Smartphone-operated affordable PCR thermal cycler for the detection of antimicrobial resistant bacterial genes.

Antimicrobial resistance (AMR) is a global public health threat. Surveillance of AMR requires affordable, rapid, and user-friendly diagnostic methods. Our aim was to develop a low-cost thermocycler to perform polymerase chain reaction (PCR). We developed a smartphone-operated PCR thermal cycler using locally available recycled materials. The thermal cycler was used for the amplification for three bacterial genes-bla-TEM, bla-CTXM and 16s rRNA in human urine samples. The performance of custom-built thermal cycler was compared with commercial thermal cycler. The thermal cycler was portable (<1kg weight), required 12 V power supply, 25 µL of solution, and cost only USD50.0. Temperature and time conditions were instructed using a custom-built smartphone application. The ramping rate of was 0.23°C for heating and 0.43°C for cooling. The reported temperatures were within ± 0.5°C of set temperature. The human urine samples were highly resistance and multi-resistant. Nearly 46% (n = 54) E. coli isolates were positive in ESBL screening test. The custom-built thermocycler was able to accurately predict the presence of bla-TEM, bla-CTXM genes, and 16s rRNA (n = 6). We developed and demonstrated a portable, low-cost, easy-to-use, and smartphone-operated PCR thermal cycler. Since it is portable, it can be used in remote location and field settings, including places without stable power supply. The use of the thermal cycler system can be extended, beyond the detection of AMR genes, e.g., in clinical diagnosis, genetics, forensic analysis, and environmental protection.

Material Properties of Traditional Handmade Paper Samples Fabricated from Cellulosic Fiber of Lokta Bushes.

Handmade papers (HPs) are fabricated from fibrous biomass of Lokta bushes and other plant species following traditional eco-friendly method in Nepal. Although HP fabricated from Lokta bushes is believed to be durable and resistant to bugs and molds, material properties of this paper are not reported in literature. In this study, we measured several material properties of 10 handmade Lokta paper samples collected from local enterprises and paper industries. The mean caliper, grammage, apparent density, equilibrium moisture content, Cobb 60, brightness, opacity, tensile strength, and tensile index values in the paper samples ranged from ∼90 to 700 µm, 50 to 150 g/m2, 0.2 to 0.4 g/cm3, 4 to 7%, 50 to 400 g/m2, 56 to 67%, 83 to 98%, 30 to 2900 N/m, and 1 to 27 Nm/g, respectively. These properties suggested that the HPs are lightweight papers with intermediate to high strength. The tensile strength was found to be significantly higher along the length direction (p < 0.05). Characteristic features of cellulose, hemicellulose, and lignin were observed in FTIR spectra. The crystalline and amorphous phases were also identified in X-ray diffraction (XRD) data. Electron microscopy images revealed a nicely cross-linked network of intact fibers having almost parallel arrangement of microfibrils. These features could provide strength and durability to the paper samples. Understanding the material properties of HPs down to the sub-microscopic level may help improve the paper quality and find novel applications in the future.

Review of drug recalls and quality of pharmaceutical products in Nepal.

OBJECTIVES: To evaluate the pattern of substandard and falsified pharmaceutical products recall in Nepal. SETTING: We analysed drug recall notices issued by the Department of Drug Administration (DDA), Nepal, and systematically reviewed peer-reviewed research articles during January 2010 to December 2020. PARTICIPANTS: This study did not include human participants. However, data were collected from 72 drug recall notices issued by DDA and four research papers. RESULTS: A total of 346 pharmaceutical products were recalled during the reported period. The number of recalled pharmaceutical products has increased significantly over the past decade in Nepal. The most frequently recalled drugs were antimicrobials followed by gastrointestinal medicines, vitamins and supplements and pain and palliative medicines among others. Number of imported recalled drugs were slightly higher (42.2%) than domestic recalled drugs (40.7%). Sixty-two percentage of recalled drugs were substandard, 11% were falsified and remaining 27% were not registered at the DDA. Similarly, higher number of modern drugs (62%) were recalled than traditional ones (35%). Hand sanitisers used to minimise COVID-19 transmission contributed significantly to the list of recalled pharmaceutical products in 2020. Most of these sanitisers contained significant amounts of methanol (as high as 75% v/v) instead of appropriate amount of ethyl or isopropyl alcohol. The peer-reviewed research papers reported issues with labelling, unregistered drugs and drugs failed in several laboratory testing. CONCLUSION: Our analysis showed that number of recalls of substandard and falsified drugs are increasing in Nepal. Since the recall data in this paper did not include number of samples tested and location of samples collected, more studies to understand the prevalence of substandard and falsified drugs in Nepal is recommended.

