical properties may help to avoid thermal spikes43. Furthermore, we have revealed that the thermal spike observed for ZrH2 can be suppressed by using the deuteride (ZrD2) instead of the hydride44. Based on these findings and the fact that YD2 is expected to have similar physical and chemical properties as YH2, YD2 is a promising material as a neutron moderator for fast spectrum reactors to transmute LLFPs.In this study, we propose yttrium deuteride  of LLFP must be larger than the production rate of LLFP in the core. The ratio of the TR to the production rate is called the support ratio (SR), and it is an important parameter to evaluate the efficiency of transmutation systems2, to the fast neutron spectrum reactor. We studied the effects of the moderator on the associated neutron energy spectra, the effective half-lives of LLFPs, transmutation rates (TRs), and SRs by simulations with the continuous-energy Monte Carlo code MVP-II and the burn-up simulation code MVP-BURN with JENDL-4.0 nuclear data library46.This paper demonstrates a transmutation method for LLFPs that does not require isotope separations. In this method, the LLFPs can be transmuted to stable or short-lived radioactive isotopes by introducing a novel moderator material, YD46. The initial isotopic compositions used in this study are listed in Table 2 in various ratios (see Method section for details).Figure 2 moderator material on the neutron spectra leaking from the core was examined. Figure 2 moderator material. The spectrum from the first row of the radial blanket region loaded with depleted UO2 pellets (without LLFP and moderator) shows a flux (n cm−2s−1lethargy−1) on the order of 1012 at 10–100 eV, whereas the spectrum with LLFP and moderator (Cs 70% + YD2 30%) shows a flux approximately one order of magnitude higher in the energy region below 100 eV. This softened spectrum is effective for transmuting LLFPs. It is also confirmed that the integrals of the microscopic capture reaction rates compared with those without the YD2 moderator are greater for all LLFPs .79Se, 99Tc, 107Pd, and 129I are sufficiently large (SR > 1) with the conditions that they are loaded in 54 assemblies in the innermost row of the radial blanket region. The SRs for these LLFPs are higher because of their relatively large neutron capture cross sections and small fission yields. In contrast, the SRs for 93Zr and 135Cs are barely more than 1.0, even though they were loaded in a total 498 assemblies in the radial blanket region (full length) and the radial shield region (core height).The SRs were calculated from the ratio of the transmuted LLFP mass to the produced LLFP mass in the core fuel. As shown in Table min) required to achieve SR > 1.0 were estimated from the number of loaded assemblies (Nloaded) divided by SR (Nmin = Nloaded/SR) , it is possible that SR > 1.0 for the simultaneous transmutation of all LLFPs. Nevertheless, the total number of assemblies required for the simultaneous transmutation exceeded 530. To improve the overall system efficiency, we focus on Cs in the next section because Cs is the most difficult LLFP to transmute.The computational simulations were performed separately for each LLFP, although the simultaneous transmutation of all LLFPs is necessary in practice. The minimum number of assemblies (NR) Table . For ins133Cs as 135Cs , the irradiation and cooling method, which consists of 2 years of irradiation and 6 years of cooling, was proposed to suppress the production of 134Cs, which is a source of 135Cs32. In this method, the weight of 135Cs gradually decreases, meaning that the method would take longer time than continuous irradiation. Nevertheless, when the “irradiation and cooling method” is applied, SRs are >1.0 for all LLFPs and the resulting total number of assemblies required for simultaneous transmutation is comparable with the total number of assemblies considered.The irradiation and cooling method (see Method section for more details) was used to study a more suitable Cs transmutation scheme. Figure Cs Table , 133Cs p2 mixing ratio will also improve the efficiency of the proposed transmutation system.The target nuclides are converted into stable or non-radioactive nuclides during transmutation, and the compositions of nuclides in the target pellets change. Therefore, LLFPs from irradiated pellets under real irradiation must be recycled. In this study, pellet recycling is not considered, although recycling will improve efficiency. Additionally, optimizing the pin and blanket configurations and the LLFP and YD2 as a moderator may improve transmutations of LLFPs in the fast spectrum reactor and can be achieved SR > 1.0 for all LLFPs. Neutron spectra in various positions in the reactor were analyzed and the LLFP transmutation capability defined by the effective half-lives, SRs and TRs of LLFPs were evaluated by the Monte Carlo code MVP-II and the burn-up simulation code MVP-BURN with JENDL-4.0 nuclear data library.We demonstrated that using YD2 was shown to be an effective moderator material for softening the neutron spectrum leaking from the core. This soft spectrum allowed LLFP transmutation in the radial blanket and shield regions. Each LLFP was loaded without isotope separation but with the initial compositions contained all isotopes. The calculations and analyses showed that the half-lives of LLFPs decreased drastically from more than 105 years to less than 102 years. The TRs for LLFPs were relatively small compared to other systems because we focused on improving the SRs. The SRs for all LLFPs were >1.0, including 135Cs, which is difficult to transmute effectively. Although the simulations were performed with assuming each LLFP was loaded separately, the proposed system achieved SR > 1.0 for all LLFPs within a total of 481 assemblies when the irradiation and cooling method of Cs transmutation was applied. The number was smaller than the 498 assemblies, which is the maximum number of assemblies by the core specification.YD47. We thus also address the improvements of these data by various way of physical descriptions49.We established an effective, efficient transmutation method for LLFPs to increase transmutation efficiency and improve SRs without isotope separations. If the core region is composed of transuranic or long-life MA nuclides and they can be burned as fuel materials, all the long-lived radioactive elements can be consumed by using the proposed system. In future work, we will examine the simultaneous transmutations of all LLFPs with SR > 1.0. Furthermore, such computational simulation study strongly depends on the nuclear data library regarding nuclear fission and reaction, such as cross section of nuclide, fission product yield, decay chain46 with JENDL-4.045 cross-section library. The number of history of neutrons was 10,000batch, the number of skipped batches was 100 for accurate source distribution, and the number of effective batches was 1,000. From this Monte Carlo simulation, the neutron spectrum and the reaction rates of LLFPs in various regions in the fast spectrum reactor were obtained. The typical