Waterborne Cryptosporidium species and Giardia duodenalis in resources of MENA: A systematic review and meta-analysis.

This review explores our understanding of Cryptosporidium species and Giardia duodenalis distribution in Middle East and North African (MENA) water resources. Results emphasize that Cryptosporidium species (sp.) and G. duodenalis (oo)cysts are present in distinct categories of water in ten MENA countries. Cryptosporidium sp. proportional prevalence in the MENA region was 24.5% (95% CI 16.3-33.8), while G. duodenalis prevalence was 37.7% (95% CI 21.9-55.1). Raw wastewater and surface water were the water categories most significantly impacted. Both parasites were reported in the various types of MENA drinking waters. The most frequent species/genotypes reported were C. hominis, C. parvum, and G. duodenalis assemblage A. Despite the high prevalence of (oo)cysts reported, we should consider the absence of waterborne outbreaks. This indicates significant underestimation and underreporting of both parasites in MENA. Stakeholders should apply water contamination legislation to eradicate Cryptosporidium sp. and G. duodenalis (oo)cysts from water resources/categories.

The impact of chlorine, ultraviolet-C, and microwave treatment on the survivability of Blastocystis sp. cysts.

Blastocystis sp. is a common widely distributed gut protozoan, with water transmission identified as one of its transmission routes. This study aimed to investigate the effect of chlorine, ultraviolet (UV)-C, and microwave (MW) treatments on the in vitro viability of cysts of Blastocystis sp. Purified Blastocystis sp. cysts were molecularly subtyped. Viable cysts were subjected to different free chlorine concentrations (1, 2, and 4 ppm), different doses of UV-C (5.13, 10.26, 20.52, and 40.47 mJ/cm2), and MW irradiation times (10, 15, 30, and 45 s). Viability reduction percentage, log10 inactivation, and micrometre-based optical microscopy examined cyst number and appearance after each disinfection trial. The three disinfectants' efficacy and application conditions were assessed. The analysed isolates of Blastocystis cysts were subtype 3, possessed varying sizes and shapes, but two identical genomes. The cysts of Blastocystis sp. were resistant to chlorine at all doses and exposure durations tested. UV-C at a dose of 40 mJ/cm2 and MW treatment for 15 s were able to completely disinfect the cysts. The MW was the most effective disinfectant against Blastocystis cysts based on all evaluated factors. MW irradiation is the most efficient water treatment method for eradicating Blastocystis cysts in an easy and safe manner.

Molecular characterization of Cryptosporidium and Giardia in environmental samples and faecal samples from biogas users in Bac Giang, Vietnam.

Little is known about Cryptosporidium and Giardia in biogas waste and humans in Vietnam. There is a potential risk of infections during or after using the biogas system. The detected protozoan genotypes are zoonotic pathogens, and contamination of vegetables may relay through runoff to the surface waters and soil. The objective of this study was to understand the role of the environment in the epidemiology of human infections in Bac Giang province, Vietnam, with a focus on investigating the presence of Cryptosporidium spp. genotypes and Giardia assemblages among 239 environmental samples and 94 faecal samples of biogas users. PCR and sequencing analysis were used to identify the occurrence and genotypes of Cryptosporidium and Giardia in these samples. Results showed that 13/333 (3.9 %) and 9/333 (2.7 %) samples were positive for Cryptosporidium oocyst and Giardia cysts, respectively. Characterization revealed the presence of Cryptosporidium scrofarum, C. suis, C. meleagridis, C. bailey and Giardia intestinalis assemblage A and E. C. scrofarum and Giardia assemblage E were identified for the first time in humans in Bac Giang. The current information from the above investigations will be valuable for protozoan source tracking and control interventions against Cryptosporidium and Giardia infection associated with biogas wastes in Vietnam.

Free-living amoebae in an oil refinery wastewater treatment facility.

Free Living Amoebae (FLA) are ubiquitous microorganisms reported from harsh environmental conditions. Oil refinery facilities consume vast volumes of water during their processes, generating a large amount of wastewater. The present study aimed to evaluate the wastewater treatment process in an oil refinery wastewater treatment facility (ORWWTF) for the presence of FLA. Water samples were collected from an oil refinery wastewater (ORWW) for nine months. After recording physical-chemical features, samples were cultivated onto non-nutrient agar (NNA). The discriminative fragments of the ribosomal RNA (rRNA) gene were amplified and sequenced to characterize the isolated FLA. Phylogenetic tree, and network analysis were employed to evaluate genetic relationships. The thermo- and osmotolerant tests were performed on the isolated FLA. Twenty-five (32.9%) samples were positive for FLA cultivation. Acanthamoeba spp., Vahlkampfiids, and Vermamoeba spp. were detected, of which Acanthamoeba species were predominant. There was no statistical correlation between pH, NH3, PO4, H2S, and TDS with the presence of FLA. A statistical correlation between the presence of FLA and the type of wastewater treatment plants (WWTPs) was significant (P-value = 0.011). All Acanthamoeba spp. isolates belonged to the genotypes T4 (17/21; 80.95%) and T11 (4/21; 19.05%). Vahlkampfiids were Naegleria spp., (7/10; 70%), Tetramitus aberdonicus (1/10; 10%), Learamoeba spp., (1/10; 10%), and Vahlkampfia spp., (1/10; 10%). All three Vermamoeba spp. were V. vermiformis. The ORWW contains toxic materials, and a few microorganisms can stay active in these environments. This is the first study which isolates FLA from such super harsh conditions. For the first time, T. aberdonicus, and Learamoeba spp., were isolated from oily wastewater. Our findings signify the concern due to the distribution of potentially pathogenic FLA to downstream lands via treated wastewater that may be released after treatment processing.

Well water sources simultaneous contamination with Cryptosporidium and Acanthamoeba in East-Southeast Asia and Acanthamoeba spp. in biofilms in the Philippines.

Cryptosporidium is the leading agent of waterborne parasitic protozoan outbreaks and is the second leading cause of infant mortality due to diarrhoea worldwide. Acanthamoeba spp. causes Acanthamoeba keratitis (AK) and a life-threatening condition known as granulomatous amoebic encephalitis (GAE). The present study aimed to assess the water quality of an indigenous and a rural community for waterborne parasitic protozoan contamination. Aquatic samples (n = 22) were processed by filtration of 500 mL portion through a 1.2 µm pore size glass microfiber filter and eluted for light microscopy, culture in non-nutrient agar, and PCR analysis. Overall, 36% (8/22) of the investigated aquatic samples were positive for either Cryptosporidium spp. oocysts (13%; 3/22) or Acanthamoeba spp., (36%; 8/22) or both (13%; 3/22). Cryptosporidium spp. oocysts were detected in 27% (3/11) of wet season samples only while Acanthamoeba spp. were detected in 18% (2/11) and 55% (6/11) of wet and dry season samples, respectively. Subsequently, molecular detection for Acanthamoeba species identified A. lenticulata and A. hatchetti with 98-99% BLAST similarity. This is the first report on the simultaneous contamination of Cryptosporidium and Acanthamoeba in well water sources in East-Southeast Asia, the first detection of Acanthamoeba spp. in biofilms in the Philippines, and the longest viability demonstrated for A. lenticulata in two-year-old water samples stored at room temperature.

Parasitological Assessment of Sewage Sludge Samples for Potential Agricultural Reuse in Tunisia.

Wastewater sludge represents an important resource for reuse in agriculture. However, potentially harmful pathogens are a main threat in this context. Thus, the aim of this study was to examine the presence of helminth ova and protozoan cysts in dried sewage sludge samples collected from ten wastewater treatment plants (WWTPs) located in eight governorates in Tunisia. Based on morphological criteria, protozoan cysts of Giardia spp., Entamoeba histolytica/dispar/moshkovskii, and Entamoeba coli, were detected in all dried sludge composite samples (N = 116) from the investigated WWTPs. The mean concentration ranged from 1.4 to 10.7 cysts per 100 g dry matter (DM). The identified helminth eggs were Ascaris spp., Strongyles, Taeniid eggs, Hymenolepis nana, Enterobius vermicularis, and hookworm species. Ascaris spp. and Taeniid eggs were detected in 56.9 and 74.1% of analyzed samples, respectively. The presence of Trichuris spp., Hymenolepis diminuta, and Toxocara spp. eggs in dried sewage sludge samples was low (0.9, 1.7, and 2.6%, respectively). The mean concentration of helminth eggs during the three-year study was less than 1 egg/100 g DM. All examined dried sewage sludge sample contents were below the WHO (2006) and US EPA (2003) recommendations, and thus, the sludge can potentially be reused in agriculture.

Cryptosporidium and Giardia in Biogas Wastewater: Management of Manure Livestock and Hygiene Aspects Using Influent, Effluent, Sewage Canal Samples, Vegetable, and Soil Samples.

Cryptosporidium and Giardia are two water- and foodborne protozoan parasites that can cause diarrheal diseases. Poor microbial quality, sanitation conditions, and hygiene practices at exposure to biogas wastewater are important risk factors for human and animal infection. This study highlights the presence and level of both parasites in the environment in relation to biogas waste reuse in Vietnam. A total of 239 samples were collected from different types of samples in the studied districts in Bac Giang province in 2020 via direct immunofluorescent detection to study the occurrence of Cryptosporidium spp. and Giardia spp. (oo)cysts. Among the samples, Cryptosporidium was found in 19 (7.9%) with concentration from 1.104 to 3.105 oocysts/100 mL, while Giardia in 40 (16.7%) with concentration from 1.104 to 2.106 cysts/100 mL, respectively. In detail, the results show that the percentages of positive detection of Cryptosporidium spp. and Giardia spp. in influent, effluent, sewage canal, and vegetables were 13.1% (11/84), 6.0% (5/83), 15.4% (2/13) and 5.9% (1/17) and 26.2% (22/84), 7.2% (6/83), 7.7% (1/13) and 5.9% (1/17), respectively. The results show a trend of decreasing Cryptosporidium and Giardia densities, without statistical significance. Although these parasites decreased after biogas treatment, the remaining loads observed in biogas effluent can reach the watercourses and soil receiving it. Further investigations are needed to contribute to a general understanding of the risk of protozoan parasites, as well as strategies to control and reduce the contamination of environmental water sources and plants and reduce the burden of the pathogens in biogas wastewater in Vietnam.

Survey and first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea, Turkey.

Infection with Acanthamoeba spp. may result in granulomatous amoebic encephalitis and Acanthamoeba keratitis. Water is an important habitat where Acanthamoeba species thrive. Therefore, studying the occurrence of this free-living amoeba in water sources will help understand the infection dynamics. The aim of the study was to survey and report on the presence of Acanthamoeba spp. in water resources from the Ordu and Giresun provinces in Black Sea. Acanthamoeba spp. was found in 1/17 natural spring water samples from Ordu and in 2/18 from Giresun. Acanthamoeba species were not detected in any of the investigated tap water samples. Sequencing of the (SSU) rDNA gene resulted in the identification of haplotype I (Acanthamoeba genotype: KJ094684). T4 (8.6%) was the only isolated genotype in both Ordu and Giresun provinces. This is the first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea. The occurrence of Acanthamoeba species in natural spring water sources should be considered as a potential risk for human infection, especially to high-risk populations.

Acanthamoeba species isolated from Philippine freshwater systems: epidemiological and molecular aspects.

Free-living amoeba (FLA) research in the Philippines is still in its infancy but has, by far, demonstrated the presence of potentially pathogenic species. Acanthamoeba may cause sight-threatening and central nervous system infections to humans, yet its epidemiologic distribution from local environmental sources is yet to be defined. The present study aimed to provide a baseline epidemiologic distribution of Acanthamoeba spp. in freshwater systems in the Philippines and establish potential pathogenicity of isolates through thermo-tolerance assay. A total of 63 water samples were collected from 13 freshwater systems all over the Philippine archipelago. The low-volume (50 ml) water samples were processed and cultured on non-nutrient agar lawned with Escherichia coli and observed for amoebic growth using light microscopy. Amoebic culture demonstrated 14.28% (9/63) positivity while further molecular testing of culture-positive plates using Acanthamoeba-specific primers demonstrated 100% (9/9) confirmation of Acanthamoeba species. Genotyping of Acanthamoeba isolates revealed T1, T3, T4, T5, T7, T11, and T15 genotypes. Thermo-tolerance assay demonstrated that T5 and T7 genotypes were potentially pathogenic strains. The evidence of environmental distribution of Acanthamoeba spp. in the freshwater systems in the Philippines and thermo-tolerance profile of isolates are significant aspects of amoeba study in public health and calls for initiatives in the dissemination of relevant information and the expansion of knowledge, awareness, and policies on pathogenic waterborne amoeba to mitigate, prevent, detect, and report cases of human infections.

Detection of Cryptosporidium and Giardia in foods of plant origin in North-Western Greece.

Giardia and Cryptosporidium are recognized as leading causes of waterborne and foodborne diarrhoeal disease with worldwide distribution. The study aimed to determine the protozoan contamination of various foods of plant origin. A total of 72 samples from 27 different varieties of fresh vegetables and fruits were collected from supermarkets and open markets in North-Western Greece and were examined using conventional diagnostic methods. Two out of 72 (2.8%) samples were found positive for Cryptosporidium oocysts, while no sample was found to be positive for Giardia cysts. The results show the presence of protozoan contamination in foods of plant origin, which may constitute a potential health hazard.

Intestinal protozoan and helminthic infections among hemodialysis and cancer patients.

Intestinal parasitic infections (IPIs) can be a severe threat to immunocompromised patients. This is particularly true for those undergoing chemotherapy and hemodialysis. The present research is aimed at identifying intestinal parasites that might be present in immunocompromised patients. In this cross-sectional study 1040 stool samples were collected from March to September 2017. Six hundred and forty-one stool samples from immunocompromised patients (279 samples from hemodialysis patients and 362 samples from chemotherapy patients) and 399 samples from the control group were collected in Guilan province, Iran. The samples were tested by direct, formalin-ether methods for protozoa and ova of intestinal parasites and Ziehl-Neelsen staining methods for coccidian parasites such as Cryptosporidium species. The overall parasitic infection rate was highest (15%) in hemodialysis patients and 11.3% in chemotherapy patients, whereas the lowest rate was observed (7.3%) in the control group. The infectivity rates were statistically significant (P = 0.008) when compared with the control group. The parasites found were Blastocystis hominis (8.9% of the cases), Entamoeba coli (1.6%), Iodamoeba butschlii (0.8%), Endolimax nana (0.6%), Chilomastix mesnili (0.5%), Strongyloides stercoralis (0.5%), and Taenia species (0.15%), whereas Giardia lamblia was detected only in the control group. There was not a correlation between prevalence of parasites with age or education levels of the infected individuals. Results of the present study suggest that periodic stool examinations in special parasitological laboratories should be included as part of routine and general medical care.

Detection of Cryptosporidium oocysts and Giardia cysts in vegetables from street markets from the Qinghai Tibetan Plateau Area in China.

Cryptosporidium and Giardia are well-known parasitic protozoans responsible for waterborne and foodborne diarrhoeal diseases. However, data are not available on market vegetables contaminated with Cryptosporidium and Giardia in China. In the present study, 642 different vegetable samples were collected from Xining City street vendors in the Qinghai Province to study the Cryptosporidium and Giardia contamination rates via PCR and sequence analyses. Cryptosporidium spp. and Giardia duodenalis were detected in 16 (2.5%) and 73 (11.4%) samples, respectively. Two species of Cryptosporidium, C. parvum (n = 11) and C. andersoni (n = 5), were identified. G. duodenalis assemblage B was identified in almost all positive samples (n = 72), except one sample that contained G. duodenalis assemblage E. We report on the rate of Cryptosporidium and Giardia contamination in vegetables for the first time from the Qinghai Tibetan Plateau Area (QTPA) in China.

Detection of Acanthamoeba spp. in two major water reservoirs in the Philippines.

Water reservoirs are important manmade structures providing water security to deliver clean and safe water for drinking and other purposes to the community. Eighty water samples were collected from Magat and Ipo water reservoirs using purposive sampling between November 2018 and January 2019. Water samples were collected in sterile containers for testing. The samples were cultured in non-nutrient agar and lawned with Escherichia coli and incubated at 33 °C. Twelve out of the 80 (15%) water samples were positive for amoebic growth. Light and scanning electron microscopy (SEM) revealed double-walled cystic stages and were initially identified as Acanthamoeba spp. based on morphological characteristic in reference to Page's established criteria. Their extracted DNAs were used in polymerase chain reaction using JDP1 and JDP2 primers and confirmed the presence of Acanthamoeba DNA in agarose gel electrophoresis. Aligned sequences from PCR products were deposited in GenBank under accession numbers MK886460, MK909919, MK905437, MK910997, MK911021 and MK886514. The presence of potentially pathogenic Acanthamoeba spp. in water reservoirs is considered a potential risk for public health, requiring appropriate processing of water in treatment plants.

Acanthamoeba spp. in river water samples from the Black Sea region, Turkey.

The present study aims to investigate the occurrence of free living amoeba (FLA) in water resources (rivers and tap water) in Samsun in the Black Sea. The presence of Acanthamoeba spp. was confirmed in 98 of 192 water samples collected from 32 sites of Samsun province (Samsun centre, Terme, Carsamba, Tekkekoy, Bafra) by PCR. Acanthamoeba spp. were found in 15/36 river samples from Samsun, in 58/90 from Terme, in 12/30 from Carsamba, in 7/18 from Tekkekoy and in 6/18 from Bafra. No Acanthamoeba species were detected in tap water samples. The highest rate in river waters contaminated with Acanthamoeba species was in Terme followed by Samsun centre (41.7%), Carsamba (40%), Tekkekoy (38.9%) and Bafra districts (33.3%), respectively. The result of the subsequent sequence analysis showed Haplotype I (A. triangularis) in 5%, Haplotype II (A. polyphaga) in 29.6%, Haplotype III (Acanthamoeba spp.) in 62% and Haplotype IV (A. lenticulata) in 3%. The most common genotype was Acanthamoeba T4 (Acanthamoeba spp., A. polyphaga, A. triangularis) and T5 genotype was also found in 3%. The T4 genotype is the most common genotype associated with Acanthamoeba keratitis (AK) worldwide; therefore, humans and animals living in the area are at risk after contact with such waters.

First detection of Echinococcus multilocularis in environmental water sources in endemic areas using capsule filtration and molecular detection methods.

Water is one of the possible transmission routes for water- and foodborne parasites to humans. Echinococcus multilocularis is a parasite, which causes alveolar echinococcosis (AE). Nevertheless, no environmental studies have been performed as yet to confirm the occurrence of E. multilocularis in water supplies. Accordingly, 105 water samples of 50 L volume were collected from surface waters (lakes, rivers, canals) and wells in the Warmia-Masuria Province (Echinococcus endemic area) and Pomerania Province (Echinococcus non-endemic area), Poland. The water was filtered and subsequently analysed with nested PCR and real-time PCR. E. multilocularis DNA was found in two (1.9%) samples, which originated from two lakes localised in the Warmia-Masuria Province. Sequencing of the positive samples confirmed that the PCR products were fragments of the E. multilocularis mitochondrial 12S rRNA gene. This is the first investigation describing E. multilocularis detection in environmental water samples, using molecular diagnostic tools. The results indicate that water could be considered as a potential source of E. multilocularis infections in humans and animals, in endemic areas.

Amoebicidal and Amoebistatic Effects of Artemisia argyi Methanolic Extracts on Acanthamoeba castellanii Trophozoites and Cysts.

PURPOSE : The present study aimed to investigate the amoebicidal and amoebistatic efects of Artemisia argyi leaf methanolic extract by testing the effects on trophozoites and on cysts. We also determined cytotoxic effect, enzymatic and non-enzymatic antioxidant activities, total phenolic, lavonoid and antioxidative contents of A. argyi. METHODS: A. argyi was harvested from various geographic sites in Ordu province in Turkey. The fresh leaves were subjected to methanolic extraction. In 100 µl culture, different concentrations of A. argyi methanolic extract (in quantities from 1.2, 2.3, 4.7, 9.4, 18.7, 37.4, 74.8 mg/ml) and the same volume of trophozoite/cyst suspension were mixed for the determination of the amoebicidal activity of the plant extract. Human bronchial epithelial cells were treated with the same concentrations of Artemisia extracts to determine cytotoxic potential. RESULTS: Total phenolic and lavonoid contents of the extract were calculated as 261 mg gallic acid/g dry extract and 29 mg quercetin/g dry extract, respectively. Total antioxidant activity was also calculated as 367 mg ascorbic acid/g dry extract. The growth of trophozoites stopped in A. argyi methanolic extract with 50% inhibitory concentrations (IC50)/8 h for 37.4 mg/ ml and 74.8 mg/ml extract solution and had stronger amoebicidal activity on the cysts with IC50/72 h. Artemisia showed stronger inhibitory effects on bronchial epithelial cells at the concentrations of 9.4, 18.7, 37.4 and 74.8 mg/ml. CONCLUSION: The study indicated that A. argyi leaf extract has cytotoxic and anti-amoebic activities.

A Case of Coenurosis in a Wild Rabbit (Lepus sinensis) Caused by Taenia serialis Metacestode in Qinghai Tibetan Plateau Area, China.

Six cystic metacestodes were found in the abdominal muscles of a wild rabbit, Lepus sinensis, in China. The coenurus contained one or more scolices armed with hooklets. Mitochondrial cox1 (1,623 bp) confirmed 98% homology with cox1 of Taenia serialis. This is the first report of T. serialis infection in an intermediate host in the Qinghai Tibetan Plateau Area, China.

Lasiopodomys fuscus as an important intermediate host for Echinococcus multilocularis: isolation and phylogenetic identification of the parasite.

BACKGROUND: Echinococcus multilocularis causes alveolar echinococcosis (AE) and is widely prevalent in Qinghai Province, China, where a number of different species have been identified as hosts. However, limited information is available on the Qinghai vole (Lasiopodomys fuscus), which is hyper endemic to Qinghai Province and may represent a potential intermediate host of E. multilocularis. Thus, L. fuscus could contribute to the endemicity of AE in the area. METHODS: Fifty Qinghai voles were captured from Jigzhi County in Qinghai Province for the clinical identification of E. multilocularis infection via anatomical examination. Hydatid fluid was collected from vesicles of the livers in suspected voles and subjected to a microscopic examination and PCR assay based on the barcoding gene of cox 1. PCR-amplified segments were sequenced for a phylogenetic analysis. E. multilocularis-infected Qinghai voles were morphologically identified and subjected to a phylogenetic analysis to confirm their identities. RESULTS: Seventeen of the 50 Qinghai voles had E. multilocularis-infection-like vesicles in their livers. Eleven out of the 17 Qinghai voles presented E. multilocularis infection, which was detected by PCR and sequencing. The phylogenetic analysis showed that all 11 positive samples belonged to the E. multilocularis Asian genotype. A morphological identification and phylogenetic analysis of the E. multilocularis-infected Qinghai voles confirmed that all captured animals were L. fuscus. CONCLUSIONS: L. fuscus can be infected with E. multilocularis and plays a potential role in the life cycle and epidemiology of E. multilocularis in the Qinghai-Tibetan Plateau of China.

First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens.

It is estimated that faecal-orally transmitted diseases are common in Afghanistan, as a consequence of poor hygienic standards of life and widespread contamination of water and food with both human and animal faeces. However, there is little information in the literature concerning infections caused by intestinal parasites in the Afghan population. In this study, we report the occurrence of Giardia intestinalis assemblages (A and B) in formalin-fixed stool samples collected from 245 Afghan schoolchildren living in Ghazni Province in eastern Afghanistan. Detection of the parasite's DNA and genotyping was performed using real-time PCR, specific to the ß-giardin gene of G. intestinalis. Positive results were recorded in 52 (21.2%) samples. Genotyping was successful in 39 faecal samples and showed the predominance of assemblage B of G. intestinalis in this population (15 assemblage A and 24 assemblage B). Co-infection with both genotypes A and B was detected in four samples. Additionally, we evaluated the effect of 10% buffered formalin fixative on the detection of G. intestinalis DNA using real-time PCR and nested PCR characterised by different lengths of PCR products (74 and 479 bp, respectively). The human faeces containing the Giardia cysts were tested for 16 weeks. Amplification of G. intestinalis DNA with real-time PCR was possible up to 6 weeks of preservation of stool sample in formalin, compared to only 2 weeks with nested PCR. This suggests that real-time PCR is a more suitable tool in cases where stool samples have to be kept in formalin for longer periods of time.

Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters.