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Fulltext Virulence gene profiles and antimicrobial susceptibility of Salmonella Brancaster from chicken

Khoo E, Roslee R, Zakaria Z, Ahmad NI

J Vet Sci, 2023 Nov;24(6):e82.
PMID: 38031519 DOI: 10.4142/jvs.23053

BACKGROUND: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years.
OBJECTIVE: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia.
METHODS: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s).
RESULTS: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [blaTEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3″)-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected.
CONCLUSION: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/veterinary
Fulltext Utilization of Small RNA Genes to Distinguish Vibrio cholerae Biotypes via Multiplex Polymerase Chain Reaction

Ahmed SA, Raabe CA, Cheah HL, Hoe CH, Rozhdestvensky TS, Tang TH

Am J Trop Med Hyg, 2019 Jun;100(6):1328-1334.
PMID: 30963989 DOI: 10.4269/ajtmh.18-0525

The diarrheal disease "cholera" is caused by Vibrio cholerae, and is primarily confined to endemic regions, mostly in Africa and Asia. It is punctuated by outbreaks and creates severe challenges to public health. The disease-causing strains are most-often members of serogroups O1 and O139. PCR-based methods allow rapid diagnosis of these pathogens, including the identification of their biotypes. However, this necessitates the selection of specific target sequences to differentiate even the closely related biotypes of V. cholerae. Oligonucleotides for selective amplification of small RNA (sRNA) genes that are specific to these V. cholerae subtypes were designed. The resulting multiplex PCR assay was validated using V. cholerae cultures (i.e., 19 V. cholerae and 22 non-V. cholerae isolates) and spiked stool samples. The validation using V. cholerae cultures and spiked stool suspensions revealed detection limits of 10-100 pg DNA per reaction and 1.5 cells/mL suspension, respectively. The multiplex PCR assay that targets sRNA genes for amplification enables the sensitive and specific detection, as well as the differentiation of V. cholerae-O1 classical, O1 El Tor, and O139 biotypes. Most importantly, the assay enables fast and cheaper diagnosis compared with classic culture-based methods.

Matched MeSH terms: Multiplex Polymerase Chain Reaction*
Fulltext Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease

Thanh TT, Anh NT, Tham NT, Van HM, Sabanathan S, Qui PT, et al.

Virol J, 2015 Jun 09;12:85.
PMID: 26050791 DOI: 10.1186/s12985-015-0316-2

BACKGROUND: Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response.
METHODS: We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients.
RESULTS: The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed.
CONCLUSION: We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*; Multiplex Polymerase Chain Reaction/standards
Fulltext Hexaplex PCR detection system for identification of five human Plasmodium species with an internal control

Chew CH, Lim YA, Lee PC, Mahmud R, Chua KH

J Clin Microbiol, 2012 Dec;50(12):4012-9.
PMID: 23035191 DOI: 10.1128/JCM.06454-11

Malaria remains one of the major killers of humankind and persists to threaten the lives of more than one-third of the world's population. Given that human malaria can now be caused by five species of Plasmodium, i.e., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and the recently included Plasmodium knowlesi, there is a critical need not only to augment global health efforts in malaria control but also, more importantly, to develop a rapid, accurate, species-sensitive/species-specific, and economically effective diagnostic method for malaria caused by these five species. Therefore, in the present study, a straightforward single-step hexaplex PCR system targeting five human Plasmodium 18S small-subunit rRNAs (ssu rRNAs) was designed, and the system successfully detected all five human malaria parasites. In addition, this system enables the differentiation of single infection as well as mixed infections up to the two-species level. This assay was validated with 50 randomly blinded test and 184 clinical samples suspected to indicate malaria. This hexaplex PCR system is not only an ideal alternative for routine malaria diagnosis in laboratories with conventional PCR machines but also adds value to diagnoses when there is a lack of an experienced microscopist or/and when the parasite morphology is confusing. Indeed, this system will definitely enhance the accuracy and accelerate the speed in the diagnosis of malaria, as well as improve the efficacy of malaria treatment and control, in addition to providing reliable data from epidemiological surveillance studies.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*; Multiplex Polymerase Chain Reaction/standards
Fulltext Comparison of three molecular methods for the detection and speciation of five human Plasmodium species

Lau YL, Lai MY, Anthony CN, Chang PY, Palaeya V, Fong MY, et al.

