Area-specific covid-19 effects on health services utilization in the Democratic Republic of the Congo using routine health information system data.

BACKGROUND: Since March 2020, the COVID-19 pandemic has shocked health systems worldwide. This analysis investigated the effects of the pandemic on basic health services utilization in the Democratic Republic of the Congo (DRC) and examined the variability of COVID effects in the capital city Kinshasa, in other urban areas, and in rural areas. METHODS: We estimated time trends models using national health information system data to replicate pre-COVID-19 (i.e., January 2017-February 2020) trajectories of health service utilization, and then used those models to estimate what the levels would have been in the absence of COVID-19 during the pandemic period, starting in March 2020 through March 2021. We classified the difference between the observed and predicted levels as the effect of COVID-19 on health services. We estimated 95% confidence intervals and p-values to examine if the effect of the pandemic, nationally and within specific geographies, was statistically significant. RESULTS: Our results indicate that COVID-19 negatively impacted health services and subsequent recovery varied by service type and by geographical area. COVID-19 had a lasting impact on overall service utilization as well as on malaria and pneumonia-related visits among young children in the DRC. We also found that the effects of COVID-19 were even more immediate and stronger in the capital city of Kinshasa compared with the national effect. Both nationally and in Kinshasa, most affected services had slow and incomplete recovery to expected levels. Therefore, our analysis indicates that COVID-19 continued to affect health services in the DRC throughout the first year of the pandemic. CONCLUSIONS: The methodology used in this article allows for examining the variability in magnitude, timing, and duration of the COVID effects within geographical areas of the DRC and nationally. This analytical procedure based on national health information system data could be applied to surveil health service disruptions and better inform rapid responses from health service managers and policymakers.

Mediators of Discrimination in Primary Care Appointment Access.

We examine how differences in questions asked and information provided by physicians' offices contribute to differences in new-patient appointment offers. Data is from a 2013-16 field experiment involving calls to a random sample of US primary care physicians on behalf of simulated new patients differentiated by race/ethnicity (Black, Hispanic, White), sex, and insurance. We find that the rates and stated reasons for denial of appointment offers differ substantially across patient groups.

Costs of insecticide-treated bed net distribution systems in sub-Saharan Africa.

BACKGROUND: Insecticide-treated nets (ITNs) are one of the most cost-effective measures for preventing malaria. The World Health Organization recommends both large-scale mass distribution campaigns and continuous distributions (CD) as part of a multifaceted strategy to achieve and sustain universal access to ITNs. A combination of these strategies has been effective for scaling up ITN access. For policy makers to make informed decisions on how to efficiently implement CD or combined strategies, information on the costs and cost-effectiveness of these delivery systems is necessary, but relatively few published studies of the cost continuous distribution systems exist. METHODS: To address the gap in continuous distribution cost data, four types of delivery systems-CD through antenatal care services (ANC) and the expanded programme on immunization (EPI) (Ghana, Mali, and mainland Tanzania), CD through schools (Ghana and mainland Tanzania), and a combined community/health facility-based distribution (Zanzibar, Tanzania), as well as mass distributions (Mali)-were costed. Data on costs were collected retrospectively from financial and operational records, stakeholder interviews, and resource use surveys. RESULTS: Overall, from a full provider perspective, mass distributions and continuous systems delivered ITNs at overlapping economic costs per net distributed (mass distributions: 4.37-4.61 USD, CD channels: 3.56-9.90 USD), with two of the school-based systems and the mass distributions at the lower end of this range. From the perspective of international donors, the costs of the CD systems were, for the most part, less costly than the mass distributions (mass distributions: 4.34-4.55 USD, Ghana and Tanzania 2017 school-based: 3.30-3.69 USD, health facility-based: 3.90-4.55 USD, combined community/health facility 4.55 USD). The 2015 school-based distribution (7.30 USD) and 2016 health facility-based distribution (6.52 USD) programmes in Tanzania were an exception. Mass distributions were more heavily financed by donors, while CD relied more extensively on domestic resource contributions. CONCLUSIONS: These results suggest that CD strategies can continue to deliver nets at a comparable cost to mass distributions, especially from the perspective of the donor.

Sustaining LLIN coverage with continuous distribution: the school net programme in Tanzania.

