Fusing Peptide Epitopes for Advanced Multiplex Serological Testing for SARS-CoV-2 Antibody Detection.

The tragic COVID-19 pandemic, which has seen a total of 655 million cases worldwide and a death toll of over 6.6 million seems finally tailing off. Even so, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise, the severity of which cannot be predicted in advance. This is concerning for the maintenance and stability of public health, since immune evasion and increased transmissibility may arise. Therefore, it is crucial to continue monitoring antibody responses to SARS-CoV-2 in the general population. As a complement to polymerase chain reaction tests, multiplex immunoassays are elegant tools that use individual protein or peptide antigens simultaneously to provide a high level of sensitivity and specificity. To further improve these aspects of SARS-CoV-2 antibody detection, as well as accuracy, we have developed an advanced serological peptide-based multiplex assay using antigen-fused peptide epitopes derived from both the spike and the nucleocapsid proteins. The significance of the epitopes selected for antibody detection has been verified by in silico molecular docking simulations between the peptide epitopes and reported SARS-CoV-2 antibodies. Peptides can be more easily and quickly modified and synthesized than full length proteins and can, therefore, be used in a more cost-effective manner. Three different fusion-epitope peptides (FEPs) were synthesized and tested by enzyme-linked immunosorbent assay (ELISA). A total of 145 blood serum samples were used, compromising 110 COVID-19 serum samples from COVID-19 patients and 35 negative control serum samples taken from COVID-19-free individuals before the outbreak. Interestingly, our data demonstrate that the sensitivity, specificity, and accuracy of the results for the FEP antigens are higher than for single peptide epitopes or mixtures of single peptide epitopes. Our FEP concept can be applied to different multiplex immunoassays testing not only for SARS-CoV-2 but also for various other pathogens. A significantly improved peptide-based serological assay may support the development of commercial point-of-care tests, such as lateral-flow-assays.

Risk factors and predictors that influence SARS-Cov-2 IgG positivity: A cross-sectional study of blood donors in Riyadh, Saudi Arabia.

OBJECTIVES: To study the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after pandemic's peak and before the vaccine enrollment in Riyadh, Saudi Arabia and further explore predictors for SARS-CoV-2 positivity. METHODS: A cross-sectional study of 515 blood donors from November 22 to December 17, 2020 was conducted at King Saud University Medical City, Riyadh, Saudi Arabia to look at SARS-CoV-2 immunoglobulin G (IgG) positivity. The participants were asked questions about their demographic characteristics, past SARS-CoV-2 infection, SARS-CoV-2-related symptoms and exposures. RESULTS: The seroprevalence in our study was 12.2% (n=63/515). Being a non-citizen was associated with significantly higher seroprevalence (OR 2.10, p=0.02). Participants with history of SARS-CoV-2 exposure or symptoms regardless of SARS-CoV-2 diagnosis had higher SARS-CoV-2 IgG positivity compared to unexposed or asymptomatic participants (OR 2.47, p=0.0008 or 11.19, p=0.0001, respectively). Blood donors who had symptomatic SARS-CoV-2 IgG infection had a higher SARS-CoV-2 IgG positivity rate (OR 5.04, p=0.008) and index value (p=0.003) than the asymptomatic. Of all the reported symptoms, cough (p=0.004) and anosmia (p=0.002) were significant predictors of SARS-CoV-2 IgG. CONCLUSION: The seroprevalence of SARS-CoV-2 among the blood donors in Riyadh, Saudi Arabia is considerably lower than the percentages necessary for herd immunity. Developing SARS-CoV-2-symptoms is the critical factor for higher seropositivity after SARS-CoV-2 exposure.

Protective effect of lyophilized sapodilla (Manilkara zapota) fruit extract against CCl4-induced liver damage in rats.

