IgM-capture Enzyme-linked Immunosorbent Assay Research Articles

Possible vertical transmission of Chikungunya virus infection detected in the cord blood samples from a birth cohort in Vietnam

BackgroundChikungunya virus (CHIKV) is an alphavirus (genus Alphavirus, family Togaviridae) that is primarily transmitted to humans by Aedes mosquitoes, and can be transmitted from mother to child. Little is known about CHIKV transmission in Vietnam, where dengue is endemic and Aedes mosquitoes are abundant. This study aimed to determine the prevalence and characteristics of vertical CHIKV infection in a birth cohort, and seroprevalence of anti-CHIKV antibodies with or without confirmation by neutralization tests among women bearing children in Vietnam. MethodsWe collected umbilical cord blood plasma samples from each newly delivered baby in Nha Trang, Central Vietnam, between July 2017 and September 2018. Samples were subjected to molecular assay (quantitative real-time RT-PCR) and serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assay, and neutralization tests). ResultsOf the 2012 tested cord blood samples from newly delivered babies, the CHIKV viral genome was detected in 6 (0.3%) samples by RT-PCR, whereas, 15 samples (0.7%) were anti-CHIKV-IgM positive. Overall, 18 (0.9%, 95% CI: 0.6–1.5) samples, including three positives for both CHIKV IgM and viral genome on RT-PCR, were regarded as vertical transmission of CHIKV infection. Of the 2012 cord blood samples, 10 (0.5%, 95% CI: 0.2–0.9) were positive for both anti-CHIKV IgM and IgG. Twenty-nine (1.4%, 95% CI: 1.0–2.1) were seropositive for anti-CHIKV IgG while 26 (1.3%, 95% CI: 0.8–1.9) of them were also positive for neutralizing antibodies, and regarded as seropositive with neutralization against CHIKV infection. ConclusionThis is the first report of a possible CHIKV maternal-neonatal infection in a birth cohort in Vietnam. The findings indicate that follow-up and a differential diagnosis of CHIKV infection in pregnant women are needed to clarify the potential for CHIKV vertical transmission and its impact in the newborn.

Burden of Chikungunya Virus Infection during an Outbreak in Myanmar.

Chikungunya virus (CHIKV) infection is a re-emerging arboviral disease with no approved vaccine, although numerous options are in development. Before vaccine implementation, disease burden, affected age group, and hospitalization rate information should be documented. In 2019, a sizeable outbreak of the East Central South African genotype of CHIKV occurred in Myanmar, and during this period, a cross-sectional study was conducted in two regions, Mandalay and Yangon, to examine the molecular and seropositivity rate of the CHIKV infection. The participants (1124) included dengue-suspected pediatric patients, blood donors, and healthy volunteers, who were assessed using molecular assays (quantitative real-time RT-PCR), serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assays), and neutralization tests. The tests confirmed the following positivity rates: 11.3% (127/1124) for the molecular assay, 12.4% (139/1124) for the anti-CHIKV IgM Ab, 44.5% (500/1124) for the anti-CHIKV IgG Ab, and 46.3% (520/1124) for the CHIKV neutralizing Ab. The highest rate for the molecular test occurred with the dengue-suspected pediatric patients. The seroprevalence rate through natural infection was higher in the healthy volunteers and blood donors than that in the pediatric patients. The results of this study will help stakeholders determine the criteria for choosing appropriate recipients when a CHIKV vaccine is introduced in Myanmar.

CHIKUNGUNYA, SCRUB TYPHUS MONO, AND CO-INFECTION AMONG PATIENTS WITH UNDIFFERENTIATED FEBRILE ILLNESS: A HOSPITAL-BASED STUDY

