Direct ELISA Method Research Articles

Survey of Common Infectious Diseases in Urban Foxes (Vulpes spp.) in Southeastern Iran.

The red fox (Vulpes vulpes) is one of the most common species of wild Canidae and is relatively abundant in Iran. Foxes (Vulpes spp.) transmit many zoonotic diseases, the most important of which are visceral leishmaniasis, rabies, hydatidosis, toxocariasis, and trichinellosis. In this study, visceral leishmaniasis, rabies, ectoparasites, canine gastrointestinal helminths, dermatophytosis, distemper, parvovirus infection, and heartworm infections were evaluated among live-trapped and rescued foxes injured by traffic road accidents referred to the teaching hospital of Kerman, Iran, veterinary faculty. Skin scraping and direct microscopic examination were used to detect ectoparasites and dermatophytosis. Immunochromatography rapid kits were used to detect dirofilariasis, parvovirus infection, and distemper. Necropsy was used to check for gastrointestinal parasites. Rabies and visceral leishmaniosis were screened for with direct fluorescent antibody test and ELISA methods, respectively. Gastrointestinal helminth infections, including Toxocara canis, Taenia taeniaeformis, Dipylidium caninum, Joyeuxiella echinorhyncoids, Toxascaris leonina, Taenia hydatigena, Echinococcus granulosus, Rictolaria spp., Oxynema spp., Macracanthorhynchus hirudinaceus, and Physaloptera spp., were detected. Skin scrapings showed dermatophytosis and various ectoparasites, including Rhipicephalus sanguineus, Ctenocephalides canis and Ctenocephalides felis, and Sarcoptes scabiei, in foxes with dermal lesions. Distemper and parvovirus infection (26.66%) were the common viral diseases, and rabies infection rate was quite high (16.66%). Dirofilariasis and leishmaniasis were detected in 10% of the population. This study showed that urban foxes which often cohabit with humans and domestic animals are carriers of many different pathogens. This interaction may facilitate indirect cross-species transmission of zoonotic disease. Periodic health monitoring and multidisciplinary cooperation for the diagnosis, control, and prevention of these zoonoses is highly recommended.

Investigation of the Precence of Entamoeba histolytica, Giardia intestinalis and Cryptosporidium spp. in Patients Who Undergone Endoscopy and Colonoscopy

In this study, it was aimed to investigate the presence of Entamoeba histolytica, Giardia intestinalis and Cryptosporidium spp. in the samples taken during the procedure from patients who underwent endoscopy and/or colonoscopy with different prediagnoses, and in the stools of the same patients, by ELISA and direct microscopy methods. A total of 88 patients' endoscopic and colonoscopic pre-washed materials, which consisted of 49 individuals who belong former group and 39 individuals to the next group, were, respectively, obtained, and the stool samples were also included to study from the same group. All the specimens were immediately transferred to the parasitology research laboratory within the same day and stored C until for the next step of ELISA applications. All the samples were examined by direct microscopy and ELISA method. In the examinations performed using the ELISA method; E. histolytica was detected in 2 (2.3%) stool samples, and G. intestinalis was found in 4 (4.5%) stool samples. In the colonoscopic wash/swab samples of the patients who underwent colonoscopy, 6 (6.8%) G. intestinalis, 1 (1.1%) Cryptosporidium spp. detected. No parasites were detected by ELISA in any of the stool samples or endoscopic washing/swab samples of the patients who underwent colonoscopy. No parasites were detected in stool and wash/swab samples by the direct examination method. When the incidence of G. intestinalis in washing/swab samples taken from patients who underwent endoscopy and colonoscopy was statistically compared, the difference was found to be significant (p<0.05). When the incidence of G. intestinalis in the stools of patients who underwent endoscopy was compared, the difference between genders was found to be significant (p<0.05). In patients with gastrointestinal complaints and undergoing endoscopy and colonoscopy, investigation of the presence of parasites by stool examination with direct microscopy may be insufficient. In addition to the direct examination of the stool sample, it is thought that the investigation of parasite antigens in the wash/swab materials that can be easily taken during the endoscopy and colonoscopy procedure is necessary and critical in the diagnosis.