The tropical fruit sapodilla (Manilkara zapota syn. Achras zapota) is a rich source of nutrients, minerals and a myriad of bioactive phytochemicals such as flavonoids and catechins. Pharmacologically, sapodilla has been shown to exhibit anti-bacterial, anti-parasitic, anti-fungal, antiglycative, hypocholesterolemic and anti-cancer effects. However, its influence on hepatic tissue and serum lipids remains obscure. To address this, we used an in vivo model of liver damage to elucidate the effect of lyophilized sapodilla extract (LSE) treatment in carbon tetra chloride (CCl4) intoxicated rats. Exposure of CCl4 resulted in elevation of serum biomarkers of liver damage (aspartate transaminase, alanine aminotransferase, γ-glutamyl transferase and alkaline phosphatase), bilirubin and dysregulation of serum lipid profile (cholesterol and triglycerides). These effects were significantly and dose-dependently reversed by LSE treatment (250 and 500 mg/kg). Administration of LSE also reduced the structural damage caused by CCl4 in the liver. Furthermore, determination of oxidative stress parameters (malondialdehyde and non-protein sulfhydryls) revealed that LSE treatment mitigated CCl4-triggered modulation of both molecules. LSE also showed a strong antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ß-carotene-linoleic acid assays. In conclusion, the present study discloses the hepatoprotective and lipid-lowering effects of lyophilized sapodilla extract against CCl4-induced liver damage, an effect, at least in part, mediated by its antioxidant activity.

Vitamin D cutoff point in relation to parathyroid hormone: a population based study in Riyadh city, Saudi Arabia.

The current recommended cutoff value for low vitamin D may result in overestimation of hypovitaminosis D. Vitamin D levels at 30.0 nmol/L can diagnose the hyperparathyroid cases leading to bone loss, with moderate accuracy, in the Saudi population. The new cutoff may help in identifying true cases that need clinical treatment and can reduce the burden on healthcare system. PURPOSE: Different regions of the world have reported varying cutoff points as optimal values for vitamin D status to maintain bone health. METHODS: A cross-sectional study comprising of interviews, anthropometrics, and blood samples was conducted in primary healthcare centers in Riyadh, Saudi Arabia. Standardized serum 25-hydroxyvitamin D [25(OH)D] and parathyroid hormone (PTH) were measured using electrochemiluminescence immunoassays. Independent sample and paired sample t test were conducted to compare the true means. Pearson correlation co-efficient was calculated to measure the association between original and standardized 25(OH)D. Software program, MedCalc, was utilized to measure the receiver operating curve (ROC) for determining the optimal threshold value for vitamin D. RESULTS: The mean standardized 25(OH)D levels for 846 males and 1285 females were (32.0 ± 14.4 nmol/L vs 31.6 ± 16.7 nmol/L) respectively. Using the gold standard PTH cutoff > 6.9 pmol/L, the ROC had an optimal criterion value for males and females at 30.0 and 24.0 nmol/L, respectively. In the males, the sensitivity and specificity were 72% and 51%, whereas in females, it was 58.2% and 66.7%, respectively. The area under the curve (AUC) was at 0.62 and 0.65 (p < 0.001), respectively. CONCLUSION: The recommended cutoff value for 25(OH)D for determining bone health in the Saudi population is at 30.0 nmol/L. The comparatively low cutoff point can significantly decrease the number of people diagnosed and treated with low vitamin D, which can also reduce the burden on the health care system.

Role of Apolipoprotein E gene polymorphism in the risk of familial hypercholesterolemia: a case-control study.

Familial Hypercholesterolemia (FH) is characterized by elevated cholesterol and based on biochemical, clinical, and genetic studies and FH disease, which was documented even with limited mutations. Earlier studies focused on Apolipoprotein E (ApoE) in variable diseases. The current study aimed to investigate the genetic association between FH disease and ApoE gene polymorphisms (rs429358 and rs7412) in the Saudi population. This case-control study was a hospital-based study performed in Saudi Arabia. Two hundred and four subjects in total were recruited and consisted of FH participants (n=104) and the controls (n=100). Common polymorphisms of ApoE gene (rs429358 and rs7412) were chosen and subjected to the genotyping using the TaqMan assay. Moreover, the ApoE risk allele E4 was proved significantly associated with FH cases when compared with controls (OR-2.24 (95%CI: 1.06-4.70); p=0.02). Lipid profile parameters were significantly associated (p<0.05); however, the ApoE alleles and lipid profiles were not correlated (p>0.05). In conclusion, the FH case-control study was associated with the E4 allele in the Saudi population. However, E4 allele was appeared as a reliable risk marker for lipid profiles, but not for ApoE alleles.

