Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages.

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

Mycobacterium bovis BCG as immunostimulating agent prevents the severe form of chronic experimental Chagas disease.

Introduction: There is currently no vaccine against Chagas disease (ChD), and the medications available confer multiple side effects. Mycobacterium bovis Bacillus Calmette-Guérin (BCG) produces balanced Th1, Th2, and Th17 modulatory immune responses and has improved efficacy in controlling chronic infections through nonspecific immunity. We aimed to improve the response to infection by inducing a stronger immune response and greater protection against the parasite by trained immunity. Methods: BALB/c mice were immunized with BCG subcutaneously, and 60 days later, they were infected with Trypanosoma cruzi intraperitoneally. An evaluation of the progression of the disease from the acute to the chronic stage, analyzing various aspects such as parasitemia, survival, clinical status, and humoral and cellular immune response, as well as the appearance of visceral megas and the histopathological description of target organs, was performed. Results: Vaccination reduced parasitemia by 70%, and 100% survival was achieved in the acute stage; although the presentation of clinical signs was reduced, there was no increase in the antibody titer or in the differential production of the isotypes. Conclusion: Serum cytokine production indicated a proinflammatory response in infected animals, while in those who received BCG, the response was balanced by inducing Th1/Th2-type cytokines, with a better prognosis of the disease in the chronic stage.

Proteome and immunogenicity differences in BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c during planktonic growth in 7H9 and Proskauer Beck media.

Bacillus Calmette-Guèrin (BCG) remains as the only vaccine employed to prevent tuberculosis (TB) during childhood. Among factors likely contributing to the variable efficacy of BCG is the modification in its antigenic repertoire that may arise from in vitro growth conditions. Our vaccine candidate, BCGΔBCG1419c, improved protection against TB in mice and guinea pigs with bacteria grown in either 7H9 OADC Tween 80 or in Proskauer Beck Tween 80 media in independent studies. Here, we compared the proteomes of planktonic cultures of BCG and BCGΔBCG1419c, grown in both media. Further to this, we compared systemic immunogenicity ex vivo elicited by both types of BCG strains and cultures when used to vaccinate BALB/c mice. Both the parental strain BCG Pasteur ATCC 35734, and its isogenic mutant BCGΔBCG1419c, had several medium-dependent changes. Moreover, ex vivo immune responses to a multiantigenic (PPD) or a single antigenic (Ag85A) stimulus were also medium-dependent. Then, not only the presence or absence of the BCG1419c gene in our strains under study affected the proteome produced in vitro but also that this was affected by culture medium, potentially leading to changes in the capacity to induce ex vivo immune responses.

BCG∆BCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice.

The efficacy of BCG vaccines against Mycobacterium tuberculosis (Mtb) strains of lineage 2 (Beijing) in preclinical models and humans has been questioned. We have developed BCG∆BCG1419c, by deletion of BCG1419c in BCG Pasteur, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the capacity of BCG and BCG∆BCG1419c to induce autophagy in murine macrophages, modify c-di-GMP content and transcript levels of BCG1416c, encoding the enzyme responsible for c-di-GMP synthesis/degradation, and of BCG1419c, encoding the phosphodiesterase involved in c-di-GMP degradation. Furthermore, we evaluated proteomic differences in vitro and compared protection against TB produced by a low dose of the HN878-Beijing strain at 3- and 6-months post-infection. We found that BCG∆BCG1419c induced more autophagy and produced different levels of c-di-GMP as well as different transcription of BCG1416c with no expression of BCG1419c. BCG∆BCG1419c differentially produced several proteins, including some involved in interaction with host cells. Vaccination with either BCG strain led to control of bacillary burden in lungs and spleen at 3- but not 6-months post-infection, whereas it reduced pneumonic areas compared with unvaccinated controls at 6 months post-infection. Vaccination with BCG∆BCG1419c delayed progression of lung necrosis as this was observed only at 6 months post-infection. Taken together, compared with BCG, BCG∆BCG1419c increased autophagy, presented different levels of c-di-GMP and transcription of BCG1416c in vitro in a growth-phase dependent manner, modified its proteome and delayed progression of lung pathology produced by a highly virulent Beijing strain.

hilD Is Required for the Active Internalization of Salmonella Newport into Cherry Tomatoes.

