Evaluation of a Rapid Detection Influenza Virus A Antigens Kit Using Paired Serum Antibody Test

Purpose

To evaluate the feasibility for gold immunochromatographic assay (GICA) in rapid detection of influenza virus A infection.

Materials and Methods

Seventy-three patients were enrolled. All patients contributed nasopharyngeal secretions and paired serum samples. Nasopharyngeal secretions was used for colloidal gold immunochromatographic rapid assay for influenza A virus immediately after the collection of specimen. Paired serum samples were used for the hemagglutination inhibition assay at the Centers for Disease Control and Prevention influenza network laboratory in Beijing.

Results

Compare GICA test to hemagglutination inhibition (HI) assay, the Kappa value was 0.402 and the p value in the paired χ² test was higher than 0.05. Therefore, the difference was not statistically significant. The sensitivity of GICA was 50.0% and the specificity was 90.2%, and the negative predictive value was 90.2%.

Conclusion

The sensitivity for Influenza A antigen detection by using GICA is relatively low, the specificity is relatively satisfactory.     

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein

Recent epidemics of West Nile virus (WNV) around the world have been associated with significant rates of mortality and morbidity in humans. To develop standard WNV diagnostic tools that can differentiate WNV from Japanese encephalitis virus (JEV), four monoclonal antibodies (MAbs) specific to WNV envelope (E) protein were produced and characterized by isotyping, reactivity with denatured and native antigens, affinity assay, immunofluorescence assay (IFA), and epitope competition, as well as cross-reactivity with JEV. Two of the MAbs (6A11 and 4B3) showed stronger reactivity with E protein than the others (2F5 and 6H7) in Western blot analysis. 4B3 could bind with denatured antigen, as well as native antigens in indirect ELISA, flow cytometry analysis, and IFA; whereas 2F5 showed highest affinity with native antigen. 4B3 and 2F5 were therefore used to establish an antigen capture-ELISA (AC-ELISA) detection system. The sensitivity of this AC-ELISA was 3.95 TCID(50)/0.1 ml for WNV-infected cell culture supernatant. Notably, these MAbs showed no cross-reactivity with JEV, which suggests that they are useful for further development of highly sensitive, easy handling, and less time-consuming detection kits/tools in WNV surveillance in areas where JEV is epidemic.     

Novel monoclonal antibodies that bind to wild and fixed rabies virus strains

Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2bκ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays.     

Comparison of the direct fluorescence assay and real-time polymerase chain reaction for the detection of influenza virus A and B in immunocompromised patients

OBJECTIVE:

This study evaluated the diagnostic performance of two methods for the detection of influenza virus in immunocompromised transplant patients.

METHODS:

A total of 475 respiratory samples, 236 from patients in a hematopoietic stem cell transplantation program and 239 from kidney transplant patients, were analyzed by a direct fluorescence assay and the Centers for Disease Control real-time polymerase chain reaction protocol for influenza A and B detection.

RESULTS:

Influenza detection using either method was 7.6% in the hematopoietic stem cell transplant group and 30.5% in the kidney transplant patient group. Influenza detection by real-time polymerase chain reaction yielded a higher positive rate compared with fluorescence than that reported by other studies, and this difference was more pronounced for influenza A. The fluorescence assay sensitivity, specificity, positive and negative predictive values, and kappa coefficient were 17.6%, 100%, 1, 0.83, and 0.256, respectively, and lower detection rates occurred in the kidney transplant patients.

CONCLUSIONS:

The real-time polymerase chain reaction performance and the associated turnaround time for a large number of samples support the choice of this method for use in different routine diagnostic settings and influenza surveillance in high-risk patients.     

Performance evaluation of the HepB Typer-Entecavir kit for detection of entecavir resistance mutations in chronic hepatitis B

Background/Aims

Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations.

Methods

The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients.

Results

Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%.

Conclusions

The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B.     