Q192R polymorphism in the PON1 gene and familial hypercholesterolemia in a Saudi population.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant condition characterized by abnormal levels of low-density lipoprotein (LDL) in the blood. FH is a risk factor for atherosclerosis and cardiovascular disease. The relationship between the paraoxonase 1 (PON1) gene, atherosclerosis and coronary artery disease has not been studied in Saudi patients. OBJECTIVE: To investigate the genetic associations of the Q192R polymorphism in the PON1 gene with FH in Saudi patients. DESIGN: Case-control study. SETTING: Tertiary care center, Riyadh. METHODS: Two hundred Saudi patients were enrolled in this study, including 100 patients with FH and 100 healthy controls, during the period from January 2012 to March 2013. Serum was separated from coagulated blood (3 mL) and used for analysis of lipid profiles. Genomic DNA was isolated from anticoagulant-treated blood (2 mL). Genotyping for the Q192R polymorphism was performed by polymerase chain reaction-restriction fragment length polymorphism analysis, followed by 3% agarose gel electrophoresis. MAIN OUTCOME MEASURE: The strength of association between the Q192R polymorphism and FH in the Saudi population. RESULTS: We confirmed that QR versus QQ (odds ratio [OR]: 1.55; 95% confidence interval [CI]: 1.05-3.43; P=.03), QR+RR versus QQ (OR: 1.98; 95% CI: 1.13-3.49; P=.01), and R versus Q (OR: 1.68; 95% CI: 1.09- 2.59; P=.01) in the Q192R polymorphism were associated with FH in the Saudi population. CONCLUSION: In conclusion, the Q192R polymorphism in the PON1 gene is associated with FH in the Saudi population. Our results confirmed that the R allele, QR, and dominant model genotypes were associated with FH. LIMITATION: Only a single variant (Q192R) was analyzed, and the medical and family histories of the patients were not known.

Diagnostic efficacy of random albumin creatinine ratio for detection of micro and macro-albuminuria in type 2 diabetes mellitus.

OBJECTIVES: To compare a less cumbersome random albumin creatinine ratio (RACR) with 24-hour urinary albumin excretion (UAE) for detection of renal damage in patients with type 2 diabetes mellitus (T2DM). METHODS: This retrospective study performed between March 2013 and June 2014 at the Department of Pathology, King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia included 122 patients (mean age 54 ± 15, 104 females and 18 males) with T2DM. Urine albumin levels of less than 30 mg/g was considered normal, from 30-300 mg/g considered as micro-albuminuria, and over 300 mg/g considered as macro-albuminuria. RESULTS: Concordance between the 2 assays was observed in 114 (93.4%) samples. The sensitivity of RACR assay was 100%, specificity was 91.3% with a positive predictive value (PPV) of 95%, and a negative predictive value (NPV) of 100% in micro-albuminuria range. For macro-albuminuria, RACR had a sensitivity of 100%, specificity of 94.1% with PPV of 94% and NPV of 100%. Receiver operating characteristic (ROC) curves analysis cut-off values of 40 mg/g-300 mg/g for micro- and greater than 300 mg/g for macro-albuminuria revealed 100% sensitivity, 97.5% specificity, 95% PPV, and 100% NPV for micro-albuminuria, and 100% sensitivity, 94% specificity, 76% PPV, and 100% NPP for macro-albuminuria. The area under the curve for micro-albuminuria was 100% and 98.2% for macro-albuminuria. CONCLUSION: Performance of RACR was comparable to 24 hour UAE assay particularly in excluding renal damage in T2DM.

Lactate dehydrogenase as a biomarker for early renal damage in patients with sickle cell disease.

