Assessment of amino acid decarboxylase activity can be conducted using tubed broth or plated agar. In this study, the test was carried out in microtitre plates containing lysine, ornithine, arginine, tyrosine, tryptophan, phenylalanine or histidine as biogenic amine precursors. Møller decarboxylase base broth (MDB) with or without 1% of a known amino acid were added to wells of a 96 well-microtitre plate. The wells were inoculated with Escherichia coli, Klebsiella pneumoniae, Acinetobacter anitratus or Staphylococcus aureus to the final concentration of 6.0 x 10(7) cfu/ml and incubated at 35ºC. The absorbance of the culture broth was read at 570 nm at 0, 1.0, 2.0, 3.0, 4.0, 5.5, 6.5 and 7.5 hour. Comparison of means of A'(570) between 0 hour and a specified incubation time was determined statistically. Positive decarboxylase activities were detected in the media inoculated with E. coli and K. pneumoniae in less than 6 hours. The current method is suitable for immediate producers of amino acid decarboxylase enzymes. It costs less as it uses less amino acid and it has the potential to be used for screening aliquots of food materials for amino acid decarboxylase activities.
Microscopic detection of active phase of lymphatic filariasis is indicated by the presence of microfilaria in whole blood. This method is not sensitive and requires relatively large amount of blood sample. PCR allows very sensitive detection of the parasite DNA using a smaller amount of blood; and the use of dried blood spots facilitates sample transportation. Nevertheless, limited studies have been reported on PCR using dried blood spot for detection of Brugia malayi. In this study, we investigated the effects of concentrating whole blood genomic DNA sample and the amplification methods [conventional PCR (C-PCR) and real-time PCR] on the detection of B. malayi DNA from dried blood spots from a very low endemic area in Malaysia. Both C-PCR and real-time PCR detected 2 out of 18 (11%) samples as positive from non-concentrated genomic DNA preparations. After the DNA samples were pooled and concentrated, both C-PCR and realtime PCR detected B. malayi DNA amplifications in 7 out of 18 (39%) samples. However one sample which showed faint band in C-PCR was detected as highly positive in real-time PCR. In conclusion, both C-PCR and real-time PCR using dried blood spots from a low endemic area demonstrated equal sensitivity for detection of B. malayi DNA.
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.
Bovine lungworm Dictyocaulus viviparus is highly endemic in temperate regions. However, the occurrence of the lungworm has not been reported in any South East Asian country. The main aim of the present study was to detect the presence of lungworm in cattle in peninsular Malaysia and to examine the morphology of the parasite. A cross-sectional study was carried out in which 602 animals from four large scale government cattle farms and one dairy smallholder farm were sampled. In addition, 283 lungs from 11 abattoirs around the country were examined. Faecal samples were examined using the Baermann technique while post-mortem examination was performed on the lungs. Approximately 5% of faecal samples and 1% of lungs were positive for lungworm. Based on the morphology of adult lungworm, eggs and first stage larvae, Malaysian bovine lungworms were D. viviparus.
The isolation of Mycoplasma hyosynoviae from a piglet with severe pneumonia is described. This is the first report of M. hyosynoviae isolation in the country. The lung sample where the isolation was made was severely consolidated, suppurative and pleurisy. The pathogenicity of the M. hyosynoviae isolated has yet to be determined.
The importance of house fly (Musca domestica L) wings in mechanical transmission of bacteria was studied. A droplet of phosphate-buffered saline containing Vibrio cholerae was rolled along one wing of each house fly. None adhered to the wings but small proportions of the bacterium were isolated from about half the wings. Vibrio cholerae was spread onto the ventral wing surfaces of each unconscious house fly which then was placed inside a bottle. When it regained consciousness, the types of activity it performed over five minutes were noted before the house fly was killed and the bacteria on its wings numerated. Control were house flies killed before inoculation. The proportion of house flies with bacteria on their wings and the mean number of bacteria remaining were significantly less on live house flies than killed controls. Among the live house flies, bacteria were detected on fewer house flies which flew (25%) than those which did not fly (81%). In addition, the mean number of bacteria on the former was significantly less than the latter (5 against 780 colonies). However, both these parameters were not significantly different between the group which performed and the group which did not perform wing grooming; takeoff and alighting over short distances, and somersaulting. Wings of unconscious house flies tethered by their thoraxes were inoculated with V. cholerae. After regaining consciousness, the house flies were allowed to move their wings in flight motions for up to 30 seconds. Small proportions of bacteria remained on all the house flies. House flies were placed in a chamber containing a liquid bait spiked with V. cholerae. After two hours, 10 were removed sequentially and cultured for V. cholerae. The bacterium was isolated from four house flies: two from the legs, and two others from their bodies minus legs and wings. In conclusion, house fly wings do not play an important role in mechanical transmission of bacteria suspended in a non-adhering liquid medium because of the low transfer rate of the bacteria to the wings and poor retention of bacteria on the wings during normal house fly activities.
