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Isoenzyme profiles and phylogenetic analysis of Giardia duodenalis isolates from Iranian patients

Rayani M, Unyah NZ, Vafafar A, Hatam GR

Environ Sci Pollut Res Int, 2020 Nov;27(32):40652-40663.
PMID: 32671708 DOI: 10.1007/s11356-020-10062-1

The main objective of this study was to characterize the Giardia duodenalis isolates from Iranian patients in Fars Province, south of Iran by biochemical and molecular methods. Fifteen mass cultivated of G. duodenalis isolates in modified TYI-S-33 medium were analyzed using isoenzyme electrophoresis and PCR genotyping. Polyacrylamide gel electrophoresis (PAGE) of five different enzyme systems was used to characterize isolates: (i) glucose-6-phosphate dehydrogenase, (ii) glucose phosphate isomerase, (iii) malate dehydrogenase, (iv) malic enzyme, and (v) phosphoglucomutase. As well, a fragment of the SSU-rDNA (292 bp) gene was amplified by PCR using the primers RH11 and RH4. The sequencing of the PCR products and phylogenetic tree were performed. The isoenzyme electrophoretic profiles divided fifteen G. duodenalis isolates into four zymodemes. G6PD, GPI, MDH, ME, and PGM enzyme systems showed 1, 2, 2, 3, and 3 enzyme pattern, respectively. G6PD isoenzyme pattern had the most homogeneity, while isoenzyme patterns of ME and PGM had the most heterogeneity in our study. Genotyping results indicated that the zymodemes 1-4 were categorized in assemblage A based on the SSU-rDNA gene. Phylogenetic analysis showed that all four zymodemes were distributed within the cluster of assemblage A. Our results indicated that both isoenzyme and DNA analyses were useful to characterize the isolates of Giardia and distinguishing various zymodemes and assemblages. It could be suggested that the genetic diversity among isoenzymes profiles of G. duodenalis may explain the variable clinical manifestations, pathogenicity, host response, drug susceptibility, and treatment efficacy of human giardiasis.
Fulltext Detection of rodent-borne parasitic pathogens of wild rats in Serdang, Selangor, Malaysia: A potential threat to human health

Tijjani M, Majid RA, Abdullahi SA, Unyah NZ

Int J Parasitol Parasites Wildl, 2020 Apr;11:174-182.
PMID: 32099788 DOI: 10.1016/j.ijppaw.2020.01.008

Rodent species, such as Rattus rattus diardii and Rattus norvegicus are invasive species of wild rats that serve as potential reservoirs of important human's pathogens. Parasitic zoonosis accounts for over 60% of all human infectious diseases worldwide. This situation arises from the recent changes in the global climate and ecosystem composition, which led to the spread of rodents and rodent-borne pathogens globally. The aim of this study was to determine the occurrence of rodent's parasites and their zoonotic potentials in some selected areas in UPM. Rodents were captured using live-traps and euthanised for helminths and protozoan recovery. Intestinal parasites were detected and identified from stool samples using formalin ethyl-acetate concentration technique (FECT), while tissue parasites were identified by histopathological examination of selected tissue sections of the liver, brain, lungs, and muscle. In this study, a total of 89 wild rats were captured. Twelve species of intestinal and tissue parasites were recorded, of which, Taenia taeniaeformis accounts for the highest infection recorded (28%) followed by Hymenolepis nana (19.5%) and Capillaria hepatica (19.1%), while Toxoplasma gondii was the least parasite (6.7%) identified. Furthermore, other parasites species observed include, Cryptosporidium spp. (21.3%), Entamoeba histolytica/Entamoeba dispar and Moniliformis moniliformis (17.9%), Angiostrongylus cantonensis (16.8%), Hymenolepis diminuta (16.1%), Giardia spp. (14.6%), Trichuris spp. (12.3%), and Sarcocystis spp. (6.74). Based on the results obtained in the present study, 17.1% and 15.4% of the rodents captured were confirmed positive for at least one species of intestinal or tissue parasites, respectively. The presence of these zoonotic parasites in the wild rats suggests the potential risk of rodent-borne zoonotic disease transmission to humans. Hence, the need to improved rats control intervention and public health awareness among the populace.
Cutaneous larva migrans: a neglected disease and possible association with the use of long socks

Hamat RA, Rahman AA, Osman M, Unyah NZ, Abdullah WO, Isa NH

Trans R Soc Trop Med Hyg, 2010 Feb;104(2):170-2.
PMID: 19732927 DOI: 10.1016/j.trstmh.2009.07.019

Cutaneous larva migrans is a common parasitic skin disease that can be easily prevented by wearing 'protective' footwear. However, this has been under-emphasized in terms of what constitutes the protective footwear. Even though the disease resolves spontaneously, the significant duration of the disease along with severity of pruritus make treatment unavoidable. Here, we present a very long-standing creeping eruption, which puzzled many attending clinicians handling the case, and the possibility of long socks as a causal effect on the development of cutaneous larva migrans infection.
Fulltext Phylogenetic analysis of Giardia lamblia human genotypes in Fars Province, southern Iran

Rayani M, Hatam G, Unyah NZ, Ashrafmansori A, Abdullah WO, Hamat RA

Iran J Parasitol, 2017 Oct-Dec;12(4):522-533.
PMID: 29317877

Background: This study is the first phylogenetic genotype analysis of Giardia lamblia in Iran. The main objective was to determine genotyping and identify the sub-assemblages of Giardia lamblia isolates involved in the transmission of giardiasis in Fars Province, south of Iran, in 2012.

