Carcinoembryonic antigen (CEA) and other surrogate inflammatory biomarkers in COVID-19

It has been two years since the global outbreak of the highly contagious and deadly corona virus disease (COVID-19) caused by SARS-CoV-2 first emerged in China. Since then, various diagnostic, prognostic and treatment strategies undertaken to address the pandemic have been dynamically evolving. Predictive and prognostic role of various biomarkers in COVID-19 has been a subject of intense exploration. We aimed to determine the association of Carcinoembryonic antigen (CEA) and various surrogate inflammatory biomarkers with the severity of COVID-19 disease. This retrospective cohort study was carried out on 98 patients admitted in Jaypee Hospital, Noida with COVID-19 disease. Information regarding demographics, laboratory parameters and clinical history was collected from Hospital Information System. Serum levels of CEA and other biomarkers such as Neutrophil-lymphocyte ratio (NLR), C-reactive protein (CRP), Interleukin-6 (IL-6), Ferritin, and Procalcitonin (PCT) were assessed. Correlation analyses were performed between the parameters and acute respiratory distress syndrome (ARDS) stages. Logistic regression and ROC curve analysis were performed to assess the various parameters for distinguishing COVID-19 patients requiring ICU admission. Mean hospital stay, NLR, CEA, IL-6, CRP, Ferritin (P< 0.0001) and PCT (P =0.01) were significantly higher in ICU patients when compared to general ward patients. NLR, median serum CEA, IL-6, and CRP levels were significantly higher in non-survivor compared to the survivors (P< 0.0001, 0.0341 and 0.0092). CEA correlated well with disease severity based upon ARDS classification and was a better marker to differentiate patient according to ARDS stages (ARDS 0 vs 2 P= 0.0006; 0 vs 3 P< 0.0001; ARDS 1 vs 2 P= 0.0183; 1 vs 3 P=0.0006). The area under the Receiver operating characteristic (ROC) curve for CEA was 0.7467 (95% CI- 0.64885- 0.84459) which revealed the potential of CEA as a biomarker to distinguish COVID-19 patients requiring ICU admission. CEA can be used to predict the severity of COVID-19 associated ARDS as well as patients requiring ICU admission. Along with routine inflammatory biomarkers (NLR, CRP, IL-6, PCT, and ferritin), CEA should be used for early identification of critical COVID-19 positive patients and for assessing prognosis.

Immunoprophylactic studies with a 43 kDa human circulating filarial antigen and a cross reactive 120 kDa Brugia malayi sodium dodecyl sulphate soluble antigen in filariasis.

Bancroftian filariasis is a major public health problem affecting about 120 million people all over the world. Immunoprophylaxis may serve as an additional adjunct along with chemotherapy and anti larval measures for successful filaria control. Circulating filarial antigen fraction (CFA2-6) containing 43 kDa antigen and adult Brugia malayi sodium dodecyl sulphate (S DS) soluble antigen fraction BmA-2 with a 120 kDa molecule were earlier shown to be reactive with endemic normal sera by immunoblotting and indirect ELISA techniques. BmA-2 was found to be highly cross reactive with CFA2-6. Sera raised against both the antigen fractions showed about 9 0 % cytotoxicity to the parasites in the presence of jird peritoneal cells in in vitro as well as by in situ micropore chamber implantation technique. Further in in vivo studies using animal model, jirds CFA2-6 and BmA-2 could induce about 90% protection to infection in immunized animals. In passive transfer studies of immunity it has been observed that BmA-2 induced protection is mainly antibody mediated.

Diagnostic use of filarial antibody and antigen isolated from hydrocele fluid for human filariasis.

Paired samples of serum and hydrocele fluid of filarial patients associated with hydrocele were analysed for filarial antibody and antigen. Sera samples showed higher titers of filarial antibody and antigen compared to their corresponding hydrocele fluid samples. HFIgG isolated from hydrocele fluid was equally useful as FSIgG isolated from serum and detected filarial antigen in 23 out of 26 microfilaraemic sera, 7 out of 10 chronic sera and 3 out of 18 endemic normal sera by inhibition ELISA. Filarial antigen was isolated from hydrocele fluid. In inhibition ELISA antigen fraction, HFA-9 (20-25 kDa) isolated by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis showed high reciprocal antigen titer of 2048. This active antigen fraction was evaluated for its diagnostic utility in comparison with Wuchereria bancrofti microfilariae excretory-secretory antigen (Wb mf ES Ag) in inhibition ELISA. Both antigen preparations detected filarial antigen in about 80% of microfilaraemic sera, 60% chronic sera and 20-30% of endemic normal sera. This study showed that antibody and antigen isolated from hydrocele fluid were equally sensitive as FSIgG and Wb mf ES Ag in the detection of filarial antigen by inhibition ELISA. Thus hydrocele fluid may be used as an alternative source for the isolation of antibody and antigen of immunodiagnostic importance.

Detection of filarial antigen using antibodies raised against Wuchereria bancrofti microfilarial SDS soluble antigen.

