Molecular detection of human Plasmodium species in Sabah using PlasmoNextm multiplex PCR and hydrolysis probes real-time PCR

BACKGROUND: Malaria is a vector borne-parasitic disease transmitted through the bite of the infective female Anopheles mosquitoes. Five Plasmodium species have been recognized by World Health Organization (WHO) as the causative agents of human malaria. Generally, microscopic examination is the gold standard for routine malaria diagnosis. However, molecular PCR assays in many cases have shown improvement on the sensitivity and specificity over microscopic or other immunochromatographic assays. METHODS: The present study attempts to screen 207 suspected malaria samples from patients seeking treatment in clinics around Sabah state, Malaysia, using two panels of multiplex PCRs, conventional PCR system (PlasmoNex™) and real-time PCR based on hydrolysis probe technology. Discordance results between two PCR assays were further confirmed by sequencing using 18S ssu rRNA species-specific primers. RESULTS: Of the 207 malaria samples, Plasmodium knowlesi (73.4% vs 72.0%) was the most prevalent species based on two PCR assays, followed by Plasmodium falciparum (15.9% vs 17.9%), and Plasmodium vivax (9.7% vs 7.7%), respectively. Neither Plasmodium malariae nor Plasmodium ovale was detected in this study. Nine discrepant species identification based on both the PCR assays were further confirmed through DNA sequencing. Species-specific real-time PCR only accurately diagnosed 198 of 207 (95.7%) malaria samples up to species level in contrast to PlasmoNex™ assay which had 100% sensitivity and specificity based on sequencing results. CONCLUSIONS: Multiplex PCR accelerate the speed in the diagnosis of malaria. The PlasmoNex™ PCR assay seems to be more accurate than real-time PCR in the speciation of all five human malaria parasites. The present study also showed a significant increase of the potential fatal P. knowlesi infection in Sabah state as revealed by molecular PCR assays

Increased detection of Plasmodium knowlesi in Sandakan division, Sabah as revealed by PlasmoNex

Background: Plasmodium knowlesi is a simian malaria parasite that is widespread in humans in Malaysian Borneo. However, little is known about the incidence and distribution of this parasite in the Sandakan division, Malaysian Borneo. Therefore, the aim of the present epidemiological study was to investigate the incidence and distribution of P. knowlesi as well as other Plasmodium species in this division based on a most recent developed hexaplex PCR system (PlasmoNex™). Methods: A total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. All patients who participated in the study were microscopically malaria positive before recruitment. Complete demographic details and haematological profiles were obtained from 85 patients (13 females and 72 males). Identification of Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene. Results: A total of 178 samples were positive for Plasmodium species by using PlasmoNex™. Plasmodium falciparum was identified in 68 samples (38.2%) followed by 64 cases (36.0%) of Plasmodium vivax, 42 (23.6%) cases of P. knowlesi, two (1.1%) cases of Plasmodium malariae and two (1.1%) mixed-species infections (i e, P. vivax/ P. falciparum). Thirty-five PlasmoNex™ positive P. knowlesi samples were misdiagnosed as P. malariae by microscopy. Plasmodium knowlesi was detected in all four districts of Sandakan division with the highest incidence in the Kinabatangan district. Thrombocytopaenia and anaemia showed to be the most frequent malaria-associated haematological complications in this study. Conclusions: The discovery of P. knowlesi in Sandakan division showed that prospective studies on the epidemiological risk factors and transmission dynamics of P. knowlesi in these areas are crucial in order to develop strategies for effective malaria control. The availability of advanced diagnostic tool PlasmoNex™ enhanced the accuracy and accelerated the speed in the diagnosis of malari

Development of a multiplex PCR detection system and identification of binding peptides for human plasmodium species / Chew Ching Hoong

Malaria is one of the most serious global health challenges. Approximately 3.3 billion people live in malaria-endemic areas and the disease threatens the lives of more than one-third of the world’s population. Malaria is caused by an eukaryotic protozoa Plasmodium and transmitted through the bite of female Anopheles mosquito. The known causative agents of human malaria include Plasmodium vivax, P. falciparum, P. malariae, P. ovale, and recently included P. knowlesi which is recognized as a zoonotic parasite. Malaria is a treatable disease, however the disease can become severe, leading to morbidity if untreated, especially for infections by two potential fatal species, i.e., P. falciparum and P. knowlesi. In the effort to improve the global health status, institution of control and surveillance of malaria, and subsequently enhancing the effectiveness of treatment management, it is critical to develop a rapid, accurate, species-specific/species-sensitive, and cost-efficient diagnostic tool for malaria detection. Therefore, a straightforward single-step multiplex polymerase chain reaction (PCR) targeting five human Plasmodium 18S small subunit ribosomal RNA (ssu rRNA) gene with an internal positive control was developed. This system is specific in detecting all five human malaria parasites with high sensitivity (i.e., 0.025 parasites/μl for P. vivax, 0.027 parasites/μl for P. ovale, 0.15 parasites/μl for P. falciparum, 0.25 parasites/μl for P. knowlesi, and 0.27 parasites/μl for P. malariae), and most significantly enables the simultaneous identification and differentiation of mixed infections at least up to the two-species level without any diagnostic constrains. In addition, the accuracy (sensitivity and specificity) of the detection system were also assured by random blind testing (n=50), clinical screening (n=246), and simulated mixed infections based on clinical samples (n=30) and clone DNA (n=60). All results were in agreement with the results from nested PCR which served as a molecular gold standard. Overall, this ABSTRACT III multiplex system will definitely enhance the accuracy and accelerate the speed in the malaria diagnosis, and improve the efficacy of malaria treatment and control. Plasmodium is an obligate intracellular parasite. Therefore, the understanding of how the parasites enter their host’s cell is of great interest and this offers an attractive target for the development of novel therapeutics. Apical membrane antigen 1 (AMA1) is the most prominent and well characterized malarial surface antigen that is essential for parasite-host cell invasion, i.e., sporozoite into hepatocyte in liver stage and merozoite into erythrocyte in asexual stage. AMA1 has long served as a potent antimalarial drug target and pivotal vaccine candidate. In the present study, recombinant AMA1 proteins of P. knowlesi (rPkAMA1) as well as P. vivax (rPvAMA1) were expressed using Escherichia coli and the binding peptides were identified using a random dodecapeptide phage display library. Phage display is a powerful and cost-effective tool that can be used for assessing the protein-protein interactions. After third rounds of biopanning, two and three phage-displayed binding peptides with affinity to rPkAMA1 and rPvAMA1 respectively were identified and validated through peptide binding assays

