Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.     

Co-infection of Giardia intestinalis and Cyclospora cayetanensis in an immunocompetent patient with prolonged diarrhea: case report

Cyclospora cayetanensis is an agent of emerging infectious disease, and a recognized cause of diarrhea in some patients. Also, the flagellated protozoan, Giardia intestinalis, induces a diarrheal illness of the small intestine. Cases of cyclosporiasis are frequently missed, primarily due to the fact that the parasite can be quite difficult to detect in human fecal samples, despite an increasing amount of data regarding this parasite. On the other hand, G. intestinalis can be readily recognized via the microscopic visualization of its trophozoite or cyst forms in stained preparations or unstained wet mounts. In this report, we describe an uncommon case of co-infection with G. intestinalis and C. cayetanensis in an immunocompetent patient with prolonged diarrhea, living in a non-tropical region of Turkey.     

Cyclospora cayetanensis: a review of an emerging parasitic coccidian

Cyclospora cayetanensis is a sporulating parasitic protozoan that infects the upper small intestinal tract. It has been identified as both a food and waterborne pathogen endemic in many developing countries. It is an important agent of Traveller's Diarrohea in developed countries and was responsible for numerous foodborne outbreaks in the United States and Canada in the late 1990s. Like Cryptosporidium, infection has been associated with a variety of sequelae such as Guillain-Barré syndrome, reactive arthritis syndrome (formally Reiter syndrome) and acalculous cholecystitis.There has been much debate as to where to place C. cayetanensis taxonomically due to its homology with Eimeria species. To date, the only genomic DNA sequences available are the ribosomal DNA of C. cayetanensis and three other species; within these a high degree of homology has been observed. This homology and the lack of sequence data from other Cyclospora species have hindered identification methods.     

Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.     

Survey for Cyclospora cayetanensis in domestic animals in an endemic area in Haiti.

From January 1997 through July 1998, we examined stool samples from 327 domestic animals, including pigs, cattle, horses, goats, dogs, cats, guinea pigs, chicken, ducks, turkeys, and pigeons in Leogane, Haiti, for the presence of Cyclospora cayetanensis infection. No coccidian oocysts morphologically compatible with C. cayetanensis were detected in any of the animal samples, despite their living in, or near, households with infected individuals. These results suggest that domestic animals are not reservoir hosts for C. cayetanensis and that in this endemic area, humans are the only natural host for this parasite.     

Clonorchiasis: an update

Clonorchis sinensis, the Chinese or oriental liver fluke, is an important human parasite and is widely distributed in southern Korea, China (including Taiwan), Japan, northern Vietnam and the far eastern part of Russia. Clonorchiasis occurs in all parts of the world where there are Asian immigrants from endemic areas. The human and animal reservoir hosts (dogs, pigs, cats and rats) acquire the infection from the ingestion of raw fish containing infectious metacercariae. The first intermediate snail hosts are mainly species of Parafossarulus and Bithynia. Numerous species of freshwater fish serve as the second intermediate hosts of C. sinensis. Extensive studies of clonorchiasis during several decades in Japan, Korea, China and other countries have shown much progress in proving its morphological features including ultrastructure, biology, pathogenesis, epidemiology, clinical manifestations and chemotherapy. The present review deals with mainly current results obtained on the epidemiological, pathological and clinical aspects, as well as control measures in endemic areas. As for the complications of clonorchiasis, formation of calculi in the intrahepatic biliary passages is one of the most characteristic pathological features. It is sometimes accompanied by suppurative cholangitis, cholecystitis, cholangiohepatitis and ultimately can cause cholangiocarcinoma. Experimental results on the relationship to the occurrence of cholangiocarcinoma are presented. Clinical diagnosis by radiological findings including cholangiography, sonography and computerized tomography as well as magnetic resonance imaging for biliary or pancreatic ducts are outlined. Current studies on immunology and molecular biology of C. sinensis were introduced. Praziquantel is the drug of choice for clonorchiasis. The most effective regimen is 25 mg kg(-1) three times daily (total dose, 75 mg kg(-1)) administered orally at 5- to 6-h intervals over a single day. Prevention and control measures are also discussed.     

Echinococcosis: an emerging or re-emerging zoonosis?