Diagnostic Performance of Faecal Calprotectin among People with Chronic Inflammatory Diseases of the Bowel in Cameroon: A Pilot Study in Sub-Saharan Africa

Background: Inflammatory bowel diseases (IBDs) are relatively common in African countries. The use of faecal markers, Calprotectin in particular, is presently of considerable interest to IBD patients. The high faecal calprotectin level has a good diagnostic accuracy in discriminating intestinal organic and functional diseases and enables the selection of patients in need of other invasive diagnosis such as endoscopy.&#x0D; Aim: To evaluate the diagnostic performance of faecal calprotectin as a useful diagnostic tool for IBD patients in Cameroon.&#x0D; Study design: This was a case control cross-sectional multicentre study conducted in major gastroenterology units of the towns of Yaoundé and Douala involving 64 participants grouped as 32 IBD positive subjects and 32 IBD negative subjects.&#x0D; Methods: Stool sample collected from participants at various recruitment sites were collected, conditioned and transported to the CIAB laboratory for analysis using a direct ELISA method. The R software was used for data analysis.&#x0D; Results: The calprotectin levels of IBD patients were significantly higher than those of the control subjects (P &lt; .001.) The area under the curve (AUC) was 0.96 [95%CI: 0.92 – 1.00; P &lt; .001]. A threshold value of 2.51µg/g was chosen to exclude the diagnosis of IBD with an 87.5% sensitivity and 100% specificity. The CRP levels correlated with those of calprotectin (r꞊0.579, P ꞊ .005). Calprotectin concentrations became abnormally elevated in all UC patients with an Endoscopy Score greater than or equal to 6 (P = .001). Correlation between the endoscopy score for Crohn's disease and calprotectin concentration did not retain significance (ρ꞊0.800; P &gt; .05).&#x0D; Conclusion: Calprotectin dosage is a sensitive test for IBD, excludes unnecessary investigations and accurately predicts disease recurrence and response to treatment.

Study of serum calcidiol level in pregnancy and its association with fetal outcome

Introduction: Presently there are no guidelines for routine screening of vitamin D deficiency in antenatal women, therefore this study was planned to find the association of maternal vitamin D level with fetal outcomes. Materials and Methods: The subjects were 100 pregnant women between 12 to 24 weeks of gestation selected from Obstetrics and Gynecology OPD. In the total number of subjects, serum 25 hydroxyvitamin D [25(OH) D] level was estimated by direct ELISA method and its association with fetal outcome - birth weight, length, head circumference and knee-heel length was studied. Results and Conclusion: In the present study a significant association was found between maternal vitamin D status and neonatal birth weight but not with other birth parameter. Keywords: Maternal Vit D, Birth weight.

Correlation between leptin and LH concentration during peri and postpartum to the onset of estrus postpartum in Bali cattle

The mechanism of the hypothalamus, pituitary, and ovaria is important in the early postpartum luteal activity. This study aims to measure levels of the leptin hormone, Luteinizing Hormone (LH) during the first-time postpartum estrus appears and to observe the signs of estrus. The study uses a prospective observational method. The samples were Bali cattle on peripartum conditionwhile postpartum estrus appeared from several sistem pertanian terintegrasi (Simantri) in Mengwi District, Badung Regency, Bali, Indonesia. The measured parameters were leptin levels, LH, and the onset of postpartum estrus. The technique of measuring hormone levels with the Direct Elisa method and Double Antibody Sandwich. The results showed there were a positive correlation (r=0.773) between leptin levels at peripartum and the onset of postpartum estrus.The higher level of leptin, the faster of postpartum estrus appears. However, a minimum leptin value is needed to increase the LH level so that expression of estrus appearance is clearly.

