A low-cost, paper-based hybrid capture assay to detect high-risk HPV DNA for cervical cancer screening in low-resource settings.

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 prevention in El Salvador: A prospective evaluation of screening and triage strategies incorporating high-resolution microendoscopy to detect cervical precancer.

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 Cáncer 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.

Towards a needle-free diagnosis of malaria: in vivo identification and classification of red and white blood cells containing haemozoin.

BACKGROUND: Optical detection of circulating haemozoin has been suggested as a needle free method to diagnose malaria using in vivo microscopy. Haemozoin is generated within infected red blood cells by the malaria parasite, serving as a highly specific, endogenous biomarker of malaria. However, phagocytosis of haemozoin by white blood cells which persist after the infection is resolved presents the potential for false positive diagnosis; therefore, the focus of this work is to identify a feature of the haemozoin signal to discriminate between infected red blood cells and haemozoin-containing white blood cells. METHODS: Conventional brightfield microscopy of thin film blood smears was used to analyse haemozoin absorbance signal in vitro. Cell type and parasite maturity were morphologically determined using colocalized DAPI staining. The ability of features to discriminate between infected red blood cells and haemozoin-containing white blood cells was evaluated using images of smears from subjects infected with two species of Plasmodium, Plasmodium yoelii and Plasmodium falciparum. Discriminating features identified by blood smear microscopy were characterized in vivo in P. yoelii-infected mice. RESULTS: Two features of the haemozoin signal, haemozoin diameter and normalized intensity difference, were identified as potential parameters to differentiate infected red blood cells and haemozoin-containing white blood cells. Classification performance was evaluated using the area under the receiver operating characteristic curve, with area under the curve values of 0.89 for the diameter parameter and 0.85 for the intensity parameter when assessed in P. yoelii samples. Similar results were obtained from P. falciparum blood smears, showing an AUC of 0.93 or greater for both classification features. For in vivo investigations, the intensity-based metric was the best classifier, with an AUC of 0.91. CONCLUSIONS: This work demonstrates that size and intensity features of haemozoin absorbance signal collected by in vivo microscopy are effective classification metrics to discriminate infected red blood cells from haemozoin-containing white blood cells. This reduces the potential for false positive results associated with optical imaging strategies for in vivo diagnosis of malaria based on the endogenous biomarker haemozoin.

In vivo microscopy of hemozoin: towards a needle free diagnostic for malaria.

Clinical diagnosis of malaria suffers from poor specificity leading to overtreatment with antimalarial medications. Alternatives, like blood smear microscopy or antigen-based tests, require a blood sample. We investigate in vivo microscopy as a needle-free malaria diagnostic. Two optical signatures, birefringence and absorbance, of the endogenous malaria by-product hemozoin were evaluated as in vivo optical biomarkers. Hemozoin birefringence was difficult to detect in highly scattering tissue; however, hemozoin absorbance was observed in increasingly complex biological environments and detectable over a clinically-relevant range of parasitemia in vivo in a P. yoelii-infected mouse model of malaria.

Saturated semiconductor optical amplifier phase modulation for long range laser radar applications.

We investigate the use of a semiconductor optical amplifier operated in the saturation regime as a phase modulator for long range laser radar applications. The nature of the phase and amplitude modulation resulting from a high peak power Gaussian pulse, and the impact this has on the ideal pulse response of a laser radar system, is explored. We also present results of a proof-of-concept laboratory demonstration using phase-modulated pulses to interrogate a stationary target.

Self-activated liquid-crystal cells with photovoltaic substrates.

We show that photovoltaic fields are capable of efficiently reorienting liquid crystals, leading to new concepts of optically addressable light modulators. Using an arrangement consisting of a liquid-crystal layer between LiNbO3:Fe photovoltaic substrates, we observed spatial filtering due to self-phase modulation in a planar-oriented cell and nonlinear transmission between crossed polarizers in a twist-oriented cell. These processes do not require an external electric field. The substrates are arranged such that light propagates along the +c axis in each substrate, allowing a secondary process of power transfer to occur through contradirectional photorefractive two-beam coupling.

Spatial distribution of power coupling in self-pumped photorefractive reflection gratings.

The spatial distribution of the power transfer achieved by contradirectional two-beam coupling using self-pumped photorefractive reflection gratings is investigated in two materials with different photorefractive gain coefficients, LiNbO3:Fe and KNbO3:Fe. Incremental portions of the volume grating are erased optically by inducing thin optical damage planes, reducing the overall two-beam coupling efficiency. By monitoring the effect of local grating disruption, the distribution of power transfer is spatially resolved throughout the crystal, and the results are found to be in agreement with our theoretical predictions.