The house fly, Musca domestica has long been considered a potential agent for disease transmission ever since its existence. The general truth of this assertion remains undisputed till the present day in spite of increasing awareness toward an improved sanitation and better hygiene. The habitual movement of house fly from filthy substrata such as human faeces, animal excreta, carcasses, garbage, etc. makes them ideal candidates for disease transmission such as cholera, shigellosis, salmonellosis and others when settling on food. Fly as a potential mechanical vector of pathogenic bacteria was elucidated in this study by examining flies from various breeding sites such as food courts, dumping ground, food processing areas and poultry farm in Peninsular Malaysia. The flies were baited with 10% sugar solution on a glass slide in the field. All materials used for collection of samples were sterile. Bacteria from fly sample were isolated using the normal isolation technique. Bacillus sp., Coccobacillus sp., Staphylococcus sp., Microccus sp., Streptococcus sp., Acinetobacter sp., Enterobacter sp., Proteus sp., Escherichia sp., Klebsiella sp. and yeast cells were isolated from feaces, vomitus, external surfaces and internal organs of house fly. Newly emerged house fly did not harbour any bacteria.
Laboratory strain of the Malaysian Culex quinquefasciatus was susceptible to Wuchereria bancrofti. Thirty three percent of the Cx. quinquefasciatus that fed on W. bancrofti patient were infective after 12-14 days. There is a possibility for W. bancrofti to occur in the urban areas of the Malaysia in the near future.
Is Acanthamoeba sp. normally found in the eyes? A study was carried out to establish the possibility of Acanthamoeba sp. as a part of the normal conjunctival flora. Conjunctiva swabbing were carried out in 286 healthy Orang Asli school children using sterile cotton swab. The swab was then inoculated onto non-nutrient agar (NN-A). Heat killed Escherichia coli that was used as food source for the growth of the amoebae was pipetted onto and away from the smear. The plates were incubated at 30 degrees C and examined daily using an inverted microscope for 14 days. Morphology of the trophozoites and cysts of the amoebae were used as the taxonomic criteria for identification. Positive-controls and negative-controls were done to check for the consistency of the technique used and monitoring of contamination respectively. None of the conjunctiva swab cultured was positive for Acanthamoeba sp. This finding may indicate that Acanthamoeba sp. is not part of normal conjunctival flora or conjunctiva swab is an insensitive technique to isolate the organism. However, a more extensive research is needed to investigate these possibilities.
A study of chikungunya virus was carried out to establish Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) as a rapid detection technique of the virus. The susceptibility of lab-colonized Aedes aegypti to chikungunya virus was also determined. Artificial membrane feeding technique was used to orally feed the mosquitoes with a human isolate of chikungunya virus. A total of 100 fully engorged female Ae. aegypti were obtained and maintained for 7 days. Seventy of them survived and then pooled at 10 individuals per pool. Total RNA was extracted from the samples and RT-PCR amplifications were carried out. Five out of 7 pools showed positive PCR band at 350-bp, indicating Ae. aegypti is a potential vector of chikungunya virus. The minimum infection rate (MIR) was 71% within these laboratory colonies. RT-PCR is a sensitive technique that is useful in detecting infected mosquitoes in epidemic areas. This technique can de used as a rapid detection method and provide an early virologic surveillance systems of chikungunya virus infected mosquitoes.
Artemisia annua L., a medicinal herb, produces secondary metabolites with antimicrobial property. In Malaysia due to the tropical hot climate, A. annua could not be planted for production of artemisinin, the main bioactive compound. In this study, the leaves of three in vitro A. annua L. clones were, extracted and two bioactive compounds, artemisinin and a precursor, were isolated by thin layer chromatography. These compounds were found to be effective in inhibiting the growth of Gram-positive and Gram-negative bacteria but not Candida albicans. Their antimicrobial activity was similar to that of antibactericidal antibiotic streptomycin. They were found to inhibit the growth of the tested microbes at the minimum inhibition concentration of 0.09 mg/mL, and toxicity test using brine shrimp showed that even the low concentration of 0.09 mg/mL was very lethal towards the brine shrimps with 100% mortality rate. This study hence indicated that in vitro cultured plantlets of A. annua can be used as the alternative method for production of artemisinin and its precursor with antimicrobial activities.