Relationship between Insulin Sensitivity and Body Fat Composition, and Leptin Sensitivity in Non-Obese and Overweight/Obese Adults

Both insulin and leptin are major contributors for the body energy balance. Obesity is a state of energy imbalance and is also associated with changes in both insulin sensitivity and leptin sensitivity. The aim of this study was to find out the relationship between insulin sensitivity and body fat composition, and leptin sensitivity in non-obese and obese adults. A total of 86 adults participated: 42 non-obese and 44 over-weight/obese. Body fat (BF) percent was determined by skinfold method. Fasting plasma glucose was analyzed by glucose oxidase-phenol and 4 aminophenazone (GOD-PAP) method using spectro-photometer, fasting serum insulin and leptin concentrations by direct sandwich ELISA method and resting energy expenditure (REE) by indirect calorimetry. Leptin sensitivity index and insulin sensitivity were expressed as REE : Leptin ratio and homeostatic model assessment-insulin resistance (HOMA-IR), respectively. It was found that median value of HOMA-IR was significantly higher [2.93 vs 1.72, p&lt;0.01] and leptin sensitivity was significantly lower [116.76 vs 265.66, p&lt;0.001] in the overweight/obese adults than the non-obese adults, indicating that insulin sensitivity and leptin sensitivity were markedly reduced in overweight/obese adults in compare to non-obese adults. There was a moderate degree of positive relationship between HOMA-IR and BF only in the overweight/obese (ρ=0.509, n=44, p&lt;0.001) and all adults (ρ=0.39, n=86, p&lt;0.001). Similarly, a weak negative relationship between leptin sensitivity index and HOMA-IR was found in the overweight/obese (ρ=-0.328, n=44, p&lt;0.05) and all adults (ρ=-0.35, n=86, p&lt;0.01). It can be concluded that the insulin sensitivity was adiposity dependent, but, it did not depend on leptin sensitivity.

Vitamin D status in exclusively breast-fed infants and their mothers

Background: India, despite being a tropical country has widespread vitamin D deficiency across all age groups. Present study was conducted to estimate the prevalence of subclinical hypovitaminosis D in exclusively breastfed infants and their mothers and also to study the correlation between the Vitamin D levels of these infants and their mothers.Methods: This cross-sectional study was conducted in 37 exclusively breastfed infants aged 6 months and below and their mothers. Under strict aseptic precautions 2 ml of blood was taken from each infant and mother and serum levels of 25(OH) D levels were measured by the direct ELISA method. Statistical analysis was done by using chi-square test, one-way ANOVA f-test and student independent t-test.Results: In present study, authors found that 78.3% of the mothers were Vitamin D deficient and 21.7% of the mothers were Vitamin D insufficient. Among the infants, authors found that 91.9% of the infants were Vitamin D deficient and 8.1% of the infants were Vitamin D insufficient. The association between dietary pattern and socio-economic class and mean maternal vitamin D levels was found to be statistically significant (p &lt;0.05).Conclusions: There is a high prevalence of hypovitaminosis D amongst exclusively breastfed infants and their mothers. Hence, public awareness needs to be created regarding the benefits of sunlight exposure. Vitamin D fortification of food products and routine Vitamin D supplementation program for antenatal and lactating mothers and their young infants may be able to address this emerging public health problem in our country.

