Qualification of a 21-valent pneumococcal urine antigen detection assay and development of clinical positivity cutoffs

Figures & data

Table 1. Clinical urine samples without known pneumococcal disease used for clinical specificity evaluation.

Co-morbidity	Patient number
Pulmonary disease	206
Cardiovascular disease	326
Diabetes	283
Healthy	269

Pulmonary disease included COPD, asthma, emphysema and chronic bronchitis. Cardiovascular disease included hypertension, congestive heart failure. Renal disease included chronic kidney disease. Note: Some patients also had multiple underlying diseases and were accounted for in each applicable category.

Table 2. IPD samples used for clinical sensitivity evaluation with serotyping results determined by blood culture isolates.

Assay serotype alignment with vaccine types	Number of samples
21-valent assay unique serotypes	32
15- and 21-valent assays shared serotypes	21
15-valent assay unique serotypes	6
15- and 21-valent assays 30 serotypes	59^†
Non-vaccine serotypes	11
Total	70

† Some samples were applicable to multiple sub-categories so that the sum of the category numbers exceeded up to the total number of 70 samples.

Figure 1. Development of the 21-valent SSUAD. (A) Schematic representation of the assay workflow, not drawn to scale. (B) Representative standard curves from four plates from selectivity testing. Vertical lines indicate the assay range.

Table 3. Assay qualification performance in 21-valent assay.

Performance characteristic	Result
Precision	Total assay precision ranged between 7.6 and 17.8% CV across the 21 serotypes
Ruggedness	PnPs concentrations differences were within 19% between analysts
Accuracy	Relative accuracy was estimated between 80% and 150% across the quantifiable range for 18 serotypes. The recoveries were around 60% for 15A, 52% for 23A and 32% for 35B, respectively, due to potential urine interference
Dilution linearity	The dilution fold-bias per tenfold dilution was <2-fold for all 21 serotypes
Selectivity	The average recovery was between 80% and 125% for each pre-dilution of urine with limited exceptions. At the lower pre-dilutions of urine, recoveries less than 80% were observed for serotypes 15A (53–73%), 23A (50–77%) and 35B (33–76%). Within these three serotypes, the low recovery results improved when urines were diluted more, which suggested a potential of urine interference with the measurement of PnPs
Specificity	Based on development and qualification data, no interference of measurements between 30 serotypes from the 21-valent and the previously published 15-valent assay [29] was observed The resulting measured concentration for the samples absent the target PnPs generated a result that was below its determined lower limit of quantitation (LLOQ) for all serotypes except for serotype 23B with geometric mean concentration of 0.053 ng/ml, just above the LLOQ 0.045 ng/ml Though the recoveries for each spiked sample were borderline of 50% for serotype 15A and 23A and were relatively low (30%) for serotype 35B, potentially due to urine interference, the data provided no evidence that any one serotype either potentiates or interferes with the measurement of another serotype No interference was observed against non-assay pneumococcal polysaccharides 6D, 7C, 13, 21, 34, 35F, 38 No interference was observed against non-pneumococcal bacterial lysates of Acinetobacter baumannii, Escherichia coli K12, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Neisseria gonorrhoeae, Clostridium difficile, Bacteroides fragilis, Staphylococcus aureus SACO1, Staphylococcus aureus, RN4220

Table 4. Consolidated table for parameters from serotype-specific urine antigen detection assay 21-valent assay qualification.