Correction: Microfluidic ELISA employing an enzyme substrate and product species with similar detection properties.

Correction for 'Microfluidic ELISA employing an enzyme substrate and product species with similar detection properties' by Basant Giri et al., Analyst, 2018, 143, 989-998, https://doi.org/10.1039/C7AN01671A.

Handmade Paper as a Paper Analytical Device for Determining the Quality of an Antidiabetic Drug.

Paper analytical devices (PADs) are a class of low-cost, portable, and easy-to-use platform for several analytical tests in clinical diagnostics, environmental pollution monitoring, and food and drug safety screening. These devices are primarily made from cellulosic paper. Considering the importance of eco-friendly and local or distributed manufacturing of devices realized during the COVID-19 pandemic, we systematically studied the potential of handmade Nepali paper to be used in fabricating PADs in this work. We characterized five different handmade papers made from locally available plant fibers using an eco-friendly method and used them to fabricate PADs for determining the drug quality. The thickness, grammage, and apparent density of the paper samples ranged from 198.6 to 314.8 µm, 49.1 to 117.8 g/m2, and 0.23 to 0.43 g/cm3, respectively. The moisture content, water filtration, and wicking speed ranged from 5.8 to 7.1%, 35.7 to 156.7, and 0.062 to 0.124 mms-1, respectively. Furthermore, the water contact angle and porosity ranged from 76.6 to 112.1° and 79 to 83%, respectively. The best paper sample (P5) was chosen to fabricate PADs for the determination of metformin, an antidiabetic drug. The metformin assay on PADs followed a linear range from 0.0625 to 0.5 mg/mL. The assay had a limit of detection and limit of quantitation of 0.05 and 0.18 mg/mL, respectively. The average amount of metformin concentration in samples collected from local pharmacies (n = 20) was 465.6 ± 15.1 mg/tablet. When compared with the spectrophotometric method, PAD assay correctly predicted the concentration of 90% samples. The PAD assay on handmade paper may provide a low-cost and easy-to-use system for screening the quality of drugs and other point-of-need applications.

Excessive iodine in iodized household salt in Nepal.

Iodine is an essential trace element required for the regulation of physiological processes involving the thyroid gland. However, inadequate and excessive intake of iodine are responsible for health problems, such as iodine deficiency disorders, hypothyroidism, hyperthyroidism, thyroiditis, thyroid papillary cancer, and thyrotoxicosis. The Universal Salt Iodization (USI) program has become successful in providing supplemental iodine at the population level globally. Packaging quality, fortification level, and transportation and storage conditions of iodized salt determine the availability of iodine. Previous studies have reported severe health issues caused by excessive iodine intake after the implementation of the USI program. To understand the levels of iodine, we collected 2117 household salt samples from seven districts of Nepal and tested them for iodine content; among them, 98.1% were iodized. Overall median concentration of iodine was 53.9 ppm (range: 43.5-61.4 ppm). The majority (67.2%) of samples had iodine in the range of 45-75 ppm. Approximately 0.9% of samples had inadequate, 13.3% contained adequate, and 83.9% had excessive iodine than the World Health Organization-recommended value. Iodine content varied among the sampling districts and seasons, to some extent. Our study confirmed that iodized salt is widely used in Nepal and is excessively iodized. Excessive intake of iodine through iodized salt requires further attention by policy makers. The iodine level may need adjustment to address the health impact.

A compact microfluidic geometry for multiplexing enzyme-linked immunosorbent assays.

