Toxoplasmosis: beyond animals to humans

The parasitic zoonosis toxoplasmosis, which was poorly understood before the advent of the HIV epidemic, has become a major clinical problem worldwide. Humans acquire toxoplasmosis from cats, from consuming raw or undercooked meat and from vertical transmission to the foetus through the placenta during pregnancy. Studies of the unique environmental factors in various communities indicate the important roles that eating habits and culture have on the transmission of this infection. The socioepidemiological aspects of toxoplasmosis are thought to be important contributing factors for the spread of this disease. Preventative measures should consider the cultures and beliefs of people in various communities more than solving poverty and giving orthodox health education.     

Development of a Rapid,Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings.     

Impact of health educational programmes on the prevalence of enterobiasis in schoolchildren in Thailand

Enterobiasis is a worldwide prevalent disease particularly in low income areas. The budget needed for the prevention, treatment and eradication of the disease has thus far frustrated the limited budgets of global public health systems. A study was undertaken to determine if education in addition to medical treatment of enterobiasis could make a difference to the rates of infection. A total of 777 children (399 male and 378 female) from 11 elementary schools in five districts of Samut Prakan Province, Thailand were examined between December 2000 and March 2002. In five of the 11 schools studied, medical treatments were applied, followed by a programme of educating the children in the prevention of infection. Children in the remaining six schools received medical treatment only. The study showed a decrease in infections among children who received supplementary education. This decrease was significant in comparison to the decrease shown among children who received medical treatment only. The study therefore showed that educating high risk individuals played a key role in the prevention of enterobiasis.     

Toxoplasma gondii infection: What is the real situation?

The prevalence of chronic Toxoplasma infections reported in the literature varies enormously. We hypothesize that one factor could be due to the different methods used in the evaluation of infections. Serological evidence of Toxoplasma infections in 450 pregnant women (PW) and 300 HIV-infected patients (HIV) were investigated by the Sabin–Feldman dye test and two other commercial ELISA kits (kit1 and kit2). Anti-Toxoplasma IgG antibodies obtained from the Sabin–Feldman dye test, ELISA kit1 and ELISA kit2 in the PW subjects were 14.7%, 29.6% and 38.7%, and in the HIV subjects were 13%, 34.7% and 36.3%, respectively. So there were significant differences in the seroprevalences when different diagnostic tests were used (P < 0.05). Regarding Sabin–Feldman dye test as the gold standard for anti-Toxoplasma antibodies detection, we found that the sensitivity and specificity of the ELISA kit1 and kit2 was in the range of their specification. However as the two ELISA kits used in our study identified a much higher prevalence of Toxoplasma infections which indicated that false positive cases were being reported. Based on results obtained, it is therefore highly recommended that research workers should be aware that the reports of serological studies in terms of high positive results should be treated with some skepticism until additional precise diagnostic tools are developed.     

Blastocystis subtype 5: Predominant subtype on pig farms,Thailand

Blastocystis is a unicellular protist most commonly detected in humans and a variety of animals. The predominant mode of its transmission is the fecal–oral route, but its zoonotic potential is not completely understood. The objective of this study was to determine the presence and genetic diversity of Blastocystis on pig farms in Nakhon Pathom Province, Central Thailand. A total of 154 human and 90 pig stool samples were collected and analyzed. Nested PCR detected Blastocystis in 35.55% of the pig samples and 6.49% of the human samples. Subtyping based on regions of the small-subunit ribosomal RNA (SSU rRNA) gene identified three Blastocystis subtypes in pigs and humans: ST1, ST3, and ST5. Blastocystis ST5 was the predominant subtype, followed by ST1 and then ST3. All the sequences from the Blastocystis-positive samples from both pigs and humans were closely related. This study reveals a possibility of low host specificity of Blastocystis STs (ST1, ST3 and ST5) on pig farms in Thailand. We tentatively suggest that close contact with or exposure to pig stools may be a significant source of Blastocystis detected in pig handlers. Further studies are required to confirm the zoonotic transmission of this organism in Thailand, because pigs may play an important role in the transmission of Blastocystis.     

Plasmodium falciparum: effect of anti-malarial drugs on the production and secretion characteristics of histidine-rich protein II

Plasmodium falciparum histidine-rich protein II (HRP2) is one of the best documented malaria proteins. However, little is known about the development of HRP2 concentrations under the influence of anti-malarial drugs. HRP2 levels were determined in cell medium mixture, cellular compartment, and in culture supernatant using a double-site sandwich ELISA specific for HRP2. Characteristic increases in the overall HRP2 levels were found during the later ring and the trophozoite stages. Throughout the later schizont development, rupture, and reinvasion, however, the HRP2 levels remained comparatively stable. When the cultures were exposed to serial dilutions of anti-malarial drugs, a distinct inhibition of HRP2 production was seen with increasing concentrations of drugs, resulting in sigmoid dose-response curves, similar to those obtained from conventional drug sensitivity assays. HRP2 therefore allows for a very accurate estimation of parasite development and its inhibition and may therefore be ideally suited for use in drug sensitivity or bioassays.     

Cryptosporidium Oocyst Detection in Water Samples: Floatation Technique Enhanced with Immunofluorescence Is as Effective as Immunomagnetic Separation Method

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 10¹, 10², and 10³ per 10 µl were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 10² per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.     

Toxoplasma gondii: Simple duplex RT-PCR assay for detecting SAG1 and BAG1 genes during stage conversion in immunosuppressed mice

Toxoplasmic encephalitis (TE) is caused by reactivation of dormant bradyzoites into rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immune-compromised hosts. Diagnosis of this life-threatening disease is complicated, since it is difficult to distinguish between these two stages. It is, therefore, mainly based on a test positive for T. gondii antibodies, and specific clinical symptoms. We developed a duplex RT-PCR to detect the expression of bradyzoite (BAG1) and tachyzoite (SAG1) specific genes simultaneously during tachyzoite/bradyzoite stage conversion. The conversion reaction was observed in many organs of experimental mice, indicated by tachyzoites in the cerebrum, cerebellum, heart and lung, beginning in week 1 after the suppression period, and continuing until the end. Bradyzoites were also detected in nearly all organs throughout the study, suggesting that during the reactivation period, bradyzoites not only escape from cysts and reinvade neighboring cells as tachyzoites, but are also driven into developing new bradyzoites. The results of our study show that duplex RT-PCR is an easy, rapid, sensitive, and reproducible method, which is particularly valuable when numerous samples must be analyzed. This technique may usefully serve as an alternate tool for diagnosing TE in severely immunocompromised patients.