Salmonella enterica is a foodborne pathogen that can be internalized into fresh produce. Most of the Salmonella virulence genes are clustered in regions denominated Salmonella Pathogenicity Islands (SPI). SPI-1 encodes a Type Three Secretion System (T3SS-1) and effector proteins that allow the internalization of Salmonella into animal cells. HilD is a transcriptional regulator that induces the expression of SPI-1 genes and other related virulence genes located outside of this island. Here, we assessed the role of hilD in the internalization of Salmonella Newport and Typhimurium into cherry tomatoes, by evaluating either an isolate from an avocado orchard, S. Newport-45 or the laboratory strain S. Typhimurium SL1344 and their isogenic mutants in hilD. The internalization of these bacteria was carried out by using a temperature gradient of 12°C. The transcription of hilD and invA was tested by qRT-PCR experiments. Our results show that S. Newport-45 hilD mutant viable cells obtained from the interior of the fruit were decreased (2.7-fold), compared with those observed for S. Typhimurium SL1344. Interestingly, at 3 days postinoculation, the cells recovered from S. Newport-45 hilD mutant were similar to those recovered from all the strains evaluated, suggesting that hilD is required only for the initial internalization of S. Newport.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 8. Impact on women's empowerment.

BACKGROUND: Indigenous Maya women in the rural highlands of Guatemala have traditionally faced constraints to decision-making and participation in community affairs. Anecdotal experiences from previous Curamericas Global projects in Guatemala and Liberia have suggested that interventions using the CBIO+ Approach (which consists of implementing together the Census-Based, Impact-Oriented Approach, the Care Group Approach, and Community Birthing Centers), can be empowering and can facilitate improvements in maternal and child health. This paper, the eighth in a series of 10 papers examining the effectiveness of CBIO+ in improving the health and well-being of mothers and children in an isolated mountainous rural area of the Department of Huehuetenango, explores changes in women's empowerment among mothers of young children associated with the Curamericas/Guatemala Maternal and Child Health Project, 2011-2015. METHODS: Knowledge, practice, and coverage (KPC) surveys and focus group discussions (FGDs) were used to explore six indicators of women's empowerment focusing on participation in health-related decision-making and participation in community meetings. KPC surveys were conducted at baseline (January 2012) and endline (June 2015) using standard stratified cluster sampling. Seventeen FGDs (9 with women, 3 with men, 2 with mothers-in-law, and 3 with health committees), approximately 120 people in all, were conducted to obtain opinions about changes in empowerment and to identify and assess qualitative factors that facilitate and/or impede women's empowerment. RESULTS: The KPC surveys revealed statistically significant increases in women's active participation in community meetings. Women also reported statistically significant increases in rates of participation in health-related decision-making. Further, the findings show a dose-response effect for two of the six empowerment indicators. The qualitative findings from FGDs show that the Project accelerated progress in increasing women's empowerment though women still face major barriers in accessing needed health care services for themselves and their children. CONCLUSION: The Project achieved some notable improvements in women's decision-making autonomy and participation in community activities. These improvements often translated into making decisions to practice recommended health behaviors. Traditional cultural norms and the barriers to accessing needed health services are not easily overcome, even when empowerment strategies are effective.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 9. Key stakeholder perspectives on strengthening the CBIO+ Approach.

BACKGROUND: Community-based health interventions have been an integral part of recent health gains globally. An innovative approach to delivering community health care combines the Census-Based, Impact-Oriented (CBIO) Approach with Care Groups and Community Birthing Centers called Casas Maternas Rurales. CBIO+ was adopted by Curamericas/Guatemala in its Maternal and Child Health Project, 2011-2015. Here, we describe the opinions of Project staff and local government health care workers about the strengths and challenges of CBIO+.  METHODS: Self-administered questionnaires, key informant interviews, and focus group discussions were used to obtain the views of 21 staff members from Curamericas/Guatemala as well as 15 local government health workers. The evaluation focused on four primary areas: (1) advisability of integrating the CBIO+ Approach into the government's rural health system, (2) staff knowledge of the CBIO+ Approach, (3) advantages, disadvantages and challenges of the CBIO+ Approach, and (4) proposed improvements to the CBIO+ Approach. The data were coded into categories and from these categories themes were derived. RESULTS: The most commonly mentioned advantage of CBIO+ was the inclusion of the community in program planning, which improved participation. Many respondents noted that the CBIO+ Approach was challenging to implement in communities with internal conflicts. Among other challenges mentioned were coordinating (both among the Project staff and with others in the communities), maintenance of a high level of community participation, and overcoming opposition of men to women's participation in Care Groups. The staff mentioned a number of possible changes, including increasing male involvement, raising salaries for community-level paid staff, providing volunteers with incentives, and improving coordination both internally and externally. There was a strong demand among the local Ministry of Public Health and Social Welfare staff for the Project to continue. CONCLUSION: The CBIO+ Approach and its implementation by Curamericas/Guatemala was overall embraced by local staff. By eliciting feedback while the project was ongoing, actionable areas for improvement were identified.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ approach of Curamericas: 2. Study site, design, and methods.

