A systematic review of CQ-resistant Plasmodium vivax malaria infections in India.

INTRODUCTION: Chloroquine (CQ) is the drug of choice for treating uncomplicated Plasmodium vivax (P. vivax) malaria in India. The knowledge about the exact burden of CQ resistance in P. vivax in India is scarce. Therefore, this systematic review aimed to assess the prevalence of CQ resistance in reported P. vivax cases from India. METHODS: PubMed, EMBASE, and Web of Science, were searched using the search string: 'Malaria AND vivax AND chloroquine AND (resistance OR resistant) AND India'. We systematically reviewed in-vivo and in-vitro drug efficacy studies that investigated the CQ efficacy of P. vivax malaria between January 1995 and December 2022. Those studies where patients were followed up for at least 28 days after initiation of treatment were included. RESULTS: We identified 12 eligible CQ therapeutic efficacy studies involving 2470 patients, Of these 2329 patients were assessed by in-vivo therapeutic efficacy methods and the remaining 141 were assessed by in-vitro methods. CQ resistance was found in 25/1787 (1.39%) patients from in-vivo and in 11/141 (7.8%) patients from in-vitro drug efficacy studies. CONCLUSION: Based on the available studies, the prevalence of CQ resistance in P. vivax was found to be relatively lower in India. However, continued surveillance and monitoring are crucial to identify the emergence of CQ resistance.

Effectiveness of Photobiomodulation Therapy on Neuropathic Pain, Nerve Conduction and Plantar Pressure Distribution in Diabetic Peripheral Neuropathy - A Systematic Review.

BACKGROUND: Diabetic peripheral neuropathy is a severe complication of type 2 diabetes mellitus. The most common symptoms are neuropathic pain and altered sensorium due to damage to small nerve fibers. Altered plantar pressure distribution is also a major risk factor in diabetic peripheral neuropathy, leading to diabetic foot ulcers. OBJECTIVE: The objective of this systematic review was to analyze the various studies involving photobiomodulation therapy on neuropathic pain and plantar pressure distribution in diabetic peripheral neuropathy. METHODS: We conducted a systematic review (PubMed, Web of Science, CINAHL, and Cochrane) to summarise the evidence on photobiomodulation therapy for Diabetic Peripheral Neuropathy with type 2 diabetes mellitus. Randomized and non-randomized studies were included in the review. RESULTS: This systematic review included eight studies in which photobiomodulation therapy showed improvement in neuropathic pain and nerve conduction velocity. It also reduces plantar pressure distribution, which is a high risk for developing foot ulcers. CONCLUSION: We conclude that photobiomodulation therapy is an effective, non-invasive, and costefficient means to improve neuropathic pain and altered plantar pressure distribution in diabetic peripheral neuropathy.

Erythrocyte miRNA-92a-3p interactions with PfEMP1 as determinants of clinical malaria.

Based on the recently added high throughput analysis data on small noncoding RNAs in modulating disease pathophysiology of malaria, we performed an integrative computational analysis for exploring the role of human-host erythrocytic microRNAs (miRNAs) and their influence on parasite survival and host homeostasis. An in silico analysis was performed on transcriptomic datasets accessed from PlasmoDB and Gene Expression Omnibus (GEO) repositories analyzed using miRanda, miRTarBase, mirDIP, and miRDB to identify the candidate miRNAs that were further subjected to network analysis using MCODE and DAVID. This was followed by immune infiltration analysis and screening for RNA degradation mechanisms. Seven erythrocytic miRNAs, miR-451a, miR-92a-3p, miR-16-5p, miR-142-3p, miR-15b-5p, miR-19b-3p, and miR-223-3p showed favourable interactions with parasite genes expressed during blood stage infection. The miR-92a-3p that targeted the virulence gene PfEMP1 showed drastic reduction during infection. Performing pathway analysis for the human-host gene targets for the miRNA identified TOB1, TOB2, CNOT4, and XRN1 genes that are associated to RNA degradation processes, with the exoribonuclease XRN1, highly enriched in the malarial samples. On evaluating the role of exoribonucleases in miRNA degradation further, the pattern of Plasmodium falciparum_XRN1 showed increased levels during infection thus suggesting a defensive role for parasite survival. This study identifies miR-92a-3p, a member of C13orf25/ miR-17-92 cluster, as a novel miRNA inhibitor of the crucial parasite genes responsible for symptomatic malaria. Evidence for a plausible link to chromosome 13q31.3 loci controlling the epigenetic disease regulation is also suggested.

