Abstract
Background:
Travel-related malaria in non-endemic areas returning from endemic areas presents important challenges to diagnosis and treatment. Imported malaria to newly malaria-free countries poses further threats of malaria re-introduction and potential resurgence. For those traveling to places with high Plasmodium falciparum prevalence, prophylaxis against this parasite is recommended, whereas causal prophylaxis against relapsing malaria is often overlooked.
Methods:
We analyzed a cluster of imported malaria among febrile patients in Shanglin County, Guangxi Province, China, who had recent travel histories to Western and Central Africa. Malaria was diagnosed by microscopy and subsequently confirmed by species- and subspecies-specific PCR. Plasmodium vivax was genotyped using a barcode consisting of 42 single nucleotide polymorphisms.
Results:
Investigations of 344 PCR-confirmed malaria cases revealed that in addition to Plasmodium falciparum being the major parasite species, the relapsing parasites Plasmodium ovale and P. vivax accounted for ~40% of these imported cases. Of the 114 P. ovale infections, 65.8% and 34.2% were P. ovale curtisi and P. ovale wallikeri, respectively, with the two subspecies having a ~2:1 ratio in both Western and Central Africa. Phylogenetic analysis of 14 P. vivax isolates using a genetic barcode demonstrated that 11 formed a distinct clade from P. vivax populations from Eastern Africa.
Conclusion:
This study provides support for active P. vivax transmission in areas with the predominant Duffy-negative blood group. With relapsing malaria making a substantial proportion of the imported malaria, causal prophylaxis should be advocated to travelers with a travel destination to Western and Central Africa.
Keywords: imported malaria, relapses, Plasmodium vivax, Plasmodium ovale, west Africa, central Africa
1. Introduction
In the past decade, malaria control efforts have led to an impressive reduction in global malaria incidence, bringing renewed promises of malaria elimination [1]. In Southeast Asia, 21 countries have allied to re-focus their national malaria control programs on malaria elimination (www.apmen.org); the six countries of the Greater Mekong Sub-region (GMS) have endorsed a regional plan of achieving malaria elimination by 2030 [2]. Within the GMS, China was the first country to achieve no autochthonous malaria in 2017 [3]. However, recent reports indicate that the number of imported malaria cases from endemic areas outside China’s borders has remained relatively consistent since 2007 [3]. In 2017, 2,670 imported cases were reported; Plasmodium falciparum accounted for 64.3%, whereas Plasmodium vivax and Plasmodium ovale cases were similarly abundant (496 and 350 cases, respectively) [3]. Interestingly, of the 2,285 cases imported from Africa, large proportions originated from Western (n=952, 35.6%) and Central (n=881, 33.0%) Africa. Given the abundance of Anopheles species that are competent vectors of human malaria in southern China [4], imported malaria cases could spark autochthonous infections and enable the establishment of new transmission foci, as observed in Greece [5]. Especially worrisome are the relapsing parasites P. vivax and P. ovale, which form dormant hypnozoites that can activate months later to cause new episodes, significantly extending the period of carriage and transmission. Thus, imported malaria remains a significant challenge for countries to maintain their malaria elimination status in the post-elimination era, requiring the implementation of tightened surveillance and prompt responses to imported malaria.
Guangxi Zhuang Autonomous Region, located in southwest China, was historically a malaria hyperendemic area, with malaria incidence peaking at 296.7 cases per 10,000 in 1954 [6]. After years of intensified control, the malaria burden in Guangxi declined sharply, and no local malaria transmission has been detected after 2012 [7]. However, since then, imported malaria cases have increased steadily in Guangxi. A malaria outbreak from May to August 2013 in Shanglin County revealed that most of the 874 malaria patients were adult males (98.6%) and gold miners (92.3%), who had returned from Ghana (99.7%) [8, 9]. Diagnosis by microscopy showed that P. falciparum and P. vivax accounted for 94.6% and 4.8% of the cases, respectively. Because Guangxi’s subtropical climate is conducive to Anopheles mosquito breeding and year-round malaria transmission, the massive importation of malaria to this malaria-free region is concerning. To promote prevention and control of imported malaria in Shanglin, we conducted an epidemiological investigation of returning overseas travelers who received care for malaria in Shanglin County Hospital from 2016–2017. Specifically, we estimate the prevalence of imported Plasmodium species detected by PCR and microscopy, describe the geographic distribution of these various species among their countries of origin, and illustrate the phylogenetic analysis of P. vivax strains in this population.