BACKGROUND: The Curamericas/Guatemala Maternal and Child Health Project, 2011-2015, included implementation research designed to assess the effectiveness of an approach referred to as CBIO+ , composed of: (1) the Census-Based, Impact-Oriented (CBIO) Approach, (2) the Care Group Approach, and (3) the Community Birthing Center Approach. This is the second paper in a supplement of 10 articles describing the implementation research and its findings. Paper 1 describes CBIO+ , the Project Area, and how the Project was implemented. OBJECTIVE: This paper describes the implementation research design and details of how it was carried out. METHODS: We reviewed the original implementation research protocol and the methods used for all data collection related to this Project. The protocol and methods used for the implementation research related to this Project were all standard approaches to the monitoring and evaluation of child survival projects as developed by the United States Agency for International Development Child Survival and Health Grants Program (CSHGP) and the CORE Group. They underwent independent peer review supervised by the CSHGP before the implementation research began. RESULTS: The study area was divided into two sets of communities with a total population of 98,000 people. Project interventions were implemented in Area A from 2011 until the end of the project in 2015 (44 months) and in Area B from late 2013 until 2015 (20 months). Thus, Area B served as a quasi-comparison area during the first two years of Project implementation. The overarching study question was whether the CBIO+ Approach improved the health and well-being of children and mothers. The outcome indicators included (1) changes in population coverage of evidence-based interventions, (2) changes in childhood nutritional status, (3) changes in the mortality of children and mothers, (4) quality of care provided at Community Birthing Centers, (5) the impact of the Project on women's empowerment and social capital, (6) stakeholder assessment of the effectiveness of the CBIO+ Approach, and (7) the potential of wider adoption of the CBIO+ Approach. CONCLUSION: The implementation research protocol guided the assessment of the effectiveness of the CBIO+ Approach in improving the health and well-being of children, mothers, and their communities.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 4. Nutrition-related activities and changes in childhood stunting, wasting, and underweight.

BACKGROUND: This is the fourth paper in our supplement on improving the health and well-being of rural indigenous Maya mothers and children in the Western Highlands of Guatemala, where the prevalence of stunting is the highest in Latin America and among the highest in the world. Reducing childhood undernutrition was one of the objectives of the Maternal and Child Health Project, 2011-2015, implemented by Curamericas/Guatemala. The implementation research portion of the Project attempted to determine if there were greater improvements in childhood nutritional status in the Project Area than in comparison areas and whether or not a dose-response effect was present in terms of a greater improvement in the Project Area with a longer duration of interventions.  METHODS: The Project provided nutrition-related messages to mothers of young children, cooking sessions using locally available nutritious foods, a lipid-based nutrient supplement (Nutributter®) for a short period of time (4 months), anti-helminthic medication, and repeated growth monitoring and nutrition counseling. Measures of height and weight for calculating the prevalence of underweight, stunting, and wasting in under-2 children were analyzed and compared with the anthropometric data for children in the rural areas of the Northwestern Region and in the Western Highlands of Guatemala. RESULTS: The prevalence of stunting declined in Area A from 74.5% in September 2012 to 39.5% in June 2015. Area A comprised approximately one-half of the Project Area and was the geographic area with the greatest intensity and duration of nutrition-related Project interventions. Minimal improvements in stunting were observed in the Northwestern Region, which served as a comparison area. Improvements in multiple output and outcome indicators associated with nutritional status were also observed in Areas A and B: infant and young child feeding practices, routine growth monitoring and counseling, and household practices for the prevention and treatment of diarrhea. CONCLUSION: The Project Area in which Curamericas/Guatemala implemented the CBIO+ Approach experienced a reduction in the prevalence of stunting and other measures of undernutrition in under-2 children. Given the burden of undernutrition in Guatemala and other parts of the world, this approach merits broader application and further evaluation.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 5. Mortality assessment.

