Continuity of care is important for child well-being in all settings where postnatal retention of mother-infant pairs in care remains a challenge. This analysis reports on completeness of patient-held infant Road to Health Booklets (RtHBs), amongst HIV exposed and unexposed infants during the first two years after the RtHB was launched country-wide in South Africa.Secondary data were analysed from two nationally representative, cross-sectional surveys, conducted in 2011-12 and 2012-13. These surveys aimed to measure early effectiveness of the national programme for preventing vertical HIV transmission. Participants were eligible for this analysis if they were 4-8 weeks old, receiving their six-week immunisation, not needing emergency care and had their RtHBs reviewed. Caregivers were interviewed and data abstracted from RtHBs. RtHB completeness across both surveys was defined as the proportion of RtHBs with any of the following indicators recorded: infant birth weight, BCG immunisation, maternal syphilis results and maternal HIV status. A partial proportional odds logistic regression model was used to identify factors associated with completeness. Survey sampling weights were included in all analyses.P<0.05) associated with survey year, marital status, socio-economic status, maternal antenatal TB screening, antenatal infant feeding counselling, delivery at a clinic or hospital and type of birth attendant.Data from 10 415 (99.6%) participants in 2011-12 and 9529 (99.2%) in 2012-13 were analysed. Overall, recording of all four indicators increased from 23.1%   = 22.2-24.0) in 2011-12 to 43.3% (95% CI = 42.3-44.4) in 2012-13. In multivariable models, expected RtHB completeness , was significantly (Routine patient-held infant health RtHB, a critical tool for continuity of care in high HIV/TB prevalence settings, was poorly completed, with less than 50% of the RtHB showing expected completeness. However, government efforts for improved usage of the booklet were evidenced by the near doubling of completeness from 2011 to 2013. Education about its importance and interventions aiming at optimising its use without violating user privacy should be continued. Since 1994, South Africa has made considerable progress in improving the health status of children. With children under the age of 5 years representing approximately 11% of the South African population, national child health policies have focused specifically on the improvement of infant and child health and nutrition . AlthougLoss to follow up of infants along child health programmes such as the immunization or the prevention of mother-to-child transmission of HIV (PMTCT) cascade renders continuity of care post-delivery difficult. Missed opportunities for early infant diagnosis of HIV status or initiation of antiretroviral treatment are often attributed to poor recording of mother’s and/or the infant’s HIV status on patient-held infant health records such as the Road to Health Book (RtHB), poor maternal knowledge about mother-to-child transmission of HIV (MTCT) and the absence of provider-initiated counselling and testing service for the undocumented, unknown, or undeclared HIV-exposed infants . The SouKey to the provision of effective and efficient health care inter alia is the ability to track and retain patients in care through a well-functioning health information system. When mothers and children move from one clinic to another or even from one service to the next within the same clinic, patient-level data (for example HIV-exposure status and HIV test results) required to deliver the necessary longitudinal health care is often of poor quality, or is unrecorded or lost .Currently, the RtHB is the only, readily available tool to be used for monitoring child growth, providing key child health and nutrition messages and tracking uptake of child health care services in South Africa. The patient-held RtHB provided to all children at birth is a simple tool for monitoring and managing these services. The RtHB used in South Africa has been updated four times with the last update being in 2010 . The curPreliminary analyses of data from the nationally representative South African PMTCT evaluation (SAPMTCTE) demonstrated that the absence of documentation of maternal HIV status on the RtHB was associated with a significantly increased risk of postnatal MTCT, and MTCT-or-death . Given these data, there is a dire need to understand and evaluate optimal use of the RtHB across the country. Using data from two large nationally representative SAPMTCTEs, this paper aims to report on the average completeness of the RtHB during the first 6 weeks postpartum and to understand factors that hinder or enable its optimal use. It will benefit future attempts to promote optimal use of the booklet and clarify our understanding as to whether RtHB incompleteness is largely a provider-related problem or certain maternal factors also play a role.Data from two nationally-representative cross-sectional South African PMTCT surveys conducted in 2011-12 (August 2011 – March 2012) and 2012-13 (October 2012 – May 2013) were used. These surveys were designed primarily to measure MTCT by 6 weeks postpartum. The surveys enrolled mother/caregiver-infant pairs at public primary health care facilities (PHCs) and community health centres (CHCs) offering immunization services. A multi-stage , stratified cluster sampling approach was used. Facility size was measured using expected number of DTP  immunisations at 6 weeks. The strata were then created by combining the determined size with known maternal HIV prevalence . Sample size at provincial and facility levels were then determined using probability proportionate to size with a target of providing reasonable provincial and national level estimates. Details of the primary studies have been published elsewhere [In each of the primary studies, trained data collectors recruited eligible mother/caregiver infant pairs consecutively or systematically, depending on facility size, until the targeted sample size was achieved. Infants aged 4-8 weeks who were receiving their six-week immunisation on the day of visit and who were not in need of emergency care were eligible for inclusion in the study following signed informed consent (administered in the participant’s preferred language). Face-to-face interviews were conducted after the routine visit activity was completed and data were captured electronically using mobile phones and then stored in an access-restricted database. Data collected through interviews included maternal socio-demographic backgrounds, antenatal care histories and early postnatal health care uptake.All enrolled participants were asked to present the RtHB during the study interview. During the survey interviews, data were extracted from the RtHB, including infant birth weight, BCG immunization, infant HIV exposure status and maternal syphilis testing results. These data are from the earliest two years after the full country-wide implementation of the RtHB (and phasing out of the RtHC) to which PMTCT indicators had been added.The primary outcome used is “completeness of the RtHB”. Therefore, the data for this study were restricted to the sample of enrolled participants who brought the RtHB during the study interview and these were N = 10 415 (99.6%) in 2011-12 and N = 9529 (99.2%) in 2012-13. We created a composite outcome variable using extracted data for four variables that should have been completed at birth, namely; infant birth weight, BCG immunization, maternal HIV status and indication of whether maternal syphilis testing was done. Therefore, the outcome variable is ordinal with counts from 0 through to 4. These indicators were simply chosen because they were of common interest to both survey aims and are also important to assess PMTCT and maternal and child health service uptake.Maternal characteristics potentially associated with uptake of health care services were assessed for association with completeness of the RtHB. These were age, educational qualification, marital status, parity, knowledge of MTCT modes and relative socioeconomic status (SES). Participants were defined as having good knowledge of MTCT if they understood the definition of MTCT and could correctly identify all three modes of MTCT viz. transmission during pregnancy, childbirth and breastfeeding, whilst those who did not understand the definition of MTCT or were not able to identify all modes were defined as having poor knowledge of MTCT. SES, grouped into quartiles, was calculated for each year using principal component analyses from household characteristics , household possessions , any food shortage and source of income . DiffereData analysis was done in STATA version SE 13 . An ordered logistic regression analyses was used for the outcome variables. Survey sampling weights were used to account for the sample design  and realisation .An additional subcategory was created for independent variables which had more than 5% of data entries in each survey year missing or with ‘don’t know’ responses to the questionnaire. This was done to minimise deviation from the correct estimates when applying the survey sampling weights which were calculated based on the actual attained sample size. The sub-category for ‘unknown’ responses was created for TB screening during delivery and for knowledge of MTCT modes.Factors associated with completeness of the RtHB were identified using a partial proportional odds (PPO) logistic regression analyses in three steps ,15. The 2 statistics and probability values for the null hypothesis that the odds of a predictor variable are proportional across the different binary groupings of the outcome variable (see Table S1 in Online Supplementary Document(Online Supplementary Document)). The possible ordered binary groupings were; completeness ≥1 vs <1, completeness ≥2 vs <2, completeness ≥3 vs <3 and completeness = 4 vs <4 recorded variables. The proportionality of odds assumption was supported in four predictor variables  and hence these were constrained accordingly in the regression analyses. The rest of the predictor variables violated the proportional odds model assumption and thus were not constrained under this assumption.The Brant test output for proportionality of odds across the categories of the outcome variable for each predictor variable are presented as χP-value <0.25 in step 1, indicating potential influence of the predictor on changes in the outcome, were included into a multivariable PPO logistic regression model (to get ‘adjusted’ estimates). In step 3, those predictor variables not included in step 2 were then included, one at a time, and if they significantly shifted the estimates of any predictor variable already in the multivariable model, by shifting the result for proportional odds assumption or the 95% confidence intervals (CI) of the odds ratio completely, then they were retained in the final model, otherwise they were not included.The PPO model was then used with a function to only constrain proportionality to those predictor variables which were consistent with the parallel regression assumptions  and leaving the rest unconstrained. In step 1, separate bivariate PPO logistic regression analyses taking into account the proportionality test results and constraining/not constraining accordingly, were