statistical error of the multiplication factor (keff) was about 0.015% in 1 46. The fission product yield data was also derived from JENDL-4.0.The core characteristics was evaluated with a continuous-neutron energy Monte Carlo Code MVP-II/MVP Burn2) into fissile material by neutron capture reactions. In this system, the radial blanket assemblies are replaced by the assemblies with pins containing the pellets of the mixture of LLFPs and moderator as an alternative for depleted UO2 pellets. In the simulations, refueling could not be simulated for the fuel assemblies and LLFPs. Therefore, the core fuel volume was virtually magnified 100-fold to obtain the constant neutron flux from the core for 20 years.The fast reactor core used in this study is a sodium-cooled fast breeder reactor. Figure 79Se, 93Zr, 99Tc, 107Pd, 126Sn, 129I, and 135Cs. These nuclides will probably dominate the long-term radioactivity of the geological disposal of SNF  was chosen because it has a higher melting point (610 °C) and chemical stability at the ordinary temperature of the fast reactor. We will address the fabrication of LLFP pellets with YD2 in a future study.The chemical forms of the LLFPs are an important factor to consider. The material design and fabrication of the irradiation target containing LLFPs and moderator material involve considerations of a wide range of phenomena that affect pellet performance, such as thermal tolerance, swelling, irradiation damage, cracking, radioactive isotope production, and gas evolution. It is difficult to treat LLFPs as the same chemical substance because they have different physical and chemical properties. Assessing a pellet as a function of temperature and irradiation history is a minimum performance standard. The metallic form is the best choice because it generally has high melting point and the space volume for loading can be minimized42. In a previous study, we fabricated Zr-based pellets containing thin Tc metal wires and simulated the homogeneous dispersion of immiscible materials42. To simplify and reduce the calculation cost, various ratios with metallic or chemical forms of the target LLFPs were investigated with homogeneous dispersion in YD2. The mixing ratios of LLFP/YD2 were varied based on the position of the radial blanket or radial shielding assemblies  were neglected because they decrease the SRs slightly.Cesium has the stable isotope The effective half-life is an important parameter for evaluating the efficiency of transmutation. The effective half-life is the half-life when transmutation is considered. For a single target nuclide, the effective half-life can be defined asOther two important parameters of the transmutation system are Transmutation Rate (TR) and Support Ratio (SR). TR is defined as the ratio of the amount of initial loaded LLFPs to the amount of transmuted LLFPs in the target assembly (per unit time) as135Cs can be approximately expressed by Eq. In this system, TR of LLFPs except for 135Cs can be approximated by Eq. SR is defined as the ratio of the amount of transmuted LLFPs to the amount of LLFPs produced in the core fuel over the same period of time in a reactor,Supplemental information


To measure the effects of information, a challenge, and a conditional cash transfer on take-up of voluntary medical male circumcision (VMMC).A randomized, controlled experiment with 4000 postcard recipients in Soweto (Johannesburg), South Africa.We examined differences in take-up of several decisions in the VMMC cascade between the control arm and each of several intervention arms using logistic regression.Logistic regression analysis indicated that the group offered US $10 as compensation and the group challenged with “Are you tough enough?” had significantly higher take-up of the VMMC procedure than did the control group . Similarly, the compensation group had significantly higher take-up of the VMMC counseling session than did the control group [odds ratio 3.76 (CI: 1.79 to 7.89)]. The analysis did not reveal significantly different take-up of either the VMMC counseling session or the procedure in the partner preference information group compared with the control group . The analysis did not reveal significantly higher take-up of the VMMC nurse hotline in any intervention group compared with the control group .Among adult males in Soweto, South Africa, compensation of US $10 provided conditional on completing the VMMC counseling session compared with no compensation offer and a postcard with a challenge, “Are you tough enough?” compared with no challenge, resulted in moderate increases in take-up of circumcision. Based on these findings, the international donor community is aiding scale-up of mass VMMC campaigns in 14 high HIV prevalence, low male circumcision prevalence priority countries in sub-Saharan Africa.4 However, take-up of this fully subsidized health service remains low.Evidence from randomized controlled trials conducted in Kenya,8 and transportation costs associated with the counseling session/procedure9 seem to be large impediments to receiving VMMC. Second, individuals are concerned about pain associated with the procedure and recovery period.10 Third, men are concerned that VMMC will reduce sexual performance and pleasure.10 Fourth, the fact that explaining to individuals that VMMC reduces the acquisition of HIV and other sexually transmitted infection (STI)s seems to increase the acceptability of VMMC10 suggests that individuals may be uninformed about possible benefits of VMMC.Existing literature on demand for VMMC highlights several factors that may be inhibiting VMMC take-up. First, opportunity costs of foregone wages during the recovery periodTo assess the magnitudes and relative importance of these barriers, we conducted a randomized controlled trial. In this study, we randomly assigned postcards offering a conditional cash transfer for completing the VMMC counseling session, a challenge message, and information on a possibly previously unknown benefit of VMMC, through 4000 postcards in Soweto township.11, and the second highest number of people living with HIV of any province in South Africa.12 We conducted our study in collaboration with the Centre for HIV and AIDS Prevention (CHAPS), which currently operates 23 VMMC sites in Gauteng. The Human Subjects Research Committee  at the University of Witwatersrand, Johannesburg and the Institutional Review Board at Reed College provided human subjects research ethical approval for our study.Soweto, our study setting, is a relatively poor township in Johannesburg, South Africa. Gauteng province, where Soweto is located, has the lowest prevalence of male circumcision, 25.2%Outreach workers distributed postcards individually sealed in envelopes in a blinded, prespecified order to households, following a random walk method. In this random walk, outreach workers began distribution at prespecified locations, leaving the next postcard at every fifth house with an adult present, using coin-flips to determine the path at each intersection. The prespecified order randomized each postcard type and