Multiplex detection of bacterial pathogens by PCR/SERS assay.

Bacterial infections are a leading cause of death globally. The detection of DNA sequences correlated to the causative pathogen has become a vital tool in medical diagnostics. In practice, PCR-based assays for the simultaneous detection of multiple pathogens currently rely on probe-based quantitative strategies that require expensive equipment but have limited sensitivity or multiplexing capabilities. Hence, novel approaches to address the limitations of the current gold standard methods are still in high demand. In this study, we propose a simple multiplex PCR/SERS assay for the simultaneous detection of four bacterial pathogens, namely P. aeruginosa, S. aureus, S. epidermidis, and M. smegmatis. Wherein, specific primers for amplifying each target gDNA were applied, followed by applying SERS nanotags functionalized with complementary DNA probes and Raman reporters for specific identification of the target bacterial pathogens. The PCR/SERS assay showed high specificity and sensitivity for genotyping bacterial pathogen gDNA, whereby as few as 100 copies of the target gDNA could be detected. With high sensitivity and the convenience of standard PCR amplification, the proposed assay shows great potential for the sensitive detection of multiple pathogen infections to aid clinical decision-making.

Evaluating the efficacy of nasal irrigation in postoperative functional endoscopic sinus surgery patients: a systematic review and meta-analysis.

PURPOSE: Functional endoscopic sinus surgery (FESS) is a mainstay surgical intervention for chronic rhinosinusitis with nasal polyposis (CRSwNP). Nasal irrigation, particularly with normal saline, is a widely recommended postoperative care modality. This systematic review and meta-analysis aimed to assess the efficacy of various nasal irrigation solutions in postoperative FESS patients. METHODS: A comprehensive search was conducted in multiple databases for randomized controlled trials investigating normal saline and various substances for nasal irrigation post-FESS. The systematic review followed PRISMA guidelines, and the meta-analysis used R software for data synthesis. Outcome measures included SNOT-22 and LKES scores. The Cochrane tool was employed to evaluate the potential for bias. RESULTS: Results from 14 studies, focusing on six each for SNOT-22 and LKES, revealed a significant reduction in symptoms and endoscopic scores with various solutions compared to normal saline. The meta-analysis using the random-effects model indicated a negative standardized mean difference (SMD) of - 0.69(95% CI [- 1.64; 0.27], p = 0.157) for symptoms and endoscopic scores (SMD = - 0.48, 95% CI [- 1.32; 0.36], z = - 1.12, p = 0.264). Subgroup analyses highlighted budesonide's efficacy over normal saline, but substantial heterogeneity and potential publication bias were noted. CONCLUSION: Nasal irrigation with various solutions postoperative FESS patients demonstrated significant improvements in patient-reported symptoms and endoscopic scores compared to normal saline. Budesonide appeared particularly effective. However, high heterogeneity and potential publication bias warrant cautious interpretation. Standardized outcome measures and further research are needed to strengthen the evidence.

COVID-19 Vaccination in a Patient With Gluten Enteropathy: A Case Report.

In India, the COVID-19 vaccination for adolescents aged 15-17 years has been started since January 2022. Gluten enteropathy, also known as celiac or nontropical sprue, can arise as an autoimmune disease of the small intestines. We report a 15-year-old female with a history of allergy to gluten-containing products who came for the first dose of the COVID-19 vaccination to adult vaccination OPD at All India Institute of Medical Sciences, Jodhpur. After taking a detailed history, she had an allergy to gluten-containing products for five years. She had no previous history of allergic reactions to injections or medicines. The first dose of Covaxin was given to this female under proper supervision, and she was followed up for any adverse events. We did not find any evidence of adverse events following the COVID-19 vaccination in people with gluten enteropathy. The patient was discharged after one hour of observation. To date, no cases of Covaxin vaccination have been reported among gluten enteropathy patients. We discuss the current evidence relating to Covaxin vaccinations, highlighting that administering the vaccine to gluten-sensitive individuals did not cause any adverse reactions. However, proper history taking and other standard procedures should be followed while administering Covaxin to any known allergies.