done between the outcome and each predictor variable (estimates ‘unadjusted’ for possible confounding by other variables). In step 2, those with Wald’s test 2 test was used to present descriptive summaries of; independent variables by survey year; completeness of each of the 4 health indicators in the RtHB by survey year; and expected completeness  by each independent variable.A χOut of all those eligible and consenting for full participation in the study, 10 415 in 2011-12 and 9529 in 2012-13 were in possession of a RtHB during the study interview.P = 0.065). Knowledge of MTCT modes was around 60% in both 2011-12 and 2012-13 but appeared to be significantly lower by 2% points in 2012-13 (P = 0.0006).Across both surveys, infants were generally brought to the clinic by their mothers (97%). The majority of mothers were between the ages of 20-34 years with a nearly 2% significant decrease in teenage mothers over time, had an education level of high school or above and the majority of mothers described themselves as either single, widowed, divorced or separated, as shown in P = 0.001). In both survey years, just more than 75% of child deliveries took place in hospitals, less than 20% at a clinic while home deliveries significantly decreased from 5.6% in 2011-12 to 3.5% in 2012-13 (P = 0.004). Approximately two thirds of deliveries were attended by nurses, midwives or community health workers. Doctor-facilitated deliveries increased significantly by nearly 2% between 2011-12 (27.3%) and 2012-13 (29.1%) while deliveries by traditional birth attendants decreased significantly from 4.3% to 3.1% during the same period (P = 0.0007).The proportion of women reporting to have been screened for TB during pregnancy was very low, below 40% across both survey years. On the other hand, most women had received infant feeding counselling during pregnancy, with a significant increase from 89.9% in 2011-12 to 92.5% by 2012-13  and the recording of maternal HIV status (49.1% (95% CI = 48.0-50.2) in 2011-12 to 67.8% (95%C = 66.8-68.8) in 2012-13, P < 0.0001). By the 2012-13 survey recording of infant birth weight and infant BCG immunisation was >89% whilst recording of maternal HIV outcome and syphilis results remained below 70%.Recording of each of the four health indicators in the RtHB were compared between the survey years (P < 0.0001).Completeness of the RtHB improved over time. Recording of only one or two of the four indicators decreased by 5.4 and 14.7 percentage points respectively whilst recording of all four indicators increased by 20.2 percentage points between the two survey years , vs incomplete , was then stratified by each independent variable for each survey year (P < 0.0001). Being married or co-habiting significantly reduced the odds of any level of completion in the RtHB by 10% (P = 0.003). Being in the lowest SES ranking also appeared to significantly reduce the odds of having 3 or all 4 indicators recorded in the booklet by 10% (P = 0.016) and 20% (P < 0.0001) respectively. Maternal education and parity were not significantly associated with completeness of the RtHB.Compared to having zero variables recorded on the RtHB, the odds of having all 4 variables recorded was at least 60% higher in 2012-13 compared to 2011-12 . Delivering at a clinic or a hospital increased the odds of expected completeness compared to delivering at home . Having a nurse, midwife or community health worker handling a child birth as opposed to a doctor significantly increased the odds of having more indicators completed in the RtHB (adjusted odds ratio AOR 1.3 (95% CI = 1.2-1.4), P<0.0001). However, there was no difference in RtHB completeness between deliveries assisted by doctors and those assisted by traditional birth attendants. Significant differences in the completeness of the RtHB at different levels were seen across provinces. Of note were very high odds of better completeness in KwaZulu Natal (AOR≥2.5) and very low odds of better completeness (AOR≤0.6) in North West provinces compared to other provinces.All four factors  directly related to the health care provider were significantly associated with completeness of the RtHB. The odds of having the expected completeness  of the RtHB vs zero, was significantly higher among women who had been screened for TB or received counselling about infant feeding compared to those who did not receive these services during pregnancy  and what were the key predictors of completion. This study shows that most infants were brought to the clinic by their mothers who were mostly between 20-34 years, single, had achieved an education level of high school or more and were multiparous. This provides a unique opportunity for health care workers to engage with mothers on the development of her child as well as to educate mothers on monitoring the child’s growth and health. A study carried out at a primary, secondary and tertiary care centres in one province in South Africa showed that 85.3% of 300 carers who brought their child for care were mothers . Turner Our analyses demonstrate that although possession of the RtHB at the infant’s first immunisation visit is high, overall completeness of the RtHB by this time point is below 50%. In particular, recording of maternal HIV outcome (67.8% in 2012-13) and mother’s syphilis result (69.7% in 2012-13), despite gradual increase overtime, was low overall. Results from a study conducted in one province of South Africa showed that out of 109 RtHBs reviewed, only 50% had the mother’s HIV outcome indicated and PCR results were only recorded for 6% of the exposed infants . HarrisoAlthough reasons for poor recording of health indicators were beyond the scope of this analysis, findings from other studies have provided possible challenges for poor monitoring of the RtHB resulting in missed opportunities for immunisation or growth monitoring. Kitenge and Govender discuss several issues expressed by health care workers in monitoring the RtHB including staff shortages, lack of equipment, work overload and understaffing of nurses, stock-out of vaccines, absence of the RtHB and poor attendance of caregivers at immunisation scheduled visits . AlthougThis study was a secondary analysis from the national PMTCT surveys; therefore data explaining why the RtHB was already incomplete as early as 6 weeks post-partum were not available. Although the simplest reasons for incompleteness of the RtHB such as the caregiver not taking it along for a health care visit (user-related) or the health care provider not asking for the booklet in order to record health information (provider-related) were not evaluated directly here, our sample of mothers suggested that close to 100% of mothers had brought the card to the 6 week immunisation visit and the majority delivered with a formal health provider, but despite this the RtHBs were incomplete. This suggests the responsibility for the largely incomplete RtHBs observed here may lie primarily with provider-related factors.Other studies conducted either in one sub-district or one province of South Africa have reported underutilization of the RtHB. These studies found that both health facility staff and mothers influence underutilization, for example, health facility staff often do not ask for the book, do not fill in the information adequately and accurately and have poor understanding and interpretation of the growth charts ,27. As aAlthough we could not infer causality due to the cross-sectional nature of this study, we observed that both maternal-related and health provider-related factors were associated with RtHB completeness. Being married or co-habiting and belonging to the low SES group lowered the odds of having all four indicators recorded. In the case of factors more related to the health care provider, mothers who had received TB screening or infant feeding counselling during pregnancy, had delivered at a clinic or hospital or had a nurse or midwife or a community health worker assisted delivery compared to doctor-assisted had significantly higher odds of achieving the expected completeness of the RtHB. This could reflect on the competence of the staff and/or their diligence with following general protocols and recommendations at the facilities used by mothers. The difference between doctors and nurses/midwives/community health workers is likely to reflect general responsibilities at the facilities, ie, the latter are usually the majority in primary health care services and hence likely to do more data recording duties compared to the former. This could lead to incomplete recording when doctors attend to clients without the nurses’ involvement.The results on low recording of maternal HIV status we found here support what was being experienced in practise during the early period after the introduction of the booklet. Although no data has been published, there are unofficial reports about the PMTCT page being torn off from the booklet . This waOur study has the following limitations. The two national evaluations were not primarily designed to evaluate the completeness of the infant RtHB within the first 6 weeks of life, therefore the data explaining why the RtHB was already incomplete as early as 6 weeks’ post-partum or the reasons for poor recording of health indicators were not available. Also, the cross-sectional nature of the study limited inference of causality on the observed associations. Infants who required emergency care at the clinic or who died prior to 4-8 weeks or who utilised mobile or private clinics or hospitals were excluded from the survey. Hence infants who received immunisations and whose RtHB was monitored at a private clinic or hospital were not part of this analysis. We do not have information which helps to answer the question of whether lack of information on the RtHB is a result of a health practitioner not asking for the booklet to record information or the caregiver did not bring the booklet. Presently, there are many health indicators that are recorded in the booklet but we were limited to those that had been evaluated in the two national PMTCT surveys done during the years 2011 to 2013.This is the first large, nationally representative study giving a good overview of the nature of use of the RtHB at the very early postnatal phase, in South Africa. Expected use of the RtHB clearly improved over time but was still unsatisfactory by 2012-13, with completeness of all four listed indicators still below 50% yet nearly all mothers brought their records to the facility. Recording of all variables requested in the RtHB, especially those introduced for PMTCT, is important for monitoring and thus preventing MTCT and improving survival. There is a need to assess the current RtHB status and see if the positive trend continued and whether steps should be taken to implement interventions for optimal usage. Since this study showed a consistent association between receipt of antenatal care TB screening, infant feeding counselling or delivery at a clinic/hospital and completeness of the RtHB, integrating promotion of the booklet with in-facility PMTCT activities could be one way to improve its completeness. It is also clear from all the findings presented in this study that interventions aiming at optimal use of the booklet need to not only focus on health providers but also on the users.


Previously, the safety and accuracy of the Eversense continuous glucose monitoring (CGM) system were characterized in three pivotal trials among individuals with type 1 diabetes (T1D) and type 2 diabetes (T2D) with a single 90- or 180-day sensor insertion–removal cycle. The Post-Market Clinical Follow-up (PMCF) registry is a prospective study evaluating the long-term safety and performance of the Eversense CGM system over multiple sensor insertion–removal cycles among adults with T1D and T2D. All patients who had a sensor subcutaneously implanted across 534 participating centers in Europe and South Africa from June 2016 to August 2018 were enrolled. Adverse events (AEs) were recorded at each visit and patients were instructed to inform their clinic if they experienced any AEs between visits. AEs were adjudicated for relatedness to the device, procedure, or drug (dexamethasone acetate). The primary safety endpoint was the rate of related serious adverse events (SAEs) through four sensor insertion–removal cycles. The registry enrolled 3023 patients. As of last follow-up, 5417 sensors had been inserted with a total of 1260 patient-years (PYs) of follow-up: 969 patients had used the system for at least 6 months and 173 patients had used the system for at least 1 year. No related SAEs were reported. The most frequently reported related AEs were sensor location site infection , inability to remove the sensor upon first attempt , and adhesive patch location site irritation . One nonserious allergic reaction to lidocaine was reported, which resolved with administration of an antihistamine. The full intended sensor life was achieved by 91% of 90-day sensors and 75% of 180-day sensors. The PMCF registry provides real-world evidence that the Eversense CGM system is safe over multiple cycles of use. The 90-day system was approved for use in the United States in June 2018. Regulatory approval in Europe and the United States was based on three pivotal clinical studies, which evaluated the accuracy and safety of the system over a single insertion–removal cycle up to 180 days (PRECISE) or up to 90 days (PRECISE II and PRECISION). These studies enrolled 206 patients who had 335 sensor insertion–removal cycles and were followed for a cumulative total of 61 patient-years (PYs).8–10The fully implantable fluorescence-based Eversense CGM system was designed to address several of the limitations of traditional CGM systems. Initial approval of the Eversense system was granted in Europe in May 2016 with an up to 90-day sensor wear time (Eversense8–10 Device- or procedure-related adverse events (AEs) were relatively infrequent (12.6% of patients). AEs were generally mild (78% of events) and transient in nature (66% of events resolved in <2 weeks), and typically resolved with either no or minimal intervention . The most common device- or procedure-related AEs in a pooled analysis of pivotal studies were pain/discomfort (2.9% of patients), redness/erythema at the sensor insertion site (2.4% of patients), incision site infection (1.5% of patients), and inability to remove the sensor on the first attempt (1.5% of patients).11 The primary limitation of the safety database from the pivotal studies is that only a single 90- or 180-day insertion–removal cycle was evaluated.The safety profile of the Eversense CGM system over a single insertion–removal cycle was favorable in all three pivotal studies and compared favorably with traditional transcutaneous CGM systems.The goal of this registry study was to evaluate the long-term safety of the Eversense CGM system in a large real-world patient population following multiple insertion–removal cycles.9 The system comprises three components: a small fully implantable fluorescence-based glucose sensor, a removable smart transmitter, and a mobile application that allows for real-time monitoring of current and historical glucose values on a mobile device. The 90- and 180-day sensors have similar components, including a silicone rubber dexamethasone acetate-eluting ring that contains 1.75 mg of dexamethasone acetate to reduce the foreign body response to the device. Sensors are placed in the subcutaneous tissue of the upper arm under local anesthesia with lidocaine. The placement site is closed using Steri-Strips™ without the need for suturing. The patient-contacting material in the adhesive for the removable smart transmitter is silicone.The Eversense CGM system has been previously described in detail.The transmitter wirelessly communicates through NFC with the sensor and sends data to the mobile application through low energy Bluetooth. The CGM system provides hypoglycemia and hyperglycemia threshold, rate-of-change, and predictive alerts. Two sensor calibrations per day with blood glucose meter readings are required. The sensor operating life is up to either 90 or 180 days, depending on the sensor, or until the fluorescence intensity data indicate that the sensor sensitivity to glucose is inadequate to maintain high accuracy.The Post-Market Clinical Follow-up (PMCF) registry was a prospective registry study among adult participants aged 18 years or older with T1D and T2D across 534 centers in Europe and South Africa. All patients who received the Eversense CGM system at the participating centers were to be enrolled until at least 100 patients had reached four sensor insertion–removal cycles. Consistent with the device label, patients were not candidates for the system if they required a planned MRI during the period of sensor wear, were critically ill or hospitalized, had a known contradiction to dexamethasone, required intravenous mannitol or mannitol irrigation solutions, or were pregnant. The PMCF registry was conducted in compliance with the Declaration of Helsinki, according to the European medical device regulations (MEDDEV 2.12–2), and applicable local requirements for prospective registry data collection. Owing to the registry nature of the study, identifying patient information  was removed before being transferred to the study database.Before the first insertion, patients were trained on the use of the device. Follow-up visits were scheduled every 90 or 180 days, depending on the sensor, for removal of the expired sensor and insertion of a new sensor in the opposite arm.Investigators documented all AEs that were thought to be potentially related to the device, procedure, or drug (dexamethasone acetate) occurring in the clinic and during home use. All current and previous sensor insertion–removal sites and the surrounding area were assessed by the health care provider at each placement and visit. Patients were asked to provide information on any AEs, health-related problems, and changes in health status at each clinic visit. Patients were also instructed to contact the clinic regarding AEs that occurred between visits.An AE was defined as any untoward medical occurrence, unintended disease or injury, or untoward clinical signs  in patients, users, or other persons, whether or not related to the medical device. A serious adverse event (SAE) was defined as an AE that led to death, led to serious deterioration in the health of the patient requiring medical assistance including emergency medical services and/or hospitalization, or led to fetal distress, fetal death, or a congenital abnormality or birth defect. AEs were adjudicated for relatedness to the device, procedure, or drug by a medical monitor.The primary safety endpoint of the study was the rate of related SAEs through four insertion–removal cycles. The primary safety endpoint was to be evaluated against an 8% performance goal using the Kaplan–Meier method. Power analysis determined that a sample size of 100 patients through four insertion–removal cycles would provide ∼80% power to evaluate the primary safety endpoint.Sensor survival through the intended sensor life was evaluated using the Kaplan–Meier method for both the 90- and 180-day sensors.n = 108, 32%), lack of medical reimbursement , and temporary discontinuation for prescription order or availability of the CGM system in their country .The PMCF registry enrolled 3023 patients across 534 participating centers in 14 European countries and South Africa from June 2016 to August 2018. As of August 2018, 5417 sensors had been inserted with a cumulative follow-up of 1260 PYs: 1320 patients (44%) had >1 sensor insertion–removal cycle and 280 patients (9%) had reached their fourth insertion–removal cycle. By last follow-up, 337 patients (11%) had discontinued use of the system. The most common reasons for discontinuation were unknown . One hundred thirty-three AEs were reported of which 117 were adjudicated as related , which met the performance goal for the primary safety endpoint . No clinically significant differences in the incidence rates of related AEs were observed over multiple sensor insertion–removal cycles.n = 29 patients [0.96%]; 2.46 events per 100 PYs). Twenty cases resolved with sensor removal with or without antibiotics, seven cases resolved with local/antibiotic treatment without sensor removal, two cases resolved without information on intervention, and information on resolution was not available in two cases. None of the cases of sensor site infection required hospitalization or administration of systemic antibiotics.The most commonly reported related AE was infection at the sensor site. Thirty-one cases of infection were reported . Twenty sensors were successfully removed during the second attempt by the attending physician or a surgeon; no general anesthesia was required for these secondary procedures. Information on the secondary removal procedure was not available in four cases despite multiple attempts by study personnel to contact the clinical sites. Investigators did not report any other AEs resulting from the unsuccessful first attempts at sensor removal.The first attempt of sensor removal was not successful in 24 cases . Fifteen events were reported as resolved and patients continued to use the CGM system, one event resolved with explant for concomitant infection, and information on resolution was not available in four cases.Adhesive patch irritation was reported in 20 cases (All other related AEs occurred in six or fewer cases at a rate of 0.20% (0.48 events per 100 PYs) or less.The Kaplan–Meier rate for sensor survival through intended sensor life was 91% for the 90-day sensor and 75% for the 180-day sensor.8–10The results of this large registry study demonstrated a favorable safety profile for the Eversense CGM system under real-world use in 3023 patients for 5417 sensor insertion–removal cycles, and for a total of 1260 PYs of device exposure. No related SAEs were reported. The incidence of related AEs over multiple insertion–removal cycles was low. The overall safety profile of the Eversense CGM system in this registry was consistent with the three pivotal trials, which evaluated safety over a single insertion–removal cycle.12 In all cases wherein information was available, complete resolution was achieved with a course of antibiotics or with sensor removal.The most commonly reported related AE, infection at the sensor site, was reported at a rate of 0.96% or 2.46 events per 100 PYs. The rate of infection observed in the registry is considerably lower than the rate of 7.3–11.5 per 100 PYs reported for insulin infusion sets.Inability to remove the sensor on the first attempt occurred in 24 cases. Although such events inconvenience the patient and health care provider, none of the events were serious and the sensor was successfully removed on the second attempt in all cases wherein information was available.13Adhesive patch irritation occurred in 20 cases or 0.66% of all patients. In all cases wherein information was available, the events resolved and the patients continued to use the system, suggesting that the benefits of the device outweighed the inconvenience of transient adhesive irritation. The transient skin reactions observed with Eversense are notably less clinically significant than the allergic contact dermatitis skin reactions that have been reported for transcutaneous CGM systems that contain isobornyl acrylate.A particular strength of this registry was the evaluation of a large number of patients under real-world use conditions. Given the self-reported nature of data collection in the registry, it is possible that some AEs that occurred between visits were not captured. However, it is unlikely that a serious event related to the device occurred without the clinic's knowledge. Another limitation of the study was the inability to collect information on resolution of some of the AEs despite multiple attempts by study personnel to contact the clinical sites.Overall, the PMCF registry demonstrated that the safety profile of the Eversense CGM system is consistent over multiple cycles of sensor insertion–removal in patients with T1D and T2D.