stratified on timing and location of distribution. Importantly, our statistical analysis compares VMMC take-up across groups of postcard recipients rather than with nonrecipients, which reduces many spillover concerns that complicate other studies. If recipients shared their postcards with those who had not gotten any postcard, and this person visited a clinic, this does not compromise the design because the second person was not in another experimental group.Men who presented a study postcard at a participating CHAPS clinic were asked to provide written informed consent.All postcards offered basic information about the HIV risk-reduction benefits of circumcision and clinic hours, and an offer of refreshments for male respondents, aged 18 years and older, who participated in a VMMC consultation. Subsets of these were also offered a conditional cash transfer , information on a possibly previously unknown benefit of VMMC , or a challenge message . Our cross-cutting randomization produced 4 study arms of equal size . Control postcards included basic information that VMMC reduces HIV acquisition by 51–76% and the names and workdays of participating clinics. The offer of light refreshments served as an incentive for the recipient  to bring the postcard with him if he chose to come to a clinic. Requiring individuals to bring the postcard to the clinic for refreshments allowed us to measure take-up of multiple decisions in the VMMC cascade and observe how this varied across study arms.“Money” postcards offered R100  as compensation for attending a VMMC counseling session at a participating clinic in addition to the information on the control postcard. “Information” postcards stated that a recent survey indicated that among partners of uncircumcised men, 2 of 3 would prefer that their partner be circumcised in addition to the statements in the control postcard. “Challenge” postcards added the statement, “Are you tough enough?,” to the control postcards to frame the statement about the 51–76% reduction in HIV acquisition.All transfers were disbursed conditional on attending a VMMC counseling session at a participating clinic. Distribution of postcards began on June 30, 2014. All postcards stated an offer expiration date of August 29, 2014, meaning each postcard had a redemption period of approximately 2 months.We collected data on 3 outcomes related to the VMMC cascade . These 3 outcomes were (1) calling or texting the VMMC hotline, (2) completing the counseling session, and (3) completing the procedure. Each type of postcard had a different phone number, which meant that each call could be linked with the type of postcard seen by the caller. For each individual who brought a postcard to a participating clinic, a nurse recorded the postcard information along with whether the individual completed the counseling session and whether he underwent the procedure. We also collected demographic information and past risky sexual behavior from individuals who brought postcards to the clinics. No individuals under age 18 presented postcards.P value of less than 0.05.The primary outcomes of interest were uptake of the counseling session and of the procedure, as well as the initial possible step of calling the VMMC hotline. For each of these 3 outcomes, the independent variable is a binary variable equal to 1 if the participant completed the step and equal to zero if otherwise. Our primary analysis uses logistic regression to compare uptake for each of these outcomes in each of the intervention groups to uptake in the control group. We used Stata Version 13.1 (StataCorp) to conduct our statistical analyses and all statistical tests were two sided with a The trial specified a sample size of 4000 postcards, divided into 4 study arms of equal size. In our power calculations, we analyzed the sample size required to test whether a single intervention generated a statistically larger take-up. We assumed a two-sided test with an alpha of 0.05 and a goal of 90% power. In addition, we assumed that 5% of control postcard recipients would obtain VMMC and that an intervention postcard  would increase this number by 5 percentage points. The required sample size in each group was 621, or 2484 summed across the 4 different study arms. We chose a sample size of 4000 to ensure that we were able to identify an even smaller intervention effect than 5 percentage points.Table Among respondents who subsequently presented a postcard at a participating clinic, 91% said they had gotten it directly from the distributor and 9% said they had gotten it from a friend or family member. The average age of men who completed the survey was 29 years. Just under one-half of respondents worked for pay in the last week, and exactly one-half did so in the last year. The average year of schooling among the respondents was roughly 11. Approximately one-half of men had at least 1 child and 67% were neither married nor living with their partner. Twenty-six percent of respondents reported that an immediate family member was HIV positive. More than three-quarters of respondents had ever been tested for HIV. Nine percent had ever been treated for any STI, and 6% currently had symptoms. Nearly all respondents reported ever having had sex (90%), with the average age of debut at 16 years. The average number of partners in the last year was nearly 2 and one-half of respondents reported using a condom the last time they had sex.Table Column 2) in Table  in TableColumn (3) in Table 13 Thus, we estimate that this intervention costs between US $450 and US $1350 per HIV infection averted, excluding clinical costs.This intervention is extremely low cost. Combined with noticeable impacts on uptake, this implies that the cost-effectiveness is relatively high. Printing and distribution of each postcard cost approximately US $2, and the cash transfer costs US $10 per consultation. Refreshments for each consultation cost approximately US $1. The high conversion rate (110 of the 123 men who came for a consultation stayed for the procedure) and only needing to pay the latter 2 costs for those who chose to use the voucher kept costs low. However, the number of postcards needed to encourage each additional circumcision may be considered high, increasing costs. The expected effect on take-up from the addition of the compensation offer on the postcard relative to the basic postcard was 2.5 percentage points. This gives a cost per additional circumcision of US $91. The fact that Soweto is a high HIV prevalence area implies that 1 VMMC generates approximately 1/15–1/5 of an HIV infection averted.The results of our randomized controlled trial indicate that offering economic compensation for the VMMC counseling session was effective at increasing take-up of the VMMC procedure. Similarly, including the challenge, “Are you tough enough?,” was also effective at increasing VMMC take-up. In contrast, providing the information that, among partners of uncircumcised men, 2 of 3 would prefer that their partner be circumcised was not effective at increasing VMMC take-up.14 The second study also