Among many complications of sickle cell disease, renal failure is the main contributor to early mortality. It is present in up to 21% of patients with sickle cell disease. Although screening for microalbuminuria and proteinuria is the current acceptable practice to detect and follow renal damage in patients with sickle cell disease, there is a crucial need for other, more sensitive biomarkers. This becomes especially true knowing that those biomarkers start to appear only after more than 60% of the kidney function is lost. The primary purpose of this study is to determine whether lactate dehydrogenase (LDH) correlates with other, direct and indirect bio-markers of renal insufficiency in patients with sickle cell disease and, therefore, could be used as a biomarker for early renal damage in patients with sickle cell disease. Fifty-five patients with an established diagnosis of sickle cell disease were recruited to in the study. Blood samples were taken and 24-h urine collection samples were collected. Using Statcrunch, a data analysis tool available on the web, we studied the correlation between LDH and other biomarkers of kidney function as well as the distribution and relationship between the variables. Regression analysis showed a significant negative correlation between serum LDH and creatinine clearance, R (correlation coefficient) = -0.44, P = 0.0008. This correlation was more significant at younger age. This study shows that in sickle cell patients LDH correlates with creatinine clearance and, therefore, LDH could serve as a biomarker to predict renal insufficiency in those patients.

Screening for genetic mutations in LDLR gene with familial hypercholesterolemia patients in the Saudi population.

Familial hypercholesterolemia (FH) is caused by genetic defects involving the low density lipoprotein-receptor (LDL-R), predisposing affected people to premature atherosclerotic cardiovascular disease and death. The aim of the present study was to assess certain exons in the LDLR gene mutation detection analysis affecting in the Saudi population with FH. This case-control study was carried out with 200 subjects; 100 were FH cases and 100 were healthy controls. Five mL of venous blood samples were collected from all the subjects and used for biochemical and genetic analysis. DNA was extracted from 2 mL of the EDTA samples, and precise primers were designed for LDL-R gene which includes Exon 3, 4 and 8. PCR was followed by DNA sequencing. In our study, we found 25 mutations in cases in Exon-3 and 2 mutations in controls, however, we have found only 5 mutations in exon 4 and none of the mutations were identified in exon 8. We conclude that screening of FH among Saudi population is very important to identify individuals who are prone to develop the disease.

Unusual presentation of arsenic poisoning in a case of celiac disease.

Arsenic poisoning may occur from sources other than drinking water such as rice, seafood, or insecticides. Symptoms and signs can be insidious, non-specific, atypical, and easily overlooked. We present a 39-year-old woman with celiac disease who was on gluten-free diet for 8 years and presented with diarrhea, headache, insomnia, loss of appetite, abnormal taste, and impaired short-term memory and concentration, but with no skin lesions. Arsenic concentration in her 24-hour urine was 682.77 micro g/g creatinine (normal < 15). She responded very well to chelation therapy with dimercaptosuccinic acid given orally and recovered within 2 weeks. The suspected source of arsenic poisoning was rice, as drink.ing contaminated ground water is not known in Saudi Arabia and she had not taken seafood. Therefore, arsenic poisoning should be suspected based on the meticulous medical history in cases of patients with celiac disease whose main food is rice and who present with unusual symptoms.

Autoantibody detection: prevailing practices at a tertiary care hospital in Riyadh.

BACKGROUND: Anti-nuclear antibody (ANA) test as the first level investigation for detection of auto-immune rheumatic disease has been recommended in a number of international guidelines. This study was performed to evaluate the local practice and trends of auto-antibody laboratory requests. METHODS: Data were collected from 249 initial laboratory requests for first level auto-antibody detection between April 2012 and April 2013 in the Immunology Unit at King Khalid University Hospital, Riyadh. This group of patients included 151 females and 98 males (mean age 40.1 +/- 21; range 4-85 years). RESULTS: Of the total requests, ANA as a single first level investigation was requested by only 32 (13%) clinicians whereas the rest of the investigations included simultaneous testing of ANA and second level extractable nuclear antigen (ENA) auto-antibody tests. Anti-double stranded DNA (anti-dsDNA) antibody was simultaneously tested with ANA in 158 patients as first level test where both the tests were positive in 44 (27.8%) patients and in 24 (15.1%) patients a negative ANA test was associated with a positive anti-dsDNA antibody test. Rheumatoid factor (RF) tested positive in 04/53 (7.5%), anti-neutrophil cytoplasmic antibody (ANCA) in 01/48 (2%) and SS-A and SS-B in 03/37 (8.1%) requests as first level tests with ANA. CONCLUSIONS: Using second level auto-antibody tests in conjunction with ANA as the first line investigation does not appear to be a cost effective approach, highlighting the importance of adherence to the guidelines. ANA negative and anti-dsDNA positive group of patients requires further assessment in a large scale study.

