Correlation of Plasma 25(OH)D3 and Vitamin D Binding Protein Levels with COVID-19 Severity and Outcome in Hospitalized Patients.

BACKGROUND: The Coronavirus Disease-19 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been declared a worldwide pandemic. The severity of COVID-19 varies greatly across infected individuals. Possible factors may include plasma levels of 25(OH)D and vitamin D binding protein (DBP), as both are involved in the host immune response. Other possible nutrition-related factors include malnutrition and/or obesity which disrupt the optimal host immune response to infections. Current literature shows inconsistent evidence about the association of plasma 25(OH)D3 and DBP on infection severity and clinical outcomes. OBJECTIVES: This study aimed to measure plasma 25(OH)D3 and DBP in hospitalized COVID-19 cases and assess their correlation with infection severity, inflammatory markers, and clinical outcome. METHODS: 167 patients were included in this analytical cross-sectional study, of which 81 were critical and 86 were non-critical hospitalized COVID-19 patients. Plasma levels of 25(OH)D3, DBP, and the inflammatory cytokines, IL-6, IL-8, IL-10, and TNF-α were assessed using the Enzyme-linked Immunosorbent Assay (ELISA). Information regarding biochemical and anthropometrical indices, hospital length of stay (LoS), and illness outcome was obtained from the medical records. RESULTS: Plasma 25(OH)D3 level was found to be significantly lower in critical compared to non-critical patients (Median = 8.38 (IQR = 2.33) vs. 9.83 (IQR = 3.03) nmol/L, respectively; p < 0.001), and it positively correlated with hospital LoS. However, plasma 25(OH)D3 did not correlate with mortality or any of the inflammatory markers. DBP on the other hand correlated positively with mortality (rs = 0.188, p = 0.015) and hospital LoS (rs = 0.233, p = 0.002). DBP was significantly higher in critical than non-critical patients (Median = 1262.18 (IQR = 463.66) vs. 1153.35 (IQR = 418.46) ng/mL, respectively; p < 0.001). Furthermore, IL-6 and IL-8 were significantly higher in critical than non-critical patients. However, no differences were found in IL-10, TNF-α, IL-10/TNF-α, TNF-α/IL-10, IL-6/IL-10, or CRP between groups. CONCLUSION: The current study found that critical COVID-19 patients had lower 25(OH)D3 than non-critical patients, yet, levels were found to be suboptimal in both groups. Further, critical patients had higher DBP levels as compared to non-critical patients. This finding may encourage future research to unravel the effects of this understudied protein that appears to have significant associations with inflammation, even though the precise mechanism is unknown.

Assessment of Proinflammatory Cytokines Among Patients with Middle East Respiratory Syndrome Coronavirus Infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with significant morbidity and mortality. This study was performed to assess the proinflammatory cytokines profile among MERS-CoV patients. A total of 46 MERS-CoV-infected patients (27 symptomatic and 19 asymptomatic) were assessed and compared with 52 normal healthy controls for plasma levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-17, IL-7, IL-6, interferon (IFN)-α, and IL-15 using a customized luminex kit. Whereas asymptomatic MERS-CoV patients and controls were no different; the mean plasma levels among MERS-CoV symptomatic patients were significantly higher than the normal controls: IL-1ß (16.89 ± 1.23 vs. 12.80 ± 0.59 pg/mL; p < 0.001), TNF-α (14.04 ± 0.93 vs. 10.35 ± 0.29 pg/mL; p < 0.0001), IL-17 (14.3 ± 0.89 vs. 11.47 ± 0.61 pg/mL; p < 0.001), IL-7 (21.56 ± 1.00 vs. 16.31 ± 0.30 pg/mL; p < 0.0001), IL-6 (156.5 ± 37.90 vs. 18.60 ± 1.59 pg/mL; p < 0.0001), and IFN-α (68.73 ± 13.06 vs. 23.57 ± 1.05 pg/mL; p < 0.0001). The mean plasma levels of IL-7 (24.81 ± 1.63 vs. 19.79 ± 0.94 pg/mL; p < 0.01), IL-6 (312.7 ± 94.67 vs. 101.2 ± 25.67 pg/mL; p < 0.01), and IFN-α (89.00 ± 18.97 vs. 51.05 ± 8.68 pg/mL; p < 0.05) were significantly elevated among MERS-CoV symptomatic patients with fatal outcome compared with MERS-CoV symptomatic patients who survived. Only IL-7 was found to have a higher risk ratio of mortality (4.76, 95% confidence interval: 1.5-14.94; p < 0.01). No differences were observed in IL-15 levels among the groups. Significantly elevated proinflammatory cytokines among symptomatic MERS-CoV-infected patients may contribute to manifestations of cytokine storm frequently observed among critically ill MERS-CoV patients and IL-7 may serve as a marker for disease activity.

