Standardization and validation of a western blot for the diagnosis of human immunodeficiency virus

Objectives: To standardize and validate a western blot test for the diagnosis of human immunodeficiency virus. Methods: A prospective observational study was carried out during 2017 and 2018. The western blot test was standardized, using the polyacrylamide gel electrophoresis technique with sodium dodecyl sulfate (SDS PAGE), being the nitrocellulose blot strips prepared with an Optimal HIV-1 antigen concentration of 2.71 µg / mm. The western blot was validated in the laboratory against 400 reference samples (300 sera and 100 plasmas): 200 positive and 200 negatives for antibodies against HIV-1, being the reference test the Immunoblot of the Fujirebio brand. Diagnostic performance parameters were estimated using Epidat v3.1 and Excel. Results: Eight important bands of the HIV-1 antigen were identified: p17, p24, p31, p39, gp41, p55, p66, and gp120. According to the Consortium for the normalization of serology for retroviruse, those that were taken as specific diagnostic bands were: p24, p31, gp41, and gp120. The sensitivity, specificity, positive and negative predictive value and validity index against sera were: 96.7%, 96.0%, 96.0%, 96.6%, 96.3%; and against plasmas: 98.0%, 100.0%, 100.0%, 98.0%, 99.0% respectively. No false positives and negatives were found, but some were undetermined. Conclusion: The development of this western blot test with proprietary technology presented similar diagnostic performance to the reference test, without showing cross-reactions, being useful for confirming HIV.

Migratory non-itchy rash

A 58-year-old man presented with 4-day history of multiple, erythematous, non-itchy, painless, patchy spots, along with fatigue and jaw pain. This rash started around the periumbilical area and then spread over his chest and right upper back (Figure 1,2). There was no involvement of face, mucous membranes, and extremities. He denied any sore throat, cough, or other symptoms. Besides the skin rash, his physical examination was unremarkable. There was no temporomandibular joint swelling, or joint tenderness. A month ago, he travelled along with the west coast of Michigan – a Lyme-endemic region of the USA and noted his exposure to mosquitoes. Shortly after his visit, he recalled having fever, chills, myalgia and a similar patchy groin rash which resolved in a few days. At that time, blood work by his family physician revealed mild transaminitis. At the current visit, repeat blood work and electrocardiogram were normal. A clinical diagnosis of early disseminated Lyme disease was made. Lyme Ab IgM and IgG were both elevated, as was his Western blot test. He was given a 10-day course of doxycycline. He reported complete resolution of his symptoms at follow up.

Dibenzofuran, 4-Chromanone, Acetophenone, and Dithiecine Derivatives: Cytotoxic Constituents from Eupatorium fortunei

Five new compounds, eupatodibenzofuran A (1), eupatodibenzofuran B (2), 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one (3), eupatofortunone (4), and eupatodithiecine (5), have been isolated from the aerial part of Eupatorium fortunei, together with 11 known compounds (6‒16). Compounds 1 and 2 featured a new carbon skeleton with an unprecedented 1-(9-(4-methylphenyl)-6-methyldibe nzo[b,d]furan-2-yl)ethenone. Among the isolates, compound 1 exhibited potent inhibitory activity with IC50 values of 5.95 ± 0.89 and 5.55 ± 0.23 μM, respectively, against A549 and MCF-7 cells. The colony-formation assay demonstrated that compound 1 (5 μM) obviously decreased A549 and MCF-7 cell proliferation, and Western blot test confirmed that compound 1 markedly induced apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways.