Assessment of trace elements in sera of patients undergoing renal dialysis.

OBJECTIVE: To assess the serum levels of copper, zinc, iron, and lead in patients on maintenance dialysis. METHODS: This cross-sectional study performed at King Khalid University Hospital, Riyadh, Saudi Arabia between September 2011 and October 2012 included 42 patients with end stage renal disease on hemodialysis (HD), 18 patients on peritoneal dialysis (PD), and 18 normal controls. Serum copper, zinc, and lead levels were determined by atomic absorption spectrophotometry, and serum iron was determined by spectrophotometric determination. RESULTS: The median serum copper level in HD patients (20.5 nmol/L; 95% confidence interval [CI]: 17.52-22.39; interquartile range [IQR]: 16.40-24.20) was higher (p=0.001) than the controls (14.30 nmol/L; 95% CI: 9.72-16.91; IQR: 9.70-17), and the PD patients (15.60 nmol/L; 95% CI: 14.17-16.66; IQR: 14.10-16.70). Although no different from PD patients` serum levels of zinc in HD patients (9.50 nmol/L; 95% CI: 7.83-12.09; IQR: 7.00-14.40) were lower than controls (13.20 nmol/L; 95% CI: 10.65-15.22; IQR: 10.58-15.35; p=0.03). Copper/zinc ratio in HD patients was 2.4, 2.5 in PD patients, and 0.88 in controls. The serum iron levels in HD patients (10 mmol/L; 95% CI: 8.03-11.96; IQR: 7-14.50; p=0.003), and PD patients (10 mmol/L; 95% CI 6.56-14.43; IQR 5.50-15; p=0.03) were lower than controls. Serum lead levels in PD patients (0.11 umol/L; 95% CI: 0.02-0.14; IQR: 0.02-0.14) were lower than HD patients (0.18 umol/; 95% CI: 0.15-0.21; IQR: 0.13-0.25; p=0.005), and controls (0.15 umol/L; 95% CI: 0.07-0.24; IQR: 0.06-0.25; p=0.04). CONCLUSION: Alterations in serum trace elements emphasize the need for monitoring trace elements in patients receiving maintenance dialysis.

Prevalence of vitamin D deficiency in Saudi newborns at a tertiary care center.

OBJECTIVE: To evaluate vitamin D levels in Saudi newborns utilizing umbilical cord samples, and to benchmark the results with international figures. METHODS: This cross-sectional study was carried out at King Khalid University Hospital, Riyadh, Saudi Arabia between November 2013 and March 2013. Vitamin D levels were assessed in the umbilical cord of healthy term neonates born above 2.5 kg from healthy pregnant mothers. Gestational age (GA), birth weight, gender, levels of sun exposure, and consumption of vitamin D rich food data were collected. Our primary outcome was the percentage of newborns with vitamin D deficiency (vitamin D level below 25 nmol/l). Association of vitamin D deficiency with sun exposure and consumption of vitamin D rich food was tested using a Chi-squared test. RESULTS: Umbilical samples of 200 newborns were obtained. The average birth weight was 3.2 kg. Deficient vitamin D levels were detected in 59% of the sample. Almost 90% of included newborns had vitamin D levels below 50 nmol/l. We found no association of vitamin D deficiency status to level of sun exposure or to consumption of vitamin D rich food. CONCLUSION: Vitamin D deficiency is very common in Saudi newborns at hospital, and is consistent with regional data. Efforts to assess and treat vitamin D deficiency during pregnancy and provide adequate supplementation to newborns are necessary to rectify such a public health concern.

