This study aimed to determine the in vivo effectiveness of the ethanolic extract of Piper betle L. leaves against Staphylococcus aureus-infected wounds in mice and its antimicrobial properties on clinical isolates of multiple drug-resistant bacterial pathogens. Twenty mice were divided into four groups. Wounds were created in all mice under anesthesia by excision from the dorsal skin down to the subcutaneous fat and inoculating with S. aureus. After 24 h, the wound of each mouse was treated once daily by application of the respective cream. Group I was treated with mupirocin antibacterial cream; Group II received a cream base containing no active ingredient; Groups III and IV were treated with 2.5% and 5.0% concentrations of P. betle cream, respectively. Further, an in vitro study was performed by adding undiluted, 1:50 and 1:100 dilutions of the four studied creams in normal saline containing 1.5 × 108 CFU/mL of the following bacteria: antimicrobial-susceptible S. aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant S. aureus, extended-spectrum β-lactamase-producing Escherichia coli, vancomycin-resistant Enterococcus, metallo-βlactamase-producing P. aeruginosa and carbapenem-resistant Klebsiella pneumoniae. The mice in Groups III and IV had significantly faster wound contraction and significantly shorter reepithelialization time than Group II (p < 0.05), which were not significantly different from Group I (p > 0.05). P. betle creams inhibited all studied bacterial strains at full concentration and at a dilution of 1:50. The inhibitory effect was more significant than Groups I and II (p < 0.05), except on S. aureus. Specifically, S. aureus inhibition was not significantly different for Groups III and IV (p > 0.05) when compared with Group I. Cream formulations derived from P. betle ethanolic extract have great potential as antimicrobial agents for the treatment of wound infection. Further clinical tests are recommended to determine the safety and efficacy of these formulations in other mammalian species.
This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.
Vibrio cholerae is a gram-negative bacterium synonymous with its namesake disease, cholera. Thus, gastrointestinal symptoms are the norm and V. cholerae is very rarely associated with skin and soft tissue infections. We describe a case of a 63-year-old Chinese woman with multiple medical comorbidities on corticosteroid therapy who developed fever and a painful swelling on her left leg after being pricked by a branch while gardening. There was no abdominal pain, vomiting or diarrhea. A diagnosis of bullous cellulitis was made clinically, and blood was sent for bacteriological culture. A beta-hemolytic commashaped gram-negative bacillus was isolated from the blood. It was also oxidase-positive and produced an acid/alkaline (A/K) reaction on triple sugar iron agar. It was identified biochemically as Vibrio cholerae. After additional testing, it was found to be of the O1 serogroup and Ogawa serotype. The infection resolved following a 10-day course of high-dose co-trimoxazole therapy.
Toxoplasmosis is a protozoal infection of zoonotic potential with worldwide geographical distribution which affects nearly all warm-blooded animals including mammals and birds. Keeping in view, this study was conducted to determine the seroprevalence of toxoplasmosis along with associated risk factors and its haematological impacts in small ruminants of district Multan, Pakistan. In this study, a total of 250 sera samples collected from sheep (n=125) and goats (n=125) from three tehsils of Multan were examined using commercially available Latex agglutination test kit for the presence of anti-T. gondii antibodies. The haematological profiles of Toxoplasma seropositive and seronegative animals were determined by using automated haematology analyser. Overall seroprevalence of toxoplasmosis in small ruminants was 42.80% with a higher prevalence rate (44.80%) in sheep as compared to goats (40.80%). Sex, existence of co-morbid conditions, feeding pattern and presence of pet cats and dogs were identified as significant (P<0.05) risk factors associated with the presence of antibodies against toxoplasmosis. The breed was found to be a significant (P=0.026) risk factor for the seroprevalence of toxoplasmosis in goats but not in sheep. Haematological analysis revealed significantly altered leukocytic counts (P<0.05) in seropositive sheep and goats as compared to seronegative ones. Our findings showed that small ruminants of the Multan District in Pakistan are toxoplasma seropositive and may pose a serious threat of public health concern in the region.