Stray dog trade fuelled by dog meat consumption as a risk factor for rabies infection in Calabar,southern Nigeria

Background

Rabies is a preventable zoonosis with the highest case fatality of any disease in the world. In the developing world, it is transmitted mainly by dog bites. In parts of southern Nigeria, dog meat is a delicacy.

Objective

To highlight trade in stray dogs as a major risk factor for rabies in animals and humans in south-south Nigeria.

Method

Patients admitted into the University of Calabar Teaching Hospital (UCTH) with a diagnosis of rabies between July and October 2012 were analysed for risk factors, post exposure prophylaxis (PEP), health seeking behaviour and outcome. Focused group interview were also conducted among traders/handlers of stray dogs.

Results

Ten cases of rabies in subjects aged 3 to 52 years were recorded in these five months period. Eight of the cases were male and apparently got infected directly or indirectly through the trade in stray dogs for human consumption. None had proper PEP and all patients died.

Conclusion

Stray dog trade, fuelled by eating of dog meat, is a risk factor for human and animal rabies in Calabar, southern Nigeria. Culling of stray dogs, control of stray dogs'' trade and public enlightenment on PEP is recommended.     

Comparison of HCV core antigen and anti-HCV with HCV RNA results

Background

The measurement of anti-HCV antibodies using immunological methods and the confirmation of viral nuclear acid based on molecular methods is important in diagnosis and follow-up of the HCV infection.

Objectives

In this study, we aimed to analyse HCV core Antigen positivity among anti-HCV antibody positive sera to determine the significance of testing of HCV core Ag for the laboratory diagnosis of HCV infection, by considering the correlation between serum HCV core Ag and HCV RNA levels.

Methods

115 patients suspected of having hepatitis C and who were positive for anti-HCV antibody were investigated using chemiluminescent and molecular methods. Anti-HCV antibody, HCV core Ag and HCV RNA levels were detected by the Vitros ECiQ immunodiagnostic system, Architect i2000 system and RT-PCR, respectively.

Results

The sensitivity, specificity, positive and negative predictive values and accuracy rate of HCV core Antigen assay were detected as 86.5%(83/96), 100%(19/19), 100%(83/83), 59.4%(19/32), 88.7%(102/115) respectively.

Conclusion

HCV core Ag assay could be used for diagnosis of HCV infection as it is easy to perform, cost-effective, has high specificity and positive predictive value. However, it should be kept in mind that it may have lack of sensitivity and negative predictive value.     

Evaluation of a Chromogenic Culture Medium for the Detection of Clostridium difficile

Purpose

Clostridium difficile (C. difficile) is an important cause of nosocomial diarrhea. Diagnostic methods for detection of C. difficile infection (CDI) are shifting to molecular techniques, which are faster and more sensitive than conventional methods. Although recent advances in these methods have been made in terms of their cost-benefit, ease of use, and turnaround time, anaerobic culture remains an important method for detection of CDI.

Materials and Methods

In efforts to evaluate a novel chromogenic medium for the detection of C. difficile (chromID CD agar), 289 fecal specimens were analyzed using two other culture media of blood agar and cycloserine-cefoxitin-fructose-egg yolk agar while enzyme immunosorbent assay and polymerase chain reaction-based assay were used for toxin detection.

Results

ChromID showed the highest detection rate among the three culture media. Both positive rate and sensitivity were higher from chromID than other culture media. ChromID was better at detecting toxin producing C. difficile at 24 h and showed the highest detection rate at both 24 h and 48 h.

Conclusion

Simultaneous use of toxin assay and anaerobic culture has been considered as the most accurate and sensitive diagnostic approach of CDI. Utilization of a more rapid and sensitive chromogenic medium will aid in the dianogsis of CDI.     