2. Methods
2.1. Patients and survey methods
We conducted an epidemiological investigation of 399 suspected malaria patients who had returned from Africa and received care at Shanglin County Hospital, Guangxi Province, from January 1, 2016 to November 30, 2017. Initial diagnosis of malaria was performed by microscopic examination of Giemsa-stained thin and thick blood smears. After obtaining informed consent from the participants, demographic information (sex, age, occupation, education, countries visited) was ascertained using a structured questionnaire, and 5 ml of venous blood was collected. The study protocol was approved by the IRB of Kunming Medical University.
2.2. Molecular diagnosis
Parasite genomic DNA was extracted from 200 μL of whole blood using the QiAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. After extraction, DNA was eluted with 50 μL of elution buffer and stored at −20°C until molecular diagnosis by PCR could be completed. Plasmodium genus-specific primers were used for the primary PCR. Nested PCR using species-specific primers was performed to identify P. falciparum, P. vivax, P. malariae, and P. ovale [10]. The two P. ovale subspecies, P. ovale curtisi and P. ovale wallikeri, were differentiated using specific primers described previously [11].
2.3. Genotyping of P. vivax cases and phylogenetic analysis
The 42 single nucleotide polymorphism (SNP) barcode for identifying geographic origins of P. vivax infections was used to genotype the P. vivax strains [12]. High-throughput genotyping of the SNPs were performed using the iPLEX chemistry coupled with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Briefly, P. vivax samples were adjusted to have a final DNA concentration of 10–30 ng/μl, and nested PCR was performed for DNA fragments covering the 42 SNPs as described earlier [12]. The PCR products were then treated with shrimp alkaline phosphatase, and a single-nucleotide primer extension assay was performed. The mass spectra of the primer extension products were acquired and analyzed with the MassARRAY® System and Typer 4.0 software (Agena Bioscience). P. vivax isolates genotyped in this study were compared with those from South America (French Guiana and Brazil); Asia (Sri Lanka) and Eastern Africa (Ethiopia) [12]. A phylogenetic tree was constructed by using the Maximum Likelihood method with 50 bootstraps implemented in MEGA version 5.1 [13].
3. Results
3.1. Demographic characteristics of patients with imported malaria
During the two-year period from January 1, 2016 to November 30, 2017, 399 febrile patients with recent travel histories to Africa were subjected for malaria diagnosis by microscopic examination of thick and thin smears, of which 320 were confirmed to be parasite-positive (Table 1). The remaining 79 patients who were parasite-negative by microscopy all had rigor-like symptoms, were treated with an oral artemisinin-based combination therapy (ACT), artesunate-amodiaquine earlier at local clinics, and attended the County Hospital within 24 h of the treatment for fear of deterioration of their conditions. At the hospital, they were presumptively treated with dihydroartemisinin-piperaquine (D-Artepp®) with symptoms resolved within two days. Although expatriates returned home throughout the year, 53% returned during a four-month period from April – July (not shown). Most patients (97.5%) were adult males with a median age of 41 (range 15–60) years; nearly all (99.4%) were farmers from rural areas of Shanglin county; the majority (97.2%) had a travel history to Western and Central Africa (Table 1); none of them took chemoprophylaxis. Consistent with P. falciparum being the predominant parasite species in Africa, 60% (192/320) of the microscopy-confirmed malaria cases were due to P. falciparum infection. The remaining patients were diagnosed with two relapsing malaria species, P. ovale (37.2%) and P. vivax (2.8%).
