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Fulltext Biomarkers of severe dengue disease - a review

John DV, Lin YS, Perng GC

J Biomed Sci, 2015;22:83.
PMID: 26462910 DOI: 10.1186/s12929-015-0191-6

Dengue virus infection presents a wide spectrum of manifestations including asymptomatic condition, dengue fever (DF), or severe forms, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) in affected individuals. The early prediction of severe dengue in patients without any warning signs who may later develop severe DHF is very important to choose appropriate intensive supportive therapy since available vaccines for immunization are yet to be approved. Severe dengue responses include T and B cell activation and apoptosis, cytokine storm, hematologic disorders and complement activation. Cytokines, complement and other unidentified factors may transiently act on the endothelium and alter normal fluid barrier function of the endothelial cells and cause plasma leakage. In this review, the host factors such as activated immune and endothelial cells and their products which can be utilized as biomarkers for severe dengue disease are discussed.
Fulltext Whole genome sequencing of Mycobacterium tuberculosis SB24 isolated from Sabah, Malaysia

Philip N, Rodrigues KF, William T, John DV

Genom Data, 2016 Sep;9:137-9.
PMID: 27556011 DOI: 10.1016/j.gdata.2016.08.007

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB) that causes millions of death every year. We have sequenced the genome of M. tuberculosis isolated from cerebrospinal fluid (CSF) of a patient diagnosed with tuberculous meningitis (TBM). The isolated strain was referred as M. tuberculosis SB24. Genomic DNA of the M. tuberculosis SB24 was extracted and subjected to whole genome sequencing using PacBio platform. The draft genome size of M. tuberculosis SB24 was determined to be 4,452,489 bp with a G + C content of 65.6%. The whole genome shotgun project has been deposited in NCBI SRA under the accession number SRP076503.
Cerebrospinal fluid inflammatory cytokine profiles of patients with neurotropic parasitic infections

John DV, Sreenivas N, Deora H, Purushottam M, Debnath M, Mahadevan A, et al.

Trop Biomed, 2023 Dec 01;40(4):406-415.
PMID: 38308827 DOI: 10.47665/tb.40.4.005

The pathogenesis of chronic parasitic central nervous system (CNS) infections, including granulomatous amoebic meningoencephalitis (GAE), cerebral toxoplasmosis (CT), and neurocysticercosis (NCC), is primarily due to an inflammatory host reaction to the parasite. Inflammatory cytokines produced by invading T cells, monocytes, and CNS resident cells lead to neuroinflammation which underlie the immunopathology of these infections. Immune molecules, especially cytokines, can therefore emerge as potential biomarker(s) of CNS parasitic infections. In this study, cerebral spinal fluid (CSF) samples from suspected patients with parasitic infections were screened for pathogenic free-living amoebae by culture (n=2506) and PCR (n=275). Six proinflammatory cytokines in smear and culture-negative CSF samples from patients with GAE (n = 2), NCC (n = 7), and CT (n = 23) as well as control (n = 7) patients were measured using the Multiplex Suspension assay. None of the CSF samples tested was positive for neurotropic free-living amoebae by culture and only two samples showed Acanthamoeba 18S rRNA by PCR. Of the six cytokines measured, only IL-6 and IL-8 were significantly increased in all three infection groups compared to the control group. In addition, TNFa levels were higher in the GAE and NCC groups and IL-17 in the GAE group compared to controls. The levels of IL-1b and IFNg were very low in all the infection groups and the control group. There was a correlation between CSF cellularity and increased levels of IL-6, IL-8, and TNFa in 11 patients. Thus, quantifying inflammatory cytokine levels in CSF might help with understanding the level of neuroinflammation in patients with neurotropic parasitic diseases. Further studies with clinico-microbiological correlation in the form of reduction of cytokine levels with treatment and the correlation with neurological deficits are needed.
Recombinant LipL32 Protein Developed Using a Synthetic Gene Detects Leptospira-specific Antibodies in Human Serum Samples

Yaakob Y, Rodrigues KF, Opook F, William T, John DV

Malays J Med Sci, 2017 Oct;24(5):44-51.
PMID: 29386971 DOI: 10.21315/mjms2017.24.5.5

Background: Synthetic biology is emerging as a viable alternative for the production of recombinant antigens for diagnostic applications. It offers a safe alternative for the synthesis of antigenic principles derived from organisms that pose a high biological risk.
Methods: Here, we describe an enzyme-linked immunosorbent assay (ELISA) using the synthetic recombinant LipL32 (rLipL32) protein expressed in Escherichia coli for the detection of Leptospira-specific antibodies in human serum samples. The rLipL32-based ELISA was compared with a microscopic agglutination test (MAT), which is currently used as the gold standard for the diagnosis of leptospirosis.
Results: Our results showed that all the MAT-positive serum samples were positive for Leptospira-specific IgG in an ELISA, while 65% (n = 13) of these samples were also positive for Leptospira-specific IgM. In the MAT-negative serum samples, 80% and 55% of the samples were detected as negative by an ELISA for Leptospira-specific IgM and IgG, respectively.
Conclusion: An ELISA using the synthetic rLipL32 antigen was able to distinguish Leptospira-specific IgM (sensitivity 65% and specificity 80%) and IgG (sensitivity 100% and specificity 55%) in human serum samples and has the potential to serve as a rapid diagnostic test for leptospirosis.
Fulltext Expression distribution of cancer stem cells, epithelial to mesenchymal transition, and telomerase activity in breast cancer and their association with clinicopathologic characteristics

Makki J, Myint O, Wynn AA, Samsudin AT, John DV

Clin Med Insights Pathol, 2015;8:1-16.
PMID: 25624778 DOI: 10.4137/CPath.S19615

A total of 167 surgically resected primary invasive breast carcinomas and 63 metastatic lymph node lesions were analyzed for immunohistochemical (IHC) localization of the CD44(+)CD24(-low) breast cancer stem cell (CSC) markers, epithelial to mesenchymal transition (EMT) markers, and telomerase activity by double-staining IHC technique, in formalin-fixed, paraffin-embedded tissue, the results were validated by double-staining immunofluorescent and flow cytometry techniques. The results showed that CSCs with CD44(+)CD24(-low) phenotype were significantly increased in node-positive tumors, high-grade tumors, and ductal carcinoma in situ (DCIS). There was a high incidence of telomerase expression in metastatic lymph node lesion. There were considerably high number of tumor cells with EMT expression in metastatic lymph node lesion, and triple-negative tumor. The occurrence of EMT phenomena was usually accompanied by the co-existence of CSCs of CD44(+)CD24(-low) phenotype. There was no association between the existence of CSCs and detection of telomerase activity in tumor cells. Increased numbers of both CSCs of CD44(+)CD24(-low) phenotype and cells underwent EMT in DCIS lesion might be an initial step in the stromal invasion and propagation of breast cancer, and occurrence of EMT in the breast tumor associated with high prevalence of CSCs, promoting tumor invasiveness and metastasis.
Fulltext Metagenomic data of bacterial community from different land uses at the river basin, Kelantan

Rupert R, Lie GJCW, John DV, Annammala KV, Jani J, Rodrigues KF

Data Brief, 2020 Dec;33:106351.
PMID: 33072827 DOI: 10.1016/j.dib.2020.106351

The data provided in the article includes the sequence of bacterial 16S rRNA gene from a high conservation value forest, logged forest, rubber plantation and oil palm plantation collected at Kelantan river basin. The logged forest area was previously notified as a flooding region. The total gDNA of bacterial community was amplified via polymerase chain reaction at V3-V4 regions using a pair of specific universal primer. Amplicons were sequenced on Illumina HiSeq paired-end platform to generate 250 bp paired-end raw reads. Several bioinformatics tools such as FLASH, QIIME and UPARSE were used to process the reads generated for OTU analysis. Meanwhile, R&D software was used to construct the taxonomy tree for all samples. Raw data files are available at the Sequence Read Archive (SRA), NCBI and data information can be found at the BioProject and BioSample, NCBI. The data shows the comparison of bacterial community between the natural forest and different land uses.
Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing

John DV, Aryalakshmi B, Deora H, Purushottam M, Raju R, Mahadevan A, et al.

Trop Biomed, 2022 Dec 01;39(4):489-498.
PMID: 36602206 DOI: 10.47665/tb.39.4.002

Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine microbiological testing. Direct PCR of such samples enables the identification of microbes that may be fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative brain abscess samples by molecular methods could enable detection and/or treatment of the source of infection.