Displaying all 16 publications

  1. Rajendran K, Anwar A, Khan NA, Shah MR, Siddiqui R
    ACS Chem Neurosci, 2019 06 19;10(6):2692-2696.
    PMID: 30970208 DOI: 10.1021/acschemneuro.9b00111
    Primary amoebic meningoencephalitis (PAM), a deadly brain infection, is caused by brain-eating amoeba Naegleria fowleri. The current first line of treatment against PAM is a mixture of amphotericin B, rifampin, and miltefosine. Since, no single effective drug has been developed so far, the mortality rate is above 95%. Moreover, severe adverse side effects are associated with these drugs. Nanotechnology has provided several advances in biomedical applications especially in drug delivery and diagnosis. Herein, for the first time we report antiamoebic properties of cinnamic acid (CA) and gold nanoparticles conjugated with CA (CA-AuNPs) against N. fowleri. CA-AuNPs were successfully synthesized by sodium borohydride reduction of tetrachloroauric acid. Size and morphology were determined by atomic force microscopy (AFM) while the surface plasmon resonance band was analyzed by ultraviolet-visible (UV-vis) spectrophotometry for the characterization of the nanoparticles. Amoebicidal and cytopathogenicity (host cell cytotoxicity) assays revealed that both CA and CA-AuNPs displayed significant anti- N. fowleri properties ( P < 0.05), whereas nanoparticles conjugation further enhanced the anti- N. fowleri effects of CA. This study established a potential drug lead, while CA-AuNPs appear to be promising candidate for drug discovery against PAM.
  2. Anwar A, Rajendran K, Siddiqui R, Raza Shah M, Khan NA
    ACS Chem Neurosci, 2019 01 16;10(1):658-666.
    PMID: 30346711 DOI: 10.1021/acschemneuro.8b00484
    Central nervous system (CNS) infections caused by free-living amoebae such as Acanthamoeba species and Naegleria fowleri are rare but fatal. A major challenge in the treatment against the infections caused by these amoebae is the discovery of novel compounds that can effectively cross the blood-brain barrier to penetrate the CNS. It is logical to test clinically approved drugs against CNS diseases for their potential antiamoebic effects since they are known for effective blood-brain barrier penetration and affect eukaryotic cell targets. The antiamoebic effects of clinically available drugs for seizures targeting gamma-amino butyric acid (GABA) receptor and ion channels were tested against Acanthamoeba castellanii belonging to the T4 genotype and N. fowleri. Three such drugs, namely, diazepam (Valium), phenobarbitone (Luminal), phenytoin (Dilantin), and their silver nanoparticles (AgNPs) were evaluated against both trophozoites and cysts stage. Drugs alone and drug conjugated silver nanoparticles were tested for amoebicidal, cysticidal, and host-cell cytotoxicity assays. Nanoparticles were synthesized by sodium borohydride reduction of silver nitrate with drugs as capping agents. Drug conjugated nanoconjugates were characterized by ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopies and atomic force microscopy (AFM). In vitro moebicidal assay showed potent amoebicidal effects for diazepam, phenobarbitone, and phenytoin-conjugated AgNPs as compared to drugs alone against A. castellanii and N. fowleri. Furthermore, both drugs and drug conjugated AgNPs showed compelling cysticidal effects. Drugs conjugations with silver nanoparticles enhanced their antiacanthamoebic activity. Interestingly, amoeba-mediated host-cell cytotoxicity was also significantly reduced by drugs alone as well as their nanoconjugates. Since, these drugs are being used to target CNS diseases, their evaluation against brain-eating amoebae seems feasible due to advantages such as permeability of the blood-brain barrier, established pharmacokinetics and dynamics, and United States Food and Drug Administration (FDA) approval. Given the limited availability of effective drugs against brain-eating amoebae, the clinically available drugs tested here present potential for further in vivo studies.
  3. Rajendran K, Anwar A, Khan NA, Siddiqui R
    ACS Chem Neurosci, 2017 12 20;8(12):2626-2630.