Most malaria-endemic countries have struggled in the past decade to establish effective national-scale continuous distribution mechanisms for long-lasting insecticidal nets (LLINs). Since the implementation of the Tanzania National Voucher Scheme in 2004 and mass-distribution campaigns in 2009-2011 and 2015-2016, Tanzania has been committed to finding new and innovative ways of achieving and maintaining universal bed net coverage. Planning for the School Net Programme (SNP) began in 2011 and in 2013, the country piloted a SNP in three regions. Nets were distributed annually to children attending schools in selected primary and secondary grades. Intra-family re-distribution was assumed, and hence the family as a whole, rather than just the children themselves, were the targeted beneficiaries. The programme has since expanded to 14 regions and has seen six rounds of annual distribution. In its fifth year, 3 million nets were distributed at a cost of USD 3.64 per net and USD 0.60 per person-year of protection (including the net). ITN access and use were maintained at a high level (~ 50-75%) over the first 4 years of distribution within selected evaluation areas, even in the absence of a mass distribution event. Net distribution through primary schools has proven to be a feasible and effective strategy for maintaining consistently high coverage in Tanzania.

Opportunities to Improve Employee Satisfaction Within State and Local Public Health Agencies.

CONTEXT: Public health agencies will likely struggle to staff at necessary levels, given ongoing workforce shortages, the potential for mass retirement, and expanding responsibility. Although the majority of public health workers are satisfied with their jobs overall, it is critical to understand the degree to which they are satisfied and identify factors that contribute to any dissatisfaction that occurs. OBJECTIVE: This study identified opportunities for public health agencies to improve work environments and, in turn, improve employee satisfaction and retention. DESIGN: Using data from the 2014 Public Health Workforce Interest and Needs Survey, we analyze responses to the survey question, "If you wish, you may provide comments below about your level of job satisfaction." The 2966 responses (2389 from state and 542 from local public health agencies) that indicated a negative disposition were examined to understand employee dissatisfaction. SETTING: The survey was administered to a representative sample of state health departments and convenience samples of local health departments. PARTICIPANTS: Responses from employees of state and local health departments are considered. MAIN OUTCOME MEASURES: The most frequently occurring themes overall were identified. In addition, responses describing weaknesses in organizational support (specifically training, communication, workload, and innovation) were summarized. RESULTS: The most frequently occurring themes were as follows: (1) salary, specifically in relation to the merit system, performance evaluation, and workload; (2) job security with emphasis on funding, organizational transformation, and politics/government; and (3) career development related to the merit system, performance evaluation, and management. Respondents also reported opportunities in the areas of training, communication, workload, and innovation to improve satisfaction levels. CONCLUSIONS: These findings serve as a call to action for leaders in health departments as well as national public health leaders to remedy the concerns raised in their responses. Some of the solutions are within the realm of public health agency leadership, but some may fall within the realm of governors and public health leaders at the federal level. It is important to share these findings so that appropriate decision makers can address public health workforce retention and recruitment issues in the interest of retaining valuable employees.

Public Health Workforce Self-Identified Training Needs by Jurisdiction and Job Type.

CONTEXT: Ensuring adequate and appropriate training of the workforce is a crucial priority for governmental public health. This is particularly important, given the diverse backgrounds of the public health workforce; the vast majority (approximately 83%) do not have formal training in public health, and those that do have formal training in public health have limited training in management and other essential organizational skills. OBJECTIVE: The purpose of this article is to identify training needs among public health workers in specific job types and settings. DESIGN AND PARTICIPANTS: This cross section study used 2014 data from the Public Health Workforce Interests and Needs Survey. Qualitative analyses were used to code open-ended responses to questions about training needs. Needs are stratified across job types and jurisdiction. RESULTS: Eight main themes or skill areas were identified with the largest proportion indicating a need for management/leadership skills (28.2%). The second most frequent need was communication skills (21.3%). Across the 9 job types examined, general management skills were either the first or second training need for 7 job types. Among individuals who already have leadership/management positions, budgeting was the most common training need. CONCLUSIONS: Findings from this study can inform targeted strategies to address training needs for specific types of employees. Such strategies can influence the efficiency and effectiveness of public health efforts and employee satisfaction. As new public health frameworks-like Public Health 3.0 and the Chief Health Strategist-are advanced nationally, it is necessary to ensure that the workforce has the skills and abilities to implement these frameworks.

