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Erythrocytes deficient in glucose-6-phosphate dehydrogenase (G6PD) is more susceptible to oxidative damage from free radical derived compounds. The hemolysis triggered by oxidative agents such as primaquine (PQ) is used for the radical treatment of hypnozoites of P. vivax. Testing of G6PD screening before malaria treatment is not a common practice in Thailand, which poses patients at risk of hemolysis. This retrospective study aimed to investigate the prevalence of G6PD in malaria patients who live in Southern Thailand. Eight hundred eighty-one malaria patients were collected for 8-year from 2012 to 2019, including 785 (89.1%) of P. vivax, 61 (6.9%) of P. falciparum, 27 (3.1%) of P. knowlesi, and 8 (0.9%) of mixed infections. The DiaPlexC genotyping kit (Asian type) and PCR-RFLP were employed to determine the G6PD variants. The result showed that 5 different types of G6PD variants were identified in 26 cases (2.9%); 12/26 (46.2%) had Mahidol (487G>A) and 11/26 (42.3%) had Viangchan (871G>A) variants, while the rest had Kaiping (1388G>A), Union (1360C>T), and Mediterranean (563C>T) variants. G6PD Songklanagarind (196T>A) variant was not found in the study. Our result did not show a significant difference in the malaria parasite densities in patients between G6PD-deficient and G6PD-normal groups. According to our findings, testing G6PD deficiency and monitoring the potential PQ toxicity in patients who receive PQ are highly recommended.

Artemisinin resistance (ART) has been confirmed in Greater Mekong Sub-region countries. Currently, C580Y mutation on Pfkelch13 gene is known as the molecular marker for the detection of ART. Rapid and accurate detection of ART in field study is essential to guide malaria containment and elimination interventions. A simple method for collection of malaria-infected blood is to spot the blood on filter paper and is fast and easy for transportation and storage in the field study. This study aims to evaluate LAMP-SNP assay for C580Y mutation detection by introducing an extra mismatched nucleotide at the 3’ end of the FIP primer. The LAMP-SNP assay was performed in a water bath held at a temperature of 56°C for 45 min. LAMP-SNP products were interpreted by both gel-electrophoresis and HNB-visualized changes in color. The method was then tested with 120 P. falciparum DNA from dried blood spot samples. In comparing the LAMP-SNP assay results with those from DNA sequencing of the clinical samples, the 2 results fully agreed to detect C580Y. The sensitivity and specificity of the LAMP-SNP assay showed 100%. There were no cross-reactions with other Plasmodium species and other Pfkelch13 mutations. The LAMP-SNP assay performed in this study was rapid, reliable, and useful in detecting artemisinin resistance in the field study.

Plasmodium vivax is usually considered morbidity in endemic areas of Asia, Central and South America, and some part of Africa. In Thailand, previous studies indicated the genetic diversity of P. vivax in malaria-endemic regions such as the western part of Thailand bordering with Myanmar. The objective of the study is to investigate the genetic diversity of P. vivax circulating in Southern Thailand by using 3 antigenic markers and 8 microsatellite markers. Dried blood spots were collected from Chumphon, Phang Nga, Ranong and, Surat Thani provinces of Thailand. By PCR, 3 distinct sizes of PvMSP3α, 2 sizes of PvMSP3β and 2 sizes of PvMSP1 F2 were detected based on the length of PCR products, respectively. PCR/RFLP analyses of these antigen genes revealed high levels of genetic diversity. The genotyping of 8 microsatellite loci showed high genetic diversity as indicated by high alleles per locus and high expected heterozygosity (HE). The genotyping markers also showed multiple-clones of infection. Mixed genotypes were detected in 4.8% of PvMSP3α, 29.1% in PvMSP3β and 55.3% of microsatellite markers. These results showed that there was high genetic diversity of P. vivax isolated from Southern Thailand, indicating that the genetic diversity of P. vivax in this region was comparable to those observed other areas of Thailand.

Background Drug resistance within the major malaria parasites Plasmodium vivax and Plasmodium falciparum threatens malaria control and elimination in Southeast Asia. Plasmodium vivax first-line treatment drug is chloroquine together with primaquine, and the first-line treatment for P. falciparum malaria is artemisinin in combination with a partner drug. Plasmodium vivax and P. falciparum parasites resistant to their respective first-line therapies are now found within Southeast Asia. The resistance perimeters may include high transmission regions of Southern Thailand which are underrepresented in surveillance efforts. Methods This study investigated blood samples from malaria centres in Southern Thailand. Genetic loci associated with drug resistance were amplified and sequenced. Drug resistance associated genes Pvmdr1, Pvcrt-o, Pvdhfr, and Pvdhps were characterized for 145 cases of P. vivax malaria, as well as the artemisinin resistance-associated Pfkelch13 gene from 91 cases of P. falciparum malaria. Results Plasmodium vivax samples from Southern Thai provinces showed numerous chloroquine and antifolate resistance-associated mutations, including SNP and Pvcrt-o K10-insertion combinations suggestive of chloroquine resistant P. vivax phenotypes. A high proportion of the C580Y coding mutation (conferring artemisinin resistance) was detected in P. falciparum samples originating from Ranong and Yala (where the mutation was previously unreported). Conclusions The results demonstrate a risk of chloroquine and antifolate resistant P. vivax phenotypes in Southern Thailand, and artemisinin resistant P. falciparum observed as far south as the Thai–Malaysian border region. Ongoing surveillance of antimalarial drug resistance markers is called for in Southern Thailand to inform case management. Electronic supplementary material The online version of this article (10.1186/s12936-019-2903-y) contains supplementary material, which is available to authorized users.

