Microscopy and RDT detection
A total of 296 blood samples from malaria suspected patients were collected (Figure 1). By using microscopy and RDT, 288 positive samples including 243 P. falciparum, 17 P. vivax, 21 P. ovale, 6 P. malariae and 1 coinfection (P. falciparum + P. ovale) were observed. Seven out of the remaining 8 cases (296 minus 288) were microscopy negative but RDT positive (Figure 1). The final case was both microscopy and RDT negative but was a suspected patient with clinical symptoms of malaria.
For the 243 microscopy-positive P. falciparum cases (Figure 2A), parasitaemia ranged from 100 to 500,000 parasite/μl, and the mean parasite density was 88,879 parasite/μl (95% CI = 71,794–105,963). These cases were divided into six groups according to their densities: Very Low (≤100 parasite/μl), Low (101–500 parasite/μl), Low-middle (501–3,000 parasite/μl), Middle (3,001–10,000 parasite/μl), Middle-high (10,001–100,000 parasite/μl) and High (>100,000 parasite/μl). From the very low group to the high group (see Figure 2B and Table 2), case numbers of P. falciparum for each density interval were 7 (2.88%), 26 (10.70%), 34 (33+1, 13.99%), 30 (12.35%), 96 (39.51%) and 50 (20.58%), respectively. For P. vivax, parasitaemia ranged from 100 to 30,000 parasite/μl and the mean parasite density was 4,600 parasite/μl (95% CI= 989–8,211). For P. ovale, parasitaemia ranged from 500 to 10,000 parasite/μl and the mean parasite density was 2,610 parasite/μl (95% CI =1,348–3,871). Of which, 23.53% (4/17) of P. vivax and 19.05% (4/21) of P. ovale had parasite densities ≤500 parasite/μl. All of the 6 P. malariae cases had parasite densities from 800 to 4,000 parasite/μl and the one mixed infection was 50,000 parasite/μl (see Figure 2A).
Table 2 Determination of Plasmodium species by microscopy examination and molecular diagnosis.
Microcopic
|
Parasitemia (No. of parasites /μl)
|
Nested PCR a
|
Real-time PCR
|
Species
|
Intensity
(parasite/ul)
|
No.
|
Species
|
No.
|
Species
|
No.
|
Microcopy
positive
|
P. falciparum
|
Very Low (≤100)
|
7
|
Pf
|
6
|
Pf
|
5
|
Low (101-500)
|
26
|
Pf
|
25
|
Pf
|
21
|
Low-middle (501-3,000)
|
33
|
Pf
|
32
|
Pf
|
32
|
Middle (3,001-10,000)
|
30
|
Pf
|
30
|
Pf
|
30
|
Middle-high (10,001-100,000)
|
96
|
Pf
|
96
|
Pf
|
96
|
High (>100,000)
|
50
|
Pf
|
50
|
Pf
|
50
|
Error correction (3,000)
|
1
|
Pf+Po
|
1
|
Pf+Po
|
1
|
P. vivax
|
From 100 to 10,000
|
15
|
Pv
|
15
|
Pv
|
15
|
Error correction
|
2
|
Pv+Po
|
2
|
Pv+Po
|
2
|
P. ovale
|
From 100 to 10,000
|
19
|
Po
|
19
|
Po
|
21
|
Error correction
|
2
|
Pf a +Po
|
2
|
Pf +Po
|
0
|
P. malariae
|
From 100 to 10,000
|
6
|
Pm
|
6
|
Pm
|
6
|
P. falciparum+P. ovale
|
50,000
|
1
|
Po
|
1
|
Po
|
1
|
Microcopy
negative
|
P. falciparumb
|
7
|
Pf
|
5
|
Pf
|
3
|
Suspected case
|
1
|
Pf
|
1
|
Pf
|
1
|
Total
|
296
|
|
291
|
|
284
|
a,The nested PCR products of these cases have been confirmed by DNA sequencing. b, These cases were RDT positive for Pf.