Development of Monoclonal Antibodies Against Human IRF-5 and Their Use in Identifying the Binding of IRF-5 to Nuclear Import Proteins Karyopherin-��1 and -��1

Purpose

IRF-5 is a direct transducer of virus-mediated and TLR-mediated signaling pathways for the expression of cytokines and chemokines which form homodimers or heterodimers with IRF-7. However, direct IRF-5-specific monoclonal antibodies (mAbs) are not available at present. These could be used to further evaluate the functions of IRF-5. In this study, we produced and characterized three mouse mAbs to human IRF-5. The binding of IRF-5 to nuclear import proteins was first identified using a mAb.

Materials and Methods

His-tagged human IRF-5 protein spanning amino acid residues 193-257 was used as an antigen and three mAbs were produced. The mAbs were tested with ELISA, Western blot analysis (WB), immunofluorescent staining (IF), and immunoprecipitation (IP). In addition, the nuclear import protein which carried phosphorylated IRF-5 was identified using one of these mAbs.

Results

MAbs 5IRF8, 5IRF10 and 5IRF24 which reacted with the recombinant His-IRF-5^193-257 protein were produced. All mAbs bound to human IRF-5, but not to IRF-3 or IRF-7. They could be used for WB, IF, and IP studies. The binding of phosphorylated IRF-5 to karyopherin-α1 and -β1 was also identified.

Conclusion

Human IRF-5-specific mAbs are produced for studying the immunologic roles related to IRF-5. Phosphorylated IRF-5 is transported to the nucleus by binding to nuclear import proteins karyopherin-α1 and -β1.     

Compliance with iron-folic acid (IFA) therapy among pregnant women in an urban area of south India

Background

Anaemia is highly prevalent among pregnant women and iron deficiency is the most important cause. Like many other countries, India has policies to give pregnant women iron supplements. Non-compliance is one important challenging factor in combating anaemia.

Objective

To estimate the compliance for IFA tablets among pregnant women and to study the social factors influencing it.

Methodology

This study included 190 pregnant women seeking ante-natal care in tertiary health Centres in the Mangalore city in South India. After Institutional Ethics Committee (IEC) approval, data was collected by personal interview. Missing ≥2 doses consecutively was considered non-compliance. The data was analyzed using SPSS (Statistical Package for Social Sciences) version 11.5.

Results

The mean age of the study population was 25.8 years (SD: 4.1). Majority of the subjects consumed mixed diet and 72.1% belonged to lower socioeconomic status. Overall, compliance with IFA tablets was 64.7%. Compliance increased with the increase in age, birth order and single daily dose. Forgetfulness and both perceived as well as experienced side effects of IFA therapy were the important factors for non-compliance.

Conclusion

There was a moderate level of Compliance towards IFA tablets with key social and demographic factors playing important role.     

Compliance with iron-folic acid (IFA) therapy among pregnant women in an urban area of south India

Background

Anaemia is highly prevalent among pregnant women and iron deficiency is the most important cause. Like many other countries, India has policies to give pregnant women iron supplements. Non-compliance is one important challenging factor in combating anaemia.

Objective

To estimate the compliance for IFA tablets among pregnant women and to study the social factors influencing it.

Methodology

This study included 190 pregnant women seeking ante-natal care in tertiary health centres in the Mangalore city in south India. After Institutional Ethics Committee (IEC) approval, data was collected by personal interview. Missing >2 doses consecutively was considered non-compliance. The data was analyzed using SPSS (Statistical Package for Social Sciences) version 11.5.

Results

The mean age of the study population was 25.8 years (SD: 4.1). Most of the subjects consumed mixed diet and 72.1% belonged to lower socioeconomic status. Overall, compliance with IFA tablets was 64.7%. Compliance increased with the increase in age, birth order and single daily dose. Forgetfulness and both perceived as well as experienced side effects of IFA therapy were the important factors for non-compliance.

Conclusion

There was a moderate level of compliance towards IFA tablets with key social and demographic factors playing an important role.     