Methods: Forty G. lamblia isolates were collected from the patient's fecal samples with gastrointestinal discomfort referred to the health centers and hospitals in Shiraz, Fars Province, south of Iran. Purification of G. lamblia cysts from fecal samples and DNA extraction were performed using monolayer of sucrose density gradient and Phenol-Chloroform-Isoamylalcohol (PCI) respectively. Semi-nested PCR and sequence analysis were then performed using the primers (GDHeF, GDHiF, and GDHiR) which amplified a 432-bp fragment of Giardia glutamate dehydrogenase (gdh) gene. Phylogenetic analysis was carried out using a neighbor-joining tree composed of the nucleotide sequences of G. lamblia isolates obtained in this study and the known sequences isolates published in GenBank.

Results: G. lamblia sub-assemblage AII was the most prevalent genotype with 80% of the cases and 20% of the cases belong to sub-assemblage BIII and BIV based on the DNA sequence of the gdh. G. lamblia isolates at Fars Province were widely distributed within assemblage A cluster (sub-assemblage AII) and the remaining isolates were dispersed throughout the assemblage B cluster (sub-assemblage BIII and BIV).

Conclusion: PCR Sequencing and phylogenetic analysis was a proper molecular method for genotyping and discriminating of the of G. lamblia sub-assemblages in fecal samples, using the glutamate dehydrogenase gene that suggests a human contamination origin of giardiasis.
Fulltext A Review on Plants and Microorganisms Mediated Synthesis of Silver Nanoparticles, Role of Plants Metabolites and Applications

Mustapha T, Misni N, Ithnin NR, Daskum AM, Unyah NZ

Int J Environ Res Public Health, 2022 Jan 07;19(2).
PMID: 35055505 DOI: 10.3390/ijerph19020674

Silver nanoparticles are one of the most extensively studied nanomaterials due to their high stability and low chemical reactivity in comparison to other metals. They are commonly synthesized using toxic chemical reducing agents which reduce metal ions into uncharged nanoparticles. However, in the last few decades, several efforts were made to develop green synthesis methods to avoid the use of hazardous materials. The natural biomolecules found in plants such as proteins/enzymes, amino acids, polysaccharides, alkaloids, alcoholic compounds, and vitamins are responsible for the formation of silver nanoparticles. The green synthesis of silver nanoparticles is an eco-friendly approach, which should be further explored for the potential of different plants to synthesize nanoparticles. In the present review we describe the green synthesis of nanoparticles using plants, bacteria, and fungi and the role of plant metabolites in the synthesis process. Moreover, the present review also describes some applications of silver nanoparticles in different aspects such as antimicrobial, biomedicine, mosquito control, environment and wastewater treatment, agricultural, food safety, and food packaging.
Phytochemicals and Potential Therapeutic Targets on Toxoplasma gondii Parasite

Abdullahi SA, Unyah NZ, Nordin N, Basir R, Nasir WM, Alapid AA, et al.

Mini Rev Med Chem, 2020;20(9):739-753.
PMID: 31660810 DOI: 10.2174/1389557519666191029105736

Identification of drug target in protozoan T. gondii is an important step in the development of chemotherapeutic agents. Likewise, exploring phytochemical compounds effective against the parasite can lead to the development of new drug agent that can be useful for prophylaxis and treatment of toxoplasmosis. In this review, we searched for the relevant literature on the herbs that were tested against T. gondii either in vitro or in vivo, as well as different phytochemicals and their potential activities on T. gondii. Potential activities of major phytochemicals, such as alkaloid, flavonoid, terpenoids and tannins on various target sites on T. gondii as well as other related parasites was discussed. It is believed that the phytochemicals from natural sources are potential drug candidates for the treatment of toxoplasmosis with little or no toxicity to humans.
Fulltext Toxoplasma gondii induced cognitive impairment in rats via dysregulation of dopamine receptors and indoleamine 2,3 dioxygenase

Wana MN, Watanabe M, Chiroma SM, Unyah NZ, Abdullahi SA, Nordin S, et al.