Differential expression of carcinoembryonic antigen-related cell adhesion molecule-5 (CEACAM5) and dipeptidyl peptidase-4 (DPP4) with detection of Middle East respiratory syndrome-coronavirus in peripheral blood.

BACKGROUND: Middle East respiratory syndrome-coronavirus (MERS-CoV) utilizes CD26 (dipeptidyl peptidase-4) and CD66e or CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5) receptors for cell infection. Peripheral blood mononuclear cells (PBMCs) play a critical role in mounting adaptive immune response against the virus. This study was performed to assess the expression of CD26 and CD66e on PBMCs and their susceptibility to MERS-CoV infection. METHODS: Surface expression of CD26 and CD66e receptors on PBMCs from MERS-CoV patients (n = 20) and healthy controls (n = 20) was assessed by flow cytometry and the soluble forms were determined by enzyme-linked immunosorbent assay (ELISA). MERS-CoV UpE and Orf1a genes in PBMCs were detected by using Altona diagnostics reverse transcription polymerase chain reaction (RT-PCR) kit. RESULTS: Mean fluorescent intensity (MFI) of CD66e was significantly higher on CD4 + lymphocytes (462.4 ± 64.35 vs 325.1 ± 19.69; p < 0.05) and CD8 + lymphocytes (533.8 ± 55.32 vs 392.4 ± 37.73; p < 0.04) from patients with MERS-CoV infection compared to the normal controls. No difference in MFI for CD66e was observed on monocytes (381.8 ± 40.34 vs 266.8 ± 20.6; p = 0.3) between the patients and controls. Soluble form of CD66e among MERS-CoV patients was also higher than the normal controls (mean= 338.7 ± 58.75 vs 160.7 ± 29.49 ng/mL; p < 0.01). Surface expression of CD26 on PBMCs and its soluble form were no different between the groups. MERS-CoV was detected by RT-PCR in 16/20 (80%) patients from whole blood, among them 8 patients were tested in PBMCs, 4/8 (50%) patients were positive. CONCLUSION: Increased expression levels of CD66e (CEACAM5) may contribute to increased susceptibility of PBMCs to MERS-CoV infection and disease progression.

Correlation of Gut Microbiota, Vitamin D Status, and Pulmonary Function Tests in Children With Cystic Fibrosis.

