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Concept: Eva Engvall


Zika virus is a flavivirus transmitted primarily by Aedes species mosquitoes, and symptoms of infection can include rash, fever, arthralgia, and conjunctivitis (1).* Zika virus infection during pregnancy is a cause of microcephaly and other severe brain defects (2). Infection has also been associated with Guillain-Barré syndrome (3). In December 2015, Puerto Rico became the first U.S. jurisdiction to report local transmission of Zika virus, with the index patient reporting symptom onset on November 23, 2015 (4). This report provides an update to the epidemiology of and public health response to ongoing Zika virus transmission in Puerto Rico. During November 1, 2015-April 14, 2016, a total of 6,157 specimens from suspected Zika virus-infected patients were evaluated by the Puerto Rico Department of Health (PRDH) and CDC Dengue Branch (which is located in San Juan, Puerto Rico), and 683 (11%) had laboratory evidence of current or recent Zika virus infection by one or more tests: reverse transcription-polymerase chain reaction (RT-PCR) or immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA). Zika virus-infected patients resided in 50 (64%) of 78 municipalities in Puerto Rico. Median age was 34 years (range = 35 days-89 years). The most frequently reported signs and symptoms were rash (74%), myalgia (68%), headache (63%), fever (63%), and arthralgia (63%). There were 65 (10%) symptomatic pregnant women who tested positive by RT-PCR or IgM ELISA. A total of 17 (2%) patients required hospitalization, including 5 (1%) patients with suspected Guillain-Barré syndrome. One (<1%) patient died after developing severe thrombocytopenia. The public health response to the outbreak has included increased laboratory capacity to test for Zika virus infection (including blood donor screening), implementation of enhanced surveillance systems, and prevention activities focused on pregnant women. Vector control activities include indoor and outdoor residual spraying and reduction of mosquito breeding environments focused around pregnant women's homes. Residents of and travelers to Puerto Rico should continue to employ mosquito bite avoidance behaviors, take precautions to reduce the risk for sexual transmission (5), and seek medical care for any acute illness with rash or fever.

Concepts: Antibody, Epidemiology, Mosquito, ELISA, ELISPOT, Aedes, Dengue fever, Eva Engvall


Background To estimate the frequency and duration of detectable Zika virus (ZIKV) RNA in human body fluids, we prospectively assessed a cohort of newly infected participants in Puerto Rico. Methods We evaluated samples obtained from 150 participants (including 55 men) in whom ZIKV RNA was detected on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay in urine or blood in an enhanced arboviral clinical surveillance site. We collected serum, urine, saliva, semen, and vaginal secretions weekly for the first month and then at 2, 4, and 6 months. All specimens were tested by means of RT-PCR, and serum was tested with the use of anti-ZIKV IgM enzyme-linked immunosorbent assay. Among the participants with ZIKV RNA in any specimen at week 4, biweekly collection continued until all specimens tested negative. We used parametric Weibull regression models to estimate the time until the loss of ZIKV RNA detection in each body fluid and reported the findings in medians and 95th percentiles. Results The medians and 95th percentiles for the time until the loss of ZIKV RNA detection were 14 days (95% confidence interval [CI], 11 to 17) and 54 days (95% CI, 43 to 64), respectively, in serum; 8 days (95% CI, 6 to 10) and 39 days (95% CI, 31 to 47) in urine; and 34 days (95% CI, 28 to 41) and 81 days (95% CI, 64 to 98) in semen. Few participants had detectable ZIKV RNA in saliva or vaginal secretions. Conclusions The prolonged time until ZIKV RNA clearance in serum in this study may have implications for the diagnosis and prevention of ZIKV infection. Current sexual-prevention guidelines recommend that men use condoms or abstain from sex for 6 months after ZIKV exposure; in 95% of the men in this study, ZIKV RNA was cleared from semen after about 3 months. (Funded by the Centers for Disease Control and Prevention.).