Comparison of objective measures and patients' perceptions of quality of services in government health facilities in the Democratic Republic of Congo.

OBJECTIVE: Examine the relationship between patients' perceptions of quality and the objective level of quality at government health facilities, and determine whether the pre-existing attitudes and beliefs of patients regarding health services interfere with their ability to accurately assess quality of care. DESIGN: Cross-sectional, visit-level analysis. SETTING: Three regions (Nord-Ubangi, Kasai/Kasai-Central and Maniema/Tshopo) of the Democratic Republic of Congo. PARTICIPANTS: Data related to the inpatient and outpatient visits to government health facilities made by all household members who were included in the survey was used for the analysis. Data were collected from patients and the facilities they visited. MAIN OUTCOME MEASURES: Patients' perceptions of the level of quality related to availability of drugs and equipment; patient-centeredness and safety serve compared with objective measures of quality. RESULTS: Objective measures and patient perceptions of the drug supply were positively associated (ß = 0.16, 95% CI = 0.03, 0.28) and of safety were negatively associated (ß = -0.12, 95% CI = -0.23, -0.01). Several environmental factors including facility type, region and rural/peri-urban setting were found to be significantly associated with respondents' perceptions of quality across multiple outcomes. CONCLUSIONS: Overall, patients are not particularly accurate in their assessments of quality because their perceptions are impacted by their expectations and prior experience. Future research should examine whether improving patients' knowledge of what they should expect from health services, and the transparency of the facility's quality data can be a strategy for improving the accuracy of patients' assessments of the quality of the health services, particularly in low-resourced settings.

Exploring the barriers to rigorous monitoring and evaluation of health systems strengthening activities: qualitative evidence from international development partners.

The number of health systems strengthening (HSS) programs has increased in the last decade. However, a limited number of studies providing robust evidence for the value and impact of these programs are available. This study aims to identify knowledge gaps and challenges that impede rigorous monitoring and evaluation (M&E) of HSS, and to ascertain the extent to which these efforts are informed by existing technical guidance. Interviews were conducted with HSS advisors at United States Agency for International Development-funded missions as well as senior M&E advisors at implementing partner and multilateral organizations. Findings showed that mission staff do not use existing technical resources, either because they do not know about them or do not find them useful. Barriers to rigorous M&E included a lack suitable of indicators, data limitations, difficulty in demonstrating an impact on health, and insufficient funding and resources. Consensus and collaboration between international health partners and local governments may mitigate these challenges. Copyright © 2016 John Wiley & Sons, Ltd.

Why Do People Work in Public Health? Exploring Recruitment and Retention Among Public Health Workers.

CONTEXT: The public health workforce is critical to the functioning of the public health system and protection of the population's health. Ensuring a sufficient workforce depends on effectively recruiting and retaining workers. OBJECTIVE: This study examines factors influencing decisions to take and remain in jobs within public health, particularly for workers employed in governmental public health. DESIGN: This cross-sectional study employed a secondary data set from a 2010 national survey of US public health workers. PARTICIPANTS: Survey respondents were included in this study if they responded to at least 1 survey item related to recruitment and retention. A total of 10 859 survey responses fit this criterion. MAIN OUTCOME MEASURES: Data examined demographics of public health workers and factors that influenced decisions to take jobs in and remain in public health. RESULTS: Job security (ß = 0.42; 95% confidence interval [CI], 0.28-0.56) and competitive benefits (ß = 0.49; 95% CI, 0.28-0.70) were significantly and positively associated with governmental employees' decisions to take positions with their current employers compared with public health workers employed by other types of organizations. The same finding held with regard to retention: job security (ß = 0.40; 95% CI, 0.23-0.57) and competitive benefits (ß = 0.53; 95% CI, 0.24-0.83). Two personal factors, personal commitment to public service (ß = 0.30; 95% CI, 0.17-0.42) and wanted a job in the public health field (ß = 0.44; 95% CI, 0.18-0.69), were significantly and positively related to governmental employees deciding to remain with their current employers. CONCLUSIONS: It is important to recognize the value of competitive benefits for both current and potential employees. Public health agencies should maintain these if possible and make the value of these benefits known to policy makers or other agencies setting these benefit policies. Job security associated with governmental public health jobs also appears to offer public health an advantage in recruiting and retaining employees.