Effect of photobiomodulation on serum neuron specific enolase (NSE) among patients with diabetic peripheral neuropathy - A pilot study.

BACKGROUND AND AIM: Photobiomodulation is an emerging therapy for Diabetic peripheral neuropathy (DPN) of which the management is still a dilemma for clinicians. Elevated Neuron Specific Enolase (NSE) is associated with neuropathy. We aimed this study to assess the effect of Low Level Laser Therapy (LLLT) on Serum Neuron Specific Enolase in Type II Diabetes Mellitus patients with DPN. METHODOLOGY: Pre post interventional study was done on 50 patients with DPN. DPN was confirmed using 10g Monofilament test, Vibration perception threshold (VPT) and Michigan Neuropathy Screening Instrument. All patients were provided with LLLT for 9 min on dorsal and plantar of foot with a dosage of 3.1 J/cm 2 for 10 days. A blood sample was collected at baseline and 4 weeks after LLLT for NSE estimation. RESULT: A significant reduction in serum NSE levels (0.006) after 4 weeks of laser therapy was observed in 42 patients when compared with baseline. A significant reduction in the vibration perception threshold (p = 0.003) and Numeric pain rating scale (p = 0.004) were observed. CONCLUSION: In this pilot study, we have assessed the effect of LLLT on serum NSE levels among patients with DPN and showed improved quality of life and decrease in serum NSE levels. These findings should be investigated in larger trials.

Effectiveness of customized insoles on maximum plantar pressure in diabetic foot syndrome: A systematic review.

BACKGROUND AND AIMS: Diabetic foot syndrome is a severe complication of type 2 diabetes mellitus with diabetic peripheral neuropathy. Increased maximum plantar pressure is a strong predictor that may be detrimental and cause a plantar ulcer. This present systematic review aims to evaluate the effectiveness of customized insoles on reducing maximum plantar pressure in diabetic foot syndrome. METHODS: We conducted a systematic review (PubMed, Cochrane Database of Systematic Reviews, CINAHL, Pedro, Scopus) to summarize the evidence on the customized insole on maximum plantar pressure in diabetic foot syndrome. Randomized and non-randomized studies were included in the review. The quality of the included studies was assessed independently by the two review authors with the Modified Downs and Black checklist for the assessment of the methodological quality of both randomized and non-randomized studies. RESULTS: A total of 1512 studies screened. After the exclusion criteria, 5 studies were included in the study. The outcome measure that was considered is maximum plantar pressure and pressure-time integral. CONCLUSIONS: We concluded that the practice of customized insoles could significantly reduce maximum plantar pressure. While developing an insole, parameters like contoured insoles with shape and pressure based, weight-bearing position, and duration with good adherence to footwear application can significantly reduce maximum plantar pressure.

Relationship Between Glycated Hemoglobin and Vibration Perception Threshold in Diabetic Peripheral Neuropathy.

Foot ulcers, infections, and deformity are some of the major sources of mortality and morbidity among the diabetic population. The relationship between glycated hemoglobin (HbA1c) and diabetic peripheral neuropathy (DPN) has been well established. There is a dearth of literature on the relationship between vibration perception threshold (VPT) and HbA1c values. So, the objective of the study was to determine the strength of linear relationship between HbA1c levels and vibration perception threshold in DPN. This cross-sectional study was conducted at Kasturba Hospital, Manipal, and diabetic foot screening camps held at various parts of Udupi district. Ethical approval was obtained from the Institutional Ethics Committee, Kasturba Hospital, Manipal (IEC:281/2017). A total of 534 participants ranging from 30 to 70 years of age and were diagnosed with type 2 diabetes mellitus on medications were included in the study. Neuropathy assessment consisting of monofilament and vibration perception threshold was done using Neurotouch beta version (Yostra Labs, Bengaluru, India). HbA1c measurement was done using turbidimetric inhibition immunoassay technique (Roche Diagnostics, Mannheim, Germany). Pearson correlation coefficient showed a moderate to good correlation between HbA1c and VPT (r = .0.753, P < .001). Linear regression result has shown a significant relationship of VPT with HbA1c (4.033 [95% confidence interval = 3.67-4.39]). The present study has concluded that there is strong relationship between HbA1c values and VPT and could be a predictor for complications in the foot following DPN.

