Rapid diagnosis of Plasmodium falciparum malaria using a point-of-care loop-mediated isothermal amplification device.

LAMP diagnosis of malaria is simple and cost-effective with acceptable sensitivity and specificity as compared to standard diagnostic modules such as microscopy, RDTs and nested PCR, and thus its deployment for onsite screening of malaria in resource-limited regions is under consideration. However, the requirement of an electricity-operated dry bath and bulky read-out unit is still a major concern. In an effort to simplify this limitation, we have developed a portable LAMP device and fluorescence readout unit which can be used in the rapid point-of-care diagnosis of malaria. We have developed a point-of-care diagnostic LAMP device that is easy to operate by a mobile application, and the results can be quantified with a fluorescent readout unit. The diagnostic performance of the device was evaluated in 90 P. falciparum-infected clinical isolates stored at 4°C for 6-7 years and 10 freshly collected isolates from healthy volunteers. The LOD and quantitative ability of LAMP in estimating parasitemia levels were revealed with laboratory-grown P. falciparum strain (3D7). The LAMP assay performed in our device was exclusive for P. falciparum detection with sensitivity and specificity determined to be 98.89% and 100%, respectively, in clinical isolates. The LOD was documented to be 1 parasite/µl at the cut-off ADC value of 20. Parasite density estimated from ADC values showed concordance with microscopically determined parasite density of the cultured P. falciparum 3D7 strain. The LAMP assay performed in our device provides a possible portable platform for its deployment in the point-of-care diagnosis of malaria. Further validation of the quantitative ability of the assay with freshly collected or properly stored clinical samples of known parasitemia is necessary for field applicability.

Optical absorbance-based rapid test for the detection of sickle cell trait and sickle cell disease at the point-of-care.

People afflicted with sickle cell disease (SCD) experience severe deterioration in quality of life. The disease is characterized by debilitating pain, anemia, and increased susceptibility to life threatening infections. This genetic disorder is endemic to many parts of the world. Extensive and accurate screening of individuals with sickle cell trait (SCT) in the population, coupled with genetic counselling can inhibit the propagation of the disease. The gold-standard techniques for the detection of sickle hemoglobin, such as capillary electrophoresis, HPLC, and genetic testing, are prohibitively expensive and time-consuming. Mass screening is usually conducted with a low-cost test called the solubility test, which does not offer high specificity. This study proposes a game-changing single-step low-cost method for rapidly yet accurately screening and diagnosing SCD and SCT. This method relies on the hitherto unexplored differences in the optical absorbance between diseased, trait, and normal blood samples, under deoxygenated conditions. The proposed method was tested in two phases of clinical validation: a pilot study and a blind study. A total of 438 patient samples were tested using the proposed method across the two phases. The proposed method offers an average accuracy, sensitivity, and specificity of 97.6%, 96.9%, and 98.6%, respectively. The proposed test has the potential to obliviate the conventional two-step process of screening and diagnostic tests as it can be used at the point-of-care with minimal training and yet yield results reliable enough to assess disability benefit claims.

Rapid diagnosis of Leishmania infection with a portable loopmediated isothermal amplification device.

L. donovani is an intracellular protozoan parasite, that causes visceral leishmaniasis (VL), and consequently, post-kala azar dermal leishmaniasis (PKDL). Diagnosis and treatment of leishmaniasis is crucial for decreasing its transmission. Various diagnostic techniques like microscopy, enzyme-linked immunosorbent assays (ELISA) and PCR-based methods are used to detect leishmaniasis infection. More recently, loop-mediated isothermal amplification (LAMP) assay has emerged as an ideal diagnostic measure for leishmaniasis, primarily due to its accuracy, speed and simplicity. However, point-of-care diagnosis is still not been tested with the LAMP assay. We have developed a portable LAMP device for the monitoring of Leishmania infection. The LAMP assay performed using our device can detect and amplify as little as 100 femtograms of L. donovani DNA. In a preliminary study, we have shown that the device can also amplify L. donovani DNA present in VL and PKDL patient samples with high sensitivity (100%), specificity (98%) and accuracy (99%), and can be used both for diagnostic and prognostic analysis. To our knowledge, this is the first report to describe the development and application of a portable LAMP device which has the potential to evolve as a point-of-care diagnostic and prognostic tool for Leishmania infections in future.