BACKGROUND: The Curamericas/Guatemala Maternal and Child Health Project, 2011-2015, implemented the Census-Based, Impact-Oriented Approach, the Care Group Approach, and the Community Birthing Center Approach. Together, this expanded set of approaches is known as CBIO+. This is the fifth of 10 papers in our supplement describing the Project and the effectiveness of the CBIO+ Approach. This paper assesses causes, levels, and risk factors for mortality along with changes in mortality. METHODS: The Project maintained Vital Events Registers and conducted verbal autopsies for all deaths of women of reproductive age and under-5 children. Mortality rates and causes of death were derived from these data. To increase the robustness of our findings, we also indirectly estimated mortality decline using the Lives Saved Tool (LiST). FINDINGS: The leading causes of maternal and under-5 mortality were postpartum hemorrhage and pneumonia, respectively. Home births were associated with an eight-fold increased risk of both maternal (p = 0.01) and neonatal (p = 0.00) mortality. The analysis of vital events data indicated that maternal mortality declined from 632 deaths per 100,000 live births in Years 1 and 2 to 257 deaths per 100,000 live birth in Years 3 and 4, a decline of 59.1%. The vital events data revealed no observable decline in neonatal or under-5 mortality. However, the 12-59-month mortality rate declined from 9 deaths per 1000 live births in the first three years of the Project to 2 deaths per 1000 live births in the final year. The LiST model estimated a net decline of 12, 5, and 22% for maternal, neonatal and under-5 mortality, respectively. CONCLUSION: The baseline maternal mortality ratio is one of the highest in the Western hemisphere. There is strong evidence of a decline in maternal mortality in the Project Area. The evidence of a decline in neonatal and under-5 mortality is less robust. Childhood pneumonia and neonatal conditions were the leading causes of under-5 mortality. Expanding access to evidence-based community-based interventions for (1) prevention of postpartum hemorrhage, (2) home-based neonatal care, and (3) management of childhood pneumonia could help further reduce mortality in the Project Area and in similar areas of Guatemala and beyond.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 3. Expansion of population coverage of key interventions.

BACKGROUND: This is the third in a series of 10 articles describing the Curamericas/Guatemala Maternal and Child Health Project, 2011-2015, and its effectiveness in improving the health and well-being of 15,327 children younger than 5 years of age and 32,330 women of reproductive age in the Department of Huehuetenango in180 communities that make up the municipalities of San Sebastian Coatán, Santa Eulalia, and San Miguel Acatán. The Project combined the Census-Based, Impact-Oriented (CBIO) Approach with the Care Group Approach and the  Community Birthing Center (Casa Materna Rural) Approach. This combined approach we refer to as CBIO+. The Project trained women volunteers every two weeks (in Care Groups) to provide health education to neighboring households. Messages focused on the promotion of maternal and newborn health, nutrition, prevention and treatment of acute respiratory infection and diarrhea in children, and immunizations. METHODS: Household knowledge, practice and coverage (KPC) surveys were executed at baseline in January 2011 and at endline in June 2015 to measure changes in levels of knowledge of danger signs, key household practices (such as Essential Newborn Care and handwashing), and health service utilization (such as antenatal care and care seeking for a child with signs of pneumonia) in two separate Project Areas (Area A with 41 months and Area B with 20 months of full intervention implementation). RESULTS: For the 24 indicators of the interventions under the Project's control, statistically significant improvements were observed for 21 in Area A and 19 in Area B. However, for some of the interventions that required support from the government's Extension of Coverage Program (immunization, family planning, and vitamin A administration) no improvements were noted because of the cessation of the program by the government after Project implementation began. In both Areas A and B one-half of the indicators improved by at least two-fold. CONCLUSION: This community-based Project has been effective in quickly achieving marked improvements in indicators for interventions that are important for the health of mothers and children. These achievements are notable in view of the challenging context in which the Project was implemented.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 10. Summary, cost effectiveness, and policy implications.