Evaluating the Diagnostic Potential of Serum Vascular Endothelial Growth Factor and Adiponectin in Diabetic Peripheral Neuropathy.

INTRODUCTION: Diabetic peripheral neuropathy (DPN) presents a formidable health challenge in type 2 diabetes mellitus (T2DM) patients. This study in eastern Uttar Pradesh aims to assess the roles of vascular endothelial growth factor (VEGF) and adiponectin in DPN, recognizing the crucial need for understanding its molecular underpinnings for enhanced diagnosis and management. METHODS: In a cross-sectional study analyzing clinical and biochemical data, 86 individuals aged 35 to 65 years were examined, including 43 with neuropathy and 43 without. Neuropathy assessment included the neuropathy symptom score (NSS), diabetes neuropathy examination (DNE) score, and nerve conduction studies. Levels of VEGF and adiponectin were correlated with motor nerve amplitude, NSS, and DNE scores. Receiver operating characteristic (ROC) curve analysis gauged diagnostic potential, and logistic regression assessed predictors for DPN. RESULTS: Patients with neuropathy exhibited significantly elevated VEGF levels compared to those without, while adiponectin showed no significant difference. VEGF demonstrated a negative correlation with motor nerve amplitude and a positive correlation with NSS and DNE scores. ROC analysis revealed strong diagnostic capability for VEGF (area under the curve: 0.807). NSS and DNE scores indicated good and moderate diagnostic accuracy, respectively. In logistic regression analysis, VEGF emerged as the sole significant predictor (odds ratio: 1.11, 95% CI (1.03, 1.20), p = 0.0092). CONCLUSION: Findings suggest VEGF's potential as a biomarker for diagnosing DPN in T2DM, associated with neuropathy severity. Adiponectin showed no significant association. The study underscores NSS and DNE scores' therapeutic relevance as valid neuropathy assessment tools.

Identifying Fast and Slow-Acting Antimalarial Compounds of Pandemic Response Box Against Blood-Stage Culture of Plasmodium falciparum 3D7.

The evolving clinical resistance in Plasmodium falciparum and the spike in malarial cases after the COVID-19 outbreak has triggered a search for new antimalarials effective against multi-drug-resistant P. falciparum strains. In this study, we assessed the timing of action, either fast or slow-acting of 13 potent compounds of Pandemic Response Box (PRB) against blood-stage Pf3D7 strain by SYBR Green-I assay. The asynchronous culture of Pf3D7 was exposed to varying concentrations of 13 compounds, and IC50 values were determined at 12, 24, 48, 72, and 96 h. We identified four fast-acting compounds (MMV000008, MMV1593541, MMV020752, MMV396785) with rapid-growth inhibitory activity having IC50 values ≤ 0.3 µM at 12 and 24 h. Similarly, we determined nine slow-acting compounds (MMV159340, MMV1634492, MMV1581558, MMV689758, MMV1593540, MMV394033, MMV019724, MMV000725, MMV1557856) having IC50 values ≤ 0.5 µM at 72 and 96 h. Furthermore, the stage-specific action of the two most potent fast-acting compounds (MMV1593541 and MMV020752) against rings, trophozoites, and schizonts at 48 h of exposure revealed that ring-stage parasites showed reduced IC50 values compared to mature stage forms. Therefore, our study demonstrates for the first time the identification of the most potent fast and slow-acting compounds from PRB against blood-stage infection, suggesting its utility in clinics and considering it as a partner drug in combination therapies.

Evaluation of a telemedicine-enabled universal eye health delivery model in rural southern India.