    PMID: 29206032 DOI: 10.1021/acschemneuro.7b00430
    The overall aim of this study was to determine whether conjugation with silver nanoparticles enhances effects of available drugs against primary amoebic meningoencephalitis due to Naegleria fowleri. Amphotericin B, Nystatin, and Fluconazole were conjugated with silver nanoparticles, and synthesis was confirmed using UV-visible spectrophotometry. Atomic force microscopy determined their size in range of 20-100 nm. To determine amoebicidal effects, N. fowleri were incubated with drugs-conjugated silver nanoparticles, silver nanoparticles alone, and drugs alone. The findings revealed that silver nanoparticles conjugation significantly enhanced antiamoebic effects of Nystatin and Amphotericin B but not Fluconazole at micromolar concentrations, compared with the drugs alone. For the first time, our findings showed that silver nanoparticle conjugation enhances efficacy of antiamoebic drugs against N. fowleri. Given the rarity of the disease and challenges in developing new drugs, it is hoped that modifying existing drugs to enhance their antiamoebic effects is a useful avenue that holds promise in improving the treatment of brain-eating amoebae infection due to N. fowleri.
  4. Rajendran K, Anwar A, Khan NA, Aslam Z, Raza Shah M, Siddiqui R
    ACS Chem Neurosci, 2020 08 19;11(16):2431-2437.
    PMID: 31347828 DOI: 10.1021/acschemneuro.9b00289
    Naegleria fowleri (N. fowleri) causes primary amoebic meningoencephalitis (PAM) which almost always results in death. N. fowleri is also known as "brain-eating amoeba" due to its literal infestation of the brain leading to an inflammatory response in the brain tissues. Currently, there is no single drug that is available to treat PAM, and most treatments are combinations of antifungal, anticancer, and anti-inflammatory drugs. Recently nanotechnology has gained attention in chemotherapeutic research converging on drug delivery, while oleic acid (OA) has shown positive effects on the human immune system and inflammatory processes. In continuation of our recent research in which we reported the effects of oleic acid conjugated with silver nanoparticles (OA-AgNPs) against free-living amoeba Acanthamoeba castellanii, in this report, we show their antiamoebic effects against N. fowleri. OA alone and its nanoconjugates were tested against the amoeba by using amoebicidal and host cell cytopathogenicity assays. Trypan blue exclusion assay was used to determine cell viability. The results revealed that OA-AgNPs exhibited significantly enhanced antiamoebic effects (P < 0.05) against N. fowleri as compared to OA alone. Evidently, lactate dehydrogenase release shows reduced N. fowleri-mediated host cell cytotoxicity. Based on our study, we anticipate that further studies on OA-AgNPs could potentially provide an alternative treatment of PAM.
  5. Anwar A, Masri A, Rao K, Rajendran K, Khan NA, Shah MR, et al.
    Sci Rep, 2019 02 28;9(1):3122.
    PMID: 30816269 DOI: 10.1038/s41598-019-39528-0
    Herein, we report green synthesized nanoparticles based on stabilization by plant gums, loaded with citrus fruits flavonoids Hesperidin (HDN) and Naringin (NRG) as novel antimicrobial agents against brain-eating amoebae and multi-drug resistant bacteria. Nanoparticles were thoroughly characterized by using zetasizer, zeta potential, atomic force microscopy, ultravoilet-visible and Fourier transform-infrared spectroscopic techniques. The size of these spherical nanoparticles was found to be in the range of 100-225 nm. The antiamoebic effects of these green synthesized Silver and Gold nanoparticles loaded with HDN and NRG were tested against Acanthamoeba castellanii and Naegleria fowleri, while antibacterial effects were evaluated against methicillin-resistant Staphylococcus aureus (MRSA) and neuropathogenic Escherichia coli K1. Amoebicidal assays revealed that HDN loaded Silver nanoparticles stabilized by gum acacia (GA-AgNPs-HDN) quantitatively abolished amoeba viability by 100%, while NRG loaded Gold nanoparticles stabilized by gum tragacanth (GT-AuNPs-NRG) significantly reduced the viability of A. castellanii and N. fowleri at 50 µg per mL. Furthermore, these nanoparticles inhibited the encystation and excystation by more than 85%, as well as GA-AgNPs-HDN only completely obliterated amoeba-mediated host cells cytopathogenicity. Whereas, GA-AgNPs-HDN exhibited significant bactericidal effects against MRSA and E. coli K1 and reduced bacterial-mediated host cells cytotoxicity. Notably, when tested against human cells, these nanoparticles showed minimal (23%) cytotoxicity at even higher concentration of 100 µg per mL as compared to 50 µg per mL used for antimicrobial assays. Hence, these novel nanoparticles formulations hold potential as therapeutic agents against infections caused by brain-eating amoebae, as well as multi-drug resistant bacteria, and recommend a step forward in drug development.
  6. Siddiqui R, Rajendran K, Abdella B, Ayub Q, Lim SY, Khan NA
    Parasitol Res, 2020 Jul;119(7):2351-2358.
