Looking for Cryptosporidium: the application of advances in detection and diagnosis

Abstract

The protozoan Cryptosporidium is a major public and animal health concern. Young children, immunocompromised people, and pre-weaning animals are especially vulnerable, but treatment options are limited and there is no vaccine. A laboratory diagnosis is required to confirm cases of cryptosporidiosis, and species and genotype determination is essential in distinguishing human from non-human sources, understanding transmission, and strengthening the epidemiological evidence for causative links in outbreaks. However, testing is not consistent, as demonstrated by investigation of a significant increase in cases in some European countries during 2012. Many methods employed are laborious and time-consuming; recent advances, translated into diagnostic assays, can improve testing and facilitate typing to support clinical and environmental investigations.

Figure 1

Schematic representation of the Cryptosporidium life-cycle and transmission emphasising elements exploited in diagnosis and detection. A population of sporulated oocysts is ingested by a host, and undergo excystation in the intestinal lumen. Four sporozoites are present in each oocyst, and emerge (A) to invade the microvilli at the brush border of the mucosal epithelium (B) and develop into trophozoites (not shown). Trophozoites undergo asexual division (merogony) to form eight merozoites within type I meronts (C), and release invasive merozoites (D) that invade adjacent host cells to form additional type I meronts or to form type II meronts (E). Merozoites released from type II meronts do not recycle but invade host cells to initiate the sexual stage. Microgametocytes released from microgamonts (F) fertilise the macrogamonts (G) to become zygotes. Most of the zygotes develop into oocysts with a thick, two-layered wall (H) but a minority only have a unit membrane surrounding the four sporozoites (I) and facilitate the autoinfective cycle that maintains infection without further ingestion of thick-walled oocysts. Sporulated, thick-walled oocysts containing four sporozoites (J) are released in faeces and transmit infection between hosts. Abbreviation: TRAPs, thrombospondin related adhesive proteins.

Figure 2

Workflow for Cryptosporidium diagnosis during investigation of gastroenteritis. Unbroken boxes represent adopted tests (see: http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/); broken lines and boxes represent reference or specialist tests; broken, grey lines and box represents alternative workflow based on multi-pathogen diagnostic PCRs. The optimisation of automated nucleic acid extraction processes and application of multiplex, real-time PCR offers great potential for streamlining diagnostic workflows.

Figure I

Relative attributes of features of diagnostic assays, ranked low to high. Abbreviations: AF, acid fast; EIA, enzyme immunoassay; FM, fluorescence microscopy; ICLF, immunochromatographic lateral flow; IFM, immunofluorescence microscopy.