Objectives: Chikungunya virus is a common arthropod-related acute febrile disease and it is transmitted by Aedes aegypti or Aedes albopictus species. On the other hand, the bacterium Orientia tsutsugamushi causes scrub typhus, which is also an acute febrile illness with multiple organ involvement. Coinfection of chikungunya and scrub typhus may lead to severe manifestation including severe respiratory and central nervous system (CNS) complications. Coinfection of chikungunya and scrub typhus may lead to severe manifestation including severe respiratory and CNS complications. Therefore, the proper diagnosis can prevent the clinical complications. The aim and objective of our study is to find the seroprevalence of chikungunya and scrub typhus and coinfection of both through medical assessment and serological research of these patients presented with acute febrile infection at Diamond Harbour Government Medical College and Hospital. Methods: A prospective study was conducted from August 2022 to January 2023 at VRDL, Department of Microbiology, Diamond Harbour Government Medical College and Hospital. Serum was collected for IgM antibody enzyme-linked immunosorbent assay (ELISA) for scrub typhus (In bios kit) and Chikungunya (NIV Chikungunya IgM Capture ELISA Kit) test. Four hundred and eighty-seven samples were tested for IgM antibody by chikungunya and scrub typhus ELISA kit. Result: The present study demonstrated that, from the month of August 2022 to January 2023, 67% of chikungunya cases, 25% cases with only scrub typhus, and 8% cases with both chikungunya and scrub typhus presented positive. A present study shows that chikungunya is slightly more prevalent in males as compared to females, where scrub typhus is equally positive in both male and female patients. Conclusion: Laboratory testing of both of the diseases can prevent the complication of other suspected disease in coinfected patients.

Prevalence of Chikungunya and Scrub Typhus Coinfection among Dengue Negative Patients in Kolkata, India-A Newly Emerging Public Health Hazard

The chikungunya virus (CHIKV) and scrub typhus infection has scattered worldwide creating human health hazards in India and Asia specific region. We aimed to identify chikungunya, scrub typhus, and their co-infection in dengue-negative samples having undifferentiated febrile illnesses. Enzyme linked immunosorbent assay (ELISA) methods were used to detect the chikungunya and scrub typhus specific IgM antibody by using chikungunya IgM capture ELISA kit and scrub typhus IgM Microlisa ELISA kit, respectively. OD value was measured with the help of BeneSphera (India) ELISA microplate reader. Among 490 suspected patients, 57 (11.63%) samples were tested positive for chikungunya IgM antibodies, while 43 (8.77%) came positive for scrub typhus IgM antibodies, but all samples tested negative for dengue IgM antibodies. Additionally, 5% of the total positive cases were positive for both chikungunya and scrub typhus infection. Our study offers a hypothesis regarding one of the possible causes of the decline in the frequency of scrub typhus and chikungunya cases reported in Kolkata and other districts of West Bengal. As an outcome, physicians treating undifferentiated febrile patients in endemic locations should look for chikungunya, scrub typhus, and existing coinfection between them to avoid delayed diagnosis and provide proper treatment against these infections.

Development and evaluation of recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (ELISA) and double antigen sandwich ELISA for detection of antibodies to Chikungunya virus

Chikungunya virus (CHIKV) reemerged and caused millions of human infections since 2004. The disease could be established, when the virus has been introduced to areas where the appropriate vectors are endemic. The differential diagnosis of CHIKV infection varies based on place of residence, travel history, and exposures. Serological tests are commonly used to diagnose CHIKV infection, but their availability and assessments of the performance of the diagnostics have been limited. To develop and evaluate antibodies detection methods for chikungunya diagnosis and serological investigation. Recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (Mac-ELISA) and double antigen sandwich ELISA (Das-ELISA) for detection of antibodies to Chikungunya virus were developed and evaluated. The repeatability was evaluated by testing of three reference sera at single dilutions in triplicated for 5 times. The sensitivity, specificity, accuracy, and agreement of the MAC-ELISA and Das-ELISA were obtained by comparing the detection results of 225 serum samples (45 positive; 180 negative) with a real-time RT-PCR assay and an IFA commercial tests manufactured by Euroimmun. The established ELISA assays were standardized by determining the optimal concentrations of the key reagents. The coefficient values of repeat testing were within 10% and 20% for intraassay and interassay precision, respectively. A sensitivity of 60.0% and 52.5%, a specificity of 96.2% and 96.8%, and an accuracy of 89.8% and 88.9% were obtained for the Mac-ELISA and Das-ELISA, respectively, when compared to a CHIKV qRT-PCR method. And a sensitivity of 100%, a specificity of 97.5% and 99.5%, and an accuracy of 97.8% and 99.6% were yielded respectively when using the IIFT as a reference method, which showed a highly consistence to the commercial IIFT assay with a Kappa value greater than 0.90. The Mac-ELISA and Das-ELISA based on recombinant E2 protein of CHIKV were developed and standardized, which could detect IgM or total antibodies against CHIKV in 2-3 hours with acceptable sensitivities and specificities. These assays can be used for laboratory diagnosis and serological investigation of CHIKV infections to evaluate the risk of CHIKV transmission.