Am J Trop Med Hyg, 2015 Jan;92(1):28-33.
PMID: 25385862 DOI: 10.4269/ajtmh.14-0309

In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [MSP]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi. The MSP-multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax. The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP-multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar's test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar's test: P = 0.0044) or real-time multiplex PCR with MSP-multiplex PCR (McNemar's test: P = 0.0012).

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext Combined PCR and MAT improves the early diagnosis of the biphasic illness leptospirosis

Philip N, Affendy NB, Masri SN, Yuhana MY, Than LTL, Sekawi Z, et al.

PLoS One, 2020;15(9):e0239069.
PMID: 32915919 DOI: 10.1371/journal.pone.0239069

The diagnosis of leptospirosis remains a challenge due to its non-specific symptoms and the biphasic nature of the illness. A comprehensive diagnosis that includes both molecular (polymerase chain reaction (PCR)) and serology is vital for early detection of leptospirosis and to avoid misdiagnosis. However, not all samples could be subjected to both tests (serology and molecular) due to budget limitation, infrastructure, and technical expertise at least in resource-limited countries. We evaluated the usefulness of testing the clinically suspected leptospirosis cases with both techniques on all samples collected from the patients on the day of admission. Among the 165 patient's blood/serum samples tested (from three hospitals in Central Malaysia), 43 (26%) showed positivity by microscopic agglutination test (MAT), 63 (38%) by PCR, while 14 (8%) were positive by both MAT and PCR. For PCR, we tested two molecular targets (lipL32 by qPCR and 16S rDNA or rrs by nested PCR) and detected lipL32 in 47 (29%) and rrs gene in 63 (38%) patients. The use of more than one target gene for PCR increased the detection rates. Hence, a highly sensitive multiplex PCR targeting more than one diagnostic marker is recommended for the early detection of Leptospira in suspected patients. When the frequencies for positivity detected either by MAT or PCR combined, leptospirosis was diagnosed in a total of 92 (56%) patients, a higher frequency compared to when samples were only tested by a single method (MAT or PCR). The results from this study suggest the inclusion of both serology and molecular methods for every first sample irrespective of the days post-onset of symptoms (DPO) collected from patients for early diagnosis of leptospirosis.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Burundi

Afolabi O, Milan B, Amoussa R, Koebnik R, Poulin L, Szurek B, et al.

