Demographics
Of the 194 participants, 105 (54.1%) were residents of Obom, a high parasite prevalence area and 89 (45.9%) were residents of Asutsuare, a low parasite prevalence area. There was no significant difference (p= 0.652) in the distribution of males between the two study sites (53% in Asutsuare and 49% in Obom) (Table 1) or in terms of age (p= 0.109). The median (IQR) age of participants from Obom was 14 (12 – 24.3) years and the median (IQR) age in Asutsuare was 16 (13 – 25.8).
Table 1. Demographics of the study participants
Parameters
|
Obom (n=105)
|
Asutsuare (n=89)
|
P-value
|
Sex
|
|
|
|
Male/Female
|
48/50*
|
43/38*
|
0.652a
|
Age (yrs)
|
|
|
|
Median
|
14
|
16
|
0.109b
|
Range
|
64
|
56
|
|
Min-Max
|
6.0-70.0
|
10.0-66.0
|
|
Temp (0C)
|
|
|
|
Median
|
35.9
|
36.5
|
|
Range
|
7.4
|
4.3
|
|
Min-Max
|
30.1-37.5
|
33.2-37.5
|
|
Parasite Positivity
|
|
|
|
Microscopy n (%)
|
20 (19.1)
|
2 (2.2)
|
0.0002a
|
HRP2 RDT n (%)
|
37 (37.8)
|
0
|
0.0001a
|
yrs=year, Min = minimum, Max = maximum, Temp = temperature, and n = total number of samples tested. a Pearson Chi-Square, b Mann Whitney (Two-tailed). *a few samples had missing gender data.
Estimation of parasite prevalence and density by microscopy
A total of 19.1% (20/105) and 2.2% (2/89) of the samples were identified containing P. falciparum by microscopy in the high (Obom) and low (Asutsuare) transmission sites respectively (Figure 2 and Table 1). One of the samples from Obom contained a mixture of P. falciparum and P. malariae (however, this was not confirmed by PCR). A higher number of P. falciparum parasite carriers were detected in the high parasite prevalence setting (Obom) relative to the low parasite prevalence setting of (Asutsuare) (Pearson Chi-Square, p= 0.0002) (Table 1). Parasite density estimated per microlitre (PD/µl) blood from Obom ranged between 32 and 5080 with a median (IQR) of 180 (80-405), whilst in Asutsuare, both samples that tested positive by microscopy had a parasite density of 40 (Figure 3).
Estimation of parasite prevalence based on antigen detection
RDT positivity rates
The HRP2-RDT identified a total of 37.8% (39/101) of the samples collected from the high transmission area as positive. RDT results were not available for 4 samples from the high parasite prevalence area. None of the samples from the low parasite prevalence setting of Asutsuare tested positive by the HRP2 RDT (Figure 2 and Table 1).
HRP2 Bead detection of Plasmodium antigen levels
Detection of the P. falciparum HRP2 antigen was significantly higher in Obom (61.9%) when compared to Asutsuare (11.2%), p< 0.0001. The P. falciparum HRP2 antigen levels of samples in Obom ranged from 226.0 pg/ml to 820,368 pg/ml, with a median of 4689.0 pg/ml and 49.4 pg/ml to 44,980 pg/ml with a median of 236.4 pg/ml in Asutsuare. The median HRP2 antigen levels in samples from Obom (4689.0 pg/ml) was significantly higher than samples from Asutsuare with median HRP2 antigen level of 236.4 pg/ml (Mann Whitney test, p< 0.0001) (Figure 3).
Estimation of parasite prevalence based on molecular tests
Nested PCR
The prevalence of P. falciparum by Nested PCR (nPCR) in Obom (71.4% (70/98)) was significantly higher (Fisher’s exact test, p = 0.0056) than parasite prevalence in Asutsuare (50.6% (42/83) (Figure 2). The nPCR could not be performed for 7 and 6 samples from Obom and Asutsuare respectively.
PET-PCR Analysis
In Obom, 60% (63/105) of the samples tested positive for P. falciparum, with CT values ranging from 23.8 to 36.3 and a median CT value of 30.5. The corresponding parasite density estimates ranged from 0.4 p/µl to 7,002 p/µl, with a median of 37.1 p/µl. In Asutsuare, 5.6% (5/89) of the samples tested positive for P. falciparum, with a CT range of 27.7 to 33.1 and a median of 31.8. The corresponding parasite density estimates range from 5.0 p/µl to 331.7 p/µl, with a median of 14.0 p/µl (Figure 3). Although a significantly higher number of parasites were detected in Obom than in Asutsuare, (Fisher’s exact test, p< 0.001) (Figure 2), there was no significant difference between the estimated parasite densities of the two sites when their median parasite density was compared (Mann Whitney test, p= 0.8879).
Illustration of relationships among sensitive detection methods by areas
A total of 84.1% (53/63) of the PET-PCR positive samples and 28.5% (12/42) of the PET-PCR negative samples from Obom tested positive by the HRP2 bead assay (Figure 4A). Whilst in Asutsuare, 80% (4/5) of the PET-PCR positive samples and 7.1% (6/84) of the PET-PCR negative samples tested positive by the HRP2 bead assay (Figure 4B).
There were 8 samples from Obom that tested positive for P. falciparum by all the five methods tested (Figure S1), whilst 8 samples were negative by the same five methods (Figure S1). In the low transmission setting, no sample was identified as positive by all the methods (Figure S1), whilst 35 samples were identified as negative by all the five tests.