Toxoplasma gondii is an obligate intracellular protozoon which causes toxoplasmosis, an important zoonotic disease that is endemic worldwide. Common sources of T. gondii infection in humans are food or water contaminated with oocysts and raw or undercooked meat with cysts. In animals, common sources of infection include feed, water, or litter contaminated with oocysts. The diagnosis and molecular characterization of T. gondii infection in humans and animals is crucial due to public and veterinary health importance. Various traditional and serological methods have been used in clinical practice for toxoplasmosis diagnosis, but interpreting the results remains a challenge. Several molecular techniques have also been used for the detection and genetic characterization of T. gondii , but primarily in research settings. In this paper, we review the techniques that are currently used for the diagnosis and genetic characterization of T. gondii in humans and animals, along with their advantages and disadvantages. The techniques reviewed have laid the groundwork for the future development of more effective and precise detection and characterization of T. gondii . These advances will contribute to a better understanding of epidemiology, prevention and control of toxoplasmosis. Thus, this review would be of particular interest to clinical physicians, veterinarians and researchers.
High-fat diet (HFD) can cause hyperlipidemia, fatty liver and cardiovascular disorders. Herein, we evaluated therapeutic effects and possible underlying mechanisms of actions of Schistosoma mansoni soluble egg antigen (SEA) against experimental HFD induced dyslipidemia, hepatic and cardiovascular pathology. Forty Swiss albino mice were divided into four groups (10 each); mice fed standard diet (SD), mice fed HFD, mice fed HFD for 8 weeks then infected by S. mansoni cercaria (HFD+I) and mice fed HFD for 8 weeks then treated with SEA (HFD+SEA), all mice were euthanized 16 weeks after starting the experiment. HFD+SEA mice showed significantly (p<0.001) reduced total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG), and significantly (p<0.05) increased high-density lipoprotein cholesterol (HDL-C) comparing to HFD mice with non-significant difference with HFD+I mice group. Doppler flowmetry showed significantly (p<0.01) lower arterial resistance and significantly (p<0.05) higher blood flow velocity in HFD+SEA and HFD+I mice groups than HFD mice. HFD+SEA mice revealed improving in liver and aortic pathology and these were better than HFD+I mice group. HFD+SEA and HFD+I mice groups had less myocardium lipid deposits, but still showing some congested blood vessels. HFD myocardium revealed strong CD34+ expression on immunohistochemistry study, while that of HFD+SEA showed weak and HFD+I mice had moderate expressions. HFD+SEA mice had significantly (p<0.01) lower serum IL-1β and vascular endothelial growth factor (VEGF) and significantly (p<0.001) higher serum transforming growth factor beta 1 (TGF-β1) and IL-10 than HFD mice with non-significant difference with HFD+I mice. In conclusion, SEA lowered serum lipids, improved aortic function, decreased liver and cardiovascular pathology in HFD mice, so, it is recommended to purify active molecules from SEA to develop anti-dyslipidemic treatment.
Chikungunya virus (CHIKV) is a neglected tropical pathogen that causes fever and long-lasting severe arthralgia. Despite its high morbidity, there is still no licensed specific therapeutic option for it. This study proposes a multi-epitope subunit vaccine candidate for CHIKV, designed using computational methods. It was based on the E2 spike glycoprotein in CHIKV, from which T- and B-cell epitopes were predicted and then refined. The pan HLA DR-binding epitope (PADRE) was added to this refined construct, then simulated compared with the native protein, where it was predicted to elicit more than twice the number of antibody titers. Thus, this construct is potentially effective against CHIKV, which further experimentation using live models would be able to verify. This study also demonstrates the feasibility of using rational tools in the future to further optimize vaccine design.
Antimicrobial resistance is a worldwide public health concern. Rise in the number of antimicrobial resistant organisms, such as extended spectrum β-lactamase- (ESBL) and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae, continue to burden millions of people worldwide. E. coli and K. pneumoniae were isolated and collected for four months from a teaching hospital in the Philippines. All isolates were subjected to ESBL and carbapenemase testing using the double disk synergy test and modified Hodge test, respectively. Their pattern of resistance among different classes of antimicrobial agents was also investigated using the Kirby-Bauer disk diffusion test. Among the 32 clinical isolates tested, 28.1% were positive for ESBL production and 6.3% were positive for carbapenemase production. Species-specific classification showed that E. coli (44.4%) has the highest rate of ESBL production whereas both E. coli (5.6%) and K. pneumoniae (7.1%) showed almost similar rates of carbapenemase production. Antimicrobial resistance pattern of drug resistant isolates showed that all organisms were resistant to ampicillin, and majority showed resistance towards ciprofloxacin, cefotaxime, ceftriaxone, and sulfamethoxazole/trimethoprim. ESBL production is seen highest among E. coli isolates while similar rates of carbapenemase production was observed to both E. coli and K. pneumoniae isolates. Overall, antimicrobial resistance continues to rise and poses a huge threat in public health worldwide. Efforts should be made in developing rapid tests for antimicrobial resistance and to search for effective treatment from infections caused by multidrug resistant organisms.
Trypanothione reductase is a key enzyme that upholds the redox balance in hemoflagellate protozoan parasites such as T. congolense. This study aims at unraveling the potency of Kolaviron against trypanothione reductase in T. congolense infection using Chrysin as standard. The experiment was performed using three different approaches; in silico, in vitro and in vivo. Kolaviron and Chrysin were docked against trypanothione reductase, revealing binding energies (-9.3 and -9.0 kcal/mol) and Ki of 0.211μM and 0.151μM at the active site of trypanothione reductase as evident from the observed strong hydrophobic/hydrogen bond interactions. Parasitized blood was used for parasite isolation and trypanothione reductase activity assay using standard protocol. Real-time PCR (qPCR) assay was implored to monitor expression of trypanothione reductase using primers targeting the 177-bp repeat satellite DNA in T. congolense with SYBR Green to monitor product accumulation. Kolaviron showed IC50 values of 2.64μg/ml with % inhibition of 66.78 compared with Chrysin with IC50 values of 1.86μg/ml and % inhibition of 53.80. In vivo studies following the administration of these compounds orally after 7 days post inoculation resulted in % inhibition of Chrysin (57.67) and Kolaviron (46.90). Equally, Kolaviron relative to Chrysin down regulated the expression trypanothione reductase gene by 1.352 as compared to 3.530 of the infected group, in clear agreement with the earlier inhibition observed at the fine type level. Overall, the findings may have unraveled the Kolaviron potency against Trypanosoma congolense infection in rats.
The present study aims to develop a system which consists of four pairs of primers that specifically detects Salmonella spp., Salmonella serovar Typhi and Salmonella serovar Paratyphi A with an internal amplification control. The system, when applied in Polymerase Chain Reaction (PCR) under specific conditions, reaction mixture and cycling temperatures produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.
A PCR-based assay that can simultaneously detect and differentiate five different types of nosocomial bacterial pathogens was developed. Six pairs of selected primers targeting femA (132 bp) and mecA (310 bp) of methicillin-resistant Staphylococcus aureus, gltA (722 bp) of Acinetobacter baumannii, phoA (903 bp) of Escherichia coli, mdh (364 bp) of Klebsiella pneumoniae and oprL (504 bp) of Pseudomonas aeruginosa were used in this study. The conditions were optimized for the multiplex PCR to ensure specific amplification of the selected targets. Sensitivity and specificity tests were also carried out using a blind test approach on 50 bacterial cultures and resulted in 100% for both positive and negative predictive values.
Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.
Specification on residual action of a possible alternative insecticide derived from plant materials is important to determine minimum interval time between applications and the environmental persistence of the biopesticides. The objective of this study is to evaluate crude acethonilic extract of Ipomoea cairica leaves for its residual and persistence effects against Culex quinquefasciatus larvae. Wild strain of Cx. quinquefasciatus larvae were used for the purpose of the study. Two test designs, replenishment of water and without replenishment of water were carried out. For the first design, a total of 10 ml of test solution containing Ip. cairica extracts was replenished daily and replaced with 10 ml of distilled water. For the second design, treatment water was maintained at 1500 ml and only evaporated water was refilled. Larval mortality was recorded at 24 hours post-treatment after each introduction period and trials were terminated when mortality rate falls below 50%. Adult emergences from survived larvae were observed and number of survivals was recorded. For the non-replenishment design, mortality rate significantly reduced to below 50% after 28 days, meanwhile for replenishment of water declined significantly after 21 days (P < 0.05). There was no adult emergence observed up to seven days for non-replenishment and first two days for replenishment of water design. The short period of residual effectiveness of crude acethonilic extract of Ip. cairica leaves with high percentage of larval mortality on the first few days, endorses fewer concerns of having excess residues in the environment which may carry the risk of insecticide resistance and environmental pollution.
Tick-borne encephalitis (TBE) is a viral infection of the central nervous system and is caused by tick bites, usually after travel to rural or forested areas. The disease is prevalent in Scandinavia, Western Europe, Central Europe and the former Soviet Union and East Asia including Japan. In Malaysia, so far there are no reported cases of TBE. In the present time, many illnesses have been attributed to traveling to other parts of the world. Thus it is important to carry out TBE prevalence study to determine whether the virus is present among Malaysian population. Samples (sera and CSF) from patients admitted to major MOH hospitals in Peninsular Malaysia and Sabah with a clinical diagnosis of encephalitis but is IgM negative for JE, were tested for TBEV IgM ELISA and TBEV IgG ELISA (DRG, Germany). Out of the 600 samples screened for TBEV IgG, all were non-reactive. In addition, out of the 100 samples screened for TBEV IgM, all the samples were also non-reactive. Our results indicate that currently TBE is not present in the Malaysian population. Among the reasons for this could be lack of the infection agent, absence of the suitable vector or subjects selected for the study did not fit the criteria of possible exposure to TBE infections. Hence we recommend that for any future study, the selection of subjects should include those who returned from tick-infested forested areas.
Third-generation cephalosporin resistant Enterobacteriaceae (TCRE) is a global concern especially in neonatal sepsis. We performed a secondary data analysis in a Thai neonatal intensive care unit to identify the risk factors for acquisition of TCRE sepsis and mortality of Enterobacteriaceae sepsis between 1991 and 2017. Multivariate logistic and Cox proportional regression were used for analysis. Numbers of neonates with TCRE and nonTCRE sepsis were 100 and 41 patients, respectively. Medians (interquartile ranges) of gestational age, birthweight, onset of sepsis and total hospital stay of neonates with Enterobacteriaceae sepsis were 32 (28, 38) weeks, 1670 (1025, 2750) grams, 11 (6, 25) days and 41 (22, 74) days, respectively. Univariate and multivariate analysis, neonates with TCRE sepsis were more likely to have birth asphyxia (adjusted odds ratio [aOR] = 2.6; 95% confidence interval [CI] 1.1-6.0; p = 0.02) and history of aminoglycoside exposure (aOR = 2.9; 95% CI 1.3- 6.7; p = 0.01). In-hospital case fatality rate from Enterobacteriaceae sepsis was 26% (36/141). In Cox regression, neonates with TCRE sepsis was not an independent risk of non-survivors, but septic shock (adjusted hazard ratio = 9.9; 95% CI 5.0-19.7, p < 0.001) increased 30-day mortality in the final model. Asphyxia and previous aminoglycoside consumption were risks of acquisition for neonatal TCRE sepsis while the burden was not a significant difference. Infection prevention and control must be strictly implemented in high multidrug-resistant area.
Appendicitis has a worldwide prevalence and affects all age groups. The aetiology of acute appendicitis is still much debated, many factors have been implicated. The pathology is likely to be due to obstruction of the lumen of the appendix. Parasites, both helminths and protozoa have been suggested to be the cause of acute appendicitis. Studies have demonstrated that parasites are present in the appendix specimens removed from surgery methods.
The Plasmodium falciparum serine repeat antigen (SERA) is one of the promising blood-stage malarial vaccine candidates. In this study, recombinant Mycobacterium bovis bacille Calmette-Guerin (rBCG) expressing the 22 kDa protein (SE22) from the 47 kDa Nterminal domain of serine repeat antigen (SERA), generated in favour of mycobacterium codon usage, elicited specific immune response in BALB/c mice with a mixed Th1/Th2 profile. Immunized sera containing high levels of specific IgG1 and IgG2a against the epitope (as determined by ELISA) were reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA). Furthermore, the lymphocyte proliferative response to SE22 antigen from rBCG-immunized mice was higher than that of controls. The expression of intracellular cytokines (IL-2, IL-4 and IFNγ) in CD4+- and CD8+-cells was also enhanced following in-vitro stimulation with SE22. These findings indicate that a rBCG-based vaccine candidate expressing a blood-stage antigen of P. falciparum could enhance both humoral and cellular immune responses, thus paving the way for the rational use of rBCG as a vaccine candidate against malaria.
Gastrointestinal nematodes can cause assorted health problems to human and other primates. The status of gastrointestinal nematodes in non-human primates remained less documented in Malaysia. This study aimed to determine the occurrence of gastrointestinal nematodes recovered from the fecal samples of captive non-human primates at the Matang Wildlife Centre (MWC), Sarawak. Fresh fecal samples were collected from 60 non-human primates of six species (i.e. Orangutan, Bornean gibbon, Silvered Leaf monkey, Slow loris, Pig-tailed macaque, and Long-tailed macaque) and processed using simple fecal floatation method and fecal sedimentation method. This study shows high prevalence of nematode infection (>=50%) and co-infection (22 from 45 infected individuals) in all species of captive non-human primates found in MWC, except one individual of young Silvered Leaf monkey was negative for nematode. From these, eight genera of 11 species and one unknown nematode larvae were recovered and among them Oesophagostomum sp., Ascaris sp., and Strongyloides sp. were the most common nematodes infecting the non-human primates. All the Bornean gibbon (n=7) were found to be infected with nematodes. Moreover, Long-tailed macaques at the centre were heavily infected by Ascaris sp. (number of total count, nt = 2132; total mean abundance, MA=113.70). This is the first report of high prevalence nematode infection on multiple species of captive non-human primates in a wildlife centre located in Sarawak. Some of the nematodes are of zoonotic potential. This information is important for health care management, both in-situ and ex-situ conservations of captive and free-ranging nonhuman primates.
Three-dimensional (3D) models of the marginal hooks, dorsal and ventral anchors, bars and haptoral reservoirs of a parasite, Sundatrema langkawiense Lim & Gibson, 2009 (Monogenea) were developed using the polygonal modelling method in Autodesk 3ds Max (Version 9) based on two-dimensional (2D) illustrations. Maxscripts were written to rotate the modelled 3D structures. Appropriately orientated 3D haptoral hard-parts were then selected and positioned within the transparent 3D outline of the haptor and grouped together to form a complete 3D haptoral entity. This technique is an inexpensive tool for constructing 3D models from 2D illustrations for 3D visualisation of the spatial relationships between the different structural parts within organisms.
Entamoeba histolytica, the causative agent of human amoebiasis remains a significant cause of morbidity and mortality in developing countries and is responsible for up to 100,000 deaths worldwide each year. Entamoeba dispar, morphologically indistinguishable from E. histolytica is more common in humans in many parts of the world. Similarly Entamoeba moshkovskii, which was long considered to be a free-living amoeba is also morphologically identical to E. histolytica and E. dispar, and is highly prevalent in some E. histolytica endemic countries. Humans are the host of infection and there would not appear to be other meaningful animal reservoirs of E. histolytica. Entamoeba. histolytica can be present in sewage and contaminated water. The infection is mainly transmitted via ingestion of water or food contaminated by faeces containing E. histolytica cysts. Clinical features of amoebiasis range from asymptomatic colonization to amoebic dysentery and invasive extraintestinal amoebiasis, which is manifested most commonly in the form of abscesses in liver and lungs. The epidemiology of amoebiasis has dramatically changed since the separation of E. histolytica and E. dispar species and the worldwide prevalence of these species has not been estimated until recently. Morever, E. moshkovskii, another morphologically indistinguishable human parasitic Entamoeba was not mentioned or considered as a contributor to the prevalence figures in endemic areas. Amoebiasis is still a major health problem especially in aboriginal settlements and amongst people living in remote area in Malaysia. However, until now there is only one data currently available to indicate the true prevalence and incidence of E. histolytica and E. dispar. Further studies are needed to determine the burden of E. histolytica, E. dispar and E. moshkovskii infections in Malaysia. In the present review, we briefly summarize all methods use in diagnosing Entamoeba species, ranging from microscopic identification to molecular detection such as culture and isoenzyme analysis, antibody detection tests, antigen detection tests, immunochromatographic assays, conventional PCR, real-time PCR and loop-mediated isothermal amplification (LAMP).