Retrospective feasibility study of Mycobacterium tuberculosis modified lipoprotein as a potential biomarker for TB detection in children

Background: Current TB diagnostic methods available have been developed for adults and development efforts have neglected the differences in disease and sampling that occur between adults and children. Diagnostic challenges are even greater in HIV co-infected children and infants. Methods and results: We established a sandwich ELISA assay to detect Mycobacterium tuberculosismodified lipoprotein (TLP) ex vivo in plasma. The study population contains plasma samples from 21 patients with active TB and 24 control samples with no TB, collected in the International Maternal Pediatric Adolescent AIDS Clinical Trails (IMPAACT) P1041 study. Retrospective analysis was performed and the result demonstrate that TLP level is associated with TB disease. Conclusions: Plasma levels of TLP associate with active TB disease in HIV positive subjects and can be used as an indicator for TB detection in children.

Effective diagnosis of schistosomiasis haematobium by Silver beads Sandwich ELISA

Schistosomiasis is a major public health problem. Diagnosis by simple and rapid immunoassays is a priority. The magnetic bead immunoassay using magnetic nanoparticles conjugated with anti-schistosomal antibody was evaluated for di­agnosing human schistosomiasis infection. The present study was to evaluate the sandwich ELISA as a simple test for the detection of schistosomal antigen (CSA) in serum and urine samples of S. haematobium patients and compare it with ELISA. Investigation conducted on eighty six cases divided to three groups, 34 were positively for Schistosoma haematobium, 32 were positively for intestinal parasites ova and negative for S. haematobium ova in urine and 20 were negative urine and stool examination (Control). Immunomagnetical bead based Enzyme-linked immunosorbent assay (ELISA) using for detected for antigen in sera and urine infected by S. haematobium. Sandwich ELISA sensitivities was 79.4% (serum) and 73.5% (urine) and which increase by used nano-sandwich ELISA to 88.2% (serum) and 82.4% (urine), respectively. Sandwich ELISA specificities was 86.4% (serum) and 80.8% (urine) and which increase by used nano-sandwich ELISA to 93.3% (serum) and 88.5% (urine). We found that, nano-sandwich ELISA assay had highly sensitive and specifically and technical method was applicably, fast, cheaper, accurate and promising diagnostic method for schistosomiasis.

Antibodies Against Hepatitis E Virus (HEV) in European Moose and White-Tailed Deer in Finland

The main animal reservoirs of zoonotic hepatitis E virus (HEV) are domestic pigs and wild boars, but HEV also infects cervids. In this study, we estimated the prevalence of HEV in Finnish cervid species that are commonly hunted for human consumption. We investigated sera from 342 European moose (Alces alces), 70 white-tailed deer (Odocoileus virginianus), and 12 European roe deer (Capreolus capreolus). The samples had been collected from legally hunted animals from different districts of Finland during 2008–2009. We analysed the samples for total anti-HEV antibodies using a double-sandwich ELISA assay. Seropositive sera were analysed with RT-qPCR for HEV RNA. HEV seroprevalence was 9.1% (31/342) in moose and 1.4% (1/70) in white-tailed deer. None of the European roe deer were HEV seropositive (0/12). No HEV RNA was detected from samples of seropositive animals. HEV seropositive moose were detected in all districts. Statistically, HEV seroprevalence in moose was significantly higher (p < 0.05) in the North-East area compared to the South-West area. The highest HEV seroprevalence (20.0%) in district level was more than six times higher than the lowest (3.1%). We demonstrated the presence of total anti-HEV antibodies in European moose and white-tailed deer in Finland. Our results suggest that HEV is circulating among the moose population. Infections may occur also in white-tailed deer. We were the first to report a HEV seropositive white-tailed deer from Europe. Further studies are needed to demonstrate the HEV genotypes in cervids in Finland and to evaluate the importance of the findings in relation to food safety.

TONGUE FUNCTION, SALIVARY FLOW RATE AND IgA, IgM AND IgG TOTAL SALIVARY LEVELS IN CHRONIC CHAGASIC PATIENTS

Although microscopic alterations have been detected in tongues and salivary glands of chagasic patients and the identification of biomarkers in saliva has proved advantageous, there are no studies evaluating tongue function and total salivary IgA, IgG and IgM levels in chronic chagasic patients. The aim of this study was to evaluate tongue function, salivary flow rate, and total salivary IgA, IgG and IgM levels comparing chronic and non-chronic chagasic individuals. 37 patients were selected: chronic cardiac chagasic patients (n=6), chronic chagasic patients with the associated form of the disease (cardiopathy and megaesophagus) (n=11), and nonchagasic individuals (n=20). The tongue function underwent a phonoaudiological evaluation.The salivary f low rate was measured by sialometry. The total salivary IgA, IgG and IgM levels were evaluated by sandwich ELISA assay. Chagasic patients with the associated form of the disease presented higher salivary flow rate and lower salivary protein levels. No significant differences were noted in the lingual function or in the total salivary immunoglobulin levels among the groups. Although patients with chagasic megaesophagus presented higher levels of salivary flow and lower salivary protein, the fact that there were no significant differencesin lingual function and total salivary immunoglobulin levels among the groups led to the conclusion that chronic chagas disease does not modify the lingual function or the total IgA, IgG and IgM salivary levels. The present study was the first to evaluate the function of the tongue and salivary total immunoglobulin levels in Chagas disease.