We have previously reported a novel approach to implementing multiplex enzyme-linked immunosorbent assay (ELISA) in connected microchannels by exploiting the slow diffusion of the enzyme reaction product across the different assay segments. This work builds on that report by implementing the noted assay in segments arranged along the circumference of a circular channel layout to reduce the footprint size and sample volume requirement. Using the current design, a 5-plex cytokine ELISA was demonstrated in a 1.5 × 1.5-cm region, which corresponded to a reduction in the footprint area by about a factor of 3 compared to that reported in our previous study. Additionally, the selective coating of our assay segments with the target molecules was realized in this work using electroosmosis instead of hydrodynamic flow as was the case in the previous report. This aspect of our experimental design is particularly significant as it permits the use of cross-sectional channel dimensions significantly shorter than those employed in the current work. Moreover, the use of an electric field for coating purposes enables the integration of functionalities such as electrokinetic preconcentration of analyte molecules during the sample incubation period that can further enhance the capabilities of our assay method.

Characterization, Behavior, and Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in the Estuary Sediments.

Polycyclic aromatic hydrocarbons (PAHs) are persistent toxic substances that have ubiquitous presence in water, air, soil, and sediment environments. The growth of PAH toxicities and related ecotoxicology risk in estuary sediment has a serious concern. Present study examined the PAHs concentration, sources, and ecological risk from selected sites in Subarnarekha River estuary (SRE) sediment deposits. The sum of toxic 16 PAHs was ranged from 36.8 to 670.8 ng/g (mean = 223.46 ± 196.35 ng/g). The total PAH concentration varied significantly among the sampling sites (range 511.3 ng/g to 233.8 ng/g) based on allochthonous contaminant loads. Among the 16 compounds, Phen had the highest concentration (40.18 ng/g), followed by Pye (31.86 ng/g), Flur (29.36 ng/g), and NA (19.33 ng/g). Most of the sampling sites contained abundant 3-ring and 4-5-ring PAHs. Based on diagnostic ratios and PCA analysis petroleum combustion, biomass, and coal-burning have been identified as the major sources. The PAHs had high mutagenic equivalent factor and toxic equivalent factor values posing great ecological threats and health risks.

Machine-Learning-Assisted Analysis of Colorimetric Assays on Paper Analytical Devices.

Paper-based analytical devices (PADs) employing colorimetric detection and smartphone images have gained wider acceptance in a variety of measurement applications. PADs are primarily meant to be used in field settings where assay and imaging conditions greatly vary, resulting in less accurate results. Recently, machine-learning (ML)-assisted models have been used in image analysis. We evaluated a combination of four ML models-logistic regression, support vector machine (SVM), random forest, and artificial neural network (ANN)-as well as three image color spaces, RGB, HSV, and LAB, for their ability to accurately predict analyte concentrations. We used images of PADs taken at varying lighting conditions, with different cameras and users for food color and enzyme inhibition assays to create training and test datasets. The prediction accuracy was higher for food color than enzyme inhibition assays in most of the ML models and color space combinations. All models better predicted coarse-level classifications than fine-grained concentration classes. ML models using the sample color along with a reference color increased the models' ability to predict the result in which the reference color may have partially factored out the variation in ambient assay and imaging conditions. The best concentration class prediction accuracy obtained for food color was 0.966 when using the ANN model and LAB color space. The accuracy for enzyme inhibition assay was 0.908 when using the SVM model and LAB color space. Appropriate models and color space combinations can be useful to analyze large numbers of samples on PADs as a powerful low-cost quick field-testing tool.

Review of analytical performance of COVID-19 detection methods.

In the recent SARS-CoV-2 pandemic, public health experts have emphasized testing, tracking infected people, and tracing their contacts as an effective strategy to reduce the spread of the virus. Several diagnostic methods are reported for detecting the coronavirus in clinical, research, and public health laboratories. Some tests detect the infection directly by detecting the viral RNA and other tests detect the infection indirectly by detecting the host antibodies. A diagnostic test during the pandemic should help make an appropriate clinical decision in a short period of time. Recently reported diagnostic methods for SARS-CoV-2 have varying throughput, batching capacity, requirement of infrastructure setting, analytical performance, and turnaround times ranging from a few minutes to several hours. These factors should be considered while selecting a reliable and rapid diagnostic method to help make an appropriate decision and prompt public health interventions. This paper reviews recent SARS-CoV-2 diagnostic methods published in journals and reports released by regulatory agencies. We compared the analytical efficiency including limit of detection, sensitivity, specificity, and throughput. In addition, we also looked into ease of use, affordability, and availability of accessories. Finally, we discuss the limitations of the methods and provide our perspectives on priorities for future test development.

