Loop-mediated isothermal amplification (LAMP), a rapid nucleic acid amplification method, was developed for the clinical diagnosis of toxoplasmosis. Three LAMP assays based on the SAG1, SAG2, and B1 genes of Toxoplasma gondii were developed. The sensitivities and specificities of the LAMP assays were evaluated by comparison with the results of conventional nested PCR. The LAMP assays were highly sensitive and had a detection limit of 0.1 tachyzoite, and no cross-reactivity with the DNA of other parasites was observed. Blood was collected from 105 individuals to test the LAMP assays: 40 patients with active toxoplasmosis, 40 negative controls, and 25 patients with other parasitic infections. The SAG2-based LAMP (SAG2-LAMP) had a greater sensitivity (87.5%) than the SAG1-LAMP (80%), B1-LAMP (80%), and nested PCR (62.5%). All the LAMP assays and nested PCR were 100% specific. This is the first report of a study which applied the LAMP method to diagnose toxoplasmosis from human blood samples. Due to its simplicity, sensitivity, and specificity, LAMP is suggested as an appropriate method for routine diagnosis of active toxoplasmosis in humans.
Four hundred and six AIDS patients were recruited in this retrospective study. The seroprevalence of toxoplasmosis among 406 AIDS patients was 208 (51.2%). Their age ranged from 17 to 74 years with a median of 35 years. The majority of patients were males 172 (82.6%), Malays 99 (47.5%), single 109 (52.4%), unemployed 99 (47.6%) and heterosexual with commercial sex workers (CSW) 97 (46.6%) as the risk marker to HIV infection. Thirty-one (14.9%) of 208 AIDS-related toxoplasmosis were diagnosed as active toxoplasmic encephalitis. The most common clinical manifestation was headache (67.7%). The CT scan findings showed most lesions to be multiple (87.5%), hypodense (66.7%), and in frontal region (41.7%). Twenty-two (71%) patients had chronic (latent) Toxoplasma infection as evidenced by seropositivity for anti-Toxoplasma (IgG) antibody. They were statistically significant in the association between CD4 count and toxoplasmic encephalitis (P = 0.019; OR = 2.6; 95% CI = 1.14-6.02). After the initial six weeks of anti-TE therapy, relapsing toxoplasmic encephalitis was detected in 9.7% in this study.
This study aims to evaluate the current seroprevalence of toxoplasmosis among indigenous communities in Peninsular Malaysia and relate its association with epidemiological data. Overall seroprevalence of Toxoplasma gondii was 37.0% with 31.0% immunoglobulin (Ig) G, 1.8% IgM, and 4.2% seropositivity for both anti-Toxoplasma antibodies. Multivariate analysis showed that age above 12 years (odds ratio [OR] = 2.70, 95% confidence interval [CI] = 1.75-4.04, P < 0.001), using untreated river and mountain water supplies (OR = 1.50, 95% CI = 1.01-2.40, P = 0.050), and close proximity with cats (OR = 1.40, 95% CI = 1.10-1.76, P = 0.010) were factors associated with toxoplasmosis. Given the high seroprevalence of toxoplasmosis among these communities who live in poor socioeconomic conditions, a comprehensive health surveillance program and screening should be initiated among women of childbearing age and pregnant women during the antenatal period for early diagnosis and treatment. The role of domestic cats and environmental contamination with oocyst in soil and water has to be highlighted and addressed in future prevention strategies for these communities.
Toxoplasmosis is an important parasitic disease in immunosuppressed patients. This prospective study was conducted to determine the seroprevalence, associated risk factors and the incidence of clinically confirmed toxoplasmosis among renal patients at the University of Malaya Medical Center, Kuala Lumpur, Malaysia. We interviewed 247 renal patients, each of whom answered an epidemiological questionnaire, and collected blood samples for measurement of anti-Toxoplasma IgG and IgM antibodies by ELISA. Overall seroprevalence of latent toxoplasmosis was observed in 126 (51%) renal patients. Race (Malays), marital status (married) and primary level of education, were all factors associated with a greater chance of Toxoplasma infection. A case of clinically confirned toxoplasmosis was diagnosed in a renal transplant recipient as a result of immunosuppression. Based on the findings obtained, this preliminary study shows a high prevalence of latent toxoplasmosis in renal patients. Risk factors may have significantly contributed to Toxoplasma acquisition in these patients. We recommend further studies be carried out to monitor for trends in toxoplasmosis among immunosuppressed patients.