Development of monoclonal antibody based IgG and IgM ELISA for diagnosis of severe fever with thrombocytopenia syndrome virus infection

IntroductionSevere fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that poses a great threat to human health because of high fatality rate. MethodsTo develop sensitive and specific sero-diagnostic systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patients serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. ResultsThe MAbs based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while detecting eight additional positive samples missed by the indirect IgM ELISA. ConclusionsThe MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Aspek Klinis dan Pemeriksaan Laboratorium untuk Diagnosis Demam Berdarah Dengue

Dengue infection is a viral infection through arthropods that is found throughout the world. This disease is caused by dengue virus which consists of four serotypes and each of these serotypes can cause disease. The pathogenesis of dengue infection, especially the mechanism of dengue hemorrhagic fever (DHF) manifestations is still not clearly understood. The results of laboratory tests on dengue hemorrhagic fever found thrombocytopenia (≤ 100,000/mm3), a 20% increase in hematocrit and a positive Rumple Leed test. Five basic serological tests used to detect antibodies in diagnosing dengue infection are haemagglutination-inhibition (HI), complement fixation (CF), neutralization test (NT), IgM capture enzyme-linked immunosorbent assay (MAC-ELISA), and indirect IgG. ELISA. RT-PCR examination in a number of studies was reported to have successfully detected the dengue virus. This examination has better specificity and sensitivity compared to virus isolation with a fast turn around timeLaboratory tests on dengue infection include hematological, serological and nucleic acid tests that play a role in supporting the rapid and accurate diagnosis of dengue hemorrhagic fever.

Development of Nipah virus-specific IgM & IgG ELISA for screening human serum samples.

Nipah virus (NiV) is a zoonotic paramyxovirus that causes fatal encephalitis in humans. Enzyme Linked Immunosorbent Assay (ELISA) is a safe, sensitive, specific, and affordable diagnostic tool that can be used during screening of large-scale epidemiological investigations. Development and evaluation of IgM and IgG ELISA for screening serum samples of NiV suspected cases would also help in planning public health interventions. An IgM capture (MAC) ELISA and an indirect IgG ELISA were developed using NiV antigen to detect IgM and IgG antibodies against NiV in human sera. The sensitivity, specificity, and cross-reactivity of the assays were evaluated using NiV IgM, IgG positive, negative human sera and measles, mumps, rubella, Crimean-Congo haemorrhagic fever, Kyasanur forest disease IgM, IgG positive sera, respectively. The developed anti-NiV IgM and IgG ELISAs have shown specificity of 99.28 per cent and sensitivity of 100 per cent compared to reference test from Centers for Disease Control and Prevention, USA. Assays demonstrated negative predictive value of 100 per cent and positive predictive value as 90 and 93.94 per cent for anti-Nipah IgM ELISA and IgG ELISA respectively with test accuracy of 99.33 per cent. Timely diagnosis of NiV is crucial for the management of cases, which could prevent further spread of infection in the community. IgM ELISA can be used as primary diagnostic tool followed by polymerase chain reaction. These assays have advantages of its applicability during outbreak investigations and surveillance activities at hospital or onsite laboratories with basic biosafety practices.