Confirmation of the parasite species with PCR-based methods
These samples were confirmed via both nested PCR and real-time PCR. After reconfirmation by nested PCR (the primers are shown in Table 1), 1.04% (3/288) microscopy-positive and 25% (2/8) microscopy-negative cases failed to be detected. But 2.78% (8/288) microscopy-positive and 50% (4/8) microscopy-negative subjects were negative by real-time PCR (Figure 1). Nested PCR products of the 7 cases (12 minus 5), which were P. falciparum positive by nested PCR but negative by real-time PCR, were sequenced, and the results proved they were all P. falciparum infections. As shown in Table 2, the 5 negative cases by nested PCR were distributed either in the three groups with relatively low P. falciparum density (Very low, Low, Low-middle) or in the microscopy-negative group. For the 12 cases that were not detected by real-time PCR, they were all caused by P. falciparum. Their parasite density distributions were highly in accordance with those in the nested PCR detection, including 2 cases in the Very low group, 5 cases in the Low group, 1 case in the Low-middle group and 4 cases in the microscopy-negative group. Additionally, both molecular tools revealed one P. falciparum plus P. ovalis mixed infection case among the microscopy-positive P. falciparum samples. The one suspected case negative by both microscopy and RDT was proven to be P. falciparum infection.
Of the 17 microscopy-positive P. vivax cases, two actually proved to be P. vivax plus P. ovalis mixed infection by both nested and real-time PCR. All 21 microscopy-positive P. ovalis cases were diagnosed as positive by real-time PCR, but two of them proved to be P. falciparum plus P. ovalis mixed infection by nested PCR and subsequent sequencing. The identification of the 6 P. malariae cases showed the same results by three methods. There was also a case that was diagnosed as P. falciparum plus P. ovalis infection by microscopy. However, both nested PCR and real-time PCR revealed the existence of P. ovalis but not P. falciparum (Table 2). Subspecies identification of the 20 single P. ovalis positive cases showed that they consisted of 10 P. ovale curtisi, 9 P. ovale wallikeri and 1 mixture of the two subspecies according to the nested PCR and sequencing. Real-time PCR revealed similar results except for 3 P. ovale curtisi negative cases (data not shown).
Diagnostic profile of Plasmodium species by the different methods
As shown in Figure 1 and Table 3, amongst the 296 samples, there were 243 (82.09%) P. falciparum, 17 (5.74%) P. vivax, 21 (7.09%) P. ovalis, 6 (2.03%) P. malariae, 1 (0.34%) mixed infection (P. falciparum + P. ovalis) and 8 (2.70%) negative cases according to microscopic observations. Based on the nested PCR, 245 (82.77%), 15 (5.07%), 20 (6.76%) and 6 (2.03%) cases were identified as single infection by P. falciparum, P. vivax, P. ovalis and P. malariae, respectively, whereas dual species mixed infections included 3 (1.01%) cases of P. falciparum + P. ovalis, 2 (0.68%) cases of P. vivax + P. ovalis and 5 (1.69%) negative cases. Real-time PCR revealed a few minor discrepant results, including 238 (80.41%) P. falciparum, 15 (5.07%) P. vivax, 22 (7.43%) P. ovalis, 6 (2.03%) P. malariae, 1 (0.34%) mixed infections of P. falciparum + P. ovalis, 2 (0.68%) cases of P. vivax + P. ovalis and 12 (4.05%) negative cases. No triple or quadruple infection were detected in these samples.
Table 3 Comparison analysis of diagnostic tools for imported Plasmodium species infection in Wuhan, China.