Malaria remains one of the most important communicable diseases. A rapid, simple and accurate method is a crucial part of malaria diagnosis. The aim of this study was to reevaluate the microwave irradiation method to extract DNA from Plasmodium falciparum and compare with six other existing DNA extraction methods such as QIAamp DNA mini kit (Qiagen), FTA elute card, phenol-chloroform, Chelex, Chelex without proteinase-K and Rapid boiling. Two different P. falciparum isolates were used: (i) Laboratory strains with 0.3% parasitemia and (ii) clinical isolate with 0.6% parasitemia. Each DNA extraction method was validated for the presence of P. falciparum by a routine nested and real time PCR. In order to evaluate the sensitivity of the DNA extraction by microwave, double serial dilution of P. falciparum from in vitro culture at parasitemia that ranged from 0.0001 to 0.17% were used to extract the DNA by microwave and the P. falciparum DNA was then detected by nested and real-time PCR. The nested and real-time PCR were able to detect. P. falciparum DNA at the parasitemia level as low as 0.0003% and 0.0001%, respectively. Our results can reproduce the results from earlier studies and reveal microwave as a rapid and simple tool to extract P. falciparum DNA and subsequent molecular diagnosis of malaria.

Summary Background The Greater Mekong subregion is a recurrent source of antimalarial drug resistance in Plasmodium falciparum malaria. This study aimed to characterise the extent and spread of resistance across this entire region between 2007 and 2018. Methods P falciparum isolates from Myanmar, Thailand, Laos, and Cambodia were obtained from clinical trials and epidemiological studies done between Jan 1, 2007, and Dec 31, 2018, and were genotyped for molecular markers (pfkelch, pfcrt, pfplasmepsin2, and pfmdr1) of antimalarial drug resistance. Genetic relatedness was assessed using microsatellite and single nucleotide polymorphism typing of flanking sequences around target genes. Findings 10 632 isolates were genotyped. A single long pfkelch Cys580Tyr haplotype (from −50 kb to +31·5 kb) conferring artemisinin resistance (PfPailin) now dominates across the eastern Greater Mekong subregion. Piperaquine resistance associated with pfplasmepsin2 gene amplification and mutations in pfcrt downstream of the Lys76Thr chloroquine resistance locus has also developed. On the Thailand–Myanmar border a different pfkelch Cys580Tyr lineage rose to high frequencies before it was eliminated. Elsewhere in Myanmar the Cys580Tyr allele remains widespread at low allele frequencies. Meanwhile a single artemisinin-resistant pfkelch Phe446Ile haplotype has spread across Myanmar. Despite intense use of dihydroartemisinin–piperaquine in Kayin state, eastern Myanmar, both in treatment and mass drug administrations, no selection of piperaquine resistance markers was observed. pfmdr1 amplification, a marker of resistance to mefloquine, remains at low prevalence across the entire region. Interpretation Artemisinin resistance in P falciparum is now prevalent across the Greater Mekong subregion. In the eastern Greater Mekong subregion a multidrug resistant P falciparum lineage (PfPailin) dominates. In Myanmar a long pfkelch Phe446Ile haplotype has spread widely but, by contrast with the eastern Greater Mekong subregion, there is no indication of artemisinin combination therapy (ACT) partner drug resistance from genotyping known markers, and no evidence of spread of ACT resistant P falciparum from the east to the west. There is still a window of opportunity to prevent global spread of ACT resistance. Funding Thailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.

To monitor drug resistance in Plasmodium vivax, a multidrug resistance 1 (Pvmdr1) gene and a putative transporter protein (Pvcrt-o) gene were used as molecular markers for chloroquine resistance. The biomarkers, the dihydrofolate reductase (Pvdhfr) gene and the dihydropteroate synthetase (Pvdhps) gene, were also used for the detection of resistance to sulphadoxine-pyrimethamine (SP), this drug is often accidentally used to treat P. vivax infections. Clinical blood samples (n = 120) were collected from patients who had been to one of eight malaria-endemic countries and diagnosed with P. vivax infection. The chloroquine resistance marker, the Pvmdr1 gene, showed F976:L1076 mutations and L1076 mutation. A K10 insertion in the Pvcrt-o gene was also found among the samples successfully sequenced. A combination of L/I57:R58:M61:T117 mutations in the Pvdhfr gene and G383:G553 mutations in the Pvdhps gene were also observed. Mutations found in these genes indicate that drug resistance is present in these eight countries. Whether or not countries are using chloroquine to treat P. vivax, there appears to be an increase in mutation numbers in resistance gene markers. The detected changes in mutation rates of these genes do suggest that there is still a trend towards increasing P. vivax resistance to chloroquine. The presence of the mutations associated with SP resistance indicates that P. vivax has had exposure to SP and this may be a consequence of either misdiagnosis or coinfections with&nbsp, P. falciparum in the past.

Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain.

Acanthamoeba, one of free-living amoebae (FLA), remains a high risk of direct contact with this protozoan parasite which is ubiquitous in nature and man-made environment. This pathogenic FLA can cause sight-threatening amoebic keratitis (AK) and fatal granulomatous amoebic encephalitis (GAE) though these cases may not commonly be reported in our clinical settings. Acanthamoeba has been detected from different environmental sources namely; soil, water, hot-spring, swimming pool, air-conditioner, or contact lens storage cases. The identification of Acanthamoeba is based on morphological appearance and molecular techniques using PCR and DNA sequencing for clinico-epidemiological purposes. Recent treatments have long been ineffective against Acanthamoeba cyst, novel anti-Acanthamoeba agents have therefore been extensively investigated. There are efforts to utilize synthetic chemicals, lead compounds from medicinal plant extracts, and animal products to combat Acanthamoeba infection. Applied nanotechnology, an advanced technology, has shown to enhance the anti-Acanthamoeba activity in the encapsulated nanoparticles leading to new therapeutic options. This review attempts to provide an overview of the available data and studies on the occurrence of pathogenic Acanthamoeba among the Association of Southeast Asian Nations (ASEAN) members with the aim of identifying some potential contributing factors such as distribution, demographic profile of the patients, possible source of the parasite, mode of transmission and treatment. Further, this review attempts to provide future direction for prevention and control of the Acanthamoeba infection.

