The AIDS epidemic in Southeast Asia has led to a marked rise in the incidence of talaromycosis (penicilliosis) marneffei. The aim of this cross-sectional study was to determine the clinico-epidemiological features and outcome predictors of talaromycosis in Malaysia. We identified Talaromycosis marneffei cases from cultures of sterile specimens from 191 human immunodeficiency virus (HIV)- infected patients. Talaromycosis increased from 20-25 (2010-2011) to 45-50 cases per year (2012-2014). Talaromycosis was the HIV-presenting illness in 117 (61.3%) patients. The incidence of talaromycosis as HIV-presenting illness showed an increasing trend from 10.7 (2010) to 26.4 (2014) cases per 1000 new HIV patients. The patients were between 19 and 74 of age (mean 37.2+9.4 years) and the male to female ratio was 7.7:1. Malay (73, 38.2%) and Chinese (70, 36.3%) were the most prevalent ethnic groups. Common clinical manifestations included loss of weight (85.9%), fever (84.8%) and cough (67%), while skin lesions were only present in 42.9% cases. Common concurrent infections were oral candidiasis (79.6%), tuberculosis (36.1%) and hepatitis C infection (20.9%). Most patients (93.7%) were anaemic with mean haemoglobin level of 9.9+2.3 g/dL, 39% had impaired liver function, and 18.8% were neutropaenic. Median CD4 cell count was 16 cells/L. Most patients (70.4%) received intravenous amphotericin B followed by itraconazole. At 8-month follow up, 148 (81.8%) patients were alive while 33 (18.2%) had died. Intravenous drug abuse, concurrent toxoplasma encephalitis and concurrent Pneumocystis jiroveci pneumonia independently predicted death outcome in both univariate and multivariate logistic regression analyses.
Toxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG1 and IgG2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.
Toxoplasmosis is caused by an obligate intracellular protozoan parasite; Toxoplasma gondii, which is one of the most important zoonotic parasite worldwide. In dogs, the sexual reproductive cycle of T. gondii is lacking, and the animals are not widely consumed as food, but they are vital in the mechanical transmission of the parasite. However, there is no present data on the exposure of stray dogs to T. gondii in Malaysia. The objective of this serological survey was to determine the prevalence of T. gondii antibodies (IgG) and associated factors in stray dogs in East and West Malaysia. Antibodies to T. gondii were determined in serum samples from 222 stray dogs from 6 different states in East and West Malaysia (Peninsular Malaysia) using an Indirect ELISA. The seroprevalence for T. gondii was 23.4% (Confidence interval: CI 17.8-29.2%). Stray dogs from Selangor and Kuala Lumpur had the highest seroprevalence (32.4%; CI 13.2-45.5%) and lowest in those from Penang and Kedah (12.5%; CI 1.3-23.5%). Gender and breed were not associated with T. gondii seropositivity. However, adult dogs were more likely to be seropositive for T. gondii (OR=2.89; CI 1.1-7.7) compared with younger dogs. These results revealed that T. gondii is prevalent in stray dogs in the studied areas in Malaysia, and indicative of the level of environmental contamination of this parasite especially in urban areas.
Toxoplasma gondii is a public health risk in developing countries, especially those located in the tropics. Widespread infection may inflict a substantial burden on state resources, as patients can develop severe neurological defects and ocular diseases that result in lifelong loss of economic independence. We tested sera for IgG antibody from 493 eye patients in Malaysia. Overall age-adjusted seroprevalence was estimated to be 25% (95% CI: [21%, 29%]). We found approximately equal age-adjusted seroprevalence in Chinese (31%; 95% CI: [25%, 38%]) and Malays (29%; 95% CI: [21%, 36%]), followed by Indians (19%; 95% CI: [13%, 25%]). A logistic regression of the odds for T. gondii seroprevalence against age, gender, ethnicity and the occurrence of six types of ocular diseases showed that only age and ethnicity were significant predictors. The odds for T. gondii seroprevalence were 2.7 (95% CI for OR: [1.9, 4.0]) times higher for a patient twice as old as the other, with ethnicity held constant. In Malays, we estimated the odds for T. gondii seroprevalence to be 2.9 (95% CI for OR: [1.8, 4.5]) times higher compared to non-Malays, with age held constant. Previous studies of T. gondii seroprevalence in Malaysia did not explicitly adjust for age, rendering comparisons difficult. Our study highlights the need to adopt a more rigorous epidemiological approach in monitoring T. gondii seroprevalence in Malaysia.