|
|
Nested PCR
|
Microscopy
|
Species
|
P. falciparum
|
P. vivax
|
P. ovale
|
P. malariae
|
P. falciparum
+ P. ovale
|
P. vivax
+ P. ovale
|
Negative
|
Total (%)
|
|
P . falciparum
|
239
|
0
|
0
|
0
|
1
|
0
|
3
|
243 (82.09)
|
|
P. vivax
|
0
|
15
|
0
|
0
|
0
|
2
|
0
|
17 (5.74)
|
|
P. ovale
|
0
|
0
|
19
|
0
|
2
|
0
|
0
|
21 (7.09)
|
|
P. malariae
|
0
|
0
|
0
|
6
|
0
|
0
|
0
|
6 (2.03)
|
|
P. falciparum+P. ovale
|
0
|
0
|
1
|
0
|
0
|
0
|
0
|
1 (0.34)
|
|
Negative a
|
6
|
0
|
0
|
0
|
0
|
0
|
2
|
8 (2.70 )
|
|
Total
|
245 (82.77)
|
15 (5.07)
|
20 (6.76)
|
6 (2.03)
|
3 (1.01)
|
2 (0.68)
|
5 (1.69)
|
296 (100.00)
|
|
Real-time PCR
|
P. falciparum
|
238
|
0
|
0
|
0
|
0
|
0
|
0
|
238 (80.41)
|
|
P. vivax
|
0
|
15
|
0
|
0
|
0
|
0
|
0
|
15 (5.07)
|
|
P. ovale
|
0
|
0
|
20
|
0
|
2
|
0
|
0
|
22 (7.43)
|
|
P. malariae
|
0
|
0
|
0
|
6
|
0
|
0
|
0
|
6 (2.03)
|
|
P. falciparum+P. ovale
|
0
|
0
|
0
|
0
|
1
|
0
|
0
|
1 ( 0.34)
|
|
P. vivax+P. ovale
|
0
|
0
|
0
|
0
|
0
|
2
|
0
|
2 (0.68)
|
|
Negative
|
7
|
0
|
0
|
0
|
0
|
0
|
5
|
12 (4.05)
|
|
Total
|
245 (82.77)
|
15 (5.07)
|
20 (6.76)
|
6 (2.03)
|
3 (1.01)
|
2 (0.68)
|
5 (1.69)
|
296 (100.00)
|
|
|
|
|
|
|
|
|
|
|
|
|
|
a, these included 7 RDT positive and 1 RDT negative but suspected cases
Diagnostic performance of microscopy and real-time PCR compared to nested PCR
Nested PCR was appointed as a reference for the diagnostic summarizing of the Plasmodium species as well as the following performance assessment of the diagnostic methods. As shown in Table 4, for microscopy, the sensitivities of identification of P. falciparum, P. vivax, P. ovalis and P. malariae were 96.77%, 100%, 88.00% and 100.00%, respectively. Specificities for P. falciparum diagnosis were 91.67% and 100.00% for the other three species. The probabilities of identification of P. falciparum, P. vivax, P. ovalis and P. malariae by microscopy from the predicted positive (PPV) and not identification from the predictive negative (NPV) samples were 98.36% and 84.62% (P. falciparum), 100.00% and 100.00% (P. vivax), 100.00 % and 98.91 % (P. ovalis), 100.00 % and 100.00% (P. malariae), respectively. For the detection of P. falciparum by real-time PCR, sensitivity, specificity, PPV and NPV were 96.37%, 100%, 100% and 84.21%, respectively. These four assessment indexes were all 100% for the other three species diagnosed by real-time PCR.
Table 4 Performance of microscopy and real-time PCR compared to the reference nested PCR, including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and disease prevalence (DP).
Nested PCR ™ as standard (Confirmed by sequencing)
|
Methodological evaluation
|
Plasmodium species
|
P. falciparum
|
P. vivax
|
P. ovale
|
P. malariae
|
Nested PCR
VS Microscopy
|
Parameters
|
Positive
|
Negative
|
Positive
|
Negative
|
Positive
|
Negative
|
Positive
|
Negative
|
Positive
|
240
|
4
|
17
|
0
|
22
|
0
|
6
|
0
|
Negative
|
8
|
44
|
0
|
279
|
3
|
271
|
0
|
290
|
Analyze
|
Percentage (95% CI)
|
Percentage (95% CI)
|
Percentage (95% CI)
|
Percentage (95% CI)
|
Sensitivity
|
96.77 (93.51-98.49)
|
100.00 (77.08-100.00)
|
88.00 (67.66-96.85)
|
100.00 (51.68-100.00)
|
Specificity
|
91.67 (79.13-97.30)
|
100.00 (98.31-100.00)
|
100.00 (98.26-100.00)
|
100.00 (98.37-100.00)
|
PPV
|
98.36 (95.58-99.47)
|
100.00 (77.08-100.00)
|
100.00 (81.50-100.00)
|
100.00 (51.68-100.00)
|
NPV
|
84.62 (71.37-92.66)
|
100.00 (98.31-100.00)
|
98.91 (96.57-99.72)
|
100.00 (98.37-100.00)
|
DP
|
83.78 (78.97-87.69)
|
5.74 (3.49-9.21)
|
8.45 (5.65-12.36)
|
2.03 (0.83-4.58)
|
|
Accurancy
|
95.95 (93.03- 97.89)
|
100 (98.76-100.00)
|
98.99 (97.07-99.79)
|
100 (98.76-100)
|
Nested PCR