A smartphone microscopic method for simultaneous detection of (oo)cysts of Cryptosporidium and Giardia.

BACKGROUND: Food and water-borne illness caused by ingestion of (oo)cysts of Cryptosporidium and Giardia is one of the major health problems globally. Several methods are available to detect Giardia cyst and Cryptosporidium oocyst in food and water. Most of the available methods require a good laboratory facility and well-trained manpower and are therefore costly. There is a need of affordable and reliable method that can be easily implemented in resource limited settings. METHODOLOGY/PRINCIPLE FINDINGS: We developed a smartphone based microscopic assay method to screen (oo)cysts of Cryptosporidium and Giardia contamination of vegetable and water samples. The method consisting of a ball lens of 1 mm diameter, white LED as illumination source and Lugols's iodine staining provided magnification and contrast capable of distinguishing (oo)cysts of Cryptosporidium and Giardia. The analytical performance of the method was tested by spike recovery experiments. The spike recovery experiments performed on cabbage, carrot, cucumber, radish, tomatoes, and water resulted in 26.8±10.3, 40.1±8.5, 44.4±7.3, 47.6±11.3, 49.2 ±10.9, and 30.2±7.9% recovery for Cryptosporidium, respectively and 10.2±4.0, 14.1±7.3, 24.2±12.1, 23.2±13.7, 17.1±13.9, and 37.6±2.4% recovery for Giardia, respectively. The spike recovery results are comparable with data obtained using commercial brightfield and fluorescence microscope methods. Finally, we tested the smartphone microscope system for detecting (oo)cysts on 7 types of vegetable (n = 196) and river water (n = 18) samples. Forty-two percent vegetable and thirty-nine percent water samples were found to be contaminated with Cryptosporidium oocyst. Similarly, thirty-one percent vegetable and thirty-three percent water samples were contaminated with Giardia cyst. CONCLUSIONS: The newly developed smartphone microscopic method showed comparable performance to commercial microscopic methods. The new method can be a low-cost and easy to implement alternative method for simultaneous detection of (oo)cysts in vegetable and water samples in resource limited settings.

Selection of appropriate protein assay method for a paper microfluidics platform.

BACKGROUND: Paper-analytical devices (PADs) have gained popularity as a simple and low-cost alternative for determining a wide range of analytes including proteins. Even though several colorimetric PADs methods for protein estimation are reported in literature, they lack justification for the chosen method and parameters therein. AIM: Major aim of this work was to thoroughly evaluate the most commonly used colorimetric protein assays and recommend the most appropriate method for PADs platform. METHOD: We performed following six colorimetric protein assays on PADs: biuret, lowry, bicinchoninic acid, bradford, bromocresol green, and tetrabromophenol blue. We obtained assay signal by analyzing images of the PADs and then assessed analytical figures of merit. RESULT: Precision, accuracy, LOD, and LOQ of PADs protein assay methods ranged from 1.2 to 6.4%, 73.3-102.4%, 0.3-3.8 â€‹mg/mL, and 1.2-12.8 â€‹mg/mL, respectively. Out of six methods, we determined bromocresol green and tetrabromophenol blue as the best methods for serum and urine samples, respectively based on their optimized parameters and analytical figures of merit. The total average serum and urine protein in human samples were found to be 94.6 â€‹± â€‹16.2 â€‹mg/mL and 2.1 â€‹± â€‹1.5 â€‹mg/mL, respectively using PADs methods. The PADs result on human samples moderately correlated with the results from spectrophotometric determination (r2 â€‹> â€‹0.6). CONCLUSION: Paper-based protein assays were easy to perform and were completed with thousand-fold less volume of samples/reagents without a spectrophotometer compared to conventional assay methods. After testing human samples, we found one protein assay method may not be appropriate for all types of samples.

