Journey From a Digital Innovation to a Sustainable Health Worker Capacity-Building App in India: Experiences, Challenges, and Lessons Learned.

Health workers, especially auxiliary nurse midwives (ANMs), are among the most critical resources in improving the quality of immunization services and reducing vaccine hesitancy under the Universal Immunization Programme (UIP) in India. To improve health worker immunization skills, UIP trainings in India are primarily conducted through instructor-led classroom, cascade trainings. However, a 2018 capacity-building need assessment revealed several challenges involved in traditional classroom training, such as a single-time exposure to new guidelines, complicated logistics arrangements, a lack of refresher training, and varying quality of training. These complexities make it difficult to meet the timely knowledge and skill needs of every health worker effectively and uniformly in a rapidly changing scenario of UIP. To meet health worker capacity-building needs and address these challenges, Rapid Immunization Skill Enhancement (RISE), a learning management system (LMS) application, was conceptualized. The RISE LMS application was developed as a human-centered, interactive, continuous, and adaptable knowledge and skill-building platform for health workers engaged in the UIP. RISE complements existing classroom-based cascade training for health workers by leveraging digital technologies for faster, easier, and more effective knowledge transfer to accommodate the fast-changing needs of a dynamic health program like UIP. In this article, we share the challenges and strategic solutions to digital training applications, lessons learned, sustainability of the application, and the impact RISE has made in India, all of which stemmed from leadership, coordinated efforts from a team of skilled professionals, government acceptance, detailed planning, and continued stakeholder engagement.

Analysis of variant gene family expression by quantitative PCR.

Real-time polymerase chain reaction (PCR), or quantitative PCR (qPCR), is a rapid, sensitive, and specific method used for a broad variety of applications including quantitative gene expression analysis, DNA copy number measurement, characterization of gene and chromosomal deletions, and genotyping. Real-time reverse transcription (RT)-PCR has largely supplanted Northern blot and RNase protection assays, as two examples, as a means of quantifying transcript levels. The method utilizes small amounts of RNA and allows efficient screening of a large number of samples. Here, we describe the materials and methods required to perform real-time RT-PCR, including RNA purification, cDNA synthesis, and real-time PCR analysis of cDNA samples.

Plasmodium falciparum STEVOR proteins impact erythrocyte mechanical properties.

Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deformability of the erythrocyte membrane. This process may facilitate parasite sequestration in deep tissue vasculature.

Frequent recombination events generate diversity within the multi-copy variant antigen gene families of Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum utilises a mechanism of antigenic variation to avoid the antibody response of its human host and thereby generates a long-term, persistent infection. This process predominantly results from systematic changes in expression of the primary erythrocyte surface antigen, a parasite-produced protein called PfEMP1 that is encoded by a repertoire of over 60 var genes in the P. falciparum genome. var genes exhibit extensive sequence diversity, both within a single parasite's genome as well as between different parasite isolates, and thus provide a large repertoire of antigenic determinants to be alternately displayed over the course of an infection. Whilst significant work has recently been published documenting the extreme level of diversity displayed by var genes found in natural parasite populations, little work has been done regarding the mechanisms that lead to sequence diversification and heterogeneity within var genes. In the course of producing transgenic lines from the original NF54 parasite isolate, we cloned and characterised a parasite line, termed E5, which is closely related to but distinct from 3D7, the parasite used for the P. falciparum genome nucleotide sequencing project. Analysis of the E5 var gene repertoire, as well as that of the surrounding rif and stevor multi-copy gene families, identified examples of frequent recombination events within these gene families, including an example of a duplicative transposition which indicates that recombination events play a significant role in the generation of diversity within the antigen encoding genes of P. falciparum.

Expression switching in the stevor and Pfmc-2TM superfamilies in Plasmodium falciparum.