VS Real Time PCR
|
Parameters
|
Positive
|
Negative
|
Positive
|
Negative
|
Positive
|
Negative
|
Positive
|
Negative
|
Positive
|
239
|
0
|
17
|
0
|
25
|
0
|
6
|
0
|
Negative
|
9
|
48
|
0
|
279
|
0
|
271
|
0
|
290
|
Analyze
|
Percentage (95% CI)
|
Percentage (95% CI)
|
Percentage (95% CI)
|
Percentage (95% CI)
|
Sensitivity
|
96.37 (92.99-98.22)
|
100.00 (77.08-100.00)
|
100.00 (83.42-100.00)
|
100.00 (51.68-100.00)
|
Specificity
|
100 (90.77-100)
|
100.00 (98.31-100.00)
|
100.00 (98.26-100.00)
|
100.00 (98.37-100.00)
|
PPV
|
100 (98.03-100)
|
100.00 (77.08-100.00)
|
100.00 (83.42-100.00)
|
100.00 (51.68-100.00)
|
NPV
|
84.21 (71.63-92.09)
|
100.00 (98.31-100.00)
|
100.00 (98.26-100.00)
|
100.00 (98.37-100.00)
|
DP
|
83.78 (78.97-87.69)
|
5.74 (3.49-9.21)
|
8.45 (5.65-12.36)
|
2.03 (0.83-4.58)
|
|
Accurancy
|
96.96 (94.31-98.60)
|
100 (98.76-100)
|
100 (98.76-100)
|
100 (98.76-100)
|
The results also revealed that P. falciparum (83.78%) infection was the most prevalent, followed by P. ovalis (8.45%), P. vivax (5.74%) and P. malariae (2.03%) among the imported malaria cases in Wuhan, China. Since RDT could only distinguish falciparum and non-falciparum Plasmodium species infection, RDT was not involved in the methodological evaluation of these samples.
Origin and years distribution of the patients
Tracing the origin of the imported malaria patients demonstrated the patients returned from 28 countries of Africa and 4 countries of Asia (Fig 3A). Of the total 291 patients confirmed by nested PCR, 112 (38.49%) malaria patients were infected in West Africa, followed by Central Africa (66 cases, 22.68%), Southern Africa (62 cases, 21.31%), East Africa (38 cases, 13.06%), SE Asia (9 cases, 3.09%) and South Asia (4 cases, 1.37%) (Fig 3B). The distribution of malaria patients caused by P. falciparum was in strong accordance with above general tendency, including 102 cases from West Africa, 55 from Central Africa, 55 from Southern Africa, 27 from East Africa and 6 from SE Asia. Amongst the P. vivax patients, 8 returned from East Africa. The remainders all returned from SE Asia (3 cases) and South Asia (4 cases), respectively. The P. ovalis patients returned from West Africa (10/20), Central Africa (8/20) and Southern Africa (2/20). Only 6 P. malariae infected patients were involved, and three returned from Southern Africa, two from Central Africa and one from East Africa. The five mixed infection patients also returned from the same three regions as the P. malariae infection group.
From 2011 to 2016 (Fig 3B), the number of imported malaria patients has obviously increased yearly (F=15.11, P=0.018). Luckily, only 20 imported malaria patients were reported by the CDC and hospitals of Wuhan in 2017. However, the number of clinical malaria patients in 2018 increased to 44 cases. It is interesting to note that the predominant species was P. falciparum throughout the surveyed period. However, it is worth mentioning no consistent increasing trends in the prevalence of P. falciparum parasites infection (F=0.078, P=0.790) was detected yearly. Other Plasmodium species, including P. vivax, P. ovalis and P. malariae single infection or mixed infection, were not identified until 2014. In the next five years (2014 –2018), patients infected with P. vivax (15 cases), P. ovalis (20 cases), P. malariae (6 cases) and mixed parasite species (5 cases) were seen. In this period (2014–2018), the total imported non-P.f. cases showed no increasing trend in prevalence by year (F=4.765, P=0.117). Neither P. vivax (F=0.217, P=0.687) nor P. ovalis (F=5.000, P=0.111) significantly increased by year.