BACKGROUND: This is the final of 10 papers that describe the implementation of the Expanded Census-Based, Impact-Oriented Approach (CBIO+) by Curamericas/Guatemala in the Cuchumatanes mountains of the Department of Huehuetenango and its effectiveness in improving the health and well-being of women and children in a population of 98,000 in three municipalities. The CBIO+ Approach consists of three components: the CBIO (Census-Based, Impact-Oriented) Approach, the Care Group Approach, and the Community Birthing Center Approach. METHODS: Each of the preceding papers was summarized. An assessment was made regarding the degree to which the initial implementation research hypotheses were confirmed. The total field cost per capita for operation of the Project was calculated. An assessment of the cost-effectiveness of the Project was made based on the estimated impact of the Project, the number of lives saved, and the number of disability-adjusted life years averted. RESULTS: The Project attained a number of notable achievements in terms of expanding the coverage of key maternal and child health interventions, improving the nutritional status of children, reducing the mortality of children and mothers, providing quality care for mothers at the Community Birthing Centers (Casas Maternas Rurales) that integrate traditional midwives (comadronas) into the care of women during childbirth at the birthing centers, as well as empowering women and building social capital in the communities. CBIO+ is an effective and affordable approach that is particularly notable for its capacity to engage communities in the process of improving the health of mothers and children. Overall, there is strong and consistent evidence in support of the research hypotheses. The findings did produce evidence of declines in under-5 and maternal mortality, but they were not as robust as had been hoped. CONCLUSION: CBIO+ is an approach that has been effective in engaging communities in the process of improving the health of their mothers and children and in reducing health inequities in this marginalized, difficult-to-reach population of Indigenous Maya people. The CBIO+ Approach is cost-effective and merits further development and broader application in Guatemala and beyond.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 1. Introduction and project description.

BACKGROUND: The Curamericas/Guatemala Maternal and Child Health Project, 2011-2015, was implemented in the Western Highlands of the Department of Huehuetenango, Guatemala. The Project utilized three participatory approaches in tandem: the Census-Based, Impact-Oriented (CBIO) Approach, the Care Group Approach, and the Community Birthing Center Approach. Together, these are referred to as the Expanded CBIO Approach (or CBIO+). OBJECTIVE: This is the first article of a supplement that assesses the effectiveness of the Project's community-based service delivery platform that was integrated into the Guatemalan government's rural health care system and its special program for mothers and children called PEC (Programa de Extensión de Cobertura, or Extension of Coverage Program). METHODS: We review and summarize the CBIO+ Approach and its development. We also describe the Project Area, the structure and implementation of the Project, and its context. RESULTS: The CBIO+ Approach is the product of four decades of field work. The Project reached a population of 98,000 people, covering the entire municipalities of San Sebastián Coatán, Santa Eulalia, and San Miguel Acatán. After mapping all households in each community and registering all household members, the Project established 184 Care Groups, which were composed of 5-12 Care Group Volunteers who were each responsible for 10-15 households. Paid Care Group Promoters provided training in behavior change communication every two weeks to the Care Groups. Care Group Volunteers in turn passed this communication to the mothers in their assigned households and also reported back to the Care Group Promoters information about any births or ï»¿deaths that they learned of during the previous two weeks as a result of their regular contact with their neighbors. At the outset of the Project, there was one Birthing Center in the Project Area, serving a small group of communities nearby. Two additional Birthing Centers began functioning as the Project was operating. The Birthing Centers encouraged the participation of traditional midwives (called comadronas) in the Project Area. CONCLUSION: This article serves as an introduction to an assessment of the CBIO+ community-based, participatory approach as it was implemented by Curamericas/Guatemala in the Western Highlands of the Department of Huehuetenango, Guatemala. This article is the first of a series of articles in a supplement entitled Reducing Inequities in Maternal and Child Health in Rural Guatemala through the CBIO+ Approach of Curamericas.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 7. The empowering effect of Care Groups.