Plant Dis, 2014 Oct;98(10):1426.
PMID: 30703943 DOI: 10.1094/PDIS-05-14-0504-PDN

On May 9, 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the panicle emergence stage at Musenyi, Gihanga, and Rugombo fields in Burundi. Affected leaves showed water-soaked translucent lesions and yellow-brown to black streaks, sometimes with visible exudates on leaf surfaces. Symptomatic leaves were ground in sterile water and the suspensions obtained were subjected to a multiplex PCR assay diagnostic for X. oryzae pathovars (3). Three DNA fragments (331, 691, and 945 bp) corresponding to X. oryzae pv. oryzicola were observed after agarose gel electrophoresis. Single bacterial colonies were then isolated from surface-sterilized, infected leaves after grinding in sterile water and plating of 10-fold dilutions of the cell suspension on semi-selective PSA medium (4). After incubation at 28°C for 5 days, each of four independent cultures yielded single yellow, mucoid Xanthomonas-like colonies (named Bur_1, Bur_2, Bur_6, and Bur_7) that resembled the positive control strain MAI10 (1). These strains originated from Musenyi (Bur_1), Gihanga (Bur_2), and Rugumbo (Bur_6 and Bur_7). Multiplex PCR assays on the four putative X. oryzae pv. oryzicola strains yielded the three diagnostic DNA fragments mentioned above. All strains were further analyzed by sequence analysis of portions of the gyrB gene using the universal primers gyrB1-F and gyrB1-R for PCR amplification (5). The 762-bp DNA fragment was identical to gyrB sequences from the Asian X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), LMG 797 and NCPPB 2921 (both Malaysia), and from the African strain MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of Bur_1 was submitted to GenBank (Accession No. KJ801400). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cvs. Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, IRBB 7, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. Bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water (1 × 108 CFU/ml). Plants were inoculated with bacterial suspensions either by spraying or by leaf infiltration (1). For spray inoculation, four plants per accession and strain were used while three leaves per plant and four plants per accession and strain were inoculated by tissue infiltration. After 15 days of incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), the spray-inoculated plants showed water-soaked lesions with yellow exudates identical to those seen in the field. For syringe-infiltrated leaves, the same symptoms were observed at the infiltrated leaf area. Re-isolation of bacteria from symptomatic leaves yielded colonies with the typical Xanthomonas morphology that were confirmed by multiplex PCR to be X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. Bur_1 has been deposited in the Collection Française de Bactéries Phytopathogènes as strain CFBP 8170 ( http://www.angers-nantes.inra.fr/cfbp/ ). To our knowledge, this is the first report of X. oryzae pv. oryzicola causing bacterial leaf streak on rice in Burundi. Further surveys will help to assess its importance in the country. References: (1) C. Gonzalez et al., Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext Quintuplex PCR to detect antibiotic resistance genes in Streptococcus pneumoniae

Navindra Kumari Palanisamy, Parasakthi Navaratnam, Shamala Devi Sekaran

Journal of Clinical and Health Sciences, 2016;1:22-28.

Introduction: Streptococcus pneumoniae is an important bacterial pathogen, causing respiratory infection. Penicillin resistance in S. pneumoniae is associated with alterations in the penicillin binding proteins, while resistance to macrolides is conferred either by the modification of the ribosomal target site or efflux mechanism. This study aimed to characterize S. pneumoniae and its antibiotic resistance genes using 2 sets of multiplex PCRs. Methods: A quintuplex and triplex PCR was used to characterize the pbp1A, ermB, gyrA, ply, and the mefE genes. Fifty-eight penicillin sensitive strains (PSSP), 36 penicillin intermediate strains (PISP) and 26 penicillin resistance strains (PRSP) were used. Results: Alteration in pbp1A was only observed in PISP and PRSP strains, while PCR amplification of the ermB or mefE was observed only in strains with reduced susceptibility to erythromycin. The assay was found to be sensitive as simulated blood cultures showed the lowest level of detection to be 10cfu. Conclusions: As predicted, the assay was able to differentiate penicillin susceptible from the non-susceptible strains based on the detection of the pbp1A gene, which correlated with the MIC value of the strains.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae

Khazani NA, Noor NZ, Yean Yean C, Hasan H, Suraiya S, Mohamad S

J Trop Med, 2017;2017:7210849.
PMID: 28386286 DOI: 10.1155/2017/7210849

Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q(10) method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext Developing new primers for molecular identification of plasmodium spp. recorded in Sabah

Chua, T.H., Stanis, C.S., Song, B.K., Lau, Y.L., Jelip, P., Lau, T.Y.

Borneo Journal of Medical Sciences, 2015;9(1):3-10.
MyJurnal

Malaria is a major public health problem in tropical and subtropical areas, caused by five
species of Plasmodium (P. falciparum, P. vivax, P. malariae, P. ovale andP. knowlesi) and is the leading cause of morbidity and mortality worldwide. We have developed molecular markers for three genes viz, Cytb, dhfr and Msp-1 gene and designed a protocol for rapid molecular diagnostics of the four malaria parasites prevalent in Southeast Asia. The new primers were used on the blood
samples containing Plasmodium parasites by conventional PCR. The result was compared with
the nested PCR of Singh et al. (2004) and the microscopy method. The result shows that the new
set of primers had successfully amplified all four human malaria parasite species. These primers
were 100% sensitive and more specific than microscopy and PCR identification using these
primers was faster than the nested PCR. These alternative primers should provide powerful and
rapid molecular diagnostic method for detecting Plasmodium species as well as providing reliable
data for epidemiology study. These primers have the potential to be combined and used in
multiplex PCR.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext HLA-B*15:02 screening in epileptic patients using a high resolution melting-real time PCR (HRM-QPCR) method

Zam Zureena Mohd Rani, Nor Azian Abdul Murad, Saberi Saimun, Sri Noraima Othman, Rahman Jamal, Sue-Mian Then, et al.

