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  1. Juvenile Pityriasis Rubra Pilaris in a 4-year-old Child Treated Successfully with Secukinumab
    Albela H, Lee WQ, Nordin SN, Gopinathan LP, A Ramli MN, Leong KF
    Indian J Dermatol, 2022;67(5):601-603.
    PMID: 36865822 DOI: 10.4103/ijd.ijd_1079_21
  2. A rapid DNA biosensor for the molecular diagnosis of infectious disease
    Chua A, Yean CY, Ravichandran M, Lim B, Lalitha P
    Biosens Bioelectron, 2011 May 15;26(9):3825-31.
    PMID: 21458979 DOI: 10.1016/j.bios.2011.02.040
    Treating patients with infectious diseases relies heavily on rapid and proper diagnosis. Molecular detection such as PCR has become increasingly important and efforts have been made to simplify these detection methods. This study reports the development of a glass fibre-based lateral flow DNA biosensor that uses capture reagents coupled to carrier beads and detector reagent bioconjugated to gold nanoparticles, for the detection of foodborne pathogen, Vibrio cholerae. The DNA biosensor contains a test line which captures target PCR amplicons, an internal amplification control (IC) line which captures IC amplicons and a control line which acts as membrane control to validate the functionality of this device. The test line captures biotin labelled DNA, while the IC line captures digoxigenin labelled DNA. The detector reagent recognizes the fluorescein haptens of the amplified DNA and produces visual red lines. Scanning electron microscopy (SEM) studies performed indicated that the capture reagents remained relatively immobile within the matrix of the membrane even after binding of the detector reagent. The DNA biosensor recorded a limit of detection (LoD) of 5 ng of target DNA. A clinical evaluation was carried out with 174 strains of V. cholerae and non V. cholerae bacteria and the DNA biosensor recorded 100% for both sensitivity and specificity when compared to conventional agarose gel detection of DNA. Thus it is a viable alternative to agarose gel analysis and is easy-to-use, disposable and do not require any specialized equipment and use of carcinogenic chemicals.
  3. Role of nitric oxide in hydroxyapatite-induced phagocytosis by murine macrophage cell line (RAW264.7)
    Gopinath VK, Musa M, Samsudin AR, Lalitha P, Sosroseno W
    Arch Oral Biol, 2006 Apr;51(4):339-44.
    PMID: 16214104
    The aim of this study was to determine the role of nitric oxide (NO) in hydroxyapatite (HA)-induced phagocytosis by a murine macrophage cell line (RAW264.7). The cells were incubated with HA particles at various incubation time and phagocytosis was assessed using phagocytic index (PI). NO production from the culture supernatants was determined by the Griess reagent. The inducible nitric oxide synthase (iNOS) expression was determined by Western blot. The particles were also incubated with cells pretreated with various concentrations of L-N(6)-(1-iminoethyl) lysine hydrochloride (L-NIL) or L-arginine. Latex beads were used as a control. Our results showed that macrophage phagocytosis induced by HA was higher than that induced by the beads. However, NO production by HA-stimulated cells was lower than that by bead-stimulated cells. iNOS expression in both bead- and HA-stimulated cells was observed expressed at 7, 15, 30, and 60 min. l-Arginine enhanced but l-NIL inhibited both phagocytosis and NO production by HA-stimulated cells. The results of the present study suggest that nitric oxide may play a crucial role in HA-induced phagocytosis by RAW264.7 cells.
  4. A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species
    Yean CY, Yin LS, Lalitha P, Ravichandran M
    BMC Microbiol, 2007 Dec 11;7:112.
    PMID: 18070365
    BACKGROUND: Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene.

    RESULTS: Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases.

    CONCLUSION: The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.

  5. Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae
    Chan M, Cheong TG, Kurunathan S, Chandrika M, Ledon T, Fando R, et al.
    Microb Pathog, 2010 Nov;49(5):211-6.
    PMID: 20558271 DOI: 10.1016/j.micpath.2010.06.001
    Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10(6) and 10(8) CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.
  6. Construction and characterization of rtxA and rtxC mutants of auxotrophic O139 Vibrio cholerae
    Cheong TG, Chan M, Kurunathan S, Ali SA, ZiNing T, Zainuddin ZF, et al.
    Microb Pathog, 2010 Feb;48(2):85-90.