A cross-sectional study was carried out in University of Malaya Medical Centre, Kuala Lumpur. Blood samples from 100 HIV-infected patients and 203 Healthy Blood Donors (HBD) were collected and anti-Toxoplasma antibodies were detected by using conventional ELISA. The seroprevalence of toxoplasmosis in HIV/AIDS and Healthy Blood Donors were found to be 21% and 28.1% respectively. There was no significant association between the seroprevalence of toxoplasmosis and various possible risk factors i.e. contact with cat, consumption of undercooked meat and history of blood transfusion in both groups. No significant differences between Toxoplasma seroprevalence in HIV/AIDS and Healthy Blood Donors in association with presence of single or multiple risk factors were found. The mean CD4 count among HIV/AIDS patients in this study was 202.23 cell/cumm. There was no significant association between CD4 count and seropositivity for Toxoplasma antibodies in HIV/AIDS patients.
A cross-sectional study was conducted to determine the seroepidemiology of Toxoplasma infection and its risk association among people having close contact with animals. A total of 312 blood samples were collected from veterinary personnel (veterinarian, technicians, and students) and pet owners from veterinary clinics and hospitals in the area of Klang Valley, Malaysia. About 4 cc of blood samples drawn from agreed participants were processed for measurement of anti-Toxoplasma IgG and IgM antibodies as well as avidity test of Toxoplasma IgG by ELISA I, II, and III kits. Meanwhile, the demographic profiles and possible risk factors of these participants were also recorded in the standardized data collection sheets. Overall seroprevalence of toxoplasmosis was observed in 62 (19.9%) participants being 7 (18.4%) in veterinarians, 15 (33.3%) in veterinary technicians, 29 (14.9%) in veterinary students, and 11 (31.4%) in pet owners. Of 19.9% Toxoplasma seropositive samples, 18.3% was positive for IgG antibody, 1.0% for IgM antibody, and 0.6% for both IgG and IgM antibodies. Of three different IgG avidity ELISA kits, ELISA III showed high avidity in all five seropositive samples (IgM and IgG/IgM antibodies) indicating chronic Toxoplasma infection which is consistent with no evidence of clinical toxoplasmosis diagnosed during the time of this study. Univariate analysis showed that age group, gender, study population, gardening, task performance, and working duration were significantly associated with Toxoplasma seropositivity. Further analysis by multivariate analysis using logistic regression showed that age group of ≥30 years old (OR = 0.34, 95% CI = 0.18-0.63, p = 0.001) and working or study duration of >10 years having close contact with animals (OR = 5.07, 95% CI = 1.80-14.24, p = 0.002) were identified as significant risks for Toxoplasma infection. Based on the results obtained, a comprehensive Toxoplasma screening and health surveillance program on toxoplasmosis should be implemented among people having close contact with animals in general and confirmed Toxoplasma seronegative individuals in particular to prevent seroconversion.
We reviewed various studies regarding human toxoplasmosis in Malaysia. They showed a varying prevalence of specific Toxoplasma antibodies among the Malaysian population. The Malays have shown the highest seroprevalence of toxoplasmosis, by most studies, when compared to other races. Demographic profiles have shown that Toxoplasma seropositivity is higher in males than females, lower in people with higher incomes, higher in the unemployed and tends to increase with age. In general, the route of transmission, such as contact with a cat, consumption of undercooked meat and blood transfusion were shown to have no significant association with Toxoplasma seropositivity (p > 0.05). The immune status (CD4 cell count < 200 cell/mm3) was strongly associated with toxoplasmic encephalitis (p < 0.05).