OBJECTIVES: To evaluate the accuracy of tele-ophthalmic examination (TOE) for common ocular conditions in comparison with the gold-standard in-person examination (IPE) for diagnosis and treatment advice. METHODS: In a prospective, diagnostic accuracy validation study, we recruited 339 consecutive new patients, aged ≥16 years, visiting a vision centre (VC) associated with Aravind Eye Hospital in south India during January and February, 2020. All participants underwent the TOE, followed by IPE on the same visit. The in-person ophthalmologist was masked to the TOE diagnosis and treatment advice. Data were analysed via the sensitivity specificity of TOE versus the gold-standard IPE. RESULTS: TOE achieved high sensitivity and specificity for identifying normal eyes with 87.4% and 93.5%, respectively. TOE had high sensitivity for cataracts (91.7%), infective conjunctivitis (72.2%), and moderate sensitivity for pterygium (62.5%), DR (57.1%), non-serious injury (41.7%), but low sensitivity for glaucoma (12.5%). TOE had high specificity ranging from 93.5% to 99.8% for all diagnoses. The sensitivity for treatment advice ranged from 58.1% to 77.2% and specificity from 96.9% to 100%. CONCLUSIONS: The TOE in VCs has acceptable accuracy to an IPE by an ophthalmologist for correctly identifying and treating major eye ailments. Through providing universal eye care to rural populations, this model may contribute to work toward achieving Universal Health Coverage, which is a linchpin of the health-related U.N. Sustainable Development Goals (SDG).

Prevalence of Internet Addiction and Impact of Internet Socialization on Professional, Academic, Social Lives and Sleep Pattern Among Students and Professionals from Various Fields Across India.

Background: Internet addiction was found to be associated with a variety of psychosocial problems, including academic failure, low self-esteem, poor diet, mental disorders like depression and anxiety, sleep deprivation, and social withdrawal. Objective: To determine the prevalence and risk factors of internet addiction and evaluate the impact of internet socialization on professional, academic, social lives and sleep pattern among students and professionals from various fields across India. Methodology: This cross-sectional, descriptive study was conducted from January 2022 to July 2023 among 772 participants who were aged ≥18 years. The study tool consisted of a questionnaire with four parts comprising questions related to demographic characteristics, pattern of use and impact of internet over academic, professional, social life, sleep pattern, and Kimberley Young's Internet Addiction Test (IAT) questionnaire. Results: The prevalence of Internet addiction was 53.6% among the participants. Participants aged above 30 years demonstrated a decreased risk of internet addiction compared to those aged below 30 years (AOR = 0.35). Participants who reported becoming restless without internet access (AOR = 4.88) and going to bed late because of the use of internet or social media (AOR = 3.01) had a significantly higher risk of internet addiction. Conclusion: Internet addiction is very common among professionals, and students and there is a high risk of internet addiction in persons who get restless when they cannot log in, spend too much time online, stay up late using the internet, and share personal information soon after meeting others on social media sites.

Methods for a population-based Comprehensive Eye care Workload Assessment (CEWA) study in Southern India.

Eye care programs, in developing countries, are often planned using the prevalence of blindness and visual impairment, often estimated from Rapid Assessment of Avoidable Blindness (RAAB) surveys. A limitation of this planning approach is that it ignores the annual overall eye care requirements for a given population. Moreover, targets set are arbitrary, often influenced by capacity rather than need. To address this lacunae, we implemented a novel study design to estimate the annual need for comprehensive eye care in a 1.2 million populations. We conducted a population-based longitudinal study in Theni district, Tamil Nadu, India. All permanent residents of all ages were included. We conducted the study in three phases, (i) household-level enumeration and enrollment, (ii) basic eye examination (BEE) at household one-year post-enrollment, and (iii) assessment of eye care utilization and full eye examination (FEE) at central locations. All people aged 40 years and above were invited to the FEE. Those aged <40 years were invited to the FEE if indicated. In the main study, we enrolled 24,327 subjects (58% aged below 40 years and 42% aged 40 years and above). Of those less than 40 years, 72% completed the BEE, of whom 20% were referred for FEE at central location. Of the people aged ≥40 years, 70% underwent FEE. Our study design provides insights for appropriate long-term public health intervention planning, resource allocation, effective service delivery, and designing of eye care services for resource-limited settings.

Repurposing Polyether Ionophores as a New-Class of Anti-SARS-Cov-2 Agents as Adjunct Therapy.

