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ABSTRACT Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14 kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500 ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n = 114), including suspect (n = 30) and positive (n = 50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n = 34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density = 0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.
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Animals , Dogs , Humans , Mice , Rabbits , Leishmaniasis/blood , Dog Diseases/blood , Cysteine Proteases/blood , Leishmania , Enzyme-Linked Immunosorbent Assay , Antibodies, Protozoan , Leishmaniasis/veterinary , Leishmania infantum , Cysteine , Leishmaniasis, VisceralABSTRACT
Purpose: The aim of this study was to determine the seroprevalence of Lymes disease in a population at risk in south India. Methods: Prospective ongoing study and included screening of forest workers and staff of Nagarahole and Bandipur forest ranges in South India for Lymes disease. Screening included a detailed questionnaire for Lymes disease, complete ocular and systemic examination by an ophthalmologist and infectious disease specialist and blood collection. ELISA for IgM and IgG antibodies for Borrelia burgdorferi were performed on the collected sera samples. Western blot confirmation was done on the seropositive samples. Ticks were also collected from these forest areas for future studies to detect if they harbor B. burgdorferi. Results: Seroprevalence of 19.9% was noted by ELISA. Western blot confirmation was seen in 15.6% of the seropositive samples. There was significant correlation between seropositivity and exposure to tick bites (P = 0.023). Conclusion: There is a high seroprevalence of infection with B. burgdorferi in the forest areas of Nagarahole and Bandipur ranges in south India.
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Context: Mycoplasma pneumoniae (M. pneumoniae) causes up to 40% of community-acquired pneumonia in children. It is impossible to identify M. pneumoniae infection on the basis of clinical signs, symptoms, and radiological features. Therefore, correct etiological diagnosis strongly depends on laboratory diagnosis. Aims: This study aims to investigate the role of M. pneumonia e in pediatric lower respiratory tract infections (LRTIs) employing enzyme-linked immunosorbent assays (ELISA) and particle agglutination (PA) test. Settings and Design: Two hundred and eighty children, age 6 months to 12 years with community-acquired LRTIs were investigated for M. pneumoniae etiology. Materials and Methods: We investigated 280 children hospitalized for community-acquired LRTIs, using ELISA and PA test for detecting M. pneumoniae immunoglobulin M (IgM) and immunoglobulin G antibodies. Statistical Analysis Used: The difference of proportion between the qualitative variables was tested using the Chi-square test and Fischer exact test. P ≤ 0.05 was considered as statistically significant. Kappa value was used to assess agreement between ELISA and PA test. Results: M. pneumoniae was positive in 51 (23.2%) <5 years and 33 (54.0%) children in ≥5 years of age group, and this difference was statistically significant (P < 0.001). Clinical and radiological findings in M. pneumoniae positive and negative groups were comparable. ELISA detected M. pneumoniae in 78 (27.8%) and PA test 39 (13.9%) patients; 33 (84.6%) ELISA positive and 6 (15.4%) ELISA negative. ELISA/PA test together detected M. pneumoniae infection in 84 (30%) children. Conclusions: Our data underline that M. pneumoniae plays an important role in children with community-acquired LRTIs and more particularly in children >5 years of age.
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Background: Blood transfusion has profound role to play in specific illness, but still due to unsafe and careless practices the peril of transfusion transmissible infections (TTIs) such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), syphilis and malaria prevails. Objective: To study the seroprevalence of TTIs in healthy blood donors in specific Kuppuswamy’s socio-economic scale at a Blood Bank of a tertiary care teaching hospital in north India, to enhance the awareness about transfusion related risks and to implement better strategic measures to prevent TTI, in high risk groups. Material and Method: Total 10,569 blood units were collected from Jan-2014 to Septmeber-2015. All donors were categorised according to the Kuppuswamy’s Socioeconomic Status Scale (KSESS) followed by screening of all sera samples for hepatitis B surface antigen (HBsAg), antibodies to HCV, HIV types 1 and 2 using enzyme-linked immunosorbent assays (ELISA) and for malaria antigen and Treponema pallidum by using immunochromatographic tests and Rapid Plasma Reagin test (RPR) respectively. All the samples found reactive for HIV, HBsAg, and HCV were again confirmed by second ELISA. Results: The overall seroprevalence was HCV 2.06 % (218/10569) > HBV 1.71% (181/10569) > HIV 0.03% (3/10569). No donor was found positive for Malaria and VDRL. The prevalence of transfusion transmissible diseases in specific socio economic class was as follows-:Upper lower class (IV) 248/2261 (10.96%) > Lower class (V) 34/483 (7.03%) > Lower Middle class (III) 97/5789 (1.67%) > Upper middle class (II) 22/1552 (1.42%) > Upper class (I) 1/484 (0.20%) and seroprevalence of transfusion transmissible diseases in each socio economic class, out of total donations was as follows-: Upper lower class (IV) 248/10569 (2.35%)> Lower middle class (III) 97/10569 (0.92%) >Lower class (V) 34/10569 (0.32%)> upper middle class (II) 22/10569 (0.21) >Upper class (I) 1/10569 (0.009%). Conclusion: Maximum positive TTIs had association with low socio-economic status people with increased medical and behavioral risk factors. Hence, we conclude that awareness among the high risk population group, strict and skillfulness selection of donors and use of effective laboratory screening tests is the prerequisite for the safe donation!!
