Rapid and sensitive detection of single cryptosporidium oocysts from archived glass slides

Abstract

In this study we report on the development and application of a novel method for efficiently extracting and detecting single Cryptosporidium oocysts from archived glass slides. Laser capture microscopy was used to extract low numbers of oocysts from archived glass slides. Highly sensitive real-time PCR methods were then developed to enable the rapid detection and identification of Cryptosporidium oocysts from these samples. The method was applied to fecal smears stained with a variety of standard oocyst stains and water samples. This application, with samples derived from both public health and water service laboratories, highlighted the strong potential of this method to be used as a rapid high-throughput screening tool for the routine monitoring of Cryptosporidium and other medically important pathogens from clinical, veterinary, and environmental water samples. Importantly, the application of our protocol could be used to type Cryptosporidium and other pathogens from stored archived glass slides in public health and water service laboratories, providing vital epidemiological updates and helping to identify and trace pathogens and their routes of infection and ultimately improve their control.

FIG. 1.

(A and B) Application of LCM for the recovery of Cryptosporidium parvum oocysts from archived fecal smears. (A) Fecal smears on glass slides were acid fast stained using the ZN stain method. This acid-fast staining resulted in clearly visible oocysts, which are indicated by the arrows. (B) The oocysts were then recovered from slides by using the LPC function of the LCM, which catapulted the oocysts into a microcentrifuge tube. The arrow indicates an area of the glass slide from which a ZN-stained oocyst was recovered using LCM.

FIG. 2.

(A through C) Detection and identification of Cryptosporidium parvum from archived clinical glass slides using real-time LightCycler PCR detection of 18S rRNA, which produced 232-bp PCR products. (A) Sensitivity of PCR detection of C. parvum, where a single oocyst was routinely detected. (B) Agarose gel (1.5%) of 232-bp PCR products from 18S rRNA. Lane 1, 100-bp DNA ladder; lane 2: negative control; lane 3, positive control, C. parvum (Moredun strain); lane 4, 50 oocysts; lane 5, 20 oocysts; Lane 6, 10 oocysts; lane 7, 7 oocysts; lane 8, 5 oocysts; lane 9, 3 oocysts; lane 10, 2 oocysts; lane 11, 1 oocyst; lane 12, no oocysts. (C) Clustal alignment of C. parvum isolate Moredun (accession no. DQ431245), C. parvum isolate ISSC6 (accesion no. DQ656104), and C. andersoni (accesion no. AY954885) sequences show the ability to genotype and differentiate between C. parvum isolates after sequencing LightCycler PCR products. Shaded gray areas indicate sequence variations between isolates, and examples of sequence variations are indicated within boxes B1 and B2.

FIG. 3.

Detection and genotyping of Cryptosporidium parvum oocysts from clinical samples (fecal glass smears) using LCM followed by real-time LightCycler PCR targeting the TRAP-C2 gene. PCR products were run on a 1.5% agarose gel, and positive results were indicated by the presence of a 369-bp PCR product. Lane l, 100-bp DNA ladder; lane 2, negative control; lane 3, patient sample Tn3 (slide number), stored for 3 years; lane 4, patient sample Tn23, stored for 2 years; lane 5, patient sample Tn24, stored for 2 years.