Single-shot circular fringe projection for the profiling of objects having surface discontinuities.

Fringe projection profilometry (FPP) is a widely used non-contact optical method for 3D profiling of objects. The commonly used linear fringe pattern in FPP has periodic intensity variations along the lateral direction. As a result, the linear fringe pattern used in FPP cannot uniquely represent the lateral shift induced by the objects having surface discontinuities. Thus, unambiguous surface profiling of objects, especially with surface discontinuities, using a single linear fringe image having a single fringe frequency, is unfeasible. This paper proposes using a radially symmetric circular fringe pattern as the structured light pattern for accurate unambiguous surface profiling of sudden height-discontinuous objects. To the best of our knowledge, this is the only method that can reconstruct discontinuous height profiles with the help of a single fringe image having a single frequency. The performance of the proposed algorithm is evaluated on several synthetic and real objects having smooth variations and discontinuities. Compared to the well-known fringe projection methods, the results depict that for a tolerable range of error, the proposed method can be applied for the reconstruction of objects with 4 times higher dynamic range and even at much lower fringe frequencies.

Recent technical advances in whole slide imaging instrumentation.

Microscopic observation of biological specimen smears is the mainstay of diagnostic pathology, as defined by the Digital Pathology Association. Though automated systems for this are commercially available, their bulky size and high cost renders them unusable for remote areas. The research community is investing much effort towards building equivalent but portable, low-cost systems. An overview of such research is presented here, including a comparative analysis of recent reports. This paper also reviews recently reported systems for automated staining and smear formation, including microfluidic devices; and optical and computational automated microscopy systems including smartphone-based devices. Image pre-processing and analysis methods for automated diagnosis are also briefly discussed. It concludes with a set of foreseeable research directions that could lead to affordable, integrated and accurate whole slide imaging systems.

Diagnosis of some diseases such as cervical cancer is done using a microscope, and this process still relies heavily on human experts. Since the need for such diagnosis is increasing at a rapid pace, it makes a lot of sense to automate the whole process. This requires automatic microscopes, which should be able to take images of a 'slide' - a glass slab with colorized human cells at its surface. These images should get analyzed by a software, resulting in a fully automated diagnosis. This article reviews recent research into this field, especially the technical advances on the hardware for automated microscopes (also known as slide imagers). It compares research reports and highlights how there's still more effort needed to build low-cost, yet clinically useful systems. It also highlights some of the emerging technologies that can be integrated into slide imagers to enable new kinds of diagnostics.

A rapid aptamer-based fluorescence assay for the detection of lipopolysaccharides using SYBR Green I.

Lipopolysaccharides (endotoxins), found on Gram-negative bacteria, can trigger a severe immune response in humans leading to septic shock and in extreme cases, even death. Therefore, the detection and neutralization of lipopolysaccharides (LPS) is of utmost importance in the pharmaceutical and medical industries. The United States Food and Drug Administration (US FDA) recommended detection method for LPS, the Limulus amebocyte lysate (LAL) assay, is expensive, time consuming, complex, and is prone to interference from proteases. As an alternative, this paper proposes a rapid, label-free fluorescence-based assay using LPS-specific aptamers and the SYBR Green DNA stain. The proposed method has a detection limit of 0.1 ng/ml, which is sufficient to detect the permissible levels of LPS in many pharmaceutical drugs and medical products. The fluorescence signal was found to be a linear function of the concentration of LPS in the range from 0.1 ng/ml to 105 ng/ml.

Pebrine diagnosis using quantitative phase imaging and machine learning.