Polyclonal antibodies raised in mouse ascitic fluid against Wuchereria bancrofti microfilarial antigens (Wb Mf SDS S Ag) were studied for their diagnostic use in bancroftian filariasis using a dip stick, enzyme-linked immunosorbent assay. In sandwich ELISA, 100% of microfilaremic sera (30 out of 30) 53% of acute filarial sera (7/13), 40% of subacute filarial sera (6 out of 15), 13% of chronic filarial sera (2/15) and 20% of endemic area normal sera (3/15) showed the presence of filarial antigen. Determination of filarial antigen titer in microfilaremic sera showed an apparent positive correlation between microfilarial density and antigen titer. The antibody raised against Wb Mf SDS S Ag was found to be cross reactive with phosphorylcholine epitopes. The filarial antigen detected by anti Wb Mf SDS S Ag antibodies in sandwich ELISA is possibly associated with the active stage (microfilaremia) of infection.

Immunoprophylaxis against filarial parasite, Brugia malayi: potential of excretory-secretory antigens in inducing immunity.

The role of excretory-secretory antigens in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell-mediated reaction as well as in vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised against Brugia malayi microfilarial and infective larval excretory-secretory antigens (Bm Mf ESA and Bm L3 ESA) promoted the adherence of peritoneal exudate cells to Brugia malayi microfilariae and infective larvae in vitro and induced cytotoxicity to the parasites within 48 h. The anti Bm Mf ESA serum was more effective than anti Bm L3 ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48–72 h. Further, Mastomys natalensis immunized against Bm Mf ESA and L3 ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretorysecretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.

Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone.

The reacting pattern of circulating filarial antigen fraction-2 from Wuchereria bancrofti and soluble antigen from adult Brugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa of Brugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa of Brugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal sera i.e .proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa of Brugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA2-8) and Brugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA2-2, 6 and 7 and BmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.

Penicillinase ELISA for detection of tubercular antigen in tuberculosis.

Stick sandwich enzyme linked immunosorbent assay (ELISA) using rabbit anti PPD-RT 23 immunoglobulins and enzyme penicillinase has been explored for detection of tubercular antigen in sera and CSF samples of pulmonary tuberculosis and tubercular meningitis (TBM) respectively. The analysis of sera showed 73.3% of pulmonary tuberculosis cases, 16.2% of healthy controls and 44.4% of Hansen's disease positive for tubercular antigen. The accuracies of positive and negative predictive values were 69% and 82% respectively. The analysis of CSF samples showed the presence of tubercular antigen in 76.4% of TBM, 16.6% of pyogenic meningitis cases, 19.4% of neurological diseases other than meningitis and 16.1% non-neurological disease controls. The accuracies of positive and negative predictive values were 48% and 94% respectively. Hence this simple test using economical and indigenous reagents can be applied for the diagnosis of pulmonary and extra-pulmonary tuberculosis.

Analysis, characterization and diagnostic use of circulating filarial antigen in bancroftian filariasis.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of circulating filarial antigen fraction-2 isolated from plasma of microfilaraemic patients with Wuchereria bancrofti infection has shown 21 bands with molecular weights ranging from 12 to 120 kDa. The gel (12 cm) was sliced at an interval of one cm and the eluates of all the gel slices viz., CFA2–1 to CFA2–12 showed the presence of filarial antigen by sandwich enzyme-linked immunosorbent assay. The low molecular weight circulating filarial antigen fractions were found to share a common epitope with Wuchereria bancrofti microfilariae excretory-secretory antigen and urinary filarial antigen. The 3 antigen fractions CFA2-1, CFA2–9 and CFA2–12 showed higher sensitivity in detecting filarial immunoglobulin Μ antibodies than immunoglobulin G antibodies. However CFA2–9 fraction was found useful in serological differentiation of microfilaraemics from those with disease manifestations when filarial immunoglobulin G antibodies were detected. The antigenic epitope of CFA2–1 appears to be a carbohydrate, whereas CFA2–9 appears to be protein in nature.

Antigenic analysis of excretory-secretory products of Wuchereria bancrofti and Brugia malayi infective larval forms by SDS-PAGE.

Excretory-secretory (ES) products of W. bancrofti and the closely related B. malayi infective larval forms were analysed for their antigenic activity by SDS-PAGE followed by Western blotting as well as by gel elution-sandwich ELISA using filarial serum immunoglobulin-G (FSIgG) as a capture antibody. In W. bancrofti infective larval ES products, the protein molecules of 66, 46, 35, 33, 30 and 14 kDa molecular wt. showed antigenic activity by immuno blotting technique. In sandwich ELISA technique eventhough all SDS-PAGE fractions except ESA 6 (55-47 kDa) showed antigenic positivity, the fractions ESA 8 (37-31 kDa) and ESA 9 (31-25 kDa) showed high reciprocal antigen titre of 262144 and 32768 respectively. In B. malayi infective larval ES products, the protein molecules of 109, 102, 97 and 77 kDa molecular wt. showed reactivity with FSIgG by blotting technique, where as in sandwich ELISA except ESA 7 (47-37kDa), all fractions showed antigenic positivity. However, these fractions failed to show high antigen titre similar to W. bancrofti ES products with FSIgG.