Heliyon, 2023 Mar;9(3):e14370.
PMID: 36950587 DOI: 10.1016/j.heliyon.2023.e14370

Toxoplasma gondii (T. gondii) is a parasite capable of residing in the brain of their host which influences behaviour changes due to alterations in the neurotransmitters. Consequently, dopamine receptors (DRD) and indoleamine 2, 3 dioxygenase (IDO) dysregulation facilitate the progression of behaviour changes in a host as a response to infection. This study tested the effect of neurotransmitter changes as a result of T. gondii infection on rats cognitive impairment. The T. gondii strain of type I, II and III from Malaysia were previously identified by standard procedures. Sporulated oocysts each of type I, II and III were inoculated separately into three groups of Wistar rats (n = 9) respectively. Two separate control groups received either phosphate buffered saline (PBS) or MK-801 (dizocilpine). Behaviour changes were evaluated at nine weeks post infection in a square box, elevated plus maze and gene expression level of DRD and IDO compounds. The study revealed increased fatal feline attraction, reduced anxiety, decreased DRD and increased IDO gene expression in the T. gondii infected groups and MK-801 compared to the PBS control group. In conclusion, T. gondii infection alter the level of neurotransmitters in rat which cause cognitive impairment. This implies that all the T. gondii strain can cause behaviour changes if human were infected.
Fulltext Susceptibility of Toxoplasma gondii to Ethanolic Extract of Tinospora crispa in Vero Cells

Sharif AA, Unyah NZ, Nordin N, Basir R, Wana MN, Alapid Ahmad A, et al.

Evid Based Complement Alternat Med, 2019;2019:2916547.
PMID: 31827548 DOI: 10.1155/2019/2916547

Background: Toxoplasmosis remains widely distributed globally and is one of the major neglected parasitic zoonotic infections. The infection is still endemic in most parts of the world due to poor control as well as challenges of the currently used medications which can be overcome by using natural products. This study evaluated the effect of ethanolic extract from the stem of Tinospora crispa (EETC) on host cell invasion and intracellular replication of Toxoplasma gondii.
Method: The stem powder of T. crispa was soaked in absolute ethanol for 72 hours. The resulting ethanolic extract was screened for the presence of phytochemicals. Vero cells monolayer in 96-well plate was infected with RH strain of T. gondii and treated with concentrations of the EETC, Veratrine alkaloid, and clindamycin ranging from 1.56 to 200 μg/mL. MTT assay was conducted after 24 hours to evaluate the cytotoxicity and antiparasitic activities of the EETC. Four and 24 hours treatment models were adapted to assess the infection index and intracellular proliferation of T.
Results: The study revealed that the EETC had no cytotoxic effects on Vero cells with IC50 = 179 μg/mL, as compared to clindamycin (IC50 = 116.5 μg/mL) and Veratrine alkaloid (IC50 = 60.4 μg/mL). The EETC had good anti-toxoplasma activities with IC50 = 6.31 μg/mL in comparison with clindamycin (IC50 = 8.33 μg/mL) and Veratrine alkaloid (IC50 = 14.25 μg/mL). The EETC caused more than 70% and 80% reduction in infection index and intracellular proliferation in both treatment models, respectively.
Conclusion: This in vitro study showed that the EETC contains promising phytochemicals effective against T. gondii and safe to the host cells.
Fulltext Investigation of Andrographolide Effect on Non-Infected Red Blood Cells Using the 1H-NMR-Based Metabolomics Approach

Alapid AAI, Abd Majid R, Ibraheem ZO, Mediani A, Ismail IS, Unyah NZ, et al.

Metabolites, 2021 Jul 28;11(8).
PMID: 34436427 DOI: 10.3390/metabo11080486

Andrographolide (AG) has been shown to have several medicinal and pharmaceutical effects, such as antimicrobial, anti-inflammatory, antioxidant, anti-diabetic, and anti-malarial activities. Moreover, studies to assess the pharmacological effect of AG on the metabolic changes of uninfected red blood cells (uRBCs) have not yet been investigated. This study aims to evaluate the pharmacological effects of AG compared to chloroquine (CQ) on the metabolic variations of uRBCs in vitro using a proton nuclear magnetic resonance (1H-NMR)-based metabolomics approach coupled with multivariate data analysis (MVDA). Forty-one metabolites were successfully identified by 1H-NMR. The results of the unsupervised data analysis principal component analysis (PCA) showed ideal differentiation between AG and CQ. PC1 and PC2 accounted for 71.4% and 17.7% of the explained variation, respectively, with a total variance of 89.10%. Based on S-plot and VIP values, a total of 28 and 32 metabolites were identified as biomarkers in uRBCs-AG and uRBCs-CQ, respectively. In uRBCs treated with AG, ten metabolic pathways were determined to be disturbed, including riboflavin metabolism, d-glutamate and d-glutamine metabolism, phenylalanine metabolism, glutathione metabolism, proline and arginine metabolism, arginine biosynthesis, citrate cycle, glycolysis/gluconeogenesis, and pyruvate metabolism as well as alanine, aspartate, and glutamate metabolism. In contrast, in CQ-treated uRBCs, nine affected metabolic pathways were determined, which involved the same metabolic pathways for uRBCs-AG, except for glutathione metabolism. These findings suggest an evident relationship between AG and CQ associated with metabolic changes in intact RBCs after being exposed to the treatment. The metabolomics results could allow useful comprehensive insights into the underlying mechanism of the action of AG and CQ on red blood cells. Consequently, the 1H-NMR-based metabolomics approach was successfully utilized to identify the pharmacological effects of AG and CQ on the metabolic variations of uRBCs.