Clinical significance of occult hepatitis B virus infection in chronic hepatitis C patients

Background/Aims

We investigated the frequency of occult hepatitis B virus (HBV) infection in anti-hepatitis C virus (HCV)-positive individuals and the effects of occult HBV infection on the severity of liver disease.

Methods

Seventy-one hepatitis B virus surface-antigen (HBsAg)-negative patients were divided according to their HBV serological status into groups A (anti-HBc positive, anti-HBs negative; n=18), B (anti-HBc positive, anti-HBs positive; n=34), and C (anti-HBc negative, anti-HBs positive/negative; n=19), and by anti-HCV positivity (anti-HCV positive; n=32 vs. anti-HCV negative; n=39). Liver biopsy samples were taken, and HBV DNA was quantified by real-time PCR.

Results

Intrahepatic HBV DNA was detected in 32.4% (23/71) of the entire cohort, and HBV DNA levels were invariably low in the different groups. Occult HBV infection was detected more frequently in the anti-HBc-positive patients. Intrahepatic HBV DNA was detected in 28.1% (9/32) of the anti-HCV-positive and 35.9% (14/39) of the anti-HCV-negative subjects. The HCV genotype did not affect the detection rate of intrahepatic HBV DNA. In anti-HCV-positive cases, occult HBV infection did not affect liver disease severity.

Conclusions

Low levels of intrahepatic HBV DNA were detected frequently in both HBsAg-negative and anti-HCV-positive cases. However, the frequency of occult HBV infection was not affected by the presence of hepatitis C, and occult HBV infection did not have a significant effect on the disease severity of hepatitis C.     

Recombinant Diabody-Based Immunocapture Enzyme-Linked Immunosorbent Assay for Quantification of Rabies Virus Glycoprotein

The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (V_H) and light chain (V_L) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human × mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r² value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P < 0.001), while the predicted potencies by the two tests did not differ significantly (P > 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.Rabies is a fatal viral infection of the nervous system affecting all mammals, including humans through bite wounds from a rabid animal, which can be prevented by vaccination coupled with administration of anti-rabies virus serum (6, 11). Rabies transmission from nonbite exposures is rare. Scratches, abrasions, open wounds, or mucous membranes contaminated with saliva or other potentially infectious material (such as brain tissue) from a rabid animal constitute nonbite exposures. Occasionally reports of nonbite exposure are such that postexposure prophylaxis is given. Inhalation of aerosolized rabies virus is also a potential nonbite route of exposure, but with the exception of laboratory workers, most people are unlikely to encounter an aerosol version of the rabies virus (5). Organ transplantations have also been credited with nonbite transmission of rabies from human to human (3). Despite significant scientific progress, rabies remains an important zoonotic disease globally. Annually, 20,000 deaths are reported in India, making rabies one of the major causes of human mortality (21). Vaccination is therefore considered one of the most viable and important methods for the prevention of rabies by way of preexposure prophylaxis in high-risk groups, postexposure prophylaxis in contact groups, and preexposure prophylaxis in pet animals that are at risk due to possible contacts with rabid animals. The most cost-effective means of prevention and control of rabies in humans is by eliminating rabies in dogs and other susceptible animals through vaccination.The NIH mouse protection test is an in vivo potency test that has been used widely by all manufacturers of rabies vaccines. The role of different immunological parameters and the presence of virus-neutralizing antibodies are not well established because of a weak correlation between the NIH potency test results and immunogenicity when vaccines containing different strains of rabies virus were tested (2). Furthermore, this method is time-consuming and expensive, requires a large number of animals, and involves the use of live rabies virus. As a result, there is increased exposure in human beings to live and virulent rabies strains. The NIH test also requires a secure biosafety level 3 (BSL-3) facility for housing and challenging the experimental animals. Therefore, for both practical and ethical reasons, replacement of this test by more rapid and reliable in vitro methods is highly desirable. Based on the fact that the rabies virus glycoprotein (RV GP) is the antigen responsible for inducing virus-neutralizing antibodies and conferring protection against a lethal intracerebral challenge, it has been suggested that the antigenicity of the rabies vaccines could be evaluated by titration of the RV GP (17).Though some laboratories have used enzyme-linked immunosorbent assay (ELISA) to assess RV GP content for determination of the potencies of inactivated vaccines, variable correlation between ELISA and the NIH test (7, 8, 9, 13, 14, 17, 18, 20) has been reported. Essentially, all these ELISAs incorporate the use of either polyclonal antibodies or hybridoma-derived monoclonal antibodies (MAbs). Although MAbs offer substantial advantages with respect to potency, reproducibility, and freedom from contaminants (4), they are difficult to prepare in a quality-assured manner.Recombinant DNA technology has been used to a great extent in the expression of antibodies/antibody fragments (12). Antibody fragments can be readily produced from the genes encoding antibody variable domains, which can be derived either from hybridomas (19) or from bacteriophage displaying antibody fragments (16). Diabodies are bivalent or bispecific antibody fragments generated by the dimerization of variable heavy (V_H)-light chain (V_L) fragments (10) as a result of reduction in the size of the linker between variable light and variable heavy chains (1), and these antibodies have many practical applications, including immunoassay and therapy.We describe for the first time the use of a recombinant diabody in the development of an ELISA for quantification of RV GP content in human rabies vaccines incorporating the PV strain of rabies virus and its comparison with the NIH mouse protection test.     