Characterization of airborne dust samples collected from core areas of Kathmandu Valley.

Kathmandu Valley is reported to be one of the highly polluted and populated cities in the world. Particulate matter is one of the major contributors of unhealthy air in Kathmandu. Although there are several reports on spatial and temporal variation of air quality of Kathmandu Valley, the morphological and mineralogical characteristics of particulate matter are very limited or none. In this study, we report on the mineralogical and morphological analysis of airborne particulate matter collected from densely populated core areas of Kathmandu Valley using spectroscopic and microscopic techniques. The Fourier Transform Infrared (FTIR) and X-ray Diffraction (XRD) data showed the presence of clay minerals, crystalline silicate mineral, carbonate minerals, and asbestiform mineral in the dust samples. The field emission scanning electron microscopic analysis confirmed the existence of particles having diverse morphology with some of the particles having aspect ratio as high as twenty; indicating the existence of asbestiform type minerals. Based on SEM-EDX data, we found that the relative distribution of elements to be different in different samples and C, O, Mg, Ca, and Si were the major elements in the dust samples. Interestingly, the XRD data analysis showed that in all the samples quartz mineral having high degree of crystallinity was present. The XRD measurement was also carried out in three different brands of cement samples. Few minerals present in dust samples were also identified in the cement samples. This observation could indicate that cement is one of the sources of minerals in the airborne particulate matter in the Kathmandu Valley.

Is research in peril in Nepal? Publication trend and research quality from projects funded by the University Grants Commission-Nepal.

Institutions of higher learning are critical in promoting a knowledge-driven economy through research and training. Nepali universities receive funding from the University Grants Commission, Nepal (UGC-N) to support for impactful research. UGC-N requires grantees to publish research results as journal articles. We reviewed papers published through UGC-N funded research projects over a 10-year period (2008-2018) to assess the trends of article publication in terms of frequency and quality (based on journal impact factor and SCImago journal ranking). At most, 17% projects (n = 325) had publications and the majority of articles were published in journals that had neither SJR rank (74%, n = 240) nor impact factor (86%, n = 279). Most importantly, 10% of articles (n = 23) published in the non-ranked journals appeared in predatory journals. Although there were increasing trends of grants and research article publications in the last 10 years, journal-level quality metrics showed no improvements and suggested decreasing trends during the last half decade. The publication output varied among grant categories. Master research grants and PhD research grants performed better than those of faculty research grants in terms of publication in quality journals. We call for an increased commitment from political and academic leadership to promote quality research in Nepal.

Optical microscopic study of surface morphology and filtering efficiency of face masks.

BACKGROUND: Low-cost face masks made from different cloth materials are very common in developing countries. The cloth masks (CM) are usually double layered with stretchable ear loops. It is common practice to use such masks for months after multiple washing and drying cycles. If a CM is used for long time, the ear loops become stretched. The loop needs to be knotted to make the mask loop fit better on the face. It is not clear how washing and drying and stretching practices change the quality of a CM. The particulate matter (PM) filtering efficiency of a mask depends on multiple parameters, such as pore size, shape, clearance, and pore number density. It is important to understand the effect of these parameters on the filtering efficiency. METHODS: We characterized the surface of twenty different types of CMs using optical image analysis method. The filtering efficiency of selected cloth face masks was measured using the particle counting method. We also studied the effects of washing and drying and stretching on the quality of a mask. RESULTS: The pore size of masks ranged from 80 to 500 µm, which was much bigger than particular matter having diameter of 2.5 µm or less (PM2.5) and 10 µm or less (PM10) size. The PM10 filtering efficiency of four of the selected masks ranged from 63% to 84%. The poor filtering efficiency may have arisen from larger and open pores present in the masks. Interestingly, we found that efficiency dropped by 20% after the 4th washing and drying cycle. We observed a change in pore size and shape and a decrease in microfibers within the pores after washing. Stretching of CM surface also altered the pore size and potentially decreased the filtering efficiency. As compared to CMs, the less frequently used surgical/paper masks had complicated networks of fibers and much smaller pores in multiple layers in comparison to CMs, and therefore had better filtering efficiency. This study showed that the filtering efficiency of cloth face masks were relatively lower, and washing and drying practices deteriorated the efficiency. We believe that the findings of this study will be very helpful for increasing public awareness and help governmental agencies to make proper guidelines and policies for use of face mask.

