METHODS: Over six months in 2018, we recruited 368 adults who met the WHO 2009 criteria for probable dengue infection. They underwent the following blood tests: full blood count, dengue virus (DENV) rapid diagnostic test (RDT), ELISA (dengue IgM and IgG), nested RT-PCR for dengue, multiplex qRT-PCR for Zika, Chikungunya and dengue as well as PCR tests for Leptopspira spp., Japanese encephalitis and West Nile virus.
RESULTS: Laboratory-confirmed dengue infections (defined by positive tests in NS1, IgM, high-titre IgG or nested RT-PCR) were found in 167 (45.4%) patients. Of these 167 dengue patients, only 104 (62.3%) were positive on rapid diagnostic testing. Dengue infection was significantly associated with the following features: family or neighbours with dengue in the past week (AOR: 3.59, 95% CI:2.14-6.00, p<0.001), cutaneous rash (AOR: 3.58, 95% CI:1.77-7.23, p<0.001), increased temperature (AOR: 1.33, 95% CI:1.04-1.70, p = 0.021), leucopenia (white cell count < 4,000/μL) (AOR: 3.44, 95% CI:1.72-6.89, p<0.001) and thrombocytopenia (platelet count <150,000/μL)(AOR: 4.63, 95% CI:2.33-9.21, p<0.001). Dengue infection was negatively associated with runny nose (AOR: 0.47, 95% CI:0.29-0.78, p = 0.003) and arthralgia (AOR: 0.42, 95% CI:0.24-0.75, p = 0.004). Serotyping by nested RT-PCR revealed mostly mono-infections with DENV-2 (n = 64), DENV-1 (n = 32) and DENV-3 (n = 17); 14 co-infections occurred with DENV-1/DENV-2 (n = 13) and DENV-1/DENV-4 (n = 1). Besides dengue, none of the pathogens above were found in patients' serum.
CONCLUSIONS: Acute undifferentiated febrile infections are a diagnostic challenge for community-based clinicians. Rapid diagnostic tests are increasingly used to diagnose dengue infection but negative tests should be interpreted with caution as they fail to detect a considerable proportion of dengue infection. Certain clinical features and haematological parameters are important in the clinical diagnosis of dengue infection.
METHODS: Consecutive GBS and MFS patients presenting to our center between 2010 and 2020 were analyzed. The clinical characteristics, electrophysiological data, and antiganglioside antibody profiles of the patients were utilized in determining the clinical classification.
RESULTS: This study classified 132 patients with GBS and its related disorders according to the new classification criteria as follows: 64 (48.5%) as classic GBS, 2 (1.5%) as pharyngeal-cervical-brachial (PCB) variant, 7 (5.3%) as paraparetic GBS, 29 (22%) as classic MFS, 3 (2.3%) as acute ophthalmoparesis, 2 (1.5%) as acute ataxic neuropathy, 2 (1.5%) as Bickerstaff brainstem encephalitis (BBE), 17 (12.9%) as GBS/MFS overlap, 4 (3%) as GBS/BBE overlap, 1 (0.8%) as MFS/PCB overlap, and 1 (0.8%) as polyneuritis cranialis. The electrodiagnosis was demyelinating in 55% of classic GBS patients but unclassified in 79% of classic MFS patients. Anti-GM1, anti-GD1a, anti-GalNAc-GD1a, and anti-GD1b IgG ganglioside antibodies were more commonly detected in the axonal GBS subtype, whereas the anti-GQ1b and anti-GT1a IgG ganglioside antibodies were more common in classic MFS and its subtypes.
CONCLUSIONS: Most of the patients in the present cohort met the criteria of either classic GBS or MFS, but variants were seen in one-third of patients. These findings support the need to recognize variants of both syndromes in order to achieve a more-complete case ascertainment in GBS.
OBJECTIVES: The aim of this study is to assess the role of Granulocyte Macrophage-Colony Stimulating Factor (GMCSF) in asthmatic airway hyper-responsiveness associated with RSV infections.
MATERIALS AND METHODS: Forty five asthmatic cases and 45 healthy individuals were studied in a cross-sectional design. All asthmatics underwent symptom score assessment.GMCSF concentrations in sputum and RSV-IgM/IgG in serum samples were measured for all participants by Enzyme Linked Immuno-Sorbent Assay (ELISA).
RESULTS: The GM-CSF concentration level was significantly higher in asthmatics (270.27± 194.87pg/mL) especially among moderate and severe disease with mean concentration of 197.33±98.47 and 521.08± 310.04 respectively, compared to healthy controls (22.20±21.27 pg/ mL) (p =0.0001). The sputum level of GM-CSF in asthmatics is highly significant associated with positive anti-RSV IgG sera which represents 35/45(77.8%) with mean GM-CSF concentration of (276.99± 86.42) compared with controls at about 31/45 (68.9%) with GM-CSF mean concentration of (22.84±23.47). On the other hand, positive anti-RSV IgM in asthma cases was 8 out of 45(17.8 %) with GM-CSF mean concentration of (307.25± 306.65). Furthermore, GM-CSF sputum level was significantly correlated with eosinophil count especially in moderate and severe asthma.
CONCLUSIONS: This study revealed that GM-CSF level is associated with eosinophilia and indicates asthma severity that might be evident during RSV infection .The distinctive GM-CSF features observed in the sputum from asthmatics with RSV may be useful as a diagnostic methods to help match patients with antibody therapy.
Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.
Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.
Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.
METHODS: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD).
RESULTS: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits.
CONCLUSION: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.
Methods: A prospective cohort study involving different populations within the Vidarbha regions of Maharashtra, India was conducted through camps organised from May 2009 to October 2015. A total of 568 serum samples were collected from high-risk people recruited as study cohorts based on inclusion criteria, additional risk factors and clinical symptoms. Samples were evaluated by indirect ELISA using the whole-cell antigens of B. abortus. The results were compared with the commercially available IgG detection ELISA kit to ascertain the specificity and sensitivity of the developed test.
Results: Fever, body ache, joint pain, lower back pain, loss of appetite and weight loss were major symptoms associated with the disease. With the cut-off of > 0.8, the positivity of brucellosis infection was at 12.32% (70/568) compared to 9.33% (53/568) as detected by the commercial kit. The in-house developed ELISA method yielded a sensitivity of 87.5% and specificity of 99.18% as compared to the commercial kits (sensitivity -80.30% and specificity -99.6%).
Discussion: The B. abortus S19-derived whole-cell protein-based ELISA is rapid and cost-effective and can be used for screening brucellosis infection in lieu of the commercially available ELISA kits.
METHODS: A total of 2598 serum samples (1417 urban samples, 1181 rural samples) were randomly collected from adults ages 35-74 y. The presence of the dengue IgG antibody and neutralising antibodies to dengue virus (DENV) 1-4 was determined using enzyme-linked immunosorbent assay and the plaque reduction neutralisation test assay, respectively.
RESULTS: The prevalence of dengue IgG seropositivity was 85.39% in urban areas and 83.48% in rural areas. The seropositivity increased with every 10-y increase in age. Ethnicity was associated with dengue seropositivity in urban areas but not in rural areas. The factors associated with dengue seropositivity were sex and working outdoors. In dengue IgG-positive serum samples, 98.39% of the samples had neutralising antibodies against DENV3, but only 70.97% of them had neutralising antibodies against DENV4.
CONCLUSION: The high seroprevalence of dengue found in urban and rural areas suggests that both urban and rural communities are vital for establishing and sustaining DENV transmission in Malaysia.