Additional file 1: Table S1. A patient information and results summary (see Table B for P. vivax results summary by specimen).

The genotype of Toxoplasma gondii has been extensively studied to determine whether its characteristics may influence the course of toxoplasmosis. Recent genotyping studies in South America have found Toxoplasma hybrid isolates or atypical strains which were more virulent and highly diverse than North American and European species. Although, the high seroprevalence of Toxoplasma infection among pregnant women and HIV-infected patients from Songklanagarind Hospital, Songkhla, Southern Thailand has been previously reported, there is no reported study of human genotyping data on T. gondii strains. This is the first genetic typing of T. gondii isolates obtained from naturally infected cats in Thailand. Five chromosomal loci were analyzed by a nested PCR-RFLP technique to determine the genotype of 13 T. gondii isolates from cat feces gathered in the South of Thailand. The PCR-RFLP patterns of SAG1, SAG2-new, SAG3, BTUB and GRA6 markers resulted in five diverse genotypes: the type I (one isolate), type III (two isolates), type II or type III (one isolate), recombinant genotypes (two isolates) and atypical genotypes (two isolates). The presence of unusual genotypes may lead to new virulent traits associated with more severe forms of human Toxoplasma infections. More evaluations are needed before conclusions could be made as to whether the oocysts from cat feces play an important role in the severity of Toxoplasma infections.

Thai medicinal plants represent a rich source of potential anti-parasitic compounds. 1-hydroxy-2-hydroxymethylanthraquinone (CFQ) purified from Coptosapelta flavescens, a plant commonly used to expel intestinal worms, indicated potential anti-giardial agent as shown in a previous study. This study aims to investigate its mechanism of action. We assessed whether CFQ was involved as an inducer of apoptosis as well as having effects on the fine structure of Giardia intestinalis trophozoites. We observed the consequences of exposing G. intestinalis trophozoites to CFQ and metronidazole, both had an IC50 of 0.42μg/ml, after 6, 12 and 24h exposure. An apoptosis in trophozoite was confirmed by the AnnexinV-FITC assay and as viewed by flow cytometry. CFQ at its IC50 induced apoptosis as early as 6h after incubation while metronidazole produced little or no apoptosis at its IC50 value. Ultrastructural analyzes at 24h demonstrated that both CFQ and metronidazole induced several physical alterations, including the appearance of wrinkled and rounded cells, membrane blebbing, ventral disc damage, electron dense precipitates in the nuclei, all of which were indicative of cell death. However, membrane rupture was found only in G. intestinalis exposed to CFQ and this proved the induction of apoptosis. Taken together, we have provided a mechanistic explanation of the action of CFQ against G. intestinalis trophozoites. These results have provided further evidence that CFQ is a new compound that has the potential for use to treat infections from G. intestinalis.

Objective To investigate the antiprotozoal activity of medicinal plant extracts and isolated active compounds from the most active plant. Methods Twenty one medicinal plants with ethnobotanical use in Thailand, which were claimed to have anti-diarrhoeal or anti-parasitic activity, were screened for their anti-intestinal protozoan activity against Entamoeba histolytica ( E. histolytica ) and Giardia intestinalis ( G. intestinalis ). The most active compound was isolated and tested against E. histolytica and G. intestinalis . Results An acetone extract of Coptosapelta flavescens was the most active against both E. histolytica and G. intestinalis (minimal inhibitory concentration=125 and 15.63 μg/mL, respectively). Two anthraquinones and one naphthoquinone were isolated. The compound 1-hydroxy-2-hydroxymethylanthraquinone was the most active chemical against E. histolytica and G. intestinalis with minimal inhibitory concentration values of 20 and 2.5 μg/mL, respectively. In time killing assay, the percentage of viable G. intestinalis , when compare to control, after expose to compound 1-hydroxy-2-hydroxymethylanthraquinone showed significantly ( P E. histolytica , its activity was comparable to metronidazole. Conclusions These observations provide preliminary evidence that 1-hydroxy-2-hydroxymethylanthraquinone from Coptosapelta flavescens can be considered to be a potential anti-parasitic agent against E. histolytica and G. intestinalis infections.

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.

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.

This study investigated the distribution of parasites as main contaminants in water environments of peninsular Malaysia (October 2011–December 2011) and the southeastern coast of Thailand (June 2012). Sixty-four water samples, 33 from Malaysia and 31 from Thailand, of various water types were examined according to U.S. Environmental Protection Agency guidelines. Drinking or household water types from both countries were free from parasitic contamination. The recreational/environmental (except a swimming pool in Malaysia) and effluent water types from these two countries were contaminated with waterborne parasites: Giardia (0.04–4 cysts/L), Cryptosporidium (0.06–2.33 oocysts/L), hookworm (6.67–350 ova/L), Ascaris (0.33–33.33 ova/L), and Schistosoma (9.25–13.33 ova/L). The most contaminated sites were recreational lake garden 3 in Malaysia and river 2 in Thailand. Higher concentrations of Giardia, Cryptosporidium, and hookworm were found in samples from Malaysia than in samples from Thailand. The presence of Giardia cysts showed a significant association with the presence of Cryptosporidium oocysts (P < 0.005).

