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Cervical cancer is a leading cause of cancer death for women in low-resource settings. The World Health Organization recommends that cervical cancer screening programs incorporate HPV DNA testing, but available tests are expensive, require laboratory infrastructure, and cannot be performed at the point-of-care. We developed a two-dimensional paper network (2DPN), hybrid-capture, signal amplification assay and a point-of-care sample preparation protocol to detect high-risk HPV DNA from exfoliated cervical cells within an hour. The test does not require expensive equipment and has an estimated cost of$3 per test without the need for batching. We evaluated performance of the paper HPV DNA assay with short synthetic and genomic HPV DNA targets, HPV positive and negative cellular samples, and two sets of clinical samples. The first set of clinical samples consisted of 16 biobanked, provider-collected cervical samples from a study in El Salvador previously tested with careHPV and subsequently tested in a controlled laboratory environment. The paper HPV DNA test correctly identified eight of eight HPV-negative clinical samples and seven of eight HPV-positive clinical samples. We then performed a field evaluation of the paper HPV DNA test in a hospital laboratory in Mozambique. Cellular controls generated expected results throughout field testing with fully lyophilized sample preparation and 2DPN reagents. When evaluated with 16 residual self-collected cervicovaginal samples previously tested by the GeneXpert HPV assay ("Xpert"), the accuracy of the HPV DNA paper test in the field was reduced compared to testing in the controlled laboratory environment, with positive results obtained for all eight HPV-positive samples as well as seven of eight HPV-negative samples. Further evaluation showed reduction in performance was likely due in part to increased concentration of exfoliated cells in the self-collected clinical samples from Mozambique compared with provider-collected samples from El Salvador. Finally, a formal usability assessment was conducted with users in El Salvador and Mozambique; the assay was rated as acceptable to perform after minimal training. With additional optimization for higher cell concentrations and inclusion of an internal cellular control, the paper HPV DNA assay offers promise as a low-cost, point-of-care cervical cancer screening test in low-resource settings.

Cervical cancer remains a leading cause of cancer death for women in low- and middle-income countries. The goal of our study was to evaluate screening and triage strategies, including high-resolution microendoscopy (HRME), to detect cervical abnormalities concerning for precancer at the point of care. Women (n = 1824) were enrolled at the Instituto de Cancer de El Salvador. All underwent screening by both human papillomavirus (HPV) testing using careHPV and visual inspection with acetic acid (VIA). Screen-positives, along with 10% of screen-negatives, were invited to return for a follow-up examination that included triage with VIA, colposcopy and HRME imaging. Biopsies were taken of any abnormalities identified. If no abnormalities were identified, then the worst scoring site by HRME was biopsied. The sensitivities of HPV testing and VIA to screen for cervical intraepithelial neoplasia Grade 2 or more severe diagnoses (CIN2+) were 82.1% and 75% (P = .77), while the specificities were 90.4% and 80.9% (P < .001), respectively. The sensitivities of VIA, colposcopy and HRME as triage tests for CIN2+ were 82.1%, 82.1% and 71.4%, respectively (P ≥ .38). HRME had a significantly higher specificity (66.7%) than VIA (51.9%) (P < .001) and colposcopy (53.3%) (P < .001). When evaluating different theoretical screening and triage strategies, screening with HPV testing followed by triage with HRME would result in more women receiving appropriate care (97%) compared to screening with VIA (75%) or HPV alone (90%). Our findings demonstrate that screening with HPV is superior to VIA, and that triage with HRME imaging increases the specificity of detecting CIN2+ at the point of care in a low-resource setting.

Cervical cancer disproportionally affects women in low- and middle-income countries, in part due to the difficulty of implementing existing cervical cancer screening and diagnostic technologies in low-resource settings. Single-board computers offer a low-cost alternative to provide computational support for automated point-of-care technologies. Here we demonstrate two new devices for cervical cancer prevention that use a single-board computer: 1) a low-cost imaging system for real-time detection of cervical precancer and 2) a low-cost reader for real-time interpretation of lateral flow-based molecular tests to detect cervical cancer biomarkers. Using a Raspberry Pi computer to provide real-time image collection and processing, we developed: 1) a low-cost, portable high-resolution microendoscope system (PiHRME); and 2) a low-cost automatic lateral flow test reader (PiReader). The PiHRME acquired high-resolution (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$4.4~\mu \text{m}$ \end{document}) images of the cervix at half the cost of existing high-resolution microendoscope systems; image analysis algorithms based on convolutional neural networks were implemented to provide real-time image interpretation. The PiReader acquired and analyzed images of a point-of-care human papillomavirus (HPV) serology test with the same contrast and accuracy as a standard flatbed high-resolution scanner coupled to a laptop computer, for less than one-fifth of the cost. Raspberry Pi single-board computers provide a low-cost means to implement point-of-care tools with automatic image analysis. This work demonstrates the promise of single-board computers to develop and translate low-cost, point-of-care technologies for use in low-resource settings., Cervical cancer disproportionally affects women in low- and middle-income countries, in part due to the difficulty of implementing existing cervical cancer screening and diagnostic technologies in low-resource settings. Here we demonstrate two new devices for cervical cancer prevention that use a single-board computer: 1) a low-cost imaging system for real-time detection of cervical precancer and 2) a low-cost reader for real-time interpretation of lateral flow-based molecular tests to detect cervical cancer biomarkers.

INTRODUCTION: Cervical cancer mortality rates remain high in low- and middle-income countries (LMICs) and other medically underserved areas due to challenges with implementation and sustainability of routine screening, accurate diagnosis, and early treatment of preinvasive lesions. AREAS COVERED: In this review, we first discuss the standard of care for cervical cancer screening and diagnosis in high- and low-resource settings, biomarkers that correlate to cervical precancer and cancer, and needs for new tests. We review technologies for screening and diagnosis with a focus on tests that are already in use in LMICs or have the potential to be adapted for use in LMICs. Finally, we provide perspectives on the next five years of technology development for improved cervical cancer screening and diagnosis in LMICs. EXPERT OPINION: Innovation toward improved molecular and imaging tests is needed to enable effective, affordable see-and-treat approaches to detect and treat cervical precancer in a single visit. Current molecular tests remain too complex and/or costly for widespread use. Especially with imaging tests, decision support may improve performance of new technologies.

Objective Variants in the SLCO1B1 gene, encoding a hepatic methotrexate (MTX) transporter, affect clearance of high-dose MTX. We tested whether in the *14 and *15 alleles of SLCO1B1 influenced the response to low-dose MTX in juvenile idiopathic arthritis (JIA) patients. Methods The study included 310 JIA patients genotyped for three single nucleotide polymorphisms (SNPs) in SLCO1B1 (rs4149056, rs2306283, and rs11045819). A patient's SLCO1B1 diplotype was determined by combining the SNPs into the *1a, *1b, *4, *5, *14, and *15 alleles. Number of active joints at follow-up (visit closest to 6 months of treatment and prior to starting a tumor necrosis factor inhibitor) was used as the dependent variable in a negative binomial regression model that included active joint count at baseline as a covariate. Results The SLCO1B1*14 allele was associated with less response to MTX (P = 0.024) and the *15 allele was not associated with response to MTX (P = 0.392). Conclusion SLCO1B1 alleles may be associated with poor response to MTX in JIA patients. The *14 allele has been associated with fast clearance (low exposure) after high-dose MTX in patients with leukemia. Thus, the SLCO1B1 gene may be informative for precision dosing of MTX in JIA patients. Patients carrying the *14 allele may require a higher dose than noncarriers to achieve a similar response to MTX.

Objective Variants in the SLCO1B1 gene, encoding a hepatic methotrexate (MTX) transporter, affect clearance of high‐dose MTX. We tested whether in the *14 and *15 alleles of SLCO1B1 influenced the response to low‐dose MTX in juvenile idiopathic arthritis (JIA) patients. Methods The study included 310 JIA patients genotyped for three single nucleotide polymorphisms (SNPs) in SLCO1B1 (rs4149056, rs2306283, and rs11045819). A patient's SLCO1B1 diplotype was determined by combining the SNPs into the *1a, *1b, *4, *5, *14, and *15 alleles. Number of active joints at follow‐up (visit closest to 6 months of treatment and prior to starting a tumor necrosis factor inhibitor) was used as the dependent variable in a negative binomial regression model that included active joint count at baseline as a covariate. Results The SLCO1B1*14 allele was associated with less response to MTX (P = 0.024) and the *15 allele was not associated with response to MTX (P = 0.392). Conclusion SLCO1B1 alleles may be associated with poor response to MTX in JIA patients. The *14 allele has been associated with fast clearance (low exposure) after high‐dose MTX in patients with leukemia. Thus, the SLCO1B1 gene may be informative for precision dosing of MTX in JIA patients. Patients carrying the *14 allele may require a higher dose than noncarriers to achieve a similar response to MTX.

Each day, approximately 830 women and 7,400 newborns die from complications during pregnancy and childbirth. Improving maternal and neonatal health will require bringing rapid diagnosis and treatment to the point of care in low-resource settings. However, to date there are few diagnostic tools available that can be used at the point of care to detect the leading causes of maternal and neonatal mortality in low-resource settings. Here we review both commercially available diagnostics and technologies that are currently in development to detect the leading causes of maternal and neonatal mortality, highlighting key gaps in development where innovative design could increase access to technology and enable rapid diagnosis at the bedside.

Background: The global COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid, accurate, and accessible diagnostics to enable timely treatment and outbreak control. However, current diagnostic tests based on RT-qPCR are insufficient to meet the global testing demand because of their high cost and complexity and supply chain shortages. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a promising alternative to RT-qPCR because of its sensitivity, speed, and robustness to sample inhibitors. Here, we describe the development and optimisation of a sample-to-answer workflow, including a simple lysis and inactivation protocol that provides results in

Recent advances in bioengineering have enabled development of sensitive and specific diagnostic tests that detect and quantify cells, proteins, and nucleic acids. However, the development of diagnostic tests for use in low-resource settings presents a major challenge due to a lack of resources, infrastructure, and trained personnel. The lack of appropriate and affordable diagnostics has significant consequences; for example, in 2015, over 85% of the 5.7 million global deaths caused by infectious and parasitic diseases occurred in low- and lower-middle-income settings (Global Health Estimates 2015: Deaths by Cause, Age, Sex, by Country and by Region, 2000-2015. Geneva, World Health Organization; 2016). In this chapter, we outline general performance criteria that diagnostic tests must meet to be useful in low-resource settings. We review recent efforts to develop appropriate diagnostic technologies for three classes of biomarker targets in resource-limited settings: (1) cell-based assays, (2) protein detection tests, and (3) nucleic acid tests. Finally, we summarize innovation gaps that remain in order to implement effective diagnostic tests to combat infectious diseases worldwide.

Major gaps in our understanding of the leukodystrophies result from their rarity and the lack of tissue for the interdisciplinary studies required to extend our knowledge of the pathophysiology of the diseases. This study details the natural evolution of changes in the CNS of the shaking pup (shp), a model of the classical form of the X-linked disorder Pelizaeus–Merzbacher disease, in particular in glia, myelin, and axons, which is likely representative of what occurs over time in the human disease. The mutation in the proteolipid protein gene, PLP1, leads to a delay in differentiation, increased cell death, and a marked distension of the rough endoplasmic reticulum in oligodendrocytes. However, over time, more oligodendrocytes differentiate and survive in the spinal cord leading to an almost total recovery of myelination, In contrast, the brain remains persistently hypomyelinated. These data suggest that shp oligodendrocytes may be more functional than previously realized and that their early recruitment could have therapeutic value.

q-Space diffusion MRI (QSI) was used to study the spinal cords of Long Evans shaker (les) rats, a model of dysmyelination, and their age-matched controls at different maturation stages. Diffusion was measured parallel and perpendicular to the fibers of the spinal cords of the two groups and at different diffusion times. The results showed that QSI is able to detect the dysmyelination process that occurs in this model in the different stages of the disease. The differences in the diffusion characteristics of the spinal cords of the two groups were found to be larger when the diffusion time was increased from 22 to 100 ms. We found that the radial mean displacement is a much better parameter than the QSI fractional anisotropy (FA) to document the differences between the two groups. We observed that the degree of myelination affects the diffusion characteristics of the tissues, but has a smaller effect on FA. All of the extracted diffusion parameters that are affected by the degree of myelination are affected in a diffusion time-dependent fashion, suggesting that the terms apparent anisotropy, apparent fractional anisotropy and even apparent root-mean-square displacement (rmsD) are more appropriate.

The brains of Long Evans shaker (les) rats, a model of dysmyelination, and their age- matched controls were studied by ex-vivo q-space diffusion imaging (QSI) and diffusion tensor imaging (DTI). The QSI and DTI indices were computed from the same acquisition. The les and the control brains were studied at different stages of maturation and disease progression. The mean displacement, the probability for zero displacement and kurtosis were computed from QSI data while the fractional anisotropy (FA) and the eigenvalues were computed from DTI. It was found that all QSI indices detect the les pathology, at all stages of maturation, while only some of the DTI indices could detect the les pathology. The QSI mean displacement was larger in the les group as compared with their age-matched controls while the probability for zero displacement and the kurtosis were both lower all indicating higher degree of restriction in the control brains. Since all the DTI eigenvalues were higher in the les brains as compared to controls, the less efficient DTI measure for discerning the les pathology was found to be the FA. Clearly, the most sensitive DTI parameter to the les pathology is λ3, i.e., the minimal diffusivity. Since the QSI and DTI data were obtained from the same acquisition, despite the somewhat higher SNR of the QSI data compared to the DTI data, it seems that the higher diagnostic capacity of the QSI data in this experimental model of dysmyelination, originates mainly from the higher diffusing weighting of the QSI data.

The Long–Evans shaker (les) rat has a mutation in myelin basic protein that results in severe CNS dysmyelination and subsequent demyelination during development. During this time,lesoligodendrocytes accumulate cytoplasmic vesicles, including lysosomes and membrane-bound organelles. However, the mechanism and functional relevance behind these oligodendrocyte abnormalities inleshave not been investigated. Using high-magnification electron microscopy, we identified the accumulations inlesoligodendrocytes as early and late autophagosomes. Additionally, immunohistochemistry and Western blots showed an increase in autophagy markers inles. However, autophagy did not precede the death oflesoligodendrocytes. Instead, upregulating autophagy promoted membrane extensions inlesoligodendrocytesin vitro. Furthermore, upregulating autophagy inlesrats via intermittent fasting increased the proportion of myelinated axons as well as myelin sheath thickness inlesand control rats. Overall, this study provides insight into the abnormalities described inlesas well as identifying a novel mechanism that promotes the survival and function of oligodendrocytes.

Macrophages represent an important therapeutic target, because their activity has been implicated in the progression of debilitating diseases such as cancer and atherosclerosis. In this work, we designed and characterized pH-responsive polymeric micelles that were mannosylated using “click” chemistry to achieve CD206 (mannose receptor)-targeted siRNA delivery. CD206 is primarily expressed on macrophages and dendritic cells and upregulated in tumor-associated macrophages, a potentially useful target for cancer therapy. The mannosylated nanoparticles improved the delivery of siRNA into primary macrophages by 4-fold relative to the delivery of a nontargeted version of the same carrier (p < 0.01). Further, treatment for 24 h with the mannose-targeted siRNA carriers achieved 87 ± 10% knockdown of a model gene in primary macrophages, a cell type that is typically difficult to transfect. Finally, these nanoparticles were also avidly recognized and internalized by human macrophages and facilitated the delivery of...

Recently, two-dimensional paper networks have been developed to enable multistep assays to be performed in a lateral flow format. These devices have been used to perform simple enzyme linked immunoassays on paper. However, these devices have yet to incorporate more complex immunoassays, including the use of streptavidin-biotin detection strategies. Here we present a modified two-dimensional paper network capable of consecutively delivering six reagents. The device requires only a single user step and delivers (i) the sample, (ii) the biotinylated detection antibody, (iii) streptavidin horseradish peroxidase, (iv) a wash buffer, (v) a colorimetric substrate, and (vi) a final wash buffer. To demonstrate the utility of this approach we designed an assay to detect the malaria protein Pf HRP2. Using this platform, we were able to achieve a limit-of-detection equivalent to that of a traditional 96-well plate sandwich ELISA. In addition to improvements in the limit-of-detection, the inclusion of streptavidin-biotin simplifies the development of similar tests for other targets.

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.

Current dogma suggests that chronically demyelinated axons are at risk for degeneration, with axonal loss resulting in permanent disability in myelin disease. However, the trophic role of the myelin sheath in long-term axonal survival is incompletely understood. Previous observations of the effect of dysmyelination or demyelination on axonal survival in the myelin mutants has been limited because of their short life span. In this study, we used the Long–Evans shaker (les) rat, which can live up to 9 months, to study axonal health and survival after chronic demyelination. At 2 weeks, ∼29% of medium and ∼47% of large fiber axons are myelinated inlesspinal cord. However, by 3 months, no medium and ∼lesrats up to 9 months. Instead, there is a profound increase in oligodendrocytes, which were found to express BDNF, NT-3, and IGF-1. Importantly, this study providesin vivoevidence that mature glial cells produce various neurotrophic factors that may aid in the survival of axons after chronic demyelination.