Serotype	LLOQ (ng/ml)	ULOQ (ng/ml)	Total precision (%CV)	Assay ruggedness to analyst (%diff. in concentration)	Accuracy range (ng/ml PnPs conc. range with 80% to ~150% recovery^†)	On target specificity (%recovery across absent serotypes)	Off target specificity (ng/ml)	Selectivity (%recovery in neat urine)	Dilutional linearity (dilution fold-bias per tenfold dilution)
3	0.0229	3.125	9.0%	6.30%	[0.012, 3.125]	0.92	<LLOQ	86%	1.55
6A	0.0898	12.5	7.8%	3.93%	[0.024, 12.5]	1.16	<LLOQ	113%	1.60
7F	0.7597	100	9.3%	5.80%	[0.195, 100]	1.05	<LLOQ	112%	1.60
8	0.1976	25	7.7%	1.43%	[0.098, 25]	1.34	<LLOQ	132%	1.64
9N	0.0898	12.5	8.4%	3.03%	[0.049, 12.5]	0.99	<LLOQ	96%	1.52
10A	0.7376	100	8.0%	5.39%	[0.195, 100]	1.23	<LLOQ	120%	1.58
11A	0.0899	12.5	11.2%	7.20%	[0.049, 12.5]	1.12	<LLOQ	106%	1.44
12F	0.0919	12.5	8.2%	8.58%	[0.049, 12.5]	1.04	<LLOQ	99%	1.48
15A	0.1953	50	14.0%	4.81%	[0.098, 50]	0.52	<LLOQ	53%	1.58
15C	0.0873	12.5	17.8%	18.69%	[0.049, 12.5]	1.09	<LLOQ	111%	1.43
16F	0.0998	12.5	8.0%	6.05%	[0.024, 25]	1.33	<LLOQ	108%	1.52
17F	0.0455	6.25	8.2%	7.15%	[0.024, 6.25]	1.13	<LLOQ	94%	1.53
19A	0.0122	3.125	7.6%	9.03%	[0.006, 6.25]	1.07	<LLOQ	89%	1.55
20A	0.0903	12.5	10.7%	7.05%	[0.049, 12.5]	1.14	<LLOQ	108%	1.39
22F	0.0453	6.25	8.1%	6.78%	[0.012, 6.25]	1.14	<LLOQ	111%	1.56
23A	0.0488	12.5	8.0%	4.84%	[0.024, 25]	0.45	<LLOQ	50%	1.46
23B	0.0447	6.25	7.6%	5.95%	[0.012, 6.25]	1.24	0.053	105%	1.57
24F	0.0864	12.5	9.3%	5.93%	[0.049, 12.5]	0.98	<LLOQ	87%	1.52
31	0.0454	6.25	8.2%	7.43%	[0.024, 6.25]	1.20	<LLOQ	102%	1.47
33F	0.0879	12.5	8.6%	6.56%	[0.049, 12.5]	0.99	<LLOQ	87%	1.53
35B	0.0682	6.25	9.5%	4.75%	[0.024, 12.5]	0.29	<LLOQ	33%	1.22

† Upper end of the accuracy range expanded from 125% to around 150%.

CV: Coefficient of variation; LLOQ: Lower limit of quantitation; ULOQ: Upper limit of quantitation.

Table 5. Clinical positivity cut-offs and assay LOQs for 30 serotypes.

Assay	Serotype	Cutoff (ng/ml)	LLOQ (ng/ml)	ULOQ (ng/ml)
15-valent assay	1	0.5245	0.0625	1
	3^†	0.0117	0.0078	1
	4	0.1656	0.0313	4
	5	0.1020	0.0313	4
	6A^†	0.2343	0.0781	5
	6B	0.2517	0.0156	1
	7F^†	3.0459	0.3125	20
	9V	0.0620	0.0156	2
	14	0.0939	0.0313	4
	18C	0.7500	0.2500	4
	19A^†	0.0234	0.0078	1
	19F	1.5000	1.0000	20
	22F^†	0.3750	0.2500	4
	23F	3.3576	0.3125	40
	33F^†	0.1875	0.1250	4
21-valent assay	8	0.2964	0.1976	25
	9N	0.1344	0.0898	12.5
	10A	2.2128	0.7376	100
	11A	0.1350	0.0899	12.5
	12F	0.1380	0.0919	12.5
	15A	1.0965	0.1953	50
	15C	0.2616	0.0873	12.5
	16F	0.1500	0.0998	12.5
	17F	0.1582	0.0455	6.25
	20	0.1356	0.0903	12.5
	23A	0.2445	0.0488	12.5
	23B	0.0672	0.0447	6.25
	24F	0.2592	0.0864	12.5
	31	0.0547	0.0454	6.25
	35B	0.1519	0.0682	6.25

† The cut-offs and LOQs from six overlapping serotypes between the 15-valent and 21-valent assays are derived from the 15-valent assay as clinical sample data were determined with the 15-valent assay.

LOQ: Limit of quantitation; LLOQ: Lower limit of quantitation; ULOQ: Upper limit of quantitation.

Figure 2. Clinical specificity and sensitivity to detect serotype-specific pneumococcal infection based on the presence of pneumococcal polysaccharides in patient urines. Clinical specificity and sensitivity were assessed for each assay individually and combined as indicated.

Rajam G, Zhang Y, Antonello JM, et al. Development and validation of a sensitive and robust multiplex antigen capture assay to quantify Streptococcus pneumoniae serotype-specific capsular polysaccharides in urine. mSphere. 2022;7:e00114–00122. doi:10.1128/msphere.00114-22

 PubMed Web of Science ®Google Scholar