    PMID: 32451717 DOI: 10.1007/s00436-020-06711-6
    Naegleria fowleri causes a deadly infection known as primary amoebic meningoencephalitis (PAM). To our knowledge, there are very few transcriptome studies conducted on these brain-eating amoebae, despite rise in the number of cases. Although the Naegleria genome has been sequenced, currently, it is not well annotated. Transcriptome level studies are needed to help understand the pathology and biology of this fatal parasitic infection. Recently, we showed that nanoparticles loaded with the flavonoid Hesperidin (HDN) are potential novel antimicrobial agents. N. fowleri trophozoites were treated with and without HDN-conjugated with silver nanoparticles (AgNPs) and silver only, and then, 50% minimum inhibitory concentration (MIC) was determined. The results revealed that the MIC of HDN-conjugated AgNPs was 12.5 microg/mL when treated for 3 h. As no reference genome exists for N. fowleri, de novo RNA transcriptome analysis using RNA-Seq and differential gene expression analysis was performed using the Trinity software. Analysis revealed that more than 2000 genes were differentially expressed in response to N. fowleri treatment with HDN-conjugated AgNPs. Some of the genes were linked to oxidative stress response, DNA repair, cell division, cell signalling and protein synthesis. The downregulated genes were linked with processes such as protein modification, synthesis of aromatic amino acids, when compared with untreated N. fowleri. Further transcriptome studies will lead to understanding of genetic mechanisms of the biology and pathogenesis and/or the identification of much needed drug candidates.
  7. Verma DK, Sood N, Paria A, Swaminathan TR, Mohan CV, Rajendran KV, et al.
    Virus Res, 2021 Nov 12;308:198625.
    PMID: 34780882 DOI: 10.1016/j.virusres.2021.198625
    The tilapia lake virus (TiLV), a highly infectious negative-sense single-stranded segmented RNA virus, has caused several outbreaks worldwide since its first report from Israel in 2014, and continues to pose a major threat to the global tilapia industry. Despite its economic importance, little is known about the underlying mechanisms in the genomic evolution of this highly infectious viral pathogen. Using phylogenomic approaches to the genome sequences of TiLV isolates from various geographic regions, we report on the pervasive role of reassortment, selection, and mutation in TiLV evolution. Our findings provided the evidence of genome-wide reassortment in this newly discovered RNA virus. The rate of non-synonymous (dN) to synonymous (dS) substitutions was less than one (dN/dS = 0.076 to 0.692), indicating that each genomic segment has been subjected to purifying selection. Concurrently, the rate of nucleotide substitution for each genomic segment was in the order of 1-3 × 10-3 nucleotide substitutions per site per year, which is comparable to the rate of other RNA viruses. Collectively, in line with the results of the previous studies, our results demonstrated that reassortment is the dominant force in the evolution and emergence of this highly infectious segmented RNA virus.
  8. Jayanthi Antonisamy A, Marimuthu S, Malayandi S, Rajendran K, Lin YC, Andaluri G, et al.
    Environ Res, 2023 Jan 15;217:114758.
    PMID: 36400225 DOI: 10.1016/j.envres.2022.114758
    The concept of zero waste discharge has been gaining importance in recent years towards attaining a sustainable environment. Fruit processing industries generate millions of tons of byproducts like fruit peels and seeds, and their disposal poses an environmental threat. The concept of extracting value-added bioactive compounds from bio-waste is an excellent opportunity to mitigate environmental issues. To date, significant research has been carried out on the extraction of essential biomolecules, particularly polysaccharides from waste generated by fruit processing industries. In this review article, we aim to summarize the different extraction methodologies, characterization methods, and biomedical applications of polysaccharides extracted from seeds and peels of different fruit sources. The review also focuses on the general scheme of extraction of polysaccharides from fruit waste with special emphasis on various methods used in extraction. Also, the various types of polysaccharides obtained from fruit processing industrial wastes are explained in consonance with the important techniques related to the structural elucidation of polysaccharides obtained from seed and peel waste. The use of seed polysaccharides as pharmaceutical excipients and the application of peel polysaccharides possessing biological activities are also elaborated.
  9. Rajendran K, Ahmed U, Meunier AC, Shaikh MF, Siddiqui R, Anwar A
    ACS Chem Neurosci, 2023 Dec 06;14(23):4105-4114.