Pebrine is the most dreaded infectious disease of the silkworm and has devastated sericulture in Europe during the 18th century. Thereafter, if it is detected, the crop is burned to prevent further dissemination. The conventional microscopic examination of moth's body fluid is erroneous and it exacerbates on Metarhizium anisopliae (MA) contaminated test samples. This is due to the resemblance of pebrine and MA spores in the microscopic examination. Therefore, this study aims to demonstrate an efficient pebrine detection technique. In the proposed method, a motorised brightfield microscope is custom-made to acquire focused and defocused images of test spores. These images are used to produce quantitative phase images of the spores by the transport of intensity equation method. The phase images' histogram of oriented gradients feature is used by a machine learning classifier to categorise the spores. This system classified 92 pebrine and 185 MA spores with an accuracy of 97% at 0.04 seconds/spore. The duration taken for image acquisition is 2.5 minutes per sample (10 fields of view covering an area of 302 × 260 µm2 ). The proposed method shows reliable results in pebrine diagnosis and would be an efficient alternative for current approaches.

Photocatalytic degradation of MB by TiO2: studies on recycle and reuse of photocatalyst and treated water for seed germination.

Photocatalysis is an effective way for treatment of wastewater and degradation of dyes. It is important to assess the reusability of photocatalyst and treated water after the treatment process. In this study, the photocatalytic activity of TiO2 (titanium dioxide) and TiO2-TMAOH (titanium dioxide-tetramethylammonium hydroxide) was analyzed for degradation of methylene blue dye. Enhanced degradation of methylene blue is observed while treated with TiO2-TMAOH with photodegradation efficiency (PDE) 80% within 20 min. A further study shows the reusability of TiO2 for degradation of dye for six cycles with a decrease in photodegradation efficiency from 90% (cycle-1) to 50% (cycle-2). Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX), and cyclic voltammetry (CV) analysis were carried out to identify the functional groups in treated water, traces of titanium, and TMAOH, respectively. Seed germination of Vigna radiata using TiO2- and TiO2-TMAOH-treated water shows equivalent and consistent growth. Water quality analysis of treated water shows improved biochemical oxygen demand (BOD) level (1.5 mg L-1), which is suitable for reusability of water for many applications. The outcomes suggest treated water can be used for irrigation and plantation purposes.

Emerging graphene-based sensors for the detection of food adulterants and toxicants - A review.

The detection of food adulterants and toxicants can prevent a large variety of adverse health conditions for the global population. Through the process of rapid sensing enabled by deploying novel and robust sensors, the food industry can assist in the detection of adulterants and toxicants at trace levels. Sensor platforms which exploit graphene-based nanomaterials satisfy this requirement due to outstanding electrical, optical and thermal properties. The materials' facile conjugation with linkers and biomolecules along with the option for further enhancement using nanoparticles results in highly sensitive and selective sensing characteristics. This review highlights novel applications of graphene derivatives for detection covering three important approaches; optical, electrical (field-effect) and electrochemical sensing. Suitable graphene-based sensors for portable devices as point-of-need platforms are also presented. The future scope of these sensors is discussed to showcase how these emerging techniques will disrupt the food detection sector for years to come.

dsDNA-templated fluorescent copper nanoparticles for the detection of lipopolysaccharides.

The introduction of lipopolysaccharides (LPS) or endotoxins that originate from Gram-negative bacteria into the human blood stream induces a severe immune response that can lead to septic shock, and even death. Hence, the accurate detection of LPS is of great importance in the medical and pharmaceutical sectors. This paper proposes a novel label-free fluorescence assay for the detection of LPS utilizing aptamers and the interference synthesis of dsDNA-templated copper nanoparticles. The assay can be performed at room temperature and does not require expensive reagents. The proposed assay has a limit of detection of 0.95 ng ml-1 of LPS, and the fluorescence emission from the copper nanoparticles was found to vary linearly with the concentration of LPS over a wide range (1 to 105 ng ml-1) with R2 = 0.9877.

Enhanced In Vitro Wound Healing Using PVA/B-PEI Nanofiber Mats: A Promising Wound Therapeutic Agent against ESKAPE and Opportunistic Pathogens.

Opportunistic skin pathogens and their resistance to pre-existing therapeutics are a challenge to normal physiological wound healing processes. Consistent development of antimicrobial agents is required to overcome the complications raised by antimicrobial resistance. An effective alternative proposed in recent research includes the use of antimicrobial nanoparticles or nanobiopolymers. Unfortunately, metallic nanoparticles that have been proven as antimicrobial agents also possess a certain level of toxicity. In this work, we demonstrate the use of a cationic polymer, branched polyethyleneimine (B-PEI), that has been electrospun to obtain a scaffold/fiber (B-PEI NF) mat resulting in a large surface area-to-volume ratio. SEM analysis revealed that the average diameter of the obtained fibers is 240 nm. The formation of nanoscaffold modulates the controlled release of the polymer from the matrix resulting in long-term effects. The antimicrobial and antibiofilm activity of the B-PEI nanofiber (B-PEI NF) was evaluated against ESKAPE pathogens (Pseudomonas aeruginosa and Staphylococcus aureus) and also against Candida albicans. Dose-dependent inhibition was observed for microbial growth and biofilm for all three test organisms, the minimum inhibitory concentration required for inhibiting P. aeruginosa, S. aureus, and C. albicans is 33.125, 26.5, and 19.875 µM, respectively, in 2 mL of bacterial/fungal broth. Crystal violet and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed significant reduction in biomass and cell viability of sessile cells, respectively, within the biofilm after treatment using B-PEI NFs. A B-PEI NF matrix promotes cell migration and wound healing processes by mimicking the extracellular matrix. In vitro wound healing studies showed a fivefold increase in cell migration and wound healing by B-PEI NFs (97% wound coverage in 17 h) when compared to B-PEI (15% wound coverage in 17 h). The in vitro wound healing assays confirmed the biocompatibility and better wound healing activity of B-PEI NF mats.

Guided filter based image enhancement for focal error compensation in low cost automated histopathology microscopic system.

Low-cost automated histopathology microscopy systems usually suffer from optical imperfections, producing images that are slightly Out of Focus (OoF). In this work, a guided filter (GF) based image preprocessing is proposed for compensating focal errors and its efficacy is demonstrated on images of healthy and malaria infected red blood cells (h-RBCs and i-RBCs), and PAP smears. Since contrast enhancement has been widely used as an image preprocessing step for the analysis of histopathology images, a systematic comparison is made with six such prominently used methods, namely Contrast Limited Adaptive Histogram Equalization (CLAHE), RIQMC-based optimal histogram matching (ROHIM), modified L0, Morphological Varying(MV)-Bitonic filter, unsharp mask filter and joint bilateral filter. The images enhanced using GF approach lead to better segmentation accuracy (upto 50% improvement over native images) and visual quality compared to other approaches, without any change in the color tones. Thus, the proposed GF approach is a viable solution for rectifying the OoF microscopy images without the loss of the valuable diagnostic information presented by the color tone.

Handheld fluorometer for in-situ melamine detection via interference synthesis of dsDNA-templated copper nanoparticles.

Fluorescent copper nanoparticles templated by dsDNA have gained significant research interest as they are inexpensive and easy to synthesize, and have found applications in the detection of a wide range of analytes. The presence of the analyte in the reaction mixture interferes with the synthesis of the copper nanoparticles and the subsequent drop in fluorescence can be correlated to the concentration of the analyte present in the solution. Analyte detection using copper nanoparticle-based assays is amenable for in-situ applications as the test does not require expensive reagents and can be performed at room temperature. However, expensive and sophisticated detection systems are required for the detection of copper nanoparticles due to the low fluorescence emission signal from these nanoparticles. This restricts the use of the technology to centralized labs. Utilizing a recently developed chemical technique for fluorescence enhancement, this paper presents the first report of a handheld fluorometer capable of detecting DNA-templated copper nanoparticles. The fluorometer is portable and constructed with low-cost, off-the-shelf components like a UV-LED and a PIN photodiode. The performance of the developed system is demonstrated through the detection of melamine in milk samples via the interference synthesis of copper nanoparticles. Melamine is an adulterant used in dairy products that is harmful to human health if present in levels above 1 ppm. The developed system is capable of detecting up to 0.1 ppm of melamine in milk samples with a linear relationship observed between the detector output and concentration of melamine in the range from 0.1 ppm to 100 ppm (R2 = 0.9979).

