Development of nucleic acid lateral flow immunoassay for duplex detection of Leishmania martiniquensis and Leishmania orientalis in asymptomatic patients with HIV
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SUBMITTER: Nawattanapaibool N
PROVIDER: S-EPMC11346928 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Quantitative nucleic acid amplification testing (NAAT) is a key enabling technology for infectious disease management, especially in instances where viral load informs therapeutic decisions. Inadequate access to quantitative NAATs remains a challenge to the successful deployment of antiretroviral therapy (ART) regimens for patients with chronic hepatitis B virus (CHB) in low resourced settings (LRS). Current field-deployable NAATs are generally qualitative (yes/no) rather than quantitative in nature, making them ill-suited for viral load monitoring programs for CHB patients. Here, we report the development of a proof-of-concept molecular diagnostic test, the semiquantitative ligation and amplification (SQLA) assay, which achieves semiquantitative detection of input target DNA at two independently tunable detection thresholds with a simple visual readout. The SQLA assay utilizes a duplex competitive thermophilic helicase-dependent amplification (tHDA) chemistry and can be performed in under 1 h.
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Project description:Since 1999, the number of asymptomatic leishmaniasis cases has increased continuously in Thailand, particularly among patients with HIV who are prone to develop symptoms of cutaneous and visceral leishmaniasis further. The asymptomatic infection could play a key role in Leishmania transmission and distribution. Understanding population structure and phylogeographic patterns could be crucially needed to develop effective diagnoses and appropriate guidelines for therapy. In this study, genetic variation and geographic distribution of the Leishmania/HIV co-infected population were investigated in endemic northern and southern Thailand. Interestingly, Leishmania orientalis was common and predominant in these two regions with common regional haplotype distribution but not for the others. Recent population expansion was estimated, probably due to the movement and migration of asymptomatic individuals; therefore, the transmission and prevalence of Leishmania infection could be underestimated. These findings of imbalanced population structure and phylogeographic distribution patterns provide valuable, insightful population structure and geographic distribution of Leishmania/HIV co-infection to empower prevention and control of transmission and expansion of asymptomatic leishmaniasis.
| S-EPMC10754904 | biostudies-literature
Project description:The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 105 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.
| S-EPMC8035659 | biostudies-literature
Project description:Leishmaniasis is a parasitic disease caused by protozoan flagellates of the genus Leishmania. Recently, Leishmania martiniquensis and Leishmania orientalis, emerging species of Leishmania, were isolated from patients in Thailand. Development of the vaccine is demanded; however, genetic differences between the two species make it difficult to design a vaccine that is effective for both species. In this study, we applied immuno-informatic approaches to design a chimeric multi-epitope vaccine (CMEV) against both L. martiniquensis and L. orientalis. We identified seven helper T lymphocyte (HTL) epitopes, sixteen cytotoxic T lymphocyte (CTL) epitopes, and eleven B-cell epitopes from sixteen conserved antigenic proteins found in both species. All these epitopes were joined together, and to further enhance immunogenicity, protein and peptides adjuvant were also added at the N-terminal of the molecule by using specific linkers. The candidate CMEV was subsequently analyzed from the perspectives of the antigenicity, allergenicity, and physiochemical properties. The interaction of the designed multi-epitope vaccine and immune receptor (TLR4) of the host were evaluated based on molecular dockings of the predicted 3D structures. Finally, in silico cloning was performed to construct the expression vaccine vector. Docking analysis showed that the vaccine/TLR4 complex took a stable form. Based on the predicted immunogenicity, physicochemical, and structural properties in silico, the vaccine candidate was expected to be appropriately expressed in bacterial expression systems and show the potential to induce a host immune response. This study proposes the experimental validation of the efficacy of the candidate vaccine construct against the two Leishmania.
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Project description:BackgroundGlobally, Latin America ranks third among regions with most cases of AIDS related cryptococcal meningitis. In 2009, a lateral flow immunoassay (LFA) for the detection of cryptococcal antigen (CrAg) was developed as a potential point-of-care test for diagnosis of cryptococcal infection. In 2011 World Health Organizations recommended on CrAg screening for HIV positive persons with CD4 below 100 cells/μL, followed by preemptive fluconazole treatment. However, in Argentina no formal recommendations for CrAg screening have been issued.MethodsHIV positive patients > = 18 years with advanced immunosuppression (CD4 counts ≤100 cells/μL within 3 months or WHO stage III/IV), who visited the hospital between April 1, 2014 and January 31, 2015, were included. The LFA was performed according to the manufacturer's instructions on all serum samples. When CrAg detection was positive, a lumbar puncture was performed to rule out cryptococcal meningitis. Patients without evidence of meningeal involvement were treated with preemptive oral fluconazole in ambulatory care.ResultsWe included 123 patients. Prevalence of CrAg-positivity was 8.1%. Among the 10 CrAg-positive patients, 6 had meningeal involvement detected through the CSF analysis (CSF India-ink testing, CSF CrAg and culture). The remaining 4 patients with positive CrAg received targeted preemptive treatment with oral fluconazole and were free of cryptococcal disease during the follow-up period. None of the 113 patients with a negative CrAg test result developed cryptococcal disease.ConclusionsThis is the first study in Argentina, to our knowledge, describing the prevalence of cryptococcosis and usefulness of CrAg screening. LFA provided early diagnosis to determine a high prevalence of CrAg in our hospital, and that screening for subclinical infection with preemptive antifungal treatment, prevented a substantial proportion of meningeal disease.
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Project description:(1) Background: Autochthonous leishmaniasis, a sandfly-borne disease caused by the protozoan parasites Leishmania orientalis (formerly named Leishmania siamensis) and Leishmania martiniquensis, has been reported for immunocompromised and immunocompetent patients in the southern province of Thailand. Apart from the recent genomes of the northern isolates, limited information is known on the emergence and genetics of these parasites. (2) Methods: This study sequenced and compared the genomes of L. orientalis isolate PCM2 and L. martiniquensis isolate PCM3 with those of the northern isolates and other 14 Leishmania species using short-read whole-genome sequencing methods and comparative bioinformatic analyses. (3) Results: The genomes of the southern isolates of L. orientalis and L. martiniquensis were 30.01 Mbp and 32.39 Mbp, and the comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different strains. Comparative proteome analysis showed six protein groups with 53 unique proteins for the strain PCM2 and 97 for the strain PCM3. Certain proteins were related to virulence, drug resistance, and stress response. (4) Conclusion: Therefore, the findings could indicate the need for more genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions.
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Project description: Not available
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Project description:Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.
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Project description:The authors describe a gold nanocage-based lateral flow strip biosensor (LFSB) for low-cost and sensitive detection of IgG. This protein was used as a model analyte to demonstrate the proof-of-concept. The method combines the unique optical properties of gold nanocages (GNCs) with highly efficient chromatographic separation. A sandwich-type of immunoreactions occurs on the GNC-based LFSB which has the attractive features of avoiding multiple incubation, separation, and washing steps. The captured GNCs on the purple test zone and control zone of the biosensor are producing characteristic purple bands, and this enables IgG even to be visually detected. Quantitatation was accomplished by reading the intensities of the bands with a portable strip reader. The LFSB fabrication and assay parameters were optimized. The biosensor displays a linear response in the 0.5 to 50 ng·mL-1 IgG concentration range, and it has a 15 min assay time. The detection limit is 0.1 ng·mL-1 of IgG, which is 2.5 times lower than that when using a gold nanoparticle-based LFSB. In our perception, this assay has a wide potential for the detection of other proteins and species for which respective antibodies are available.
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