found similar effects of conditional compensation of approximately US $11 on VMMC uptake in Kenya.15At least 2 other studies have conducted randomized controlled trials testing the effect of conditional economic compensation on VMMC take-up. The magnitude of the main effect reported in the first study is very similar to the effect of economic compensation that we report in the current analysis. That is, in Kenya, an offer of US $8.50 in food vouchers disbursed conditional on completing the VMMC procedure increased VMMC uptake relative to the control arm more than 4-fold.10 and (expected) diminished sexual performance as major factors limiting demand for VMMC.10 Although many largely nonexperimental studies propose that explaining to individuals that VMMC reduces the acquisition of HIV and other STIs increases the acceptability of VMMC,10 our results indicate that the responsiveness of VMMC take-up to further information campaigns, or at least information campaigns citing partner preference for circumcised men, may be limited.The finding that posing the challenge, “Are you tough enough?,” was effective at increasing VMMC take-up provides scarce experimental evidence for the largely nonexperimental literature citing (concern about) painFocus group discussions among study participants point to a possible explanation for the effectiveness of what was relatively small financial compensation: many of the men reported that they were thinking of taking up the VMMC procedure anyway. The extremely high rate of conversion  also supports this explanation. If this explanation is true, then it suggests that the use of financial compensation in the context of VMMC may not be coercive. Men induced to complete the VMMC procedure may be likely to be considering the VMMC decision seriously already and compensation simply may be a nudge. In this study, payment conditional on the consultation rather than the procedure provided additional assurance against coercion.Interventions generated VMMC procedures that would not have occurred during redemption period in the absence of the interventions. The ratio of procedures conducted among men who would have not received the procedure in the absence of the US $10 offer to those who would have performed so anyway was approximately 5 to 1. For the challenge intervention, this ratio was slightly less than 3 to 1.This study has a few limitations. First, respondents were only given between 1 and 2 months to redeem their vouchers. A longer redemption period may have generated higher take-up. Second, because we chose not to conduct a baseline survey of postcard recipients , we do not know background characteristics of recipients. Without a baseline survey, we cannot know which postcard recipients were uncircumcised at baseline, and we cannot compare the demographic characteristics of those recipients who did and did not visit a clinic. Although this lack of a baseline may limit the nuances we can uncover, the randomized design removes concerns about omitted variables, reinforcing the credibility of the study findings.


Background: In a previous study on encephalitis lethargica, we identified a virus related to enterovirus in autopsy brain material. Transmission electron microscopy (TEM), immunohistochemistry (IHC) and molecular analysis were employed.  Our present objective was to investigate, using a similar approach, as to whether virus-like particles (VLP) and enterovirus antigen are present in Parkinson’s disease (PD) brainstem neurons.Methods: Fixed tissue from autopsy specimens of late onset PD and control brainstem tissue were received for study. The brain tissue was processed for TEM and IHC according to previous published methods.Results:  We observed VLP in the brainstem neurons of all the cases of PD that were examined.  In the neurons’ cytoplasm there were many virus factories consisting of VLP and endoplasmic reticulum membranes. In some neurons, the virus factories contained incomplete VLP. Complete VLP in some neurons’ virus factories had an average diameter of 31 nm, larger than control brain ribosomes. In the nuclei, there were VLP with an average diameter of 40 nm. In cases of human poliomyelitis, there were cytoplasmic virus factories and intranuclear virus particles similar to those observed in PD. On preparing PD brain sections for IHC there was positive staining using anti-poliovirus antibody and anti-coxsackie antibody. This result was statistically significant.Conclusions: We present evidence for an enterovirus infection in PD.  For future studies, virus isolation and molecular analysis are suggested. We have applied the methodology used to characterise encephalitis lethargica by means of TEM of fixed brain tissues and IHC. Our aim is to identify and characterise virus particles and viral products in PD brain.Following our study on encephalitis lethargica2, and it remains a common cause of debilitation and death. Two hundred years later, we present evidence suggesting an enterovirus infection is present in the brainstem in PD.PD is widespread, especially among the elderly. It was called ‘the shaking palsy’ by Parkinson in 18173. This syndrome was termed ‘post-encephalitic parkinsonism’ and more popularly in England ‘the sleepy sickness’. It was very common and often conflated with PD. By the end of the 20th century every patient who suffered from post-encephalitic parkinsonism had died, whereas PD continued to occur.Following the pandemic of encephalitis lethargica during World War 1, many of the survivors of the acute phase of the disease developed a syndrome similar to PD, but in which the patients became somnolent. They developed tremor and oculogyric crises during which their eyes rolled uncontrollablyet al.4 discussed the neurological sequelae of infection by influenza virus, as well as that of other viruses known to induce parkinsonism, including Coxsackie, Japanese encephalitis B, St. Louis, West Nile and HIV viruses. Janget al. in anin vivo study5 showed that a pathogenic influenza virus can enter the central nervous system causing neuroinflammation and induce neurodegeneration. Zhouet al.6 in a literature review suggested that viruses may be associated with neurodegenerative diseases, including Alzheimer’s disease, PD and multiple sclerosis. Duvoisin7 reported in a review the evidence for association of PD with infectious diseases. Hawkeset al.8 proposed a dual hit theory for the pathogenesis of infection in PD, in which channels for virus infection pass through the nasal mucosa and the intestinal epithelium. Svenssonet al.9 and Liuet al.10 found a reduced incidence of PD in patients who underwent total vagotomy, suggesting a pathway for infection via the vagus nerve. Gibbs and Gajdusek11 carried out experiments designed to isolate a virus in PD usingin vitro cell cultures co-cultivated with PD brain tissue. Direct inoculation of PD brain tissue to non-human primates was also carried out. No evidence of PD developed in the inoculated animals and no evidence was found of virus replication in the cell cultures. Wetmuret al.12 did not detect nucleic