Concepts: Sexual intercourse, ELISA, ELISPOT, Assay, Eva Engvall, Immunoassay, Multiplex, Body fluids


Background The West African outbreak of Ebola virus disease has caused more than 8500 deaths. A vaccine could contribute to outbreak control in the region. We assessed a monovalent formulation of a chimpanzee adenovirus 3 (ChAd3)-vectored vaccine encoding the surface glycoprotein of Zaire ebolavirus (EBOV), matched to the outbreak strain. Methods After expedited regulatory and ethics approvals, 60 healthy adult volunteers in Oxford, United Kingdom, received a single dose of the ChAd3 vaccine at one of three dose levels: 1×10(10) viral particles, 2.5×10(10) viral particles, and 5×10(10) viral particles (with 20 participants per group). Safety was assessed over the next 4 weeks. Antibodies were measured on enzyme-linked immunosorbent assay (ELISA) and T-cell responses on enzyme-linked immunospot (ELISpot) and flow-cytometry assays. Results No safety concerns were identified at any of the dose levels studied. Fever developed in 2 of the 59 participants who were evaluated. Prolonged activated partial-thromboplastin times and transient hyperbilirubinemia were observed in 4 and 8 participants, respectively. Geometric mean antibody responses on ELISA were highest (469 units; range, 58 to 4051; 68% response rate) at 4 weeks in the high-dose group, which had a 100% response rate for T cells on ELISpot, peaking at day 14 (median, 693 spot-forming cells per million peripheral-blood mononuclear cells). Flow cytometry revealed more CD4+ than CD8+ T-cell responses. At the vaccine doses tested, both antibody and T-cell responses were detected but at levels lower than those induced in macaques protected by the same vaccine. Conclusions The ChAd3 monovalent vaccine against EBOV was immunogenic at the doses tested. (Funded by the Wellcome Trust and others; number, NCT02240875 .).

Concepts: Antibody, Microbiology, Immunology, ELISA, ELISPOT, Assay, Eva Engvall, Ebola


Aflatoxin, a mycotoxin found commonly in maize and peanuts worldwide, is associated with liver cancer, acute toxicosis, and growth impairment in humans and animals. In Tanzania, sunflower seeds are a source of snacks, cooking oil, and animal feed. These seeds are a potential source of aflatoxin contamination. However, reports on aflatoxin contamination in sunflower seeds and cakes are scarce. The objective of the current study was to determine total aflatoxin concentrations in sunflower seeds and cakes from small-scale oil processors across Tanzania. Samples of sunflower seeds (n = 90) and cakes (n = 92) were collected across two years, and analyzed for total aflatoxin concentrations using a direct competitive enzyme-linked immunosorbent assay (ELISA). For seed samples collected June-August 2014, the highest aflatoxin concentrations were from Dodoma (1.7-280.6 ng/g), Singida (1.4-261.8 ng/g), and Babati-Manyara (1.8-162.0 ng/g). The highest concentrations for cakes were from Mbeya (2.8-97.7 ng/g), Dodoma (1.9-88.2 ng/g), and Singida (2.0-34.3 ng/g). For seed samples collected August-October 2015, the highest concentrations were from Morogoro (2.8-662.7 ng/g), Singida (1.6-217.6 ng/g) and Mbeya (1.4-174.2 ng/g). The highest concentrations for cakes were from Morogoro (2.7-536.0 ng/g), Dodoma (1.4-598.4 ng/g) and Singida (3.2-52.8 ng/g). In summary, humans and animals are potentially at high risk of exposure to aflatoxins through sunflower seeds and cakes from micro-scale millers in Tanzania; and location influences risk.

Concepts: Aflatoxin, ELISA, ELISPOT, Assay, Eva Engvall, Peanut, Tanzania, Sunflower


The use of pesticides in Mexican agriculture creates an interest in learning about the presence of these substances in different environmental matrices. Glyphosate (GLY) is an herbicide widely used in the state of Campeche, located in the Mayan zone in the western Yucatan peninsula. Despite the fact that GLY is considered a non-toxic pesticide to humans, its presence in water bodies through spillage, runoff, and leaching are a risk to human health or biota that inhabit these ecosystems. In the present study, glyphosate residues were determined in groundwater, bottled drinking water, and the urine of subsistence farmers from various localities of the Hopelchén municipality in Campeche. Determination of GLY was carried out using Enzyme-Linked Immunosorbent Assay (ELISA). The highest concentrations of GLY were observed in the groundwater (1.42 μg/L) of Ich-Ek and urine (0.47 μg/L) samples of subsistence farmers from the Francisco J. Mújica communities. The glyphosate concentrations in groundwater and bottled drinking water indicate an exposure and excessive use of glyphosate in these agricultural communities. This is one of the first studies that reports glyphosate concentration levels in human urine and bottled drinking water in México and in the groundwater in the Yucatan Peninsula as part of a prospective pilot study, to which a follow-up will be performed to monitor this trend over time.