Background: Children with cystic fibrosis (CF) are expected to have suboptimal serum vitamin D status and altered gut microbiota. The altered gut microbiota is hypothesized to have a pro-inflammatory effect that further complicates the existing respiratory inflammation. Emerging evidence suggests an association between vitamin D and gut microbiota. The aim of this study was to assess the relationships between 25-hydroxyvitamin D [25(OH)D] status, pulmonary function, and fecal bacteria in children with CF. Methods: In this cross-sectional study, a total of 35 children with CF (8.7 ± 2.83 years) and 24 controls without CF (9 ± 2.7 years) were included in this study. Serum 25(OH)D status was measured using the Elecsys vitamin D total II assay. In the CF group, gut microbiota composition was assessed using real-time PCR analysis. Pulmonary function tests (PFTs) were measured using spirometry. Comparisons between the CF and non-CF controls were conducted using the independent sample t-test. In the CF group, one-way analysis of variance (ANOVA) was used to assess differences in PFTs and gut microbiota composition across the three vitamin D subgroups. The correlations between 25(OH)D status and PFTs, or gut microbiota composition, and PFTs with gut microbiota composition were analyzed using the Pearson's correlation coefficient test. Results: Children with CF had significantly lower serum 25(OH)D levels compared with children without CF (44.3 ± 22.4 vs. 59 ± 25.5, respectively, P = 0.026). Children with CF with optimal serum 25(OH)D level had significantly higher levels of Bacteroidetes, Firmicutes, and total bacteria (P = 0.007, P = 0.007, and P = 0.022, respectively). The level of Firmicutes was found to be significantly higher in mild forced expiratory volume in 1 s (FEV1) compared with moderate FEV1 (P = 0.032), whereas the level of the other bacteria species was comparable across FEV1 severity groups. Conclusion: Our findings may encourage studies that target and modify gut microbiota to potentially achieve better outcomes in terms of respiratory function in CF.

Disseminated histoplasmosis in a heart transplant recipient from Saudi Arabia: A case report.

Histoplasma is endemic in North and Central America. We describe a case of disseminated histoplasmosis in a heart transplant recipient outside the known endemic areas. A 68-year-old gentleman known to have dilated cardiomyopathy. He underwent left ventricular assist device (LVAD) implantation in India and 2 years later did heart transplant in King Faisal Specialist and Research Center Hospital. Six weeks post-transplant he presented with headache and fever. All investigations were negative, and he was discharged home. Four days after discharge he presented with headache, fever, blurred vision, and an episode of loss of consciousness. Examination showed an ill looking patient who is highly febrile. Repeated work up showed pancytopenia. A repeat LP was negative. Bone marrow biopsy showed Small intracellular organisms. Extended work up revealed a positive Histoplasma urinary antigen, positive Histoplasma PCR from the bone marrow biopsy. Patient was started on Liposomal Amphotericin followed by Itraconazole with marker clinical improvement. This is the first reported case of disseminated Histoplasmosis in Saudi Arabia. We postulate that the patient had reactivation of a latent infection acquired at the time of LVAD insertion in India rather than donor derived infection by the negative fungal culture and PCR done on the donor's lung granuloma tissue.

Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing.

BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None.

Ten-year experience of Q fever endocarditis in a tertiary cardiac center in Saudi Arabia.

BACKGROUND: Q fever endocarditis (QFE) is considered rare in the Middle East, with only a few cases reported in Saudi Arabia. The aim of this study is to report on the experience of our centre on QFE. METHODS: We searched the medical records for cases of QFE at our cardiac center from 2009-2018. Demographic data, clinical features, serology and echocardiography results, treatments, and outcomes were assessed. RESULTS: Five hundred and two cases of infective endocarditis were detected over the 10 years period. Among the 234 patients with blood culture-negative endocarditis (BCNE), 19 (8.10%) had QFE. All patients had a previously diagnosed congenital heart disease except for one patient with rheumatic heart disease. Eleven patients had received a bovine jugular vein-related implant, e.g., a Melody valve (seven patients) or Contegra conduit (four patients). Coinfection was detected in three patients, and immunologic and embolic phenomena were observed in five patients. All patients received a combination of hydroxychloroquine and doxycycline, with good outcomes. Only two patients required surgery while on treatment. Two patients died several months after treatment; the cause of death was not identified. CONCLUSION: This study indicates that Q fever exists in our population. The majority of the patients had congenital heart disease (CHD) and underwent bovine jugular vein implants. Patients with CHD are at increased risk of infective endocarditis. Bovine jugular vein implants increase the risk of infective and possibly QFE. Proper exclusion of Q fever is warranted in all BCNE and possibly in culture-positive endocarditis cases in areas endemic to Q fever. KEY POINTS: We presented the largest series of Q fever endocarditis cases in Saudi Arabia. We showed that Q fever is not rare in the Middle East and suggest that it should be considered in all blood culture-negative endocarditis cases.