Low level laser therapy for the patients with painful diabetic peripheral neuropathy - A systematic review.

BACKGROUND: Low Level Laser Therapy (LLLT) is an emerging treatment modality for management of neuropathic pain. It works by triggering biochemical changes with in cells. OBJECTIVE: This systematic review focused on finding evidence on the effectiveness of LLLT on treatment of painful diabetic neuropathy. METHODS: We conducted a systematic review (PubMed, Web of Science, CINAHL and Cochrane) to find the evidence on effectiveness of LLLT on treatment of painful diabetic neuropathy. Randomized and non-randomized studies were included in the review. RESULTS: A total of 627 studies were screened. After the exclusion criteria (duplicate, animal studies, LLLT for treating other neuralgias) 6 studies were included in the study. The outcome measure that were considered were the difference in pain score and nerve conduction velocity test and quality of life questionnaire. CONCLUSION: The evidence obtained shows LLLT has a positive effect in controlling diabetic neuropathic pain.

2,5-Dimethyl-4-hydroxy-3(2H)-furanone as an Anti-biofilm Agent Against Non-Candida albicans Candida Species.

BACKGROUND: The predominance of non-Candida albicans Candida (NCAC) species causing healthcare-associated infections has increased over the last decade pertaining to their ability to form biofilms on medical devices. These biofilm-associated infections are challenging to treat as they are resistant to antifungal agents and evade host-immune response resulting in a high risk of device failure or biomaterial removal. Thus, to minimize the risk of biofilm-associated infections, preventing biofilm formation is the best approach which is mediated by the quorum quenching process. METHODS: The present study investigated the modulatory effect of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) on NCAC biofilm formation and also assessed the effect of the DMHF-coated catheters on biofilm formation of NCAC. The NCAC isolates studied were Candida tropicalis, Candida glabrata and Candida krusei isolated from catheter tip, urine and blood, respectively. RESULTS: DMHF at a concentration of 30 µg/mL showed an inhibitory effect against NCAC biofilms at various stages and was statistically significant (p ≤ 0.05) against the various concentrations (50-5 µg/mL) tested and also among the three phases of experiment. The furanone content on coated catheters ranged from 170 to 750 µg and release of furanone from the coated catheter was about 15 µg for 30 days. The effect of DMHF-coated catheters on NCAC biofilm formation was observed by the scanning electron microscopy which revealed the absence of NCAC adherence on DMHF-coated catheters. DISCUSSION: This study provides a design to develop furanone-coated biomaterials which could be implemented in healthcare settings to reduce medical device-associated infections. The excellent biological performance, combined with their antimicrobial properties, suggests that 2,5-dimethyl-4-hydroxy-3(2H)-furanone could be an effective anti-infective coating for implantable devices.

Auxanographic Carbohydrate Assimilation Method for Large Scale Yeast Identification.

INTRODUCTION: The auxanographic carbohydrate assimilation had been an important method for differentiation of yeasts. Prevailing methods described in the literature for carbohydrate assimilation has limited scope for use in large scale yeast identification. AIM: To optimize the large scale auxanographic carbohydrate assimilation method for yeast identification. MATERIALS AND METHODS: A modified auxanographic carbohydrate assimilation method was developed and a total of 35 isolates of Candida species comprising of four ATCC (American Type Culture Collection) Candida strains (Candida albicans ATCC 90028, Candida tropicalis ATCC 90018, Candida parapsilosis ATCC 750, Candida krusei ATCC 6258) and 31 clinical isolates of Candida tropicalis (n=13), Candida krusei (n=7), Candida glabrata (n=3), Candida kefyr (n=3), Candida albicans (n=5) were validated. The carbohydrates tested were Glucose, Sucrose, Maltose, Lactose, Cellubiose, Raffinose, Trehalose, Xylose, Galactose and Dulcitol. RESULTS: A total of 35 Candida species were tested for their carbohydrate assimilative property and the results were consistent with the existing standard protocols. A well circumscribed opaque yeast growth indicated assimilation of the test carbohydrate and translucent to opalescent growth with the outline of initial inoculum alone indicated lack of assimilation. The control plate indicated no growth of the Candida species. CONCLUSION: The carbohydrate assimilation tests finds utility for yeast diversity studies exploring novel ecological niches. The technique described here facilitates testing of an extended range of carbohydrates and yeasts in a cost effective manner.

Genetic and epigenetic changes in host ABCB1 influences malaria susceptibility to Plasmodium falciparum.

Multiple mechanisms such as genetic and epigenetic variations within a key gene may play a role in malarial susceptibility and response to anti-malarial drugs in the population. ABCB1 is one of the well-studied membrane transporter genes that code for the P-glycoprotein (an efflux protein) and whose effect on malaria disease predisposition and susceptibility to drugs remains to be understood. We studied the association of single nucleotide variations in human ABCB1 that influences its function in subjects with uncomplicated and complicated malaria caused by Plasmodium falciparum (Pf). Global DNA methylation and ABCB1 DNA promoter methylation levels were performed along with transcriptional response and protein expression in subjects with malaria and healthy controls. The rs2032582 locus was significantly associated with complicated and combined malaria groups when compared to controls (p < 0.05). Significant DNA methylation difference was noticed between case and control (p < 0.05). In addition, global DNA methylation levels of the host DNA were inversely proportional to parasitemia in individuals with Pf infection. Our study also revealed the correlation between ABCB1 DNA promoter methylation with rs1128503 and rs2032582 polymorphisms in malaria and was related to increased expression of ABCB1 protein levels in complicated malaria group (p < 0.05) when compared to uncomplicated malaria and control groups. The study provides evidence for multiple mechanisms that may regulate the role of host ABCB1 function to mediate aetiology of malaria susceptibility, prognosis and drug response. These may have clinical implications and therapeutic application for various malarial conditions.

New molecular detection methods of malaria parasites with multiple genes from genomes.

For the effective control of malaria, development of sensitive, accurate and rapid tool to diagnose and manage the disease is essential. In humans subjects, the severe form of malaria is caused by Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) and there is need to identify these parasites in acute, chronic and latent (during and post-infection) stages of the disease. In this study, we report a species specific and sensitive diagnostic method for the detection of Pf and Pv in humans. First, we identified intra and intergenic multiloci short stretch of 152 (PfMLS152) and 110 (PvMLS110) nucleotides which is present up to 44 and 34 times in the genomes of Pf and Pv respectively. We developed the single-step amplification-based method using isolated DNA or from lysed red blood cells for the detection of the two malaria parasites. The limit of detection of real-time polymerase chain reaction based assays were 0.1copyof parasite/µl for PfMLS152 and PvMLS110 target sequences. Next, we have tested 250 clinically suspected cases of malaria to validate the method. Sensitivity and specificity for both targets were 100% compared to the quantitative buffy coat microscopy analysis and real-time PCR (Pf-chloroquine resistance transporter (PfCRT) and Pv-lactate dehydrogenase (PvLDH)) based assays. The sensitivity of microscopy and real-time PCR (PfCRT and PvLDH primers) assays were 80.63%; 95%CI 75.22%-85.31%; p<0.05 and 97.61%; 95%CI 94.50%-99.21%; p<0.05 in detecting malaria infection respectively when compared to PfMLS152 and PvMLS110 targets to identify malaria infection in patients. These improved assays may have potential applications in evaluating malaria in asymptomatic patients, treatment, blood donors and in vaccine studies.

Categorical complexities of Plasmodium falciparum malaria in individuals is associated with genetic variations in ADORA2A and GRK5 genes.