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Background: Parvovirus B19 infection is associated with clinical symptoms that vary in the spectrum from trivial to severe. The important clinical manifestations are erythema infectiosum or the fifth disease, transient aplastic anemia in patients with hemoglobinopathies, acute polyarthralgia syndrome in adults, hydrops fetalis, spontaneous abortion and stillbirth. Acute infection in nonimmune pregnant women can lead to fetal hydrops. In view of the many complications that can result from acute parvovirus B19 infections during pregnancy, documenting the seroprevalence of anti-parvovirus B19 IgG and its association with the history of abortion in an Iranian population of pregnant women would be of value. Materials and Methods: Serum samples from 86 pregnant women were collected between May and September 2011 in West Azerbaijan province of Iran. Every pregnant woman completed a questionnaire which included age, history of tattooing, blood transfusion, and abortion. Anti-B19 specific IgG was detected by using commercial enzyme-linked immunosorbent assays. Results: Anti-B19- specific IgG antibody was detected in (65/86, 75.6%) of pregnant women. The mean age was 25.56 ± 5.30 years and three women had a documented history of blood transfusion (2 of them tested seropositive for B19). 16/18 (88.8%) of women with a history of abortion were IgG positive. The frequency of abortion sessions in the seropositive group (25 sessions of abortion: 11 women experienced once, 2 twice, 2 thrice and one 4 times) was 4.03 times greater than abortion in seronegative group (2 abortions/21 seronegative women). Conclusion: Our study reaffirms previous reports regarding the higher frequency of abortion among anti-B19 IgG seropositive pregnant women and a possible role of this viral infection in the pathogenesis of abortion.
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The ORFK8.1 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant 0RFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 ug/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein's specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.
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PURPOSE: Mycoplasma pneumoniae is a common respiratory pathogen responsible for acute respiratory infections in young children. The standard laboratory methods for the specific diagnosis of M. pneumoniae infection have been isolation in culture and serological methods. The objective of this study was to compare the performance of enzyme-linked immunosorbent assays (ELISA) for the detection of M. pneumoniae specific IgG and IgM antibodies and polymerase chain reaction (PCR) in diagnosis of M. pneumoniae pneumonia. METHODS: For a 1-year period, 111 patients admitted to Severance Hospital and Yongdong Severance Hospital with clinical features of pneumonia and radiographically defined pneumonia were included. Serum specimens and throat swab specimens were obtained at the time of admission. Patients who showed M. pneumoniae antibody titer 1:320 or greater or a fourfold increase in M. pneumoniae antibody titer between acute and convalescent sera obtained 5 days to 3 weeks after the onset of illness were diagnosed as having M. pneumoniae pneumonia. PCR and ELISA were also performed. RESULTS: The sensitivity, specificity, false positivity, and false negativity of PCR were 40.6 percent, 63.3 percent, 69.1 percent, and 27.5 percent, respectively. The sensitivity, specificity, false positivity, and false negativity of ELISA IgM were 9.4 percent, 100 percent, 0 percent, and 26.9 percent, respectively. The sensitivity, specificity, false positivity, and false negativity of the use of PCR and ELISA in combination were 46.9 percent, 63.3 percent, 65.9 percent, and 25.4 percent, respectively. CONCLUSION: These observations suggest that the use of PCR and ELISA in addition to the detection of serum antibody to Mycoplasma pneumoniae using microparticle agglutination would allow the maximal number of diagnoses to be made at a very early phase of infection.
Subject(s)
Child , Humans , Agglutination , Antibodies , Diagnosis , Enzyme-Linked Immunosorbent Assay , Immunoglobulin G , Immunoglobulin M , Mycoplasma pneumoniae , Mycoplasma , Pharynx , Pneumonia , Pneumonia, Mycoplasma , Polymerase Chain Reaction , Respiratory Tract Infections , Sensitivity and SpecificityABSTRACT
Aim To discuss whether specific Egg yolk antibody(IgY) can be used for snake venom antigens detection.Methods Chickens(white Leghorn) were immunized with detoxicated king cobra venom by formaldehyde.Egg yolk antibody were isolated from egg yolk,and labeled with horse radish peroxidase(HRP).Snake venom antigens samples,including king cobra venom,cobra venom,bungrarus fasciatus venom,bungrarus multicinctus venom,agkistrodon actus venom and Guangdong viper venom,were detected using ELISA,and sensitivity,precision and specificity were tested,respectively.Results At about 32 ?g?L~(-1),the king cobra antigens were detected using the method;Linear relation was better(r=0.963) when the concentration of kingcobra venom was within 32～750?g?L~(-1).The method had good specificity and no cross reactivity was observed among the reagents and agkistrodon acutus guenther venom and vipera russelli siamensis smith venom;little cross reactivity was shown with bungarus multicinctus blyth venom and bungarus fasciatus chmeider venom;cross reactivity was obvious with cobra venom;the average intra-assay coefficient of variation(CV) was 1%～3%,and the inter-assay CV was within 8%.Conclusions The study indicates that IgY can be good reagents for snake venom antigens detecton,and the study provides foundation for the development of the diagnosis kits of snakebites.
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Objective To obtain the prevalence data on human T Lymphotropic virus (HTLV) infection in 2 339 blood donors of an endemic coastal region of Fujian, China. Methods Serum antibodies to HTLV in the donors were detected by a home made double antigen sandwitch ELISA kit. All the ELISA positive samples were further confirmed by Western blot (WB) and/or polymerase chain reaction (PCR) combined with sequencing. Results Nine samples were confirmed as HTLV 1 infection among the 2 339 donors. A man whose wife is one of the nine was also detected to infect by WB and PCR. Conclusion The prevalence of HTLV infection was 0.38% in the endemic coastal region of Fujian, China.