Colonic basidiobolomycosis presenting with intestinal obstruction and a normal eosinophil count.

Gastrointestinal basidiobolomycosis (GIB), caused by Basidiobolus ranrum, is a rare fungal infection with a limited geographic distribution. The majority of the cases are reported from the warm areas of Arizona in USA, Saudi Arabia and Iran. We report a middle aged patient who was admitted to hospital with suspected metastatic colonic carcinoma. He presented with constipation, anorexia and weight loss. Computed tomography scan disclosed a mass involving the mid and distal sigmoid colon and hypodense lesion in hepatic segment IV. Excised tissue during a Hartmann's surgery showed an extensive eosinophil-rich transmural inflammation with mural necrotizing granulomas and several broad septated fungal hyphae. He was commenced on voriconazole following surgery. The diagnosis of basidiobolomycosis was established by histopathological examination. Since the diagnosis was not suspected preoperatively tissue culture for fungi was not collected. However molecular testing confirmed the diagnosis of GIB. Therapy involved a combination of surgical resection of the mass and prolonged voriconazole treatment. Increased awareness among physicians is needed for early diagnosis and treatment of GIB.

Disseminated angioinvasive basidiobolomycosis with a favourable outcome.

Basidiobolomycosis, a rare fungal infection, is of worldwide distribution but areas commonly involved include the tropical areas of Africa, USA and South East Asia. 88% of the cases are reported among patients younger than 20 years. Many of the case reports in Saudi Arabia are from Tohama area where our patient lives. The diagnosis tends to be overlooked as the presentation may mimic colonic carcinoma in adults or inflammatory bowel diseases and tuberculosis in both children and adults. Angioinvasion seen in our patient is extremely rare suggesting the diagnosis of mucormycosis and resulting in a delay in choosing the most appropriate treatment. We report this case to remind physicians and surgeons to consider this diagnosis in patients from endemic area presenting with such conditions.

In-vitro anticancer and antimicrobial activities of PLGA/silver nanofiber composites prepared by electrospinning.

In the present work, a series of 0, 1 and 7 wt% silver nano-particles (Ag NPs) incorporated poly lactic-co-glycolic acid (PLGA) nano-fibers were synthesized by the electrospinning process. The PLGA/Ag nano-fibers sheets were characterized using SEM, TEM and DSC analyses. The three synthesized PLGA/silver nano-fiber composites were screened for anticancer activity against liver cancer cell line using MTT and LDH assays. The anticancer activity of PLGA nano-fibers showed a remarkable improvement due to increasing the concentration of the Ag NPs. In addition to the given result, PLGA nano-fibers did not show any cytotoxic effect. However, PLGA nano-fibers that contain 1 % nano silver showed anticancer activity of 8.8 %, through increasing the concentration of the nano silver to 7 % onto PLGA nano-fibers, the anticancer activity was enhanced to a 67.6 %. Furthermore, the antibacterial activities of these three nano-fibers, against the five bacteria strains namely; E.coli o157:H7 ATCC 51659, Staphylococcus aureus ATCC 13565, Bacillus cereus EMCC 1080, Listeria monocytogenes EMCC 1875 and Salmonella typhimurium ATCC25566 using the disc diffusion method, were evaluated. Sample with an enhanced inhibitory effect was PLGA/Ag NPs (7 %) which inhibited all strains (inhibition zone diameter 10 mm); PLGA/Ag NPs (1 %) sample inhibited only one strain (B. cereus) with zone diameter 8 mm. The PLGA nano-fiber sample has not shown any antimicrobial activity. Based on the anticancer as well as the antimicrobial results in this study, it can be postulated that: PLGA nanofibers containing 7 % nano silver are suitable as anticancer- and antibiotic-drug delivery systems, as they will increase the anticancer as well as the antibiotic drug potency without cytotoxicity effect on the normal cells. These findings also suggest that Ag NPs, of the size (5-10 nm) evaluated in the present study, are appropriate for therapeutic application from a safety standpoint.