Quantitative Detection of Pork Contamination in Cooked Meat Products by ELISA

Recent news of many cases of adulteration of meats with pork has bolstered the need for a way to detect and quantify the unwanted contamination of pork in other meats. To address this need, Microbiologique, Inc. has produced a sandwich ELISA assay that can rapidly quantify the presence of pork in cooked horse, beef, chicken, goat, and lamb meats. We carried out a validation study and showed that this assay has an analytical sensitivity of 0.00014 and 0.00040% (w/v) for cooked and autoclaved pork, respectively, and an analytical range of quantitation of 0.05–3.2% (w/v) in the absence of other meats. The assay can measure pork contamination down to 0.1% (w/w) in the presence of cooked horse, beef, chicken, goat, and lamb meats. The assay is quick and can be completed in 1 h and 10 min.

Aspartyl (Asparaginyl) β-Hydroxylase AABH as a serum biomarker for colorectal cancer.

86 Background: Colorectal Cancer (CRC) is the third most common type of cancer diagnosed in the US and Canada. WHO, Canadian Cancer Society (CCS), the National Comprehensive Cancer Network (NCCN) and American Cancer Society (ACS) recommend that men and women begin CRC screening at age 50 or younger if at high risk. Recommended screening procedures: Annual occult fecal blood test (OFBT), a colonoscopy every 5 years, OFBT and colonoscopy every 5 years, or a colonoscopy every 10 years. According to The Surveillance, Epidemiology, and End Results (SEER) Program, only 39% of CRC are diagnosed in stage I, 36% are diagnosed in Stage II, 19% are diagnosed with metastasis. The corresponding 5-year survival rates are 89.8%, 67.7%, and 10.3%. Neither the CCS nor the ACS recommends a blood test be done as part of screening. This is due to the fact that, until now, there has not been a blood test with adequate sensitivity or specificity for screening. Methods: In this study we discovered that Aspartyl (Asparaginyl) β-Hydroxylase (AABH) measurement in serum can be used as an screening test for CRC. AABH has been detected by immunohistochemical staining (IHC) on the cell surface of different cancers including CRC. It has been detected by IHC in > 97% of tumor specimens tested (n > 200) but has not been found in tissue samples from normal individuals. Results: This observation and the observation that AABH is found in the serum of patients with cancer, but not in n0n cancer patients, led us to develop a Sandwich ELISA Assay to measure AABH in serum. In the current study we have quantified AABH levels in CRC patients and compared it with normal individual. Increased levels of AABH were found in the serum of 91.5% of patients with CRC in all different stages of Cancer (n = 60). In normal individuals, AABH was essentially undetectable in serum (n = 30). AABH was identified in serum from patients with CRC irrespective of cancer stage. All serum AABH levels for stages I, II, III and IV were more than 3.3 ng/mL. Conclusions: Thus, our data indicate that by measuring AABH in the serum, we should be able to detect CC at an earlier stage than it is currently detected, resulting to a much better 5-year survival for CC.

Detection of circulating antigens in serum samples of mice experimentally infected with Trichinella spiralis by a sandwich ELISA based on IgY

In the present study, a sandwich ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating antigens (CAg) in sere of mice experimentally infected with Trichinella spiralis. The IgY-sandwich ELISA assay involved the use of chicken antibody IgY against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae as a capture antibody and mouse polyclonal antibody IgG to ES antigens as a detecting antibody. This method was able to detect as little as 3 ng/ml of ES antigens added to normal mouse serum. A group of sixteen mice was orally inoculated with 500 T. spiralis muscle larvae per animal. The serum samples from the infected mice were taken during 1–35 days post-infection (dpi). The CAg was detectable as early as 8 dpi in the sera of infected mice. The level of CAg increased dramatically during 13–15 dpi and reached a peak at 22 dpi and remained a plateau for 3 days, then declined gradually. Another peak of CAg occurred at 31 dpi. The anti-Trichinella antibodies was first detected in 14.3 % of the infected mice at 2 weeks post-infection (wpi), and reached a peak positive rate of 100 % at 5 wpi. Moreover, the infected mice were treated with abendazole at 5 wpi and the serum CAg levels increased significantly during 2–6 days posttreatment (dpt) and then declined rapidly during 8–14 dpt. By 42 dpt, the CAg levels decreased to the undetected level, but the detection rate of antibodies was still 100 %. The IgY-sandwich ELISA appears to be a sensitive for detection of antigenemia of T. spiralis and valuable to judge the efficacy of chemotherapy in trichinellosis.