acids complementary to herpes simplex type 1 DNA or influenza A/NWS RNA in the brain of patients with sporadic PD. Elizanet al.13 reported that tests were negative for arbovirus antibody using sera from cases of encephalitis lethargica and PD, and in another study14 Elizanet al. searched for antibodies to herpes simplex virus 1, measles and rubella in the serum and cerebral spinal fluid of post-encephalitic parkinsonism patients with negative results. Schwartz and Elizan15 did not detect virus particles by means of transmission electron microscopy (TEM) in PD brain tissue nor in cell cultures co-cultivated with PD brain tissue. The possible involvement of enterovirus in PD was not considered in these studies15.A number of previous studies have proposed a virus etiology for PD. A review by Jang16. Jucker and Walker17 found that misfolded α-synuclein can develop disease propagating properties. Human gut microbiota from PD patients induced enhanced motor dysfunction in mice18, suggesting that gut microbiota play a part in the pathogenesis of PD.Immature dopamine neurons grafted to PD patients showed that Lewy bodies developed in the grafts19, reported that the LRRK 2 gene (PARK 8) is the most common genetic cause of familial (early onset) PD. The frequency of LRRK 2 mutations in patients with a family history of PD is 5–7%. The heterozygous mutation, 2877510 G>A is the most commonly described, accounting for the majority of familial cases and up to 1.6% of cases of sporadic PD. Moket al.20 found that, in a combined analysis of genome-wide association data of 9,387 cases of PD, eight cases were reported with deletions at the 22q1.2 chromosome site. Age of onset of PD of the patients carrying these deletions was lower  than those who did not carry the deletions . In the present study, all the PD cases are from the late age of onset.Davie, in a review of PD21 and Barker and Williams-Grey22, who showed that Lewy bodies were found in 6–8% of autopsies of patients with no clinical evidence of brain disease. The finding of Lewy bodies is the histopathological hallmark of PD; they carry an accumulation of α-synuclein. Barker and Williams-Grey22 reviewed the literature defining α-synuclein as the core protein of Lewy bodies. They are present in PD, in dementia with Lewy bodies, and in multiple system atrophy. Josephs and Parisi23 found that brain tissue from post-encephalitic parkinsonism cases did not carry α-synuclein protein, thus differentiating encephalitis lethargica and the subsequent syndrome, post-encephalitic parkinsonism, from PD.Pathological studies on the incidence of Lewy bodies in the brain were carried out by Herva and Spillantini24. Thalidomide may worsen existing PD25.Drug induced parkinsonism was described as an acute/subacute syndrome in which tremor is infrequent compared to PD. Most cases of drug induced parkinsonism were caused by both classic and second generation neuroleptics and calcium channel blockers. Resolution by 75% of drug induced parkinsonism occurred six months after withdrawal of the drug1. Fifty nm sections were cut on a Reichart ultramicrotome, stained with 1% uranyl acetate, then stained with Reynold’s lead citrate, and examined in a JEM1400 TEM (JEOL UK) at Public Health England, Colindale, UK. Images were acquired with an AMT XR60 digital camera . VLP in the digital images of cytoplasmic virus factories of PD cases and ribosomes in the cytoplasm of control cases were measured using the measurement facility of the AMT image acquisition software (Deben Ltd. UK). Calliper measurements were also made of each of 30 particles in magnified paper prints of each image (Brain tissue from 14 sporadic cases of PD and seven control cases was examined by TEM in order to identify virus-like particles (VLP) in the neurons of the PD brainstem. None of the control cases had poliomyelitis. The method used for TEM of autopsy brain that had been fixed and embedded in paraffin blocks for histology has been previously reportedh image . Biased Clinical reports of the PD cases received from the Armed Forces Institute of Pathology (USA) and the John Radcliffe Hospital (UK) stated that Lewy bodies were found by the examining neuropathologists in the brainstem neurons of all the PD cases studied, indicating that PD was present at the time of death.1. Rabbit polyclonal anti-polio antiserum raised against polio types 1, 2, and 3, and goat anti-coxsackie polyclonal antiserum raised against a coxsackie virus isolate were donated by the Department of Virology, National Institute for Biological Standards and Control, Potters Bar, UK.IHC testing was carried out by exactly the same technique described previouslyMethod for preparing the polyclonal antibodiesThe inoculation of one rabbit with Poliovirus type 1 for the production of polyclonal antibody:10 pfu/ml) 1:5 dilution.Virus/adjuvant injected subcutaneously into loose skin, 0.25ml into 4 sites [total 1ml]. Poliovirus Sabin type 1 .TEM changes were observed in the brainstem neurons in the PD cases and in the spinal cord motor neurons in the poliomyelitis cases. TEM of PD neurons at low magnification showed advanced apoptosis. There were almost ‘empty’ nuclei with clumped chromatin and multiple cytoplasmic virus factories. Few cytoplasmic organelles remained . VLP werfactory . VLP werfactory . The avefactory . CytoplaIntranuclear VLP were found in all 14 cases of PD. The VLP were lined along the internal membrane of neurons and to a lesser degree throughout the nuclei . The intIn the cases of human poliomyelitis, motor neurons of the spinal cord showed cytoplasmic virus factories and apoptosis similar to that shown in PD neurons . IntranuA Lewy body is illustrated from a case of PD . In this22, in that Lewy bodies were found in the brain of a proportion of control cases. The case reported here could represent a preclinical PD event.In the control cases, the neurons showed little apoptosis. Nissl bodies were present consisting of rough endoplasmic reticulum . Free riStatistical analysis section).IHC staining of PD brain sections showed 13 positive results and 8 negative results using rabbit anti-polio antiserum and 13 positive and 7 negative results using goat anti-coxsackie antiserum; and for control brain sections, there were 10 negative results and 2 positive results for poliovirus antiserum and there were 13 negative results and 1 positive result for coxsackie antiserum. The data for the IHC tests are reported inThe results were negative for IHC staining of PD tissue using mouse monoclonal anti-enterovirus antibodies as well as monoclonal and polyclonal anti-parvovirus antibody staining. The absence of reaction of enterovirus monoclonal antibodies to PD tissue sections may be attributed to the fact that the solitary epitope to which the antibodies were raised was inappropriate for the staining of PD neurons carrying the putative enterovirus antigen.A light microscopy image of an area of the inferior olive of a case of PD is shown that was stained for poliovirus antigen . There ihttp://turner.faculty.swau.edu/mathematics/math241/materials/contablecalc/.The