Toxoplasma gondii, the etiologic agent of toxoplasmosis, infects about 30 - 50% of the world population. The currently available anti-Toxoplasma agents have serious limitations. The present study aimed to investigate the effects of two antimalarials; buparvaquone (BPQ) and chloroquine (CQ), on immunocompromised mice with chronic cerebral toxoplasmosis, using spiramycin as a reference drug. The assessed parameters included the estimation of mortality rates (MR) among mice of the different study groups, in addition to the examination of the ultrastructural changes in the brain tissues by transmission electron microscopy. The results showed that only CQ treatment could decrease the MR significantly with zero deaths, while both spiramycin and BPQ caused an insignificant reduction of MR compared to the infected non-treated group. All the used drugs decreased the number of mature ruptured cysts significantly compared to the infected non-treated group, while only CQ increased the number of atrophic and necrotic cysts significantly. Furthermore, both spiramycin and BPQ improved the microvasculopathy and neurodegeneration accompanying the infection with different degrees of reactive astrocytosis and neuronal damage with the best results regarding the repair of the microvascular damage with less active glial cells, and normal neurons in the CQ-treated group. In conclusion, this study sheds light on CQ and its excellent impact on treating chronic cerebral toxoplasmosis in an immunocompromised mouse model.
One of the most important routes of transmission for Toxoplasma gondii infection is the ingestion of foods contaminated with cat feces containing sporulated oocysts. The diagnosis of T. gondii infection by fecal microscopy is complicated, as other similar coccidian oocysts are often present in the same fecal specimen. This study aimed to identify T. gondii oocysts in cat feces using a novel PCR technique. Feline fecal specimens (n = 254) were screened for coccidian oocysts by light microscopy using the Sheather's flotation method. PCR analysis performed on the same specimens targeted a 529 bp repeat element and internal transcribed spacer-1 (ITS-1) regions were used to confirm the presence of Toxoplasma oocysts. By light microscopy, 49/254 (19.3%) of specimens contained coccidian oocysts. PCR analysis demonstrated 2/254 (0.8%) and 17/254 (6.7%) positive results using Tox and ITS-1 primers, respectively. However, coccidian oocysts were not identified on microscopic examination of specimens that were PCR-positive by Tox primers. Coccidian oocysts were identified on microscopic examination of 6/17 (35.3%) of the PCR-positive fecal specimens using ITS-1 primers. The BLAST results of 16 ITS-1 sequences were identified as T. gondii (n = 12; 4.7%) and Hammondia hammondi (n = 4; 1.6%). There was slight agreement between the 529 bp and ITS-1 PCR results (κ = 0.148). This is the first report of the detection of Toxoplasma oocysts using PCR analysis on feline fecal specimens from Southern Thailand. The ITS-1 region has potential as an alternative marker to identify T. gondii oocysts in feline fecal specimens.
The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.
Toxoplasma gondii is an intra-cellular parasite that infects humans through vertical and horizontal transmission. The cysts remain dormant in the brain of infected humans and can reactivate in immunocompromised hosts resulting in acute toxoplasmic encephalitis which may be fatal. We determined the onset and progression of brain cysts generation in a mouse model following acute toxoplasmosis as well as the ability of brain cysts to reactivate in vitro. Male Balb/c mice, (uninfected control group, n = 10) were infected orally (study group, n = 50) with 1000 tachyzoites of T. gondii (ME49 strain) and euthanized at 1, 2, 4, 8 and 16 weeks post infection. Brain tissue was harvested, homogenized, stained and the number of brain cysts counted. Aliquots of brain homogenate with cysts were cultured in vitro with confluent Vero cells and the number of cysts and tachyzoites counted after 1 week. Brain cysts but not tachyzoites were detected at week 2 post infection and reached a plateau by week 4. In vitro Vero cells culture showed similar pattern for cysts and tachyzoites and reactivation of cyst in vitro was not influenced by the age of the brain cysts.
The inhibitory effect of active fractions of Eurycoma longifolia (E. longifolia) root, namely TAF355 and TAF401, were evaluated against Toxoplasma gondii (T. gondii). In our previous study, we demonstrated that T. gondii was susceptible to TAF355 and TAF401 with IC₅₀ values of 1.125 µg/mL and 1.375 µg/mL, respectively. Transmission (TEM) and scanning electron microscopy (SEM) observations were used to study the in situ antiparasitic activity at the IC₅₀ value. Clindamycin was used as positive control. SEM examination revealed cell wall alterations with formation of invaginations followed by completely collapsed cells compared to the normal T. gondii cells in response to the fractions. The main abnormality noted via TEM study was decreased cytoplasmic volume, leaving a state of structural disorganization within the cell cytoplasm and destruction of its organelles as early as 12 h of treatment, which indicated of rapid antiparasitic activity of the E. longifolia fractions. The significant antiparasitic activity shown by the TAF355 and TAF401 active fractions of E. longifolia suggests their potential as new anti-T. gondii agent candidates.
Toxoplasma gondii infection causes toxoplasmosis, an infectious disease with worldwide prevalence. The limited efficiency of drugs against this infection, their side effects and the potential appearance of resistant strains make the search of novel drugs an essential need. We examined Eurycoma longifolia root extract and fractions as potential sources of new compounds with high activity and low toxicity. The main goal of this study was to investigate the anti-T. gondii activity of crude extract (TACME) and four fractions (TAF 273, TAF 355, TAF 191 and TAF 401) from E. longifolia, with clindamycin as the positive control.
A review of the various studies on toxoplasmosis in peninsular Malaysia is presented. The period of review spanned between 1973 and 1980 during which a number of serological surveys were carried out for the presence of Toxoplasma gondii antibody in Malaysians, using either the indirect hemagglutination (I.H.A.) or the indirect fluorescent antibody (I.F.A.) tests. The prevalence rates of Toxoplasma antibody were consistently foundhighest among Malays, followed by Indians, Orang Aslis (Aborigines) and lowest among Malays, followed by Indians, Orang Aslia (Aborigines) and lowest among Chinese, the 4 major ethnic groups living in Malaysia. Positive titres, present in all age groups, showed an increase with age but no difference due to sex. However, higher prevalence of positive cases was recorded among rural dwellers and the lower socioeconomic group than from urban dwellers. The possible routes of infection among the ethnic groups were discussed. Among animal populations, the presence of Toxoplasma antibody was detected in buffaloes, swine, goats, cattle, cats and dogs. The epidemiological importance of the findings are discussed and suggestions made for future studies.
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.
During pregnancy, Toxoplasma gondii can be transmitted from mother to foetus and trigger a primary infection that may be symptomatic. It is important to distinguish between recently acquired and past infections to ensure proper treatment to minimize irreversible foetal injury. We used PCR of the B1 gene to evaluate the accuracy of T. gondii IgG antibody avidity testing in discriminating recent from past infection. In a cross-sectional study, T. gondii IgG and IgM antibodies were detected by enzyme linked fluorescence assay (ELFA) in 2120 serum samples from pregnant women referred to Karaj medical laboratories, February 2013 through March 2015 with 40 samples found positive. IgM-positive samples were evaluated by IgG avidity testing and PCR to amplify the B1 gene. Avidity studies indicated 20 samples with high IgG avidity, 15 with low IgG avidity, and five showing borderline values. The B1 gene was amplified in the borderline samples, with nine of the 15 showing low avidity. The B1 gene was not amplified in the high avidity sera. Our findings suggest that IgG avidity alone may not be sufficient to discriminate recent from past T. gondii infection and should not be used as the sole confirmatory test in pregnant women with IgG and IgM T. gondii antibodies. IgG avidity testing in combination with PCR may be more reliable for distinguishing between high- and low-risk infection and decrease the frequency of unnecessary treatment of pregnant women.
We have developed an ELISA that employs monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in patients' serum samples. Using serum spiked with Toxoplasma soluble and with SAG1 recombinant proteins, the detection limits were 31.25 ng/mL and 62.50 ng/mL, respectively. We obtained positive results in 28% (21/75) and 11% (23/206) of probable active and chronic toxoplasmosis serum samples, respectively. Western blot analysis on pooled antigen-positive serum samples showed antigenic bands of molecular weights 25 and 75 kDa from sera of probable active infection and five antigenic bands ranging in size from 26 to 33 kDa from chronic infection sera. This assay would be useful as an initial serum selection step in developing a Toxoplasma antigen detection test and for characterization studies.
In this study we have cloned unreported gene fragments of Toxoplasma gondii GRA7 and SAG1 and expressed the corresponding recombinant proteins, followed by evaluation of their usefulness for the serological diagnosis of toxoplasmosis. Both recombinant proteins were expressed efficiently in insoluble form, purified by single step Ni-NTA affinity chromatography and their antigenicity to detect toxoplasma specific IgG antibodies were determined by immunoblotting. A total of 60 serum samples from three groups of individuals based on their anti-toxoplasma antibody profiles were tested, namely (I) IgM+, IgG+ (n=20), (II) IgM-, IgG+ (n=20) and (III) IgM-, IgG- (n=20). Both recombinant proteins exhibited high sensitivity (100%) with sera from Group I. rGRA7 and rSAG1 reacted 40% and 80% respectively with Group II sera. The specificity of the recombinant proteins based on reactivities with Group III sera were 100% and 80% with rGRA7 and rSAG1 respectively. Thus rGRA7 was found to be better at discriminating probable acute from chronic phases of toxoplasmosis, and it also showed higher specificity.
Toxoplasmosis can cause serious disease in immunocompromised patients and to congenitally infected foetuses. Appropriate laboratory investigations in potential cases of acute Toxoplasma infection are important. Excretory secretory antigen (ESA) is immunogenic during both human and experimental infections, therefore is considered as a good candidate for investigation into new infection markers. In this study, ESA was prepared from in vitro cultures of Toxoplasma gondii to identify T. gondii ESA antigenic component(s) that is/are most reactive with serum samples from probable acute cases of toxoplasmosis. Serum samples were obtained from several categories of individuals with the following Toxoplasma serology: Group I: IgM+ IgG+ (low IgG avidity) or IgM+ IgG- from sera of patients who had clinical query of toxoplasmosis (n=35). Group II: IgM- IgG+ (high IgG avidity) from chronically infected individuals (n=30). Group III: normal/healthy individuals with anti-Toxoplasma IgMIgG- (n=20). Group IV: individuals with other infections who had anti-Toxoplasma IgM- IgG- (n=10). The ESA was subjected to SDS-PAGE, followed by Western blot analysis using the above sera and probed with peroxidase conjugated anti-human IgM and IgA antibodies. The blots were then developed using chemiluminescence substrate. The selected antigenic band was excised from the gel after two dimensional electrophoresis and sent for mass spectrometry analysis using MALDI TOF-TOF. The most promising antigenic band was a 10 kDa protein which showed sensitivity of 80% in both IgM and IgA blots, and specificity of 96.7% with sera from other infections and healthy controls. The two best identifications for the 10 kDa band were ubiquitin (ribosomal protein CEP52 fusion protein) and polyubiquitin.
In vitro culture of Toxoplasma gondii can provide tachyzoites which are active, viable and with desirable purity. Thus the aim of this study was to optimize the cell culture method for T. gondii propagation to obtain a consistent source of parasites with maximum yield and viability, but minimum host cell contamination for use in production of excretory-secretory antigen. Tachyzoites with seed counts of 1x10(6), 1x10(7) and 1x10(8) harvested from infected mice were added to VERO cells of different degrees of confluence, namely 50%, 85% and 100%, and examined periodically using an inverted microscope. When the maximum release of the tachyzoites was observed from the host cells, the culture supernatant was removed and the tachyzoites harvested. Using a Neubauer chamber, the percentages of viable tachyzoites and host cell contamination were determined using trypan blue stain. Parameters that gave the best yield and purity of viable tachyzoites were found to be as follows: VERO cells at 85% confluence in DMEM medium and inoculum comprising 1x10(7) tachyzoites. After about 3 days post infection, the tachyzoites multiplied 78x, with a yield of ~7.8x10(8) per flask, 99% viability and 3% host cell contamination. This study has successfully optimized the method of propagation of T. gondii tachyzoites in VERO cells which produce parasites with high yield, purity and viability.
During the period 1996-1998, 134 patients suspected of having ocular toxoplasmosis were seen in the Ophthalmology Clinic of the University Hospital, Kuala Lumpur. Clinical presentations in these patients ranged from poor vision to severe retinal detachment. Of these patients, 72% were confirmed positive for Toxoplasma gondii infection by serological methods. Chorioretinjtis and vitritis were found to be the most apparent symptoms, both having 100%correlation with serological positivity, This was followed by uveitis, floaters, and retinal detachment with correlation at 78%, 75%and 75%, respectively. However, there was no correlation between level of serotitre and ocular presentations. KEYWORDS: Toxoplasmosis, serology, chorioretinitis, uveitis
Toxoplasmosis is an opportunistic infection caused by the protozoan parasite Toxoplasma gondii. T. gondii is widespread globally and causes severe diseases in individuals with impaired immune defences as well as congenitally infected infants. The high prevalence rate in some parts of the world such as South America and Africa, coupled with the current drug treatments that trigger hypersensitivity reactions, makes the development of immunotherapeutics intervention a highly important research priority. Immunotherapeutics strategies could either be a vaccine which would confer a pre-emptive immunity to infection, or passive immunization in cases of disease recrudescence or recurrent clinical diseases. As the severity of clinical manifestations is often greater in developing nations, the development of well-tolerated and safe immunotherapeutics becomes not only a scientific pursuit, but a humanitarian enterprise. In the last few years, much progress has been made in vaccine research with new antigens, novel adjuvants, and innovative vaccine delivery such as nanoparticles and antigen encapsulations. A literature search over the past 5 years showed that most experimental studies were focused on DNA vaccination at 52%, followed by protein vaccination which formed 36% of the studies, live attenuated vaccinations at 9%, and heterologous vaccination at 3%; while there were few on passive immunization. Recent progress in studies on vaccination, passive immunization, as well as insights gained from these immunotherapeutics is highlighted in this review.