Sero-molecular Prevalence of Zika Virus among Pregnant Women Attending Some Public Hospitals in Lagos State, Nigeria

Zika virus (ZIKV) is one of the arboviruses implicated in febrile illness, microcephaly and other neurological disorders in babies whose mothers were infected during pregnancy. Information on ZIKV in Nigeria is limited. Hence, this study was aimed at investigating the seroprevalence of Zika virus among pregnant women in Lagos State. In a cross-sectional study, blood samples collected from 352 randomly selected pregnant women in four hospitals in Lagos State were separated and plasma analyzed using Zika virus IgG and IgM capture Enzyme-linked immunosorbent assay (Demeditec Diagnostics, Germany). The optical densities were read using Precision Microplate reader (Molecular devices) and cut-off calculated according to manufacturer’s guide. Parameters and symptoms such as history of fever, rashes on the body, exposure to mosquito were extracted from the questionnaire and analyzed. IgM seropositive samples were screened for ZIKV RNA on RT-qPCR. Out of 352 samples screened, 7(2.0%) and 5(1.4%) of the pregnant women tested positive for IgG and IgM respectively. None tested positive for both IgG and IgM markers. Statistical analysis showed that there is no significant relationship between the symptoms analyzed in this study at 95% Confidence interval except conjunctivitis. None of the ZIKV IgM seropositive samples tested positive for ZIKV RNA on RT-qPCR. The results show that there is evidence of exposure to Zika virus among the population studied in Lagos, Nigeria. Also, the low level seroprevalence of the virus in the population studied indicates that there is lack of herd immunity of Zika virus infection in Lagos, Nigeria.

Comparative analysis of NS1 antigen card test and ELISA of clinically suspected dengue fever cases in a tertiary care hospital, Mumbai

Dengue, mosquito borne acute febrile viral illness has evolved as a global life threatening public health concern in South east Asia affecting around 2.5 billion individuals in more than 100 countries. The study was conducted to compare commercially available NS1 Ag card test with IgM Capture ELISA. A total of 521 clinically suspected cases of dengue were tested by Rapid Card test and Enzyme Linked Immunosorbent Assay (ELISA). Confirmed dengue positive cases by these methods were 228.A total of 222 cases were found to be positive by Card test. Out of these 222 positive samples, 61 (27.5%) were positive for NS1 antigen only. A total of 123 cases were positive for IgM by ELISA (99 for IgM only and 24 for IgM + IgG) as compared to 117 cases by rapid card test. Thus six cases (2.6%) of IgM positivity were missed by the rapid card test. Immunochromatographic testing (ICT) can prove to be a very useful tool for especially in resource poor settings, such as in the peripheral health care center. On the other hand, in tertiary care hospitals, where a large number of samples are to be tested and the technical expertise is available, ELISA can be very effective in diagnosis of dengue infection.

Evaluation of the diagnostic performance and its associated factors of a commercial anti-EV-A71 IgM-capture ELISA kit in hospitalized children with clinical diagnostic HFMD.

Enterovirus A71 (EV-A71) is the main pathogen of severe hand, foot, and mouth disease (HFMD). Commercial enzyme-linked immunosorbent assays (ELISAs) are widely used in Chinese hospitals for the rapid diagnosis of acute EV-A71 infections. We present an evaluation of the diagnostic performance of a commercial anti-EV-A71 IgM-capture ELISA kit. A prospective, hospital-based HFMD cohort was established in Henan Children's Hospital (February 2017 - February 2018). Stool and blood specimens were collected from 1413 participants for diagnosing EVA71 by quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and anti-EV-A71 ELISA. Detection yields of EV-A71 IgM increased from 6.5 % (95 % CI:3.3 %-11.4 %) at 0∼24 h, to 42 % (95 % CI:28.3 %-57.8) at 120∼144 h from onset to sampling, and stabilized at ∼40 % after 144 h. With increased time from onset to sampling, the sensitivity of the commercial ELISA increased from 0.54 (95 % CI:0.25-0.81) to 0.74 (95 % CI:0.43-0.66), while specificity decreased from 0.97 (95 % CI:0.93-0.99) to 0.80 (95 % CI:0.69-0.89), and PPV decreased from 0.96 (95 % CI:0.92-0.99) to 0.84 (95 % CI:0.73-0.92). Multivariate analysis found age, EV-A71 vaccination, previous HFMD/Herpangina infection, disease severity, infection during peak EV-A71 season, and sampling time after symptom onset were significantly associated with the diagnostic performance of this anti-EV-A71 IgM-capture ELISA. Achieving satisfactory specificity and sensitivity scores, this commercial anti-EV-A71 IgM-capture ELISA kit is suitable for clinical EV-A71 diagnosis, particularly in resource-poor areas. However, clinicians should interpret results in the context of patient history and epidemiological setting.

