Alzheimer’s disease is among the challenging diseases to social and healthcare systems because no treatment has been achieved yet. Although the ambiguous pathological mechanism underlying this disorder, ion channel dysfunction is one of the recently accepted possible mechanism. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play important roles in cellular excitability and synaptic transmission. Ivabradine (Iva), an HCN blocker, is acting on HCN channels, and is clinically used for angina and arrhythmia. The current study aimed to investigate the therapeutic effects of Iva against scopolamine (Sco) induced dementia. To test our hypothesis, Sco and Iva injected rats were tested for behavioural changes, followed by ELISA and histopathological analysis of the hippocampus. Induced dementia was confirmed by behavioural tests, inflammatory cytokines and oxidative stress tests and histopathological signs of neurodegeneration, multifocal deposition of congo red stained amyloid beta plaques and the decreased optical density of HCN1 immunoreactivity. Iva ameliorated the scopolamine-induced dysfunction, the hippocampus restored its normal healthy neurons, the amyloid plaques disappeared and the optical density of HCN1 immunoreactivity increased in hippocampal cells. The results suggested that blockage of HCN1 channels might underly the Iva therapeutic effect. Therefore, Iva might have beneficial effects on neurological disorders linked to HCN channelopathies. Rather than acute inflammation, the brain in Alzheimer's disease is characterised by gradual memory loss, chronic inflammation with an oxidative stress accompanied by higher levels of inflammatory cytokines3.Dementia is the most common neurodegenerative disease from which the Alzheimer’s disease (AD) is the most common among the elderly. AD is characterized by neuropsychiatric symptoms such as progressive memory impairment, personality changes, cognitive dysfunction, and language disorders, which seriously disturb the patient’s quality of life4. Since Alzheimer's disease was first identified at the beginning of the twentieth century, diagnosing it has been extremely difficult5. The molecular and clinical events, including amyloid accumulation, neuroinflammation, tau accumulation, neural degeneration, cognitive impairment, and behavioural psychological symptoms, develop along with AD progression6.The affected population is increasing rapidly for unknown reasons. Although the huge efforts to advent a medication, no effective cure for AD has been discovered yet7. The HCN channels are encoded by four genes (HCN1–4). These four subtypes of HCN channels, can be assembled into different combinations and conformations8. In the voltage range of activation, HCN channels carry an inward current, termed If in the heart and Ih in neurons. Ih currents, independent of synaptic plasticity changes mediated by N-methyl-d-aspartate (NMDA) receptor-dependent excitability changes, appear to modulate neuronal excitability9. HCN channels are widely distributed in the hippocampus, the associative cortices, and subcortical structures and may participate in the etiology or treatment of AD by affecting neuronal excitability and regulating Aβ generation12.The HCN channels are members of the voltage-gated pore loop channel family. They play an important role in the generation of neuronal and cardiac automaticity12. Recently, Iva has been shown to have central effects such as anticonvulsant, antioxidant, and neuroprotective properties13 and is effective at reducing seizure susceptibility14. Iva had an inhibitory effect, not only, on neuropathic pain, but also on inflammatory responses15. So, Iva’s effect may be important in the initial stage of inflammation. Furthermore, it was reported that Iva has anticonvulsant and neuroprotective effects against PTZ- and PICRO-induced seizures. It reveals a significant antioxidant effect in the prefrontal cortex (PFC), hippocampus and striatum (ST) by determination of MDA, GSH and nitrite levels. Also, it markedly downregulated the apoptotic marker (cleaved caspase-3) in the hippocampus13.Ivabradine (Iva) is a broad-spectrum HCN cation blocker, which blocks the HCN-mediated current. It is already in use as a heart-rate-reducing agent in the clinic16, that has been validated to have neurosupportive effects against many neurodegenerative diseases17 and improves the cholinergic and cognitive functions of patients with AD. It was the first-line anti-AD treatment medication and it has been shown to be effective and safe18. In view of the relationship between HCN channels and neuronal excitability in the modulation of cognitive function, the present study hypothesized that Iva could possess a therapeutic effect against Sco models of dementia, based on HCN blockage mechanisms in cerebral areas related to cognitive functions. We used Donepezil as a positive control, to compare the effects of administration of high and low dose of Iva, because it improves cognition and/or behavior, and is FDA approved for use for AD patients, although, does not alter the progression of the AD19.Donepezil, a selective and reversible AChE inhibitorRats of different groups didn't show a significant difference in initial latency (IL) during the acquisition phase Fig. A. On theRats of different experimental groups showed a gradual decrease in the mean escape latency (to the hidden platform) throughout the 6 successive days of the acquisition trials, reflecting acquisition (learning) Table . During As shown in Fig. E, there Compared to normal control, ES group demonstrated significantly higher hippocampal levels of inflammatory cytokines such as TNF-α, IL-2 and IL-6  and Group III (ES group 4 mg/kg/day for 28 days): there was a decrease in the weight of the brain in group II and group III, marked decrease in group III, when compared with that of the control  for further analysis, after cessation of Sco administration. Group IV, which received low dose of Iva 5 mg/kg/day for 14 days; group V, which received high dose of Iva 10 mg/kg/day for 14 days; and group VI, which received Donepezil, one of the already validated medication for Alzheimer disease (0.5 mg/kg/day for 14 days).In the three groups, the brain weight was restored in group IV, V and VI to the healthier average, when compared to group III Table . This waThe pyramidal layer of CA1, CA2, CA3 and hilum in the three groups (IV-VI) showed more normal healthy cells and less deformed cells than group III Fig. A–L. In GMoreover, the severely affected rat exhibited DG with no hilum and the granular cells had been drastically reduced and became condensed, irregular and surrounded by large space. CA3 disappeared and few vacuoles were seen instead. CA2 pyramidal cells had been deformed with irregular shapes. Moreover, large space and many nuclei of glia cells had been seen in between CA2 cells. CA1 pyramidal cells were dramatically decreased and arranged in one row and became intensively stained.In the three groups, there was a significant increase in the thickness of the granular layer of the DG Table A,C,E,G,I21. After administration of Sco in group II, merely one plaque (diameter 30 µm) was observed compared to the control group which has no plaque. Whereas ES in group III, resulted in multifocal deposition of amyloid beta plaques (8 plaques) with variable sizes  were observed  and reduced in size (from 20 to 40 µm) when compared to those in G-III. Interestingly, in case of administration of higher dose of Iva in group V for the same period, the deposition of amyloid beta plaques disappeared in all stained slides. The donepezil administrated group showed comparable results to those of the higher dose of Iva, where the deposition of amyloid beta plaques disappeared.All hippocampal sections were processed simultaneously, and images were captured using fixed microscope parameters. Briefly, at least 10 different HCN1-immunoreactive cells in different subfields of the hippocampus from 3 different sections of the three replicates were measured. The mean and maximum grey intensity were measured using Image J software and the optical density of HCN1 immunoreactivity was calculated according to the following equation: OD = log (max gray intensity/mean gray intensity). We found that the OD of HCN1 immunoreactivity in group II, after 3 weeks of Sco injection, was comparable to that of the control group in different subfields of the HC Table A–D. HoweWe used scopolamine to induce dementia model (4 mg/kg for 28 days), then we investigated the effect of ivabradine (for the consecutive 15 days) on cognitive functions. To ensure that the amelioration was due to either Iva or Donepezil, there was a group administrated scopolamine (4 mg/kg and 28 days) and left for 15 days without administrating any medication. The behavioural tests were performed then sacrificed, biochemical and the conventional histopathological examination were assessed. We found that this group did not recover to the level of the control group (not published data).18); the passive avoidance, Morris water maze, and novel object recognition. Our study showed that the injection of Sco has impaired the ability of learning and memory. This was detected by behavioural tests and confirmed by ELISA and histopathology.In this study, the therapeutic effects of Iva, a known antianginal and antiarrhythmic agent, had been investigated and compared to that of donepezil, the already validated medication for AD. Three behavioural tests were used for evaluating amnesia  doses of 150–200r X‐rays31 which has been attributed to impairment of hippocampal neurogenesis23.In the present study, the cognitive impairment was associated with reduction of pyramidal neurons numbers and thinning of the pyramidal layer along the entire CA after Sco administration. Similar findings have been reported in the hippocampal CA1 and CA3 regions after global brain ischemia32 and, in a recent study, after high doses of caffeinated energy drinks in adult male albino rats33. The severely affected rat resembled those described in stroke model of dementia 34, in cadmium subcutaneously injected rat35 and in oral administration of AlCl3 to rats36. In those models, neurons of CA and DG underwent marked distortion, reduced cellular density, loose arrangement, and misalignment, disappeared cell outline, shrunken cytoplasm, obscured nuclear boundary and the nucleolus disappeared. Additionally, some cells shrank and exhibited vacuolated eosinophilic cytoplasm and hyperchromatic pyknotic shrunken nuclei (apoptotic changes) associated with the appearance of pericellular haloes. The aforementioned results revealed that the ES (group III) induced more significant defects of memory accompanied by many histopathological signs of AD validating the success of AD model establishment. Therefore, we continued the evaluation of the Iva administration on ES rats (group III).In the present study, especially after ES, vigorous signs of neurotoxicity appeared