Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection

Chan, Wai Sing; Au, Chun Hang; Leung, Sau Man; Ho, Dona N.; Wong, Elaine Yue Ling; To, Man Yan; Ng, Man Kin; Chan, Tsun Leung; Ma, Edmond Shiu Kwan; Tang, Bone Siu Fai

doi:10.1186/s13000-020-00960-w

Table 1 Details of clinical specimens and results of Nanopore sequencing

From: Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection

Patient	Specimen		Routine infectious disease workups		Nanopore sequencing		Possible pathogens^# (abundance)	Remarks
Patient	Specimen	N^*	Test	Result^§	Time^†	Abundance^¶	Possible pathogens^# (abundance)	Remarks
1	BAL	5	Culture	C. albicans C. glabrata	4 min 4 min	61% 22%	/	/
2	Sputum	2	Culture	Candida sp.^(M)	4 min	61%	/	/
3	Sputum	6	Culture	S. pyogenes^(H)	9 min	59%	/	/
4	Sputum	5	Culture	H. influenzae^(H)	22 min	41%	/	/
5	BAL	7	Culture Microscopy	C. albicans^(M) P. jirovecii	5 min 5 min	93% 0.1%	/	/
6	Sputum	5	Culture	S. pneumoniae^(H)	5 min	6%	/	S. pneumoniae antigen (urine): negative
7	BAL	7	Culture PCR	C. albicans^(S) L. pneumophila	5 min 4 min	98% 98%	/	/
8	BAL	7	Culture	C. albicans^(S)	2 min	85%	/	/
9	NPS	1	PCR	B. parapertussis	32 min	0.2%	/	/
10	Sputum	3	Culture	M. abscessus	27 min	0.3%	/	AFB smear: negative
11	BAL	5	Culture	M. avium complex Raoultella sp.^(S) S. aureus^(S)	ND ND 2 min	ND ND 0.5%	/	AFB smear: negative
12	NPS	2	PCR Serum IgG	C. pneumoniae	2 min	0.8%	**S. pneumoniae(80%)**	Bacterial culture: not performed *Consensus sequence showed 100% identity toS. pneumoniae***
13	Sputum	3	PCR	M. pneumoniae	10 min	99%	/	/
14	NPA	3	Culture	C. pseudodiphtheriticum^(H)	4 min	4%	/	/
15	NPS	2	Culture	M. catarrhalis^(M)	16 min	98%	/	/
16	Sputum	2	Culture	M. catarrhalis^(M)	13 min	98%	/	/
17	Lung Bx	5	Culture	C. neoformans	3 min	67%	/	/
18	Sputum	4	PCR	P. jirovecii	12 min	97%	**L. pneumophila(11%)**	**L. pneumophilaPCR: not performed Consensus sequence showed 99% identity toL. pneumophila** P. jirovecii DNA: 3.13 × 10⁶ copies/mL
19	Sputum	6	PCR Culture Culture	P. jirovecii S. aureus^(S) C. albicans^(S)	8 min 43 min 8 min	0.001% 0.1% 74%	/	P. jirovecii DNA: 1.77 × 10⁶ copies/mL
20	NPS	2	PCR	C. pneumoniae	1 min	0.03%	/	/
21	BAL	2	NGS	R. mucilaginosa	1 min	67%	/	*Culture-negativeR. mucilaginosa* Shotgun NGS by MiSeq Consensus sequence showed 99% identity toR. mucilaginosa**
22	BW	5	PCR Culture	M. tuberculosis complex C. dubliniensis	9 min 3 min	0.01% 2%	/	AFB smear: positive
23	NPS	1	PCR	B. pertussis	40 min	0.02%	/	/
24	BAL	7	Culture Culture PCR	C. albicans P. kudriavzevii P. jirovecii	1 min ND 1 min	31% ND 33%	**L. wadei(64%)**	*Culture-negativeL. wadei* Consensus sequence showed 99% identity toL. wadei** P. jirovecii DNA: 3.43 × 10⁷ copies/mL
25	ETA	4	Culture	Candida sp.^(S)	ND	ND	/	/
26	BAL	4	PCR	P. jirovecii	2 min	50%	/	P. jirovecii DNA: 8.82 × 10⁴ copies/mL
27	TA	2	Culture	C. dubliniensis^(S)	1 min	90%	/	/
28	NPS	2	PCR Serum IgM	C. pneumoniae	1 min	84%	/	/
29	BW	3	Culture	S. maltophilia^(S) C. albicans^(S) C. glabrata^(S)	1 min 1 min 1 min	7% 51% 37%	/	/
30	Sputum	6	PCR	M. pneumoniae	1 min	65%	/	/
31	Sputum	7	PCR	M. pneumoniae	1 min	53%	/	/
32	BAL	6	PCR	M. pneumoniae C. albicans	2 min ND	1% ND	/	Fungus culture: negative
33	Sputum	5	PCR	M. pneumoniae	1 min	90%	/	/
34	BAL	7	Culture	C. albicans^(S)	2 min	86%	/	Serum beta-D-glucan: negative
35	Sputum	6	Culture	Burkholderia sp.^(M)	1 min	46%	/	/
36	Sputum	5	PCR	M. pneumoniae	1 min	12%	/	/
37	Sputum	7	Culture Urine Ag	S. pneumoniae^(M)	3 min	0.6%	**C. albicans(85%)**	Fungus culture: not performed *Consensus sequence showed 100% identity toC. albicans***
38	Sputum	2	PCR	P. jirovecii	ND	ND	**C. striatum(84%) C. haemulonis(70%)**	P. jirovecii DNA: < 760 copies/mL Bacterial/ fungus culture: not performed *Consensus sequence showed 99 and 100% identity toC. striatumandC. haemulonis, respectively.*
39	Sputum	2	PCR	M. pneumoniae	1 min	47%	/	/
40	NPS	1	PCR	M. pneumoniae	ND	ND	/	PCR Cp value = 37.06 (45 cycles)
41	NPS	1	PCR	M. pneumoniae	2 min	0.9%	/	/
42	Sputum	1	Culture	Normal flora	22 min	/	/	16S data revealed reads from normal flora dominated by N. perflava (19.55%) with H. influenzae colonization (1.68%). ITS sequencing: not performed
43	Sputum	6	Culture	Normal flora	87 min	/	/	16S data revealed reads from normal flora dominated by L. buccalis (46.08%). ITS data revealed no fungal reads. Fungus culture: negative

Summary
○ Average number of routine tests taken for bacterial/ fungal etiology: 4 (1–7)
○Average elapsed sequencing time of the first target read: 7 min (1–43 min; pre-sequencing preparation: 5 h)
○ Concordance with routine test results: 47/54 = 87.04%
○ Possible pathogens detected by Nanopore workflow: 7 from 6 patients
^*Number of tests taken for bacterial/ fungal etiology
^§Scanty^(S), moderate^(M) and heavy^(H) growth correspond to microbial growth up to primary, second and third sector of culture agar plate, respectively
^†Elapsed Nanopore sequencing time of first target reads
^¶Bacterial and fungal abundance were calculated separately
^#Possible pathogens not detected by routine workups but identified by Nanopore sequencing

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ISSN: 1746-1596

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