Neurology Asia, 2018;23(2):137-144.
MyJurnal

Background: The HLA-B*15:02 polymorphism in epileptic patients is known to be associated with carbamazepine-induced Stevens-Johnson syndrome (SJS). The prevalence of HLA-B*15:02 polymorphism seemed to be ethnic-specific with a higher frequency of HLA-B*15:02 in Asian compared to the Europeans. This study was performed to determine the frequency of the HLA-B*15:02 polymorphism in epileptic patients at the Chancellor Tuanku Muhriz Hospital-UKM Medical Centre (HCTM-UKMMC) using high resolution melting-real time PCR (HRM-QPCR) method.
Methods: We performed a fast and effective in-house high resolution melting-real time polymerase chain reaction method and compared it with the conventional multiplex-PCR method. The specificity and sensitivity of each test were also determined using DNA from saliva.
Results: Using the conventional multiplexPCR approach for screening, 25 out of 64 (39.1%) epileptic patients were positive for HLA-B*15:02. However, using the HRM-QPCR technique, 24/64 (37.5%) of the patients were positive. The one patient who tested positive by the multiplex-PCR but negative using the HRM-QPCR turned out to be negative by DNA sequencing. The HRM-QPCR and DNA sequencing showed 100% sensitivity and specificity. The multiplex-PCR showed 100% sensitivity and 98.4% specificity compared to both HRM-QPCR and DNA sequencing. The HRM-QPCR is also more cost-effective (multiplex-PCR ($25.15 USD/test).
Conclusion: Our result suggested that multiplex PCR, HRM-QPCR and Sanger sequencing can be used for detection of HLA-B*15:02. However, a qualitative method such as multiplex PCR should be confirmed with other quantitative methods such as HRM-QPCR and Sanger sequencing.
Keywords: Epilepsy, carbamazepine-induced Steven Johnson syndrome, multiplex-polymerase chain reaction, high resolution melting-real time polymerase chain reaction (HRM-QPCR), DNA sequencing

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext Detection of Vibrio cholerae in street food (satar and otak-otak) by Loop-Mediated Isothermal Amplification (LAMP), multiplex Polymerase Chain Reaction (mPCR) and plating methods

Tang, J-Y-H., Farhana Sakinah, M.R., Nakaguchi, Y., Nishibuchi, M., Chai, L-C., New, C.Y., et al.

Food Research, 2018;2(5):447-452.
MyJurnal

This goal of this study was to investigate the presence of Vibrio cholerae in street food,
namely satar and otak-otak, using Loop-Mediated Isothermal Amplification (LAMP),
multiplex Polymerase Chain Reaction (mPCR) and conventional plating on Thiosulphate
Citrate Bile-Salt Sucrose (TCBS) agar methods. A total of 78 satar and 35 otak-otak were
purchased from different districts of Terengganu (Besut, Setiu, Kuala Terengganu and
Kemaman). V. cholerae was found in satar with LAMP (10.3%), mPCR (10.3%) and
plating (0%). No V. cholerae was found in otak-otak using the three methods. This might
be due to V. cholerae able to survive in satar after grilling due to its thickness which may
contribute to undercooking. This study concluded that low presence of V. cholerae in satar
and otak-otak can be detected by molecular methods but not the conventional plating
method. LAMP assay is a useful tool for rapid detection of pathogens in food due to its
simplicity, highly sensitive and visual interpretation capability. Though the prevalence of
V. cholerae was low in the samples, proper handling of this food will help in reducing the
risk of acquiring infection from V. cholerae in contaminated samples.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Dual panel multiplex PCR assay for rapid detection of medically important fungi and resistant species of candida and aspergillus

Jacinta Santhanam, Mohd Hanif Jainlabdin, Ang LC, Tzar Mohd Nizam

Sains Malaysiana, 2018;47:489-498.