histologically, which reached to a severe deformed hippocampus in one rat. The balloon cells (dark pyknotic nuclei with intense vacuolations) in the CA with cortical disorganization are associated also with drug-resistant epilepsy35 and mesenchymal stem cells treatment36.Grossly, Iva and donepezil improved the Sco induced atrophy of the brain in group IV, V and VI. There was an increase in the weight of the brain when compared to group III. This could be confirmed by quantification of the CSA and perimeter of the sagittal section of the BEC and the hippocampus, which significantly increased when compared to those of group III (ES). Treatment with Iva and donepezil reduced the degenerative changes and restored the normal hippocampal cytoarchitecture including the cell populations and the regular distribution. Similar effects have been reported for quercetin treatment37 and Myricetin38 after intracerebroventricular injection of streptozotocin (ICV STZ) and for triazine derivative, after STZ39. Likewise, in these models and ours, the number of hippocampal CA3 pyramidal neurons raised and the learning and memory have been improved. Luckily, there was a significant increase in the thickness of the granular layer of the DG after high Iva and after donepezil administration, as that described for topiramate40. Similar effects have been reported for melatonin treatment41 and for Centella asiatica after d-gal and AlCl3 induced neurodegeneration36. This might be due to promoting aberrant neuron regeneration and the survival of new-born neurons in the hippocampus40.In the current study, the morphometric data revealed restoration of the pyramidal layer thickness of CA1 and CA3 especially after high Iva and donepezil. A similar neuroprotective effect has been reported for Luteolin42. It impairs learning and memory through increasing activities of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), adenosine deaminase (ADA), and lipid peroxidation with a concomitant decrease in levels of nitric oxide (NO), reduced glutathione (GSH), SOD, glutathione S-transferase (GST), and catalase activities43. Moreover, Sco increased the expression of inflammatory mediators such as cyclooxygenase2 (COX2), nuclear factor kappa B (NF-kB), TNF-α44.Sco is an antimuscarinics, which works by blocking the acetylcholine (inhibitor) in the central nervous system (CNS)43. It regulates endocytic trafficking of amyloid precursor protein (APP), via up-regulation of sorting nexin protein-33 SNX33 expression45. Our study confirmed its therapeutic effect which lead to restoration of the normal healthy neurons and cytoarchitecture and the HCN1 expression, which indicates a new mechanism for donepezil.Donepezil can inhibit the activity of BuChE, and adenosine deaminase (ADA), significantly decreases lipid peroxidation and increases levels of NO and antioxidant status46. Furthermore, the decrease in HCN1 and HCN2 expression in medial temporal lobe epilepsy with hippocampal sclerosis has been suggested to increase the susceptibility to seizures because it reduces the density of Ih currents and enhance the excitability of neurons47. The pathogenetic mechanism underlies the neuronal discharge activity inconsistency is one of the main discussed hypotheses of the neurodegenerative diseases48.In the present study, the optical density of HCN1 immunoreactivity decreased significantly after ES in group III in different subfields of the hippocampus, indicating the involvement these channels in the Sco-induced neurodegeneration, similar to the effect of chronic cerebral hypoperfusion in rath), is effective at reducing seizure susceptibility14. Iva, at present, has been approved for use in clinical practice as an adjunct in the treatment of select patients with symptomatic , as it reduces heart rate by direct action on the sinus node and improve outcomes in HF49. Till now, the relationship between HF and AD remains largely unclear. Alzheimer’s disease and HF often occur together and thus increase the cost of care and health resource utilization50. A recently proposed risk factor for AD is HF. Decreased cerebral blood flow and neurohormonal activation due to heart failure may contribute to the dysfunction of the neurovascular unit and cause an energy crisis in neurons. This leads to the impaired clearance of amyloid beta and hyperphosphorylation of tau protein, resulting in the formation of amyloid beta plaques and neurofibrillary tangles51.Iva, the broad-spectrum HCN blocker which blocks the HCN-mediated current 57. The important link to the levels of expression of Ih and HCN protein have recently been shown to be regulated by neuronal activity58. Since the most consistent effect of ACh on hippocampal pyramidal neurons appears to be a pronounced membrane depolarization coupled with increased membrane resistance59. Therefore, the resulting lower neuronal activity of pyramidal neurons caused by Scopolamine might be the cause for lower HCN expression, and the restoration of neuronal activity in case of Donepezil might lead to the restoration of HCN expression.Fortunately, we found that both of Iva and donepezil has improved the Sco-induced memory impairment and cognitive deficits in rats and ameliorated the histopathological alterations. Notably, we also realized, from our data, a significant amelioration in the behavioural and histopathological signs of AD after high dose of Iva which is comparable to those of donepezil. It has been reported that there was possible causal relationships between key markers associated with AD . i.e., decreased expression of HCN channels is correlated to ADTaken together, our results supported the hypothesis that the dysfunction of HCN1 channels can underlie neuroinflammation and resulting neuronal dysfunction. In addition, donepezil and Iva can markedly ameliorate the Sco-induced neuronal toxicity by modulating HCN1 expression and function. Furthermore, Iva has a potential neurosupportive therapeutic value for the treatment of insulted neurons in dementia and could be a candidate for a new medication to relieve cognitive dysfunctions which might be partly attributed to blocking of HCN1 channels. This adds new benefits for this drug in patients with HF associated with AD which opens a possible new concern for its neuroprotective effect. However, further studies are essential before considering Iva an effective anti-AD drug.Adult male Wistar rats weighing 350 ± 20 g were supplied by the animal house facility, Faculty of Medicine, Assiut University and were housed in groups of four rats per cage at a temperature of 22 ± 2 °C on a 12 h light/dark cycle. Food and water were provided ad libitum. The research was conducted in accordance with the internationally accepted principles for Guide for the Care and Use of Laboratory Animals  and approved by Assiut University Institutional Animal Ethics Committee . This animal study was performed in accordance with ARRIVE guidelines.To analyze the effects of Iva on rat models of Sco induced dementia, animals were randomly divided into 6 groups, each has 8 rats. Sco hydrobromide , Donepezil  and Iva hydrochloride . First, we validated the Sco induction of dementia. To this end, we used 3 groups; group I the control, which was injected with i.p. saline in equal volumes and regimens to Sco.; group II, Sco-treated group which received i.p Sco, 6 mg/kg/day for 21 days; and group III, Extended Sco-treated (ES) which received i.p Sco for longer period, 4 mg/kg/day for 28 days. Second, we chose the most affecting time/dose protocol to cause obvious demented brain for further analysis of the effect of Iva. To this end, we used 3 other groups after having been administered ES Sco and showed behavioural signs of dementia; group IV, low Iva which received low dose of Iva (5 mg/kg/day) for 2 weeks; group V, high Iva which received high dose of Iva (10 mg/kg/day) for 2 weeks; and group VI, which received donepezil (0.5 mg/kg/day) for 2 weeks. During this study, we analyzed each fifth section for at least three different representative sections. See the timeline graphical representation Fig. .Figure 160, Morris water maze (MWM)61 and Novel object recognition test62.With the researchers blinded to treatment conditions, behavioural tests were performed as described before; Passive avoidance testAfter completion the behavioural tests, overnight fasted animals were anesthetized with thiopental sodium and transcardially perfused by 0.9% cold saline. The whole brain was carefully isolated from each rat and divided into hemispheres. The brain was isolated from each rat and bisected into hemispheres. The left hemispheres were fixed in 10% neutral-buffered-formalin (PFA) for 48 h, followed by washing twice with phosphate buffered saline (PBS). These hemispheres were further processed for paraffine embedding. We analysed each fifth section for at least three different representative rats. The hippocampi of the right hemispheres were immediately dissected on dry ice, wet tissues were blotted dry with a filter paper and stored at − 80 °C to be used for ELISA.63. Evaluation of hippocampal oxidative stress biomarkers was measured as described before, such lipid peroxidase 64, superoxide dismutase (SOD)65 and total antioxidant capacity (TAC)66.Measurement of the hippocampal level of pro-inflammatory cytokines (tumor necrosis factor-α (TNF- α), interleukin (IL)-2, and IL-6) was performed as described before21. A modified Highman's Congo red stain has been used. The technique was performed briefly, paraffine sections were dewaxed in xyline 3 times 5 min each then hydrated in descending grades of ethanol 3 min each. Sections were incubated in congo red solution (100 ml ethanol 50% + 0.6 g congo red) for 40 min, rinsed in distilled water, differentiated quickly in alkaline alcohol solution (1 M NaOH in saturated NaCl solution in 37% ethanol) and rinsed in tap water for 1 min. Sections were counterstain with hematoxylin for 20 s, rinsed for 2 min. Sections were dehydrated in ethanol, cleared in xylene, and finally covered using DPX mounting medium. The amyloid deposits were stained red, and the nuclei were stained blue  and the sections were further stained with eosin and haematoxylin for investigating the histological changes. Congo red stain were applied to investigate the amyloid plaques on formalin-fixed, paraffin-embedded tissue sectionsned blue .67 however the secondary antibody was Ultra Tek HRP Anti-polyvalent kit  was purchased from ScyTek (USA).Immunohistochemical analysis was also conducted against HCN1 antibody, , to investigate the impact of Iva treatments on the Sco-induced demented brain. The techniques was performed as described byData are expressed as the mean ± standard error of mean (SEM). Statistical analysis was performed by a one-way and two way analysis of variance (ANOVA), using GraphPad Prism 5.03 . For all statistical comparisons, a P-value of < 0.05 was considered statistically significant.Supplementary Information.