Hand-Powered Elastomeric Pump for Microfluidic Point-of-Care Diagnostics.

The pumping of fluids into microfluidic channels has become almost an unavoidable operation in all microfluidic applications. Such a need has seen an outburst of several techniques for pumping, out of which the majority of techniques involve complicated fabrication, as they require the introduction of electrodes, valves, piezoelectric materials, acoustic transducers, etc., into the microfluidic device. In addition to the complexity, this also escalates the cost incurred per device. Further, the use of stable external power supplies to produce such a pumping action adds to the bulkiness of the pumps, making them unsuitable for point-of-care diagnostic (POCD) applications. This paper reports a technique of pumping that is simple to realize and does not require external electric/magnetic power, but exploits the elastic properties of materials to achieve the pumping action. This mechanism of pumping ensured the cost per pump to less than 4 USD and can be used for at least 500 times. Several simulations, validation, and characterization experiments were performed on the developed pump to establish its functionality and suitability for use in POCD applications.

Microfluidic In-Flow Decantation Technique Using Stepped Pillar Arrays and Hydraulic Resistance Tuners.

Separating the particles from the liquid component of sample solutions is important for several microfluidic-based sample preparations and/or sample handling techniques, such as plasma separation from whole blood, sheath-free flow focusing, particle enrichment etc. This paper presents a microfluidic in-flow decantation technique that provides the separation of particles from particle-free fluid while in-flow. The design involves the expansion of sample fluid channel in lateral and depth directions, thereby producing a particle-free layer towards the walls of the channel, followed by gradual extraction of this particle-free fluid through a series of tiny openings located towards one-end of the depth-direction. The latter part of this design is quite crucial in the functionality of this decantation technique and is based on the principle called wee-extraction. The design, theory, and simulations were presented to explain the principle-of-operation. To demonstrate the proof-of-principle, the experimental characterization was performed on beads, platelets, and blood samples at various hematocrits (2.5%-45%). The experiments revealed clog-free separation of particle-free fluid for at least an hour of operation of the device and demonstrated purities close to 100% and yields as high as 14%. The avenues to improve the yield are discussed along with several potential applications.

Enhancement of the fluorescence properties of double stranded DNA templated copper nanoparticles.

The weak fluorescence emission from dsDNA templated copper nanoparticles necessitates the use of high-end detectors like photomultiplier tubes for their detection. This sets limitations on their applicability to in-situ analyte detection and point-of-care applications which utilize comparatively low cost and less sensitive detectors. In this article, a technique to improve the fluorescence properties of copper nanoparticles templated on dsDNA is reported. The fluorescence enhancement is achieved by introducing a modification in the conventional synthesis technique by using a combination of sodium ascorbate and Taq buffer. When compared to the existing methods, the proposed method achieves 11 times higher fluorescence signal intensity from the dsDNA templated copper nanoparticles and 4 times faster attainment of maximum fluorescence signal. The effect of the ionic strength of the individual constituent components of Taq buffer on the fluorescence emission from the copper nanoparticles is also studied here. The utility of this enhancement strategy for analyte measurement is demonstrated with the example of melamine detection from milk samples. A linear relationship was observed between the fluorescence intensity from the copper nanoparticles and the concentration of melamine in the range from 0.5 ppm to 100 ppm (R2 = 0.9919), with a limit of detection of 0.1 ppm. The reported fluorescence enhancement technique also results in 2.95 times improved sensitivity of detection when compared to the conventional technique.