Invasive fungal infections (IFIs) have risen dramatically in recent years among high risk immunocompromised patients.
Rapid detection of fungal pathogens is crucial to timely and accurate antifungal therapy. Two multiplex polymerase
chain reaction (PCR) assays were developed to detect major fungal species that cause invasive infections and identify
resistant species. Genus specific primers for Candida, Aspergillus, Fusarium and species specific primers for Candida
glabrata, Candida krusei and Aspergillus terreus which are known to be clinically resistant species, were designed from
the internal transcribed spacer (ITS) regions of ribosomal ribonucleic acid (rRNA) gene complex. Both assays were
performed simultaneously to promote rapid detection of fungal isolates based on distinct amplicon sizes. Inclusion of the
universal fungal primers ITS 1 and ITS 4 in the genus specific assay produced a second amplicon for each isolate which
served to confirm the detection of a fungal target. The limit of detection for the genus specific assay was 1 nanogram
(ng) deoxyribonucleic acid (DNA) for Aspergillus fumigatus and Candida albicans, 0.1 ng DNA for Fusarium solani, while
the species-specific assay detected 0.1 ng DNA of A. terreus and 10 picogram (pg) DNA of C. krusei and C. glabrata. The
multiplex PCR assays, apart from universal detection of any fungal target, are able to detect clinically important fungi
and differentiate resistant species rapidly and accurately, which can contribute to timely implementation of effective
antifungal regime.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Evaluation of new multiplex PCR primers for the identification ofPlasmodium species found in Sabah, Malaysia

Stanis CS, Song BK, Chua TH, Lau YL, Jelip J

Turk J Med Sci, 2016 Jan 05;46(1):207-18.
PMID: 27511356 DOI: 10.3906/sag-1411-114

BACKGROUND/AIM: Malaria is a major public health problem, especially in the Southeast Asia region, caused by 5 species of Plasmodium (P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi). The aim of this study was to compare parasite species identification methods using the new multiplex polymerase chain reaction (PCR) against nested PCR and microscopy.
MATERIALS AND METHODS: Blood samples on filter papers were subject to conventional PCR methods using primers designed by us in multiplex PCR and previously designed primers of nested PCR. Both sets of results were compared with microscopic identification.
RESULTS: Of the 129 samples identified as malaria-positive by microscopy, 15 samples were positive for P. falciparum, 14 for P. vivax, 6 for P. knowlesi, 72 for P. malariae, and 2 for mixed infection of P. falciparum/P. malariae. Both multiplex and nested PCR identified 12 P. falciparum single infections. For P. vivax, 9 were identified by multiplex and 12 by nested PCR. For 72 P. malariae cases, multiplex PCR identified 58 as P. knowlesi and 10 as P. malariae compared to nested PCR, which identified 59 as P. knowlesi and 7 as P. malariae.
CONCLUSION: Multiplex PCR could be used as alternative molecular diagnosis for the identification of all Plasmodium species as it requires a shorter time to screen a large number of samples.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext Quintuplex PCR to detect antibiotic resistance genes in Streptococcus pneumoniae

Navindra Kumari Palanisamy, Parasakthi Navaratnam, Shamala Devi Sekaran

Journal of Clinical and Health Sciences, 2016;1(2):22-28.
MyJurnal

Introduction: Streptococcus pneumoniae is an important bacterial pathogen, causing respiratory infection. Penicillin resistance in S. pneumoniae is associated with alterations in the penicillin binding proteins, while resistance to macrolides is conferred either by the modification of the ribosomal target site or efflux mechanism. This study aimed to characterize S. pneumoniae and its antibiotic resistance genes using 2 sets of multiplex PCRs. Methods: A quintuplex and triplex PCR was used to characterize the pbp1A, ermB, gyrA, ply, and the mefE genes. Fifty-eight penicillin sensitive strains (PSSP), 36 penicillin intermediate strains (PISP) and 26 penicillin resistance strains (PRSP) were used. Results: Alteration in pbp1A was only observed in PISP and PRSP strains, while PCR amplification of the ermB or mefE was observed only in strains with reduced susceptibility to erythromycin. The assay was found to be sensitive as simulated blood cultures showed the lowest level of detection to be 10cfu. Conclusions: As predicted, the assay was able to differentiate penicillin susceptible from the non-susceptible strains based on the detection of the pbp1A gene, which correlated with the MIC value of the strains.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext Malaria diagnosis using a combined system of a simple and fast extraction method with a lyophilised Dual-LAMP assay in a non-endemic setting