Concepts: Human, Agriculture, Water, Water pollution, ELISA, ELISPOT, Eva Engvall, Mexico


Background  A disintegrin and metalloprotease (ADAM) 12 is one of the metalloproteinase-type ADAMs and possesses extracellular metalloprotease and cell-binding functions. ADAM12 is expressed in two alternative forms, such as a membrane-anchored form (ADAM12-L) and a short secreted form (ADAM12-S). Objective  To investigate the clinical significance of serum ADAM12-S levels in systemic sclerosis (SSc). Methods  Serum ADAM12-S levels were determined by a specific enzyme-linked immunosorbent assay in 61 SSc patients and 18 healthy controls. Results  Serum ADAM12-S levels were significantly increased in diffuse cutaneous SSc (dcSSc) patients than in healthy controls (0.417 ± 0.389 vs. 0.226 ± 0.065 ng/mL; P < 0.05), while being comparable between limited cutaneous SSc (0.282 ± 0.258 ng/mL) and healthy controls. Serum ADAM12-S levels significantly elevated in dcSSc patients with disease duration of ≤6 years (0.537 ± 0.449 ng/mL, P < 0.05), but not in dcSSc with disease duration of >6 years (0.225 ± 0.049 ng/mL), compared to healthy controls. Furthermore, in dcSSc patients with disease duration of ≤6 years, serum ADAM12-S levels correlated positively with modified Rodnan total skin thickness score, ground glass score, and serum C-reactive protein values, while showed inverse correlation with fibrosis score. Conclusion  Elevated serum ADAM12-S levels are associated with elevated serum inflammatory marker, severity of skin fibrosis, and activity of interstitial lung disease in dcSSc, suggesting the possible contribution of ADAM12-S to the pathological events in this disorder.

Concepts: Inflammation, ELISA, ELISPOT, Assay, Skin, Eva Engvall, Scleroderma, Systemic scleroderma


Abstract Background: Of postpartum women, 15%-20% retain≥5 kg of their gestational weight gain, increasing risk for adult weight gain. Postpartum women are also in a persistent elevated inflammatory state. Both factors could increase the risk of obesity-related chronic disease. We hypothesized that breastfeeding women randomized to a Mediterranean-style (MED) diet for 4 months would demonstrate significantly greater reductions in body weight, body fat, and inflammation than women randomized to the U.S. Department of Agriculture’s (USDA) MyPyramid diet for Pregnancy and Breastfeeding (comparison diet). Methods: A randomized, controlled dietary intervention trial was conducted in 129 overweight (body mass index [BMI] 27.2±4.9 kg/m(2)), mostly exclusively breastfeeding (73.6%) women who were a mean 17.5 weeks postpartum. Dietary change was assessed using a validated Food Frequency Questionnaire (FFQ) before and after intervention as well as plasma fatty acid measures (gas chromatography/flame ionization detector [GC/FID]). Anthropometric measurements and biomarkers of inflammation, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), also were assessed at baseline and 4 months via enzyme-linked immunosorbent assay (ELISA). Results: Participants in both diet groups demonstrated significant (p<0.001) reductions in body weight (-2.3±3.4 kg and -3.1±3.4 kg for the MED and comparison diets, respectively) and significant (p≤0.002) reductions in all other anthropometric measurements; no significant between-group differences were shown as hypothesized. A significant decrease in TNF-α but not IL-6 was also demonstrated in both diet groups, with no significant between-group difference. Conclusions: Both diets support the promotion of postpartum weight loss and reduction in inflammation (TNF-α) in breastfeeding women.