In the erythrocytes, malaria parasite entry and infection is mediated through complex membrane sorting and signaling processes. We investigated the effects of single-locus and multilocus interactions to test the hypothesis that the members of the GPCR family genes, adenosine A2a receptor (ADORA2A) and G-protein coupled receptor kinase5 (GRK5), may contribute to the pathogenesis of malaria caused by Plasmodium falciparum (Pf) independently or through complex interactions. In a case-control study of adults, individuals affected by Pf malaria (complicated n=168; uncomplicated n=282) and healthy controls (n=450) were tested for their association to four known SNPs in GRK5 (rs2230345, rs2275036, rs4752307 and rs11198918) and two in ADORA2A (rs9624472 and rs5751876) genes with malaria susceptibility, using techniques of polymerase chain reaction-restriction fragment length polymorphisms and direct DNA sequencing. Single-locus analysis showed significant association of 2 SNPs; rs5751876 (OR=3.2(2.0-5.2); p=0.0006) of ADORA2A and rs2230345 (OR=0.3(0.2-0.5); p=0.0006) of GRK5 with malaria. The mean of the serum creatinine levels were significantly higher in patients with variant GG (p=0.006) of rs9624472 in ADORA2A gene compared to AA and AG genotypes in complicated Pf malaria cases, with the G allele also showing increased risk for malaria (OR=1.3(1.1-1.6); p=0.017). Analyses of predicted haplotypes of the two ADORA2A and the four GRK5 SNPs have identified the haplotypes that conferred risk as well as resistance to malaria with statistical significance. Molecular docking analysis of evolutionary rs2230345 SNP indicated a stable activity of GRK5 for the mutant allele compared to the wild type. Further, generalized multifactor dimensionality reduction to test the contribution of individual effects of the six polymorphisms and higher-order interactions to risk of symptoms/clinical complications of malaria suggested a best six-locus model showing statistical significance. The study provides evidence for the role of ADORA2A and GRK5 that might influence the etiology of malaria infection.

Host genetic variations in glutathione-S-transferases, superoxide dismutases and catalase genes influence susceptibility to malaria infection in an Indian population.

Antioxidant enzymes can contribute to disease susceptibility or determine response to therapy in individuals with malaria. Genetic variations due to polymorphisms in host genes encoding antioxidant enzymes such as glutathione S-transferases-theta, mu, pi (GSTT, GSTM, GSTP), superoxide dismutases (SOD) and catalase (CAT), may therefore, influence inter-individual response to malaria pathology and propensity of infection caused by Plasmodium vivax (Pv) and Plasmodium falciparum (Pf). Therefore, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing, we investigated the association of deletions of GSTT1 and GSTM1, single nucleotide polymorphisms (SNPs) of GSTP1 (rs1695), SOD1 (rs2234694), SOD2 (rs4880, rs1141718), SOD3 (rs2536512) and CAT (rs1001179) in individuals infected with Pf (n = 100) and Pv (n = 100) against healthy controls (n = 150). Our data suggest a significant role for GSTM1 deletions in complicated Pv (p = 0.0007) malaria with ODDs ratio 3.8 [with 95 % confidence interval (CI) 1.9-7.4]. The results also indicated that polymorphisms present in GSTP1, SOD1 and CAT genes may be associated with malaria susceptibility (p < 0.05), whereas SOD3 polymorphism may play a role in malarial resistance (p < 0.05). In addition, we observed significant SNP-SNP interactions with synergistic genetic effects in SOD2, SOD3 and CAT genes for Pv and in SOD2 and SOD3 genes for Pf. In conclusion, our results provide convincing evidence for a relationship between polymorphisms in host antioxidant enzymes and susceptibility to malaria infection.

Hot and cold: coexistent Graves' disease and Hashimoto's thyroiditis in a patient with Schmidt's syndrome.

A 37-year-old housewife presented with generalised fatigue, palpitations and weight loss over the past 3 months. Physical examination revealed signs of hyperthyroidism. Thyroid function tests confirmed the presence of thyrotoxicosis. Pertechnetate radionuclide imaging of the thyroid showed diffusely increased radiotracer uptake consistent with Graves' disease and a cold nodule in the right lobe. Needle aspiration from the nodule yielded evidence of Hashimoto's thyroiditis. The patient also tested strongly positive for antithyroid peroxidase antibodies. Simultaneous laboratory evaluation revealed primary adrenal failure and probable pernicious anaemia, thus producing a diagnosis of Schmidt's syndrome. The patient was initiated on appropriate medical therapy for endocrinopathy. Graves' disease was treated with radioablation.

Simultaneous occurrence of internal capsule infarct and cerebellar haemorrhage in a patient with hemiplegia.