statistics were calculated using Microsoft Excel 10. The Fisher test results were checked using the ‘R’ statistical package, version 3.3.3. The chi-squared tests were checked against the calculator onThe first null hypothesis, based on the data inA t-test for two independent sample means without assuming equality of variances was used. The resulting probabilities are shown in-4. The null hypothesis is thus again rejected.The next null hypothesis tested, based on the data for the TEM of PD tissue and of controls was that the positive TEM result is independent of whether the cases are of PD or else controls. Using a chi-squared test for categorical data, the test statistic was calculated at 16.8. With one degree of freedom, the null hypothesis was rejected at the 1% significance level. Using the Fisher exact test, the hypergeometric probability of the results being obtained if the null hypothesis holds is 1.3 × 10-4, so that the null hypothesis is again rejected at 1% significance level. For the anti-poliovirus antibody, the hypergeometric probability is calculated at 0.014, so the null hypothesis cannot be rejected at the 1% significance level, but is rejected at 5%.The third null hypothesis tested was that the observed positive immunohistochemical staining of sections using anti-coxsackie antibody or anti-poliovirus antibody is independent of whether the cases are of PD or else controls. Again using a chi-squared test for categorical data, the test statistic was calculated at 11.4 for anti-coxsackie antibody, which rejects the null hypothesis at the 1% significance level; and 6.3 for anti-poliovirus antibody, which does not reject the null hypothesis at the 1% significance level but does at 5%. Using the Fisher exact test, the hypergeometric probability of the anti-coxsackie antibody results being obtained if the null hypothesis holds is 8.1 × 101. The finding of virus particles in the nuclei of cells infected with an RNA virus was a novel observation. Virus factory has been described as a specific intracellular compartment where viral components concentrate by Novoaet al.27. Virus factories often include membrane components that are involved in virus replication. This has been demonstrated in our PD study .The TEM images presented here are listed in the legends by the author’s TEM serial number followed by the donor’s autopsy number. Copies of the images are stored at the Public Health England electron microscopy laboratory. They may be freely obtained by contacting Dr. Matthew Hannah at PHE. et al. Dr. Dourmashkin is a highly qualified electron microscopist with decades of experience. The difference he finds between patient samples and controls is impressive and may lead to treatments that include attempts to prevent viral replication in patients early in disease development.  The paper is inclusive but I was unclear whether any blinded studies were performed.  Were their blinded samples given to the authors and could they identify patient vs. control sample?  Perhaps I missed this in the paper but of course the question is of great interest.I have now read the paper by Dourmashkin I approve the paper for publication but do believe that the ability to predict disease vs control material is of great importance.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Ref. 16 refers to Treatment of children with human cadaver-derived growth hormone (hGH) contaminated with prions resulted in iatrogenic transmission of CreutzfeldtJakob disease (iCJD).  I was unable to find any reference to ‘immature dopamine neurons grafted to PD patients” and Lewy bodies in Ref. 16.  Need correct reference or further explanation.Fig. 4 - would be helpful to use arrows to indicate several VLPs which were used in measurements.Fig. 7 - need arrow - VLP In a PD neuron, close to the internal face of the nuclear membrane.Re:  Immunohistochemistry Methods:  Was antiserum raised against polio types 1, 2, and 3 or…only poliovirus type 1?Fig. 11 - How were these VLP particles identified as intranuclear?  Are smaller particles also seen - i.e. the cluster of particles in the quadrant directly above the putative VLP?Fig. 12 - what are the particles seen inside the Lewy body?Fig. 16 - Shows only IHC for rabbit anti-polio.  Would be useful to show an example of IHC for anti-coxsackie.Table 3 - IHC for anti-coxsackie and anti-polio - nearly all dual positives, only one is specific for coxsackie only.Is this result expected?Comments/minor revisionsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. General comments Ultrastructural and immunohistochemical analysis of brain tissue from Parkinson’s disease patients could provide important data on the pathogenesis of the disease. Electron microscopy of autopsy tissue, particularly after formalin fixation and embedding for light microscopy, is challenging as preservation is far from optimal.  Despite this difficulty the authors have provided persuasive evidence of cytoplasmic structures that may be viral. However, although the authors have convincingly shown differences between Parkinson’s and control brain tissue I feel they should be more cautious in their rather definitive interpretation that there are enterovirus structures antigenically related to polio and coxsachie virus present. In addition I think the discussion should be expanded as indicated below.Major specific commentsThe ICH showed labelling for both coxsachie and polio virus antigen. What evidence is there in the literature that these viruses have common antigens which may explain their findings? In their studies was either pre-immune or non-immune serum used as a control? Does binding of the coxsachie antiserum block subsequent labelling with the polio virus antiserum and vice versa? et al 1965 Virology26, 389)   should be cited.The micrographs of the cytoplasmic virus factories in the Parkinson’s tissue are persuasive of enterovirus infection, particularly in view of the similarity to cytoplasmic structures in polio brain tissue and the absence in controls. However, the presence of nuclear virus particles would not be expected of an enterovirus infection. This observation should be more extensively discussed. In addition the extensive early em studies in the 1960s by authors such as Dales (DalesIs it possible to use haemotoxylin to stain nuclei in the IHC to confirm or not the presence of nuclear viral antigen?   Table 3 summarises the em and IHC data showing that for 13 samples there was both em and IHC. All 13 were positive for virus factories by em but 8/13 and 7/13 were positive for staining with coxsachie and poliovirus antiserum respectively. Given that it may be expected that IHC is more sensitive since a larger area of tissue may be examined the authors should comment on the difference between the em and IHC findings.Minor comments I presume that the tissue was fixed in formalin, this should be stated in methodsFor the reader it would be helpful if a micrograph could be shown in which both ribosomes and VLPs are indicatedFigure 7 legend states that an arrow indicates the nuclear membrane, I cannot see an arrow on this micrograph.There appears to be a core in one of the nuclear particles shown in figure 11, indicated by the arrow, can the authors comment.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The amendments made by the authors have satisfied my concerns. Steven Patterson


