Cyclospora

C. cayetanensis are obligate intracellular parasites of the human small intestine. Oocysts of C. cayetanensis are spherical and 8–10µm in diameter. The oocysts have a 50 nm-thick wall and a bilayer threadlike outer coat(3). Infection rates in the general population of up to 41.6% have been reported(20)

The oocyst is an undifferentiated sphere containing a morula  when it is first shed in the faeces, which is not infective (meaning the direct faecal–oral route utilised by Cryptosporidium does not occur). It may take days to weeks (5–12 days at 26–30°C) for sporulation of the oocysts to occur, depending on the environmental conditions, when the sporont divides into two sporocysts (4 by 6.3 μm) and stieda and substieda bodies. Each sporocyst contains two elongated 1.2 by 9 μm sporozoites that are folded in two with a membrane-bound nucleus and micronemes. When the sporulated oocysts of C. cayetanensis are ingested, the oocyst excsyts, freeing the sporozoites, typically in the jejunum of the small intestine. Once they invade the intestinal epithelial cells, the schizogony process (asexual multiplication and sexual development) starts, where a trophozoite forms that grows into a mature schizont. This first generation contains 8–12 merozoites (asexual form) which are then released, likely through cell rupture, to invade other epithelial cells where the process replicates.

After several cycles of the schizogony process, type II meronts (sexual forms) develop, with each cell containing four merozoites. Some of these then form single macrogametes while others divide multiple times to form microgametes. A microgamete fertilizes a macrogamete upon being released, which develops into a zygote. It is the zygote which develops into an oocyst with an environmentally resistant wall. The oocyst passes into the environment in the faeces, as a non-sporulated and immature (non-infectious) oocyst, where it may survive for extended periods in the environment, given the marked seasonality of infection in areas where the disease is endemic(1).

Once in the environment, the oocyst sporulates, due to higher concentrations of atmospheric oxygen, and becomes infectious for humans. Prior to sporulation, the oocyst is non-infective, which is why direct human-to-human transmission does not occur. Evidence suggests that oocysts are continuously excreted during infection, with contamination of food or drinking water leading to human ingestion and infection. The infectious dose has not been determined, but based on outbreak investigations and extrapolations from other coccidians is thought to be relatively low(4), possibly as low as between 10 and 100 oocysts(7).

Life cycle of Cyclospora

Molecular methods including molecular markers such as intervening transcribed spacer regions can identify different genotypes of C. cayetanensis. DNA extraction followed by PCR amplification, PCR sequencing and computer database homology comparison can also be used. Restriction fragment length polymorphism analysis of amplicons can be used to determine genus, species and strain(13).

Stool samples may be sent for microscopy. Up to three samples may be necessary due to the intermittent excretion of this parasite(19). Fluorescent microscopy is a rapid, sensitive, and inexpensive method of diagnosis as Cyclospora oocysts autofluoresce). Trichrome stain and optical brighteners are used to detect spores in faeces, urines, respiratory secretions and other aspirates. Electron microscopy remains an important diagnostic method but its sensitivity is relatively poor. Haematoxylin stain alone for 15 minutes on biopsy specimens by light rather than electron microscopy can detect tissue stages of Cyclospora (15).

Assessment of six different procedures that included Giemsa, trichrome, chromotrope, Gram-chromotrope, acid-fast and safranin stains indicated that heating of faecal smears prior to safranin-based staining yielded a uniform, fast, reliable and easy-to-perform procedure that was superior to acid-fast staining; however the modified Ziehl Neelsen technique produced a variable stain of  Cyclospora oocysts. The small size, intracellular nature and poor staining properties with many histological stains contributes to the under-reporting of Cyclospora infections.

• In 1990 in a Chicago (US) hospital’s physicians' dormitory, 10 cases were attributed to an infected water source.
• In 1994 in a waterborne outbreak in Nepal, 12 out of a group of British expatriates became ill after drinking out of the military water supply. The mix of river and municipal water had been chlorinated and pumped into tanks beside the houses. Cyclospora oocysts were found in the chlorinated tanks.

Agriculture

Farm management practices to reduce the occurrence of Cyclospora:

• Procedures associated with primary production should be conducted under good hygienic conditions and should minimize potential hazards to health due to the contamination of fresh fruits and vegetables.(17)
• A risk management approach (e.g. HACCP) should be utilised, with Cyclospora included in the assessment when assessing the risk and requirement/decision on the technologies to be used.

• Identify the source and distribution of water used and assess the risks for the water being contaminated with protozoa. Sources of water contamination should be assessed and controlled to the extent feasible.
• Follow good agricultural practices that minimize the potential for contaminated water to contact the edible portion of the crop e.g. during irrigation.
  • Use appropriate wash methods.
  • Vigorous washing of produce not subject to bruising or injury may increase the likelihood of pathogen removal with brush washing being more effective than washing without brushes.
• For some types of produce (apples, celery, tomatoes) the temperature of wash water should be greater than that of the produce or the resultant pressure differential may cause water to be pulled into the plant material. Pathogens present on the produce surface or in the water may become internalized therefore temperature of the wash water must be considered for certain types of produce.

• Optimise management and storage of manure and slurry to reduce the infectivity of oocysts through raised temperature and ammonia levels.
• Reduce run off from animal farms into drinking water supplies, for example by planting strips of grass or vegetation. This may help to trap sediment and therefore reduce the organic matter contamination.
• Use potable water for making up insecticides and pesticides.

Processing including water suppliers, industries or sectors that use fresh product and operations in which contaminated process or waste water could be used:

• All water that is to be used in direct contact with food and food contact surfaces must be of potable quality and should be free of pathogenic microorganisms.
• A consistent supply of food-safe water for use in direct contact with foods and food contact surfaces is required.
• When water to be used for drinking, bottling or vending can be treated prior to use, microfiltration membranes or similar can be incorporated in conjunction with other measures (such as UV treatment, reverse osmosis or pasteurisation/distillation) to reduce further the likelihood of Cyclospora contamination.
• Water to be used as an ingredient (juices, beer and so on) or in the making of baby formula should be filtered and /or other treatment systems used.
• A risk management approach (e.g. HACCP) should be utilised and Cyclospora included in the assessment when assessing the risk and requirement/decision on the technologies to be used, taking into account:
  • Water source
  • pH (naturally acidity, carbonation)
  • Temperature of product
  • Presence of preservatives
  • Final consumer
  • Whether chemical technologies are sufficient or physical removal methods are required

Recommendations for food handlers with Cyclospora infection

Recommendations for food handlers with diarrhoeal illness:

1. Exclude food handlers for 48 hours after the first normal stool.
2. Refer for appropriate diagnostic testing

Recommendations for food handlers with diarrhoeal illness and laboratory confirmed Cyclospora infection:

1. Exclude from work until diarrhoea resolves;
2. Refer for antimicrobial therapy :
3. Per routine, re-emphasize the importance of proper hand washing before food preparation and after using the toilet; and
4. Check local regulations to see if other measures are required.

For further information see the Food Standards Agency guidance ‘Fitness to Work - Regulatory guidance and best practice advice for Food Business Operators (2009)’ (http://www.food.gov.uk/multimedia/pdfs/publication/fitnesstoworkguide09v3.pdf [1])