Martín Ramírez A, Barón Argos L, Lanza Suárez M, Carmona Rubio C, Pérez-Ayala A, Hisam SR, et al.

Pathog Glob Health, 2024 Feb;118(1):80-90.
PMID: 37415348 DOI: 10.1080/20477724.2023.2232595

Malaria is a parasitic disease distributed in tropical areas but with a high number of imported cases in non-endemic countries. The most specific and sensitive malaria diagnostic methods are PCR and LAMP. However, both require specific equipment, extraction procedures and a cold chain. This study aims to solve some limitations of LAMP method with the optimization and validation of six LAMP assays, genus and species-specific, using an easy and fast extraction method, the incorporation of a reaction control assay, two ways (Dual) of result reading and reagent lyophilization. The Dual-LAMP assays were validated against the Nested-Multiplex Malaria PCR. A conventional column and saline extraction methods, and the use of lyophilized reaction tubes were also assessed. A new reaction control Dual-LAMP-RC assay was designed. Dual-LAMP-Pspp assay showed no cross-reactivity with other parasites, repeatability and reproducibility of 100%, a significant correlation between parasite concentration and time to amplification and a LoD of 1.22 parasites/µl and 5.82 parasites/µl using column and saline extraction methods, respectively. Sensitivity and specificity of the six Dual-LAMP assays reach values of 100% or close to this, being lower for the Dual-LAMP-Pm. The Dual-LAMP-RC assay worked as expected. Lyophilized Dual-LAMP results were concordant with the reference method. Dual-LAMP malaria assays with the addition of a new reaction control LAMP assay and the use of a fast and easy saline extraction method, provided low limit of detection, no cross-reactivity, and good sensitivity and specificity. Furthermore, the reagent lyophilization and the dual result reading allow their use in most settings.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Genotype-Phenotype Study of β-Thalassemia Patients in Sabah

Suali L, Mohammad Salih FA, Ibrahim MY, Jeffree MSB, Thomas FM, Siew Moy F, et al.

Hemoglobin, 2022 Nov;46(6):317-324.
PMID: 36815306 DOI: 10.1080/03630269.2023.2169154

β-thalassemia is a serious public health problem in Sabah due to its high prevalence. This study aimed to investigate the effects of different types of β-globin gene mutations, coinheritance with α-globin gene mutations, XmnI-Gγ, and rs368698783 polymorphisms on the β-thalassemia phenotypes in Sabahan patients. A total of 111 patients were included in this study. The sociodemographic profile of the patients was collected using a semi-structured questionnaire, while clinical data were obtained from their medical records. Gap-PCR, ARMS-PCR, RFLP-PCR, and multiplex PCR were performed to detect β- and α-globin gene mutations, as well as XmnI-Gγ and rs368698783 polymorphisms. Our data show that the high prevalence of β-thalassemia in Sabah is not due to consanguineous marriages (5.4%). A total of six different β-globin gene mutations were detected, with Filipino β°-deletion being the most dominant (87.4%). There were 77.5% homozygous β-thalassemia patients, 16.2% compound heterozygous β-thalassemia patients, and 6.3% β-thalassemia/Hb E patients. Further evaluation on compound heterozygous β-thalassemia and β-thalassemia/Hb E patients found no concomitant α-globin gene mutations and the rs368698783 polymorphism. Furthermore, the XmnI-Gγ (-/+) genotype did not demonstrate a strong impact on the disease phenotype, as only two of five patients in the compound heterozygous β-thalassemia group and two of three patients in the β-thalassemia/Hb E group had a moderate phenotype. Our findings indicate that the severity of the β-thalassemia phenotypes is closely related to the type of β-globin gene mutations but not to the XmnI-Gγ and rs368698783 polymorphisms.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Fulltext Detection of Respiratory Viruses from ARTI Patients by xTAG RVP Fast v2 Assay and Conventional Methods

