Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis

Figures & data

Table 1 Sequencing confirmed clinical samples and isolates used for validating specificity

Organisms	No. of clinical samples	No. of isolates	Total
Target strains used to test the specificity N=303
Herpes simplex virus types 1	19	2	21
Herpes simplex virus types 2	23	0	23
Chlamydia trachomatis	30	0	30
Treponema pallidum	16	0	16
Neisseria gonorrhoeae	18	26	44
Trichomonas vaginalis	18	0	18
Mycoplasma hominis	35	0	35
Ureaplasma urealyticum	41	0	41
Mycoplasma genitalium	27	0	27
Ureaplasma parvum	48	0	48
Haemophilus ducreyi	0	0	0
Other species used to test the specificity N=65
Human Immunodeficiency Virus	15	1	16
Human papillomavirus types 6	2	0	2
Human papillomavirus types 11	2	0	2
Human papillomavirus types 16	3	0	3
Human papillomavirus types 18	3	0	3
Human papillomavirus types 31	2	0	2
Human papillomavirus types 33	3	0	3
Human papillomavirus types 45	1	0	1
Human papillomavirus types 52	2	0	2
Human papillomavirus types 58	2	0	2
Neisseria lactamica	0	4	4
Neisseria polysaccharea	0	5	5
Neisseria meningitidis	0	2	2
Moraxella catarrhalis	3	1	4
Klebsiella pneumoniae	3	1	4
Mycoplasma pneumoniae	2	1	3
Pseudomonas aeroginosa	2	1	3
Staphylococcus aureus	3	1	4

Table 2 Limit of detection of each target gene using regression probit analysis

Target pathogen	Target	No. of replicates (%) for positive standards, copies/reactions					Limit of detection, copies/reactions
		100	50	10	5	1
Herpes simplex virus types 1	gD	12/12 (100)	12/12 (100)	12/12 (100)	10/12 (83)	6/12 (50)	7.079
	gG	12/12 (100)	12/12 (100)	12/12 (100)	6/12 (50)	0/12 (0)	7.215
Herpes simplex virus types 2	gD	12/12 (100)	12/12 (100)	12/12 (100)	11/12 (92)	9/12 (75)	5.692
	gG	12/12 (100)	12/12 (100)	12/12 (100)	10/12 (83)	5/12 (42)	6.927
Chlamydia trachomatis	cryptic plasmid	12/12 (100)	12/12 (100)	12/12 (100)	12/12 (100)	7/12 (58)	3.230
	omp1	12/12 (100)	12/12 (100)	12/12 (100)	11/12 (92)	9/12 (75)	5.692
Treponema pallidum	polA	12/12 (100)	12/12 (100)	12/12 (100)	12/12 (100)	11/12 (92)	1.739
	tpp47	12/12 (100)	12/12 (100)	12/12 (100)	11/12 (92)	7/12 (58)	5.732
Neisseria gonorrhoeae	opa	12/12 (100)	12/12 (100)	12/12 (100)	7/12 (58)	6/12 (50)	10.009
	porA	12/12 (100)	12/12 (100)	12/12 (100)	11/12 (92)	5/12 (42)	5.630
Trichomonas vaginalis	beta-tubulin	12/12 (100)	12/12 (100)	12/12 (100)	11/12 (92)	6/12 (50)	5.688
	repeated DNA target	12/12 (100)	12/12 (100)	12/12 (100)	11/12 (92)	9/12 (75)	5.692
Mycoplasma hominis	gap	12/12 (100)	12/12 (100)	12/12 (100)	4/12 (33)	0/12 (0)	7.961
	YidC	12/12 (100)	12/12 (100)	12/12 (100)	9/12 (75)	7/12 (58)	8.428
Ureaplasma urealyticum	ureA	12/12 (100)	12/12 (100)	12/12 (100)	10/12 (83)	5/12 (42)	6.927
	ureC	12/12 (100)	12/12 (100)	12/12 (100)	12/12 (100)	6/12 (50)	3.369
Mycoplasma genitalium	16S rRNA IR	12/12 (100)	12/12 (100)	12/12 (100)	11/12 (92)	8/12 (67)	5.744
	magB	12/12 (100)	12/12 (100)	12/12 (100)	10/12 (83)	4/12 (33)	6.762
Ureaplasma parvum	rpmA	12/12 (100)	12/12 (100)	12/12 (100)	10/12 (83)	6/12 (50)	7.079
	ureD	12/12 (100)	12/12 (100)	12/12 (100)	10/12 (83)	2/12 (17)	6.381
Haemophilus ducreyi	16S rRNA IR	12/12 (100)	12/12 (100)	12/12 (100)	6/12 (50)	0/12 (0)	7.215
	recD	12/12 (100)	12/12 (100)	12/12 (100)	10/12 (83)	2/12 (17)	6.381

Table 3 Samples with discordant results by the STI-MS method after arbitration testing

Sample ID	STI-MS results	Confirmed results
M32	UU	Negative
M33	MH	Negative
M34	CT	Negative
M35	NG	Negative
M36	UU, NG	UU
M37	UU, MH, HSV1	UU, MH
M38	MH, HSV1,HSV2	HSV1, HSV2
M39	UU, CT, MH, NG	UU, CT, MH
M40	TP, UU, CT, MG, MH, NG, HSV2	TP, UU, CT, MG, MH, NG

Abbreviations: UU, Ureaplasma urealyticum; MH, Mycoplasma hominis; CT, Chlamydia trachomatis; NG, Neisseria gonorrhoeae; TP, Treponema pallidum; HSV1, Herpes simplex virus types 1; HSV2, Herpes simplex virus types 2; MG, Mycoplasma genitalium.