The emergence of SARS-CoV-2 and its variants have posed a significant threat to humankind in tackling the viral spread. Furthermore, currently repurposed drugs and frontline antiviral agents have failed to cure severe ongoing infections effectively. This insufficiency has fuelled research for potent and safe therapeutic agents to treat COVID-19. Nonetheless, various vaccine candidates have displayed a differential efficacy and need for repetitive dosing. The FDA-approved polyether ionophore veterinary antibiotic for treating coccidiosis has been repurposed for treating SARS-CoV-2 infection (as shown by both in vitro and in vivo studies) and other deadly human viruses. Based on selectivity index values, ionophores display therapeutic effects at sub-nanomolar concentrations and exhibit selective killing ability. They act on different viral targets (structural and non-structural proteins), host-cell components leading to SARS-CoV-2 inhibition, and their activity is further enhanced by Zn2+ supplementation. This review summarizes the anti-SARS-CoV-2 potential and molecular viral targets of selective ionophores like monensin, salinomycin, maduramicin, CP-80,219, nanchangmycin, narasin, X-206 and valinomycin. Ionophore combinations with Zn2+ are a new therapeutic strategy that warrants further investigation for possible human benefits.

Prevalence of Depression Among Undergraduate Medical Students in India: A Systemic Review and Meta-Analysis.

BACKGROUND: Systematic reviews have proved that there is a high rate of depression among medical students when compared with their age-matched peers. Very few studies have evaluated the pooled prevalence of depression among medical students in India. OBJECTIVES: To determine the pooled prevalence of depression among medical students in India. MATERIALS AND METHODS: This review was done by searching databases like PubMed, Google Scholar, and Scopus for available original articles published between 2019 and 2022 on depression among Indian medical (MBBS) undergraduate students using PRISMA guidelines. RESULTS: A total of 19 original research articles were included in this review, involving students at different medical colleges from various regions of India. The pooled prevalence of depression among 5944 medical students was 50.0% (95% CI: (31%-70%)) based on the random effect model. This meta-analysis also found that the pooled prevalence of depression among females (pooled prevalence: 38.0%, 95% CI: 20.0 to 58.0) was slightly higher than among males (pooled prevalence: 34.0%, 95% CI: 15.0 to 55.0). CONCLUSION:  The high prevalence of depression among medical students demands regular screening for depression along with counselling services. It shows that there is a need to raise awareness among students and other stakeholders, such as parents and medical educators, concerning symptoms and signs of depression among medical students.

Unraveling the Role of Tumor Necrosis Factor-Alpha in Diabetic Peripheral Neuropathy: A Systematic Review and Meta-Analysis.

Diabetic peripheral neuropathy (DPN) is a prevalent and debilitating complication of diabetes mellitus, leading to sensory abnormalities, decreased balance, and increased risk of foot problems. Although tumor necrosis factor-alpha (TNF-α) has emerged as a potential factor in the pathogenesis of DPN, its role remains contested. This study intends to thoroughly analyze the association between TNF-α and DPN by combining data from various global studies. This systematic review and meta-analysis adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and included 23 articles investigating TNF-α levels in DPN patients for systematic review and 11 articles for meta-analysis. Data were extracted, and heterogeneity was examined. A random-effect model was chosen due to high heterogeneity. The major outcome measure across studies was serum TNF-α levels. The meta-analysis found a significant mean difference of 15.2464 (95% confidence interval = 4.4963; 25.9965) under the random-effect model due to the substantial heterogeneity (I2 = 98.1%) among included studies. The meta-analysis indicates a consistent elevation in TNF-α levels in individuals with DPN compared to those without neuropathy. This underlines the potential of TNF-α as a biomarker and contributor to diabetic neuropathy. Despite heterogeneity, the study's extensive scope and systematic approach enhance the trustworthiness and generalizability of the findings.

Mental Health Morbidities and their Correlates among the Adolescents in Kamrup (Metro), Assam: A School-Based Study.

Background: India has the largest adolescent population worldwide, and among this group, mental health issues account for 16% of the total global burden of disease and injury. Disturbingly, approximately half of these health conditions manifest before the age of 14 years. To assess the prevalence of depression, anxiety, and stress along with its correlates among the adolescents in the schools of Kamrup (Metro) district, Assam. Methods and Material: This cross-sectional study was conducted in 10 randomly selected high schools in the study area among the adolescents selected from 8th to 10th standards in the age group of 14 to 17 years. A pre-designed, pre-tested interview schedule, the modified Kuppuswamy scale, and 'Depression Anxiety Stress Scale-21 Items' (DASS 21) questionnaire were used for data collection. Results: The mean ± 2SD age of the adolescents was 14.74 ± 1.58 years. Among the adolescents, the overall prevalence of depression, anxiety, and stress was found to be 22.2%, 24.4%, and 6.9%, respectively. Conclusions: The occurrence of mental health morbidities among adolescents is notably widespread, yet researchers have largely overlooked this aspect. This underscores the need for comprehensive investigations into the factors that contribute to these mental health issues.