Effect of aldosterone on the amplification of oncolytic vaccinia virus in human cancer lines

Background/Aims

JX-594 is an oncolytic virus derived from the Wyeth vaccinia strain that causes replication-dependent cytolysis and antitumor immunity. Starting with a cross-examination of clinical-trial samples from advanced hepatocellular carcinoma patients having high levels of aldosterone and virus amplification in JX-594 treatment, we investigated the association between virus amplification and aldosterone in human cancer cell lines.

Methods

Cell proliferation was determined by a cell-counting-kit-based colorimetric assay, and vaccinia virus quantitation was performed by quantitative polymerase chain reaction (qPCR) and a viral plaque assay. Also, the intracellular pH was measured using a pH-sensitive dye.

Results

Simultaneous treatment with JX-594 and aldosterone significantly increased viral replication in A2780, PC-3, and HepG2 cell lines, but not in U2OS cell lines. Furthermore, the aldosterone treatment time altered the JX-594 replication according to the cell line. The JX-594 replication peaked after 48 and 24 hours of treatment in PC-3 and HepG2 cells, respectively. qPCR showed that JX-594 entry across the plasma membrane was increased, however, the changes are not significant by the treatment. This was inhibited by treatment with spironolactone (an aldosterone-receptor inhibitor). JX-594 entry was significantly decreased by treatment with EIPA [5-(N-ethyl-N-isopropyl)amiloride; a Na⁺/H⁺-exchange inhibitor], but aldosterone significantly restored JX-594 entry even in the presence of EIPA. Intracellular alkalization was observed after aldosterone treatment but was acidified by EIPA treatment.

Conclusions

Aldosterone stimulates JX-594 amplification via increased virus entry by affecting the H⁺ gradient.     

Prevalence of Chlamydia trachomatis infection among patients attending infertility and sexually transmitted diseases clinic (STD) in Kano,North Western Nigeria

Background

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the world with severe complications. The aim of this study was to determine the prevalence and possible risk factors of C. trachomatis in Kano. There is dearth of information on this subject in this locality.

Method

Urine samples, Endocervical swabs and Urethral swab were collected from consecutive patients attending the Infertility and STD clinics in Aminu Kano Teaching Hospital (AKTH) between June and December 2012, after administering a questionnaire by the attending physician and also obtaining an informed consent.Samples were analyzed using Diaspot Chlamydia kit, a rapid immunoassay test for the detection of genital chlamydial antigen in urinogenital samples.