Concepts: Nutrition, Obesity, Mass, Fat, Body mass index, ELISA, Eva Engvall, Body weight


Contamination of milk and dairy products with aflatoxin M1 (AFM1) presents a risk for human health. The aim of this study was to investigate the presence of AFM1 in pasteurized milk samples in Fariman, located in the province of Khorasan Razavi, Iran, by enzyme-linked immunosorbent assay (ELISA). Forty-five samples of pasteurized milk from different supermarkets were collected during 3 months in summer (July to September, 2012). AFM1 contamination was detected in all of milk samples. The mean concentration of aflatoxin M1 was 27.2 ng/l. The range of AFM1 content was 8.8-64 ng/l. Thirteen (28.8 %) of the samples had AFM1 levels exceeding the maximum levels (50 ng/l) accepted by the European Union. Due to the fact that milk is used by all the age groups including infants and children in Fariman city, it is necessary to minimize the health risk from AFM1 contamination in milk. For this reason, the level of its precursor, aflatoxin B1 (AFB1), in dairy feeds must be reduced, requiring constant aflatoxin monitoring of relevant agricultural commodities.

Concepts: Milk, ELISA, ELISPOT, Assay, Eva Engvall, Immunoassay, Pasteurization, Dairy product


The aim of the study was to evaluate the adequacy of enzyme-linked immunosorbent assay (ELISA) in the post-exposure determination of the β-agonists clenbuterol and salbutamol in animal plasma and serum. Experimental guinea pigs (n = 20) were treated with two doses (0.25 and 2.5 mg/kg) of clenbuterol (n = 10) and salbutamol (n = 10) for seven days, whereas the control animal group (n = 10) was left untreated. Validation of the applied method yielded acceptable recovery (mean > 70%) and repeatability rates, showing ELISA to be applicable for the semi-quantitative determination of both analytes in both matrices, preferably in plasma. In both matrices, clenbuterol concentrations were proven to be significantly (14-fold) higher than those of salbutamol. Concentrations of both analytes were higher in plasma than in serum. The application of a 10-fold higher clenbuterol and salbutamol dose (2.5 mg/kg) resulted in concentrations 3- to 4-fold higher for clenbuterol and 2- to 3-fold higher for salbutamol, indicating a different release rate of these two β-agonists.

Concepts: Antibody, ELISA, ELISPOT, Assay, Eva Engvall, Immunoassay, Multiplex, Magnetic immunoassay


Abstract Background: Interleukin-6 (IL-6) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases such as coronary artery disease (CAD). The -174 IL-6 G/C promoter polymorphism influences mRNA levels and protein expression and is implicated in CAD. The Indian population in South Africa, unlike the black community, has a high prevalence of premature CAD. This polymorphism has not been fully explored in this population. The present study assessed the -174 IL-6 G/C polymorphism in young Indian patients with angiographically documented CAD and compared them with age- and gender-matched Indian and black control subjects. Methods: Polymorphic variants were assessed by polymerase chain reaction-restriction fragment length polymorphism, and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). Results: The -174 IL-6 C allele was found with a higher frequency (23%) in the total Indian group compared to 2% in the black participants [P<0.0001, odds ratio (OR)=0.05, 95% confidence interval (CI) 0.018-0.14). The difference in frequency was more pronounced when Indian controls were compared to black controls (29% vs. 2%, respectively) (P<0.0001, OR=0.05, 95% CI 0.02-0.17). A significant association between the -174 IL-6 G allele and CAD was found in Indian patients compared to Indian controls (84% in cases vs. 71% in Indian controls; P=0.043, OR=0.47 95% CI 0.23-0.95). Levels of IL-6 in circulation were higher in black controls (6.62±0.63 pg/mL) compared to Indian controls (2.51±0.57 pg/mL) and CAD patients (1.46±0.36 pg/mL) (P<0.0001). Levels of IL-6 were higher in all groups with homozygous -174 IL-6 C alleles, but only significant in the healthy Indian control group (GG 3.73±0.94 pg/mL vs. GC/CC 0.89±0.5 pg/mL, P=0.0001). Conclusion: The presence of the IL-6 -174 G allele influences levels of IL-6 and increases the risk of CAD in South African Indians.

Concepts: Atherosclerosis, Coronary artery disease, Heart, Black people, ELISA, ELISPOT, South Africa, Eva Engvall