The Multistage Antimalarial Compound Calxinin Perturbates P. falciparum Ca2+ Homeostasis by Targeting a Unique Ion Channel.

Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, 'Calxinin'. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials.

Clinico-Epidemiological Profiles of COVID-19 Elderly Patients in Guwahati City, Assam, India: A Cross-Sectional Study.

Background As of November 14, 2021, coronavirus disease 2019 (COVID-19 has affected more than 3,44,00,000 individuals in India and resulted in more than 4,60,000 deaths in India.Symptoms of COVID-19 include cough, fever, dyspnea, diarrhea, fatigue, expectoration, myalgia, hemoptysis, abdominal pain, and anorexia.Associated comorbidities such as hypertension, diabetes, cardiovascular illness, and respiratory sicknesses influence the severity and prognosis of the COVID-19. Therefore, this study was conducted to determine the factors associated with the severity and outcome of elderly Indian people diagnosed with COVID-19. Methodology This hospital-based descriptive cross-sectional study was conducted among elderly patients with confirmed COVID-19 who were admitted to Gauhati Medical College Hospital from July 21, 2020, to January 15, 2021. The demographic data, exposure history, clinical symptoms and signs, underlying comorbidity, severity of COVID-19, and outcome data of each elderly patient were obtained and analyzed using SPSS software (Version 25.0, IBM Corp., Armonk, NY). The Fisher exact test, chi-square test, and binary logistic regression analysis were used for different study variables. Result A total of 165 hospitalized COVID-19 elderly patients were included in this study, and their mean age was 68.4 years. The most common symptoms were cough (34.5%), fever (28%), breathing difficulty (22%), weakness (13.1%), and chest pain (3.6%). Those with breathing difficulty (adjusted OR [aOR]: 7.293, 95% CI: 2.229-23.860, p=0.001), loose stool (aOR: 12.142, 95% CI: 1.052-140.209, p=0.045), hypertension (aOR: 2.703, 95% CI: 1.023-7.139, p=0.045), and severity of COVID-19 (aOR: 7.691, 95% CI: 2.870-20.607, P<0.001) had increased risk of poor outcome among the COVID-19 elderly. Being hypertensive (aOR: 2.474, 95% CI: 1.060-5.774, p=0.036) had an increased risk of severity of COVID-19. Conclusion The most common symptoms of COVID-19 elderly patients were fever, cough, and breathing difficulty. In elderly COVID-19 patients, hypertension played a crucial role in determining the severity of COVID-19, whereas breathing difficulty, loose stool, hypertension, and moderate-to-severe COVID-19 elderly patients had a poor outcome.

A comprehensive review on classifying fast-acting and slow-acting antimalarial agents based on time of action and target organelle of Plasmodium sp.

The clinical resistance towards malarial parasites has rendered many antimalarials ineffective, likely due to a lack of understanding of time of action and stage specificity of all life stages. Therefore, to tackle this problem a more incisive comprehensive analysis of the fast- and slow-acting profile of antimalarial agents relating to parasite time-kill kinetics and the target organelle on the progression of blood-stage parasites was carried out. It is evident from numerous findings that drugs targeting food vacuole, nuclear components, and endoplasmic reticulum mainly exhibit a fast-killing phenotype within 24 h affecting first-cycle activity. Whereas drugs targeting mitochondria, apicoplast, microtubules, parasite invasion, and egress exhibit a largely slow-killing phenotype within 96-120 h, affecting second-cycle activity with few exemptions as moderately fast-killing. It is essential to understand the susceptibility of drugs on rings, trophozoites, schizonts, merozoites, and the appearance of organelle at each stage of the 48-h intraerythrocytic parasite cycle. Therefore, these parameters may facilitate the paradigm for understanding the timing of antimalarials action in deciphering its precise mechanism linked with time. Thus, classifying drugs based on the time of killing may promote designing new combination regimens against varied strains of Plasmodium falciparum and evaluating potential clinical resistance.