Furthermore, the same layers contain a rich archaeological and palaeontological record, confirming that knapping was taking place in situ and that stone tools were used for butchering hippo carcasses at the site. The site gives direct information on hominin landscape use at 0.7 Ma and may provide fresh perspective on the childhood of our ancestors.We report the occurrence at 0.7 million years (Ma) of an ichnological assemblage at Gombore II-2, which is one of several archaeological sites at Melka Kunture in the upper Awash Valley of Ethiopia, 2000 m asl. Adults and children  We report on a tracksite at Gombore II-2 (Melka Kunture) in the Upper Awash Valley of Ethiopia  a French Mission led by Jean Chavaillon started the research in 1965. From 1999 onwards the responsibility for investigations has been held by the Italian Archaeological Mission at Melka Kunture and Balchit and is now directed by Margherita Mussi of Sapienza University of Rome2. Scientific interest at the time was focused on the association of hippopotamus bones with lithics, suggesting that it was a butchering site4. From 2013 to 2015, some of the authors excavated over ~35 m2 more . Sands filled the tracks. In the central-eastern part of the excavated area, sand lenses accumulated to a maximum thickness of 0.1 m over ~20 m2. To the west, the sands are residual, and the trampled sandy silt layer outcropped over further ~15 m2  were plastic and humid enough to allow the production and preservation of fossil footprints11.The Acheulean layer is covered by ~0.50 m-thick sandy and silty channel deposits and ~2.0 m massive silty clay interbedded by sands, interpreted as overbank flood deposits, with palaeosol development. Stratified sands interbedded with silty-sandy sediments with fossil and lithic remains accumulated above the uppermost palaeosol, suggesting recurrent anthropogenic activity2 16. However no 3D data was collected at this site before it was destroyed and there are few published 2D images of the children’s tracks. Perhaps the best preserved, to date, children’s tracks are the Holocene tracks from Namibia17  dating to 1 Ma (Homo heidelbergensis). Here we used modern growth curves derived from those developed by WHO24, based on 2D foot lengths, following Ashton et al.16 to give a first approximation and to determine potential ages. Foot length growth curves for Homo sapiens are dependent on the individual’s sex as well as their ethnicity25 and crucially the levels of nutrition26. It is also worth noting that WHO growth curves are based on direct foot measurements, and not on 3D tracks made in soft and compliant substrates. Also in dealing with larger tracks, the potential for overlap between adolescents and adult females, given sexual dimorphism, needs to be considered27. To complicate this further the applicability of growth curves based on Homo sapiens to earlier hominin species is also potentially problematic. For example, Dean and Smith28 suggest that typical growth curve for early Homo erectus was more like that of a modern chimpanzees, than that of Homo sapiens, although unique to itself. The age estimates made here . In addition, a first phalanx of Equus sp. was found. Most of the skeletal elements (n = 63) could not be attributed to Family/Order level, but only to animal size classes. Other bones (n = 137) could not even be attributed to size classes due to their high degree of fragmentation and surface concretion.The associated faunal assemblage consists of 247 bone and tooth remains  are covered. Size class 3 represents medium-sized bovids  and Equids. With size class 2 the small bovids (such as Gazella and Antidorcas) are represented. Finally, size class 1 equates to mammals below the size of Gazella. All animal size classes were documented with the exception of size class 1. Hippos  are almost exclusively represented by elements of the axial skeleton, . Cranial elements are exclusively teeth, mainly canines and incisors. The humerus is almost always represented by shaft fragments, in contrast to the good preservation of femurs and tibiae, represented by complete bones or intact epiphyses with parts of the diaphysis attached. A minimum of three hippo individuals is identified on the basis of three left scapulae.When possible the taxonomically indeterminable material was sorted according to size classes based on bone-thickness, taking into account the variety of taxa identified by GeraadsHippopotamus cf. amphibius and size class 5, 23 elements showed gnawing damage. This accounts for almost 32% of the assemblage. The general features of the gnawing damage31 suggest that the bones were subject to hyena scavenging. What is particularly striking are the homogeneous gnawing patterns evident for example on the scapulae which is best illustrated on scapula A-35  allows recognizing an under-representation of spirally fractured long-bone shaft fragments compared to all other size classes. Characteristic however are elements displaying bending breaks (n = 9) indicating fresh bone breakage. Bending breaks almost exclusively characterize rib fragments that could be attributed to size class 5 animals. Bone flakes (n = 2) are equally present in the assemblage, resulting from long bone fracturing by either hominins or carnivores. Carnivore modification was particularly noted in bones of size class 5. Of the 71 skeletal elements attributed to -35 Fig. . The cra-35 Fig. .Figure 632. For 157 faunal remains weathering stages could be determined, not all of which could be attributed to size  classes. The preservation of bone fragments of all size classes is mainly characterised by weathering stages 1 and 2 (n = 138). Weathering stages 3 und 4 were recorded for a further 8 specimens, representing size classes 2, 4 and 5. The documented weathering stages indicate that bones from differing size classes were buried relatively soon after the death of the animals. The assemblage composition was also considered according to the susceptibility to fluvial transport, expressed in the so-called “Voorhies groups”. Ribs, vertebrae, sternum and sacrum are subsumed under Group I as these bones are immediately moved by water. Scapulae, phalanges and ulnae belong to the intermediate Group I/II. Group II summarizes bones which are gradually removed, while the cranium and the mandible represent Group III, considered to represent a lag deposit32.Overall, the faunal assemblage was affected by coatings of carbonate concretion (n = 79) which covered the bone surfaces to different degrees for all size classes . This strongly supports the assumption that the burial milieu was comparable for bones of all size classes. To estimate exposure time between animal death and final burial, bone weathering stages were documented, following Behrensmeyer’s work33, the presence of Group I bones indicates a non-fluvially winnowed assemblage. The degree of rounding was determinable only on a limited number of fragments (see Supplementary data) but it seems to correlate with bone fragment size and the degree of mineralization. Accordingly, we interpret small bone fragments as sediment components, as expected in a watering hole environment such as Gombore II-2.At Gombore II-2 skeletal elements of Groups I-III have been recorded, but most of the bones belong to Groups I and I/II. According to Behrensmeyer13. Exceptional preservation does happen if tracks are buried quickly in a low-energy environment and in these cases, they may record biological events happening just before they were infilled11. This contrasts with the archaeological record, which accumulates over time-lengths that are difficult to determine with precision. In the deeper and therefore earlier lithic- and fossil- bearing deposits at Gombore II-2 chronostratigraphic sequence disturbance is clear. For example, the 0.875 Ma Middle Acheulean layer was heavily reworked by water activity7. This contrasts with the archaeological evidence reported here, which shows little or no disturbance. What is clear from the sequence as a whole is that hominins repeatedly exploited the area ever since they returned during a favourable climatic episode of the Mid Pleistocene Transition, as suggested by Mussi et al.5. The interpretive challenge here is to reconcile different types of record; a footprint does not move and provides reliable evidence of in situ activity10, a snapshot into past life, while the archaeological record does not always provide this being more time integrated or averaged.Footprints do not last long at the surface when exposed to natural processes and anatomically diagnostic features may disappear within hours or daysGombore II-2 contains evidence of hominin tracks in association with a range of other animal tracks. The archaeological and faunal record provides evidence of the preparation of stone tools and carcass butchery. There are potential hominin tracks that due to their size may indicate the presence of children. The anatomy of these is not particularly distinct which we attribute to standing individuals rather than walking and that the ground was heavily trampled both before and after the specific track-making event.13. Specifically desiccated tracked areas around small water bodies like this tend to become brecciated very quickly when exposed for more than a couple of days. Blocks break from upstanding elements of tracks and infill the lower parts. Significantly no breccia was recovered as part of the track infill at this site. Furthermore, the ichnosurface is littered with archaeological and palaeontological material (lithics and bones) that do not appear to have been re-orientated by water flow. Bone preservation suggests that animal remains accumulated over a relatively short time interval, without significant displacement. Finally, lithic implements and bone fragments are impaled into some tracks and also overlying them. We suggest that the balance of probability therefore indicates that the footprints, knapping and butchery were all produced on site during a relatively short time interval and certainly within one season.At Gombore II-2, the track-bearing surface is slightly hollowed, and most impressions surround the depressed area which is assumed to have contained water Fig. . The acc17  toe impressions; (2) nail scrapes associated with foot withdrawal; (3) rounded heels; and (4) peripheral outlines consistent with the correct anatomically position of toes and a medial longitudinal arch. This is supported by the quantitative comparison of the tracks with the children’s tracks from Namibia toe impr35. We speculate that the presence of children’s tracks in direct stratigraphic association with such activities suggests that this learning occurred from a very young age.The hominin tracks at Gombore II-2 add to the growing number of early track sites discovered in East Africa. The co-association of the hominin tracks with other activities, such as carcass butchery, and the production of the lithic tools is a novel feature when compared to other track sites identified to date. The interpretation is based on a powerful synergy of archaeology, geoarchaeology, zooarchaeology and ichnology data that points to methodological good practice which historically may not always have been followed. The butchered hippopotami and the association of a diverse fauna based on the ichnological record, confirm the richness of the area in terms of food availability. The presence of possible hominin tracks made by children as young as 1 year old, would imply that children had to tag along the adults in a mobile group and to learn first-hand information about hunting and butchering. Tool making, tool using and butchery behaviours are all inferred to be learnt skills14, where the preserved tracks, attributed to Homo erectus, tell a story of mobile all male hunting/foraging groups, with the females and juveniles, one assumes, left to other tasks in other locations. The implication of such a reconstruction is that adult foraging groups were perhaps the norm, with a division of labour, potentially along gender lines. Our observations of a later hominin species rather suggest mixed-age groups at least in certain contexts. Gombore II-2 illustrates the complimentary nature of ichnology and archaeology and its potential to allow a more comprehensive and inclusive approach to past life.The observations reported here complement those made at Ileret in Kenya8. The deposit was removed progressively one thin layer at a time, following the excavation method known as “décapage horizontal”. Scalpels and small brushes were used to isolate and when needed, empty the footprints observed on the ground. Photogrammetric models of the tracks were constructed in Agisoft Photoscan (http://www.agisoft.com/). Analysis, visualization and contour outputs were executed in DigTrace (www.digtrace.co.uk). Zooarchaeological analyses focused on the identification of taxa, skeletal element representation and biotic and abiotic surface modifications. Bone surfaces were studied using a Dino-Lite PRO digital microscope with a magnification of up to 200x. All traces were registered per bone and recorded by anatomical position. Diagnostic criteria were used to identify hominin induced cut-marks36 and anthropogenic fractures38.In the nineties of the last century a French team, led by Chavaillon, established a general excavation grid at Gombore II-2, with each square meter identified by a progressive letter and number. We followed that grid and the related nomenclature as documented in the literaturehttp://morphosource.org.The archaeological collections, including the faunal remains, are kept at the National Museum of Addis Ababa. All the footprint data and scans from the excavation will be made available on the 3D sharing site Ethical approval for the modern analogue studies of children’s footprints prints is provided by Bournemouth University.Supplementary information