Evaluation of secondary metabolites, antioxidant activity, and color parameters of Nepali wines.

We evaluated the quality of wines produced in Nepal in terms of phenolic, flavonoid, anthocyanin and tannin content, antioxidant capacity, and color parameters using spectrophotometric methods. The total phenolic content, total flavonoid content, and total antioxidant activities in Nepali wines ranged from 85.5 to 960.0 (mean = 360.5 ± 268.7) mg/L GAE, 40.9-551.3 (mean = 188.9 ± 161.5) mg/L QE, and 66.6-905.0 (mean = 332.8 ± 296.5) mg/L AAE, respectively. These parameters were significantly higher in red wines compared to white wines. The phenolic and flavonoid content showed strong correlation with each other as well as with antioxidant activities. Additional parameters measured included various color parameters and carbohydrates. The wine color showed strong correlation with phenol, flavonoid, and antioxidant activity, whereas this correlation was not significant with anthocyanin content. Multivariate analysis was carried out to better describe and discriminate the wine samples. Finally, we compared Nepali wines with wines from other countries.

Microfluidic ELISA employing an enzyme substrate and product species with similar detection properties.

The requirement for an enzyme label to carry out a chemical reaction directly at the signaling region of the enzyme substrate in order to produce a large change in its detectability places a significant constraint on the scope of enzyme-linked immunosorbent assays (ELISAs). In particular, this requirement limits the kinds of enzyme label-substrate couples employable in ELISAs and prevents their independent optimization with respect to the enzyme reaction and the detectability of the enzyme reaction substrate/product. The detection limit and multiplexing capabilities of the assay are consequently restricted in addition to rendering the technique applicable to a narrow range of assay conditions/samples. Attempting to address some of these limitations, the current article describes a microfluidic ELISA method that does not require the enzyme label to act around the signaling region of the substrate molecule. A highly detectable rhodamine based substrate was synthesized to demonstrate the reported assay which upon cleavage by the enzyme label, alkaline phosphatase, transformed from a monoanionic to a monocationic species, both of which had nearly identical fluorescence properties. These species were later separated based on their charge difference using capillary zone electrophoresis in an integrated device yielding a quantitative measure for the analyte (human TNF-α) in our sample. Impressively, the noted approach not only enabled the use of a new kind of enzyme substrate for ELISAs but also allowed the detection of human TNF-α at concentrations over 54-fold lower than that possible on commercial microwell plates primarily due to the better detectability of the rhodamine dye.

Advances in paper-analytical methods for pharmaceutical analysis.

Paper devices have many advantages over other microfluidic devices. The paper substrate, from cellulose to glass fiber, is an inexpensive substrate that can be readily modified to suit a variety of applications. Milli- to micro-scale patterns can be designed to create a fast, cost-effective device that uses small amounts of reagents and samples. Finally, well-established chemical and biological methods can be adapted to paper to yield a portable device that can be used in resource-limited areas (e.g., field work). Altogether, the paper devices have grown into reliable analytical devices for screening low quality pharmaceuticals. This review article presents fabrication processes, detection techniques, and applications of paper microfluidic devices toward pharmaceutical screening.

Status of chemistry lab safety in Nepal.

Chemistry labs can become a dangerous environment for students as the lab exercises involve hazardous chemicals, glassware, and equipment. Approximately one hundred thousand students take chemistry laboratory classes annually in Nepal. We conducted a survey on chemical lab safety issues across Nepal. In this paper, we assess the safety policy and equipment, protocols and procedures followed, and waste disposal in chemistry teaching labs. Significant population of the respondents believed that there is no monitoring of the lab safety in their lab (p<0.001). Even though many labs do not allow food and beverages inside lab and have first aid kits, they lack some basic safety equipment. There is no institutional mechanism to dispose lab waste and chemical waste is disposed haphazardly. Majority of the respondents believed that the safety training should be a part of educational training (p = 0.001) and they would benefit from short course and/or workshop on lab safety (p<0.001).