Prevalence of Polycystic Ovarian Syndrome in India: A Systematic Review and Meta-Analysis.

Stein-Leventhal syndrome, often known as polycystic ovarian syndrome (PCOS), is a syndrome that affects women's reproductive health. PCOS is one of the most common endocrine and metabolic disorders in women of reproductive age. The etiology of PCOS remains unknown mainly, and the estimation of PCOS burden in a specific geographical location will impact disease control strategies. Hence, this study estimated the pooled prevalence of PCOS in Indian women. Databases such as PubMed, CINHAL, Scopus, and Google Scholar were thoroughly searched. Only those published Indian studies that reported the prevalence of PCOS from 2010 to 2021 and had at least one of the following diagnostic PCOS criteria were included in the systematic review: the National Institutes of Health (NIH), Rotterdam's criteria, or/and Androgen Excess Society (AES). MetaXL version 5.3 software was used for data analysis. The risk of bias was assessed using modified Joanna Briggs Institute criteria for cross-sectional studies. Out of 17132 articles, 11 articles were selected for systematic review and meta-analysis. The pooled prevalence of PCOS was 11.33(7.69-15.59) using the random effect. The proportion of Hirsute using the Ferriman-Gallwey score was highly variable, ranging from 1.6% to 37.9% (n=6). The prevalence rate of PCOS is high among Indian women. The pooled prevalence of PCOS was close to 10% using Rotterdam's criteria and AES criteria, while it was 5.8% using NIH criteria. The study's overall finding emphasizes the need for more acceptable and uniform diagnostic criteria for screening PCOS. At the same time, policy-makers should consider giving more importance to PCOS in their effort to control non-communicable diseases.

Highly specific detection of KRAS single nucleotide polymorphism by asymmetric PCR/SERS assay.

The molecular diagnosis of KRAS mutations has become crucial for clinical decision-making in colorectal cancer (CRC) treatments. Currently, the common methods for detecting mutations are based on quantitative PCR, DNA sequencing and droplet digital PCR (ddPCR), which require expensive specialized equipment and testing reagents. Herein, we propose a simple and specific strategy by integrating asymmetric PCR with surface-enhanced Raman spectroscopy (Asy-PCR/SERS) for the detection of KRAS G12V mutation, one of the most common driver mutations in CRC. To discriminate mutant targets from non-targets, Asy-PCR was applied to obtain single-stranded DNA (ssDNA) with unequal amounts of forward and reverse primers, subsequently, detection of the target mutant ssDNA amplicons was attempted by hybridization with Raman reporter-coded and allele-specific oligonucleotide-functionalized gold nanoparticles (SERS nanotags). The oligo encoding of the KRAS G12V mutant sequence could be identified by using a portable Raman spectrometer where the characteristic spectra of SERS nanotags indicate the presence of mutant targets. The Asy-PCR/SERS method showed high specificity and sensitivity for identifying as few as 0.1% mutant alleles of KRAS G12V mutation from non-target sequences. Using colorectal polyp biopsies, we demonstrated that Asy-PCR/SERS assay could distinguish KRAS G12V (c.35G > T) and KRAS G12D (c.35G > A) which occur at the same nucleotide location. As KRAS G12V is a driver oncogene in other cancers including lung, pancreatic, ovarian and endometrial cancers, the proposed assay shows great potential for application in additional tumor streams.

Mammalian host microRNA response to plasmodial infection: role as therapeutic target and potential biomarker.