Cryptosporidium and Giardia are two important pathogenic parasites that have caused many waterborne outbreaks which affected hundreds of thousands of people. Contamination from effluent discharged by sewage treatment plants have been implicated in previous waterborne outbreaks of Cryptosporidium and Giardia. This study evaluated the reduction of Cryptosporidium and Giardia (oo)cysts in two sewage treatment plants (STPA and STPB) in Malaysia which employed different treatment processes for a period of a year. Raw sewage influents and treated sewage effluents were concentrated by repeated centrifugation, subjected to sucrose density flotation and concentrated to a minimal volume depending upon the levels of contaminating debris. Cryptosporidium oocysts and Giardia cysts were enumerated using epifluorescence microscopy. The parasite concentrations in raw sewage were 18-8480 of Giardia cysts/litre and 1-80 of Cryptosporidium oocysts/litre. In treated sewage, the concentration of parasites ranged from 1-1462 cysts/litre and 20-80 oocysts/ litre for Giardia and Cryptosporidium respectively. Statistical analysis showed that sewage treatment process which employed extended aeration could reduce the concentration of Cryptosporidium and Giardia (oo)cysts significantly but treatment process which encompasses aerated lagoon could only reduce the concentration of Giardia cysts but not Cryptosporidium oocysts significantly. This phenomenon is of great concern in areas whereby effluent of sewage treatment plants is discharged into the upstream of rivers that are eventually used for abstraction of drinking water. Therefore, it is important that wastewater treatment authorities rethink the relevance of Cryptosporidium and Giardia contamination levels in wastewater and watersheds and to develop countermeasures in wastewater treatment plants. Further epidemiological studies on the occurrence and removal of pathogenic organisms from excreta and sewage are also recommended, in order that the public health risks can be defined and the most cost effective sewage treatment options developed.
Toxoplasma gondii infects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of whole Toxoplasma lysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressed T. gondii dense granular protein-5 (GRA5) in Escherichia coli and isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronic T. gondii infections (sensitivities of 46.8% and 61.2%, resp.).
Dengue fever and dengue hemorrhagic fever have been known to be endemic and reportable diseases in Malaysia since 1971. Major outbreaks occurred in 1973, 1982 and in 1998. For the past few decades until now. many studies have been performed to investigate the importance of these two diseases in Malaysia. A retrospective study was carried out in Hospital Tengku Ampuan Rahimah Klang to find the prevalence of these diseases. The data was collected from the record department of this hospital starting from the year 1999 until 2003 (5 years). A total of 6,577 cases of dengue fever and 857 cases of dengue hemorrhagic fever were reported. From the year 2000 onwards, cases of dengue fever had increased tremendously. However for the year 2001, there was a slight decrease in the reported cases. Most cases occurred in 2003, increasing from 674 in 1999 to 2,813 in 2003. Highest incidence was seen in Malay males more than 12 years of age. However, the cases of dengue hemorrhagic fever declined tremendously throughout the years. Most cases occurred in 1999 with 674 cases, then declining to only one in the year 2001 before it increased to 60 and 72 in the years 2002 and 2003, respectively. Most cases occurred in patients above 12 years old, the majority of which were Malay males.
Toxoplasma gondii is a protozoan parasite that is capable of causing a zoonotic disease, known as toxoplasmosis. Vertical transmission of T. gondii from the mother to the fetus, during pregnancy may cause severe complications to the developing fetus. This current study aimed to determine the seroprevalence and investigate the associated risk factors of Toxoplasma infection in pregnant women (n=219) visiting the antenatal clinic at UMMC. While the elevated level of anti-Toxoplasma IgG and IgM antibodies indicates the presence of infection, it fails to differentiate between a past and a recent infection. Thus, the study also demonstrates the usefulness of IgG avidity in validating the timing of infection. The serum samples were tested for the presence of anti-Toxoplasma IgG and IgM antibodies by ELISA test, and the seropositive samples for both anti-Toxoplasma IgG and IgM antibodies were further evaluated by IgG avidity. The results showed that the overall prevalence of T. gondii seropositivity was 34.7%. Of these, 30.6% (67/219) were positive for anti-Toxoplasma IgG antibody only, 2.3% (5/219) were positive for anti-Toxoplasma IgM only, and the remaining 1.8% (4/219) was positive for both anti-Toxoplasma IgG and IgM antibodies. All of the pregnant women who were positive for both anti-Toxoplasma IgG and IgM antibody were found to have past infection when evaluated by IgG avidity. In this study, Malay ethnicity and the number of existing previous children were significantly associated with T. gondii seropositivity (p<0.05). Based on these findings, information and education on the transmission and prevention of congenital toxoplasmosis are very crucial as a public health effort towards a healthier society.
Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans, is still an issue in Southeast Asia (SEA). This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays.
Garcinia mangostana extract (GME) has severe pharmacokinetic deficiencies and is made up of a variety of bioactive components. GME has proven its anti-Acanthamoeba effectiveness. In this investigation, a GME-loaded niosome was developed to increase its potential therapeutic efficacy. A GME-loaded niosome was prepared by encapsulation in a mixture of span60, cholesterol, and chloroform by the thin film hydration method. The vesicle size, zeta potential, percentage of entrapment efficiency, and stability of GME-loaded niosomes were investigated. The values for GME-loaded niosome size and zeta potential were 404.23 ± 4.59 and -32.03 ± 0.95, respectively. The delivery system enhanced the anti-Acanthamoeba activity, which possessed MIC values of 0.25-4 mg mL-1. In addition, the niosomal formulation decreased the toxicity of GME by 16 times. GME-loaded niosome must be stored at 4 °C, as the quantity of remaining GME encapsulated is greater at this temperature than at room temperature. SEM revealed the damage to the cell membrane caused by trophozoites and cysts, which led to dead cells. In light of the above, it was found that GME-loaded niosomes had better anti-Acanthamoeba activity. The study suggested that GME-loaded niosomes could be used as an alternative to Acanthamoeba's therapeutic effects.
COVID-19, caused by the SARS-CoV-2 virus, can lead to massive inflammation in the gastrointestinal tract causing severe clinical symptoms. SARS-CoV-2 infects lungs after binding its spike proteins with alveolar angiotensin-converting enzyme 2 (ACE2), and it also triggers inflammation in the gastrointestinal tract. SARS-CoV-2 invades the gastrointestinal tract by interacting with Toll-like receptor-4 (TLR4) that induces the expression of ACE2. The influx of ACE2 facilitates cellular binding of more SARS-CoV-2 and causes massive gastrointestinal inflammation leading to diarrhea. Diarrhea prior to COVID-19 infection or COVID-19-induced diarrhea reportedly ends up in a poor prognosis for the patient. Flavonoids are part of traditional remedies for gastrointestinal disorders. Preclinical studies show that flavonoids can prevent infectious diarrhea. Recent studies show flavonoids can inhibit the multiplication of SARS-CoV-2. In combination with vitamin D, flavonoids possibly activate nuclear factor erythroid-derived-2-related factor 2 that downregulates ACE2 expression in cells. We suggest that flavonoids have the potential to prevent SARS-CoV-2 induced diarrhea.
Plants with medicinal properties have been used in the treatment of several infectious diseases, including Acanthamoeba infections. The medicinal properties of Cambodian plant extracts; Annona muricata and Combretum trifoliatum were investigated against Acanthamoeba triangularis. A total of 39 plant extracts were evaluated and, as a result, 22 extracts showed positive anti-Acanthamoeba activity. Of the 22 extracts, 9 and 4 extracts showed anti-Acanthamoeba activity against trophozoites and cysts of A. triangularis, respectively. The minimum inhibitory concentration of A. muricata and C. trifoliatum extracts against trophozoites and cysts was 500 and 1,000 μg/mL, respectively. The combination of A. muricata at 1/4×MIC with chlorhexidine at 1/8×MIC demonstrated a synergistic effect against trophozoites, but partial synergy against cysts. A 40% reduction in trophozoites and 60% of cysts adhered to the plastic surface treated with both extracts at 1/2×MIC were noted comparing to the control (P < 0.05). Furthermore, a reduction of 80% and 90% of trophozoites adhered to the surface was observed after pre-treatment with A. muricata and C. trifoliatum extracts, respectively. A 90% of cysts adhered to the surface was decreased with pre-treatment of A. muricata at 1/2×MIC (P < 0.05). A 75% of trophozoites and cysts from Acanthamoeba adhered to the surface were removed after treatment with both extracts at 4×MIC (P < 0.05). In the model of contact lens, 1 log cells/mL of trophozoites and cysts was significantly decreased post-treatment with both extracts compared to the control. Trophozoites showed strong loss of acanthopodia and thorn-like projection pseudopodia, while cysts demonstrated retraction and folded appearance treated with both extracts when observed by SEM, which suggests the potential benefits of the medicinal plants A. muricata and C. trifoliatum as an option treatment against Acanthamoeba infections.
The objective of this study was to characterize the polypeptides associated with cysts of Blastocystis hominis. This form is believed to be infective and plays a role in parasite resistance to anti-B. hominis drugs currently used for treatment of Blastocystis associated diarrhea. Cysts were induced through in vitro culture of the parasite in complete medium supplemented with bacterial extract with trypticase, metronidazole or doxycycline. SDS-PAGE analysis showed almost similar polypeptide patterns of parasite extracts obtained from in vitro cultured parasites before and after exposure with the three supplements. Polypeptide bands at 76, 58.5, 48, 45, 40, 38, 32, 25 and 22 kDa were constantly seen in all antigenic preparations and no specific cyst-associated polypeptide was present. However, on immunoblot analysis, 3 out of 16 blastocystosis human sera identified a cyst-associated polypeptide at 60 kDa in all parasite extracts prepared from cultures with the three supplements. In addition, there were associated morphological changes detected in these parasites stained with acridine orange and observed under fluorescence microscopy. Metronidazole induced cyst forms (reddish cells) as early as 12 hours post-exposure; more cyst production (with stronger immunoblot bands) occurred after 24 hours exposure. However, cysts rupture with release and destruction of B. hominis daughters cells occurred after 48 hours exposure. Doxycycline induced less cyst-like forms at 24 hours (weaker 60 kDa band) and less destruction of the cysts (60 kDa band still present at 72 hours post exposure). Bacterial extract and trypticase also induced cysts at 12 hours with increasing numbers up to 72 hours exposure (corresponding increase in intensity of 60 kDa band from samples harvested at 12 to 72 hours post exposure) without any sign of deleterious effect on the parasite.
Acanthamoeba keratitis infection extends due to the growing number of contact lens users. Indigenous plants including Garcinia mangostana play a vital role in human health and well being. Many species of this plant have been reported with myriads of potent medicinal properties. However, the aims of this study were, for the first time, to isolate compounds from the flower of G. mangostana and to test their anti-Acanthamoeba and anti-adhesion activity against Acanthamoeba triangularis. Powdered flowers of G. mangostana were extracted and chromatographed on a silica gel column. The structures of the compounds were established with the aid of 1H NMR. More so, the anti-Acanthamoeba and anti-adhesion properties were tested on a 96-well polystyrene microtiter plate and soft contact lenses. Scanning electron microscope (SEM) was used to determine the features of A. triangularis on contact lenses. Eight pure compounds were obtained, namely 9-hydroxycalabaxanthone, tovophillin A, garcinone E, garcinone B, α-mangostin, gartinin, 8-deoxygartinin and γ-mangostin. The extract and pure compounds exhibited anti-Acanthamoeba activity with MIC values in the range of 0.25-1 mg/mL. In addition, the extract and α-mangostin displayed significant activity against the adhesion of A. triangularis trophozoites both in polystyrene plate and in contact lenses at 0.5 × MIC (0.25 mg/mL). Furthermore, α-mangostin has the potential to remove A. triangularis adhesion in contact lenses similar to a commercial multipurpose solution (MPS). SEM study confirmed that crude extract and α-mangostin are effective as solutions for contact lenses, which removed A. triangularis trophozoites within 24 h. Alpha-mangostin was non-toxic to Vero cells at a concentration below 39 μM in 24 h. Crude extract of G. mangostana flower and its α-mangostin serve as candidate compounds in the treatment of Acanthamoeba infection or as lens care solution, since they can be used as a source of natural products against Acanthamoeba and virulence factor associated with the adhesion of A. triangularis.
Peganum harmala, a well-known medicinal plant, has been used for several therapeutic purposes as it contains numerous pharmacological active compounds. Our study reported an anti-parasitic activity of P. harmala seed extract against Acanthamoeba triangularis. The stress induced by the extract on the surviving trophozoites for Acanthamoeba encystation and vacuolization was examined by microscopy, and transcriptional expression of Acanthamoeba autophagy-related genes was investigated by quantitative PCR. Our results showed that the surviving trophozoites were not transformed into cysts, and the number of trophozoites with enlarged vacuoles were not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of tested AcATG genes, i.e., ATG3, ATG8b, and ATG16, was at a basal level along the treatment. However, upregulation of AcATG16 at 24 h post treatment was observed, which may indicate an autophagic activity of this protein in response to the stress. Altogether, these data revealed the anti-Acanthamoeba activity of P. harmala extract and indicated the association of autophagy mRNA expression and cyst formation under the extract stress, representing a promising plant for future drug development. However, further identification of an active compound and a study of autophagy at the protein level are needed.
Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public.