Borrelia miyamotoi DNA in a patient suspected of Lyme borreliosis

Background Clinical manifestations in infection caused by B. miyamotoi can mimick highly variable symptoms of Lyme disease. The aim of our studies was to detect DNA of B. miyamotoi spirochetes in clinical materials from patients suspected of neuroborreliosis(retrospectively).Methods Samples of blood serum and cerebrospinal fluid were collected from 133 patients with clinical manifestations of neuroborreliosis. Diagnosis was established by detection of IgM and / or IgG specific antibodies to B. burgdorferi with ELISA in both sera and CSF. Specificity of positive ELISA results in sera were confirmed with Western-blot test. Bacterial DNA from the collected material was extracted, amplified and sequenced.Results Among 133 patients with clinical manifestations of neuroborreliosis recognized in the years 2010-2018., DNA of B. miyamotoi was detected in CSF from 1 (0.8%) patient with extraocular optic neuritis of the left eye (GenBank accession No. MK674170 and MK674171).Conclusion Detection of B. miyamotoi in patients with central nervous system infections, will allow a better understanding of the epidemiology of infections caused by Borrelia sp. spirochetes. Patients with neurological symptoms and questionable serological findings are a serious diagnostic problem, due to failure to meet the criteria for neuroboreliosis. This indicates the need for further studies of patients with signs of CNS infection.

Borrelia miyamotoi DNA in a patient suspected of Lyme borreliosis

Background Clinical manifestations in infection caused by B. miyamotoi can mimick highly variable symptoms of Lyme disease. The aim of our studies was to detect DNA of B. miyamotoi spirochetes in clinical materials from patients suspected of neuroborreliosis(retrospectively).Methods Samples of blood serum and cerebrospinal fluid were collected from 133 patients with clinical manifestations of neuroborreliosis. Diagnosis was established by detection of IgM and / or IgG specific antibodies to B. burgdorferi with ELISA in both sera and CSF. Specificity of positive ELISA results in sera were confirmed with Western-blot test. Bacterial DNA from the collected material was extracted, amplified and sequenced.Results Among 133 patients with clinical manifestations of neuroborreliosis recognized in the years 2010-2018., DNA of B. miyamotoi was detected in CSF from 1 (0.8%) patient with extraocular optic neuritis of the left eye (GenBank accession No. MK674170 and MK674171).Conclusion Detection of B. miyamotoi in patients with central nervous system infections, will allow a better understanding of the epidemiology of infections caused by Borrelia sp. spirochetes. Patients with neurological symptoms and questionable serological findings are a serious diagnostic problem, due to failure to meet the criteria for neuroboreliosis. This indicates the need for further studies of patients with signs of CNS infection.

Tick exposure and prevalence of Borrelia burgdorferi antibodies among hunters and other individuals exposed to vector ticks in eastern Poland

Background. Lyme borreliosis is the most frequent tick-borne disease in Europe and North America, and the number of registered cases is on the increase. Frequent presence in the habitats of ticks enhances the risk of tick bites and possible infection with Borrelia burgdorferi spirochetes. Objective. The aim of the study was to assess the risk of B. burgdorferi infection posed to hunters and other individuals exposed to activity-related contact with ticks. Material and methods. The study was carried out in the northern part of the Lublin Province (eastern Poland) and involved 150 individuals exposed to tick bites (110 hunters and 40 individuals exposed to activity-related contact with ticks). The analysis of sera for the presence of B. burgdorferi IgM and IgG antibodies was carried out. All 150 individuals were tested with the ELISA assay, and positive and borderline results of the assay were verified with the Western blot test. All study participants completed a questionnaire, which provided information about exposure to ticks, application of prophylactic measures, and awareness of Lyme borreliosis. Results. The ELISA assay revealed a positive or borderline result in at least one of the classes of B. burgdorferi antibodies in 63.3% (95/150) of the individuals (IgM 14.0%, IgG 63.3%). Verification carried out with the Western blot test showed a positive or borderline result in at least one of the antibody classes in 38.0% (57/150) of the examined persons (IgM 2.7%, IgG 36.7%). Abdomen (56.0%) and legs (53.7%) were the most frequently bitten body regions. Tick bites on the abdomen were significantly more frequently declared by hunters. Inspection of the body after returning from natural areas was more popular prophylactic method than use of repellents. Inspection of the body was significantly more often used in the group of the hunters. Conclusions. The risk of B. burgdorferi infection among hunters and other individuals undertaking activities associated with exposure to tick bites in the study area is high.

