Study population
The median age of the 143 patients was eleven years old, and 52.4% were male. Among them, 52 (36.4%) were HIV-infected, most (80.8%) presented viral load (VL) above 1000cp/ml, and only half (52.4%) were ART experienced at sampling (Table 2).
Table 2
Characteristics of the study population from Kinshasa (DRC).
|
With paired DBS/plasma
(%)
|
Only with DBS
(%)
|
Total
|
Total available samples
|
42 (100)
|
101 (100)
|
143 (100)
|
Male gender
|
19 (45.2)
|
56 (55.4)
|
75 (52.4)
|
Median age at sampling [IQR]
|
14 [12-17]
|
9 [3-12.8]
|
11 [5-14]
|
< 1
|
1 (2.4)
|
18 (17.8)
|
19 (13.3)
|
≥1-5
|
0
|
12 (11.9)
|
12 (8.4)
|
>5-10
|
5 (11.9)
|
22 (21.8)
|
27 (18.9)
|
>10-15
|
16 (38.1)
|
32 (31.7)
|
48 (33.5)
|
>15
|
18 (42.9)
|
16 (15.8)
|
34 (23.8)
|
Unknown
|
2 (4.7)
|
1 (1)
|
3 (2.1)
|
Confirmed HIV status
|
|
|
|
HIV+
|
38 (90.5)
|
14 (13.9)
|
52 (36.4)
|
HIV-
|
4 (9.5)
|
87 (86.1)
|
91 (63.6)
|
ARV experience and VL
|
|
|
|
ARV experience
|
39 (92.9)
|
23 (22.8)
|
75 (52.4)
|
≥1000 cp/ml
|
31 (73.8)
|
11 (10.9)
|
42 (29.4)
|
Mother’s HIV status
|
|
|
|
HIV+
|
17 (40.5)
|
43 (42.6)
|
60 (41.9)
|
HIV-
|
4 (9.5)
|
24 (23.7)
|
28 (19.6)
|
Unknown
|
21 (50)
|
34 (33.7)
|
55 (38.5)
|
Information extracted from clinical files. DRC, the Democratic Republic of the Congo; DBS, dried blood samples, IQR, interquartile range; ART, antiretroviral treatment; cp/ml, HIV-1 RNA copies per millilitre of plasma corrected from the DBS cp/dot considering the patient’s haematocrit(23); VL, viral load; Viral load quantified by Cobas®v2.0 (Roche), limit of quantification 20cp/ml. Age in years. |
Immunization level in plasma of the paediatric population.
Protective IgG levels greatly varied for each pathogen in the study cohort. Different protection coverage was found in the 42 children and adolescents with available plasma for rubella (93%), diphtheria (71%), mumps (69%), measles (64%), tetanus (7%) and pertussis (2%) in plasma (Figure 1a). Therefore, we found that 93% of children/adolescents did not show IgG protection against pertussis, 43% against tetanus, 29% against measles and 21% against mumps. The percentage of patients with indeterminate results in plasma was variable, ranging from 2% for rubella to 50% for tetanus (Figure 1a). Indeterminate values were excluded from the final calculation.
Cut-off calculation for each pathogen using DBS.
To evaluate the validity of DBS to monitor the immune protection, we analysed the presence of protective IgG against the six pathogens in the 42 paired DBS/plasma specimens (Figure 1).
The sensitivity of each VirClia®IgG test for the detection of antibodies in DBS, considering the same cut-off for plasma (gold-standard sample) established by the manufacture’s, was 100% for pertussis, diphtheria and mumps, 97.4% for rubella, lower for measles (81.8%) and tetanus (66.7%). Specificity was very high for pertussis (97.6%), but decreased for tetanus (87.2%), measles (40%), mumps (7.7%), diphtheria and rubella (0% each). The PPV ranged from 28.6% (tetanus) to 92.7% (rubella)
A new cut-off was calculated for each test, to achieve optimal sensitivity and/or specificity in the detection of IgG using DBS. The application of optimal cut-off for each pathogen allowed to increase the PPV and NPV of the tests from the study cohort when using DBS. (Table 3).
Table 3
Results of VirClia®IgG test for the detection of protective IgG against six pathogens responsible for vaccine-preventable diseases using dried blood samples from 42 paediatric patients.