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

During 2009 to 2010, a total of 408 blood samples collected from malaria patients in Ranong (149) and Yala (259) Provinces, Thailand were investigated for Plasmodium spp using microscopic examination. There are no statistical differences in the prevalence of P. falciparum and P. vivax in samples collected from Ranong and Yala (46% vs 52%, and 54% vs 45%, respectively). Single nucleotide polymorphism of codon 86 in pfmdr1 (encoding P. falciparum multidrug resistance protein 1) was investigated among 75 samples of P. falciparum and 2 samples of P. knowlesi. A pfmdr1 N86Y mutation was detected in 1 out of 29 samples and 45 out of 46 samples obtained from Ranong and Yala Provinces, respectively. It is interesting that pfmdr1 was detected in two P. knowlesi DNA samples obtained previously from Ranong Province which was 99% homologous to pfmdr1 obtained from falciparum parasites in the same area but the mutation was not observed. The difference in multidrug resistance protein in Plasmodium obtained from those two border areas of Thailand will be of use in monitoring drug resistance in these border regions of the country.

Background 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. Methods A total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia (53), Thailand (120), the Philippines (33), and Vietnam (15). A physicochemical analysis was conducted. The water samples were processed in accordance with the US Environmental Protection Agency’s methods 1622/1623.1, microscopically observed and subsequently screened using qPCR assays. Results Cryptosporidium oocysts were detected in treated water samples from the Philippines (1/10), with a concentration of 0.06 ± 0.19 oocyst/L, and untreated water samples from Thailand (25/93), Malaysia (17/44), and the Philippines (11/23), with concentrations ranging from 0.13 ± 0.18 to 0.57 ± 1.41 oocyst/L. Giardia cysts were found in treated water samples from the Philippines (1/10), with a concentration of 0.02 ± 0.06 cyst/L, and in untreated water samples from Thailand (20/93), Vietnam (5/10), Malaysia (22/44), and the Philippines (16/23), with concentrations ranging from 0.12 ± 0.3 to 8.90 ± 19.65 cyst/L. The pathogens C. parvum and G. lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene, respectively. C. parvum was detected in untreated water samples from the Philippines (1/23) and Malaysia (2/44), whilst, G. lamblia detected was detected in treated water samples from the Philippines (1/10) and in untreated water samples from Thailand (21/93), Malaysia (12/44), and the Philippines (17/23). Nitrate concentration was found to have a high positive correlation with (oo)cyst (0.993). Conclusion The presence of (oo)cysts in the water samples means that there is potential risk for zoonotic disease transmission in the studied countries. Detection using qPCR is feasible for quantifying both pathogenic C. parvum and G. lamblia in large water samples.

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.

Toxoplasmosis is one of the most common opportunistic parasitic diseases in patients living with HIV/AIDS. This study aimed to determine the seroprevalence of Toxoplasma infection in HIV-infected patients and to identify associated risk factors in Toxoplasma seropositive patients. This study was conducted at a regional public hospital in Hat Yai, southern Thailand during October 2009 to June 2010. Blood samples were collected from 300 HIV-infected patients. Each subject also answered a socio-demographic and risk factors associated with Toxoplasma infection. The prevalence of anti-Toxoplasma IgG antibodies in HIV-infected patients was 109 (36.3%), of which 83 (76.2%) had past infection and 26 (23.9%) had recently acquired Toxoplasma infection as indicated by their IgG avidity. Multivariate analysis using logistic regression showed that gender difference (adjusted OR = 1.69, 95% CI = 1.05–2.72) was the only factor associated with Toxoplasma infection. From the results obtained, these HIV-infected patients could be at high risk of developing clinical evidence of severe toxoplasmosis. Therefore, it is necessary to introduce primary behavioral practices to prevent Toxoplasma infection among HIV-infected patients.

Toxoplasma gondii is an important parasite in pregnant women. This case-controlled study assessed the seroprevalence of toxoplasmosis in 640 pregnant women in southern Thailand and identified their associated risk factors. The overall seroprevalence of toxoplasmosis was 181 (28.3%). Of this, 138 (21.6%) were positive for only anti-Toxoplasma immunoglobulin G (IgG) antibody, 43 (6.7%) were positive for both IgG and IgM antibodies, and none were positive for IgM antibody. Multivariate analysis revealed that increasing age (adjusted odds ratio [OR] = 1.64, 95% confidence interval [CI] = 1.01–2.67), living outside Songkhla province (adjusted OR = 1.56, 95% CI = 1.08–2.24), parity (adjusted OR = 1.65, 95% CI = 1.01–2.68), contact with cats (adjusted OR = 1.70, 95% CI = 1.20–2.43), and drinking of unclean water (adjusted OR = 1.70, 95% CI = 1.08–2.68) were factors associated with Toxoplasma seroprevalence. On the basis of the results obtained, a health surveillance program should be initiated as a primary preventive measure for congenital toxoplasmosis and focus on educating women of the child-bearing age group to avoid contact with cats and to strictly practice personal hygiene.

The aim of this study was to develop a simple and reliable method to determine the viability of Giardia intestinalis after incubation with an anti-giardial agent by using a colorimetric method. Factors that may affect the optical density value were systematically evaluated. The most suitable conditions were obtained when G. intestinalis trophozoites, 5 × 105 cells/ml were incubated with the anti-giardial agent for 48 h. The culture medium was removed and trophozoites were immediately fixed by immersing the whole plate in absolute methanol for 2 min. The fixed trophozoites were then stained with 0.1% w/v methylene blue for 10 min, washed once by immersing the whole plate into distilled water. The dye was released by adding 0.1 M hydrochloric acid solution (300 μl) and the optical density was read at 655 nm. The 50% inhibitory concentration values (IC50) of metronidazole, ornidazole and furazolidone obtained from our proposed method (0.41 ± 0.06, 0.18 ± 0.01, 0.26 ± 0.13 μg/ml, respectively) were comparable to the IC50 values obtained by the current conventional method (0.14 ± 0.05, 0.15 ± 0.04, 0.14 ± 0.02 μg/ml, respectively). This new method did provide a convenient and reliable way to screen for potential anti-giardial agents.

Cyclization of oxime with different aldehydes and ketones under basic condition led to the formation of new dioxazole derivatives and the structure was elucidated by spectral data. The effects of diaoxazoles on the inhibition of growth of Entamoeba histolytica and Giardia intestinalis in vitro have been determined, and selected compounds further investigated for their toxicity. SAR showed that the compounds with 5-nitrothiophene group at the 3-postion of the diaoxazole ring were more active than those with the p-toluene group at the same position. It is interesting to note that the compounds found active against E. histolytica were not found active against G. intestinalis. Toxicity studies showed that the compound 8 and 9 were non-toxic against Vero cell line ATCC CCL-81.

A new anthraquinone, morinquinone, together with 18 known anthraquinones were isolated from the stems of Morinda elliptica Ridl. Their structures were elucidated on the basis of spectroscopic data. They each showed weak inhibitory activity against a susceptible strain of Staphylococcus aureus and a methicillin-resistant S. aureus. Damnacanthal was effective against Microsporum gypseum (MIC 1 μg/mL). Lucidin was active against Entamoeba histolytica (MIC 31.25 μg/mL) and Giardia intestinalis (MIC 7.8 μg/mL).

This study evaluated the anti-giardial activity of chloroform, methanol and water extracts of 12 medicinal plants (39 extracts), commonly used as self medication by AIDS patients in southern Thailand. The plant extracts and a standard drug, metronidazole, were incubated with 2x10(5) trophozoites of Giardia intestinalis per millilitre of growth medium in 96-well tissue culture plates under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and the minimum inhibitory concentration and the IC50 value for each extract was determined. The chloroform extracts from Alpinia galanga, Boesenbergia pandurata, Eclipta prostrata, Piper betle, Piper chaba, Zingiber zerumbet, and the methanol extracts from B. pandurata and E. prostrata were classified as "active", i.e. with an IC50 of100 microg/ml, whereas the chloroform extract from Murraya paniculata was classified as being "moderately active". This study shows that extracts from some medicinal plants have potential for use as therapeutic agents against G. intestinalis infections.

The anti-amoebic effects of crude methanol extracts of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall against Entamoeba histolytica infecting the caecum of mice were studied. Caecal amoebiasis in mice was induced by injection of Entamoeba histolytica trophozoites directly into the caecum. The mice were then treated orally with the extract, a standard drug (metronidazole), or vehicle p.o. for five consecutive days, beginning 24 h after the infection and were examined on the sixth day. At a dose of 1000 mg/kg per day, the extracts of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall had a curative rate of 100, 40 and 26%, respectively. At a concentration of 500 and 250 mg/kg/day, extract from Piper longum fruit was still effective in 93 and 46% of the cases, respectively, while extract from Piper sarmentosum root at a dose of less than 1000 mg/kg per day did not cure any mice from amoebiasis. Extract of Quercus infectoria nut gall at a concentration of 500 and of 250 mg/kg per day cured 26 and 13% of mice, respectively. Metronidazole at a concentration of 125 and of 62.5 mg/kg per day had a curative rate of 100 and 60%, respectively. The severity of caecal wall ulceration was reduced in mice which received the extract and metronidazole as compared to the control animals.

Background Toxoplasma gondii, an obligate intracellular protozoan parasite, causes a disease called toxoplasmosis which can sometimes be acquired congenitally by a newborn from an infected mother. This study aimed to determine the seroprevalence of Toxoplasma infection and its associated risks among 219 and 215 pregnant women from Malaysia and Myanmar, respectively. Methods Anti-Toxoplasma IgG and IgM antibodies were screened by using standard commercial ELISA kits. The socio-demographic, obstetrics and risk factors associated with Toxoplasma infection data were compared between the two countries. Results The overall prevalence of Toxoplasma infection in Malaysian pregnant women (42.47%; 95% CI = 36.11-49.09) was significantly higher (p < 0.05) than Myanmar pregnant women (30.70%; 95% CI = 27.92-37.16). By univariate analysis, this study identified that age group, education, parity, awareness on toxoplasmosis and consumption of undercooked meat were significantly associated (p < 0.05) with Toxoplasma seropositive Malaysian pregnant women but none of these factors associated with Toxoplasma seropositive Myanmar pregnant women. In comparison using univariate analysis between the two countries, it was found that Toxoplasma seropositive Malaysian pregnant women was associated with aged 30 years and above, secondary or lower-secondary level of education, the third trimester of pregnancy, having one child or more, lacking awareness of toxoplasmosis, absence of bad obstetrics history, having no history of close contact with cats or soil, living on a farm and also consumption of undercooked meat, unpasterized milk or untreated water. Avidity measurement was used to confirm the stages of Toxoplasma infection in pregnant women who were positive for both IgG and IgM antibodies and found all were infected in the past. Conclusion From our study, Toxoplasma screening and its risk measurement in pregnant women is firmly recommended for monitoring purposes and assisting proper management, including diagnosis and treatment during antenatal period. Also, it is necessary to initiate preventive measures for Toxoplasma infection among reproductive-age women in general and seronegative pregnant women in particular. Avidity measurement should be incorporated in Toxoplasma routine screening, especially with the availability of a single serum sample to assist in the diagnosis. Electronic supplementary material The online version of this article (doi:10.1186/s13071-014-0564-9) contains supplementary material, which is available to authorized users.

Toxoplasma gondii, is one of the infectious agents of congenital TORCH infections, causes severe clinical outcomes in fetus and newborns. Nevertheless this life-threatening parasitic disease is preventable by simple preventive measures related to lifestyle during pregnancy. We aim to study on the knowledge about toxoplasmosis and practices that prevents this infection among the pregnant women. Total of 2598 pregnant women from Malaysia, Philippines, and Thailand were randomly surveyed to determine the knowledge and their practices on Toxoplasma infection. The questionnaire covered respondents' general information and knowledge on plausible risks factors, symptoms, timing of infection, prevention knowledge, and preventive behavior regarding Toxoplasma infection. Majority of these pregnant women were in their age group of 20–29 years (50.9%), completed secondary level of education (51.7%), in their second trimester of pregnancies (38.1%), non-parous (36.6%), and had no history of abortion (90.4%). Based on this survey, only 11% of these pregnant women had read, heard, or seen information regarding toxoplasmosis and 3.5% of them were aware of being tested for the infection. A small percentage of these pregnant women knew that T. gondii were shed in the feces of infected cats (19.4%) and sometimes found in the raw or undercooked meat (11.0%). There was 16.1% of responding women knew that toxoplasmosis is caused by an infection. Demographic profiles such as age group, level of education, pregnancy term, and number of children of the pregnant women showed significant association with their responses toward prevention knowledge and preventive behavior related questions (P < 0.05). Thus, it is suggested that health education on toxoplasmosis and primary behavioral practices should be consistently offered to reproductive age women in general and pregnant women in particular. This information could help to reduce vertical transmission of Toxoplasma infection during pregnancy.

Background Despite the amount of awareness created, waterborne disease still poses threat, especially in developing countries. Due to the scarcity of reported data on waterborne parasites, the consumption of unsafe water prolongs. Thus, the occurrences of waterborne parasites from various samples were investigated from one of the Southeast Asian country, the Philippines. Methods A total of thirty three samples, each consisting of twelve liters, were collected and processed to obtain the sediment. Ten liters of sample each was processed to detect Cryptosporidium spp. and Giardia spp. using an immunomagnetic separation method prior to enumeration via fluorescence microscope. Meanwhile, the remaining two liters were cultured to detect Acanthamoeba and Naegleria through microscopy examination and polymerase chain reaction (PCR) analysis. Results Twelve samples (36.4%) from river (5), swimming pool (1), pond (3), rain tank (1), and natural lake (2) were positive for Cryptosporidium spp., 17 (45.5%) samples from river (9), pond (2), swimming pool (1), rain tank (1), and natural lake (4) were positive for Giardia spp. while, 13 (33.3%) samples from river (3), swimming pool (2), pond (2), dispenser (1), well (1), tap (2) and natural lake (2) were positive for Acanthamoeba spp. and 5 (18.2%) samples from river (1), natural lake (1), tap (1), dispenser (1) and mineral (1) were Naegleria spp. positive. Physical parameters such as temperature, conductivity, total dissolved solid (TDS), salinity, dissolved oxygen (DO), pH, and turbidity and chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite were also measured. The highest chemical contamination was observed at pond 2. A good correlation was observed between Giardia and nitrite (r = 0.736, p

Parasites cause a significant burden of disease globally and the majority of parasitic infections fall into the category of Neglected Tropical Diseases (NTDs). The emergence of parasitic diseases, including zoonotic infections, is associated with the process of rapid urbanization, exponential population growth, borderless trade between countries and climate change. To adopt new methods of control, a number of important issues must be addressed such as environmental-based interventions, novel drug discovery, the next generation sero-markers and development of new strategies for parasite control, and so forth. In this special issue on parasites: from source to vector and human, we have invited a few papers that address such issues. The first paper of this special issue addresses serine proteinase activities in excretory-secretory products of Toxocara canis second stage larvae (TES) related to the known sequences of TES components for example, TES26 and MUC4. The authors use protein modelling to identify molecules that can be incorporated into diagnostic and therapeutic techniques ultimately leading to better methods of control. The second paper on Chagas' disease describes extensively on aspects of transmission, association with pregnant women and congenital infection, clinical manifestations, diagnosis, treatment, control strategies, case follow-up and disease complications in Latin America. The third paper of this special issue presents a brief overview of the helminth-associated immune response, the currently available helminth secretome data, some major secretome-derived immunomodulatory molecules, their potential mode of actions and the applicability of helminth-derived therapeutic proteins in the treatment of allergic and autoimmune inflammatory disease. The fourth paper addresses the greater risk of exposure to tick borne pathogens (Anaplasma phagocytophilum and Ehrlichia chaffeensis) among rural residents using serological mass screening in China. Significant risk factors associated with infection are: having contact with animals, planting crops, having more employment time and having a history of fever. This study also proposes safety measures required to reduce zoonotic transmission. The fifth paper shows the effectiveness of different concentrations of two fractions of Curcuma longa cortex rich in turmerones and their respective liposomal formulations for treating promastigote forms of Leishmania amazonensis. The hexane fraction from the turmeric cortex, incorporated in liposomal formulation (LipoRHIC), presents a promising result for a new anti-leishmanial agent. The sixth paper uses climate modelling for global warming to indicate geographical spread, increasing number of seasonal generations and epidemiological changes of Dirofilaria spp. In addition to dirofilariosis, it also covers other mosquito-borne diseases in Russia and its neighbouring countries. The final paper of this special issue reports on the next generation sero-marker for toxoplasmosis with improved specificity and sensitivity using Toxoplasma gondii dense granular protein-5 (GRA5) on human serum samples. Veeranoot Nissapatorn Nongyao Sawangjaroen Rogan Lee Subhash Chandra Parija

The objective of this study was to assess the physico-chemical parameters and waterborne parasites in selected recreational lakes from Malaysia. Samples were collected from seven stations of Recreational Lake A (RL-A) and six stations of Recreational Lake B (RL-B). The samples were processed to detect the presence of Giardia spp. and Cryptosporidium spp. using immunomagnetic separation kit, helminth eggs or ova by bright field microscopy and Acanthamoeba spp. by cultivation in non-nutrient agar. Chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite and physical parameters such as dissolved oxygen, electrical conductivity, pH, salinity, temperature and total dissolved solid were also measured. Both lakes were freshwater with salinity ranging from 0.05 to 0.09 ppt. Most stations of these lakes were contaminated with Cryptosporidium spp., Giardia spp., Ascaris spp. and hookworm. Schistosoma spp. was found in RL-B only, while Acanthamoeba spp. was found in all stations. Of all sampling sites, station 5 of RL-B is the most contaminated. Linear regression and correlation analysis revealed that Giardia spp. and Schistosoma spp. showed a significant negative correlation with turbidity (p

After 30 years of the human immunodeficiency virus (HIV) epidemic, parasites have been one of the most common opportunistic infections (OIs) and one of the most frequent causes of morbidity and mortality associated with HIV-infected patients. Due to severe immunosuppression, enteric parasitic pathogens in general are emerging and are OIs capable of causing diarrhoeal disease associated with HIV. Of these, Cryptosporidium parvum and Isospora belli are the two most common intestinal protozoan parasites and pose a public health problem in acquired immunodeficiency syndrome (AIDS) patients. These are the only two enteric protozoan parasites that remain in the case definition of AIDS till today. Leishmaniasis, strongyloidiasis and toxoplasmosis are the three main opportunistic causes of systemic involvements reported in HIV-infected patients. Of these, toxoplasmosis is the most important parasitic infection associated with the central nervous system. Due to its complexity in nature, toxoplasmosis is the only parasitic disease capable of not only causing focal but also disseminated forms and it has been included in AIDS-defining illnesses (ADI) ever since. With the introduction of highly active anti-retroviral therapy (HAART), cryptosporidiosis, leishmaniasis, schistosomiasis, strongyloidiasis, and toxoplasmosis are among parasitic diseases reported in association with immune reconstitution inflammatory syndrome (IRIS). This review addresses various aspects of parasitic infections in term of clinical, diagnostic and therapeutic challenges associated with HIV-infection.

Cyclization of oxime with different aldehydes and ketones under basic condition led to the formation of new dioxazole derivatives and the structure was elucidated by spectral data. The effects of diaoxazoles on the inhibition of growth of Entamoeba histolytica and Giardia intestinalis in vitro have been determined, and selected compounds further investigated for their toxicity. SAR showed that the compounds with 5-nitrothiophene group at the 3-postion of the diaoxazole ring were more active than those with the p-toluene group at the same position. It is interesting to note that the compounds found active against E. histolytica were not found active against G. intestinalis. Toxicity studies showed that the compound 8 and 9 were non-toxic against Vero cell line ATCC CCL-81.

Walsura robusta Roxb. (Family: Meliaceae) is a well-known multi-purpose medicinal plant, and has been employed for a wide range of disease conditions without documented scientific data. In the current study, four pure isolated compounds, 3,4,5-trimethoxyphenyl beta-D-glucopyranoside (1), turpinionoside A (2), (+)-lyoniresinol 3alpha-O-beta-D-glucopyranoside (3) and (-)-lyoniresinol 3alpha-O-beta-D-glucopyranoside (4), were isolated from the leaves and twigs of W. robusta. Biological evaluation for free radical scavenging, antibacterial and antigiardial activities was performed. We investigated antioxidant effects of the crude extracts as well as the isolated compounds using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), hydroxyl radical (OH), and superoxide anion (O(2)) scavenging assays. Three phenolic glucosides (1, 3 and 4) were found to possess strong antioxidant activity. They scavenged DPPH(*) with IC(50) values in the range of 51.5-86.6 microM. We also detected the superoxide dismutase-like activities in compounds 3 and 4 which are lignan glucosides, demonstrating potent superoxide scavenging activity with IC(50) values in the range of 0.8 and 0.7 microM, respectively. Other biological activities including antibacterial and antigiardial assays were carried out. Preliminary results demonstrated that most extracts, except the diethyl ether extract, exhibited inhibition zones against all Gram-positive bacteria including Bacillus cereus, Staphylococcus aureus, Streptococcus mutans, and S. pyogenes. Aqueous extracts of this plant species could inhibit Gram-positive and some Gram-negative bacteria such as Escherichia coli, Salmonella typhi and Shigella sonnei. However, the determination of minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of W. robusta on all tested bacterial strains showed only weak activity, and their MBCs were greater than 25 mg mL(-1). For antigiardial activity, incubation with 2 x 10(5) trophozoites mL(-1) of the culture medium with the crude extracts at concentration ranged from 31.25 to 1000 microg mL(-1) demonstrated no activity (MIC > 1000 microg mL(-1)).

The anti-amoebic activities of chloroform, methanol and water extracts from 12 Thai medicinal plants (39 extracts) commonly used by AIDS patients in southern Thailand were screened, at a concentration of 1,000 microg/ml, against Entamoeba histolytica strain HTH-56:MUTM and strain HM1:IMSS growing in vitro. The extracts were incubated with 2x10(5) E. histolytica trophozoites/ml of medium at 37 degrees C under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and scored (1-4) according to the appearance and numbers of the trophozoites. The extracts that caused inhibition were selected and retested using the same conditions but with concentrations that ranged from 31.25 to 1,000 microg/ml using E. histolytica strain HM1:IMSS, and the IC(50) values for each extract were calculated. The chloroform extracts from Alpinia galanga (IC(50) 55.2 microg/ml), Barleria lupulina (IC(50) 78.5 microg/ml), Boesenbergia pandurata (IC(50) 45.8 microg/ml), Piper betle (IC(50) 91.1 microg/ml) and Piper chaba (IC(50) 71.4 microg/ml) and the methanol extract from B. pandurata (IC(50) 57.6 microg/ml) were all classified as "active", i.e. with an IC(50) of less than 100 microg/ml, whereas those from Murraya paniculata (IC(50) 116.5 microg/ml) and Zingiber zerumbet (IC(50) 196.9 microg/ml) were classified as being "moderately active". The IC(50) of a standard drug, metronidazole, was 1.1 microg/ml.

The activities of n-hexane, dichloromethane and methanol extracts from five anti-diarrheic Thai medicinal plants, Acacia catechu (Fabaceae) resin, Amaranthus spinosus (Amaranthaceae) whole plant, Brucea javanica (Simaroubaceae) seed, Piper longum (Piperaceae) fruit and Quercus infectoria (Fagaceae) nut gall were tested against the in vitro growth of fresh isolates of the intestinal protozoan parasite, Blastocystis hominis. The extracts at concentrations that ranged from 62.5 to 2000 microg/mL, were incubated with several isolates of Blastocystis hominis for 48 h. The activities were classified as killed, inhibited, moderately inhibited and not-inhibited. Dichloromethane and methanol extracts from the Brucea javanica seed and a methanol extract from Quercus infectoria nut gall showed the highest activity. At a concentration of 2000 microg/mL, the three extracts killed 82, 75 and 67% of the Blastocystis hominis samples tested and inhibited 94, 100 and 76% of them, respectively. Metronidazole, used as a reference antiprotozoan drug, at a concentration of 40 microg/mL, killed 97% of the Blastocystis hominis isolates and inhibited all samples tested at concentrations that ranged from 1.25 to 20 microg/mL.

The hookworm Ancylostoma caninum induces human eosinophilic enteritis (EE), probably via allergic responses to its secretions. Cysteine and metallo-proteinases may be the components of these secretions that elicit hypersensitivity reactions. In order to characterize genes encoding cysteine proteinases (CP) secreted by A. caninum, an adult hookworm cDNA library was constructed and screened with a cloned fragment of a hookworm CP gene. This fragment was obtained using consensus oligonucleotide, CP-gene-specific primers in the polymerase chain reaction. cDNAs encoding two CPs were obtained from the library and sequenced. The first gene, AcCP-1, encoded a cathepsin B-like zymogen CP of 343 amino acids (aa), predicted to be processed in vivo into a mature CP of 255 aa. Closest nucleotide identities were to Haemonchus contortus cysteine protease (61%) and to human cathepsin B (60%). The second gene, AcCP-2, encoded a mature CP of 254 aa, that showed 86% identity to AcCP-1, and 58% and 47% identity to bovine cathepsin B and human cathepsin B, respectively. Rabbit antisera raised against recombinant AcCP-1 reacted with esophageal, amphidial and excretory glands in formalin-fixed, paraffin embedded sections of both male and female adult hookworms, and with an antigen of approx. 40 kDa in Western blot analysis of excretory/secretory products from adult hookworms. Together, these immuno-hybridization results strongly suggest that the CP encoded by the AcCP-1 gene is secreted by hookworms. These are the first reported CP genes from hookworms. Proteinases encoded by these genes may be responsible for the CP activity that we have shown previously to be secreted by adult A. caninum.

This paper reports the first survey in Australia to use faecal culture to detect Dientamoeba fragilis in patients with diarrhoea. Of 3 different protozoal culture media evaluated on a case of known infection, modified Boeck & Drbohlav's medium was the most suitable. The organism could be grown from faeces stored for up to 24 h at room temperature, but for only 10 h at 4 °C. Culture was then used, in combination with microscopy of smears fixed with polyvinyl alcohol and trichrome-stained, to examine single stool specimens from 260 consecutive patients with diarrhoea in the city of Brisbane. D. fragilis was detected in 4 (1·5%) specimens, only 2 of which were positive by microscopy. Other protozoa were found in 36 (13·8%) specimens: Blastocystis hominis in 28 (10·8%), Giardia duodenalis in 4 (1·5%), Endolimax nana in 3 (1·2%), and Entamoeba coli in one (0·4%). One strain of Dientamoeba was cryopreserved in liquid nitrogen and recultured successfully 10 d later. Culture was more sensitive than microscopy in diagnosing D. fragilis infection and the organism, of dubious pathogenicity, was not common in patients with diarrhoea in this community. Prevalence surveys of intestinal protozoan infections should use faecal culture, and specimens should be less than 12 h old and not refrigerated. Dientamoeba strains isolated by culture can be cryopreserved.