Comparison among sensitive detection methods by areas
In the high transmission setting, parasite prevalence estimated by Nested PCR was significantly higher than that estimated by PET-PCR and the HRP2 bead assay (Pearson Chi square=13.06 and 6.76 respectively, p< 0.001 for both), but parasite prevalence estimated by the HRP2 bead assay and PET-PCR were similar (Pearson Chi square=31.89 and p> 0.05) (Table S3).
In the low transmission setting, parasite prevalence estimated by the HRP2 bead assay was significantly higher than that recorded by PET-PCR (Fisher’s Exact Test p< 0.000) (Table S3) and the difference between parasite prevalence estimated by both Nested PCR and PET-PCR on the one hand and Nested PCR and the HRP2 bead assay on the other were similar (Fisher’s Exact Test p=1.000 and 0.156 respectively).
Comparison between antigen detection methods by areas
The HRP2 bead assay detection of HRP2 antigen levels identified a significantly higher number of positive samples compared to the HRP2 based RDT in the high malaria transmission setting (Pearson Chi-Square=17.22, p< 0.001) (Figure 2 and 5B). Comparisons could not be made in the low transmission site, as no sample tested positive by HRP2 RDT (Figure 5D, Table 2 and Table S3).
Comparison between nucleic acid detection methods by areas
Nested PCR identified a significantly higher number of positive samples compared to PET-PCR in both the high transmission setting, Obom (Pearson Chi-Square=13.06, p< 0.001) (Figure 2, 5A and 5C) and the low transmission setting - Asutsuare. In comparing diagnostic methods that measure similar parasite features, HRP2 antigen (RDT and the HRP2 bead assay) and parasite DNA (Nested PCR and PET-PCR), fair and significant agreements were observed only for the samples collected from the high transmission setting (Obom) (Table 2). A crosstabulation analysis between PET-PCR and the HRP2 bead assay found that the two methods agreed substantially and significantly (Cohen kappa value = 67.9%, p= 0.004).
Table 2. Inter-rater agreement between similar detection tools
|
N
|
Kappa value
|
P-value
|
Antigen Methods (RDT / HRP2 bead assay)
|
|
Obom
|
97
|
0.262
|
0.004
|
Asutsuare
|
81
|
-
|
-
|
Overall
|
178
|
0.407
|
0.001
|
DNA Methods (PET-PCR / Nested PCR)
|
|
Obom
|
98
|
0.348
|
0.001
|
Asutsuare
|
83
|
0.022
|
0.665
|
Overall
|
181
|
0.272
|
0.001
|
N, number of samples used in the analysis. The differences in the total number of samples used in the analysis is due to some samples missing results from one test or the other. No statistics could be computed for RDT vs the HRP2 bead assay in Asutsuare because no RDT positive samples were identified in Asutsuare.
Agreement between diagnostic tests
Microscopy is generally referred to as the gold standard diagnostic test for malaria. When results from the microscopy read out by the microscopists used in this study was set as the gold standard (reference test)(Table 3), the level of agreement between microscopy and the PET-PCR and the HRP2 bead assay tests in Obom was poor, with a fair agreement observed between results obtained by microscopy and RDT. In Asutsuare, the interrater agreement between microscopy and both PET-PCR and Nested PCR was poor but the agreement between microscopy and the HRP2 bead assay was fair. All the poor agreements were not significant, whilst the fair agreements were significant. There was no agreement between microscopy and nPCR in both Obom and Asutsuare (Table 3).
When Cohen’s kappa analysis (Table 3) was repeated with Nested PCR set as the gold standard (reference), there was a poor agreement between Nested PCR and RDT but fair agreement between Nested PCR and PET-PCR and the HRP2 bead assay in Obom, whilst in Asutsuare, all the agreements were poor. All the agreements in Obom were significant whilst those in Asustuare were not significant. There was no agreement between the results obtained by Nested PCR that was compared to microscopy in Obom (Table 3).
Table 3. Agreement analysis between microscopy/Nested PCR and other diagnostic tests
Parameter
|
Obom
|
Asutsuare
|
Overall
|
|
Kappa (P-value)
|
Kappa (P-value)
|
Kappa (P-value)
|
Microscopy
|
|
|
|
Microscopy vs RDT
|
0.212 (0.021)*
|
-
|
0.239 (0.001)*
|
Microscopy vs PET-PCR
|
0.055 (0.452)
|
0.061 (0.135)
|
0.161 (0.010)
|
Microscopy vs HRP2 bead assay
|
0.027 (0.698)
|
0.218 (0.028)*
|
0.156 (0.009)*
|
Microscopy vs Nested PCR
|
-0.014 (0.808)
|
-0.034 (0.768)
|
0.048 (0.262)
|
|
|
|
|
Nested PCR
|
|
|
|
Nested PCR vs RDT
|
0.186 (0.019)*
|
-
|
0.182 (0.001)*
|
Nested PCR vs PET-PCR
|
0.348 (0.001)*
|
0.022 (0.665)
|
0.272 (0.001)*
|
Nested PCR vs HRP2 bead assay
|
0.248 (0.012)*
|
0.117 (0.084)
|
0.266 (0.001)*
|
Nested PCR vs Microscopy
|
-0.009 (0.874)
|
0.047 (0.157)
|
0.045 (0.204)
|
*, significant p value; vs, versus;
67.8%, however, there was also a significant discordance (P<0.001) between the methods.