BE patients undergo periodic endoscopic surveillance with biopsies to detect dysplasia and EAC, but this strategy is imperfect owing to sampling error and inconsistencies in the diagnosis and grading of dysplasia, which may result in an inaccurate diagnosis or risk assessment for progression to EAC. The desire for more accurate diagnosis and better risk stratification has prompted the investigation and development of potential biomarkers that might assist pathologists and clinicians in the management of BE patients, allowing more aggressive endoscopic surveillance and treatment options to be targeted to high-risk individuals, while avoiding frequent surveillance or unnecessary interventions in those at lower risk. It is known that progression of BE to dysplasia and EAC is accompanied by a host of genetic alterations, and that exploration of these markers could be potentially useful to diagnose/grade dysplasia and/or to risk stratify BE patients. Several biomarkers have shown promise in identifying early neoplastic transformation and thus may be useful adjuncts to histologic evaluation. This review provides an overview of some of the currently available biomarkers and assays, including p53 immunostaining, Wide Area Transepithelial Sampling with Three-Dimensional Computer-Assisted Analysis (WATS It is defined as endoscopically visible columnar epithelium containing goblet cells . Although the American Gastroenterological Association has not specified a length requirement1, the American College of Gastroenterology requires extension at least 1 cm proximal to the gastroesophageal junction4. BE is a genetically unstable metaplastic epithelium that accumulates an increasing number of genetic and chromosomal abnormalities as it progresses to low-grade dysplasia (LGD), high-grade dysplasia (HGD), and eventually EAC10. Because dysplasia is currently the primary clinical biomarker used to identify patients who are at an increased risk for EAC, clinical guidelines recommend that BE patients undergo periodic endoscopic surveillance with biopsies to detect dysplasia4. This allows for risk stratification and management of BE patients based upon the presence and grade of dysplasia prior to the development of EAC. The detection of HGD usually prompts endoscopic therapy, generally in the form of radiofrequency ablation (RFA) with or without endoscopic mucosal resection (EMR), due to its more frequent association with EAC compared with LGD16. However, a significant number of BE patients with dysplasia (~20%) are resistant to endoscopic therapy, and recurrences or progression to EAC during endoscopic therapy are not uncommon23. As for LGD, although continued surveillance every 12 months is an acceptable approach, there has been a shift toward endoscopic ablation therapy in recent years26. Subsequently, there is greater emphasis on optimization of the diagnosis of dysplasia as well as identification of patients who are more likely to progress to HGD/EAC and/or have a poor response to endoscopic therapy. However, considering the annual cancer risk for patients with non-dysplastic BE (NDBE) is low (0.1–0.5% per year)29, identification of reliable biomarkers of low risk to allow prolongation of surveillance intervals compared with the current recommendations of repeating endoscopy every 3–5 years in those with NDBE4 remains an important goal of biomarker research.Barrett’s esophagus (BE) is a pre-neoplastic condition that is associated with an increased risk of developing dysplasia and esophageal adenocarcinoma (EAC)35. In fact, there is evidence that the current surveillance protocols may not be effective in reducing mortality from EAC, with one study demonstrating that patients with fatal disease were nearly as likely to have received surveillance (55%) as were controls (60%)36. Furthermore, the rate of missed HGD/EAC  is high (19–24%)37, suggesting that early repeat endoscopy, ideally within 1 year of an initial BE diagnosis, may be crucial, although the cost-effectiveness of this approach remains to be determined. Notably, in a recent meta-analysis of 24 cohort studies of NDBE or LGD patients followed for at least 3 years after index endoscopy, Visrodia et al. reported that ~25% of EACs and 27% of HGD/EACs were classified as missed37. When only NDBE patients were considered, the rates of missed EACs and HGD/EACs were 24% and 19%, respectively37.Even though endoscopic therapy has revolutionized the treatment of BE patients, the current surveillance protocols based on the histologic classification of BE have several shortcomings, including limited sampling of the affected BE segment , sampling error of potentially neoplastic lesions, and interobserver variability among pathologists in the diagnosis and grading of dysplasia, particularly for LGD42, Wide Area Transepithelial Sampling with Three-Dimensional Computer-Assisted Analysis (WATS3D)50, TissueCypher58, mutational load analysis (BarreGen)62, fluorescence in situ hybridization (FISH)67, and DNA content abnormalities as detected by DNA flow cytometry77 have been extensively evaluated. Although none of these studies have comprehensively evaluated the potential utility of these biomarkers in reducing mortality from EAC compared with the current surveillance standards, they have demonstrated a potential benefit when used in combination with histologic findings to assist in the diagnosis and/or risk stratification of BE and dysplasia. As such, this review provides an overview of these biomarkers and tests that appear most promising based on the availability of multiple published results and/or on their commercial availability.Consequently, there is an increased interest in ancillary tests that could (1) improve the diagnostic accuracy of dysplasia (and its grading) in challenging situations to avoid a repeat endoscopic examination with biopsies ; (2) predict which NDBE or LGD patients are at a higher risk for developing HGD/EAC (including missed lesions) so that such patients can be identified early and successfully treated with endoscopic therapy to prevent progression to EAC; (3) identify patients who are less likely to develop HGD/EAC so that the surveillance of low-risk patients can be reduced; and/or (4) predict those more likely to have a poor response to endoscopic therapy. In this regard, a variety of biomarkers and assays, such as p53 immunostaining78. The rationale for its use as the primary clinical biomarker is based on the premise that EAC in BE patients develops through a sequence of molecular  and morphologic changes that begin with intestinal metaplasia and then progress from LGD to HGD, and ultimately to EAC82. This is also supported by multiple outcome studies demonstrating a strong correlation of higher EAC rates with increasing levels of dysplasia. While the annual cancer risk for NDBE patients is low (0.1–0.5% per year)29, HGD is considered a key premalignant step that is associated with a greater risk of either already having EAC or developing it on follow-up (16–100%)88. The natural history of LGD is more controversial, with variable progression rates ranging from 0.4 to 13.4% per year91. It is worth emphasizing that it is not often possible to distinguish true progression from missed lesions in these outcome studies. In other words, a patient with LGD may progress from an unsuspected HGD in the same site or elsewhere in the esophagus. In such a case, HGD/EAC detected on follow-up may represent either true progression or a delayed/missed diagnosis.Currently, dysplasia is the primary clinical biomarker used for risk assessment in the surveillance and management of BE patients. Morphologically, dysplasia is defined as unequivocal neoplastic epithelium that remains confined within the basement membrane of the epithelium from which it developed, and it is classified as (1) negative for dysplasia, (2) indefinite for dysplasia (IND), (3) LGD, or (4) HGD92, most endoscopists do not adhere to this protocol and take too few biopsies, compounding the problem of sampling error. Second, consistent diagnosis and grading of dysplasia by histology is challenging, as exemplified by a relatively high degree of interobserver variability in the histologic classification of BE among pathologists, particularly toward the lower end of the spectrum93. The most pronounced variability is linked to the diagnosis of LGD, with a recent study illustrating sub-optimal interobserver agreement for LGD (kappa = 0.11) even among gastrointestinal (GI) pathologists32. In another study, up to 85% of LGD cases were downgraded to NDBE or IND following expert pathology review91. Even though an excellent interobserver agreement for HGD among GI pathologists has been reported in earlier studies31, a more recent study demonstrated that upon review of 485 HGD samples from both academic and private centers by experienced GI pathologists, up to 40% of these cases were reinterpreted as LGD, IND, NDBE, or no BE93. Consequently, both the American College of Gastroenterology and the American Gastroenterological Association strongly recommend that all potential dysplasia cases be confirmed by at least one experienced GI pathologist before embarking on a management plan94. This recommendation is further supported by several studies demonstrating a strong correlation between the number of pathologists who agree with a diagnosis of dysplasia and the rate of neoplastic progression. For instance, Skacel et al. showed that the rate of progression was 80% when three GI pathologists agreed on a diagnosis of LGD, while the rate was 41% when two GI pathologists agreed95. Finally, even if the issues stated above could be resolved, there are no observable histologic features in NDBE or LGD on hematoxylin and eosin (H&E) staining that can accurately identify those patients most likely to develop HGD/EAC versus remain stable for years. Indeed, recent studies have suggested that many EACs develop through a more direct, accelerated pathway in which TP53 mutation is followed by doubling of the whole genome, rapidly resulting in genomic instability, oncogenic amplifications, and EAC, rather than through the stepwise accumulation of tumor suppressor alterations97. This accelerated pathway to EAC might explain in part why endoscopic surveillance is sometimes unsuccessful in detecting dysplasia before the development of EAC in some BE patients36. Overall, these results suggest that additional or alternative biomarker(s) may be useful to better risk stratify BE patients.Unfortunately, dysplasia has a number of limitations as a biomarker. First, dysplasia is often focal and may not be endoscopically visible, so sampling error is a major issue as most surveillance techniques sample only a minority of the BE segment. Although Reid et al. reported that four-quadrant biopsies taken every 1 cm in the BE segment  can consistently detect early cancers arising in HGD98. The TP53 gene encodes p53, which prevents mutations. Normal cells have low levels of this protein in their nuclei, but the gene and protein are upregulated in the presence of DNA damage or stress, resulting in DNA repair, growth attenuation, and apoptosis. In dysplastic cells and EAC, mutations in TP53 lead to aberrant nuclear accumulation of abnormal p53 protein  that can be detected on immunostaining  for p53 to confirm a dysplasia diagnosis or predict likelihood of progression to EAC is of interest but has limitations, as summarized by othersing Fig. . Alternaern Fig. . Light aype Fig. . However99. The recommendation seemed to reflect studies directed at predicting progression of NDBE to HGD/EAC rather than establishing an initial diagnosis. In one study, scoring p53 immunostaining as “significant” in the presence of strong or absent staining versus “not significant” resulted in kappa scores on the order of 0.6 (strong reproducibility), whereas scoring morphologic features as “negative for dysplasia” versus “IND” versus “LGD” versus “HGD” (4 categories) resulted in kappa scores of 0.3, an unsurprising result since grouping cases into 2 categories versus 4 produces greater observer variability ab initio41. In fact, when the authors grouped the morphologic interpretation into only two categories as they had done for p53, namely “definite dysplasia” versus “no dysplasia” on H&E, they achieved a comparable kappa score of 0.55 for morphology alone, diminishing their conclusions concerning p53 considerably. Nonetheless, the results of many studies have supported the use of p53 IHC as a marker of the likelihood of progression to HGD/EAC in patients whose biopsies show H&E findings of negative for dysplasia, IND, or LGD. The latter studies are summarized by Srivastava et al.38.Pathologists in many institutions, particularly in the UK and Europe, have advocated for the use of universal p53 IHC in BE cases to detect dysplasia that might be otherwise overlooked, to the point that the British Society of Gastroenterologists endorsed adding it reflexively in routine practice42. The authors used a retrospective set of over 500 BE patients with or without known progression from negative for dysplasia, IND, or LGD to HGD/EAC. To establish their IHC scoring system  to essentially eliminate the IND category with excellent prediction of outcome103. Ten Kate et al. also used p53 staining alone and achieved similar results to those afforded by use of H&E alone, with some synergy for the two combined but probably not enough to support reflex testing102. Years ago, two of us (GYL and EAM) were part of a group that also achieved excellent prediction of outcome using H&E alone despite imperfect interobserver variability104. We would also point out that the Kaplan–Meier curves for progression of NDBE with and without abnormal p53 staining from the study by Redston et al. do not differ dramatically because so few patients without histologic dysplasia progress regardless of p53 immunostaining status42.In a 2018 study, Ten Kate et al. reported that simply refining histologic criteria for diagnosis of LGD identified BE patients likely to progress42, although pathologists might be motivated by payments to add p53 staining to all BE samples that lack dysplasia or show LGD. Overinterpretation of normal staining, however, might result in unnecessary surveillance or ablation procedures.Incorporating reflex IHC for p53 is not terribly expensive in the individual patient, and reimbursement is readily obtained. The 2021 Medicare fee schedule listed a 2021 figure of $99.82 for a global code of 88342  and modified it to $106.07 , whereas H&E global code (88305) affords $66.76 , which was updated to $71.52 . This means that adding a p53 stain increases the cost per biopsy by two and a half fold. This might be prohibitively expensive if p53 staining is added to every single esophageal biopsy demonstrating intestinal metaplasia. No cost analysis was provided by Redston et al.104, and many gastroenterologists request second opinions for diagnoses of LGD and HGD since either is currently an indication for ablation of the affected segment99. The updated 2021 Medicare reimbursement fee for the code for outside consultation (88321) is $102.49, which is slightly cheaper than a p53 stain. Adding p53 may have some value in assessing LGD or adding diagnostic precision for cases regarded as IND103. However, using positive p53 immunostaining to justify endoscopic therapy in IND or LGD patients, when the concordance between IHC and mutation analysis is less than perfect (~90%), may mean overtreatment in ~10% of patients.Most laboratories have used p53 immunostaining for years in evaluation of samples from several organ systems. In esophageal biopsies, however, in expert hands, p53 staining is not necessary to diagnose HGD in BE, which is itself an excellent marker for high risk for progression to EAC3D or Wide Area Transepithelial Sampling with Three-Dimensional Computer-Assisted Analysis  is an adjunct test to targeted and random four-quadrant esophageal biopsies using three-dimensional computer-assisted tissue analysis. As discussed previously, the current screening and surveillance guidelines for BE and associated dysplasia require sampling of any visible mucosal abnormality followed by systemic random four-quadrant forceps biopsies obtained at 1–2 cm intervals (Seattle protocol). However, this recommended protocol is time-consuming, labor intensive, and subject to sampling error. As such, the rationale for using WATS3D is to overcome these inherent problems associated with extensive blind sampling43. In WATS3D, abrasive brushes are used to circumferentially sample the esophageal mucosa. The sampling consists of individual cells as well as mucosal strips reported to measure up to 150 μm in thickness. The material is first analyzed by an imaging system using a neural network optimized for evaluation of the esophageal mucosa. The computer system scans, analyzes, and integrates up to fifty 3-μm optical slices. Ultimately, the system builds three-dimensional images of esophageal glands, and flags goblet cells and dysplastic cells that are displayed for confirmation by a pathologist , increasing the overall detection of dysplasia by 242%46. WATS3D also identified 2570 additional BE cases , increasing the rate of BE detection from 13.1 to 33%. However, among the purported 213 “new” dysplasia cases, 128 (60%) were in fact classified as IND rather than as dysplasia by WATS3D, significantly reducing the reported increased detection rate of dysplasia. Furthermore, the increased detection rate of BE was based on the diagnosis of intestinal metaplasia, but the possibility that the cardia was sampled could not be excluded, further weakening the validity of the results.Several studies have demonstrated a significant increase in the detection rates of BE and dysplasia when WATS3D  47. In a multicenter, prospective, randomized trial of referred BE patients at 16 medical centers, Vennalaganti et al. reported that the addition of WATS3D to biopsy sampling yielded additional 23 cases of HGD/EAC. Among these 23 patients, 11 were classified by biopsy as NDBE and 12 as LGD/IND. However, the vast majority of these patients (91.3%) had been previously diagnosed with HGD/EAC on prior biopsies, and most (78%) were confirmed to have HGD/EAC on follow-up biopsies. Also, this study was performed in a high-risk BE population at referral centers, and thus it is not representative of community GI practices and of the BE population at large. Nonetheless, this series is important in that it demonstrates an increased detection rate of high-grade lesions by WATS3D, and the biopsy diagnoses had been confirmed by GI pathologists.Only a small series involving 160 BE patients tackled the issue of HGD/EAC detection by WATS3D are limited. In a study of over 4000 BE patients who had two WATS3D separated by ≥12 months, Shaheen et al. reported that individuals without dysplasia on WATS3D had a very low risk of progression to HGD/EAC (0.08% per patient-year), while those with LGD had a higher rate of 5.79% per patient-year50. However, as noted by the authors, no comparison could be made to the progression rate of LGD detected by microscopy alone. Interestingly, the authors also evaluated the category of crypt dysplasia and reported its risk of progression (1.42% per patient-year) higher than those with no dysplasia but lower than those with LGD. The heterogeneous nature of this diagnostic category likely explains the results.Long-term outcome studies of dysplasia diagnosed solely on WATS3D. First, although a study examining the interobserver agreement among pathologists using WATS3D found substantial agreement for LGD, HGD, and no dysplasia48, the diagnostic criteria used in WATS3D have not been independently tested. Also, the data used to construct the algorithm that creates three-dimensional images of esophageal glands and adequacy criteria used for this test are not well delineated. Furthermore, regenerative epithelial changes in deep glands could be easily misinterpreted as HGD on a single plane of analysis as in WATS3D, an issue that merits additional evaluation49. Another hindrance to the full validation of this technology is that this commercial test is interpreted by a limited group of pathologists. Independent reproduction and evaluation of WATS3D diagnoses in academic settings with expert reviews of all forceps biopsies by specialized GI pathologists  would go a long way to address some of these criticisms. Finally, as noted above, whether the progression rate of dysplasia detected by WATS3D differs from that of dysplasia identified by forceps biopsy and microscopy alone remains to be established.There are limitations with WATS3D has not been extensively evaluated. The cost of WATS3D has been reported to be in the range of $700–800 by our endoscopist colleagues, but whether using this commercial test as an adjunct to traditional endoscopic surveillance is cost effective in the long-term management of BE patients remains to be thoroughly evaluated. Also, the potential value of WATS3D in the era of ever improving advanced endoscopic imaging techniques has not been examined. As endoscopists improve their ability to detect ever more subtle lesions previously described as ‘invisible’, it may lessen the need for broad blind sampling, such as WATS3D.The issue of additional cost of performing WATSThe objective of TissueCypher is to evaluate samples from BE patients diagnosed as negative for dysplasia, IND, or LGD on routine histologic evaluation to identify those patients most likely to progress to HGD/EAC so that intensified screening or ablation can be offered to them. Similarly, the technique is intended to identify patients who are unlikely to progress such that their surveillance can be reduced.58. Hoechst dye allows fluorescent detection of DNA105, thereby permitting image analysis software to identify nuclei as discrete objects in tissue. It also allows the software to assess nuclear area, solidity, and DNA content. Some of the markers are combined on the same slide52. The slides are then used to perform image analysis with an image analysis algorithm. The image analysis algorithm quantifies 15 different “image features”  samples for p16, AMACR, p53, HER2, CK20, CD68, COX-2, HIF-1α, and CD45RO, together with Hoechst staining dye , IND (n = 23), and LGD (n = 18). The samples were from 58 patients who progressed to HGD/EAC , and from 210 patients who did not progress (median surveillance time of 7 years). In this study, the prevalence-adjusted proportions of patients scoring low, intermediate, and high risk using the TissueCypher method were 84.2%, 9.4%, and 6.4%, respectively. The sensitivity and specificity of the test at 5 years for the 3-tier TissueCypher classification  were 29% and 86%, respectively, and 40% and 86%, respectively, for the 2-tier classification (low and intermediate/high risk combined). By comparison, the sensitivity and specificity of an expert diagnosis of LGD were 19% and 88%, respectively, and the sensitivity and specificity of the initial community diagnoses of LGD  were 26% and 66%, respectively. Of 51 patients who progressed within 5 years, 14 scored high risk, 6 scored intermediate risk, and 31 scored low risk. Among 210 patients who did not progress, 13 scored high risk, 18 scored intermediate risk, and 179 scored low risk. Using the TissueCypher test, the prevalence-adjusted positive predictive value (PPV) was 23%; i.e., 23% of patients who score high risk would progress to HGD/EAC within 5 years. The prevalence-adjusted negative predictive value (NPV) was 96.4%. The risk prediction test also showed improved risk stratification when compared to p53 alone using the automated scoring.This method offers the advantage of using a variety of markers with a consistent interpretation, thus eliminating interobserver variability, although this does not necessarily mean an accurate interpretation. Using the company’s platform, a risk score for progression is stratified as low, intermediate, or high, but there is some advantage to combining the intermediate and high risk scores. Although similar data are reported in all studies from the TissueCypher teamOverall, expert pathologists’ diagnosis of LGD outperformed TissueCypher in specificity and PPV, but TissueCypher was more sensitive. However, this was not the case for samples from patients with no dysplasia. Patients without dysplasia as confirmed by expert pathologists who scored high risk were at about 5-fold increased risk of progression as compared to patients without dysplasia who scored low risk using TissueCypher. The adjusted PPV for the test in expert pathologist-confirmed NDBE was 26%, indicating that 26% of patients without dysplasia but with a high risk score using TissueCypher will progress within 5 years, a rate similar to that associated with an expert diagnosis of LGD.57. It does not change initial evaluation of patients by routine histology. It has been assigned a CPT code by Medicare (0108U). However, because of the high “up front” costs of TissueCypher, most insurers do not cover the testing. Also, the roughly similar performance characteristics of TissueCypher to histologic evaluation may not justify changing surveillance intervals based on the results, and thus the suggested cost benefit may not materialize, especially given its high cost. As noted above, based on the company’s data, a diagnosis of LGD by an expert pathologist offers more specificity than the test, and obtaining an expert opinion is certainly substantially cheaper. Regardless, the test is consistent, not subject to human observer variation, and outperforms pathologists in identifying patients without dysplasia who are likely to progress to HGD/EAC.TissueCypher is a send-out test  and costs about $5,000. A cost analysis sponsored by the company claimed that it would be cost-effective after 5 years by reducing the number of patients requiring surveillance and reducing EAC-associated deaths62. It can be assessed using a commercially available test , with its main objective being to detect dysplasia and risk stratify BE patients. To perform this assay, H&E-stained slides are first evaluated to identify relevant histologic targets  that are micro-dissected from 1 to 3 unstained FFPE sections (4 μm in thickness)106. Greater than 90% of each micro-dissected target should contain epithelial cells, from which purified DNA is prepared. Polymerase chain reaction (PCR) and quantitative capillary electrophoresis methods are performed on all micro-dissected areas. ML specifically assesses the presence and extent  of loss of heterozygosity (LOH) and new alleles consistent with microsatellite instability (MSI) for each micro-dissected target. The following 10 loci are examined, with associated tumor suppressor genes in parentheses: 1p , 3p , 5q , 9p (CDKN2A), 10q , 17p (TP53), 17q , 18q , 21q , and 22q (NF2). LOH is categorized as either high (> 75% of the DNA has LOH) or low clonality (50–75% of the DNA has LOH) and assigned values of 1 and 0.5, respectively. The value of the first MSI at a genomic locus is 0.75, and each additional MSI is assigned a value of 0.5. The highest weighted value at each locus is determined based on the values for low and high clonality LOH and MSI at that locus . The sum of all weighted values for all 10 genomic loci is defined as the ML for that micro-dissected target (range: 0–10).Mutational load (ML) analysis provides a measure of cumulative genetic aberrations and instability at 10 key genomic loci by assessing DNA damage around tumor suppressor genes associated with progression to HGD/EAC61. A mean ML score was significantly higher in progressors (ML = 2.2) than non-progressors (ML = 0.4) (p < 0.001).61 No progressor had a ML of 0 at baseline compared with 54% of non-progressors. Sensitivity was 100% at ML ≥ 0.5, and specificity was 96% at ML ≥ 1.5. Similarly, in a retrospective study of 28 IND patients, patients who progressed to HGD had higher levels of genomic instability (ML ≥ 1.5)62. At this threshold, the risk of progression to HGD was 33% , with a sensitivity of 100% and a specificity of 85%. Overall, these results indicate that genomic alterations as measured by ML often predate the development of HGD/EAC, potentially allowing ML to be a useful biomarker for predicting disease progression. In addition, Ellsworth et al. demonstrated a signficant correlation of higher ML with increasingly severe histologic grade of BE-associated lesions: ML = 1.1 for IND, 2.2 for LGD, and 3.3 for HGD (p < 0.001)59. These results suggest that ML may serve as an adjunctive test in patients with equivocal histology.In a case-control study involving 69 BE patients (including 23 progressors and 46 non-progressors), ML assessment was able to risk stratify patients with NDBE or LGD at baseline with respect to progression to HGD/EAC within a mean follow-up time of 4 years106. In fact, using crude lysate, there was no difference in mean ML between progressors (ML = 0.73) and non-progressors (ML = 0.74) (p = 0.93) in a nested case-control study (involving 48 progressors and 101 non-progressors), failing to validate the previous finding that ML could be useful in risk stratifying BE patients. Finally, as noted above, BarreGEN is not fully validated for commercial use at this time, and it is unclear when this test will be available for clinical use.The biggest appeal of ML assessment is that it allows a direct correlation with morphology and provides an objective quantitative measure of the presence and extent of molecular alterations associated with development of dysplasia and EAC, eliminating human interobserver variability. However, similar to other PCR-based tests, ML assessment may be hampered by insufficient amounts and/or poor quality of DNA as is often the case in FFPE mucosal biopsies. Also, micro-dissection of tiny histologic targets seen on H&E slides is susceptible to sampling error. Another limitation is that purified DNA rather than crude lysate should be used whenever possible, as ML signal in crude lysate could be “muted”MYC), 9p21 , 17q12 , and 20q13 (ZNF217)67. Each cell is categorized as either normal  or abnormal   is a technique that utilizes fluorescently labeled DNA probes to detect chromosomal abnormalities. To explore its potential utility in the diagnosis and risk stratification of dysplasia in BE patients, several studies, all conducted at Mayo Clinic, utilized a 4 locus-specific probe set targeting 8q24 (p < 0.001), whereas single locus gain was most commonly observed in LGD (28% versus 12% of NDBE versus 8% of HGD)7. Also, in a recent multicenter study, if ≥10% of cells had polysomy in the specimen, FISH was able to differentiate between HGD/EAC and the remaining histologic diagnoses with a sensitivity of 80% and a specificity of 88%66. Furthermore, in a retrospective analysis of 245 BE patients with a history of HGD, using a cutoff of more than 4 of 100 cells demonstrating polysomy, the risk of EAC was significantly higher within 2 years among patients with a polysomic FISH result (14.2%) compared with those without a polysomic result (1.4%) (p < 0.001)65. In addition, Timmer et al. demonstrated that in BE patients with HGD or intramucosal adenocarcinoma (IMC)  treated with ablation with or without preceding EMR, polysomy was associated with a lower probability of achieving complete eradication of HGD/IMC  in a univariate analysis67. Given its high diagnostic accuracy for identifying HGD/EAC and potential to detect HGD/EAC on follow-up, polysomy in combination with histology may be able to serve as a confirmatory marker of HGD/EAC and screening tool to identify patients at highest risk for subsequent detection of HGD/EAC compared with those with non-polysomy.In a FISH analysis of a range of histologic lesions from 10 esophagectomy specimens from BE patients, polysomy was found to be more prevalent in HGD (88%) and EAC (100%) than in NDBE (<10%) and LGD (57%)  loss, which often do not cause noticeable morphologic abnormalities. Thus, a positive  FISH result does not always indicate the presence of dysplasia.Despite these promising results, no reference laboratory currently offers this test (apparently due to the lack of demand), although in the past it was available at Mayo Clinic and Neogenomics. However, many academic centers and commercial laboratories routinely run FISH, which can be completed in a few days, and the commercial availability of these probes  allows these laboratories to validate and bring up the same assay if needed. Also, FISH may be more sensitive than other tests such as DNA flow cytometry (described below) by virtue of having the low threshold for a positive polysomy result 77. Although its availability has been limited to few medical centers due to perceived technical demands and use of fresh tissue in earlier studies71, subsequent studies have successfully employed FFPE tissue for DNA flow cytometric analysis to generate high-quality DNA content histograms, demonstrating the feasibility of this methodology77. For optimal results, the computer program Multicycle  should be used to analyze DNA content histograms77. The published consensus guidelines for clinical DNA flow cytometry should be followed108. Most epithelial cells are normally in the G0/G1 phase of the cell cycle and have diploid (2 N) DNA content, while less than 6% of cells have tetraploid (4 N) DNA content (G2)  fraction greater than 6% (with DNA index of 1.9–2.1) is also classified as abnormal due to its strong association with neoplasia  Fig. . Aneuploeak Fig. . The presia Fig. 5,69,71,777. As a diagnostic marker of HGD, the estimated sensitivity and specificity of abnormal DNA content were 95% and 85%, respectively. Interestingly, DNA flow cytometry also identified a subset of LGD and IND patients who are at higher risk for subsequent detection of HGD/EAC, with the univariate hazard ratios (HRs) of 7.0 and 20.0, respectively (p < 0.001)77. Considering that endoscopic therapy is increasingly being recommended for LGD patients26, abnormal flow cytometric results at baseline LGD or IND could potentially enable clinicians to recommend endoscopic therapy, whereas continued surveillance may be an acceptable approach in the setting of normal flow cytometric results. Furthermore, Bowman et al. recently demonstrated that abnormal DNA content in baseline HGD/IMC can serve as a predictive marker of persistent/recurrent neoplasia following endoscopic therapy, with the univariate and multivariate HRs of 3.8 (p = 0.007) and 6.0 (p = 0.003), respectively76. This suggests that the detection of DNA content abnormalities in baseline HGD/IMC may help to identify high-risk BE patients who may benefit from alternative therapeutic strategies  as well as long-term follow-up with shorter surveillance intervals following endoscopic therapy.A recent retrospective study analyzed 80 FFPE BE samples with HGD, 38 LGD, 21 IND, and 14 NDBE and reported that the frequency of DNA content abnormalities (aneuploidy or elevated 4 N fraction) increases with increasing histologic grade of dysplasia: 0% of NDBE, 9.5% of IND, 21.1% of LGD, and 95% of HGD110, and features potentially altering the histologic interpretation  do not cause aneuploidy or elevated 4 N fraction, which can be very helpful in evaluating IND cases111. In fact, many genetic and chromosomal abnormalities detected in BE  tend to occur early and frequently throughout large areas of BE114, even before the first histologic sign of dysplasia, limiting their utility as a diagnostic or prognostic marker of dysplasia in BE patients.There are some advantages of using DNA flow cytometry. First, DNA flow cytometry is an inexpensive send-out test  that can be completed within 2–3 days. Second, DNA flow cytometric markers of dysplasia or progression (aneuploidy or elevated 4 N fraction) are usually absent in NDBE3D, TissueCypher, mutational load assessment (BarreGen), FISH, and DNA content abnormalities as detected by DNA flow cytometry have been demonstrated as ways to support a dysplasia diagnosis and aid in risk assessment for the development of HGD/EAC (Table In conclusion, as the current surveillance methods based on the histologic diagnosis and classification of dysplasia imperfectly assess the risk of BE patients, especially those with IND or NDBE histology, there is an increasing demand for ancillary tests to aid in the diagnosis/grading of dysplasia and risk stratification of BE patients. In cases with equivocal histology, one may argue that a repeat endoscopic examination with biopsies may provide the answer without the need of an ancillary test. However, this approach is likely to be more expensive than most ancillary tests. In this regard, several biomarkers and assays, including p53 IHC, WATS


Next-generation sequencing technologies have accelerated the pace of helminth DNA metabarcoding research, enabling species detection in bulk community samples. However, finding suitable genetic markers with robust species-level resolution and primers targeting a broad species range among parasitic helminths are some of the challenges faced. This study aimed to demonstrate the potential use of the mitochondrial 12S and 16S rRNA genes for parasitic helminth  DNA metabarcoding. To demonstrate the robustness of the 12S and 16S rRNA genes for DNA metabarcoding, we determined the proportion of species successfully recovered using mock helminth communities without environment matrix and mock helminth communities artificially spiked with environmental matrices. The environmental matrices are human fecal material, garden soil, tissue, and pond water. Our results revealed the robustness of the mitochondrial rRNA genes, through the high sensitivity of the 12S rRNA gene, and the effectiveness of the 12S and 16S primers targeting platyhelminths. With the mitochondrial rRNA genes, a broad range of parasitc helminths were successfully detected to the species level. The potential of the mitochondrial rRNA genes for helminth DNA metabarcoding was demonstrated, providing a valuable gateway for future helminth DNA metabarcoding applications like helminth detection and biodiversity studies. Inhabiting a diverse range of habitats and hosts, parasitic helminths are well-known to cause diseases in humans, animals, and plants, and the secondary consequences of helminthic diseases can impede the development of various economic and industrial sectors globally4. Albeit the public health and economic importance of parasitic helminths, species identification through morphology remains the gold standard. Recently, the popularity of DNA metabarcoding has been gaining traction, with applications in many diverse fields of biology, including the detection of helminths in the environment8. The term DNA metabarcoding was first introduced by Teberlet et al. (2012) to designate high throughput multi-species identification9. Species identification can be performed from bulk samples of entire organisms or environment samples via next-generation sequencing (NGS) technologies. Though DNA metabarcoding, it (1) decreases the reliance on taxonomic expertise and ambiguous morphological characters, (2) allows for simultaneous detection of more than one species in a single reaction, thus saving cost, (3) increases the sensitivity for detection allowing trace amounts of the target organism’s DNA to be detected, and (4) allows for a non-invasive method for organism surveillance13.The disparity between the projected estimate of helminth species and those described is huge, with approximately only 15% currently defined out of 100,000 projected helminth species16. Although DNA metabarcoding holds vast potential and is a promising alternative for species identification, its application for parasitic helminths is still at an early stage. Various considerations like performing prior evaluation via mock communities, having a suitable helminth isolation and DNA extraction strategy, selecting a suitable genetic marker, sensitivity, and specificity of available primers, and type of bioinformatics analysis, are crucial for a successful DNA metabarcoding approach18.The applications of helminth DNA metabarcoding are immense, including research on biodiversity and conservation of helminths in various habitats, biomonitoring of environments through helminth diversity, and the non-invasive detection and surveillance of parasitic helminthsc oxidase subunit I (COI) gene21. DNA metabarcoding of soil and marine nematodes has been performed using the nuclear 18S rRNA gene through primers specifically targeting soil and marine nematodes19. However, the use of the 18S rRNA gene as a DNA metabarcoding marker is limited by its taxonomic resolution due to the high sequence conservation of the genetic marker. Its specificity remains an issue as studies have revealed that only a small proportion of sequence reads are specific to nematodes23. In addition to environmental samples, the non-invasive sampling of gastrointestinal nematodes in animal hosts through fecal samples emphasizes the potential of helminth DNA metabarcoding25. Studies have investigated the impact of routine anti-parasitic treatment on parasitic nematode communities of cattle, illustrating the power of DNA metabarcoding in monitoring anti-parasitic resistance in livestock26. The detection of gastrointestinal nematodes of livestock using the ITS2 region, more commonly known as ‘nemabiome’ is also popular21. Although the species-level taxonomic resolution is robust using the ITS2 region, its high variability and varying lengths between diverse nematode taxa make it a challenging genetic marker to use. Similarly, the high sequence variability of the mitochondrial COI gene in helminths restricts its use as an optimal DNA metabarcoding marker despite having better species-level resolution than the nuclear 18S rRNA gene28. PCR amplification bias is prominent, selectively amplifying only some species, thus limiting the scope of species detection for DNA metabarcoding studies.Various genetic markers have been utilized for helminth DNA metabarcoding, and popular ones include the nuclear 18S ribosomal RNA (rRNA) genes, the nuclear internal transcribed spacer 2 (ITS2) region, and the mitochondrial Cytochrome 30. The potential of using the mitochondrial rRNA genes for helminth molecular-based studies was illustrated by its sensitivity to detect various life-cycle stages of parasitic helminths with robust species-level taxonomic resolution and the ability to amplify DNA for a broad range of nematodes and trematodes. To date, most helminth DNA metabarcoding studies have focused on characterizing nematode communities in soil and marine environments with the nuclear 18S rRNA gene. Moreover, studies are few and far between for DNA metabarcoding of platyhelminths. Of late, Douchet et al. (2021) revealed the power of the mitochondrial 16S rRNA gene for environment DNA (eDNA) metabarcoding of parasitic trematode communities in freshwater systems, further substantiating the potential of the mitochondrial rRNA genes for parasitic helminth DNA metabarcoding31.Utilizing the mitochondrial 12S and 16S rRNA genes, primers targeting parasitic nematodes and trematodes were recently developed32. Moreover, as there is currently no available DNA metabarcoding strategy collectively targeting parasitic nematodes and platyhelminths together, we provide an avenue for, as well as alternative genetic markers for DNA metabarcoding of parasitic helminths.Here, we aim to demonstrate the potential use of the mitochondrial 12S and 16S rRNA genes for parasitic helminth  DNA metabarcoding. We determine the robustness of the 12S and 16S rRNA genes and primers for species identification using mock helminth communities in artificially spiked environment matrices. Using mock helminth communities presents a beneficial opportunity to evaluate the potential suitability of the mitochondrial rRNA genes for DNA metabarcoding. With known species in mock communities, it can aid to determine PCR amplification efficiency or detect possible bias against certain species, and also test the sensitivity and robustness of these new primersFrom the 20 sets of mock communities for the two genetic markers , 2,439,972 raw sequence reads were generated in total. Of these, 565,734 reads, 972,922 reads, and 901,316 reads were obtained from 12S-platyhelminth, 12S-nematode, and 16S-helminth, respectively. After merging the raw sequence reads that passed quality filtering, 254,393 reads were recovered for 12S-platyhelminth, 254,624 reads for 12S-nematode, and 404,123 reads for 16S-helminth. Among the three replicates of the no environment matrix mock community per genetic marker, the number of raw sequence reads and final filtered sequences obtained were similar, showing limited amounts of variability among PCR replicates. Comparing the proportion of raw sequence reads obtained between the 12S and 16S rRNA genes, more were obtained from the 12S rRNA gene. The number of raw sequence reads obtained and the percentage of target-specific sequences recovered for each type of mock helminth community are presented in Supplementary Table The percentage of target-specific sequences (platyhelminths and nematodes) recovered was calculated for each mock helminth community to determine the specificity and robustness of the 12S and 16S primers for helminth DNA metabarcoding. Using the 12S-platyhelminth primers, 100% of the filtered sequences obtained belonged to platyhelminths for each type of mock community. A similar result was observed with the 16S platyhelminth primers, where more than 94% of the sequences were platyhelminth specific (except the mock tissue community). For the 12S nematode primers, a low percentage of nematode-specific sequences were obtained. However, the 16S nematode primers performed significantly better than the 12S nematode primers, where more than 50% of the sequences were nematode specific (except the pond water mock community).With five types of mock helminth communities, the proportion of helminth species successfully recovered was obtained to determine the effectiveness and accuracy of the mitochondrial rRNA genes for helminth DNA metabarcoding. Figure Gnathostoma spinigerum, where it was not recovered for the 16S rRNA gene in replicates 2 and 3, whereas G. spinigerum was recovered in replicate 1. As presented in Fig. Twenty representative parasitic helminth species, comprising 10 platyhelminths and 10 nematodes, were selected to make up the no environment matrix mock helminth community. Three PCR replicates were performed for each genetic marker to determine whether PCR amplification bias could be present. The results revealed no significant difference between the replicates, with similar species recovered. The only exception was the larva of Trichinella spiralis, Trichinella papuae, Trichuris trichiura, and Gnathostoma spinigerum. Angiostrongylus cantonesis was detected with the 12S rRNA gene, while Paracapillaria sp. was detected with the 16S rRNA gene. However, Strongyloides stercoralis, Haemonchus contortus, Oesophagostomum dentatum, and Globocephalus sp. were not recovered by either genetic marker. The results suggest the lower sensitivity of the nematode primers compared with the platyhelminth primers and agree with the low percentage of nematode-specific sequences obtained after analysis , where the high sensitivity of the mitochondrial 12S and 16S primers was revealed by successfully amplifying all representative trematode specimens of various life-cycle stages for DNA barcoding30. As research on platyhelminth DNA metabarcoding remains relatively unexplored, apart from the study by Douchet et al. (2021), having a highly sensitive primer for platyhelminth detection can be helpful31.Second, compared to nematodes, a higher proportion of platyhelminths was recovered for the mitochondrial 12S and 16S rRNA genes. Among all the mock helminth communities with both genetic markers, only the metacercaria of 33. Having taxa-specific primers are valuable, especially for eDNA metabarcoding, where primers should efficiently amplify target taxa and not amplify non-target DNA present in the environment41.Aside from the high sensitivity of the 12S and 16S platyhelminth primers, another measure of effective DNA metabarcoding primers lies in their specificity. Based on our sequences obtained from the platyhelminth primers, more than 94% were platyhelminth specific  to accommodate for Illumina sequencing and degraded eDNA present in environmental samples, the high sequence conservation of the 18S rRNA gene can limit its use as there might not be sufficient informative sites for accurate species identification44. Thus, the mitochondrial rRNA genes possessing sufficient sequence variation to assign taxa to the species level further augment their potential for helminth DNA metabarcoding.Finally, apart from having high sensitivity and specificity, the mitochondrial rRNA genes possess additional advantages contributing to their potential for helminth DNA metabarcoding. Species-level taxonomic resolution of the mitochondrial rRNA genes is more robust than the nuclear 18S rRNA gene. Using the nuclear 18S rRNA gene for species identification  could be challenging due to its low sequence variability. In an evaluation of three 18S primers for nematodes, Ahmed et al. (2020) showed that some taxa present in mock communities could only be taxonomically assigned to the family or genus level30. Through targeting a broad range of helminth species added in the mock communities, we validated that these primers can be applicable for various types of helminth DNA metabarcoding studies targeting different hosts and environment types. The species included in the mock communities can be found not only in humans, but also in intermediate and definitive hosts such as snails, fishes, crustaceans, rodents, pigs, and ruminants.The mitochondrial 12S and 16S rRNA gene primers targeted a broad range of parasitic nematodes, trematodes, and cestodes. We showed that parasitic nematodes belonging to the four clades , trematodes belonging to three orders , and cestodes in two orders (Cyclophylllidea and Diphyllobothridea) were able to be successfully detected. Our results are congruent with Chan et al. , where the same parasitic nematodes and trematodes were able to be identified via Sanger sequencing using the mitochondrial rRNA gene primersThus, the validation of the mitochondrial rRNA gene primers through mock helminth communities supports their universality for broad helminth species, host types, and host habitats, enhancing their potential as DNA metabarcoding markers.We also provide a highly sensitive, accurate, and specific assay for the concurrent detection of the three main groups of parasitic helminths . Helminth DNA metabarcoding studies that have been conducted thus far usually focus on each helminth group. Here, three groups of parasitic helminths can be targeted by utilizing three and four pairs of primers for the 16S and 12S rRNA genes, respectively. Although we targeted nematodes, trematodes, and cestodes for the 16S rRNA gene in a single Illumina sequencing run, the sensitivity of the 16S assay can potentially be improved if nematodes and platyhelminths were targeted separately. Nonetheless, our study provides a valuable resource demonstrating the feasibility of DNA metabarcoding for the main groups of parasitic helminths.Taking these additional advantages into consideration – having a robust taxonomic level resolution, primers targeting broad species range within helminths, and an assay to simultaneously detect both nematodes and platyhelminths, the mitochondrial 12S and 16S rRNA genes has the edge over other genetic markers frequently utilized for parasitic helminth DNA metabarcoding.8. A possible factor contributing to unsuccessful species recovery lies in the DNA extraction step, where if a complex mixture of specimens is present, reduced efficiency in obtaining maximum DNA yield for each specimen can result46. This study used only one replicate per mock helminth community and did not account for DNA extraction bias.Despite the promising potential use of the mitochondrial rRNA genes for helminth DNA metabarcoding, we observed some drawbacks that could be further improved on. Firstly, not all species were successfully detected in the mock helminth communities. The unsuccessful species recovery was prominent for nematodes compared to platyhelminths, especially with an increased number of species present in the mock helminth community. Moreover, the same species not detected via Illumina sequencing were able to be successfully amplified when single-species PCR was performed. A similar observation was also seen by Porazinska et al. (2009) using the nuclear rRNA genes, where not all species were accounted for in the mock nematode communityP. heterotremus were not detected and the larva of S. stercoralis could only be detected in some mock helminth communities. Therefore, we suggest that DNA extraction procedures should be optimized, especially when helminths of various life-cycle stages are present. For example, tissue homogenization should be thoroughly done with a tissue lyser via bead-beating, particularly for small specimens, and to aid in mechanically disrupting the cuticle of nematodes or eggshells. The thick wall of trematode metacercaria can also be broken down with the addition of enzymes during DNA extraction48. Also, DNA extraction is commonly performed using commercial kits, with varied kit performance depending on the sample type. Waeyenberge et al. (2019) evaluated 15 DNA extraction procedures in nematode mixtures and found varying degrees of DNA yield25. Additionally, replicate sampling can be performed on a particular community to detect as many species as possible50.Similar to the above, helminth immatures such as the metacercaria of COI gene for helminths, the small number of sequences available in reference databases restricts their ability for taxonomic assignment. Without a comprehensive reference sequence library, the chances of sequences being assigned to the species level decrease. Although different sequences can be delimited based on the degree of sequence variation and phylogenetic analysis, the species status of the sequence will remain ‘unknown’ without any taxonomic assignment. The limited number of reference sequences available for the mitochondrial rRNA genes can be circumvented by first generating reference sequences for individual species through Sanger sequencing31. Generating single sequences per species using the mitochondrial rRNA genes will also aid in the build-up of a comprehensive reference library for future users and accelerate the genetic markers’ popularity.Lastly, as the mitochondrial rRNA genes are not as widely used as the nuclear 18S rRNA gene or the mitochondrial natural validation of the DNA metabarcoding approach and expanding the reference library of the mitochondrial rRNA genes for helminths.In conclusion, we revealed the potential use of the mitochondrial 12S and 16S rRNA genes for DNA metabarcoding of parasitic nematodes, trematodes, and cestodes using mock helminth communities with artificially spiked environment matrices. The mitochondrial rRNA genes as alternative genetic markers for helminth DNA metabarcoding may be beneficial and contribute to future applications such as non-invasive sampling of parasitic helminths and helminth biodiversity assessments in the environment. Future research perspectives include 30. Five types of mock helminth communities were simulated through spiking of helminth communities with environmental samples. They comprise–no environment matrix, human fecal material, garden soil, tissue, and pond water, mimicking examples of environmental samples commonly used to detect parasitic helminths. Three PCR replicates of the no environment matrix mock community were performed, with each replicate comprising 20 helminth species. Table Mock helminth communities were prepared using the archived specimens at the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Thailand. Ethical clearance and experimental protocols were approved by the Animal Care and Use Committee, Faculty of Tropical Medicine, Mahidol University, Bangkok (No. FTM-ACUC 016/2021E). Helminths were identified to the species level based on morphological characters and preserved in 70% ethanol at − 20 °C. For the mock communities, helminths that had available partial 12S and 16S rRNA gene sequences from previous studies were selected2 piece of the tissue was removed for platyhelminths. Due to the small size of helminth immatures, the whole specimen  was directly used in the mock communities.Adult helminths were individually separated and washed thoroughly with sterile distilled water. A 1 mm piece of the tissue was removed for nematodes, while a flat 1 mmOnce the helminth specimens were prepared, they were spiked into each type of environment, followed by DNA extraction. One specimen per species was used for each mock community. Each mock community was homogenized with silica beads in lysis buffer using a TissueLyser LT  before DNA extraction.Human fecal material and garden soil were individually sieved using a 40 μm wire sieve mesh, and approximately 0.25 g of environmental samples were placed in a 1.7 ml microcentrifuge tube for DNA extraction with their respective helminth mock community. Total genomic DNA was then isolated from each mock community using the QIAmp® PowerFecal® DNA kit , following the manufacturer’s recommendations.Representative helminths were added into 15 ml of pond water and filtered through an 11 μm Whatman filter paper . After filtration, the tip of the filter paper was cut (approximately 1 cm in length) and placed in a 1.7 ml microcentrifuge tube for DNA extraction. Total genomic DNA was isolated using the DNeasy® Blood & Tissue kit , following the manufacturer’s recommendations.The selected specimens were placed directly in a 1.7 ml microcentrifuge tube, and total genomic DNA was isolated using the DNeasy® Blood & Tissue kit  following the manufacturer’s recommendations. The metacercaria and larva stages of representative trematodes and nematodes were used in the tissue environment to mimic the parasitic helminth immature stages that are commonly found in animal tissues. As helminths are usually isolated during animal tissue examination prior to molecular analyses, the mock tissue environment was assumed to be similar to the no environment matrix mock environment.30 that targeted parasitic nematodes and trematodes. We designed new primers targeting the 12S rRNA gene for cestodes in this study. Briefly, the primers were designed using the complete 12S rRNA gene sequences of 35 selected cestode species from the NCBI database. PCR were performed on representative cestode specimens to test the newly designed primers for its sensitivity and specificity. Reference sequences for the 12S rRNA gene of the cestode specimens used in this study were also generated by Sanger sequencing . For DNA metabarcoding, all the 12S and 16S primers were tagged with the Illumina adaptor overhang sequence . As more than one primer pair was used in the PCR reaction, multiplex PCR optimization was performed before use. PCR amplification was performed in a final volume of 30 μl containing 15 μl of 2X i-Taq™ mastermix , 5 μm to 10 μm of each primer, and 2 μl of DNA template. The PCR was conducted in a thermocycler  with the following profiles: 94 °C for 2 min of initial denaturation, followed by 94 °C for 30 s, 52 °C to 60 °C for 45 s, and 72 °C for 1 min, followed by a final extension step at 72 °C for 5 min. The PCR primers and annealing temperatures are listed in Table Three sets of PCR reactions were performed for each mock community . For the 12S rRNA gene, two PCR reactions with primers targeting each helminth group (12S-platyhelminth and 12S-nematode) were conducted per mock community, while for the 16S rRNA gene, one PCR reaction with primers targeting both platyhelminths and nematodes (16S-helminth) was performed. We used the 12S and 16S locus-specific primers from Chan et al. Library preparation was performed using the 16S metagenomic sequencing library preparation protocol with the Herculase II Fusion DNA Polymerase Nextera XT V2 kit . The library was sequenced on an Illumina MiSeq platform.https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html). The quality of the raw sequence reads obtained was checked via FastQC through the command line51. The raw sequence reads were imported into Geneious Prime® 2022.0.2 (http://www.geneious.com), where the Illumina overhang adaptors, primer sequences, and low-quality sequences below Phred score of 20 were trimmed via BBDuk. The subsequent filtered raw sequence reads were merged and sequences generated. The resulting sequences were then filtered based on the target amplicon size per genetic marker for each helminth group  and 379 bp (platyhelminth) for 16S-helminth). After filtering based on the target amplicon size, contigs were generated and exported to FASTA format. The target sequences generated per sample were aligned in ClustalX52 together with the 12S and 16S rRNA gene sequences of representative helminth species obtained from the NCBI GenBank database. The sequences of the reference helminth species used were from the complete mitochondrial genomes and the partial 12S and 16S rRNA gene sequences obtained by Chan et al.  previously30 .Supplementary Tables.