Entomological impact of indoor residual spraying with pirimiphos-methyl: a pilot study in an area of low malaria transmission in Senegal

BackgroundScaling-up of effective anti-malarial control strategies in Central-West region of Senegal has resulted in the sharp decline in malaria prevalence in this area. However, despite these strategies, residual malaria transmission has been observed in some villages (hot spots). The objective of this study was to assess the impact of indoor residual spraying (IRS) with pirimiphos-methyl on malaria transmission in hot spot areas.MethodsThe malaria vector population dynamics were monitored in each of the six selected villages (4 of which used IRS, 2 were unsprayed control areas) using overnight human landing catches (HLC) and pyrethrum spray catches (PSC). The host source of blood meals from freshly fed females collected using PSC was identified using the direct ELISA method. Females caught through HLC were tested by ELISA for the detection of Plasmodium falciparum circumsporozoite protein and Anopheles gambiae complex was identified using PCR.ResultsPreliminary data shown that the densities of Anopheles populations were significantly lower in the sprayed areas (179/702) compared to the control. Overall, malaria transmission risk was 14 times lower in the intervention zone (0.94) compared to the control zone (12.7). In the control areas, three Anopheles species belonging to the Gambiae complex (Anopheles arabiensis, Anopheles coluzzii and Anopheles melas) maintained the transmission, while only An. coluzzii was infective in the sprayed areas.ConclusionThe preliminary data from this pilot study showed that IRS with the CS formulation of pirimiphos-methyl is likely very effective in reducing malaria transmission risk. However, additional studies including further longitudinal entomological surveys as well as ecological and ethological and genetical characterization of vectors species and their populations are needed to better characterize the entomological impact of indoor residual spraying with pirimiphos-methyl in the residual transmission areas of Senegal.

A study of serum Vitamin D level and its association with hypertension.

Background:Evidence suggests that low levels of Vitamin D may adversely affect the cardiovascular (CV) system. Several studies have been done regarding the relation and possible causative role of Vitamin D in CV disorders and its well-known risk factors; however, there are limited studies in this part of the world. The aims were as follows: (1) To study the relation between serum Vitamin D level between nonhypertensive and hypertensive patients. (2) To study the relation of serum Vitamin D levels in patients with isolated systolic hypertension (ISH), isolated diastolic hypertension, systolo-diastolic hypertension, and their comparison vis-à-vis nonhypertensives.Materials and Methods:A cross-sectional study was conducted with 154 patients attending medicine outpatient department of a tertiary care hospital of North Bengal from June 2012 to May 2013. The Vitamin D was measured by direct ELISA method. Blood pressure (BP) measurements were done. Statistical analysis was done by using SPSS 16.0 for Windows.Results:The Vitamin D level in the hypertensive group was 22.36 ± 12.64; ISH Group was 22.04 ± 14.26; the isolated diastolic hypertension (IDH) Group was 18.82 ± 0.00; and the systolo-diastolic hypertensives (SDH) Group was 22.67 ± 12.51. Then, the mean value of Vitamin D in nonhypertensive Group (27.47 ± 13.43) was significantly (P < 0.05) higher than IDH, SDH, and the hypertensive as a whole groups. The relation with ISH Group also reached near significance (P = 0.074). There was a negative correlation with BP and serum Vitamin D. This remained statistically significant (P = 0.044) for systolic BP (SBP) and near significant (P = 0.075) for mean arterial pressure. In population having serum Vitamin D <30 ng/ml (deficient or insufficient), the negative correlation relationship between SBP and serum Vitamin D remains statistically significant (P = 0.010).Conclusion:Among the hypertensives, SDH shows significantly lower levels of serum Vitamin D. The patients with ISH show a trend, though not statistically significant, toward a lower level of Vitamin D compared to the nonhypertensive population.

Humoral immune response against two surface antigens of Chlamydia pecorum in vaccinated and naturally infected sheep.