A Prospective Study Conducted Among Participants Reporting Acute Febrile Illness in a Cohort of Periurban and Rural Cambodians

Abstract The objective of this prospective cohort study was to assess the incidence of dengue, chikungunya, respiratory viruses, and diarrheal etiologies among periurban and rural Cambodians with confirmed acute febrile illness. During a 2-year period (2016–2017), 612 participants aged 6 months to 30 years who exhibited vector-borne febrile illness (median [interquartile range] age, 8.0 [4–11] years), in which 288 (48.0%) were female, were tested for acute dengue virus and chikungunya virus infection. A total of 67 (11.2%) clinical specimens were positive for dengue virus by either nucleic acid detection (n = 23 [34.3%]; dengue serotype 1 [n = 5] and dengue serotype 2 [n = 18]) or dengue IgM capture enzyme-linked immunosorbent assay (n = 44 [65.7%]), and only 8 specimens were both positive. Clinical presentations included fever (100%), headache (74.1%), muscle aches (27.2%), and joint pain (17.3%). Forty-two of the 612 participants were diagnosed with chikungunya (7.0%) by anti–chikungunya virus enzyme-linked immunosorbent assay (IgM) or chikungunya-specific reverse transcription polymerase chain reaction. By understanding the incidence of diseases causing morbidity and mortality in rural areas within Cambodia, mitigating strategies can be developed to reduce infections.

Evaluation of a Newly Developed Rapid Automated Fluorescent Lateral Flow Immunoassay to Detect IgG and IgM Antibodies to Chikungunya Virus.

The ichroma Chikungunya virus (CHIKV) IgG/IgM (Boditech Med Inc., Chuncheon, Korea) is a newly developed rapid lateral flow immunoassay for detection of anti- CHIKV-IgG/IgM. This study conducted with thirty-six anti-CHIKV IgG positive sera, 57 anti-CHIKV IgM positive sera and 163 anti-CHIKV IgG/IgM negative sera which were confirmed by commercial enzyme-linked immunosorbent assays (ELISAs) (Inbios CHIKjj Detect™ IgM Capture ELISA, Inbios CHIKjj Detect™ IgG ELISA (InBios International Inc., Seattle, WA, USA), Anti-CHIKV ELISA (IgM), Anti- CHIKV ELISA (IgG) (Euroimmun, Lübeck, Germany)). The ichroma detected all 36 anti-CHIKV IgG and 57 anti-CHIKV IgM positivity (100% sensitivity). For 163 anti-CHIKV IgG/IgM negative sera, the ichroma showed one false positive for IgM (99.4% specificity). The ichroma showed no cross-reactivity and no interference. The ichroma demonstrated good diagnostic performance compared to the current ELISAs.

Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M

The incidence of chikungunya virus (CHIKV) infection has increased dramatically in recent decades. Effective diagnostic methods must be available to optimize patient management. IgM-capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) is routinely used for the detection of specific CHIKV IgM. This method requires inactivated CHIKV viral lysate (VL). The use of viral bioparticles such as Virus-Like Particles (VLPs) and Pseudotyped-Particles (PPs) could represent an alternative to VL.Bioparticles performances were established by MAC-ELISA; physico-chemical characterizations were performed by field-flow fractionation (HF5) and confirmed by electron microscopy.Non-purified PPs give a detection signal higher than for VL. Results suggested that the signal difference observed in MAC-ELISA was probably due to the intrinsic antigenic properties of particles.The use of CHIKV bioparticles such as VLPs and PPs represents an attractive alternative to VL. Compared to VL and VLPs, non-purified PPs have proven to be more powerful antigens for specific IgM capture.