Continuous stacking computational approach based automated microscope slide scanner.

Cost-effective and automated acquisition of whole slide images is a bottleneck for wide-scale deployment of digital pathology. In this article, a computation augmented approach for the development of an automated microscope slide scanner is presented. The realization of a prototype device built using inexpensive off-the-shelf optical components and motors is detailed. The applicability of the developed prototype to clinical diagnostic testing is demonstrated by generating good quality digital images of malaria-infected blood smears. Further, the acquired slide images have been processed to identify and count the number of malaria-infected red blood cells and thereby perform quantitative parasitemia level estimation. The presented prototype would enable cost-effective deployment of slide-based cyto-diagnostic testing in endemic areas.

An Efficient Microscale Technique for Determining the Erythrocyte Sedimentation Rate.

The erythrocyte sedimentation rate (ESR) is a commonly used test to screen for inflammatory conditions such as infections, autoimmune diseases, and cancers. However, it is a bulk macroscale test that requires a relatively large blood sample and takes a long time to run. Moreover, it provides no information regarding cell sizes or interactions, which can be highly variable. To overcome these drawbacks, we developed a microfluidic microscopy-based protocol to dynamically track settling red blood cells (RBCs) to quantify velocity of cell settling, as a surrogate for the ESR. We imaged individual cells in a vertical microfluidic channel and applied a hybrid cell detection and tracking algorithm to compute settling velocities. We combined eigenvalue background subtraction and centroid detection together with the Kalman filter and Hungarian assignment solver algorithms to increase accuracy and computational speed. Our algorithm is designed to track settling RBCs/aggregates in high cellularity samples rather than single cells in suspension. Detection accuracy was 79.3%, which is comparable to state-of-the-art cell-tracking techniques. Compared with conventional ESR tests, our approach has the advantages of being automated, using microliter volumes of blood samples, and rapid turnaround.

Rapid sensing of melamine in milk by interference green synthesis of silver nanoparticles.

A highly sensitive, selective, and rapid interference green synthesis based determination of potential milk adulterant melamine has been reported here. Melamine is a nitrogenous compound added to milk for mimicking proteins, consumption of which leads to kidney stones and renal failures. Melamine interacts with ascorbic acid (AA) through strong hydrogen-bonding interactions, thus resulting in an interference/interruption in the formation of silver (Ag) nanoparticles which was confirmed by UV-Vis spectroscopy and Transmission Electron Microscopy (TEM). The corresponding benchmark validations for melamine spiked milk samples were performed using High Performance Liquid Chromatography (HPLC). This interference in the formation of Ag nanoparticles resulted in color change that varies with concentration of melamine, thereby enabling in-situ rapid sensing of melamine from milk to a lower limit of 0.1ppm with a linear correlation coefficient of 0.9908.

Cytopathological image analysis using deep-learning networks in microfluidic microscopy.

Cytopathologic testing is one of the most critical steps in the diagnosis of diseases, including cancer. However, the task is laborious and demands skill. Associated high cost and low throughput drew considerable interest in automating the testing process. Several neural network architectures were designed to provide human expertise to machines. In this paper, we explore and propose the feasibility of using deep-learning networks for cytopathologic analysis by performing the classification of three important unlabeled, unstained leukemia cell lines (K562, MOLT, and HL60). The cell images used in the classification are captured using a low-cost, high-throughput cell imaging technique: microfluidics-based imaging flow cytometry. We demonstrate that without any conventional fine segmentation followed by explicit feature extraction, the proposed deep-learning algorithms effectively classify the coarsely localized cell lines. We show that the designed deep belief network as well as the deeply pretrained convolutional neural network outperform the conventionally used decision systems and are important in the medical domain, where the availability of labeled data is limited for training. We hope that our work enables the development of a clinically significant high-throughput microfluidic microscopy-based tool for disease screening/triaging, especially in resource-limited settings.