BACKGROUND: While there is extensive published evidence regarding the effectiveness of the Care Group Approach in promoting community-wide health behavior change, there is no published evidence regarding its empowering effect on its participants. Our study aimed to understand if the Care Group Approach as applied in the Curamericas/Guatemala Maternal and Child Health Project in isolated rural mountainous communities in Guatemala produced evidence of empowerment among the female participants. This is the seventh of 10 papers describing the expanded Census-Based, Impact-Oriented (CBIO+) Approach in improving the health and well-being of mothers and children in the rural highlands of the Department of Huehuetenango, Guatemala. METHODS: We conducted semi-structured individual and group interviews with 96 female Care Group participants -including Level-1 Care Group Promoters, Care Group Volunteers, and Self-Help Group participants. The participants were from six communities - two from each of the three municipalities making up the Project Area. Data were analyzed both using deductive thematic and by exploring the following social constructs: perceived social status, self-efficacy, decision-making autonomy, and formation of social capital. RESULTS: The findings supported the hypothesis that Care Group participation was an empowering process. The primary themes that emerged included increased respect accorded to women in the community, women's willingness and ability to make decisions and their confidence in making those decisions, and the development of stronger bonds among Care Group members, with other community members, and with community leaders. CONCLUSION: Through increased theoretical and practical knowledge about important maternal and child health matters and through the social experience of obtaining this knowledge and sharing it with other community members, participation in the Care Group Approach empowered participants to make positive health behavior changes for themselves and for their children and families. This, in turn, led many participants to become more engaged in community activities for improved health and beyond, thereby enhancing social capital in the community. We conclude that the Care Group Approach, as applied in this setting, has made it possible for marginalized indigenous women living in a male-dominated society to become more empowered.

Reducing inequities in maternal and child health in rural Guatemala through the CBIO+ Approach of Curamericas: 6. Management of pregnancy complications at Community Birthing Centers (Casas Maternas Rurales).

BACKGROUND: In Guatemala, Indigenous women have a maternal mortality ratio over twice that of non-Indigenous women. Long-standing marginalization of Indigenous groups and three decades of civil war have resulted in persistent linguistic, economic, cultural, and physical barriers to maternity care. Curamericas/Guatemala facilitated the development of three community-built, -owned, and -operated birthing centers, Casas Maternas Rurales (referred to here as Community Birthing Centers), where auxiliary nurses provided physically accessible and culturally acceptable clinical care. The objective of this paper is to assess the management of complications and the decision-making pathways of Birthing Center staff for complication management and referral. This is the sixth paper in the series of 10 articles. Birthing centers are part of the Expanded Census-based, Impact-oriented Approach, referred to as CBIO+. METHODS: We undertook an explanatory, mixed-methods study on the handling of pregnancy complications at the Birthing Centers, including a chart review of pregnancy complications encountered among 1,378 women coming to a Birthing Center between 2009 and 2016 and inductively coded interviews with Birthing Center staff. RESULTS: During the study period, 1378 women presented to a Birthing Center for delivery-related care. Of the 211 peripartum complications encountered, 42.2% were successfully resolved at a Birthing Center and 57.8% were referred to higher-level care. Only one maternal death occurred, yielding a maternal mortality ratio of 72.6 maternal deaths per 100,000 live births. The qualitative study found that staff attribute their successful management of complications to frequent, high-quality trainings, task-shifting, a network of consultative support, and a collaborative atmosphere. CONCLUSION: The Birthing Centers were able to resolve almost one-half of the peripartum complications and to promptly refer almost all of the others to a higher level of care, resulting in a maternal mortality ratio less than half that for all Indigenous Guatemalan women. This is the first study we are aware of that analyzes the management of obstetrical complications in such a setting. Barriers to providing high-quality maternity care, including obtaining care for complications, need to be addressed to ensure that all pregnant women in such settings have access to a level of care that is their fundamental human right.

Genome sequences of BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c.