    PMID: 37983556 DOI: 10.1021/acschemneuro.3c00258
    Naegleria fowleri is one of the free-living amoebae and is a causative agent of a lethal and rare central nervous system infection called primary amoebic meningoencephalitis. Despite the advancement in antimicrobial chemotherapy, the fatality rate in the reported cases is more than 95%. Most of the treatment drugs used against N. fowleri infection are repurposed drugs. Therefore, a large number of compounds have been tested against N. fowleri in vitro, but most of the compounds showed high toxicity. To overcome this, we evaluated the effectiveness of naturally occurring terpene compounds against N. fowleri. In this study, we evaluated the antiamoebic potential of natural compounds including Thymol, Borneol, Andrographolide, and Forskolin againstN. fowleri. Thymol showed the highest amoebicidal activity with IC50/24 h at 153.601 ± 19.6 μM. Two combinations of compounds Forskolin + Thymol and Forskolin + Borneol showed a higher effect on the viability of trophozoites as compared to compounds alone and hence showed a synergistic effect. The IC50 reported for Forskolin + Thymol was 81.30 ± 6.86 μM. Borneol showed maximum cysticidal activity with IC50/24 h at 192.605 ± 3.01 μM. Importantly, lactate dehydrogenase release testing revealed that all compounds displayed minimal cytotoxicity to human HaCaT, HeLa, and SH-SY5Y cell lines. The cytopathogenicity assay showed that Thymol and Borneol also significantly reduced the host cell cytotoxicity of pretreated amoeba toward the human HaCaT cell line. So, these terpene compounds hold potential as therapeutic agents against infections caused by N. fowleri and are potentially a step forward in drug development against this deadly pathogen as these compounds have also been reported to cross the blood-brain barrier. Therefore, an in vivo study using animal models is necessary to assess the efficacy of these compounds and the need for further research into the intranasal route of delivery for the treatment of these life-threatening infections.
  10. Rajendran K, Ahmed U, Meunier AC, Shaikh MF, Siddiqui R, Anwar A
    ACS Omega, 2024 Mar 12;9(10):11597-11607.
    PMID: 38497026 DOI: 10.1021/acsomega.3c08844
    Pathogenic Naegleria fowleri (N. fowleri) are opportunistic free-living amoebae and are the causative agents of a very rare but severe brain infection called primary amoebic meningoencephalitis (PAM). The fatality rate of PAM in reported cases is more than 95%. Most of the drugs used againstN. fowleri infections are repurposed drugs. Therefore, a large number of compounds have been tested againstN. fowleri in vitro, but most of the tested compounds showed high toxicity and an inability to cross the blood-brain barrier. Andrographolide, forskolin, and borneol are important natural compounds that have shown various valuable biological properties. In the present study, the nanoconjugates (AND-AgNPs, BOR-AgNPs, and FOR-AgNPs) of these compounds were synthesized and assessed against both stages (trophozoite and cyst) ofN. fowleri for their antiamoebic and cysticidal potential in vitro. In addition, cytotoxicity and host cell pathogenicity were also evaluated in vitro. FOR-AgNPs were the most potent nanoconjugate and showed potent antiamoebic activity againstN. fowleriwith an IC50 of 26.35 μM. Nanoconjugates FOR-AgNPs, BOR-AgNPs, and AND-AgNPs also significantly inhibit the viability of N. fowleri cysts. Cytotoxicity assessment showed that these nanoconjugates caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 μg/mL, while also effectively reducing the cytopathogenicity of N. fowleri trophozoites to the HaCaT cells. The outcomes of our experiments have unveiled substantial potential for AND-AgNPs, BOR-AgNPs, and FOR-AgNPs in the realm of developing innovative alternative therapeutic agents to combat infections caused by N. fowleri. This study represents a significant step forward in the pursuit of advanced strategies for managing such amoebic infections, laying the foundation for the development of novel and more effective therapeutic modalities in the fight against free-living amoebae.
  11. Singh S, Murali Sundram B, Rajendran K, Boon Law K, Aris T, Ibrahim H, et al.
    J Infect Dev Ctries, 2020 09 30;14(9):971-976.
    PMID: 33031083 DOI: 10.3855/jidc.13116
    INTRODUCTION: The novel coronavirus infection has become a global threat affecting almost every country in the world. As a result, it has become important to understand the disease trends in order to mitigate its effects. The aim of this study is firstly to develop a prediction model for daily confirmed COVID-19 cases based on several covariates, and secondly, to select the best prediction model based on a subset of these covariates.

    METHODOLOGY: This study was conducted using daily confirmed cases of COVID-19 collected from the official Ministry of Health, Malaysia (MOH) and John Hopkins University websites. An Autoregressive Integrated Moving Average (ARIMA) model was fitted to the training data of observed cases from 22 January to 31 March 2020, and subsequently validated using data on cases from 1 April to 17 April 2020. The ARIMA model satisfactorily forecasted the daily confirmed COVID-19 cases from 18 April 2020 to 1 May 2020 (the testing phase).

    RESULTS: The ARIMA (0,1,0) model produced the best fit to the observed data with a Mean Absolute Percentage Error (MAPE) value of 16.01 and a Bayes Information Criteria (BIC) value of 4.170. The forecasted values showed a downward trend of COVID-19 cases until 1 May 2020. Observed cases during the forecast period were accurately predicted and were placed within the prediction intervals generated by the fitted model.

    CONCLUSIONS: This study finds that ARIMA models with optimally selected covariates are useful tools for monitoring and predicting trends of COVID-19 cases in Malaysia.

  12. Venkatramanan M, Sankar Ganesh P, Senthil R, Akshay J, Veera Ravi A, Langeswaran K, et al.
    ACS Omega, 2020 Oct 13;5(40):25605-25616.
    PMID: 33073086 DOI: 10.1021/acsomega.0c02483
    Chromobacterium violaceum (C. violaceum) is a Gram-negative, rod-shaped facultatively anaerobic bacterium implicated with recalcitrant human infections. Here, we evaluated the anti-QS and antibiofilm activities of ethyl acetate extracts of Passiflora edulis (P. edulis) on the likely inactivation of acyl-homoserine lactone (AHL)-regulated molecules in C. violaceum both by in vitro and in silico analyses. Our investigations showed that the sub-MIC levels were 2, 1, and 0.5 mg/mL, and the concentrations showed a marked reduction in violacein pigment production by 75.8, 64.6, and 35.2%. AHL quantification showed 72.5, 52.2, and 35.9% inhibitions, inhibitions of EPS production (72.8, 36.5, and 25.9%), and reductions in biofilm formation (90.7, 69.4, and 51.8%) as compared to a control. Light microscopy and CLSM analysis revealed dramatic reduction in the treated biofilm group as compared to the control. GC-MS analysis showed 20 major peaks whose chemical structures were docked as the CviR ligand. The highest docking score was observed for hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester bonds in the active site of CviR with a binding energy of -8.825 kcal/mol. Together, we found that hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester remarkably interacted with CviR to inhibit the QS system. Hence, we concluded that hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester of P. edulis could likely be evaluated for treating C. violaceum infections.
  13. Tan YY, Wong LS, Nyam KL, Wittayanarakul K, Zawawi NA, Rajendran K, et al.
    Molecules, 2023 Sep 22;28(19).
    PMID: 37836592 DOI: 10.3390/molecules28196749
    Zinc oxide nanoparticles have high levels of biocompatibility, a low impact on environmental contamination, and suitable to be used as an ingredient for environmentally friendly skincare products. In this study, biogenically synthesized zinc oxide nanoparticles using Dendrobium anosum are used as a reducing and capping agent for topical anti-acne nanogels, and the antimicrobial effect of the nanogel is assessed on Cutibacterium acne and Staphylococcus aureus. Dendrobium anosmum leaf extract was examined for the presence of secondary metabolites and its total amount of phenolic and flavonoid content was determined. Both the biogenically and chemogenic-synthesized zinc oxide nanoparticles were compared using UV-Visible spectrophotometer, FE-SEM, XRD, and FTIR. To produce the topical nanogel, the biogenic and chemogenic zinc oxide nanoparticles were mixed with a carbomer and hydroxypropyl-methyl cellulose (HPMC) polymer. The mixtures were then tested for physical and chemical characteristics. To assess their anti-acne effectiveness, the mixtures were tested against C. acne and S. aureus. The biogenic zinc oxide nanoparticles have particle sizes of 20 nm and a high-phase purity. In comparison to chemogenic nanoparticles, the hydrogels with biogenically synthesized nanoparticles was more effective against Gram-positive bacteria. Through this study, the hybrid nanogels was proven to be effective against the microbes that cause acne and to be potentially used as a green product against skin infections.
  14. Law KB, Peariasamy KM, Gill BS, Singh S, Sundram BM, Rajendran K, et al.
    Sci Rep, 2020 12 10;10(1):21721.
    PMID: 33303925 DOI: 10.1038/s41598-020-78739-8
    The susceptible-infectious-removed (SIR) model offers the simplest framework to study transmission dynamics of COVID-19, however, it does not factor in its early depleting trend observed during a lockdown. We modified the SIR model to specifically simulate the early depleting transmission dynamics of COVID-19 to better predict its temporal trend in Malaysia. The classical SIR model was fitted to observed total (I total), active (I) and removed (R) cases of COVID-19 before lockdown to estimate the basic reproduction number. Next, the model was modified with a partial time-varying force of infection, given by a proportionally depleting transmission coefficient, [Formula: see text] and a fractional term, z. The modified SIR model was then fitted to observed data over 6 weeks during the lockdown. Model fitting and projection were validated using the mean absolute percent error (MAPE). The transmission dynamics of COVID-19 was interrupted immediately by the lockdown. The modified SIR model projected the depleting temporal trends with lowest MAPE for I total, followed by I, I daily and R. During lockdown, the dynamics of COVID-19 depleted at a rate of 4.7% each day with a decreased capacity of 40%. For 7-day and 14-day projections, the modified SIR model accurately predicted I total, I and R. The depleting transmission dynamics for COVID-19 during lockdown can be accurately captured by time-varying SIR model. Projection generated based on observed data is useful for future planning and control of COVID-19.
  15. Sood N, Verma DK, Paria A, Yadav SC, Yadav MK, Bedekar MK, et al.
    Fish Shellfish Immunol, 2021 Apr;111:208-219.
    PMID: 33577877 DOI: 10.1016/j.fsi.2021.02.005
    Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture species farmed worldwide. However, the recent emergence of tilapia lake virus (TiLV) disease, also known as syncytial hepatitis of tilapia, has threatened the global tilapia industry. To gain more insight regarding the host response against the disease, the transcriptional profiles of liver in experimentally-infected and control tilapia were compared. Analysis of RNA-Seq data identified 4640 differentially expressed genes (DEGs), which were involved among others in antigen processing and presentation, MAPK, apoptosis, necroptosis, chemokine signaling, interferon, NF-kB, acute phase response and JAK-STAT pathways. Enhanced expression of most of the DEGs in the above pathways suggests an attempt by tilapia to resist TiLV infection. However, upregulation of some of the key genes such as BCL2L1 in apoptosis pathway; NFKBIA in NF-kB pathway; TRFC in acute phase response; and SOCS, EPOR, PI3K and AKT in JAK-STAT pathway and downregulation of the genes, namely MAP3K7 in MAPK pathway; IFIT1 in interferon; and TRIM25 in NF-kB pathway suggested that TiLV was able to subvert the host immune response to successfully establish the infection. The study offers novel insights into the cellular functions that are affected following TiLV infection and will serve as a valuable genomic resource towards our understanding of susceptibility of tilapia to TiLV infection.
  16. Rashid SA, Nazakat R, Muhamad Robat R, Ismail R, Suppiah J, Rajendran K, et al.
    Front Public Health, 2023;11:1208348.
    PMID: 37965510 DOI: 10.3389/fpubh.2023.1208348
    Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with inadequate ventilation. There has been no standard recommended method of determining the virus in air due to limitations in pre-analytical and technical aspects. Furthermore, the presence of low virus loads in air samples could result in false negatives. Our study aims to explore the feasibility of detecting SARS-CoV-2 ribonucleic acid (RNA) in air samples using droplet digital polymerase chain reaction (ddPCR). Active and passive air sampling was conducted between December 2021 and February 2022 with the presence of COVID-19 confirmed cases in two hospitals and a quarantine center in Klang Valley, Malaysia. SARS-CoV-2 RNA in air was detected and quantified using ddPCR and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The comparability of two different digital PCR platforms (QX200 and QIAcuity) to RT-PCR were also investigated. Additionally negative staining transmission electron microscopy was performed to visualize virus ultrastructure. Detection rates of SARS-CoV-2 in air samples using ddPCR were higher compared to RT-PCR, which were 15.2% (22/145) and 3.4% (5/145), respectively. The sensitivity and specificity of ddPCR was 100 and 87%, respectively. After excluding 17 negative samples (50%) by both QX200 and QIAcuity, 15% samples (5/34) were found to be positive both ddPCR and dPCR. There were 23.5% (8/34) samples that were detected positive by ddPCR but negative by dPCR. In contrast, there were 11.7% (4/34) samples that were detected positive by dPCR but negative by ddPCR. The SARS-CoV-2 detection method by ddPCR is precise and has a high sensitivity for viral RNA detection. It could provide advances in determining low viral titter in air samples to reduce false negative reports, which could complement detection by RT-PCR.
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