The appearance of increasing drug resistance in apicomplexan intracellular Plasmodium falciparum presents a significant challenge. P. falciparum infection results in cerebral malaria (CM), causing irreversible damage to the brain leading to high mortality cases. To enhance the clinical outcome of the disease, further research is required to identify new molecular targets involved in disease manifestations. Presently, the role of non-coding microRNAs (miRNAs) derived from different cells implicated in CM pathogenesis is still barely understood. Despite the absence of miRNA machinery in Plasmodium, host-parasite interactions can lead to disease severity or impart resistance to malaria. Cytoadherence and sequestration of parasitized RBCs dysregulate the miRNA profile of brain endothelial cells, leukocytes, monocytes, and platelets, disrupting blood-brain barrier integrity and activating inflammatory signaling pathways. The abundance of miRNA in blood plasma samples of CM patients directly correlates to cerebral symptoms compared to non-CM patients and healthy individuals. Moreover, the differential host-miRNA signatures distinguish P. falciparum from P. vivax infection. Here, we review the diverse functions of host-miRNA, either protective, pathogenic, or a combination of the two, which may act as prognostic markers and novel antimalarial drug targets.

Smartphone technology facilitates point-of-care nucleic acid diagnosis: a beginner's guide.

The reliable detection of nucleic acids at low concentrations in clinical samples like blood, urine and saliva, and in food can be achieved by nucleic acid amplification methods. Several portable and hand-held devices have been developed to translate these laboratory-based methods to point-of-care (POC) settings. POC diagnostic devices could potentially play an important role in environmental monitoring, health, and food safety. Use of a smartphone for nucleic acid testing has shown promising progress in endpoint as well as real-time analysis of various disease conditions. The emergence of smartphone-based POC devices together with paper-based sensors, microfluidic chips and digital droplet assays are used currently in many situations to provide quantitative detection of nucleic acid targets. State-of-the-art portable devices are commercially available and rapidly emerging smartphone-based POC devices that allow the performance of laboratory-quality colorimetric, fluorescent and electrochemical detection are described in this review. We present a comprehensive review of smartphone-based POC sensing applications, specifically on microbial diagnostics, assess their performance and propose recommendations for the future.

Multiplex detection of ctDNA mutations in plasma of colorectal cancer patients by PCR/SERS assay.

Molecular diagnostic testing of KRAS and BRAF mutations has become critical in the management of colorectal cancer (CRC) patients. Some progress has been made in liquid biopsy detection of mutations in circulating tumor DNA (ctDNA), which is a fraction of circulating cell-free DNA (cfDNA), but slow analysis for DNA sequencing methods has limited rapid diagnostics. Other methods such as quantitative PCR and more recently, droplet digital PCR (ddPCR), have limitations in multiplexed capacity and the need for expensive specialized equipment. Hence, a robust, rapid and facile strategy is needed for detecting multiple ctDNA mutations to improve the management of CRC patients. To address this significant problem, herein, we propose a new application of multiplex PCR/SERS (surface-enhanced Raman scattering) assay for the detection of ctDNA in CRC, in a fast and non-invasive manner to diagnose and stratify patients for effective treatment. Methods: To discriminate ctDNA mutations from wild-type cfDNA, allele-specific primers were designed for the amplification of three clinically important DNA point mutations in CRC including KRAS G12V, KRAS G13D and BRAF V600E. Surface-enhanced Raman scattering (SERS) nanotags were labelled with a short and specific sequence of oligonucleotide, which can hybridize with the corresponding PCR amplicons. The PCR/SERS assay was implemented by firstly amplifying the multiple mutations, followed by binding with multicolor SERS nanotags specific to each mutation, and subsequent enrichment with magnetic beads. The mutation status was evaluated using a portable Raman spectrometer where the fingerprint spectral peaks of the corresponding SERS nanotags indicate the presence of the mutant targets. The method was then applied to detect ctDNA from CRC patients under a blinded test, the results were further validated by ddPCR. Results: The PCR/SERS strategy showed high specificity and sensitivity for genotyping CRC cell lines and plasma ctDNA, where as few as 0.1% mutant alleles could be detected from a background of abundant wild-type cfDNA. The blinded test using 9 samples from advanced CRC patients by PCR/SERS assay was validated with ddPCR and showed good consistency with pathology testing results. Conclusions: With ddPCR-like sensitivity yet at the convenience of standard PCR, the proposed assay shows great potential in sensitive detection of multiple ctDNA mutations for clinical decision-making.