What Matters in Recruiting Public Health Employees: Considerations for Filling Workforce Gaps.

We examined factors that influence the decision to join the public health workforce. In this cross-sectional study, we used 2010 secondary data representing 6939 public health workers. Factors influencing the decision to take jobs in public health were significantly associated with specific previous employment settings. Respondents generally rated organizational factors as more influential than personal factors in terms of their decision to work in governmental public health. Leaders should consider tailoring recruitment efforts to maximize job uptake and enhance the potential for long-term retention.

Disparities in access to appointments for contraceptive services among Black, Hispanic, White, and recently incarcerated women in Alabama, Louisiana, and Mississippi.

OBJECTIVE: To measure differences in access to contraceptive services based on history of incarceration and its intersections with race/ethnicity and insurance status. DATA SOURCES AND STUDY SETTING: Primary data were collected from telephone calls to physician offices in Alabama, Louisiana, and Mississippi in 2021. STUDY DESIGN: We deployed a field experiment. The outcome variables were appointment offers, wait days, and questions asked of the caller. The independent variables were callers' incarceration history, race/ethnicity, and insurance. DATA COLLECTION METHODS: Using standardized scripts, Black, Hispanic, and White female research assistants called actively licensed primary care physicians and Obstetrician/Gynecologists asking for the next available appointment for a contraception prescription. Physicians were randomly selected and randomly assigned to callers. In half of calls, callers mentioned recent incarceration. We also varied insurance status. PRINCIPAL FINDINGS: Appointment offer rates were five percentage points lower (95% CI: -0.10 to 0.01) for patients with a history of incarceration and 11 percentage points lower (95% CI: -0.15 to -0.06) for those with Medicaid. We did not find significant differences in appointment offer rates or wait days when incarceration status was interacted with race or insurance. Schedulers asked questions about insurance significantly more often to recently incarcerated Black patients and recently incarcerated patients who had Medicaid. CONCLUSIONS: Women with a history of incarceration have less access to medical appointments; this access did not vary by race or insurance status among women with a history of incarceration.

Impact of a free care policy on routine health service volumes during a protracted Ebola virus disease outbreak in the Democratic Republic of Congo.

BACKGROUND: In response to the DRC's 10th Ebola Virus Disease (EVD) outbreak, the government subsidized routine health services in select health zones with the goal of maintaining routine service volumes. We assess the impact of the initial and revised Free Care Policies (FCP) on total clinic visits, uncomplicated malaria, simple pneumonia, fourth antenatal care clinic visits, and measles vaccinations, testing the hypothesis that routine services would not significantly decrease during the FCP. METHODS AND FINDINGS: We used data from the DRC's national health information system spanning January 2017 to November 2020. Intervention facilities were those that were initially and secondarily enrolled in the FCP, which occurred in August 2018 and November 2018, respectively. Comparison facilities were limited to the North Kivu Province and were from health zones that recorded at least one case of Ebola. A controlled interrupted time series analysis was conducted. The FCP appeared to have a positive effect in increasing overall clinic attendance rates, uncomplicated malaria case rates, and simple pneumonia case rates in those health zones where the policy was enacted relative to comparison sites. The longer-term effects of the FCP were mostly non-significant or, if significant, relatively modest in nature. Rates for measles vaccinations and fourth ANC clinic visits appeared to be unaffected or minimally affected, respectively, by the implementation of the FCP and relative to comparison sites. We did not observe the decrease in measles vaccinations that has been observed elsewhere. The study is limited in that we were unable to account for health facility bypassing and service volumes at private health facilities. CONCLUSIONS: Our findings provide evidence that FCPs can be used to maintain routine service provision during outbreaks. Additionally, the study design demonstrates that routinely reported health information from the DRC are sensitive enough to detect changes in health policy.

Disparities in Access to Primary Care Appointments Among Asian American Subgroups, and the Impact of Concordance.