Biotechnology for obtaining hybrid positive control samples for immunoassay for detecting antibodies against Chlamydia trachomatis

The enzyme-linked immunosorbent assay (ELISA) is the most informative and versatile method of serological diagnostics. The possibility of detection by ELISA of specific antibodies of different classes allow one to differentiate primary infectious processes and their remission, exacerbation and chronic disease (holding of differential diagnosis). This approach is implemented in the methodology for evaluation of patients for the presence of humoral immune response against TORCH-infections pathogens (toxoplasmosis, rubella, cytomegalovirus, herpes simplex viruses infections, and some others). Therefore, testing for the presence of specific IgG and IgM antibodies against TORCH-infection pathogens in blood serum is an important element of mother and child protection. The essential problem in the production of IgM-capture ELISA diagnostic kits is obtaining positive control. The classic version of positive control is human blood serum (plasma) containing specific antibodies. But specific IgM-positive sera are insignificant raw material. This fact can significantly limit the production of diagnostic kits, especially in case of large-scale manufacture. We have suggested a methodological approach to the use of synthetic positive controls in IgM-capture ELISA kits based on conjugate of normal human IgM and monoclonal antibodies against horseradish peroxidase. It was found that it is possible to realize such a task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), reductive amination-mediated conjugation (by sodium periodate) and glutaraldehyde-mediated conjugation. It was found that conjugates of normal human IgM and monoclonal antibodies against horseradish peroxidase obtained using NHS ester-mediated maleimide conjugation and periodate method are homogeneous in molecular weight, whereas conjugate synthesized by glutaraldehyde method comprises at least three types of biopolymers with close molecular weight. It was found that synthetic positive control obtained by different methods are characterized by higher titre compared to IgM-positive high-titre serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics. We have suggested a methodological approach to the use of synthetic positive controls in indirect ELISA kits based on conjugate of normal human IgM (IgA) and monoclonal antibodies against major outer membrane protein of Ch. trachomatis. It was found that it is possible to realize such task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and reductive amination-mediated conjugation (by sodium periodate). It was found that synthetic positive control obtained by different methods are characterized by higher titre compared to IgM- and IgA-positive high-titre serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics, both at the release time and after a week’s storage at 37 °C.

Evaluation of the Diasorin Liaison® XL Zika Capture IgM CMIA for Zika virus serological testing

Due to the increase of Zika virus (ZIKV) transmission throughout the world, many commercial kits have recently become available to aid in laboratory diagnosis of ZIKV infections in clinical samples. Here, we analyze the fully automated Liaison® XL Zika Capture immunoglobulin M (IgM) assay against the recommended IgM-capture enzyme-linked immunosorbent assay.

Dengue virus infections among blood donors in Guangxi of China, 2013-2014.

An unexpected, large dengue outbreak, spreading to 20 cities of Guangdong province, occurred in July 2014. Considering the overwhelming influence of this seasonal epidemic, whether this dengue virus outbreak has an significant impact on the blood donors of the ajacent areas in Guangdong requires at least a preliminary investigation. Liuzhou City of Guangxi was chosen as a good representative of the neighbouring geographical location of the dengue virus (DENV) outbreak areas in this research. We therefore investigated qualified blood donations during potential dengue epidemic seasons and thus provided critical information for public health and donation policy making. A total of 1685 donations from 2013 to 2014 were collected at the Guangxi blood centre. Anti-DENV testing was performed using enzyme-linked immunosorbent assay (ELISA). Dengue IgG indirect ELISA and Dengue IgM capture ELISA were used to detect anti-DENV IgM and IgG. Repeat-reactive samples with ELISA test and 1685 donations with 421 pools were screened for dengue RNA by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) using an in-house assay. There was a total of 13 seropositive donors, including 6 IgM seropositive and 7 IgG seropositive. The total seropositive rate was 0·78%, with IgM 0·36% and IgG 0·42%. Seropositive donors were identified mostly between April and November, with a peak in November. .All samples in reactive pools and individual seropositive samples were negative in the follow-up RT-PCR test. Our results suggested that the Guangdong dengue outbreak had limited impact on blood safety in surrounding areas under our current testing system.