The Corona pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) calls on the Saudi government to take action to control the infection. The government closed borders, prohibited travel, limited outdoor movements, and told primary and secondary care facilities to reduce all regular non-urgent health services. It is not known whether these measures have impacted the prevalence of parasitic intestinal infections. This study has therefore been carried out to investigate this issue. Dataset of 217 stool samples submitted to the King Faisal Medical Complex (KFMC) Microbiology Laboratory in Taif, Saudi Arabia for parasitological examination during the pandemic (January-June 2020) and 649 samples submitted during the corresponding months of the previous year (January-June 2019) were extracted and analyzed. Overall, 24.1% (209/866) of samples were parasitespositives; 26.6% (173/649) before and 16.5% (36/217) during the pandemic, with 79% reduction. There was a significant difference in gender-parasitism between the two periods where the majority of parasitism were for males (p<0.001). Infections were frequent in patients aged 5- 14 years both before (84/649; 12.9%) and during (12/217; 5.5%) the pandemic, with significant difference observed between the two cohorts (p<0.002). Moreover, the majority of infected patients were non-Saudi (67.9%; 142/209), with a significant difference in nationality reported, (p=0.024). Protozoa were identified in 21.8% (189) of all samples investigated, of which, Blastocystis hominis, Entamoeba coli, Giardia lamblia, Entamoeba histolytica/dispar and Cryptosporidium species were identified in 6.1% (53), 5.4% (47), 5.0% (44), 2.8% (25), and 2.3% (20), respectively. Helminths were diagnosed in 2.3% (20/866) of samples. Eggs of hookworm, Ascaris, Taenia spp, and Hymenolepis nana were detected in 0.9% (8), 0.5% (5), 0.3% (3) and 0.4% (4), respectively. In parallel with our research hypothesis, a substantial decrease in the burden of intestinal parasitic infections was recorded with the lock-down measures taken during the Corona pandemic.
Loop-mediated isothermal amplification (LAMP) test is widely used in molecular diagnostics as a point-of-care technique alternative to traditional PCR especially in resource-limited countries. LAMP has been recently used to diagnose leptospirosis. Therefore, we undertook a systematic review and meta-analysis to compare the accuracy of LAMP with PCR in the diagnosis of leptospirosis. Sixty-one studies were extracted from three international databases and analyzed throughout using the PRISMA guideline. The pooled sensitivity of LAMP and PCR technique was 0.80 (95% CI: 0.58-0.90) and 0.54 (95% CI: 0.35-0.67) respectively indicating that LAMP is more sensitive than PCR. The Q* value of LAMP and PCR-based technique is 274.61 and 397.95, respectively. Among the analyzed studies, significant heterogeneity was observed where I2 is 90.90% for LAMP-based and 86.18% for PCR-based. Our study suggests that LAMP has better diagnostic accuracy than PCR. However, future work should be carried out to reduce heterogeneity as well as to improve and develop effective intervention strategies.
The purpose of this study was to investigate the composition of throat microbiota in pulmonary tuberculosis patients (PTB) in comparison to healthy tuberculin skin test positive (TSTp) and negative (TSTn) individuals. Throat swabs samples were collected, and the microbiota was characterized. Richer operational taxonomic units (OTUs) were present in PTB group, compared to TSTp and TSTn. Regarding alpha diversity analysis there was a higher community diversity in TSTn compared to TSTp. Beta diversity analysis showed different species composition in TSTp compared to TSTn and PTB. There was higher presence of Firmicutes in PTB and TSTn compared to TSTp group at phylum level. At the genus level, Leuconostoc and Enterococcus were higher in TSTn compared to TSTp and Pediococcus, Chryseobacterium, Bifidobacterium, Butyrivibrio, and Bulleidia were higher in PTB compared to TSTn. Streptococcus was higher in TSTn compared to PTB and Lactobacillus in PTB compared to TSTp. At species level, Streptococcus sobrinus and Bulleidia moorei were higher in PTB compared to TSTn individuals, while Lactobacillus salivarius was higher in PTB compared to TSTp. The differences in the microbiome composition could influence the resistance/susceptibility to Mtb infection.
Wet India ink mounts of cerebrospinal fluid (CSF) are useful in the laboratory diagnosis of cryptococcal meningitis. Pseudo-cryptococcal artefacts in such mounts have been attributed to leucocytes in CSF but their mode of formation has not been explained. This report describes the reproduction of such an artefact in cryptococcus free CSF-leucocyte mixtures that had been subjected to high speed centrifugation. The viscosity of DNA that could provide a morphological pseudo-capsule, and the yellow-green fluorescence of the pseudo-capsular material on staining with acridine-orange, suggest that lymphocytic nuclear DNA, which possibly leaked out after damage to the lymphocyte membrane by centrifugation, was responsible for this artefact.
Reports of zoonotic infections with Onchocerca japonica (Nematoda: Filarioidea), which parasitizes the Japanese wild boar, Sus scrofa leucomystax, have recently increased in Japan. To predict the occurrence of infection in humans, it is necessary to determine the prevalence of O. japonica infection in the natural host animals. We investigated the presence of adult worms in the footpads, and of microfilariae in skin snips, taken from the host animals, between 2000 and 2018. Onchocerca japonica was found in 165 of 223 (74%) Japanese wild boars in Honshu and Kyushu. Among the nine regions studied, the highest prevalence of O. japonica infection was found in Oita, Kyushu, where 47 of 52 (90.4%) animals were infected. The ears were the predilection sites for O. japonica microfilariae. Adult worms of O. japonica were found more frequently in the hindlimbs than in the forelimbs of the host animals. Onchocerca takaokai was found in 14 of 52 (26.9%) Japanese wild boars in Oita. In Kakeroma Island among the Nansei Islands, both O. japonica and O. takaokai were isolated from the Ryukyu wild boar, S. s. riukiuanus. These observations could help predict future occurrences of human zoonotic onchocercosis in Japan.
Plasmodium knowlesi, a common parasite of macaques, is recognised as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in vitro model to study the parasite. We have assembled a reference genome for the PkA1-H.1 line using PacBio long read combined with Illumina short read sequence data. Compared with the H-strain reference, the new reference has improved genome coverage and a novel description of methylation sites. The PkA1-H.1 reference will enhance the capabilities of the in vitro model to improve the understanding of P. knowlesi infection in humans.
The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and soman-inhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.
An investigation was undertaken for screening and isolating nematophagous-fungi from the faecal samples of various grazing animals and soils in Malaysia. Total of 111 faeces and 50 soil samples were collected and the samples were cultured on 2% water agar plates. The growth of nematophagous-fungi was stimulated by sprinkling-baiting technique. The conidia of suspected nematophagous-fungi were inoculated on 2% water agar plates. All isolated were maintained on 2% cornmeal agar plates. Verticillium spp., Fusarium spp. and Arthrobotrys spp. were identified from the faecal and soil samples. 62.5% of the faecal samples and 100% of the soil samples were shown to be positive with nematophagous-fungi. This study highlights the present of nematophagous-fungi population in faecal and soil samples. Much study remains to be done to better understanding some fungi especially their mode of action and their predatory behaviour against parasitic nematodes.
Evidence of influenza A virus (IAV) infection in dogs, a major companion animal of humans, suggests the possibility that they may constitute a new source for transmission of novel influenza viruses to humans. The potential public health risk posed by this possibility of interspecies spread of IAV between dogs and humans necessitated surveillance for the virus in dogs and their human contacts. Sera from 239 asymptomatic pet and hunting dogs in Oyo state, Nigeria were screened for anti-IAV nucleoprotein antibodies using competitive enzyme-linked immunosorbent assay (ELISA) while haemagglutination inhibiting (HI) antibodies in the positive sera were detected using influenza virus H3 and H5 subtypespecific antigens. Suspensions prepared from 239 and 39 nasal swabs from dogs and human contacts, respectively were tested for presence of the highly conserved IAV matrix gene by reverse transcriptase-polymerase chain reaction (RT-PCR). Only 4 (1.7%) of the 239 sera tested were positive by the ELISA. The HI test confirmed the presence of H3 influenza virus subtype-specific antibodies in one (25.0%) of the 4 ELISA-positive sera with a titre of 1:128 while none was positive for H5 subtype-specific antibodies. All the nasal swabs assayed by RT-PCR were negative for IAV nucleic acid. The detection of IAV antibodies in pet and hunting dogs in this study, although at a low rate, suggests that these dogs could play a crucial role in the zoonotic transmission of influenza viruses especially considering the close interaction between them and their human contacts. Continuous surveillance for IAV among dog populations in Oyo State (and Nigeria) is therefore advocated to facilitate early detection of infection or emergence of novel influenza virus strains that could be potentially harmful to humans and or animals.
Matched MeSH terms: Influenza A virus/isolation & purification*