Table 1.
Selected characteristics of patients with symptomatic malaria returning from Africa admitted to Shanglin County Hospital, Guangxi Province, China, 2016–2017
| Patient characteristics (n=399) | No. (%) |
|---|---|
| Gender | |
| Male | 389 (97.5) |
| Female | 10 (2.5) |
| Age in years (median, range) | 41 (15–60) |
| Occupation | |
| Farmers | 397 (99.4) |
| Others | 2 (0.6) |
| Education | |
| Primary school | 140 (35.1) |
| Middle and high school | 259 (64.9) |
| Regions visited | |
| West Africa (Ghana, Liberia, Sierra Leone, Ivory Coast, | 280 (70.3) |
| Central Africa (Cameroon, Central African Republic, | 89 (22.4) |
| West and Ce ntral Africa | |
| Ghana-Cameroon | 11 (2.8) |
| Ghana-ROC | 3 (0.7) |
| Ghana-DRC | 3 (0.7) |
| Liberia-DRC | 1 (0.3) |
| Others | |
| Uganda-DRC | 1 (0.3) |
| Ghana-Tanzania | 2 (0.5) |
| Ghana-Mozambique | 1 (0.3) |
| Zimbabwe-Ghana | 1 (0.3) |
| Mozambique-Cameroon | 1 (0.3) |
| Ghana-Zimbabwe-Cameroon | 1 (0.3) |
| Cambodia-Ghana | 1 (0.3) |
| Libya | 1 (0.3) |
Abbreviations: ROC – Republic of the Congo, DRC – Democratic Republic of the Congo.
3.2. PCR confirmation of Plasmodium infections
Nested PCR was conducted to confirm the diagnosis by microscopy. Of the 399 cases analyzed, 344 tested positive by nested PCR, among which 201, 90, 15, and 1 were identified as infections by P. falciparum, P. ovale, P. vivax and P. malariae, respectively (Table 2). In addition, 33 were co-infections of P. falciparum with either P. ovale, or P. vivax, or P. malariae; three were P. vivax/P. ovale co-infections; and one was P. falciparum/P. vivax/P. ovale co-infection. Consistent with increased sensitivity of nested PCR, 34 microscopy-negative, suspected malaria cases were identified to be Plasmodium positive, including 25 P. falciparum, one P. vivax, five P. ovale, and three P. falciparum/P. ovale mixed infections. Lastly, six P. falciparum and four P. ovale cases diagnosed by microscopy were PCR negative. For the 79 microscopy-negative cases, 45 were also PCR-negative, which left 354 of the 399 febrile patients as confirmed malaria cases. Since all the 45 patients had been treated with an ACT before attending the hospital, which might have reduced the parasitemia below the detection limits, and all responded well to presumptive ACT treatment, these cases were considered “suspected malaria cases.”
Table 2.
Prevalence of single and co-infections of Plasmodium species detected by PCR and microscopy
| PCR results (No.) | Microscopy (No.) | |||
|---|---|---|---|---|
| Pf | Pv | Po | ND | |
| Pf (201) | 167 | 0 | 9 | 25 |
| Pv (15) | 1 | 5 | 8 | 1 |
| Pm (1) | 0 | 0 | 1 | 0 |
| Po (90) | 7 | 3 | 75 | 5 |
| Pf/Pv (7) | 4 | 1 | 2 | 0 |
| Pf/Po (23) | 5 | 0 | 15 | 3 |
| Pf/Pm (3) | 2 | 0 | 1 | 0 |
| Pv/Po (3) | 0 | 0 | 3 | 0 |
| Pf/Pv/Po (1) | 0 | 0 | 1 | 0 |
| ND (55) | 6 | 0 | 4 | 45* |
| Total | 192 | 9 | 119 | 79 |
Abbreviations: Pf – P. falciparum, Pv – P. vivax, Po – P. ovale, Pm – P. malariae, ND – not detected.
