Panagiotis Karanis

Waterborne transmission of protozoan parasites: review of worldwide outbreaks - an update 2011-2016

This review provides a comprehensive update of worldwide waterborne parasitic protozoan outbreaks that occurred with reports published since previous reviews largely between January 2011 and December 2016. At least 381 outbreaks attributed to waterborne transmission of parasitic protozoa were documented during this time period. The nearly half (49%) of reports occurred in New Zealand, 41% of the outbreaks in North America and 9% in Europe. The most common etiological agent was Cryptosporidium spp., reported in 63% (239) of the outbreaks, while Giardia spp. was mentioned in 37% (142). No outbreaks attributed to other parasitic protozoa were reported. The distribution of reported outbreaks does not correspond to more broadly available epidemiological data or general knowledge of water and environmental conditions in the reporting countries. Noticeably, developing countries that are probably most affected by such waterborne disease outbreaks still lack reliable surveillance systems, and an international standardization of surveillance and reporting systems has yet to be established.

Genetic polymorphism in Cryptosporidium species: An update

Cryptosporidia, widely distributed protozoan parasites of vertebrates, have attracted increasing interest due to several serious waterborne outbreaks, the life-threatening nature of infection in immunocompromised patients, and the realization of economic losses caused by these pathogens in livestock. Genetic polymorphism within Cryptosporidium species is being detected at a continuously growing rate, owing to the widespread use of modern molecular techniques. The aim of this paper is to review the current status of taxonomy, genotyping and molecular phylogeny of Cryptosporidium species. To this date, 20 Cryptosporidium species have been recognized. Two named species of Cryptosporidium have been found in fish, 1 in amphibians, 2 in reptiles, 3 in birds, and 12 in mammals. Nearly 61 Cryptosporidium genotypes with uncertain species status have been found based on SSUrRNA sequences. The gp-60 gene showed a high degree of sequence polymorphism among isolates of Cryptosporidium species and several subtype groups and subgenotypes have been identified, of which the Cryptosporidium parvum IIa and IId subtype groups were found to be zoonotic. This review describes considerable progress in the identification, genetic characterization, and strain differentiation of Cryptosporidium over the last 20 years. All the valid species, genotypes and zoonotic subtypes of Cryptosporidium reported in the international literature are included in this paper with respect to the taxonomy, epidemiology, transmission and morphologic-genetic information for each species.

Giardia taxonomy, phylogeny and epidemiology: Facts and open questions

Giardia duodenalis (synonymous Giardia lamblia and Giardia intestinalis) is a flagellated protozoan parasite that reproduces in the small intestine causing giardiasis. It is a cosmopolitan pathogen with a very wide host range, including domestic and wild animal species, as well as human beings. In this paper the current knowledge about the taxonomy and phylogeny of G. duodenalis is summarized from the international literature and data on the detection and epidemiology are also reviewed concentrating on the last 20 years. Authors highlighted the current knowledge and some aspects on G. duodenalis in particular, water transmission and in vitro cultivation. The review sheds light on the difficulties of the strain differentiation and multilocus molecular analysis of Giardia strains especially when applied to water samples containing low numbers of cysts and components complicating the problem of tracking sources of contamination. Genetic elements determining or conferring traits such as infectivity, pathogenicity, virulence, and immune interaction contributing to clearance are currently not well established, if at all. These should be useful and important topics for future research.

Development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of Cryptosporidium oocysts in fecal and water samples

ABSTRACT A loop-mediated isothermal amplification (LAMP) procedure for the detection of Cryptosporidium in environmental and fecal samples was developed and evaluated. This is the first demonstration of LAMP applied to detection of Cryptosporidium. Due to its specificity and simplicity, the method could become a useful diagnostic tool for epidemiologic studies of Cryptosporidium presence.

Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing

Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptosporidium DNA in 36 of 107 cattle (33.64%), in 26 of 85 sheep (30.5%) and in 17 of 78 horses (21.79%). The HSP LAMP specific to C. muris and C. andersoni, amplified Cryptosporidium DNA in one cow (0.9%), five sheep (5.8%) and seven horses (8.9%). The gp60 LAMP assay, shown to detect C. parvum produced no amplified Cryptosporidium DNA, likely due to low sample DNA concentrations. The specificity of LAMP assays was confirmed by sequencing of the LAMP products generated in positive samples. Sequence products from the three LAMP assays showed high identity to the target gene sequences confirming the specificity of LAMP. In this study, the LAMP procedure was clearly superior to nested PCR in the detection of Cryptosporidium species DNA. Use of LAMP is proposed as an efficient and effective tool for epidemiologic survey studies including screening of healthy animals in which Cryptosporidium oocyst shedding is characteristically low and likely below the detection limit of PCR in conventional sample concentrates.

Molecular characterization of Cryptosporidium from animal sources in Qinghai province of China

The presence of Cryptosporidium oocysts in 20 zoo animals of the Xining Zoo, 16 farm yaks and 42 farm goats in Qinghai province, China was investigated by an immunofluorescence test (IFT). The species and/or genotypes were determined by nested polymerase chain reaction (PCR) and sequence analysis of a fragment of the small subunit (SSU) rRNA gene. Cryptosporidium oocysts were found in 16 zoo animals, 2 yaks, and 15 goats by IFT. The IFT positive samples were further investigated by PCR, and 16 of them were found to be positive by that method also. Sequence analysis of the PCR products derived from Cryptosporidium oocysts from Black leopard (Panthera pardus), Heijing He (Grus nigricollis), Barbary sheep (Ammotragus lervia), Takin (Budorcas taxicolor), Lesser panda (Ailurus fulgens), and White-eared pheasant (Crossoptilon crossoptilon) fecal samples matched that of Cryptosporidium parvum mouse genotype. Sequence analyses of other PCR products were consistent with cervine genotype Cryptosporidium from Ibex (Capra ibex), a novel Cryptosporidium genotype from a wild yak (Bos mutus), C. bovis–like genotype from one goat sample and also a novel Cryptosporidium genotype from one other separate goat sample. The present work reports the first data on Cryptosporidium infections in animals from the Qinghai province of mountainous central western China and the first findings of the ‘cervine’ genotype in Capra ibex, C. bovis–like genotype and the new Cryptosporidium spp. in farm goat and in wild yak.

Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: Would it come to clinical reality as a point-of-care test?

Loop-mediated isothermal amplification (LAMP) is a novel molecular method that accelerates and facilitates DNA amplification and detection under isothermal conditions. It represents a revolution in molecular biology by reducing the high cost, turnaround time and technicality of polymerase chain reaction and other amplification methods. It has been applied for the diagnosis of a variety of viral, bacterial, parasitic and other diseases in the biomedical field. LAMP has been involved in studies concerning the diagnosis of malaria which is still a major cause of morbidity and mortality in different parts of the world. For the success attained with this technology to diagnose human malaria, is it time to think that LAMP-based point-of-care diagnostics come to application to support the diagnosis of clinical malaria cases? The present review deals with the use of LAMP in the diagnosis of malaria and related investigations to make a view on what has been investigated and highlights the future perspectives regarding the possible applications of LAMP in diagnosis of the disease.

Detection and characterisation of Giardia and Cryptosporidium in Hungarian raw, surface and sewage water samples by IFT, PCR and sequence analysis of the SSUrRNA and GDH genes.

We investigated the prevalence of Giardia and Cryptosporidium species and analysed the genotypes in 36 samples collected from different water sources and various geographic areas in Hungary. Samples were collected from drinking water and sewage treatment plants and from the recreation area of Lake Balaton. The (oo)cysts were purified according to the US EPA 1623 method and they were detected by immunofluorescence test (IFT). Genomic DNA was extracted from all samples and then the GDH target gene for Giardia and the SSUrDNA for both Giardia and for Cryptosporidium species were amplified by PCR. 24 out of 36 samples (67%) were Giardia positive and 15 (42%) were Cryptosporidium positive by IFT. PCR confirmed that 13 out of 36 samples (36%) were Giardia positive and 10 (28%) contained Cryptosporidium. Twelve Giardia and two Cryptosporidium PCR products were successfully sequenced. In seven samples G. lamblia Assemblage A and in one sample Assemblage B and in four cases Assemblages A and B have been found. In one sample C. parvum and in the other separate sample C. meleagridis were detected. Sequence analysis revealed a new subtype of G. duodenalis complex, clustered close to the Assemblage A group. This study provides the first report on simultaneous detection and genotyping of G. duodenalis and Cryptosporidium species from water supplies in Hungary.

Rapid identification of Giardia duodenalis by loop-mediated isothermal amplification (LAMP) from faecal and environmental samples and comparative findings by PCR and real-time PCR methods.

The detection of two human-pathogenic Giardia duodenalis assemblages A and B in faecal and water samples by loop-mediated isothermal amplification (LAMP) has been evaluated. The LAMP reaction is reproducible, rapid and specific for the detection G. duodenalis and has lower costs compared to the other molecular assays. This is the first application of LAMP for Giardia detection.

LAMP – a powerful and flexible tool for monitoring microbial pathogens

Loop-mediated isothermal amplification (LAMP) is one of the nucleic acid amplifications tests (NATs) available for organism identification applications in various fields such as infection diagnosis. It has been commonly described as a novel method, yet over 250 publications have appeared in less than 10 years since its original description [1]. LAMP has been applied to produce highly specific and sensitive amplification of DNA or RNA from virtually every corner of the biological world, including prokaryotes and eukaryotes, plant and animal tissue.