While Asian Americans experience disparate access to health services, little is known about the extent to which providers discriminate against Asian American patients. Further, research on Asian American health disparities tends to group Asian American ethnicities together, overlooking potential within-group differences. We deployed a field experiment to assess whether Asian American ethnic sub-groups experience discrimination in appointment scheduling. We further explored the impact of racial concordance between Asian patients and physicians. Overall, we did not detect significant differences in appointment offer rates between White and Asian American patients. However, we found that Asian Americans experienced longer wait times driven primarily by the treatment of patients of Chinese and Korean descent. Physician offices, surprisingly, offered concordant Asian patients appointments at significantly lower rates. The disparities Asian Americans experience relative to White Americans through longer waits for primary care appointments are not consistent across sub-groups. Increased attention to the unique experiences of people of Asian descent in accessing health services is warranted.

The gap in contraceptive knowledge and use between the military and non-military populations of Kinshasa, DRC, 2016-2019.

INTRODUCTION: The objective of this study is to assess change over time in the modern contraceptive prevalence rate (MCPR) and related variables among married women of reproductive age (15-49 years) in the military population in Kinshasa, Democratic Republic of Congo, compared to women in the non-military population, based on cross-sectional surveys in 2016 and 2019. METHODS: Data among women living in military camps were collected as a special study of contraceptive knowledge, use, and exposure to FP messaging, for comparison to women in the non-military population from the annual PMA2020 survey. Both used a two-stage cluster sampling design to randomly select participants. This analysis is limited to women married or in union. Bivariate and multivariate analysis was used to compare the military and non-military populations. RESULTS: The socio-demographic profile of women in the military camps differed between 2016 and 2019, which may reflect the more mobile nature of this population. In both populations, knowledge of modern contraceptive methods increased significantly. Similarly, use of a modern contraceptive method also increased significantly in both, though by 2019 women in the military camps were less likely to use modern contraception (24.9%) than their non-military counterparts (29.7%). Multivariate analysis showed no significant difference in the amount of increase in MCPR for the two populations. Among contraceptive users in both populations, the implant was the leading method. Potential effects of FP programming were evident in the military population: exposure to FP messaging increased (in comparison to a decrease among the non-military population). Moreover, women who had lived in the camps for 4+ years had a higher MCPR than those living in the camps for less than four years. CONCLUSIONS: This study demonstrates the feasibility and importance of collecting data in military camps for better understanding contraceptive dynamics among this specialized population.

Association of Simulated Patient Race/Ethnicity With Scheduling of Primary Care Appointments.

IMPORTANCE: Racial and ethnic disparities in access to health care may result from discrimination. OBJECTIVES: To identify differences in the rates at which patients belonging to racial/ethnic minority groups are offered primary care appointments and the number of days they wait for their primary care appointment and to understand the mechanisms by which discrimination occurs. DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional study used 7 simulated black, Hispanic, and white patient callers to request appointments from 804 randomized primary care offices in 2 urban centers in Texas from November 2017 to February 2018. Data analysis was conducted between February and December 2018. EXPOSURES: Research assistants called randomly assigned offices to schedule an appointment, supplying the same basic information. Race and ethnicity were signaled through callers' names and voices. MAIN OUTCOMES AND MEASURES: Appointment offer rates, days to appointment, and questions asked during the call. RESULTS: Of the 7 callers (age range, 18-29 years), 2 (28.6%) self-identified as non-Hispanic black, 3 (42.9%) self-identified as non-Hispanic white, and 2 (28.6%) self-identified as Hispanic. Of the 804 calls they made, 299 (37.2%) were from simulated white callers, 215 (26.7%) were from simulated black callers, and 290 (36.1%) were from simulated Hispanic callers. Overall, 582 callers (72.4%) were offered appointments. In unadjusted models, black and Hispanic callers were more likely to be offered an appointment than white callers (black callers, 32.2 [95% CI, 25.1-39.3] percentage points more likely; P < .001; Hispanic callers, 21.1 [95% CI, 13.7-28.5] percentage points more likely; P < .001). However, after adjusting for whether insurance status was revealed, this statistical significance was lost. In adjusted models, black callers were 44.0 (95% CI, 36.2-51.8) percentage points more likely to be asked about their insurance status than white callers (P < .001), and Hispanic callers were 25.3 (95% CI, 17.1-33.5) percentage points more likely to be asked about their insurance status (P < .001) than white callers. Black and Hispanic callers received appointments further in the future than white callers (black callers: marginal effect estimate, 3.650; 95% CI, 0.579 to 6.721; P = .08; Hispanic callers: marginal effect estimate, 2.644; 95% CI, -0.496 to 5.784; P = .02). CONCLUSIONS AND RELEVANCE: In this study, black and Hispanic patients were more likely to be offered an appointment, but they were asked more frequently about their insurance status than white callers. Black and Hispanic callers experienced longer wait times than white patients, indicating a barrier to timely access to primary care.