These patients were parasite-negative by microscopy and PCR, but were presumptively treated for malaria.
There were considerable discrepancies between the diagnostic results by microscopy and PCR. Of the 192 P. falciparum microscopy-confirmed cases, 17 were identified as either P. ovale or P. vivax infections by PCR, suggesting that these should have been treated with anti-relapsing therapies. Conversely, 25 suspected and nine P. ovale infections were identified to be P. falciparum infections by PCR.
3.3. Origins of the PCR-confirmed Plasmodium infections
Of the 344 PCR-confirmed malaria cases, 231 (67.1%) and 46 (13.4%) were imported from Ghana and Cameroon, respectively (Figure 1). P. ovale mono-infections accounted for a substantial proportion of all confirmed cases from both Western Africa (25.2%) and Central Africa (23.0%). P. vivax was detected in 7.6% (26/344) of PCR-positive samples; of these, seven were mono-infections among Ghana-returned and four were mono-infections among the Democratic Republic of the Congo (DRC)-returned travelers (Figure 1). These data corroborate the presence of P. vivax transmission in both Western and Central Africa, where the Duffy-negative blood group is predominant among populations in these regions [14, 15].
Fig. 1.
Map of Africa showing the origins of PCR confirmed malaria cases. The pie charts show the proportions of each Plasmodium species and the two P. ovale subspecies.
3.4. Prevalence of P. ovale curtisi and P. ovale wallikeri
Of the PCR-confirmed Plasmodium infections, 33.1% (114/344) were P. ovale, among which 90 were P. ovale single-species infections, whereas 24 were co-infections with either P. falciparum or P. vivax or both. Molecular identification revealed that 65.8% and 34.2% of the 114 P. ovale parasites were P. ovale curtisi and P. ovale wallikeri, respectively (Table 3). Both subspecies, P. ovale curtisi (70.7% and 21.3%) and P. ovale wallikeri (59.0% and 20.5%), originated in Western and Central Africa, respectively. Among the 76 P. ovale parasites from Western Africa, 69.7% and 30.3% were P. ovale curtisi and P. ovale wallikeri, respectively. The composition of the two subspecies among 24 P. ovale parasites that originated from Central Africa was 66.7% P. ovale curtisi and 33.3% P. ovale wallikeri, similar to that from Western Africa. The P. ovale curtisi/P. ovale wallikeri ratio was also similar among those from Ghana and Cameroon.
Table 3.
Distribution of P. ovale subspecies among countries of origin
| Region | P. ovale curtisi | P. ovale wallikeri | Total | ||
|---|---|---|---|---|---|
| No. (%) | Country of origin (N) | No. (%) | Country of origin (N) | No. (%) | |
| Western | 53 (70.7) | Ghana (52) | 23 (59.0) | Ghana (22) | 76 (66.7) |
| Africa | Sierra Leon (1) | Cote D’ivoire (1) | |||
| Central | 16 (21.3) | DRC (1) | 8 (20.5) | DRC (1) | 24 (21.1) |
| Africa | ROC (3) | ROC (2) | |||
| Cameroon (11) | Cameroon (4) | ||||
| Cameroon-Angola (1) | Cameroon-DRC (1) | ||||
| Others* | 6 (8.0) | Ghana-Cameroon (2) | 8 (20.5) | Ghana-Cameroon (1) | 14 (12.3) |
| Ghana-ROC (3) | DRC-Uganda (1) | ||||
| Ghana-Zimbabwe- | Ghana-Mozambique (1) | ||||
| Cameroon (1) | DRC-Liberia (1) | ||||
| Libya (1) | |||||
| Total | 75 (65.8) | 39 (34.2) | 114 | ||
Those who traveled to more than one African region. DRC, Democratic Republic of Congo; ROC, Republic of Congo.
3.5. Phylogeny of P. vivax isolates from Western and Central Africa
To study the evolutionary relationship of P. vivax isolates from Western and Central Africa, P. vivax parasites were genotyped using the 42-SNP barcode [12]. Using the Sequenom MassARRAY platform, 14 P. vivax cases were successfully genotyped. Phylogenetic analysis was performed based on the 42-SNP barcode using the Maximum Likelihood algorithm (Figure 2). Consistent with earlier findings, the 42 SNP clearly separated parasites from South America, Asia (Sri Lanka) and Eastern Africa (Ethiopia) into three distinct clades. Except for three parasite isolates originating from Ghana and the DRC and falling into the same clade as the Eastern African parasites, the remaining 11 parasite isolates from Western and Central Africa formed a distinct clade, which was more closely related to the Asian parasites from Sri Lanka. It is interesting to note that the barcode, to a large extent, distinguished the Western African (Ghana) from the Central African (Republic of the Congo – ROC and DRC) parasites. These data demonstrated that P. vivax isolates in Western and Central Africa were rather distantly related to those from Eastern Africa.
Fig. 2.
Phylogeny of P. vivax isolates from South America, Asia, Eastern Africa, and those identified from this study (originated from Western and Central Africa). The Maximum Likelihood tree was constructed using the 42-SNP barcode for each parasite isolate. DRC, Democratic Republic of Congo; ROC, Republic of Congo.
4. Discussion
The number of imported malaria cases has remained relatively steady in China in the past decade, posing a serious threat to the recently achieved malaria-free status – with no autochthonous infections [3]. In southern China, where malaria was formerly rampant, the abundance of local vector species emphasizes the importance of a surveillance system for timely monitoring of imported cases to prevent the establishment of new transmission foci. Our epidemiological investigation demonstrates that the majority of patients with imported malaria had returned from western and central Africa (92.7%), specifically Ghana (67.7%) and Cameroon (11.5%). Of concern, approximately 40% of returned travelers were infected with P. ovale or P. vivax. Because of the earlier malaria outbreak among Ghana-returned expatriates in Shanglin County [8], health messages delivered to subsequent travelers in Shanglin County may have enabled them to recognize the risk of malaria infections, use preventive measures such as bed nets, and even carry antimalarial treatments (mostly Artesun-Plus® tablets from Guilin Pharma, which contain artesunate and amodiaquine) during their travel. Thus, many of the malaria cases occurring after their returns to Guangxi, China, may reflect the incomplete treatment of infections (as many were self-treated without a proper diagnosis) or misdiagnosis at local clinics due to challenges in diagnosing P. ovale [16]. In addition, since artesunate-amodiaquine only clears the blood-stage infections, the large proportion (~40%) of the cases containing P. ovale and P. vivax infections should represent relapses. In addition to the confirmed cases, >10% (45/399) of the febrile patients were presumptively treated for malaria without parasite detection by microscopy and PCR. Although these patients were considered as “suspected malaria cases” since they had all been pre-treated with an ACT at local village and township clinics before attending the County Hospital, all had malaria-related symptoms, and all responded well to malaria treatment at the hospital, there was a possibility that these were not malaria cases. Future training of local clinic-level for malaria diagnosis needs to be strengthened to ensure evidence-based malaria treatment.
Several studies of imported malaria in China documented an increasing number of P. ovale infections over the years [17, 18]. Compared with the 2013 malaria outbreak in Shanglin County when only one of the 874 cases was identified as P. ovale infection by microscopy [8, 9], in our study, 33% of PCR-confirmed cases were due to P. ovale single or mixed infections. While this may not indicate the increasing prevalence of P. ovale infection in the countries of origin, it is more likely linked to disease relapse since radical cure of the initial infections was not implemented due to self-medication and potential misdiagnosis. In this study, there were similar proportions of P. ovale cases from Western and Central Africa. Given that most travelers went to Ghana and Cameroon, the relative proportions of the P. ovale subspecies may reflect the same of what occurs locally in these regions. From the destination countries of the expatriates, it can be surmised that P. ovale infections occur in the majority of African countries, although their geographical distribution and prevalence may differ. The widespread distribution of P. ovale in Africa, the potential of this parasite to cause severe diseases [19, 20], and increasing proportions of expatriates contracting P. ovale infections emphasize the need for greater attention to this parasite in malaria elimination settings.