Evaluation of caprine arthritis-encephalitis virus transmission in newborn goat kids

ABSTRACT: Caprine arthritis encephalitis causes considerable losses in goat production. The main form of the caprine arthritis encephalitis virus transmission is through the ingestion of colostrum or milk from infected females. However, some transmissions cannot be explained in this manner. Therefore, this study aimed to evaluate transplacental transmission of caprine arthritis encephalitis virus. Blood samples were collected from 283 newborn kids of Anglo-Nubian and Saanen breeds born from seropositive and seronegative goats. Samples were collected immediately after birth and analyzed with agarose gel immunodiffusion and western blot. All samples were negative in the agarose gel immunodiffusion. However, the western blot test demonstrated that four kids were born positive for caprine arthritis encephalitis virus. This result indicates that although in a low frequency (1.4%), there is a possibility of transplacental transmission of small ruminant lentivirus.

A competitive enzyme-linked immunosorbent assay for rapid detection of antibodies againstTrichinella spiralisandT. britovi– one test for humans and swine

Infection with parasites from theTrichinellagenus occurs in many vertebrates but disease only occurs in humans (trichinellosis). Humans are infected after the consumption of raw or undercooked meat from infected wild or domestic animals (usually swine or horses). Using the monoclonal antibody (mAb) 7C2C5, specific for an epitope unique to the muscle larvae of the genusTrichinella, we have developed a competitive enzyme-linked immunosorbent assay (c-ELISA) that enables the rapid detection ofTrichinella-specific antibodies in sera originating from two different host species (human, swine) infected with eitherTrichinella spiralisorTrichinella britovi. This novel c-ELISA exhibited 100% specificity and sensitivity, as confirmed by a Western blot test. The assay is easy to use (one incubation step), and the time required for the procedure (45 min) is shorter than in any other ELISA format. This test could be useful for both the detection and surveillance ofTrichinellainfections.

mTOR REGULATES THE PROLIFERATION AND DIFFERENTIATION OF TENDON STEM CELLS: AN IN VITRO STUDY

Objectives: The mechanistic target of rapamycin (mTOR) controls cell growth and proliferation via translation regulation in eukaryotes. The present study investigated the effects of mTOR on the proliferation and differentiation of tendon stem cells (TSCs). Methods: The proliferation and differentiation ability of TSCs was tested in response to antagonist (MHY1485), and a depressor of mTOR (Rapamycin and KU0063794). CCK test was performed to test cell proliferation; quantitative real-time PCR (RT-PCR) and Western blot test were performed to evaluate the differentiation of TSCs. Results: Blocking of mTOR1 inhibited the proliferation of TSCs and blocking of mTOR2 enhanced the proliferation of TSCs; however, the effects of mTOR1 surpassed the effects of mTOR2. Blocking of mTOR1 or activation of mTOR2 induced the expression of TNC, and blocking of mTOR2 inhibited the expression of TNC. Blocking of mTOR1 by rapamycin decreased the expression of ap2. Both blocking of mTOR1 or mTOR2 had little effects on the expression of Runx2 and Sox9; however, activation of mTOR2 induced the expression of Runx2 and Sox9. Moreover, the Western blot test showed that blocking of mTOR1 by Rapamycin or the blocking of both mTOR1 and mTOR2 by KU-0062794 enhanced the expression of TNC; in addition, blocking of mTOR1 by Rapamycin enhanced the expression of c-EBPα and Sox9. However, activation of mTOR1 and mTOR2 by MHY1485 increased the expression of Runx2. Conclusions: mTOR played important roles in the proliferation and differentiation of TSCs. Furthermore, mTOR1 and mTOR2 played different roles on the proliferation and differentiation of TSCs. Blocking mTOR1 inhibited the proliferation of TSCs and played a dominant function.Blocking of mTOR1 enhanced the expression of tenocyte related genes; however, blocking of mTOR2 inhibited the expression of TNC. Blocking of mTOR1 by rapamycin decreased the expression of ap2 and activation of mTOR2 induced the expression of Runx2.