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Isolation and characterization of ssDNA aptamers against BipD antigen of Burkholderia pseudomallei

Selvam K, Najib MA, Khalid MF, Yunus MH, Wahab HA, Harun A, et al.

Anal Biochem, 2024 Aug 28;695:115655.
PMID: 39214325 DOI: 10.1016/j.ab.2024.115655

BACKGROUND: Melioidosis is difficult to diagnose due to its wide range of clinical symptoms. The culture method is time-consuming and less sensitive, emphasizing the importance of rapid and accurate diagnostic tests for melioidosis. Burkholderia invasion protein D (BipD) of Burkholderia pseudomallei is a potential diagnostic biomarker. This study aimed to isolate and characterize single-stranded DNA aptamers that specifically target BipD.
METHODS: The recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay.
RESULTS: The fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a Kd value of 1.0 μM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria.
CONCLUSION: AptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis.
Fulltext Performance of Immunodiagnostic Tests for Typhoid Fever: A Systematic Review and Meta-Analysis

Najib MA, Mustaffa KMF, Ong EBB, Selvam K, Khalid MF, Awang MS, et al.

Pathogens, 2021 Sep 13;10(9).
PMID: 34578216 DOI: 10.3390/pathogens10091184

Typhoid fever, also known as typhoid, is a life-threatening bacterial infection that remains a global health concern. The infection is associated with a significant morbidity and mortality rate, resulting in an urgent need for specific and rapid detection tests to aid prevention and management of the disease. The present review aims to assess the specificity and sensitivity of the available literature on the immunodiagnostics of typhoid fever. A literature search was conducted using three databases (PubMed, ProQuest and Scopus) and manual searches through the references of identified full texts to retrieve relevant literature published between 1 January 2011 and 31 December 2020. Of the 577 studies identified in our search, 12 were included in further analysis. Lipopolysaccharides (LPS) and hemolysin E (HlyE) were the most frequently studied antigens. The specimens examined in these studies included serum and saliva. Using blood culture as the gold standard, anti-LPS IgA gave the highest sensitivity of 96% (95% CI: 93-99) and specificity of 96% (95% CI: 93-99) for distinguishing between typhoid cases and healthy controls, whereas the combination of anti-LPS and anti-flagellin total IgGAM gave the highest sensitivity of 93% (95% CI: 86-99) and specificity of 95% (95% CI: 89-100) for distinguishing typhoid cases and other febrile infections. A comparably high sensitivity of 92% (95% CI: 86-98) and specificity of 89% (95% CI: 78-100) were shown in testing based on detection of the combination of anti-LPS (IgA and IgM) and anti-HlyE IgG as well as a slightly lower sensitivity of 91% (95% CI: 74-100) in the case of anti-50kDa IgA. Anti-50kDa IgM had the lowest sensitivity of 36% (95% CI: 6-65) against both healthy and febrile controls. The development of a rapid diagnostic test targeting antibodies against lipopolysaccharides combined with flagellin appeared to be a suitable approach for the rapid detection test of typhoid fever. Saliva is added benefit for rapid typhoid diagnosis since it is less invasive. As a result, further studies could be done to develop additional approaches for adopting such samples.
A label-free electrochemical DNA biosensor used a printed circuit board gold electrode (PCBGE) to detect SARS-CoV-2 without amplification

Zambry NS, Awang MS, Beh KK, Hamzah HH, Bustami Y, Obande GA, et al.

Lab Chip, 2023 Mar 14;23(6):1622-1636.
PMID: 36786757 DOI: 10.1039/d2lc01159j

The emergence of coronavirus disease 2019 (COVID-19) motivates continuous efforts to develop robust and accurate diagnostic tests to detect severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Detection of viral nucleic acids provides the highest sensitivity and selectivity for diagnosing early and asymptomatic infection because the human immune system may not be active at this stage. Therefore, this work aims to develop a label-free electrochemical DNA biosensor for SARS-CoV-2 detection using a printed circuit board-based gold substrate (PCBGE). The developed sensor used the nucleocapsid phosphoprotein (N) gene as a biomarker. The DNA sensor-based PCBGE was fabricated by self-assembling a thiolated single-stranded DNA (ssDNA) probe onto an Au surface, which performed as the working electrode (WE). The Au surface was then treated with 6-mercapto-1-hexanol (MCH) before detecting the target N gene to produce a well-oriented arrangement of the immobilized ssDNA chains. The successful fabrication of the biosensor was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). The DNA biosensor performances were evaluated using a synthetic SARS-CoV-2 genome and 20 clinical RNA samples from healthy and infected individuals through EIS. The developed DNA biosensor can detect as low as 1 copy per μL of the N gene within 5 minutes with a LOD of 0.50 μM. Interestingly, the proposed DNA sensor could distinguish the expression of SARS-CoV-2 RNA in a patient diagnosed with COVID-19 without any amplification technique. We believe that the proposed DNA sensor platform is a promising point-of-care (POC) device for COVID-19 viral infection since it offers a rapid detection time with a simple design and workflow detection system, as well as an affordable diagnostic assay.
A portable label-free electrochemical DNA biosensor for rapid detection of Salmonella Typhi

Zambry NS, Awang MS, Hamzah HH, Mohamad AN, Khalid MF, Khim BK, et al.

Anal Methods, 2024 Jul 16.
PMID: 39011785 DOI: 10.1039/d4ay00888j

A highly accurate, rapid, portable, and robust platform for detecting Salmonella enterica serovar Typhi (S. Typhi) is crucial for early-stage diagnosis of typhoid to avert and control the outbreaks of this pathogen, which threaten global public health. This study presents a proof-of-concept for our developed label-free electrochemical DNA biosensor system for S. Typhi detection, which employs a printed circuit board gold electrode (PCBGE), integrated with a portable potentiostat reader. Initially, the functionalized DNA biosensor and target detection were characterized using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) methods using a benchtop potentiostat. Interestingly, the newly developed DNA biosensor can identify target single-stranded DNA concentrations ranging from 10 nM to 20 μM, achieving a detection limit of 7.6 nM within a brief 5 minute timeframe. Under optimal detection conditions, the DNA biosensor exhibits remarkable selectivity, capable of distinguishing a single mismatch base pair from the target single-stranded DNA sequence. We then evaluated the feasibility of the developed DNA biosensor system as a diagnostic tool by detecting S. Typhi in 50 clinical samples using a portable potentiostat reader based on the DPV technique. Remarkably, the developed biosensor can distinctly distinguish between positive and negative samples, indicating that the miniaturised DNA biosensor system is practical for detecting S. Typhi in real biological samples. The developed DNA biosensor device in this work proves to be a promising point-of-care (POC) device for Salmonella detection due to its swift detection time, uncomplicated design, and streamlined workflow detection system.
Fulltext Duplex TaqMan Hydrolysis Probe-Based Molecular Assay for Simultaneous Detection and Differentiation of Burkholderia pseudomallei and Leptospira spp. DNA

Mohd Ali MR, Lih Huey L, Foo PC, Goay YX, Ismail AS, Mustaffa KMF, et al.

Biomed Res Int, 2019;2019:9451791.
PMID: 31355287 DOI: 10.1155/2019/9451791

Melioidosis and leptospirosis, caused by two different bacteria, Burkholderia pseudomallei and Leptospira spp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection of B. pseudomallei and Leptospira spp. DNA. The performance of the assay was evaluated on 20 B. pseudomallei isolates, 23 Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies) Leptospira spp. DNA and 0.2 pg (~ 25.6 copies) B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection of B. pseudomallei & Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.
Fulltext Isolation and characterization of DNA aptamers against the HlyE antigen of Salmonella Typhi

Ahmad Najib M, Winter A, Mustaffa KMF, Ong EBB, Selvam K, Khalid MF, et al.

Sci Rep, 2024 Nov 18;14(1):28416.
PMID: 39557915 DOI: 10.1038/s41598-024-78685-9

Aptamers have emerged as prominent ligands in clinical diagnostics because they provide various advantages over antibodies, such as quicker generation time, reduced manufacturing costs, minimal batch-to-batch variability, greater modifiability, and improved thermal stability. In the present study, we isolated and characterized DNA aptamers that can specifically bind to the hemolysin E (HlyE) antigen of Salmonella Typhi for future development of typhoid diagnostic tests. The DNA aptamers against Salmonella Typhi HlyE were isolated using systematic evolution of ligands by exponential enrichment (SELEX), and their binding affinity and specificity were assessed utilizing enzyme-linked oligonucleotide assay (ELONA). A total of 11 distinct aptamers were identified, and the binding affinities and species selectivities of the three most probable aptamers were determined. Kd values were obtained in the nanomolar range, with the highest affinity of 83.6 nM determined for AptHlyE97. In addition, AptHlyE11, AptHlyE45 and AptHlyE97 clearly distinguished S. Typhi HlyE from other tested bacteria, such as Salmonella Paratyphi A, Salmonella Paratyphi B, Shigella flexneri, Klebsiella pneumonia and Escherichia coli, therefore displaying desirable specificity. These novel aptamers could be used as diagnostic ligands for the future development of inexpensive and effective point-of-care tests for typhoid surveillance, especially in developing countries of the tropics and subtropics.
Identification of carriers among individuals recruited in the typhoid registry in Malaysia using stool culture, polymerase chain reaction, and dot enzyme immunoassay as detection tools

Chua AL, Aziah I, Balaram P, Bhuvanendran S, Anthony AA, Mohmad SN, et al.

Asia Pac J Public Health, 2015 Mar;27(2):NP2740-8.
PMID: 23000800 DOI: 10.1177/1010539512458521

Chronic carriers of Salmonella Typhi act as reservoirs for the organism and become the agents of typhoid outbreaks in a community. In this study, chronic carriers in Kelantan, Malaysia were first identified using the culture and polymerase chain reaction method. Then, a novel serological tool, designated Typhidot-C, was evaluated in retrospect using the detected individuals as control positives. Chronic carriage positive by the culture and polymerase chain reaction method was recorded at 3.6% (4 out of 110) among individuals who previously had acute typhoid fever and a 9.4% (10 out of 106) carriage rate was observed among food handlers screened during outbreaks. The Typhidot-C assay was able to detect all these positive carriers showing its potential as a viable carrier screening tool and can be used for efficient detection of typhoid carriers in an endemic area. These findings were used to establish the first carrier registry for S Typhi carriers in Malaysia.
Fulltext Pneumococcal conjugate vaccine implementation in middle-income countries

Tricarico S, McNeil HC, Cleary DW, Head MG, Lim V, Yap IKS, et al.

Pneumonia (Nathan), 2017;9:6.
PMID: 28702308 DOI: 10.1186/s41479-017-0030-5

BACKGROUND: Since 2000, the widespread adoption of pneumococcal conjugate vaccines (PCVs) has had a major impact in the prevention of pneumonia. Limited access to international financial support means some middle-income countries (MICs) are trailing in the widespread use of PCVs. We review the status of PCV implementation, and discuss any needs and gaps related to low levels of PCV implementation in MICs, with analysis of possible solutions to strengthen the PCV implementation process in MICs.
MAIN BODY: We searched PubMed, PubMed Central, Ovid MEDLINE, and SCOPUS databases using search terms related to pneumococcal immunization, governmental health policy or programmes, and MICs. Two authors independently reviewed the full text of the references, which were assessed for eligibility using pre-defined inclusion and exclusion criteria. The search terms identified 1,165 articles and the full texts of 21 were assessed for suitability, with eight articles included in the systematic review. MICs are implementing PCVs at a slower rate than donor-funded low-income countries and wealthier developed countries. A significant difference in the uptake of PCV in lower middle-income countries (LMICs) (71%) and upper middle-income countries (UMICs) (48%) is largely due to an unsuccessful process of "graduation" of MICs from GAVI assistance, an issue that arises as countries cross the income eligibility threshold and are no longer eligible to receive the same levels of financial assistance. A lack of country-specific data on disease burden, a lack of local expertise in economic evaluation, and the cost of PCV were identified as the leading causes of the slow uptake of PCVs in MICs. Potential solutions mentioned in the reviewed papers include the use of vaccine cost-effectiveness analysis and the provision of economic evidence to strengthen decision-making, the evaluation of the burden of disease, and post-introduction surveillance to monitor vaccine impact.
CONCLUSION: The global community needs to recognise the impediments to vaccine introduction into MICs. Improving PCV access could help decrease the incidence of pneumonia and reduce the selection pressure for pneumococcal antimicrobial resistance.