Detection and Identification of Enterocytozoon bieneusi and Encephalitozoon Species in Stool and Urine Specimens by PCR and Differential Hybridization

Daan W. Notermans; Ron Peek; Menno D. de Jong; Ellen M. Wentink-Bonnema; René Boom; Tom van Gool

doi:10.1128/JCM.43.2.610-614.2005

DOI: 10.1128/JCM.43.2.610-614.2005

Article Figures & Data

Tables

TABLE 1.

Serial dilutions of plasmids with inserts of five different microsporidial species after PCR and hybridization with the general microsporidium probe and the specific probe for the respective species

Species	Probe	ECL signal (LU) with plasmid input in copies/PCR^a							R²^b
Species	Probe	1	10	10²	10³	10⁴	10⁵	10⁶	R²^b
E. bieneusi	General	ND	360	2,499	20,471	13,599	25,094	35,454	0.87
	Specific	ND	397	3,078	16,185	20,047	33,565	46,897	0.96
E. intestinalis	General	207	597	2,604	8,929	25,744	24,823	34,808	0.91
	Specific	243	707	3,026	12,964	31,264	22,031	40,824	0.86
E. cuniculi	General	ND	3,347	5,860	26,559	38,664	33,716	47,189	0.89
	Specific	ND	3,006	7,245	26,007	33,094	29,034	45,180	0.89
E. hellem	General	ND	383	2,436	8,217	29,660	37,503	37,407	0.91
	Specific	ND	475	4,015	15,412	31,662	44,121	47,330	0.97
V. corneae	General	ND	261	1,207	5,886	18,264	20,383	25,804	0.94

↵a Values represent ECL signal expressed in luminosity units (LU). ND, not done.
↵b Correlation (squared Pearson's correlation coefficient) between log₁₀-transformed input copy number and ECL signal.

TABLE 2.

Spiked feces with serial dilutions of cultured spores from four different microsporidial species after PCR and hybridization with the general microsporidium probe and the specific probe for the respective species compared with Uvitex 2B stain and light microscopy

Species^a	Probe	ECL signal (LU) with spore input per g of feces (gene copies per PCR) of^b:				R²^c
Species^a	Probe	3.5 × 10² (17)	3.5 × 10³ (1.7 × 10²)	3.5 × 10⁴ (1.7 × 10³)	3.5 × 10⁵ (1.7 × 10⁴)	R²^c
E. intestinalis	General	215	3,032	13,527	22,266	0.96
	Specific	234	4,996	17,642	35,372	0.94
	Uvitex	Neg.	Neg.	Sporadic	Some
E. cuniculi	General	562	4,277	26,987	27,967	0.87
	Specific	564	4,305	35,538	35,786	0.84
	Uvitex	Neg.	Neg.	Sporadic	Few
E. hellem	General	568	4,146	4,972	30,171	0.72
	Specific	650	4,951	6,492	45,498	0.71
	Uvitex	Neg.	Neg.	Neg.	Sporadic
V. corneae	General	280	3,780	8,077	13,523	0.99
	Uvitex	Neg.	Neg.	Neg.	Sporadic

↵a E. bieneusi spores were not available, as they cannot be maintained in long-term culture.
↵b Values represent ECL signal expressed in LU.
↵c Correlation (squared Pearson's correlation coefficient) between log₁₀-transformed input copy number and ECL signal.

TABLE 3.

Two urine samples and PBS after PCR and hybridization with the general microsporidium probe and the specific probes for the respective species

Sample^a	Probe	ECL Signal (LU)
		E. intestinalis (DNA copies/PCR)			E. cuniculi (DNA copies/PCR)			E. hellem (DNA copies/PCR)
		2.5 × 10⁶	2.5 × 10⁴	2.5 × 10²	5.0 × 10⁶	5.0 × 10⁴	5.0 × 10²	1.9 × 10⁶	1.9 × 10⁴	1.9 × 10²
Urine I	General	23,590	18,142	3,972	29,368	18,913	461	25,558	15,805	872
	Specific	28,465	25,477	5,183	34,359	19,990	478	30,089	19,733	626
Urine II	General	29,912	7,539	11,916	36,322	28,507	2,095	30,847	27,737	2,884
	Specific	34,117	11,929	14,124	35,581	29,362	2,755	33,581	31,292	3,971
PBS	General	23,458	15,467	864	28,507	2,082	1,809	24,136	15,957	235
	Specific	33,459	18,557	1,182	29,362	19,568	2,484	33,906	17,567	277