Purpose We determined whether the chronic lack of optic nerve myelination and subsequent axon loss is associated with optical coherence tomography (OCT) changes in the retinal nerve fiber layer (RNFL), and whether this models what occurs in multiple sclerosis (MS) and confers its use as a surrogate marker for axon degeneration. Methods Using an animal model of Pelizaeus-Merzbacher disease (shp) bilateral longitudinal measurements of the peripapillary RNFL (spectral-domain OCT), electroretinograms (ERG), and visual evoked potentials (VEP) were performed in affected and control animals from 5 months to 2 years and in individual animals at single time points. Light and electron microscopy of the optic nerve and retina and histomorphometric measurements of the RNFL were compared to OCT data. Results Of the shp animals, 17% had an average reduction of OCT RNFL thickness on the superior retinal quadrant compared to controls (P < 0.05). Electroretinograms showed normal photopic A- and B-waves but flash VEPs were disorganized in shp animals. Morphologically, the shp retinas and optic nerves revealed significant RNFL thinning (P < 0.001) without retinal ganglion cell (RGC) loss, decrease total and relative retinal axonal area, and loss of optic nerve axons. There was strong positive correlation between OCT and morphometric RNFL thickness measurements (r = 0.878, P = 0.004). Conclusion The loss of optic nerve axons demonstrated in the shp model resulted in moderate thinning of the RNFL confirmed by OCT and histology. These results indicate that OCT-derived RNFL measurement can be a useful surrogate biomarker of optic nerve axon loss and potentially disease progression in demyelinating diseases.

q-Space diffusion MRI (QSI) was used to study the spinal cords of Long Evans shaker (les) rats, a model of dysmyelination, and their age-matched controls at different maturation stages. Diffusion was measured parallel and perpendicular to the fibers of the spinal cords of the two groups and at different diffusion times. The results showed that QSI is able to detect the dysmyelination process that occurs in this model in the different stages of the disease. The differences in the diffusion characteristics of the spinal cords of the two groups were found to be larger when the diffusion time was increased from 22 to 100 ms. We found that the radial mean displacement is a much better parameter than the QSI fractional anisotropy (FA) to document the differences between the two groups. We observed that the degree of myelination affects the diffusion characteristics of the tissues, but has a smaller effect on FA. All of the extracted diffusion parameters that are affected by the degree of myelination are affected in a diffusion time-dependent fashion, suggesting that the terms apparent anisotropy, apparent fractional anisotropy and even apparent root-mean-square displacement (rmsD) are more appropriate.

Smad-interacting protein-1 (SIP1) has been implicated in the development of Mowat-Wilson syndrome whose patients exhibit Hirschsprung disease, an aganglionosis of the large intestine, as well as other phenotypes. We have identified and cloned two sip1 orthologues in zebrafish. Both sip1 orthologues are expressed maternally and have dynamic zygotic expression patterns that are temporally and spatially distinct. We have investigated the function of both orthologues using translation and splice-blocking morpholino antisense oligonucleotides. Knockdown of the orthologues causes axial and neural patterning defects consistent with the previously described function of SIP1 as an inhibitor of BMP signaling. In addition, knockdown of both genes leads to a significant reduction/loss of the post-otic cranial neural crest. This results in a subsequent absence of neural crest precursors in the posterior pharyngeal arches and a loss of enteric precursors in the intestine.

Objective To develop and evaluate a paper-based point-of-care HPV serology test to determine if an individual has received two or more HPV immunizations. Methods The paper-based immunoassay was constructed using a nitrocellulose lateral flow strip with adsorbed HPV16 virus-like particles serving as the capturing moiety. Three capture zones containing virus-like particles were placed in series to allow for visual discrimination between high and low HPV16 plasma antibody concentrations. A plasma separation membrane was used to allow whole blood to be applied directly to the assay. All reagents were dried on glass fiber pads during device fabrication and were rehydrated with buffer at the time of use. A pilot study consisting of 35 subjects with a history of zero, one, two or three HPV vaccines was conducted to evaluate the immunoassay. The completed paper-based immunoassays were scanned for visual interpretation by three researchers who were blinded to the true results and separately evaluated quantitatively using MATLAB. Results For the 28 tests valid for analysis, fifteen subjects reported receiving two or more HPV vaccines, three reported receiving one, and ten reported having no HPV vaccinations. The paper-based immunoassays for all fifteen subjects who reported having received two or more HPV vaccines were judged positive by all researchers. Twelve of the thirteen tests from individuals reporting one or zero vaccinations were deemed negative by all observers. One test from an unvaccinated individual was judged positive by two out of three reviewers. Quantitatively, all tests were correctly separated between the two groups. Conclusions We successfully designed and tested a HPV serology test amenable to the point-of-care. The device showed promising results in a pilot study for discriminating between those who received two or more HPV vaccinations and those who did not. Furthermore, this device offers a platform for producing other semi-quantitative point-of-care serological tests.