A 68-year-old woman with hypertension with no history of cerebrovascular events presented with a left-sided hemiplegia which had developed acutely 2 days ago. She was not on maintenance therapy with antiplatelets or anticoagulants. A CT scan showed acute ischaemic infarction of the right internal capsule and cerebellar haemorrhage. Cardiac evaluation was normal. Doppler ultrasonography of the extracranial carotid and vertebral arteries showed diffuse arteriosclerotic changes, but did not reveal any haemodynamic occlusion. The simultaneous development of dual strokes was considered to be an extension of the same arteriosclerotic process to the intracranial carotid and basilar arteries.

Interstitial lung disease probably caused by imipramine.

Drugs are rarely associated with causing interstitial lung disease (ILD). We report a case of a 75-year-old woman who developed ILD after exposure to imipramine. To our knowledge, this is one of the rare cases of ILD probably caused due to imipramine. There is need to report such rare adverse effects related to ILD and drugs for better management of ILD.

Primary presentation of chronic calcific pancreatitis with massive unilateral pleural effusion.

We described a 45-year-old previously healthy man presenting with progressively worsening breathlessness for 10 days. Physical examination was suggestive of a left-sided pleural effusion. A chest X-ray was confirmatory. Analysis of aspirated fluid showed a lymphocytic exudate with grossly elevated amylase and lipase levels. CT revealed chronic calcific pancreatitis as the underlying cause of effusion. Retrospective questioning failed to identify classical symptoms of chronic pancreatitis including abdominal pain and steatorrhoea. The patient was managed with intercostal drainage and supportive care. Although unusual, chronic pancreatitis should be kept as a differential diagnosis in patients with unilateral exudative pleural effusion. Elevated fluid levels of amylase and lipase are useful clues to this uncommon diagnosis.

A novel subtype of myeloproliferative disorder? JAK2V617F-associated hypereosinophilia with hepatic venous thrombosis.

We report the case of a 27-year-old man, presenting with one episode of massive haematemesis and a history of persistent eosinophilia for the past 8 months. An evaluation revealed hepatic cirrhosis with portal hypertension, secondary to chronic Budd-Chiari syndrome. Further investigations confirmed a diagnosis of hypereosinophilic syndrome. Molecular genetic analysis was negative for FIP1L1-PDGFRA gene rearrangement, but positive for JAK2V617F mutation.

Single nucleotide polymorphisms of ADRB2 gene and their association with susceptibility for Plasmodium falciparum malaria and asthma in an Indian population.

The essential route to blood parasitaemia in malaria, erythrocyte invasion is facilitated by activation of the G-protein coupled receptor signaling pathway mediated by the ß2-adrenoreceptor as one of the proteins on the surface of red blood cells. The effectiveness of bronchodilators and inhaled corticosteroids in the clinical treatment for asthma patients also depend on polymorphisms in the ß2-adrenoreceptor gene (ADRB2). In a case control study, individuals affected by Plasmodium falciparum malaria, asthma and controls were tested for association of six ADRB2 single nucleotide polymorphisms (SNPs) viz. rs1042711, rs1801704, rs1042713, rs1042714, rs1042717 and rs1042718, by direct DNA sequencing. The rs1801704 locus was significantly associated with malaria when compared against controls. The rs1042713 polymorphism was associated with forced expiratory flow between 25% and 75% of the FVC in asthma patients, pre (p=0.048) and post (p=0.038) treatment measurements. Predicted haplotype of the six SNPs computed from genotype data showed T-T-A-C-G-C conferred significant risk of malaria (p=0.02) whereas T-T-A-C-G-A was associated with risk of asthma (p=0.02). The haplotype T-T-G-C-G-C was protective against both malaria (p=0.02) as well as asthma (p=0.026) and C-C-G-G-G-C was protective uniquely for asthma (p=0.04). A significant outcome was that all variant alleles at the SNP loci were part of the haplotype conferring resistance to malaria disease and asthma, except rs1042713 and rs1042718 which showed very high frequency in asthma. The pairwise linkage disequilibrium (LD) estimates showed a distinct LD block of all SNP loci (D'=1 or >0.8) in malaria patients. This characteristic haplotype block was disrupted in the controls due to non-significant pairwise LD of the SNP loci; and a more extensive disruption of the block was noted in asthma patients. The study provides evidence for the proposed role of ß2-adrenoreceptor mediated molecular mechanisms in etiology of malaria, as well as asthma disease and drug response, for further clinical and therapeutic application studies.