Systematic review and meta-analysis of the cost and cost-effectiveness of distributing insecticide-treated nets for the prevention of malaria.

Insecticide-treated nets are one of two core vector control interventions recommended by the World Health Organization for deployment in malaria-endemic regions around the world, especially sub-Saharan Africa. Although there are many factors that influence the type of distribution strategy chosen, among the most important considerations for the type of distribution strategy chosen is cost, both in terms of total expenditure required and in terms of relative cost-effectiveness. This research attempted to inform these decisions by conducting a systematic review and meta-analysis of the literature on the cost and cost-effectiveness of ITN distribution. The analysis compared the relative cost and cost-effectiveness of distribution strategies. Findings suggest that mass campaigns have lower average distribution costs per net compared with continuous/health facility distribution or sale/vouchers, although the relationship between distribution channel and cost were not statistically significant in the multivariate regression models. Continuous/health facility distribution channels were found to be more cost-effective than mass campaigns for averting DALYs, death, and cases of malaria. Those who design and budget for malaria programs should base decisions about distribution channels more on operational and epidemiological considerations than on cost per net, as the costs per net between distribution channels are not statistically different.

The influence of health facility-level access measures on modern contraceptive use in Kinshasa, DRC.

Expanding access to family planning (FP) is a principal objective of global family planning efforts and has been a driving force of national family planning programs in recent years. Many country programs are working alongside with the international family planning community to expand access to modern contraceptives. However, there is a challenging need for measuring all aspects of access. Measuring access usually requires linking information from multiple sources (e.g., individual women and facilities). To assess the influence of access to family planning services on modern contraceptive use among women, we link four rounds of individual women and service delivery points survey data from PMA2020 in Kinshasa. Multilevel logistics regression on pooled data is performed to test the influence of facility-level access factors on individual-level contraceptive use. We add variables tailored from a conceptual framework to cover elements of access to family planning: administrative access, geographic or physical access, economic access or affordability, cognitive access, service quality, and psychological access. We find that the effect of community and facility-level access factors varies extensively but having fewer stocked-out facilities and more facilities with long-acting permanent methods (LAPM) increases the odds of using modern contraceptives among women in Kinshasa. Our study shows that reliable supply chain with a broad array of method mix will increase the odds of modern contraceptive use at community level among women in Kinshasa. Using to community-oriented practices and service delivery along with empowering women to make health-related decisions should become a priority of family planning programs and international stakeholders in the country.

Factors That Influence the Recruitment and Retention of Nurses in Public Health Agencies.

OBJECTIVE: Given challenges to recruiting nurses to public health and the growth in national policies focused on population health, it is crucial that public health agencies develop strategies to sustain this important group of employees. The objective of this study was to examine factors that influence nurses' decisions to work in public health agencies. METHODS: This cross-sectional study examined perspectives of nurses who worked in state and local public health departments and responded to the 2010 Council on Linkages Between Academia and Public Health Practice's survey of public health workers. We calculated the mean rating of each recruitment and retention factor for nurses and non-nurses separately and compared differences by using t tests. We then used multivariate regression analysis to examine differences in ratings by role (ie, nurse or non-nurse). RESULTS: After controlling for personal and organizational characteristics, the influence of 5 recruitment factors was significantly stronger among nurses than among non-nurses: flexibility of work schedule ( P < .001), autonomy/employee empowerment ( P < .001), ability to innovate ( P = .002), specific duties and responsibilities ( P = .005), and identifying with the mission of the organization ( P = .02). The influence of 5 retention factors was stronger among nurses than among non-nurses : autonomy/employee empowerment ( P < .001), flexibility of work schedule ( P < .001), specific duties and responsibilities ( P < .001), opportunities for training/continuing education ( P = .03), and ability to innovate ( P = .008). CONCLUSIONS: Some factors that influence nurses to begin and remain working in local governmental public health agencies, such as flexible schedules and employee autonomy, are factors that governmental public health agencies can design into positions and highlight when recruiting from health care organizations, private industry, and academia.

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.