The aim of this review is a critical discussion of factors actually or potentially contributing to persistence or emergence of echinococcosis in humans. Alveolar echinococcosis (AE), a life-threatening infection of humans, is caused by a larval stage of Echinococcus multilocularis. The adult parasite inhabits the intestine of foxes and other carnivores and has a wide distribution in the northern hemisphere (North America and northern and central Eurasia). Recent surveys in central Europe have extended the known geographical occurrence of E. multilocularis in foxes from four countries at the end of the 1980s to at least 11 countries in 1999. Cases of human AE previously regularly reported from only four countries are now recorded from seven countries, but the annual incidences are low. Since adequate information from earlier surveys is not available, it is not possible to conclude if the new findings reflect a recent extension of the parasite's range or just the first identification of hitherto unnoticed endemic areas. Evidence of parasite spreading has been reported from North America and Japan. Factors with the potential of enhancing the infection risk for humans in the future include increasing fox populations and parasite prevalences, progressing invasion of cities by foxes, the establishment of urban cycles of the parasite, and the spill-over of the E. multilocularis infection from wild carnivores to domestic dogs and cats. In view of the potential severity and fatality of AE in humans health authorities should initiate internationally coordinated countermeasures. Although control programmes against human cystic echinococcosis (CE), caused by E. granulosus, have been established in some countries and effective control strategies are available, the parasite has still a wide geographical distribution affecting many countries of all continents. Thus, human CE is persisting in many parts of the world with high incidences, and in some areas it is a re-emerging problem. For example, alarming increases of the number of human cases have been reported from Bulgaria and Kazakhstan, and the People's Republic of China. Progress in control can only be expected if health authorities attribute a higher priority to this disease and if all modern diagnostic and control options (for example vaccination of intermediate host animals) can be used.     

Waterborne transmission ofCampylobacter enteritis

Campylobacter jejuni is an important cause of human diarrheal disease throughout the world and likeSalmonella enteritidis, has a large animal reservoir which includes most of man's domestic animals. Until recently, it has been difficult to trace the chain of transmission from animals to man because of inadequate environmental sampling techniques and means to distinguish strains. Recent improvements in these techniques have made environmental studies more feasible in 2 water-related out-breaks.In 1 study,C. jejuni was found to be an important cause of sporadic, summertime diarrheal disease among hikers in national wilderness areas of Wyoming. In this setting, illness was significantly associated with drinking untreated surface water. SubsequentlyC. jejuni was isolated from surface water, including mountian streams, and from animals in the area. Some of the environmental isolates were serotypically identical to strains isolated from humans.A second study occurred as a result of an outbreak of Campylobacter enteritis in a community in northern Illinois which was epidemiologically associated with the community water system.Campylobacter jejuni was isolated from several surface water sources and from the implicated water system. These studies demonstrate that environmental isolation ofC. jejuni is now possible and may add to our understanding of disease transmission.     

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.     

Cholera Threat to Humans in Ghana Is Influenced by Both Global and Regional Climatic Variability

Cholera, an acute diarrheal illness, is caused by infection of the intestine with the bacterium Vibrio cholerae after ingestion of contaminated water or food. The disease had disappeared from most of the developed countries in the last 50 years, but cholera epidemics remain a major public health problem in many developing countries, most often localized in tropical areas. Cholera is an infectious disease for which a relationship between disease temporal patterns and climate has been demonstrated, but only in an endemic context and for local areas of Asia and South America. Until now, similar studies have not been done in an epidemic context, on the African continent, although the largest number of cholera cases has been reported for those countries by the World Health Organization. The wavelet method was used in order to explore periodicity in (i) a long-time monthly cholera incidence in Ghana, West Africa, (ii) proxy environmental variables, and (iii) climatic indices time series, from 1975 to 1995. Cross-analysis were done to explore links between these time series, i.e., between cholera and climate. Results showed strong statistical association (coherency) from the end of the 1980s, between cholera outbreak resurgences in Ghana and the climatic/environmental parameters under scrutiny. Further examination of the existence of common spatial and temporal patterns in infectious diseases on the continent of Africa will permit development of more effective treatment of disease.     

High infection rates at low transmission potentials in West African onchocerciasis

Onchocerciasis has been successfully controlled for many years in endemic countries but more than 120 million people are still at risk. Factors which stabilise the persistence of the parasite in the population must be studied to minimise the future risk of re-infection. Among these factors, the relationship between the annual transmission potential and the parasite establishment rate is a main determinant which has to date not been quantified. Using entomological information and palpation data collected by the Onchocerciasis Control Programme in West Africa prior to the initiation of control activities, we derive annual transmission potential-dependent estimates of the parasite establishment rate from statistical analyses and computer simulations. Even at very low transmission intensities, the filarial parasite Onchocerca volvulus can efficiently establish in the human population, originating from an infection process which is strongly limited with respect to the annual transmission potential. Implementing the estimates into a simplified transmission model predicts that the critical annual biting rate, below which transmission is not possible, is much lower than previously assumed. We conclude that under the current strategy of mass distribution of microfilaricides without additional measures of vector control, the risk of re-infection is higher than previously assumed.     

Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation -- a review

Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.     

Cryptosporidium parvum and Cyclospora cayetanensis: a review of laboratory methods for detection of these waterborne parasites

Cryptosporidium and Cyclospora are obligate, intracellular, coccidian protozoan parasites that infest the gastrointestinal tract of humans and animals causing severe diarrhea illness. In this paper, we present an overview of the conventional and more novel techniques that are currently available to detect Cryptosporidium and Cyclospora in water. Conventional techniques and new immunological and genetic/molecular methods make it possible to assess the occurrence, prevalence, virulence (to a lesser extent), viability, levels, and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne oocysts. These steps have been optimized to such an extent that low levels of naturally occurring Cryptosporidium oocysts can be efficiently recovered from water. The filtration systems developed in the US and Europe trap oocysts more effectively and are part of the standard methodologies for environmental monitoring of Cryptosporidium oocysts in source and treated water. Purification techniques such as immunomagnetic separation and flow cytometry with fluorescent activated cell sorting impart high capture efficiency and selective separation of oocysts from sample debris. Monoclonal antibodies with higher avidity and specificity to oocysts in water concentrates have significantly improved the detection and enumeration steps.To date, PCR-based detection methods allow us to differentiate the human pathogenic Cryptosporidium parasites from those that do not infect humans, and to track the source of oocyst contamination in the environment. Cell culture techniques are now used to examine oocyst viability. While fewer studies have focused on Cyclospora cayetanensis, the parasite has been successfully detected in drinking water and wastewater using current methods to recover Cryptosporidium oocysts. More research is needed for monitoring of Cyclospora in the environment. Meanwhile, molecular methods (e.g. molecular markers such as intervening transcribed spacer regions), which can identify different genotypes of C. cayetanensis, show good promise for detection of this emerging coccidian parasite in water.     

Plasmodium vivax gametocytes and transmission

Malaria elimination means cessation of parasite transmission. At present, the declining malaria incidence in many countries has made elimination a feasible goal. Transmission control has thus been placed at the center of the national malaria control programs. The efficient transmission of Plasmodium vivax from humans to mosquitoes is a key factor that helps perpetuate malaria in endemic areas. A better understanding of transmission is crucial to the success of elimination efforts. Biological delineation of the parasite transmission process is important for identifying and prioritizing new targets of intervention. Identification of the infectious parasite reservoir in the community is key to devising an effective elimination strategy. Here we describe the fundamental characteristics of P. vivax gametocytes - the dynamics of their production, longevity, and the relationship with the total parasitemia - as well as recent advances in the molecular understanding of parasite sexual development. In relation to malaria elimination, factors influencing the human infectivity and the current evidence for a role of asymptomatic carriers in transmission are presented.     

Helminth parasite proteomics: from experimental models to human infections

Schistosomiasis is a major human helminth infection endemic in developing countries. Urogenital schistosomiasis, caused by S. haematobium, is the most prevalent human schistosome disease in sub-Saharan Africa. Currently control of schistosome infection is by treatment of infected people with the anthelmintic drug praziquantel, but there are calls for continued efforts to develop a vaccine against the parasites. In order for successful vaccine development, it is necessary to understand the biology and molecular characteristics of the parasite. Ultimately, there is need to understand the nature and dynamics of the relationship between the parasite and the natural host. Thus, my studies have focused on molecular characterization of different parasite stages and integrating this information with quantitative approaches to investigate the nature and development of protective immunity against schistosomes in humans. Proteomics has proved a powerful tool in these studies allowing the proteins expressed by the parasite to be characterized at a molecular and immunological level. In this review, the application of proteomic approaches to understanding the human-schistosome relationship as well as testing specific hypotheses on the nature and development of schistosome-specific immune responses is discussed. The contribution of these approaches to informing schistosome vaccine development is highlighted.     

Gymnophalloides seoi eggs from the stool of a 17th century female mummy found in Hadong, Republic of Korea

It was previously reported that paleoparasitological clues for parasites infecting humans could be found in the feces of mummies of the Joseon Dynasty (1392-1910) in the Republic of Korea. Here, we report the presence of trematode eggs, including Clonorchis sinensis, Metagonimus yokogawai, and Gymnophalloides seoi (a human parasite known in Korea since 1993) in the feces of a recently excavated female mummy in Hadong, Republic of Korea. This is the first report of the discovery of a G. seoi infection in a human mummy. Since Hadong is currently not an endemic area for G. seoi, we speculate that the parasite might have occurred frequently along coastal areas of the Korean peninsula several hundred years ago and that the endemic areas contracted to, more or less, restricted regions since that time.     

Highly sensitive and specific PCR assay for reliable detection of Cyclospora cayetanensis oocysts

Multiple outbreaks of food-borne gastroenteritis caused by the coccidian parasite Cyclospora cayetanensis have been reported annually in North America since 1995. Detection of C. cayetanensis contamination typically relies on laborious and subjective microscopic examination of produce washes. Molecular detection methods based on nested PCR, restriction fragment length polymorphism, or multiplex PCR have been developed for C. cayetanensis; however, they have not been adequately validated for use on food products. Further challenges include reliably extracting DNA from coccidian oocysts since their tough outer wall is resistant to lysis and overcoming PCR inhibitors in sample matrices. We describe preliminary validation of a reliable DNA extraction method for C. cayetanensis oocysts and a sensitive and specific novel PCR assay. The sensitivity and repeatability of the developed methods were evaluated by multiple DNA extractions and PCR amplifications using 1,000-, 100-, 10-, or 1-ooycst aliquots of C. cayetanensis oocysts in water or basil wash sediment. Successful PCR amplification was achieved on 15 and 5 replicates extracted from aliquots containing 1,000 oocysts in water and basil wash, respectively. All 45 replicates of the 100-oocyst aliquots in water and 5 in basil wash were amplified successfully, as were 43/45 and 41/45 of the 10- and 1-oocyst aliquots in water and 9/15 and 2/15 in basil wash, respectively. The developed primers showed no cross-reactivity when tested against bacteria, nematodes, and protozoans, including Eimeria, Giardia, and Cryptosporidium. Our results indicate that these methods are specific, can reliably detect a single oocyst, and overcome many of the limitations of microscopic diagnosis.     

Contemporary environmental correlates of endemic bird areas derived from meteorological satellite sensors

The present-day distribution of centres of endemism is the result of an interplay between historical biogeography and contemporary environmental conditions. The relative importance of these two factors has never been established, however, for want of information on both the distributions themselves and the continental-scale measurement of environmental variables. Recently published maps of avian endemism in Africa, and the increasing availability of continental-scale surrogates of climatic conditions derived from Earth-orbiting satellites, has allowed this problem to be addressed directly. In this paper, temporal-Fourier-processed surrogate meteorological data derived from the National Oceanic and Atmospheric Administration''s series of polar-orbiting meteorological satellites and from the geostationary Meteosat satellites are used within a discriminant analytical framework to describe and predict areas of bird endemism in East Africa. The technique predicts endemic bird areas (EBAs) with an accuracy of 89% (mean 85%, range 70 to 89%). Contemporary environmental conditions, ultimately determined by climate, therefore appear to account for a substantial fraction of the observed variation in the distribution of EBAs. On the basis of these results, several hypotheses proposed to explain the distribution of centres of avian endemism are reviewed.