Elimination of falsely reactive results in a commercially-available West Nile virus IgM capture enzyme-linked immunosorbent assay by heterophilic antibody blocking reagents

All sera initially reactive in the Focus Diagnostics West Nile virus IgM capture enzyme-linked immunosorbent assay (WNV IgM ELISA) must be retested with background subtraction to identify falsely-reactive (FR) samples due to antibodies that bind to immunoglobulins of other animal species (heterophilic antibodies). In some settings, such as pre-transplant testing of organ donors, the reporting delay associated with retesting can have an adverse impact on donor procurement and organ placement. We sought to determine if inclusion of heterophilic antibody blockers in assay conjugate could eliminate the nonspecific reactivity of FR samples. Of 6 blocking reagents evaluated using a well-characterized FR sample, immunoglobulin inhibiting reagent from Bioreclamation (IIR) and blocker from Fitzgerald Industries (BFI) were superior in their ability to inhibit false reactivity; these 2 blockers were then used to evaluate 20 additional FR and 21 truly-reactive (TR) samples. Both blockers eliminated the reactivity of 20/21 FR samples, whereas all 21 TR samples remained reactive; further, all 13 truly non-reactive (NR) samples evaluated remained non-reactive when using blocker-containing conjugate. A subset of 22 samples were tested in parallel using the initial lot and a second lot of IIR and BFI; with one exception, all samples showed the same qualitative result using both lots of a given blocker. These findings demonstrate that modification of the Focus WNV IgM screening ELISA to include heterophilic antibody blocker IIR or BFI in assay conjugate eliminates the reactivity of most FR samples, markedly reducing the number of samples requiring further testing by background subtraction.

Clinical profiles and some associated factors of Japanese encephalitis in Bali

Background Japanese Encephalitis (JE) is a mosquito-borne vi-ral disease that can affect the central nervous system and causeseveral complications and death. In Indonesia, particularly in Bali,studies of JE in humans are still very limited.Objective To assess the clinical manifestations and outcome ofJE in Bali and to identify some possible associated factors andmonthly distribution of admission to the hospitals.Methods A cross-sectional study was conducted in 8 district hospi-tals, army hospital, and Sanglah central hospital, as surveillance cen-ters in Bali, from July 2001 through January 2003. Subjects less than12 years of age and residences of Bali province who were suspectedof having acute viral encephalitis, aseptic meningitis, and acute flac-cid paralysis referred from all health facilities were included in thisstudy. The diagnosis of JE was established by detection of virus-spe-cific IgM in CSF and serum, with IgM capture enzyme-linkedimmunosorbent assay (MAC ELISA)Results During the study, 158 subjects with encephalitis were in-cluded; 55 (34.8%) were caused by JE and 103 (65.2%) were causedby non-JE. It showed that pig owning and rice farming were signifi-cantly associated with JE (PR= 3.02, 95% CI 1.29; 7.10; P= 0.009and PR= 2.86, 95% CI 1.30; 6,31, P= 0.008, respectively). Mostsubjects (46.2%) were under 2 years old with mean age of 37.24(SD 32.24) months. Forty-two (26.6%) subjects had nuchal rigidity,and all of them had aseptic meningitis. Denpasar had the highestprevalence of non-JE cases. Bangli and Klungkung had the lowestprevalence of JE and non-JE. The highest prevalence of JE was inGianyar. Peak prevalences of JE were in April and May 2002. Peakprevalences of non-JE were during August-October 2001 and inMarch-April 2002. Four (7.3%) subjects of JE group and 25 (24.3%)subjects of non-JE died. There were 25 (45.5%) out of 55 subjectswho survived with disability. Twenty-four (43.6%) JE subjects and34 (33.0%) non-JE subjects had good outcome.Conclusions Around one third of the subjects were positively di-agnosed as JE. The highest prevalence of JE was found in Gianyar.Peak prevalences of JE are in April and May 2002. Pig owning andrice farming are associated factors. Almost half of JE subjects sur-vived with disability

Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.