The acquisition of vivax malaria from Western and Central Africa indicates the ongoing transmission of P. vivax in these regions. Historically, the rarity or even “lack” of P. vivax in these regions has been attributed to the dominance of the Duffy-negative blood group in these populations, a receptor required for the invasion of erythrocytes by P. vivax [21]. Documentation of P. vivax infections in residents of Central and Western Africa as well as international travelers returning from these regions to malaria-free countries suggests widespread P. vivax transmission in Africa [14, 15]. The identification of P. vivax strains able to infect Duffy-negative individuals in Eastern Africa [22–25], Central Africa [26–28], and Western Africa [29–33] suggests that P. vivax may have evolved to exploit alternative pathways to invade Duffy-negative reticulocytes [34]. Since P. vivax in Africa has been associated with asymptomatic infections in Duffy-negative people from cross-sectional surveys [15], this study indicates that such “silent” P. vivax reservoirs can readily infect Duffy-positive people traveling to such areas. Relapses caused by the liver hypnozoites of these parasites can occur months or even a year after the primary attack [35], providing the parasites an extended period of transmission. Thus, special attention needs to be given to manage imported P. vivax and P. ovale parasites. Using a genetic barcode consisting of 42 SNPs distributed in the P. vivax genome, we found that the Central and Western African P. vivax isolates mostly fell into closely related clades, which are distinctive from parasites from South America and Eastern Africa, but more closely related to parasites from Asia (Sri Lanka) [36]. This analysis suggests that P. vivax parasites circulating in Duffy-negative blood predominant in human populations of Western and Central Africa are genetically different from those in other parts of the world.
With the steady number of imported malaria cases in China, the government has strengthened multi-level medical alliances to promote public health education of those traveling to malaria-endemic regions and ensure effective management of malaria cases from the expatriates. Printed education materials disseminated to these international travelers include detailed descriptions of malaria, transmission by mosquitoes, symptoms, diagnosis, treatment, and personal prevention measures emphasizing bet net usage. However, chemoprophylaxis (e.g., causal prophylaxis with daily primaquine or weekly tafenoquine) has been overlooked and should be advocated in the future.
5. Conclusions
The relapsing parasites P. ovale and P. vivax were detected in ~40% of the imported malaria cases in a southern China county with a recent travel history to Western and Central Africa. The presence of active transmission of P. vivax and P. ovale in many African regions demonstrates the need for accurate diagnosis, proper treatment with anti-relapse therapies, and even causal prophylaxis for travelers to Africa.
Highlights.
We investigated a cluster of imported malaria cases in a southern Chinese county
Patients had travel histories to malaria-endemic areas of West and Central Africa
Relapsing malaria P. ovale and P. vivax accounted for ~40% of PCR-confirmed cases
The two P. ovale subspecies had a ~2:1 ratio in both West and Central Africa
Some P. vivax strains formed a separate group different from East African parasites
Acknowledgments
The authors thank all the patients for volunteering to participate in the study. We thank Dr. H. G. Ngassa Mbenda for helping with the phylogenetic analysis.
Funding
LC was supported by a grant (U19AI089672) from the National Institutes of Health, USA. ZY was supported by National Science Foundation of China (31860604 and U1802286), and Major Science and Technology Project of Yunnan Province (2018ZF0081). CL, XC and YZ were supported by Yunnan Applied Basic Research Projects-Union Foundation (2017FE468-185, 2018FE001-190, 2015FB034, respectively). YQ was supported by Guangxi Zhuang Autonomous Region Health Commission of Scientific Research Project (Z20190892).
Footnotes
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Conflicts of interest
The authors affirm that they have no conflict of interest.
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