Chlamydia pecorum is a globally recognised livestock pathogen due to the significant clinical and economic impact it poses to livestock producers. Routine serological diagnosis is through a complement fixation test (CFT), which is often criticised for cross-reactivity, poor sensitivity and specificity. Although serology remains the preferred method in veterinary diagnostic laboratories, serological assays based on surface antigens of C. pecorum have not been established until now. In this study, we evaluated the use of two chlamydial recombinant protein antigens (PmpG and MOMP-G) by a direct IgG ELISA method for detection of ovine anti-chlamydial antibodies. Using the Pepscan method we then identified B cell epitopes across PmpG and MOMP-G proteins, in lambs with (a) naturally occurring asymptomatic C. pecorum infections (b) C. pecorum-associated polyarthritis and (c) recombinant PmpG and MOMP-G vaccine. Plasma IgG antibodies to PmpG in natural infection of lambs were detected earlier in infection than CFT and served as an acute phase marker. Antibodies to MOMP-G IgG were significantly heightened in lambs with C. pecorum-associated polyarthritis. PmpG and MOMP-G specific B-cell epitope mapping revealed epitope responses in immunised lambs cluster with some of the epitope responses in naturally infected lambs. B-cell epitope mapping further revealed that lambs with polyarthritis recognised several unique PmpG (50% frequency, peptide 8, 25, 40, 41 and 50) and MOMP (50% frequency, peptide 50) epitopes in comparison to asymptomatic infections. The findings of this study will have implications towards improved serodiagnosis of C. pecorum infections in livestock and inform the downstream development of alternative peptide-based antigens for future C. pecorum vaccine studies.

Was the increase in culture-confirmed Campylobacter infections in Denmark during the 1990s a surveillance artefact?

In 1991, 1999 and 2006, randomly selected individuals from the Danish Central Personal Register provided a serum sample. From individuals aged 30 years and above, 500 samples from each year were analysed for Campylobacter IgG, IgA and IgM antibodies using a direct ELISA method. We applied a seroincidence calculator available from the European Centre for Disease Prevention and Control to perform a mathematical back-calculation to estimate the annual Campylobacter seroincidence in the Danish population. The estimated Campylobacter seroincidence did not differ significantly between the 1991, 1999 and 2006 studies although the reported number of culture-confirmed cases of Campylobacter infection increased 2.5 fold from 1993 to 1999 among individuals aged 30 years and above. This suggests that Campylobacter was widely present in the Danish population before the increase in poultry-associated clinical Campylobacter infections observed from 1993 to 2001 among individuals of this age groups.

A hospital based prospective study of vitamin D deficiency in a selected group of apparently healthy children one to five years of age

Objective: To study the presence of vitamin D deficiency in a selected group of apparently healthy children 1-5 years of age Method: A hospital based prospective cross-sectional study was carried out at the Sri Guru Ramdas Institute of Medical Sciences and Research (SGRDIMSR), Amritsar on a selected group of apparently healthy children of age group 1 to 5 years, attending the paediatrics outpatient department for minor ailments or immunization from December 2012 to June 2014. Venous samples from these children were collected in two separate red topped vials (with clot activator), one for vitamin D and the other for alkaline phosphatase and calcium. These vials were sent to the Biochemistry Department of SGRDIMSR hospital. Data so obtained was statistically analysed. Results : Out of a total of 100 apparently healthy children, 78 were found to be deficient in Vitamin D. Vitamin D levels were tested by the Direct ELISA method. Vitamin D deficiency was found most commonly in the age group 4–5 years. Gender wise, 79.4% of boys and 75.7% of girls were found to be vitamin D deficient. Of breast fed children 76.6% were found deficient in vitamin D. Of the children who spent less than 30 minutes per day in the sun 73.1% were vitamin D deficient. Most (80%) cases of vitamin D deficiency were found in the upper middle class. Urban population was found to be more deficient in vitamin D as compared to rural. Fifty four percent of the children with vitamin D deficiency were of wheatish complexion. Dietary insufficiency was found in 67% of the children, of whom 82% were found to be vitamin D deficient. Clinical features of vitamin D deficiency found in our study were frontal bossing, parietal bone prominences, widening of wrist epiphyses and pot belly. Conclusion: In the selected group of apparently healthy children 1-5 years of age vitamin D deficiency was found in 78%. Sri Lanka Journal of Child Health, 2015; 44(3): 158-162

The Levels of Tau Isoforms Containing Exon-2 and Exon-10 Segments Increased in the Cerebrospinal Fluids of the Patients with Sporadic Creutzfeldt-Jakob Disease.

The alteration of protein tau in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) has been widely evaluated, possessing a significant diagnostic value for CJD. With the biotin-labeled tau-exon-specific mAbs, direct ELISA methods were established and the levels of tau isoforms containing exon-2 and exon-10 segments in CSF of the patients with various human prion diseases and in brain tissues of scrapie-infected animals were evaluated. The results showed that the levels of tau, especially containing four repeats in microtubule binding domain, were increased in the CSF samples of the patients with sporadic CJD (sCJD). Using the unlabeled (cold) mixed exon-specific mAbs, a competitive tau ELISA was conducted based on a commercial tau kit. It revealed that the majority of the increased tau in the CSF of sCJD cases was derived from the tau isoforms with exon-2 and exon-10 segments. Increases of CSF tau isoforms with exon-2 and exon-10 segments were also observed in the patients of E200K and T188K genetic CJD (gCJD), but not in the cases of fatal familiar insomnia (FFI). The increasing levels of tau isoforms with exon-2 and exon-10 segments in the group of sCJD correlated well with the positive 14-3-3 in CSF. Additionally, the similar alterative profiles of tau isoforms with exon-2 and exon-10 segments were also observed in the brain tissues of scrapie-infected rodents and a sCJD patient. Our data here propose the tau isoforms with exon-2 and exon-10 segments increase in CSF of sCJD and some types of gCJD, which may help to understand the physiological metabolism and pathological significance of various tau isoforms in the pathogenesis of prion diseases.

Lipoprotein (a) plasma levels and risk of breast cancer

Dyslipidaemia has been associated with many morbidities including cancer. This study was carried out to assess the influence of dyslipidaemia as evidenced by hyperapolipoproteinaemia (a) on breast cancer in Indian subjects. The effect of chemotherapy on these indices of dyslipidaemia was also assessed. A total of thirty histologically confirmed cases of breast cancer as well as an equal number of age- and BMI-matched controls were enrolled for the study. Their lipid profile was analyzed on a fully automated AU 400 clinical chemistry analyzer using standard reagents. Lipoprotein (a) was determined by the solid phase direct sandwich ELISA method (Diaclore, France). The levels of various lipoproteins like cholesterol, triacylglycerol, HDL, LDL and Lp(a) were higher in cases in comparison to controls. A significant increase was observed in cases with advanced stages. The mean value of lipoprotein (a) decreased in cases after chemotherapy which was statistically significant. This study shows that level of Lipoprotein (a) was significantly elevated in confirmed cases of breast cancer and levels and decreased in patients showing response to chemotherapy. Hence, it can be used as a surrogate marker for determining disease progress or efficacy of treatment.

Serum levels of tumor necrosis factor -alpha and hsCRP in patients with breast cancer and correlation with histological parameters

Background: Breast cancer is now the second most commonly cancer diagnosed in women after cervical cancer in India. Presentation at late stage further aggravates the problem. The outcome of breast cancer is usually determined by multiple factors.Aims and Objectives:This study was designed with the aim to investigate any correlation between serum tumor necrosis factor alpha and hsCRP in breast cancer with histological parameters of tumor behavior. Materials and Methods: A total of 30 histological confirmed cases of locally advanced breast cancer were enrolled for study. Total duration of study was two years. HsCRP was determined by solid phase direct sandwich ELISA method (Diaclone, France).Similarly TNF-α was also determined by Enzyme-linked immunosorbent method. Three-dimensional tumor size was determined radiologically through mammography. CT scan and MRI scan were taken at the time of diagnosis to detect metastasis. The data on tumor size, estrogen receptors status, lymph node status and TNM staging were reviewed and recorded. Results: Levels of TNF-α and hsCRP in patients with more advanced TNM staging, more advanced lymph node status and high histological grade were significantly raised. Similarly, their levels were significantly raised with increasing grade of adipose tissue invasion. Levels of TNF-α and hs CRP were also significantly raised in patients with estrogen receptor status positive whereas increase in levels of these markers was not significant in progesterone receptor status positive. Conclusion: Preoperative evaluation of tumor necrosis factor alpha and high sensitivity C-reactive protein may be valuable parameters for reflecting the severity of invasive breast cancer.DOI: http://dx.doi.org/10.3126/ajms.v6i6.12714 Asian Journal of Medical Sciences Vol.6(6) 2015 6-10

Prevalence of Giardia lamblia in Asymptomatic Patients by Direct Examination and ELISA Methods

Stool samples from 182 asymptomatic patients were collected from two hospitals in Baghdad (Al-Yarmok and Central Child) during the period from the beginning of March to the end of June/2014 and subjected to microscopic and ELISA diagnostic techniques for Giardia lamblia detection; the sensitivity and specificity for ELISA were calculated. Cysts of G. lamblia were observed in 37 and 49 stool samples of asymptomatic patients from a total 182 examined samples with prevalence recorded 20.32, 26.92% by direct examination and ELISA respectively. Of 145 samples showed negative result in microscope examination, 12 samples were positive in ELISA, sensitivity and specificity of ELISA were 75.51 and 100%. Higher prevalence rates were observed in asymptomatic children aged 2-8 years old 27.84% (22+ve/79) in contrast with 26.21% prevalence rate in asymptomatic adults aged 21-57 years old (27+ve /103). According to gender, males showed higher prevalence 28.94% (33+ve/114) while females showed lower prevalence rate 23.52% (16+ve/68). No differences in the level of infection in both age and gender were recorded. Conclusion: The superior sensitivity of the rapid enzyme assay in detecting asymptomatic cases of giardiasis when a single stool specimen is analyzed, the availability of an immunodiagnostic assay which can detect small amounts of antigens in the feces which have a potential tools to improve the diagnosis.

Challenges in keeping pace with immunogenicity advances throughout the clinical development of a biological therapeutic.

Progress in the assessment of immunogenicity over the past decade has been rapid; therapeutics in late-phase clinical studies today are subject to antidrug antibody analyses that have evolved considerably since the start of their clinical development. Data from bridging and direct ELISA methods used for the analysis of antidrug antibodies generated in response to an antibody therapeutic will be presented. The case study will highlight some of the challenges faced and the learning points gained from supporting immunogenicity analysis for these therapeutics.

Evaluation of monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) for antigen detection of foot-and-mouth disease virus using clinical samples.

A monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) method was previously developed for foot-and-mouth disease (FMD) viral antigen detection. Here we evaluated the sensitivity and specificity of two FMD viral antigen detection MSD-ELISAs and compared them with conventional indirect sandwich (IS)-ELISA. The MSD-ELISAs were able to detect the antigen in saliva samples of experimentally-infected pigs for a longer term compared to the IS-ELISA. We also used 178 RT-PCR-positive field samples from cattle and pigs affected by the 2010 type-O FMD outbreak in Japan, and we found that the sensitivities of both MSD-ELISAs were about 7 times higher than that of the IS-ELISA against each sample (P<0.01). In terms of the FMD-positive farm detection rate, the sensitivities of the MSD-ELISAs were about 6 times higher than that of the IS-ELISA against each farm (P<0.01). Although it is necessary to conduct further validation study using the other virus strains, MSD-ELISAs could be appropriate as a method to replace IS-ELISA for FMD antigen detection.

Comparison of Direct Microscopy, Culture, ELISA and Molecular Methods for the Diagnosis of Entamoeba histolytica

Amebiasis, a parasitic infection caused by Entamoeba histolytica, is one of the most common parasitic infections worldwide. Since it is still an important public health problem in developing countries, rapid differential diagnosis of amebiasis is crucial in terms of treatment. The most frequently used method for laboratory diagnosis is direct microscopy, however more reliable and specific methods are needed in order to differentiate the apathogenic Entamoeba dispar under the microscope. This study was conducted to compare the results of different methods namely, direct microscopy, culture, ELISA and PCR for the detection of E.histolytica in stool samples and to evaluate the performances of those methods. A total of 1049 stool samples collected from pediatric and adult patients who were admitted to hospital with diarrhea complaint between January 2011-March 2013, and randomly selected samples from primary school children, were included in the study. Direct microscopic examination was performed by native-lugol, physiological saline, modified formol-ethyl acetate sedimentation and trichrome staining methods. The stool samples were also inoculated into TYI-S-33 media for axenic cultivation of amoeba. The presence of amebic antigens in the samples were screened by a commercial ELISA kit (TechLab, E.histolytica II, USA). For the molecular diagnosis, a multiplex tandem real-time PCR (MT-PCR) kit (AusDiagnostics Pty Ltd, Australia) was used, after the extraction of DNAs with QIAamp DNA Stool Mini Kit (Qiagen, USA). A total of 354 samples which could be evaluated by all of the methods, were included in the study. Of the 354 stool samples, 84 (23.7%) were found E.histolytica/E.dispar positive by direct microscopy, 61 (17.2%) by trichrome staining, 46 (12.9%) by culture, 31 (8.7%) by ELISA and 9 (2.5%) by MT-PCR. Of direct microscopy positive samples 54.7% (46/84) were also positive with trichrome staining, 39.3% (33/84) with culture, 15.5% (13/84) with ELISA and 7.1% (6/84) with MT-PCR methods. On the other hand, of the nine MT-PCR positive samples, six were positive with direct microscopy, four with trichrome staining and culture, and one with ELISA. It was remarkable that only one (0.3%) sample yielded positive results with all of the diagnostic methods used. When MT-PCR was considered as the reference method, the sensitivity and specificity values of direct microscopy, trichrome staining, culture and ELISA methods were estimated as; 66.7% and 77.4%, 44.4% and 83.5%, 44.4% and 87.8%, 11.1% and 91.3%, respectively. In conclusion, if the circumstances allow, the use of all methods in combination and evaluation together with the clinical symptoms seems to be the best approaches for the laboratory diagnosis of patients with amebiasis.

The Utility of Pro-inflammatory Cytokines-TNF Alpha and CRP as Indicators of Response to Chemotherapy in Patients with Breast Carcinoma

Objective: Breast cancer is the most commonly occurring female cancer in the world the incidence of which is more than double that of the second ranked cancer (cervical cancer). Not many studies have assessed the relationship between biomarkers of inflammation and long-term survival in breast cancer patients. The aim of this study was to determine whether circulating markers of inflammation (CRP and TNF-α), measured before and after chemotherapy, predict response to therapy. Material: A total of 30 histological confirmed cases of breast cancer were enrolled for study. Total duration of study was two years. HsCRP was determined by solid phase direct sandwich ELISA method (Diaclore, France). Similarly TNF-α was also determined by Enzyme-linked immunosorbent method. Three-dimensional tumor size was determined radiologically through mammography. CT scan and MRI scan were taken at the time of diagnosis to detect metastasis. Result: The mean values of hsCRP and TNF-α in patients of breast tumor decreased after three cycles of chemotherapy and this decrease was highly significant in patients with partial/complete response to chemotherapy. Similarly, levels of hsCRP and TNF-α were high in patients with estrogen receptor positive status than in estrogen receptor negative status. No significant correlation was observed between levels of hsCRP and TNF-α with progesterone receptor status and Her 2 neu status. Conclusion: This study shows serum hsCRP and TNF-α levels were significantly elevated in confirmed cases of breast cancer and levels decreased after chemotherapy in patients showing response to it. So hsCRP and TNF-α can be used as a surrogate.