Kuan CS, Yew SM, Hooi PS, Lee LM, Ng KP

Malays J Med Sci, 2017 Oct;24(5):33-43.
PMID: 29386970 MyJurnal DOI: 10.21315/mjms2017.24.5.4

Introduction: Acute respiratory tract infections (ARTIs) are a major cause of morbidity and mortality in paediatric patients. Therefore, early detection of the viral aetiologies of ARTIs is essential for patient management and infection control. In this study, we evaluated the performance of a new multiplex polymerase chain reaction (PCR) assay (xTAG Respiratory Viral Panel [RVP] Fast v2) in the detection of respiratory viruses by comparing it with that of viral culture and direct immunofluorescence (IF) staining.
Methods: Nasopharyngeal swab and aspirate samples were collected prospectively from 199 patients who presented with ARTIs at the University Malaya Medical Centre (UMMC) in Kuala Lumpur, Malaysia during a 10-month period. The PCR assay was conducted in parallel with conventional culture and direct IF staining methods.
Results: The positive rate of the xTAG RVP Fast v2 assay (78.4%) in detecting respiratory viruses was higher than that of the viral isolation (7.5%) and direct IF (23.1%) methods. Using the xTAG RVP Fast v2 assay, human enterovirus/human rhinovirus (HEV/HRV) was the most frequently detected (46.2%). The xTAG RVP Fast v2 assay revealed mixed infection caused by two or three respiratory viruses in 40 specimens, and these were undetected by the viral isolation and direct IF methods.
Conclusion: The xTAG RVP Fast v2 assay was superior to conventional methods in the identification of common respiratory viruses, with higher sensitivity and shorter turnaround times for laboratory results.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Rapid detection of Mycobacterium tuberculosis in clinical samples by multiplex polymerase chain reaction (mPCR)

Tang TH, Ahmed SA, Musa M, Zainuddin ZF

World J Microbiol Biotechnol, 2013 Dec;29(12):2389-95.
PMID: 23807412 DOI: 10.1007/s11274-013-1407-0

Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.

Matched MeSH terms: Multiplex Polymerase Chain Reaction*
Prevalence and antibiotic resistance of Salmonella Enteritidis and Salmonella Typhimurium in raw chicken meat at retail markets in Malaysia

Thung TY, Mahyudin NA, Basri DF, Wan Mohamed Radzi CW, Nakaguchi Y, Nishibuchi M, et al.

Poult Sci, 2016 Aug 01;95(8):1888-93.
PMID: 27118863 DOI: 10.3382/ps/pew144

Salmonellosis is one of the major food-borne diseases in many countries. This study was carried out to determine the occurrence of Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in raw chicken meat from wet markets and hypermarkets in Selangor, as well as to determine the antibiotic susceptibility profile of S. Enteritidis and S. Typhimurium. The most probable number (MPN) in combination with multiplex polymerase chain reaction (mPCR) method was used to quantify the Salmonella spp., S. Enteritidis, and S. Typhimurium in the samples. The occurrence of Salmonella spp., S. Enteritidis, and S. Typhimurium in 120 chicken meat samples were 20.80%, 6.70%, and 2.50%, respectively with estimated quantity varying from <3 to 15 MPN/g. The antibiogram testing revealed differential multi-drug resistance among S. Enteritidis and S. Typhimurium isolates. All the isolates were resistance to erythromycin, penicillin, and vancomycin whereas sensitivity was recorded for Amoxicillin/Clavulanic acid, Gentamicin, Tetracycline, and Trimethoprim. Our findings demonstrated that the retail chicken meat could be a source of multiple antimicrobial-resistance Salmonella and may constitute a public health concern in Malaysia.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/veterinary

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