Results

A total of 125 consecutive samples were collected, comprising 69 females and 56 males aged between 14 – 55 years. Twelve samples tested positive for C. trachomatis antigen giving a prevalence rate of 9.6%. The age group prevalence were as follows 25 – 29 yrs (17.1%), 20 – 24 (16.7%), 15 – 19 (12.5%), 30 – 34 (11.1%) and > 49 years (9.0%). Married patients were associated with higher infection rate than single (8.3%), and divorced patients (33.3%). A higher percentage of the patients (95.2%) were not aware of the existence of C. trachomatis infection and its complications. Previous STD exposure was associated with increased risk of Chlamydia infection.

Conclusion

C. trachomatis infection if unchecked will continue to pose a threat to reproductive life with its established complications. Since asymptomatic cases are common in the population regular screening should be encouraged for every adult especially before commencement of marital life.     

Monoclonal antibody binding to the major outer membrane protein of Campylobacter coli

Campylobacter species are major enteric pathogens causing diarrhea illness in humans and animals. Immunological tests are needed for accurate and rapid identification of C. coli, in conjunction with the use of standard biochemical tests. We initiated the creation of monoclonal antibodies (MAbs) using whole C. coli cells as antigen. Four positive clones were identified, namely MAb2G6, MAb3B9, MAb4A10 and MAb5B9. Dot-blot assay and ELISA revealed that only MAb2G6 did not cross react with C. jejuni and other Campylobacter isolates. As demonstrated by dot-blot assay, MAb2G6 reacted with all 23 C. coli isolates tested but did not react with 29 isolates of C. jejuni, 3 other Campylobacter spp. isolates and 19 non-Campylobacter isolates, with the lowest detection limit was in the range of 10³ to 10⁴ bacteria. Western blots and dot blots showed that the antigen of MAb2G6 was a native protein, with immunoprecipitation assay showed that MAb2G6 bound to a protein band of approximately 43 kDa in size, corresponding to major outer membrane protein (MOMP) of C. coli revealed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Immunofluorescence assay (IFA) showed that MOMP of C. coli was indeed the antigen of MAb2G6, with immunogold-electron microscopy demonstrated that MAb2G6 conjugated with immunogold particles bound to all over the surface of C. coli cells. MAb2G6 also showed potential usage in direct detection of C. coli in faecal samples.     

Characterization of Monoclonal Antibodies to Junin Virus Nucleocapsid Protein and Application to the Diagnosis of Hemorrhagic Fever Caused by South American Arenaviruses

Junin virus (JUNV), Machupo virus, Guanarito virus, Sabia virus, and Chapare virus are members of New World arenavirus clade B and are the etiological agents of viral hemorrhagic fevers that occur in South America. In this study, we produced three monoclonal antibodies (MAbs) to the recombinant nucleocapsid protein of JUNV, designated C6-9, C11-12, and E4-2. The specificity of these MAbs was examined by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, and an epitope-mapping method. Using these MAbs, we developed antigen (Ag) capture ELISA systems. We showed that by using MAb C6-9, JUNV Ag was specifically detected. On the other hand, by using MAb C11-12 or E-4-2, the Ags of all human pathogenic South American arenaviruses were detected. The combined use of these Ag capture ELISA systems in the present study may be useful for the diagnosis of acute-phase viral hemorrhagic fever due to infection by a South American arenavirus.The South American arenaviruses Junin virus (JUNV), Machupo virus (MACV), Guanarito virus (GTOV), Sabia virus (SABV), and Chapare virus (CHPV) are members of New World arenavirus clade B. JUNV, MACV, GTOV, and SABV are the etiological agents of Argentine hemorrhagic fever (AHF), Bolivian hemorrhagic fever (BHF), Venezuelan hemorrhagic fever (VHF), and Brazilian hemorrhagic fever, respectively (4). CHPV was also recently shown to be associated with cases of hemorrhagic fever in Bolivia (5). AHF emerged in the 1950s, and since then, outbreaks have occurred annually without interruption (4). The mortality rate for AHF is estimated to be 15 to 30%, but early treatment with immune plasma reduces the rate to less than 1% (6). The region at risk has been progressively expanding into northern central Argentina, and almost 5 million people are currently considered to be at risk for AHF (6, 13). Phylogenetic analysis indicates that JUNV is more closely related to MACV than to SABV or CHPV, whereas SABV and CHPV are more closely related to each other than to other New World arenaviruses (5).Arenaviruses are enveloped and contain a bisegmented RNA genome. The genome consists of two ambisense single-stranded RNA molecules, one designated L, which encodes the RNA-dependent RNA polymerase and a zinc-binding matrix protein, Z, and the other designated S, which encodes the major structural components of the virion, i.e., the nucleocapsid protein (NP) and the envelope glycoprotein precursor (15). The arenavirus NP is the most abundant protein among the viral structural proteins both in infected cells and in virions (2) and is commonly used as a target for detecting viral antigens (Ags) (20). Moreover, arenavirus NPs have been known to be the most conserved among the same virus species and, to some extent, among different arenavirus species (3, 8). Therefore, it seems likely that monoclonal antibodies (MAbs) raised against the NP of an arenavirus would also be useful for detecting other arenaviruses (20). Recently, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed by using a recombinant NP (rNP) of JUNV, obtained from a recombinant baculovirus system, and was proposed to be useful for etiologic confirmation of AHF in seroepidemiological studies (20, 26). It is considered that an Ag capture ELISA using MAbs specific for viral Ags allows rapid diagnosis of the acute phase of viral hemorrhagic fever by detecting viral Ags in blood or tissue homogenates (20). In this study, we produced MAbs to the rNP of JUNV. These MAbs were characterized by ELISA, indirect immunofluorescence assay (IFA), and an epitope-mapping method. Ag capture ELISAs were developed by using these MAbs that are specific for JUNV and that are broadly applicable for the detection of human pathogenic New World arenaviruses.     

Direct fluorescent antibody assay and polymerase chain reaction for the detection of Chlamydia trachomatis in patients with vernal keratoconjunctivitis

OBJECTIVES:

To identify Chlamydia trachomatis via polymerase chain reaction and a direct fluorescent antibody assay in patients with vernal keratoconjunctivitis while comparing the efficacies of both tests for detecting Chlamydia trachomatis in these conditions.

METHODS:

Conjunctival scraping samples were obtained from 177 patients who were divided into two groups: a vernal keratoconjunctivitis group (group A) and a control group (group B). The polymerase chain reaction and a direct fluorescent antibody assay were performed. Sensitivity, specificity, receiver operating characteristic curves, and areas under the curve were calculated for both tests in groups A and B. Receiver operating characteristic curves were plotted using a categorical variable with only two possible outcomes (positive and negative).

RESULTS:

Statistical analysis revealed a significant association between vernal keratoconjunctivitis and Chlamydia trachomatis infection detected by a direct fluorescent antibody assay with high sensitivity and specificity. All patients in group A with positive polymerase chain reactions also presented with positive direct fluorescent antibody assays.

CONCLUSION:

The association between vernal keratoconjunctivitis and Chlamydia trachomatis infection was confirmed by positive direct fluorescent antibody assays in 49.4% of vernal keratoconjunctivitis patients and by positive polymerase chain reactions in 20% of these patients. The direct fluorescent antibody assay detected Chlamydia trachomatis in a higher number of patients than did the polymerase chain reaction. Although the diagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients. Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggest considering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergic disease.     

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

Purpose

Cockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105.

Methods

Recombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining.

Results

The rPer a 1.0105 (~13 kDa) had 100%, 98% and ≥90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues⁹⁹ QDLLLQLRDKGV¹¹⁰ contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 ℃g per gram of feces.

Conclusions

The novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.