Table 2.

Clinical case comparative study results of the targeted NGS assay and the routine methods.

Case/Specimen	Autopsy finding or diagnosis	Laboratory findings
		Targeted NGS	Routine methods (PCR, bacterial culture, or ELISA)
1–Spinal cord	Myeloencephalopathy, chronic bacterial cyst.	EHV-1.	EHV1 (Ct 32.9).
	Multifocal hepatic necrosis.
2–Pooled cerebrum, brainstem, and spinal cord	WNV encephalomyelitis.	WNV; EHV-2; EHV-5 (few reads).	WNV (Ct 25.2). Negative for EEEV and EHV-1.
3–Fetal tissue pool	Equine herpesviral abortion.	EHV-1.	EHV-1 (Ct 17). Negative for Leptospira sp. by PCR.
4–Fetal tissue pool	Equine herpesviral systemic disease; perinatal death.	EHV-1.	EHV-1 (Ct 20).
5–Fetal tissues pool	Equine herpesviral abortion.	EHV-1.	EHV-1 (Ct 15).
6–Feces	Not available.	N. risticii (few reads).	N. risticii (Ct 29.23).
7–Feces	Acute necrotizing enterocolitis.	C. difficile A and B toxin genes.C. perfringens alpha toxin gene.Salmonella sp., E. coli, adenovirus-1.	C. difficile A and B toxin genes Ct 29.8).
			C. perfringens (Ct 29.9).
			E. coli and C. difficile on colon culture.
			Negative for coronavirus, rotavirus, L. intracellularis, N. risticii, Salmonella sp.
8–Pooled duodenum/ileum scraping	Duodenal perforation, peritonitis.	C. perfringens CPE and alpha toxins genes.	Negative for L. intracellularis and Salmonella sp.
	Chronic eosinophilic enterocolitis.
9–Feces	Mesenteric pyogranuloma.	No sequences detected.	Negative for rotavirus and coronavirus.
	Mild histiocytic and neutrophilic hepatitis, myocarditis, interstitial nephritis; pyogranulomatous pneumonia.
10–Pooled ileum/colon scraping	Mycotic pneumonia.	No sequences detected.	Rotavirus (Ct 37.7), C. difficile (Ct 37.1), C. perfringens (Ct 37.2). Negative for L. intracellularis, Salmonella sp., N. risticii, coronavirus.
	Cecocolonic mucosal hyperemia.
11–Pooled small intestine/large intestine scraping	Moderate locally extensive erosive colitis.	C. perfringens alpha and beta toxins (few reads).	C. difficile A and B toxins positive via ELISA and PCR (Ct 29.6). C. difficile and C. perfringens were not isolated on colon and/or small intestine culture.
	Moderate to marked vascular congestion is present in all layers of the small intestine.
	Neonatal encephalopathy.		Negative for coronavirus, rotavirus, L. intracellularis, Salmonella sp., N. risticii, C. perfringens.
12–Intestine scraping	Moderate, multifocal to coalescing, gastric ulcers.	C. perfringens alpha toxin gene, E. coli Hly virulence factor.	C. perfringens (Ct 20.3).
	Necrotizing enterocolitis.
13–Intestine scraping	Marked necrohemorrhagic enterocolitis with mild intestinal nematodiasis and mesenteric lymph node hyperplasia.	C. perfringens alpha toxin gene (few reads).	C. perfringens (Ct 34.7). Negative for C. difficile, L. intracellularis, Salmonella sp.
		E. coli CNF-1 and eae; Cryptococcus (few reads).
14–Intestinal contents	Clostridial enterocolitis.	C. perfringens alpha, beta, netF2, beta 2, and CPE toxin genes.	C. perfringens genotype A (Ct 15.2), C. difficile (Ct 36.5). C. difficile was not isolated from colon and/or small intestine tissues.
15–Intestinal contents	Bacterial colitis.	No sequences detected.	Negative for C. difficile, C. perfringens, Salmonella sp., N. risticii
16–Intestinal contents	Enteritis.	Actinobacillus sp.	Negative for Salmonella sp.
17–Intestinal contents	Pseudomonas aeruginosa septicemia.	P. aeruginosa, Streptococcus sp.	N. risticii (Ct 30.8).
	Ulcerative duodenitis.		C. perfringens isolated on small intestine culture.
	Fibrinonecrotic typhlocolitis.		P. aeruginosa isolated on spleen culture.
18–Respiratory swab	Not available.	S. equi, EHV-2.	S. equi (Ct 32). Not tested for other pathogens.
19–Respiratory swab	Not available.	No sequences detected.	EHV-5 (Ct 37.0). Negative for EHV-2.
20–Respiratory wash	Not available.	Pseudomonas sp., K. pneumoniae.	Negative for EHV-5.
21–Respiratory swab	Not available.	No sequences detected.	Negative for EHV-2, EHV-5.
22–Respiratory swab	Not available.	EHV-5.	EHV-5 (Ct 25.1), EHV-2 (Ct 39.8).
23–Lung tissue from 50-d-old foal	Severe bronchointerstitial pneumonia with pyogranulomas and hyaline membranes.	R. equi, P. aeruginosa, EHV-2.	R. equi, S. equi subsp. zooepidemicus.
24–Respiratory swab	Not available.	P. aeruginosa.	EHV-5 (Ct 35.7), EHV-2 (Ct 37.0).
25–Lung tissue from 60-d-old foal	Bronchopneumonia.	R. equi, EHV-2.	R. equi isolated on lung culture.
26–Lung tissue	Necrotizing bronchopneumonia.	S. aureus.	S. aureus isolated on lung culture.
27–Wound swab	Not available.	S. aureus, Streptococcus sp., Actinobacillus sp.	S. aureus, Streptococcus dysgalactiae subsp. equisimilis, E. coli.