Plasmodium falciparum possesses two multigenic families, var and rif, the products of which are expressed on the surface of infected erythrocytes, where via antigenic variation they contribute to malaria pathogenesis and evasion of antibody-mediated host immunity. The products of two smaller gene families, stevor and Pfmc-2TM, also localize to the erythrocyte membrane, although it is not known if they undergo antigenic switching. Herein we use gene-specific quantitative reverse transcription polymerase chain reaction (RT-PCR) to investigate the transcription pattern of the stevor and Pfmc-2TM gene families, in both primary and second generation clonal lines of the P. falciparum isolate, NF54. We show that: (i) the expression of stevor and Pfmc-2TM families is clonally variant, (ii) the expression of stevor and Pfmc-2TM families undergoes switching, and (iii) switching rates vary among different variants and different isogenic clones at rates higher than 2% per generation. In addition, we show that chromosomal telomeric deletions are common in clonal lines and result in a spectrum of deletion genotypes. These findings provide evidence that the stevor and Pfmc-2TM gene families play a role in P. falciparum antigenic variation.

Hypervariability within the Rifin, Stevor and Pfmc-2TM superfamilies in Plasmodium falciparum.

The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a Pexel/VTS erythrocyte-trafficking motif. This catalog includes large families of predicted 2 transmembrane (2TM) proteins, including the Rifin, Stevor and Pfmc-2TM superfamilies, of which each possesses a region of extensive sequence diversity across paralogs and between isolates that is confined to a proposed surface-exposed loop on the infected erythrocyte. Here we express epitope-tagged versions of the 2TM proteins in transgenic NF54 parasites and present evidence that the Stevor and Pfmc-2TM families are exported to the erythrocyte membrane, thus supporting the hypothesis that host immune pressure drives antigenic diversity within the loop. An examination of multiple P.falciparum isolates demonstrates that the hypervariable loop within Stevor and Pfmc-2TM proteins possesses sequence diversity across isolate boundaries. The Pfmc-2TM genes are encoded within large amplified loci that share profound nucleotide identity, which in turn highlight the divergences observed within the hypervariable loop. The majority of Pexel/VTS proteins are organized together within sub-telomeric genome neighborhoods, and a mechanism must therefore exist to differentially generate sequence diversity within select genes, as well as within highly defined regions within these genes.

Nitric oxide synthase-3 overexpression causes apoptosis and impairs neuronal mitochondrial function: relevance to Alzheimer's-type neurodegeneration.

Dementia in Alzheimer's disease (AD) is correlated with cell loss that is mediated by apoptosis, mitochondrial (Mt) dysfunction, and possibly necrosis. Previous studies demonstrated increased expression of the nitric oxide synthase 3 (NOS3) gene in degenerating neurons of AD brains. For investigating the role of NOS3 overexpression as a mediator of neuronal loss, human PNET2 central nervous system-derived neuronal cells were infected with recombinant adenovirus vectors that expressed either human NOS3 or green fluorescent protein cDNA under the control of a CMV promoter. NOS3 overexpression resulted in apoptosis accompanied by increased levels of p53, p21/Waf1, Bax, and CD95. In addition, NOS3 overexpression impaired neuronal Mt function as demonstrated by the reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and nicotinamide adenine dinucleotide (reduced form)-tetrazolium reductase activities and MitoTracker Red fluorescence. These adverse effects of NOS3 were associated with increased cellular levels of reactive oxygen species and impaired membrane integrity and were not produced in cells that were transfected with a cDNA encoding catalytically inactive NOS3. Importantly, modest elevations in NOS3 expression, achieved by infection with low multiplicities of adenovirus-NOS3 infection, did not cause apoptosis but rendered the cells more sensitive to oxidative injury by H(2)O(2) or diethyldithiocarbamate. In contrast, treatment with NO donors did not enhance neuronal sensitivity to oxidative injury. These results suggest that NOS3-induced neuronal death is mediated by Mt dysfunction, oxidative injury, and impaired membrane integrity, rather than by NO production, and that neuroprotection from these adverse effects of NOS3 may be achieved by modulating intracellular levels of oxidative stress.