We conducted a pilot study to assess the degree to which an intervention led by community health advisors (CHAs) to promote cancer screening was delivered as intended and to estimate the potential effect of the intervention on receipt of screening. In contrast to previous studies and to maximize its potential public health impact, the intervention targeted 4 screening tests and only participants who were not up to date with screening guidelines for at least 1 cancer. Because CHAs had to both determine baseline adherence and provide counseling on 4 screening tests, the protocol was complex. Complex protocols can reduce implementation fidelity.In partnership with health ministries at 9 African American churches in South Los Angeles, we conducted a 1-group pretest–posttest pilot study to assess the feasibility of implementing the intervention. CHAs recruited and obtained consent from church members aged 50 to 75 years; assessed adherence to national screening guidelines for breast, cervical, colorectal, and prostate cancer; and provided evidence-based strategies  to encourage screening for tests that were overdue.We assessed implementation fidelity by reviewing baseline screening assessments and counseling scripts completed by CHAs. We estimated potential effect of the intervention on receipt of screening by using data from 3-month follow-up surveys, conducted by the research team, of participants who were nonadherent at baseline.From June 2016 to June 2018, 44 CHAs conducted baseline assessments of 775 participants, of whom 338 (44%) were nonadherent to national guidelines for 1 or more cancer screening tests. CHAs provided counseling to most nonadherent participants. At follow-up, about one-third of participants reported that they had discussed cancer screening with their provider and a smaller proportion reported receipt of a screening test; 13% of men and 25% of women reported receipt of colorectal cancer screening.This study demonstrates that with training and ongoing technical assistance, CHAs at African American health ministries can implement complex research protocols with good fidelity. Church–academic partnerships hold promise for reducing cancer disparities, and church-based programs are feasible and acceptable in many populations, including African Americans.We examined the extent to which trained community health advisors (CHAs) implemented a complex intervention that included assessing adherence to screening guidelines and counseling for up to for 4 cancers.Counseling for multiple cancer screening tests can increase the potential public health impact of the intervention. However, complex protocols can reduce implementation fidelity. This study demonstrates that with training and ongoing technical assistance, CHAs at African American health ministries can implement complex research protocols with good fidelity.African Americans have a disproportionate burden of cancer. They are more likely to develop and die of cancer than members of any other racial or ethnic group. Leading causes of cancer death among African Americans are lung, colorectal, breast, and prostate cancer . Cancer Church–academic partnerships hold promise for reducing cancer disparities  and churwww.thecommunityguide.org).Several interventions have promoted cancer screening in collaboration with African American churches. Although most interventions promoted screening for only 1 cancer site –19, a reGiven this body of literature and to maximize the potential public health impact of our intervention, we decided to partner with health ministries at African American churches to promote cancer screening for multiple sites  and to focus our efforts on church members aged 50 to 75 who were nonadherent to the national cancer screening guidelines for at least 1 of these sites. As part of capacity building efforts, we trained CHAs to assess adherence to cancer screening guidelines rather than having the research team conduct the assessment.This article describes the pilot implementation of a protocol by trained CHAs. The protocol consisted of 2 main components: 1) a baseline assessment to determine adherence to screening guidelines for multiple cancers and 2) the implementation of up to 3 intervention strategies to promote adherence to guidelines that were not met. Compared with previous research protocols in African American churches ,17,19, oImplementation fidelity is critical to successful translations of evidence-based strategies into practice , but fidIn partnership with 9 churches in South Los Angeles, we conducted a 1-group pretest–posttest pilot study  from JunSimilar to other health promotion studies in African American churches ,20, our Our intervention approach is consistent with the Social Ecological Framework, which recognizes that individuals are part of families that belong to organizations and social groups, all of which can influence health behavior ,30. AlthLeveraging strong ties of research team members to African American Christian churches in South Los Angeles, we invited 11 churches to partner with us in a study to promote cancer screening. All churches agreed to participate and identified 5 (and in one larger church 9) people to serve as a CHA. Each CHA was asked to recruit at least 10 participants, to assess their screening status, and to counsel those who were not up to date with screening. Two of the 11 churches dropped out immediately after the training because of competing activities. Each of the remaining 9 churches committed to partner with us for 12 months and received a $2,000 stipend. Each CHA received up to $500 in stipends to incentivize their participation.We provided two 4-hour training workshops for CHAs at each church to familiarize CHAs with the study protocol, cancer risk factors and screening guidelines, and the rules governing research with human participants. More details on the training and the CHAs and an evaluation of the training are described elsewhere . By usinEach church team discussed strategies to inform people attending their church about the study and to promote screening churchwide. With guidance from the research team, each team of CHAs developed SMART objectives  for the next 6 to 12 months to promote cancer screening. SMART objectives centered on publishing personal interest stories ; organizing a worship service that featured a health theme in the sermon or dedicating 1 Sunday per month to health promotion; and making presentations to inform church members and visitors about the study. An example of a SMART objective is “The Health Council will organize a 15-minute talk on Father’s Day  by [name of presenter] to inform parishioners about the study and to introduce the CHAs to the congregation.”After completion of the 2 training workshops, the research team conducted debriefing sessions with CHAs at each church every 6 to 8 weeks to answer questions, collect completed study documents (baseline surveys and counseling scripts with CHA notes), and provide CHAs with additional study materials, as needed .To assist CHAs in implementing the one-on-one education in a standardized fashion, we provided a 5-part counseling script for each cancer site .Part 1. Each script started with an interesting fact pertaining to the African American community to raise interest in the topic and to explain its importance. For example:Among cancers that affect both men and women, colorectal cancer is the second leading cancer killer in the US. According to studies, African Americans are at a higher risk for the disease than other populations. Research shows that African Americans are being diagnosed at a younger age and death rates from colorectal cancer are higher among African Americans than any other group in the United States.Part 2. Next, CHAs asked if the participant ever had a particular screening test and explained each test, if needed, using print materials from the National Cancer Institute or the American Cancer Society. The materials included pictures of the test.Part 3. Counseling on barriers to screening started with an open-ended question about any reasons why the participant did not have the test according to the guidelines. CHAs checked off the barriers that were mentioned and probed for other barriers that were not mentioned. For each barrier, a short response was provided for CHAs to use during the counseling. For example, if the barrier was “no time,” the CHA responded with, “It is important to make time for your health. The things you are putting off now because you ‘don’t have time’ are going to cost you much more than time if you wait until it is too late.”Part 4. In this part, the goal-setting section, the CHA asked participants if they were ready to ask their doctor about getting the cancer screening test that was overdue. CHAs also asked respondents to set a goal for the time frame.Part 5. The conversation ended with the CHA asking for permission to call again in 3 or 4 weeks to see if the participant had more questions or needed more help in getting the test.We selected evaluation methods to assess implementation fidelity, the acceptability of the CHA-led intervention, and the potential effect on cancer screening promotion. We developed brief assessments, keeping in mind the burden they posed to CHAs and respondents.all recommended cancer screening guidelines” or “not adherent with at least one recommended cancer screening guideline.”The 1-page baseline assessment asked for name, age, address, and telephone number of the participant and whether the participant had received any of the following: stool blood test, sigmoidoscopy, colonoscopy; mammogram, Pap test, human papilloma virus test (women only); and PSA test  . In addiImplementation fidelity. CHAs documented their counseling efforts with each participant in the notes section of the counseling script, including the barrier they discussed and whether or not they distributed print information and issued another reminder 3 or 4 weeks after the initial counseling session. Implementation fidelity  was also assessed in the 3-month follow-up survey of study participants.Three-month follow-up survey of participants who were nonadherent at baseline. Participants who were nonadherent at baseline to at least 1 screening test were contacted again for a 3-month follow-up survey. This survey assessed receipt of needed screening test(s) and discussion of cancer screening with a health care provider during the follow-up period (both of these outcomes were encouraged by CHAs); receipt of intervention components (fidelity of intervention implementation as reported by participants); and satisfaction with the educational session and information on demographic characteristics and access to care. The 3-month survey was conducted by telephone by 1 member of the research team who was not involved in the intervention, rather than by multiple CHAs. This reduced social desirability bias, decreased the workload for CHAs, and facilitated a more consistent administration of the survey. The follow-up survey took 20 minutes on average. Participants who completed the 3-month follow-up survey received a $20 store gift card.Twelve-month debriefing of CHAs. The debriefings were conducted during the end-of-study meeting and celebration at 8 churches. During this 2-hour meeting, we discussed implementation of each SMART objective and barriers to implementation and presented certificates of participation to CHAs; we then hosted a catered lunch to show our appreciation.2 tests for categorical variables and 2-sample t tests for continuous variables.We conducted all analyses by using SAS 9.4 (SAS Institute Inc). We used descriptive statistics to describe demographic information and access to care and receipt of intervention components. We compared the sex, age, zip code, and baseline screening rates of participants who completed the 3-month follow-up with the characteristics of those who did not. We also compared the characteristics of men and women who completed the 3-month follow-up survey. Both comparisons used χWe assessed the potential effect size of the intervention for 2 outcomes: 1) discussion of cancer screening with a provider during the follow-up period  and 2) receipt of a specified screening test during the follow-up period (asked only of participants nonadherent at baseline who needed a specific test). We assessed these 2 outcomes in 2 ways: “as reported” by those who completed the 3-month follow-up survey and as a “worst-case” scenario. The worst-case scenario included all participants who completed the baseline assessment; in this scenario, missing information in the follow-up survey was coded as “not discussed” or “not screened.” Analyses on discussion and receipt of a Pap test included only women aged 50 to 65 who did not report having had a hysterectomy.During the study period, 44 CHAs from 9 African American churches conducted baseline assessments of 775 participants, of whom 338 (44%) were nonadherent to national screening guidelines for 1 or more cancer screening tests. More than 90% of baseline assessments and counseling sessions were conducted face-to-face and the rest by telephone. Of the 338 participants who were nonadherent at baseline, 253 completed follow-up telephone surveys, for a retention rate of 75% . Only 20Churches developed 4 to 8 SMART objectives . One chuBarriers to implementation included being too busy with other activities, having a small congregation and not enough volunteers to support planned activities , discontinuing an activity because it was not successful , and choosing an activity that sounded exciting and creative but was too difficult to implement .According to completed counseling scripts, CHAs provided counseling on 1) colorectal cancer screening to 185 of 226 participants who were overdue, 2) breast cancer screening to 77 of 110 women, 3) cervical cancer screening to 48 of 60 women aged 50 to 65 years, and 4) PSA informed decision making to 74 out of 102 men . The barAccording to 3-month follow-up surveys, most participants said that they had discussed their cancer screening history and which screening test they needed with a CHA (80%), that the CHA had recommended they discuss cancer screening with their physician (91%) and obtain a cancer screening test (72%), and that the CHA provided print materials on cancer screening (65%). These data, provided by participants, were consistent with the implementation data derived from counseling scripts completed by CHAs.Although 44 CHAs performed baseline assessments, only 38 CHAs provided counseling, according to counseling scripts: 11 CHAs performed counseling for 1 to 4 overdue screening tests, 13 CHAs performed counseling for 5 to 10 overdue screening tests, and 14 of 44 CHAs performed counseling for more than 10 overdue screening tests.P = .001), to have a regular doctor (P < .001), to ever go to a physician for a medical checkup (P < .001), and to have had a routine checkup in the past 12 months (P = .03).Of the 338 participants who were nonadherent at baseline to at least 1 national cancer screening guideline, 124 were men and 214 were women aged 50 to 75. Of the 253 participants who completed the 3-month follow-up survey, 95 were men and 158 were women aged 50 to 75; mean age was approximately 60, and education ranged from less than high school graduate to college graduate . Women wP = .56), sex (P = .57), or age (P = .93). However, participants who completed the 3-month follow-up survey were significantly more likely than noncompleters to report at baseline that they had ever had a colonoscopy , mammogram , or Pap test . Men who completed the 3-month follow-up survey and noncompleters did not differ in ever having had a PSA test  or discussing a PSA test with a physician .We found no significant differences between the participants who completed the 3-month follow-up survey and noncompleters for residence  and prostate cancer screening (28%) with a physician and a substantial proportion of women discussed colorectal cancer screening (36%), breast cancer screening (43%), and cervical cancer screening (33%) with a physician .Smaller percentages of participants reported receipt of a screening test. Overall, 13% of men and 25% of women reported receipt of colorectal cancer screening. Among the 84 men who were nonadherent to colorectal cancer screening at baseline, 4 (5%) reported receipt of a stool blood test and 7 (8%) reported receipt of a colonoscopy at 3-month follow-up. Among women who were nonadherent at baseline, 16% (23 of 142) reported receipt of a stool blood test, 10% (14 of 142) receipt of a colonoscopy, 24% (27 of 111) receipt of a mammogram, and 17% (10 of 60) receipt of a Pap test at 3-month follow-up.“Early detection can save your life.”“I need to take better care of my health.”“I never heard of stool blood test; it is easier than colonoscopy.”“I learned about increased risk for cancer with age.”“Remember to get screened even if you feel healthy.”“Getting checked is better than trying to avoid the doctor.”A CHA from one of the churches stated, “Each person I talked to . . . I felt I was really doing something.” That quote summarizes the sentiments expressed by several CHAs and indicates that the intervention was acceptable to CHAs as well.At 3-month follow-up, most participants stated that they would recommend CHA counseling to a friend (94%) and that they would attend a counseling session on another topic if it was offered (83%). Among those who stated that they had discussed cancer screening with a CHA, 91% said that they learned something important from their discussion and 98% felt comfortable asking questions. The following quotes illustrate some of the things participants learned:Although CHAs did not systematically collect information on how many people refused to participate in the intervention, they estimated the proportion to be less than 10% of the people they approached. This and the fact that almost half (47%) of African Americans attend religious services at least once per week  suggest Although we attempted to keep all study materials simple, several CHAs found it difficult to assess adherence to 3 tests for colorectal cancer screening, each with a different recommendation on frequency. Regular debriefing sessions at each church provided the opportunity to discuss completed assessments and to clarify questions about adherence to national cancer screening guidelines.CHAs conducted a substantial number of counseling sessions, and records indicated very good implementation of one-on-one education. However, print materials were distributed only as needed , because we did not want to burden participants with unwanted materials. CHAs also had some problems reaching participants by telephone to issue reminders. We learned that many seniors in South Los Angeles scan their incoming calls and do not accept calls from unknown numbers because of widespread telemarketing and fear of scams. Providing the full dose of an intervention  is a challenge in real-life settings. In another study, which offered 3 CHA-led educational workshops to promote cancer screening in African American churches, only 24% of participants attended all 3 workshops, thereby receiving the full dose . BecauseMost CHAs were able to conduct one-on-one counseling, but only about one-third of CHAs provided counseling for more than 10 overdue screening tests. Thus, although the burden of serving as a CHA was manageable, only a few CHAs acquired the skills developed by repeat counseling. Overall, implementation required intensive training and monitoring. Although implementation of this intervention was feasible, modifications to simplify and reduce data collection protocols may be required to sustain the effort.All data were based on self-reports by CHAs and participants, which are susceptible to social desirability bias. Because we lacked a control group, we do not know how many of the screening tests reported at follow-up can be attributed to the intervention. In addition, estimates for potential effect size may have been influenced by selection bias and differential drop-out rates; participants who did not discuss screening or did not obtain a needed test may have been more likely to drop out . Our worst-case analysis attempted to account for some of these limitations. Overall, the potential effect size is similar to that reported by other studies that partnered with African American churches ,33. Our Our study supports the feasibility and acceptability of promoting cancer screening in partnership with health ministries in African American churches. We reported on one of the first studies in African American churches in which CHAs, rather than research team members, assessed screening history and in which CHAs promoted screening for multiple cancers. Our study demonstrates that with training and ongoing technical assistance, CHAs can implement complex research protocols with good fidelity.


During outbreaks of emerging viruses, such as the Zika outbreak in 2015–2016, speed and accuracy in detection of infection are critical factors to control the spread of the disease; often serological and diagnostic methods for emerging viruses are not well developed and validated. Thus, vaccines and treatments are difficult to evaluate due to the lack of comparable methods. In this study, we show how the 1st WHO International Standard for anti-Zika antibody was able to harmonize the neutralization titres of a panel of serological Zika-positive samples from laboratories worldwide. Expression of the titres in International Unit per millilitre reduced the inter-laboratory variance, allowing for greater comparability between laboratories. We advocate the use of the International Standard for anti-Zika virus antibodies for the calibration of neutralization assays to create a common language, which will permit a clear evaluation of the results of different clinical trials and expedite the vaccine/treatment development. Protocols for achieving fast and effective detection of Zika virus (ZIKV) infections during outbreaks are limited by the cross-reactivity of immunoassays and the inter-laboratory variability of results, which may adversely affect response planning in the field and slow the development of new diagnostic tools and therapies. Giada Mattiuzzo and colleagues now applied the WHO International Standard for ZIKV-specific antibodies to harmonize the neutralization titres generated from a panel of serological ZIKV-positive samples analysed in multiple laboratories across the world. The use of the International Standard, which includes a standard sample (a defined pool of sera from confirmed ZIKV-positive cases) and an agreed International Unit, reduced the inter-laboratory variance in neutralization assay results performed on ZIKV of Asian lineage, and enabled a better comparison and integration of the multisite data. This work establishes the first International Standard for an Asian lineage ZIKV-specific antibody for facilitating ZIKV diagnostics, prevention and treatment. Aedes species mosquitoes. ZIKV was discovered in 1947 and was only known as a cause of sporadic mild disease in Africa and Asia.2 The first major outbreak occurred in 2007 in Yap Island with an attack rate as high as 70% of the population.3 In 2013, during an outbreak in French Polynesia with a similarly high attack rate, the possible association with Guillain–Barré Syndrome was uncovered.4 The outbreak of ZIKV in the Latin American region in 2015–2016 was the largest recorded epidemic of Zika disease as of early 2019. Although the majority of Zika infections are asymptomatic or result in mild symptoms, the large number of cases in Latin America provided evidence for a correlation between Zika infection and increased cases of microcephaly and other neurological diseases.6 This association prompted the World Health Organization (WHO) to declare the outbreak a Public Health Emergency of International Concern (PHEIC) in February 2016.7 WHO called on the global research and product development (R&D) communities to prioritize the development of vaccines together with improved diagnostics, and innovative vector control strategies for ZIKV R&D. Although the PHEIC was declared over by the WHO Director-General in November 2016, ZIKV remains an enduring public health challenge requiring continued action, as new outbreaks may occur.8 Many uncertainties remain with regard to disease epidemiology and transmission, and, therefore, projecting the future evolution of the ZIKV epidemic and further spread based on the current knowledge remains difficult. In February 2018, WHO published “Zika Vaccine Development Technology Roadmap” to support development of a vaccine for outbreak use with the characteristics proposed within the Target Product Profile.10 In that context, standardization of virologic and immunologic assays for ZIKV vaccine development was identified as one of the priority areas. There is no licensed vaccine or treatment available, but development of these products is ongoing. According to the current knowledge on the transmission of ZIKV and experiences with past disease outbreaks, WHO has prioritized the development of vaccines suitable for use in an emergency or outbreak scenario. There are more than 40 vaccine candidates being developed using different platforms. Most of them are in the pre-clinical stage of development, but there are a few candidates which are the subject of clinical trials.11Zika virus (ZIKV) is a flavivirus mainly transmitted by 13 Indeed, despite ZIKV infection occurring through the bite of infected mosquitoes of the genus Aedes, the virus can also be passed from mother to foetus and through exchange of body fluids.16 Molecular tests for ZIKV are the most reliable and specific, and there are several diagnostic tools authorized by the United States Food and Drug Administration under Emergency Use Authorization (EUA), while others are included in the WHO Emergency Use Assessment and Listing.17 However, ZIKV in blood/serum is short-lived (3–5 days), and up to 10 days in urine;18 therefore, a serological assay to measure immune exposure by antibody detection is suggested 7 days post onset of symptoms.17 A major problem with detection of anti-ZIKV antibodies is cross-reactivity with other flaviviruses, such as dengue virus (DENV).21 Specifically, the envelope glycoprotein E contain regions highly conserved between ZIKV and DENV, which are targets for binding and neutralizing antibodies.22 At the time of the outbreak in 2015/16, the only authorized EUA test was the Zika Mac-ELISA (enzyme-linked immunosorbent assay), which addresses the antibody cross-reactivity with other flaviviruses by requiring a 4-fold higher antibody titre in a plaque reduction neutralization assay against ZIKV than other flaviviruses to confirm infection.17 Recently, to avoid cross-reactivity, serological assays have been developed targeting antibodies specific to the non-structural protein 1 (NS1), which is secreted by ZIKV-infected cells into the body fluids.24Accurate diagnosis is essential to monitor and control the spread of ZIKV infection, as well as to provide guidance for pregnant women, or those planning to have children.26 Standardized assay methods have their limitations, however, due to variations in operator performance, different implementations among laboratories, and a lack of consistency over time as new refinements and technologies are introduced. Instead, the use of a biological standard that is common to all assays allows for calibration and harmonization of the data from different laboratories worldwide and bridges through advances in technology. The WHO International Standards (WHO ISs) are the highest order of reference materials and allow comparison of biological measurements (activity/potency) by defining an internationally agreed unit, the International Unit (IU27). In-house or kit working standards should be calibrated using the WHO IS and the results reported in IU. Use of WHO IS has an impact on product development, for example, the majority of the molecular assays for hepatitis C virus (HCV) detection and quantification are calibrated to the WHO IS for HCV RNA;28 furthermore, the potency of Yellow Fever vaccines is expressed in IU29 by calibration of the assay to the 1st International Standard for Yellow Fever vaccine.30Standardized, validated assays are of utmost importance not only for diagnosis of ZIKV infection and surveillance but also for vaccine development as anti-ZIKV antibody responses will most likely be used as immune markers for the evaluation of vaccine efficacy when large clinical trials with immunological/safety endpoints may be possible, but not clinical efficacy endpoints.A multi-centre international collaborative study was organized to evaluate whether a reference preparation of sera or plasma from ZIKV-infected individuals could help harmonize serological assays for ZIKV antibody and the main outcomes of the study are presented in this article.Nine laboratories  or endpoint titre estimates.All laboratories identified correctly all the positive samples, these being either the pools of convalescent plasma/serum  antibodies were detected in qualitative  targeting the E protein of ZIKV (Brazil 2016 isolate). The samples could be ranked similarly to their neutralization titres Table  with theThe stability of the candidate IS (sample S80) was assessed by accelerated thermal degradation testing. Ampoules were placed into storage at 45, 37, 20, 4, and −20 °C, and retrieved at the following time points: 2 weeks, 1 month, 3 months, 6 months, and 1 year and stored at −20 °C until assayed. The freeze-dried preparations were reconstituted as per instruction for use and tested concurrently in triplicate by in-house ELISA. Data are reported as relative to the baseline temperature −20 °C  produced more comparable results with a reduction of inter-laboratory variance  contains ZIKV-specific antibodies. However, due to the presence of cross-reactive anti-DENV antibodies, the IS for anti-ZIKV antibodies cannot be used for the validation of specificity of ZIKV serological assays.It is important to highlight that all pools of convalescent serum/plasma in this study had some reactivity to DENV. Anti-DENV reference materials from NIBSC  were included in the study to assess assay specificity. Although cross-reactivity against DENV of neutralizing antibodies elicited by ZIKV infection has been reported, the lower titre (4–10-fold) allows for discrimination of the ZIKV infection in a DENV-immune patient.39 The IS is available from the NIBSC catalogue (www.nibsc.org/), code 16/352, in ampoule numbers anticipated to be available for 5–10 years; stability studies have shown that the preparation is also stable for long-term storage. Furthermore, sample S14, the pool of convalescent plasma (S14) from Boca Biolistics, is also available in the NIBSC on-line catalogue (code 16/320) as a working reagent calibrated to the 1st WHO IS in this collaborative study. The calculated value of 16/320 is 2756 IU/mL, with 95% confidence limits of 2003 to 3792.The outcome of this study was that the NHSBT pool of convalescent serum sample (Sample Code S80) was established by the WHO Expert Committee on Biological Standardization in October 2018 as the 1st WHO IS for anti-Asian lineage ZIKV antibody to be used in the standardization and assessment of neutralization assays with an assigned unitage of 250 IU per ampoule.This study has shown how normalization of the data from different assays by the IS allows for comparison and harmonization of results from laboratories worldwide. The use of the IS reduced the inter-laboratory variation, albeit there is still up to a 10-fold difference in the titres reported in IU/mL. Use of standardized methods with a validated standard operation procedure and sharing of the same reagents could be a further step to reduce inter-laboratory variations; however, this is beyond the scope of this work and could be evaluated by the scientific community and regulatory bodies working on Zika. Nevertheless, calibration of assays using the IS for ZIKV antibodies will allow for the consistency of antibody measurements among laboratories, the comparative analysis of candidate vaccines in clinical trials, which aim to determine protective antibody levels in human or animal models, leading to a batch/lot release specification; therefore, resulting in expedited licensing and subsequent availability of vaccines.In conclusion, WHO standardization activities led by NIBSC resulted in the development and establishment of the 1st IS for anti-Asian lineage ZIKV antibody. This is an important achievement for the scientific community across ZIKV diagnostics, prevention, and treatment. It is expected that the above-mentioned standard will be used in the clinical development of ZIKV vaccines with the aim of reporting results of the assessment of the immune response in IUs. There have been examples of missed opportunities in using the ISs for development of new vaccines. There are several reasons, but the most important are: (1) lack of understanding among principal investigators of the benefits that the IS provide in the interpretation of data from clinical trials and (2) misunderstandings about assay standardization that less experienced vaccine developers are facing when establishing testing procedures for vaccines under development. Multiple vaccine candidates have shown robust protection against ZIKV challenge in animal models, but the demonstration of the protection in humans remains a challenging task. For the comparability of clinical trial results, it is critical to use WHO IS as a basis for assay standardization and optimization. Correct interpretation of the results from clinical trials is one of the essential elements to assure quality, safety, and efficacy of vaccines and diagnostics. Feedback from users will help WHO and its Collaborating Centre, NIBSC, to advance the further use of this standard as well as the development of other standards and reagents that may improve standardization of assays used in the clinical evaluation of Zika vaccines.Nineteen participants with an anti-ZIKV antibody assay in use in their laboratories completed the study. Prerequisites for participation in the collaborative study are: (1) availability of reliable assay for detection of Zika antibody and (2) willingness to participate. This approach provides a platform to evaluate the suitability of the standard to harmonize as wide a range of assay types as possible, which is essential for robustness of WHO standards. The participants were from six countries: Germany (1), UK (2), Korea (1), Trinidad and Tobago (1), Denmark (1), and USA (13). All laboratories are referred to by a code number allocated at random and are not reflected in the order of listing at the end of the paper. Participating labs included 11 government research, public health, medical counter-measure, and regulatory organizations; three university and research organizations; five developers of biologics, assays, and reagents.The project was approved by the NIBSC Human Material Advisory Committee (project 16/005MP). Plasma and sera were donated to NIBSC anonymized by the organizations listed in Table 40 to inactivate blood-borne viruses, as on initial screening these were found to be positive for HIV RNA, HBV antigen or HCV RNA. Post treatment testing for the presence of RNA was negative.Collaborative study samples were provided to the participants blind coded and are listed in Table 31 immunized with ZIKV immunogens comprising either inactivated virus , plasmid DNA coding full-length ZIKV E protein , or the naive control (sample S6).41 Upon receipt at NIBSC, the purified Tc bovine IgG samples were diluted to a target protein concentration of 2.5 mg/mL in sterile phosphate-buffered saline (PBS)/Ca2+/Mg2+ supplemented with 5% human serum albumin and aliquoted into 0.1 mL volumes and stored at −20 °C.Additional samples were human anti-ZIKV IgG antibodies purified from plasma collected from Tc cattleNegative human plasma and serum were from single donation packs from the UK; all packs were tested for blood borne markers  and found to be negative.42Anti-DENV serotype antibodies  were sourced from NIBSC.Assays used by participants are summarized in Table 2SO4. For the analysis, absorbance from each well was subtracted from the average of absorbance of the diluent-only wells. An optical density >0.1 after subtraction with the correspondent negative control was arbitrarily chosen as a positive result.Nunc Maxisorp 96-well plates were coated overnight at 4 °C with ZIKV (Asian strain—PRVABC59)-containing supernatant from VERO cells diluted in PBS. Conditioned media from uninfected VERO cells was used as a negative control. Virus was fixed with 0.1 mL per well of 4% paraformaldehyde diluted in PBS for 30 min at room temperature. Plates were washed three times with PBS, and then blocked with 0.2 mL of 10% foetal bovine serum in PBS for 1 h at room temperature. Plates were washed three times with PBS containing 0.05% Tween-20 (PBST). A measure of 0.1 mL of serum samples diluted 1/400 in blocking buffer were added to the plates in triplicate and incubated at room temperature for 1 hour. Wells were washed with PBST and 0.1 mL of anti-human horse radish peroxidase conjugate antibody  diluted 1:5000 in blocking buffer were added to each well. After 1 h incubation at room temperature, plates were washed with PBST and developed by adding 3,3′,5,5′-Tetramethylbenzidine Substrate Systems . Reactions were stopped after 10 min by the addition of 1 M HParticipants were requested to test the samples using the method(s) in use in their laboratories for the detection of antibodies to ZIKV. Participants were asked to perform three independent assays on different days. An Excel reporting sheet was provided with suggested dilutions for assaying each study sample. For each assay, participants were requested to make at least two independent series of dilutions of the study samples and assay all samples concurrently if feasible.50 provided by the participants. GMs were calculated only when more than half of the results for a sample produced a positive response.For the neutralization assays, the GM of the potency of each sample was calculated from the endpoint titres or PRNT43 Model fit was assessed visually, and non-parallelism was assessed by calculation of the ratio of fitted slopes for the test and reference samples under consideration. The samples were concluded to be non-parallel when the slope ratio was outside the range 0.80–1.25. Relative potency estimates from all valid assays were combined to generate an unweighted GM for each laboratory and assay type, with these laboratory means being used to calculate overall unweighted GMs for each analyte.Quantitative ELISA data were analysed using a parallel line model with untransformed or log-transformed responses. Calculations were performed using the European Directorate for the Quality of Medicines & Healthcare (EDQM) software CombiStats™.s − 1] × 100%, where s is the standard deviation of the log10-transformed estimates). Further assessment of agreement in GM results for each pair of laboratories was performed by calculating Lin’s concordance correlation coefficient with log-transformed data, although these values are only based on a small number of samples . Calculations were performed using the R package DescTools.44Variability between laboratories has been expressed using geometric coefficients of variation (GCV = [10Further information on research design is available in the Supplementary TablesReporting Summary


Staphylococcus aureus (MRSA) originating from a surgical ward. Whole-genome sequencing (WGS) identified the outbreak clone as spa type t267, sequence type ST97, and SCCmec IVa. Prompted by the finding of four patients within 6 months in the same ward with this unusual MRSA type, an outbreak was suspected. Subsequent MRSA screening in the ward in February 2017 identified three-additional patients and two health care workers (HCWs) with t267/ST97-IVa. WGS linked these 9 isolates to 16 previous isolates in our WGS database and the outbreak thus included 23 patients and two HCWs. Twenty-one patients had a connection to the surgery ward during the period 2013–2017, but half of them had MRSA diagnosed in the community long after discharge. The community debut of several patients MRSA infections weeks to months after hospital discharge made the identification of a hospital source difficult and it was the SNP relatedness of the isolates that led us to identify the common denominator of hospitalization. An index patient was not identified, but our hypothesis is that HCWs with unrecognized long-term MRSA colonization could have caused sporadic nosocomial transmission due to intermittent breaches in infection prevention and control practice.This study describes a sporadically occurring 4-year outbreak of methicillin-resistant  Staphylococcus aureus (MRSA) has been a global medical challenge since its emergence in 1961, 2 years after methicillin was introduced to treat penicillin-resistant S. aureus . DNA extraction were performed on all MRSA isolates and libraries were made with 2 × 150 bp paired-end Nextera XT DNA sample preparation kit  and sequenced on a MiSeq . The reads were mapped to a USA300 reference sequence (US300_TCH1516) using stampy  that detects the presence of g stampy  with an g stampy  for singg stampy  mpileup g stampy  or Spadeg stampy . PhylogePermission to link the sequencing of MRSA from routine clinical samples to patient data without individual patient consent was obtained from the Danish Data Protection Agency . Permission to look up the patients admission data without individual consent was obtained from the Hospital Board (no. WZ17038300-2018-14).mec type, namely IVa. Thus, the suspicion of an MRSA outbreak was confirmed. This led to a several week-long screening of patients, HCWs and other staff members in the ward resulting in the finding of three-additional patients and two HCWs with t267/ST97-IVa. Furthermore, screening at the ward revealed that two HCWs carried other MRSA types  and one patient with known contact to pigs carried the livestock-associated t034/ST398-V.In January 2017, an MRSA infected patient was found in one of the hospital’s surgery wards. From the sample of abdominal pus two distinct MRSA types were found based on antimicrobial susceptibility patterns. WGS of the two isolates identified a t267/ST97-IVa and a t002/ST5-IVg. Looking 6 months back in our MRSA WGS database, we identified three other patients admitted to the same ward that had tested positive for MRSA t267/ST97. Searching our WGS database back to 2013 we found this to be an unusual MRSA type in our region with just 34 isolates from 2013 to January 2017 (0.7%). A neighbor-joining tree was then constructed with all 34 isolates showing 20 isolates with a close connection of ≤50 SNPs. These isolates also shared the same SCCFigure 1) we ended up with 25 persons. Eighteen had been admitted to the surgical ward, two were HCWs, two were family members of MRSA positive patients, and one had shared a room at another ward in another hospital with an MRSA positive patient, who had previously been admitted to the surgery ward. Finally, two patients had no relation either to another patient or to the ward to the best of our knowledge. We have data on hospitalization at the surgery ward for most patients but the surgery ward has many sub-departments and we lack data on room allocations as well as the exact department admitted to. Our data show that 13 patients had an overlap in admission period with at least one other patient .In our final outbreak cluster (Table 1).Nine patients had been diagnosed with MRSA t267/ST97-IVa at their General Practitioner and three more had been diagnosed at the Emergency Room, resulting in 12 cases first identified in the community, here defined as community-onset MRSA (CO-MRSA). Eight patients had their MRSA diagnosed at hospital wards HA-MRSA. Five persons had been diagnosed by screening of the hospital ward (including the two HCWs) . Our database contained 266 t002/ST5 isolates of which 40 harbored SCCmec IVg .Due to the finding of other MRSA types among our HCWs, we also studied the phylogenetic tree of all the t045/ST5-IVc/e and t002/ST5-IVg isolates in our WGS database. There were only three t045/ST5 isolates and the two others had another SCCThe 25 t267/ST97/IVa isolates showed a low diversity with a maximum of 50 SNP differences over the 4-year period. Within our outbreak cluster, a further sub-cluster could be distinguished with 15 isolates with a maximum of 11 SNPs. This sub-cluster consisted of 13 patients admitted in 2016 and 2017 as well as the two HCWs.spa type t267 and ST97 except for two isolates with an unnamed ST that was a single locus variant (SLV) of ST97. All isolates had SCCmec IVa and no isolates had Panton–Valentine leukocidin (PVL) or the arginine catabolic mobile element (ACME). The 14 non-outbreak related isolates of t267/ST97 had SCCmec IVc/e (mec IVa (mec V (All outbreak isolates belonged to ec IVc/e , SCCmec (mec IVa , and SCCa (mec V . The outAn outbreak group with representatives from the surgical ward, the Department of Cleaning, the Department of Clinical Microbiology, and the Infection Control Organization was established. In order to exclude shortcomings of general infection control precautions, various initiatives were launched. The ward had daily visits by the infection control and prevention nurse, where behavior was observed and adherence to procedures monitored, and on this basis an increased focus was placed on the use of protective equipment such as gloves and plastic aprons, which among other things led to an increased availability of protective equipment in the department. Furthermore, focus was on infection control precautions for both HCWs and patients and in particular on how patients could be motivated for better hand hygiene.The ward was cleaned by standard hospital cleaning, followed by manual disinfection with bleach. The HCWs with the outbreak clone were successfully decolonized.It is a global trend that patients are hospitalized for increasingly shorter time periods. Therefore, hospital acquisition of an MRSA might not be suspected or identified due to clinical onset long after discharge. Healthcare-associated MRSA outbreaks are rare in Denmark  and havemec IVa, that was discovered as the MRSA was found in four patients in the same ward within 6 months. This, for us, rare MRSA has sporadically been found around the world and has been described both as a CA-MRSA (mec, which also indicates another clone.Here, we report a 4-year long outbreak of MRSA type t267/ST97 SCC CA-MRSA  and as a CA-MRSA . We have CA-MRSA  and more CA-MRSA . The remSince the outbreak was ongoing for such a prolonged period at one of our busiest surgical wards, the actual number of people infected or colonized with the MRSA clonal isolate is probably higher than the rather modest number we report. Only individuals in the community who had a clinical infection would have been identified through a sample so MRSA carriers can very well have eluded the system. There could be various explanations to the increase in the number of positive MRSA patients in 2016–2017 in this study. Of course, we found quite a few through the screening in 2017 that might not have been found if the screening had not been performed. Another aspect could be that there were unknown MRSA positive HCWs that had quit the ward before the screening in 2017, but contributed to the increase in cases in 2016 and 2017. Nevertheless, the finding of relatively few patients over a 4-year period indicates there is relatively little spread between HCWs and patients. Previous studies have concluded that nosocomial outbreaks caused by HCWs represent rare events, and therefore screening of personnel should not be performed regularly . AnotherTo automatically detect potential outbreaks the combination of detailed epidemiological data together with WGS is crucial . In the Whole-genome sequencing can enhance the detection of prolonged hospital outbreaks of MRSA. Furthermore, we bring to light the fact that increasingly shorter hospital stays delays outbreak detection if continuous analysis of WGS is not performed. In this study, we identified an MRSA outbreak in February 2017 and with the use of WGS we could trace the outbreak 4 years back in time.We hypothesize that HCWs with an unknown MRSA carrier-state might cause sporadic transmission and sustain an unknown outbreak over many years. However, we believe that HCWs with a known MRSA carrier-state usually do not cause transmission due to their personally increased awareness of the importance of infection control standard precautions.TH and PW performed the bioinformatics analyses. All authors contributed to the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.