Diverse Profiles of Biofilm and Adhesion Genes in Staphylococcus Aureus Food Strains Isolated from Sushi and Sashimi

Abstract: Staphylococcus aureus is able to form multilayer biofilms embedded within a glycocalyx or slime layer. Biofilm formation poses food contamination risks and can subsequently increase the risk of food poisoning. Identification of food-related S. aureus strains will provide additional data on staphylococcal food poisoning involved in biofilm formation. A total of 52 S. aureus strains isolated from sushi and sashimi was investigated to study their ability for biofilm formation using crystal violet staining. The presence of accessory gene regulator (agr) groups and 15 adhesion genes was screened and their associations in biofilm formation were studied. All 52 S. aureus strains showed biofilm production on the tested hydrophobic surface with 44% (23/52) strains classified as strong, 33% (17/52) as moderate, and 23% (12/52) as weak biofilm producers. The frequency of agr-positive strains was 71% (agr group 1 = 21 strains; agr group 2 = 2 strains; agr group 3 = 12 strains; agr group 4 = 2 strains) whereas agr-negative strains were 29% (15/52). Twelve adhesion genes were detected and 98% of the S. aureus strains carried at least one adhesion gene. The ebps was significantly (p <.05) associated with strong biofilm producing strains. In addition, eno, clfA, icaAD, sasG, fnbB, cna, and sasC were significantly higher in the agr-positive group compared to the agr-negative group. The results of this study suggest that the presence of ebps, eno, clfA, icaAD, sasG, fnbB, cna, and sasC may play an important role in enhancing the stage of biofilm-related infections and warrants further investigation. Practical Application: This work contributes to the knowledge on the biofilm formation and the distribution of agr groups in S. aureus strains as well as microbial surface components in recognizing adherence matrix molecules of organisms isolated from ready-to-eat sushi and sashimi. The findings provide valuable information to further study the roles of specific genes in causing biofilm-related infections

Whole genome sequencing of methicillin-resistant Staphylococcus aureus clinical isolates from Terengganu, Malaysia, indicates the predominance of the EMRSA-15 (ST22-SCCmec IV) clone

Abstract Despite the importance of methicillin-resistant Staphylococcus aureus (MRSA) as a priority nosocomial pathogen, the genome sequences of Malaysian MRSA isolates are currently limited to a small pool of samples. Here, we present the genome sequence analyses of 88 clinical MRSA isolates obtained from the main tertiary hospital in Terengganu, Malaysia in 2016–2020, to obtain in-depth insights into their characteristics. The EMRSA-15 (ST22-SCCmec IV) clone of the clonal complex 22 (CC22) lineage was predominant with a total of 61 (69.3%) isolates. Earlier reports from other Malaysian hospitals indicated the predominance of the ST239 clone, but only two (2.3%) isolates were identified in this study. Two Indian-origin clones, the Bengal Bay clone ST772-SCCmec V (n = 2) and ST672 (n = 10) were also detected, with most of the ST672 isolates obtained in 2020 (n = 7). Two new STs were found, with one isolate each, and were designated ST7879 and ST7883. From the core genome phylogenetic tree, the HSNZ MRSA isolates could be grouped into seven clades. Antimicrobial phenotype-genotype concordance was high (> 95%), indicating the accuracy of WGS in predicting most resistances. Majority of the MRSA isolates were found to harbor more than 10 virulence genes, demonstrating their pathogenic nature

The Plasmidomic Landscape of Clinical Methicillin-Resistant <i>Staphylococcus aureus</i> Isolates from Malaysia

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority nosocomial pathogen with plasmids playing a crucial role in its genetic adaptability, particularly in the acquisition and spread of antimicrobial resistance. In this study, the genome sequences of 79 MSRA clinical isolates from Terengganu, Malaysia, (obtained between 2016 and 2020) along with an additional 15 Malaysian MRSA genomes from GenBank were analyzed for their plasmid content. The majority (90%, 85/94) of the Malaysian MRSA isolates harbored 1–4 plasmids each. In total, 189 plasmid sequences were identified ranging in size from 2.3 kb to ca. 58 kb, spanning all seven distinctive plasmid replication initiator (replicase) types. Resistance genes (either to antimicrobials, heavy metals, and/or biocides) were found in 74% (140/189) of these plasmids. Small plasmids (ermC gene that confers resistance to macrolides, lincosamides, and streptogramin B (MLSB) identified in 63 MRSA isolates. A low carriage of conjugative plasmids was observed (n = 2), but the majority (64.5%, 122/189) of the non-conjugative plasmids have mobilizable potential. The results obtained enabled us to gain a rare view of the plasmidomic landscape of Malaysian MRSA isolates and reinforces their importance in the evolution of this pathogen

Decomate II: un modelo unificado para una biblioteca digital distribuida

This paper describes the Decomate II distributed digital library system