Table 4 Results of confirmatory tests of positive samples by the STI-MS assay and resolution of discordant results following arbitration testing

Target pathogen	No. (%) of positive samples by STI-MS	No. of samples confirmed by confirmatory tests/No. of positive samples by STI-MS	Verification rate (%) of confirmatory tests	No. of discordant samples confirmed by arbitration testing	No. of overall confirmed samples	Overall verification rate (%)
Herpes simplex virus types 1	19 (10.8)	12/19	63.2	6	18	94.7
Herpes simplex virus types 2	14 (8.0)	8/14	57.1	5	13	92.9
Chlamydia trachomatis	44 (25)	37/44	84.1	6	43	97.7
Treponema pallidum	2 (1.1)	2/2	100	0	2	100
Neisseria gonorrhoeae	28 (15.9)	21/28	75	4	25	89.3
Trichomonas vaginalis	NA	NA	NA	NA	NA	NA
Mycoplasma hominis	40 (22.7)	35/40	87.5	3	38	95
Ureaplasma urealyticum	56 (31.8)	50/56	89.3	5	55	98.2
Mycoplasma genitalium	31 (17.6)	24/31	77.4	7	31	100
Ureaplasma parvum	2 (1.1)	2/2	100	0	2	100
Haemophilus ducreyi	NA	NA	NA	NA	NA	NA
Total^a	121 (68.8)	81/121	66.9	31	112	92.6

Note: ^aThe total number of clinical samples is 176.

Abbreviation: NA, not available.

Table 5 Clinical performance of the STI-MS assay for organism identification compared to that of monoplex real-time PCR

Target organism	Sample types	No. of samples with the following result					Sensitivity (%) (95% CI)	Specificity (%) (95% CI)	PPV (%) (95% CI) ^c	NPV (%) (95% CI) ^c
		TP	FP	FN	TN	Total
Herpes simplex virus types 1	Urine	1	0	0	116	117	100 (5.5–100)	100 (96.0–100)	100 (5.5–100)	100 (96.0–100)
	Swabs ^a	1	1	0	122	124	100 (5.5–100)	99.2 (94.9–100)	50 (2.7–97.3)	100 (96.2–100)
Herpes simplex virus types 2	Urine	1	1	0	115	117	100 (5.5–100)	99.2 (94.6–100)	50 (2.7–97.3)	100 (96.0–100)
	Swabs ^a	1	1	0	122	124	100 (5.5–100)	99.2 (94.9–100)	50 (2.7–97.3)	100 (96.2–100)
Chlamydia trachomatis	Urine	3	0	0	114	117	100 (3.1–100)	100 (96.0–100)	100 (31.0–100)	100 (96.0–100)
	Swabs ^a	12	2	0	110	124	100 (69.9–100)	98.2 (93.1–99.7)	85.7 (56.2–97.5)	100 (95.8–100)
Treponema pallidum	Urine	0	0	1	116	117	0 (0–94.5)	100 (96.0–100)	NA	99.1 (94.6–100)
	Swabs ^a	0	0	1	123	124	0 (0–94.5)	100 (96.2–100)	NA	99.2 (95.0–100)
Neisseria gonorrhoeae	Urine	6	3	1	107	117	85.7 (42.0–99.3)	97.3 (91.6–99.3)	66.7 (31.0–91.0)	99.1 (94.2–100)
	Swabs ^a	2	1	0	121	124	100 (19.8–100)	99.2 (94.8–100)	66.7 (12.5–98.2)	100 (96.2–100)
Trichomonas vaginalis	Urine	0	0	0	117	117	NA	100 (96.0–100)	NA	100 (96.0–100)
	Swabs ^a	1	0	0	123	124	100 (5.5–100)	100 (96.2–100)	100 (5.5–100)	100 (96.2–100)
Mycoplasma hominis	Urine	5	0	0	112	117	100 (46.3–100)	100 (95.9–100)	100 (46.3–100)	100 (95.9–100)
	Swabs ^a	14	2	0	108	124	100 (73.2–100)	98.2 (93.0–99.7)	87.5 (60.4–97.8)	100 (95.7–100)
Ureaplasma urealyticum	Urine	8	6	0	103	117	100 (59.8–100)	94.5 (87.9–97.7)	57.1 (29.6–81.2)	100 (95.5–100)
	Swabs ^a	11	6	0	107	124	100 (67.9–100)	94.7 (88.3–97.8)	64.7 (38.6–84.7)	100 (95.7–100)
Mycoplasma genitalium	Urine	2	2	0	113	117	100 (19.8–100)	98.3 (93.2–99.7)	50 (9.2–90.8)	100 (96.0–100)
	Swabs ^a	2	0	0	122	124	100 (19.8–100)	100 (96.2–100)	100 (19.8–100)	100 (96.2–100)
Ureaplasma parvum	Urine	2	1	0	114	117	100 (19.8–100)	99.1 (94.5–100)	66.7 (12.5–98.2)	100 (96.0–100)
	Swabs ^a	46	6	0	72	124	100 (90.4–100)	92.3 (83.4–96.8)	88.5 (75.9–95.2)	100 (93.7–100)
Haemophilus ducreyi	Urine	0	0	0	117	117	NA	100 (96.0–100)	NA	100 (96.0–100)
	Swabs ^a	0	0	0	124	124	NA	100 (96.3–100)	NA	100 (96.3–100)

Note: ^aUrethral/cervical swab.

Abbreviations: TP, true positive; FP, false positive; FN, false negative; TN, true negative; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value; NA, not available.