Association of angiotensin converting enzyme gene insertion/deletion polymorphism and familial hypercholesterolemia in the Saudi population.

BACKGROUND: The study of the association between genotype and phenotype is of great importance for the prediction of multiple diseases and pathophysiological conditions. The relationship between angiotensin converting enzyme (ACE) Insertion/Deletion (I/D) polymorphism and Familial Hypercholesterolemia (FH) has been not fully investigated in all the ethnicities. In this study we sought to determine the frequency of I/D polymorphism genotypes of ACE gene in Saudi patients with FH. RESULTS: This is a case-control study carried out purely in Saudi population. Genomic DNA was isolated from 128 subjects who have participated in this study. ACE gene I/D polymorphism was analyzed by polymerase chain reaction in 64 FH cases and 64 healthy controls. There was no statistically significant difference between the groups with respect to genotype distribution. Furthermore, we did not find any significant difference in the frequency of ACE I/D polymorphism in FH subjects when stratified by gender (p = 0.43). CONCLUSION: Our data suggest that ACE gene I/D polymorphism examined in this study has no role in predicting the occurrence and diagnosis of FH.

Prognostically significant fusion oncogenes in Pakistani patients with adult acute lymphoblastic leukemia and their association with disease biology and outcome.

BACKGROUND AND OBJECTIVES: Chromosomal abnormalities play an important role in genesis of acute lymphoblastic leukemia (ALL) and have prognostic implications. Five major risk stratifying fusion genes in ALL are BCR-ABL, MLL-AF4, ETV6-RUNX11, E2A-PBX1 and SIL-TAL1. This work aimed to detect common chromosomal translocations and associated fusion oncogenes in adult ALL patients and study their relationship with clinical features and treatment outcome. METHODS: We studied fusion oncogenes in 104 adult ALL patients using RT-PCR and interphase-FISH at diagnosis and their association with clinical characteristics and treatment outcome. RESULTS: Five most common fusion genes i.e. BCR-ABL (t 9; 22), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (Del 1p32) were found in 82/104 (79%) patients. TCF3-PBX1 fusion gene was associated with lymphadenopathy, SIL-TAL positive patients had frequent organomegaly and usually presented with a platelets count of less than 50 x10(9)/l. Survival of patients with fusion gene ETV6-RUNX1 was better when compared to patients harboring other genes. MLL-AF4 and BCR-ABL positivity characterized a subset of adult ALL patients with aggressive clinical behaviour and a poor outcome. CONCLUSIONS: This is the first study from Pakistan which investigated the frequency of 5 fusion oncogenes in adult ALL patients, and their association with clinical features, treatment response and outcome. Frequencies of some of the oncogenes were different from those reported elsewhere and they appear to be associated with distinct clinical characteristics and treatment outcome. This information will help in the prognostic stratification and risk adapted management of adult ALL patients.

Zinc and antioxidant vitamin deficiency in patients with severe sickle cell anemia.

BACKGROUND: Patients with severe sickle cell anemia (SCA) have a higher potential for oxidative damage due to chronic redox imbalance in red blood cells that often leads to hemolysis, endothelial injury and recurrent vaso-occlusive episodes. This study evaluated the plasma levels of vitamins A, C and E as indicators of antioxidant status. In addition, serum levels of zinc and copper were also estimated. PATIENTS AND METHODS: Twenty-five adult patients with severe sickle cell anemia (12 males and 13 females aged 29.72+/-12.94 years) and 25 matched controls were studied. Plasma levels of vitamins A, C and E were measured by HPLC technique. Serum zinc and copper levels were measured by atomic absorption spectrometry. RESULTS: There was a significant decrease in plasma levels of vitamins A, C and E and in serum levels of zinc in patients with SCA as compared with controls (P<0.0001). Serum copper levels were signficantly elevated compared with controls (P<0.0001). CONCLUSION: These findings emphasize the significant deficiencies of the antioxidant vitamins A, C and E and the trace element zinc along with the significant elevation of serum copper in patients with severe sickle cell disease. Further studies are needed to find out whether supplementation of antioxidant vitamins and zinc may ameliorate some sickle cell disease complications.