Purpose: To evaluate the corneal topographical changes in vernal keratoconjunctivitis (VKC) subjects using OCULUS Pentacam.Design: This was a cross-sectional study.Methods: VKC patients and normal subjects who fulfilled the inclusion and exclusion criteria were recruited by convenience sampling into the study. Subjects underwent a best-corrected visual acuity measurement with a Snellen chart, retinoscopy, and corneal topography (OCULUS Pentacam®), followed by anterior segment and fundus examination and intraocular pressure measurement. Data were collected and analyzed using SPSS version 26.0 for Windows . A p-value <0.05 was considered statistically significant.Results: A total of 78 eyes of 43 VKC patients and 84 eyes of normal subjects were included in the study. Most of the VKC subjects were Malay males aged 10 years or less. A majority (71.8%) had palpebral VKC of five years duration or less (57.7%) and presented between the ages of six and 10 years (44.9%). Central corneal curvature and astigmatism were significantly higher in VKC subjects compared to the normal population (p < 0.05). The minimal pachymetry was significantly lower with a longer duration of VKC (p < 0.05). Older age of presentation of VKC was associated with higher central corneal curvatures and thinner minimal pachymetry (p < 0.05). There was no association between the type of VKC and corneal topography changes. The prevalence of keratoconus and subclinical keratoconus among VKC subjects was 10.3% and 11.5%, respectively.Conclusion: Longer duration and older age of presentation of VKC are associated with significant corneal topographical changes, thus exposing them to a higher risk of the future development of keratoconus. Vernal keratoconjunctivitis (VKC) is a subtype of allergic conjunctival disease affecting the ocular surface and is characterized by chronic, bilateral inflammation of the upper tarsal and or limbal conjunctiva . VKC is Corneal involvement in VKC involves injury to the superficial and basal epithelial layers and anterior stroma . CornealEarly detection of these subtle corneal changes is vital to help arrest these changes in the early stages before developing further complications. These changes can be measured accurately by corneal topography, making it the gold standard in the screening of keratoconus and other corneal ectasias . OCULUS Most of the studies on VKC subjects were done using Placido-based corneal topographers. These studies showed the incidence of keratoconus-like topography (KLT), keratoconus, and subclinical keratoconus by corneal topography to be significantly higher than those diagnosed clinically -16. The In a study using slit-scanning topography by Baretto et al., eyes with VKC had higher elevation peaks both anteriorly and posteriorly with thinner corneas, increased central corneal curvatures, and more abnormal pachymetric indices compared to normal eyes. Twenty percent of VKC subjects had keratoconus, whereas a further 14% were found to have subclinical keratoconus .To our knowledge, no studies on VKC were conducted using Scheimpflug photography-based corneal topographers, and there are limited data on elevation-based corneal changes in VKC subjects. There are also scarce data on the corneal topographical changes in VKC based on type, duration, and age of presentation, which is valuable in clinical practice. There are no studies to determine the prevalence of keratoconus and subclinical keratoconus among VKC patients in Malaysia. Hence, we embarked on this study to determine the corneal topographical changes and prevalence of keratoconus and subclinical keratoconus in VKC among our local population by using OCULUS Pentacam®, identify those who are at a higher risk for the development of keratoconus, and establish the necessity for early corneal topography in these patients. Early detection of corneal topographical changes may allow more aggressive treatment options to prevent progression and significant ocular morbidity in these patients.This was a cross-sectional study conducted between October 2021 and January 2022 at the ophthalmology clinic of Hospital Kuala Lumpur (HKL). VKC patients on follow-up and normal subjects who presented to the ophthalmology clinic of HKL who fulfilled the inclusion criteria were recruited. The study commenced after obtaining research approval from the Medical Research and Ethical Committees of both Universiti Kebangsaan Malaysia (UKM PPI/111/8/JEP-2020-816) and the Ministry of Health (KKM/NIHSEC/P20-2311(11), KKM/NIHSEC/P20-2311) and conducted following the Declaration of Helsinki.Convenient sampling was performed for VKC patients and normal subjects (age and gender matched). The diagnosis of VKC had already been established by the consultant corneal ophthalmologist. The allergens were established with either immunoglobulin E immunoassay or skin testing. Types of VKC were divided into palpebral, limbal, and mixed based on the clinical classification by Emmert. Normal subjects were those with no ocular or systemic comorbidities, no allergies, no atopy, and with a best-corrected visual acuity (BCVA) of 6/6 and a refractive error ≤-3.00D sphere and ≤-1.75D cylinder. The inclusion criteria included all VKC patients and a comparative group of normal subjects who presented to the ophthalmology clinic of HKL during the study period. Exclusion criteria included patients unable to maintain the position for corneal topography and slit lamp examination, patients with contact lens usage within two weeks of examination, active ocular infection, and past history of ocular surgery or trauma, pregnant or lactating patients as well as patients with poor ocular surface resulting in an inability to obtain an "OK" signal via OCULUS Pentacam®. VKC subjects with acute allergic conjunctivitis, previous history of shield ulcer or corneal scars, additional ocular pathology other than VKC and keratoconus, and systemic illnesses other than those related to atopy were also excluded.A sample size of 73 eyes per group was calculated using two population means formulae based on the mean central corneal curvature (diopters) being 46.1 and 43.2 in the VKC and normal groups, respectively, with an assumption of the power of the study to be 80% at the α level of 5% . Both VKDuring the study visit, the subject’s complete medical and ocular history was obtained, followed by a complete ophthalmic examination which included BCVA with a Snellen chart , retinoscopy (Keeler), corneal topography , anterior and posterior segment examination with slit lamp biomicroscopy  which was performed together with a consultant corneal ophthalmologist and intraocular pressure measurement using Goldman applanation tonometer mounted on the slit lamp biomicroscope under topical anesthesia. Two corneal topography measurements were taken by the same two investigators, and the results were averaged. Patients with poor ocular surfaces were treated with artificial tears for two weeks before corneal topography. Only patients with scans obtaining acceptable "OK" signals on OCULUS Pentacam® were included.The parameters that were measured were the flattest meridian in the central 3 mm zone (K1), the steepest meridian in the central 3 mm zone (K2), mean corneal power in the 3 mm zone (Km), astigmatism, corneal thickness at the thinnest point of the cornea , front difference elevation (FDE), and BDE. The best-fit sphere (BFS) is the central 8 mm optical zone of the cornea, which is the commonly used reference surface to measure elevation data. An "enhanced BFS" subtracts a 4 mm zone centered on the thinnest point of the cornea from the BFS, thus maximizing the elevation difference between the apex of the cone and the rest of the peripheral cornea. The anterior and posterior elevations were thus measured by subtracting the difference in elevation between the "enhanced BFS" and the BFS.Keratoconus was defined as an eye with a characteristic slit lamp and keratometric or retinoscopic findings of keratoconus with characteristic corneal topographic abnormalities: FDE ≥ 12 μm or BDE ≥ 20 μm with corneal thickness <500 μm and Km >47 D . An eye Statistical analysis was performed using Statistical Package for Social Science (SPSS),version 26.0 for Windows . Seventy-eight eyes of 43 VKC patients and 84 eyes of 43 normal subjects were included in the final analysis.All variables were defined by descriptive analysis. The analysis of quantitative variables included a calculation of the median and interquartile range (IQR) as the data were not normally distributed. Categorical data, which were gender, age, ethnicity, laterality, and incidence of keratoconus and subclinical keratoconus, were expressed as frequencies (n) and percentages (%). The corneal topographical changes between VKC and normal subjects were analyzed using the Mann-Whitney test. The association between corneal topographical changes and type, duration, and age of presentation of VKC were analyzed using the Kruskal-Wallis test. A p-value of <0.05 was considered statistically significant.There was a total of 78 eyes of 43 VKC patients and 84 eyes of 43 normal subjects recruited in the study with no dropout. The median age of VKC and normal subjects was 14 and 15 years, respectively. Most subjects in both groups were aged 10 years or less. The age range of the subjects was 7-39 and 6-39 years for VKC and normal groups, respectively. Males outnumbered females in both groups. A majority of the subjects in both groups were Malay, followed by Chinese, Indian, and "others" which comprised one Kadazan and one Dusun subject each. There was equal distribution between the right and left eyes in both groups , followed by mixed type 20.5%). The limbal type of VKC was the least common (7.7%). The duration of VKC was indirectly proportional to the number of patients. Most of the VKC subjects had VKC for five years or less (57.7%) and first presented between the ages of six and 10 years (44.9%). Only 6.4% of patients had VKC for more than 15 years , Km (p = 0.021), and astigmatism (p < 0.001). VKC subjects had a higher steep K2, Km, and astigmatism than normal subjects Table .The limbal type of VKC consistently showed the steepest K1, K2, and Km; the highest FDE and BDE; and the thinnest minimal pachymetry. In contrast, the mixed type of VKC showed the highest corneal astigmatism. However, none of these results were statistically significant Table .Longer duration of VKC was associated with a thinner minimal pachymetry (p = 0.039). Although subjects with more than 15 years of VKC showed the highest K1, K2, FDE, and astigmatism, and subjects with 6-10 years of VKC showed the highest Km and BDE, these results were not statistically significant (Table VKC subjects who presented late between the ages of 21 and 25 years had significantly steeper K1 (p = 0.040), K2 (p = 0.028), and Km (p = 0.014) and thinner minimal pachymetry p = 0.006) than those who presented younger , followed by the limbal type (1.3%). There were no cases of keratoconus among the mixed type of VKC. Similarly, the prevalence of subclinical keratoconus was highest among the palpebral type (6.4%). However, there was a higher proportion of subclinical keratoconus among the mixed type (3.8%) compared to the limbal type (1.3%) Table .Although male VKC subjects outnumbered females by 2:1, the prevalence of keratoconus among females  was slightly higher than among males . However, in contrast, male VKC subjects  with subclinical keratoconus greatly outnumbered females  by 8:1.Due to VKC being a chronic conjunctival disease with corneal involvement, corneal topography has emerged as an essential tool for screening and detecting early changes and identifying subjects at risk prior to more progressive, irreversible changes. The ease of use and accuracy of modern corneal topographers, including Schiempflug topographers, allow quick and comfortable readings to be taken with minimal patient discomfort, thus facilitating the management of these VKC patients.Males with VKC (53) outnumbered females 25) by 2:1 in our study. Furthermore, most VKC subjects were aged 10 years or less (33.3%), followed by 11-15 years (26.9%). The number of VKC subjects steadily reduced as the subjects’ age increased. This was consistent with the clinical course of VKC itself, which usually resolves by puberty [5 by 2:1 The palpebral type of VKC was the most common in our study 71.8%), followed by the mixed 20.5%) and limbal types. A similar distribution was reported in an epidemiological study conducted in Malaysia where the palpebral type was predominant, followed by mixed and none had limbal type alone [.5% and l.8%, follGreater than half of the study subjects had a VKC duration of five years or less (57.7%), with 82.1% being 10 years or less. This is unsurprising as VKC is a self-limiting condition that usually resolves by puberty, with only a few progressing to the chronic adult form of atopic keratoconjunctivitis [Most subjects presented between the ages of six and 10 years (44.9%). This was consistent with the epidemiology of VKC itself, which predominantly affects males younger than 10 years ,3.VKC subjects showed significantly higher steep K2 (p = 0.001), Km (p = 0.021), and astigmatism (p < 0.001) compared to normal subjects. This concurs with other studies using both Placido-based and slit-scanning topographers ,15,17. TSubgroup analysis of the corneal topographical changes based on the type of VKC showed no significant results despite the limbal type consistently demonstrating steeper corneal curvatures, thinner pachymetry, and higher front and back elevation peaks. This could be attributed to the small sample size available for the limbal type (six eyes) compared to the palpebral (56 eyes) and mixed (16 eyes) types. The only other study with a similar analysis showed the same conclusion due to the limitation of sample size after stratification . NeverthA longer duration of VKC was directly proportional to thinner corneas (p = 0.039), whereby subjects with VKC of more than 15 years had a minimal pachymetry of 501 μm IQR 68) when compared with those with VKC for only five years or less who had a minimal pachymetry of 546 μm (IQR 45). This is in contrast to a study in Israel which showed no significant difference regardless of the duration of VKC despite having a similar sample size to ours ,8. Howev when comVKC subjects with older age of presentation were associated with steeper K1 (p = 0.040), K2 (p = 0.028), and Km (p = 0.014) and thinner minimal pachymetry (p = 0.006). These may be attributed to the longer duration of untreated VKC leading to more corneal damage. Since there were differences in the corneal topography between the different subtypes of VKC, future studies should involve patients of only one subtype for a more reliable comparison.87.5% (seven eyes) of keratoconus patients belonged to the palpebral type of VKC, followed by the limbal type with 12.5% (one eye). This finding mirrored a study by Chingu et al. in Turkey, where 60% of keratoconus subjects with VKC had the palpebral type, followed by 28.5% and 11.4% with mixed and limbal types, respectively  Table 99. The raThe limitation of this study was that eyes with more severe forms of VKC were excluded due to very poor ocular surface disease, the presence of corneal scars, or previous surgical intervention, as they would adversely affect the corneal topography.Longer duration and older age of VKC presentation are associated with more significant corneal topographical changes, thus leading to a higher risk of keratoconus. The prevalence of keratoconus and subclinical keratoconus among VKC subjects is similar at 10%. Corneal topography of at-risk VKC subjects offers a cost-effective modality in reducing future morbidity with education and tailored treatment.