BACKGROUND: Bacillus Calmette-Guérin (BCG) remains the only vaccine to prevent tuberculosis (TB) during childhood, with relatively low to no efficacy against pulmonary TB in adolescents and adults. BCG consists of close to 15 different substrains, where genetic variations among them might contribute to the variable protective efficacy afforded against pulmonary TB. We have shown that the vaccine candidate, BCGΔBCG1419c, which is based on BCG Pasteur, improved protection against chronic TB in murine models, as well as against pulmonary and extrapulmonary TB in guinea pigs. Here, to confirm deletion of the BCG1419c gene and to detect possible genetic variations occurring as a consequence of the spontaneous mutations that may arise during in vitro culture of mycobacteria, the genomes of BCG Pasteur ATCC 35734 and its isogenic derivative, BCGΔBCG1419c, were sequenced and subjected to a comparative analysis between them and against BCG Pasteur 1173P2. RESULTS: The complete catalog of variants in genes relative to the reference genome BCG Pasteur 1173P2 (GenBank NC008769) showed that the parental strain BCG Pasteur ATCC 35734, from which the mutant BCGΔBCG1419c originated, showed five synonymous mutations, three missense mutations, and five codon insertions, whereas the BCGΔBCG1419c mutant reported the same changes. When BCG Pasteur ATCC 35734 and BCGΔBCG1419c were compared, we confirmed that the latter was devoid of the BCG1419c gene, with only one unanticipated SNP at position 2, 828, 791  which we consider has no role in vaccine properties reported thus far. CONCLUSION: We provide evidence that the mutagenesis performed to remove BCG1419c from BCG Pasteur ATCC 35734 solely deleted this gene, and that compared with the reference strain BCG Pasteur 1173P2, few changes were present confirming that they are BCG Pasteur strains, and that changes in immunogenicity or efficacy observed thus far in BCGΔBCG1419c are most likely derived solely from the elimination of the BCG1419c gene.

Recent Developments in Mycobacteria-Based Live Attenuated Vaccine Candidates for Tuberculosis.

Vaccination is an excellent approach to stimulating the host immune response and reducing human morbidity and mortality against microbial infections, such as tuberculosis (TB). Bacillus Calmette-Guerin (BCG) is the most widely administered vaccine in the world and the only vaccine approved by the World Health Organization (WHO) to protect against TB. Although BCG confers "protective" immunity in children against the progression of Mycobacterium tuberculosis (Mtb) infection into active TB, this vaccine is ineffective in protecting adults with active TB manifestations, such as multiple-, extensive-, and total-drug-resistant (MDR/XDR/TDR) cases and the co-existence of TB with immune-compromising health conditions, such as HIV infection or diabetes. Moreover, BCG can cause disease in individuals with HIV infection or other immune compromises. Due to these limitations of BCG, novel strategies are urgently needed to improve global TB control measures. Since live vaccines elicit a broader immune response and do not require an adjuvant, developing recombinant BCG (rBCG) vaccine candidates have received significant attention as a potential replacement for the currently approved BCG vaccine for TB prevention. In this report, we aim to present the latest findings and outstanding questions that we consider worth investigating regarding novel mycobacteria-based live attenuated TB vaccine candidates. We also specifically discuss the important features of two key animal models, mice and rabbits, that are relevant to TB vaccine testing. Our review emphasizes that the development of vaccines that block the reactivation of latent Mtb infection (LTBI) into active TB would have a significant impact in reducing the spread and transmission of Mtb. The results and ideas discussed here are only based on reports from the last five years to keep the focus on recent developments.

BCGΔBCG1419c increased memory CD8+ T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG in a model of chronic tuberculosis.

Previously, we reported that a hygromycin resistant version of the BCGΔBCG1419c vaccine candidate reduced tuberculosis (TB) disease in BALB/c, C57BL/6, and B6D2F1 mice infected with Mycobacterium tuberculosis (Mtb) H37Rv. Here, the second-generation version of BCGΔBCG1419c (based on BCG Pasteur ATCC 35734, without antibiotic resistance markers, and a complete deletion of BCG1419c) was compared to its parental BCG for immunogenicity and protective efficacy against the Mtb clinical isolate M2 in C57BL/6 mice. Both BCG and BCGΔBCG1419c induced production of IFN-γ, TNF-α, and/or IL-2 by effector memory (CD44+CD62L-), PPD-specific, CD4+ T cells, and only BCGΔBCG1419c increased effector memory, PPD-specific CD8+ T cell responses in the lungs and spleens compared with unvaccinated mice before challenge. BCGΔBCG1419c increased levels of central memory (CD62L+CD44+) T CD4+ and CD8+ cells compared to those of BCG-vaccinated mice. Both BCG strains elicited Th1-biased antigen-specific polyfunctional effector memory CD4+/CD8+ T cell responses at 10 weeks post-infection, and both vaccines controlled Mtb M2 growth in the lung and spleen. Only BCGΔBCG1419c significantly ameliorated pulmonary inflammation and decreased neutrophil infiltration into the lung compared to BCG-vaccinated and unvaccinated mice. Both BCG strains reduced pulmonary TNF-α, IFN-γ, and IL-10 levels. Taken together, BCGΔBCG1419c increased memory CD8+T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG.