Cyclospora cayetanensis: Molecular diagnosis (PCR); Molecular detection in fruits and vegetables (PCR).
Información 25-08-2018.
Cyclospora cayetanensis is a protozoan of the Apicomplexa phylum belonging to the group of coccidia, considered an emerging pathogen that causes important gastroenteritis in humans, both in immunocompetent individuals and in immunocompromised patients. It was initially described as the cause of prolonged watery diarrhea in humans in Papua New Guinea (1979). The designation of the species Cyclospora cayetanensis was from 1994 when it was described in Peru.
After ingesting and infecting the host, this protozoan follows a schizogonic (asexual) and sporogonic (sexual) cycle in the intestinal mucosa, which eventually leads to the elimination of immature oocysts with the faeces. These immature oocysts should sporulate over a period of one to several weeks at a temperature of 23°C to 27°C, under conditions of adequate moisture to be infective. When they are infective, they contain sporozoites, which, when the oocysts are ingested, are released into the intestinal lumen and infect the cells of the mucosa to regenerate the schizogonic and sporogonic cycles characteristic of coccidia.
There have been diarrheas associated with C. cayetanensis in both developing and developed countries, suggesting the cosmopolitan distribution of this organism. In many of the cases described in developed countries, there is an epidemiological background of recent stays in developing countries or the intake of water or food contaminated with oocysts. Various outbreaks have been reported in North America, South America, Asia, Africa, Australia, the United Kingdom and Western Europe associated with the consumption of contaminated food products such as: raspberries, salads, basil and raw clams.
Cyclospora cayetanensis is considered primarily a cause of traveler's diarrhea, especially in individuals who have traveled to countries where it is endemic such as Nepal, Peru, Indonesia and Guatemala, among others. Its diagnosis is not included in routine laboratory diagnostic procedures of gastroenteritis for immunocompetent patients, unless there is a specific reason such as prolonged diarrhea or a history of travel to an endemic region. In these geographic areas there seems to be a seasonal association between the appearance of diarrheal outbreaks produced by this organism and the warm months of the year. For example, in Nepal, this phenomenon coincides with monsoon rains, and when the temperature oscillates between 20 and 25ºC, that is to say in the months of June to August, while cases are infrequent during the dry periods of the year. On the other hand, this parasite has been identified in both immunocompetent and immunosuppressed symptomatic patients and in healthy individuals, which seems to highlight the existence of asymptomatic carriers. The proportion of carrier individuals, in tropical areas, varies and ranges from 2 to 10% of the population.
In immunocompetent individuals, the symptomatic infection presents characteristics similar to those of any non-invasive pathogen of the small intestine, with a syndromic profile indistinguishable from that of other intestinal coccidia. The incubation period is variable, from 1 to 14 days, although on average it is usually one week. Prodromes (initial symptoms), lasting 1 to 2 days, consist of malaise and low-grade fever. The state phase is characterized by the abrupt appearance of watery diarrhea, with 5 to 10 bowel movements daily, accompanied by asthenia, anorexia, nausea, vomiting, flatulence and, occasionally, abdominal pain, steatorrhea, D-xylose malabsorption, and myalgias. The initial diarrhea lasts three to four days, but after a few days, and for several weeks, intermittent diarrheal episodes appear, of variable intensity, which can determine a loss of body mass of 5 to 10%. In non-diarrheal periods, asthenia and anorexia remain. The duration of diarrhea is very variable, ranging from 4 to 107 days, with an average of four to nine weeks. The resolution of symptoms occurs abruptly and is associated with the disappearance of fecal oocysts.
In immunosuppressed patients, symptomatic courtship is similar to that observed in immunocompetent patients, although, unlike these, in which the process is self-limiting, in the former it is more insidious and prolonged in time, with a tendency to chronification, and always having a greater severity. Although C. cayetanensis is a primary intestinal pathogen, it has also been related to the production of non-lithiasic cholecystitis in patients infected with the human immunodeficiency virus, and with the Reiter and Guillain-Barré syndromes. A bronchopulmonary infection in a fecal excretory patient of oocysts has also been described.
Normally, the laboratory diagnosis of cycloporiosis is direct and based on the microscopic observation of immature oocysts in wet preparations, or in differential stains, made from fresh or preserved faeces. For this, fecal concentration, by centrifugation, modified Ritchie, or flotation, is advisable with Sheather's sucrose flotation method in serial stool samples, given the low or moderate, and even discontinuous, excretion of oocysts. Differential stains are used to demonstrate the characteristic acid-alcohol resistance of the oocysts of this organism. However, they are laborious methods, which require a lot of time and experience on the part of the specialist who carries them out, in addition to the fact that the use of differential stains poses problems of differentiation with the parasitic Cryptosporidium species of man, C. parvum and C. muris, especially the latter. Molecular techniques, therefore, seem to provide a solution to these disadvantages, detecting the presence of C. cayetanensis quickly, specifically and sensitively. However, it continues to be a challenge for many laboratories since the usual DNA extraction procedures used in laboratories from faeces or products that may contain oocysts in many cases are not effective. Therefore, it is necessary to follow an adequate protocol for washing the sample to recover them.
Tests performed in IVAMI:
(Based on the Bacteriological Analytical Manual method Chapter 19a: Detection of Cyclospora and Cryptosporidium from fresh produce).
- Application of separation methods to elute the oocysts of Cyclospora cayetanensis from their adhesion to fruits or vegetables.
- Concentration of the oocysts.
- Molecular detection by PCR with suitable extraction methods to obtain their DNA.
Recommended sample:
- Patient feces.
- Vegetables, red fruits (raspberrys) or others that are considered contaminated.
Conservation and shipment of the sample:
- Refrigerated (preferred) for less than 2 days.
- Frozen: more than 2 days.
Delivery of results:
- Molecular diagnosis in feces of patients: 24 to 48 hours.
- Molecular detection (PCR) for gene 18S C. cayetanensis: in fruits or vegetables: 3 to 5 days.
Cost of the test:
- Molecular detection (PCR): consult ivami@ivami.com.