    PMID: 19900531 DOI: 10.1016/j.micpath.2009.11.001
    Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/rtxA mutated vaccine candidate against O139 V. cholerae.
  7. Enzyme-linked amperometric electrochemical genosensor assay for the detection of PCR amplicons on a streptavidin-treated screen-printed carbon electrode
    Yean CY, Kamarudin B, Ozkan DA, Yin LS, Lalitha P, Ismail A, et al.
    Anal Chem, 2008 Apr 15;80(8):2774-9.
    PMID: 18311943 DOI: 10.1021/ac702333x
    A general purpose enzyme-based amperometric electrochemical genosensor assay was developed wherein polymerase chain reaction (PCR) amplicons labeled with both biotin and fluorescein were detected with peroxidase-conjugated antifluorescein antibody on a screen-printed carbon electrode (SPCE). As a proof of principle, the response selectivity of the genosensor was evaluated using PCR amplicons derived from lolB gene of Vibrio cholerae. Factors affecting immobilization, hybridization, and nonspecific binding were optimized to maximize sensitivity and reduce assay time. On the basis of the background amperometry signals obtained from nonspecific organisms and positive signals obtained from known V. cholerae, a threshold point of 4.20 microA signal was determined as positive. Under the optimum conditions, the limit of detection (LOD) of the assay was 10 CFU/mL of V. cholerae. The overall precision of this assay was good, with the coefficient of variation (CV) being 3.7% using SPCE and intermittent pulse amperometry (IPA) as an electrochemical technique. The assay is sensitive, safe, and cost-effective when compared to conventional agarose gel electrophoresis, real-time PCR, and other enzyme-linked assays for the detection of PCR amplicons. Furthermore, the use of a hand-held portable reader makes it suitable for use in the field.
  8. Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples
    Foo PC, Chan YY, See Too WC, Tan ZN, Wong WK, Lalitha P, et al.
    J Med Microbiol, 2012 Sep;61(Pt 9):1219-1225.
    PMID: 22556327 DOI: 10.1099/jmm.0.044552-0
    Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.
  9. Development of a dry reagent-based triplex PCR for the detection of toxigenic and non-toxigenic Vibrio cholerae
    Chua AL, Elina HT, Lim BH, Yean CY, Ravichandran M, Lalitha P
    J Med Microbiol, 2011 Apr;60(Pt 4):481-485.
    PMID: 21183596 DOI: 10.1099/jmm.0.027433-0
    Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24 °C. The limit of detection of the thermostabilized triplex PCR assay was 2×10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free.
  10. Detection of Cryptosporidium parvum in HIV-infected patients in Malaysia using a molecular approach
    Zaidah AR, Chan YY, Asma HS, Abdullah S, Nurhaslindawati AR, Salleh M, et al.
    Southeast Asian J Trop Med Public Health, 2008 May;39(3):511-6.
    PMID: 18564692
    This cross-sectional study determined the prevalence of cryptosporidiosis in HIV-infected patients using polymerase chain reaction (PCR). Stool specimens were collected from HIV infected patients who were admitted to Hospital Raja Perempuan Zainab II, Kota Bharu, Malaysia, for various indications from December 2004 to December 2005. A modified acid-fast stain was performed on the direct stool smears, then the stool specimens were further tested using nested PCR targeting the 18S rRNA gene of Cryptosporidium parvum, with a built-in internal control (IC). Out of 59 samples, 11 were positives. Nested PCR identified a total of nine samples (16%) compared to microscopy, which identified only three samples. All PCR negative results showed IC amplicons, suggesting that these samples were true negatives and were not due to inhibition of PCR. This study highlights the importance of molecular diagnosis in determining the true prevalence and epidemiology of C. parvum.
  11. Comparison of histopathological features of Vibrio cholerae O1 El Tor and O139 Bengal infections in rabbit intestinal mucosa
    Amin A, Ali A, Kurunathan S, Cheong TG, Al-Jashamy KA, Jaafar H, et al.
    Histol Histopathol, 2009 05;24(5):559-65.