|
MEASLES
|
MUMPS
|
RUBELLA
|
Cut-off
|
Sen
|
Spe
|
PPV
|
NPV
|
Cut-off
|
Sen
|
Spe
|
PPV
|
NPV
|
Cut-off
|
Sen
|
Spe
|
PPV
|
NPV
|
PLASMA
|
>1.1
|
100%
|
100%
|
100%
|
100%
|
>1.1
|
100%
|
100%
|
100%
|
100%
|
>1.1
|
99%
|
100%
|
100%
|
100%
|
DBS with PLASMA
cut-off
|
>1.1
|
81.8%
|
40%
|
71%
|
54.6%
|
>1.1
|
100%
|
7.7%
|
70.7%
|
100%
|
>1.1
|
97.4%
|
0%
|
92.7%
|
0%
|
DBS
|
Max Sen
|
≥0,73
|
100%
|
13.3%
|
67.5%
|
100%
|
>1.1
|
100%
|
7.7%
|
70.7%
|
100%
|
>1.04
|
100%
|
0%
|
95%
|
0%
|
Max Esp
|
≥2.83
|
55.6%
|
100%
|
100%
|
55.6%
|
≥6.18
|
37.9%
|
100%
|
100%
|
41.9%
|
≥3.97
|
43.6%
|
100%
|
100%
|
12%
|
Optimal Sen and Spe
|
≥2.7
|
59.3%
|
93%
|
94%
|
56%
|
≥5.19
|
55.2%
|
76.9%
|
84%
|
43%
|
≥1.84
|
92.3%
|
66.7%
|
97.3%
|
40%
|
|
DHIPTHERIA
|
PERTUSSIS
|
TETANUS
|
Cut-off (IU/ml)
|
Sen
|
Spe
|
PPV
|
NPV
|
Cut-off
(IU/ml)
|
Sen
|
Spe
|
PPV
|
NPV
|
Cut-off
(IU/ml)
|
Sen
|
Esp
|
PPV
|
NPV
|
PLASMA
|
>0,01
|
100%
|
100%
|
100%
|
100%
|
>120
|
100%
|
100%
|
100%
|
100%
|
>0.2
|
96%
|
100%
|
100%
|
100%
|
DBS with PLASMA cut-off
|
>0.01
|
100%
|
0%
|
71,4%
|
0%
|
>120
|
100%
|
97.6%
|
50%
|
100%
|
>0.2
|
66.7%
|
87.2%
|
28.6%
|
97.1%
|
DBS
|
Max Sen
|
≥0.03
|
100%
|
25%
|
76,9%
|
≥586
|
100%
|
100%
|
100%
|
100%
|
100%
|
46.2%
|
12.5%
|
100%
|
100%
|
100%
|
Max Spe
|
≥0.1
|
36.7%
|
100%
|
100%
|
≥586
|
100%
|
100%
|
100%
|
100%
|
33.3%
|
100%
|
10%
|
95.1%
|
95.1%
|
95.1%
|
Optimal Sen and Spe
|
≥0.09
|
46.7%
|
83.3%
|
78.5%
|
≥586
|
100%
|
100%
|
100%
|
100%
|
66.7%
|
89.7%
|
33.3%
|
97.2%
|
97.2%
|
97.2%
|
IU, international units; ml, millilitre; PPV, positive predictive value; NPV, negative predictive value; DBS, dried blood spots sample; Max, maximum; Sen, sensitivity; Spe, specificity. The cut-offs provided by VirCell for plasma have been taken as reference |
Comparison of immunization results in DBS vs. plasma in the 42 children and adolescents with paired samples.
The agreement in the immune protection rate for the 6 pathogens under study in the 42 children with paired DBS/plasma specimens varied according to the considered cut-off value showed in Table 3.
When using the plasma cut-off for DBS, the percentage of immunized children in DBS for all pathogens was higher than when using plasma (Figure 2 options A vs. B), with significant differences for 2 of them (+28.6% for mumps and diphtheria). By contrast, the observed protection was only +2.4%,for pertussis, +4.8% for rubella and +9.5% for tetanus and measles.
When we applied in DBS the cut-off providing maximum (100%) specificity (Figure 2 option C), the rate of protected children for all pathogens was underestimated from 2-3 fold times vs. values provided in plasma with VirClia cut-off for 5 pathogens: rubella (-50%), diphtheria (-45.2%), mumps (-42.9%), measles (-28.6%) and tetanus (-4.8%). The only exception was pertussis, showing a similar low number of protected subjects than in plasma.
When the optimal cut-off showed in Table 3 for each pathogen was applied in DBS (Figure 2 option D), the percentage of immunized among these 42 children was lower than using plasma to diphtheria (-35.7%), mumps and measles (-23.8%) and rubella (-4.8%), was slightly higher (+2.4%) to tetanus, and similar to pertussis.
Estimated immunization level in DBS of 143 children and adolescents.
To study the protection coverage rates to 6 vaccine-preventable diseases in the whole study population from Kinshasa, we evaluated the percentage of children with protective IgG against each pathogen under study in the 143 DBS using cut-off providing maximum (100%) specificity (corresponding to 0% false positives) described at Table 3. We decided to use this cut-off to guarantee that all patients identified as immunized in the whole study cohort were protected, avoiding overestimation in the percentage of immunized patients. We did not use the plasma cut-off due to the previously observed overestimation of the percentage of immunized for all pathogens (Figure 2 option B).
With this 100% specificity cut-off, protective IgG were detected in less than half of the 143 studied patients: 46.9% for rubella, 45.5% for measles, 36.4% for diphtheria, 24.5% mumps, and 0.7% for pertussis. As it happened with the immunization values obtained when comparing the 42 paired samples DBS vs. plasma, immunization rates were between 2 and 3 times lower than those observed in plasma (gold standard) comparing data from the 143 DBS vs. 42 plasma (15.4% vs.7.1%), except for tetanus, where the percentage of immunized patients doubled (Figure 3).