    PMID: 19283664 DOI: 10.14670/HH-24.559
    Vibrio cholerae is the causative agent of the infectious disease, cholera. The bacteria adhere to the mucosal membrane and release cholera toxin, leading to watery diarrhea. There are >100 serovars of V. cholerae, but the O1 and O139 serovars are the main causative agents of cholera. The present study aimed to compare the severity of intestinal mucosal infection caused by O1 El Tor and O139 V. cholerae in a rabbit ileal loop model. The results showed that although the fluid accumulation was similar in the loops inoculated with O1 and O139 V. cholerae, the presence of blood was detected only in the loops inoculated with the O139 serovar. Serosal hemorrhage was confirmed by histopathological examination and the loops inoculated with O139 showed massive destruction of villi and loss of intestinal glands. The submucosa and muscularis mucosa of the ileum showed the presence of edema with congested blood vessels, while severe hemorrhage was seen in the muscularis propria layer. The loops inoculated with O1 El Tor showed only minimal damage, with intact intestinal villi and glands. Diffuse colonies of the O139 serovar were seen to have infiltrated deep into the submucosal layer of the intestine. Although the infection caused by the O1 serovar was focal and invasive, it was more superficial than that due to O139, and involved only the villi. These observations were confirmed by immunostaining with O1 and O139 V. cholerae-specific monoclonal antibodies. The peroxidase reaction demonstrated involvement of tissues down to the submucosal layer in O139 V. cholerae infection, while in O1 El Tor infection, the reaction was confined mainly to the villi, and was greatly reduced in the submucosal region. This is the first reported study to clearly demonstrate the histopathological differences between infections caused by the O139 Bengal and O1 El Tor pathogenic serovars of V. cholerae.
  12. Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholerae
    Lalitha P, Siti Suraiya MN, Lim KL, Lee SY, Nur Haslindawaty AR, Chan YY, et al.
    J Microbiol Methods, 2008 Sep;75(1):142-4.
    PMID: 18579241 DOI: 10.1016/j.mimet.2008.05.001
    A PCR assay has been developed based on a lolB (hemM) gene, which was found to be highly conserved among the Vibrio cholerae species but non-conserved among the other enteric bacteria. The lolB PCR detected all O1, O139 and non-O1/non-O139 serogroup and biotypes of V. cholerae. The analytical specificity of this assay was 100% while the analytical sensitivity was 10 pg/microL and 10(3) CFU/mL at DNA and bacterial level respectively. The diagnostic sensitivity and specificity was 98.5% and 100% respectively.
  13. Construction and evaluation of a O139 Vibrio cholerae vaccine candidate based on a hemA gene mutation
    Ravichandran M, Ali SA, Rashid NH, Kurunathan S, Yean CY, Ting LC, et al.
    Vaccine, 2006 May 1;24(18):3750-61.
    PMID: 16102875
    In this paper, we describe the development of VCUSM2, a live metabolic auxotroph of Vibrio cholerae O139. Auxotrophy was achieved by mutating a house keeping gene, hemA, that encodes for glutamyl-tRNA reductase, an important enzyme in the C5 pathway for delta-aminolevulenic acid (ALA) biosynthesis, which renders this strain dependent on exogenous ALA for survival. Experiments using the infant mouse and adult rabbit models show that VCUSM2 is a good colonizer of the small intestine and elicits greater than a four-fold rise in vibriocidal antibodies in vaccinated rabbits. Rabbits vaccinated with VCUSM2 were fully protected against subsequent challenge with 1 x 10(11) CFU of the virulent wild type (WT) strain. Experiments using ligated ileal loops of rabbits show that VCUSM2 is 2.5-fold less toxic at the dose of 1 x 10(6) CFU compared to the WT strain. Shedding of VCUSM2 in rabbits were found to occur for no longer than 4 days and its maximum survival rate in environmental waters is 8 days compared to the greater than 20 days for the WT strain. VCUSM2 is thus a potential vaccine candidate against infection by V. cholerae O139.
  14. Fulltext Identification of Entamoeba histolytica trophozoites in fresh stool sample: comparison of three staining techniques and study on the viability period of the trophozoites
    Tan ZN, Wong WK, Nik Zairi Z, Abdullah B, Rahmah N, Zeehaida M, et al.
    Trop Biomed, 2010 Apr;27(1):79-88.
    PMID: 20562817 MyJurnal
    Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.
  15. Dry-reagent gold nanoparticle-based lateral flow biosensor for the simultaneous detection of Vibrio cholerae serogroups O1 and O139
    Yu CY, Ang GY, Chua AL, Tan EH, Lee SY, Falero-Diaz G, et al.
    J Microbiol Methods, 2011 Sep;86(3):277-82.
    PMID: 21571011 DOI: 10.1016/j.mimet.2011.04.020
    Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.
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