• View in gallery

    Location of the Plasmodium vivax studies in Cameroon in the present paper. (A) Plasmodium vivax samples were collected in febrile patients in the district hospitals in Santchou, Dschang, and Kyé-Ossi in Cameroon. (B) Table shows the altitude, temperature, rainfall, and malaria transmission in Santchou, Dschang, and Kyé-Ossi.

  • View in gallery

    Sequencing of the PvMSP1 gene in Plasmodium vivax isolated in Dschang. To further confirm the specificity of P. vivax, six samples that were positive for P. vivax by N-PCR were selected. Sequencing was performed on these six samples for the MSP1 gene. (A) Multiple sequence alignment for five isolates shows high similarity with the reference MSP1 sequence from P01 (Papua Indonesia). (B) The DNA sequence of the five isolates were translated into protein sequence, and alignment was compared with the reference protein sequence for MSP1 sequence from P01 (Papua Indonesia). The schematic diagram is the full length PvMSP1 gene and shows the region in which the DNA sequencing was obtained. aa = amino acid; SP = signal peptide; GPI = glycosylphosphatidylinositol.

  • View in gallery

    Determination of the Duffy genotype in Plasmodium vivax–infected people in Dschang. Duffy genotyping was determined using PCR product in the Duffy promoter region which was digested by the Sty I restriction enzyme. The expected fragment size of 82, 65, 64, and 12 bp of the digested product corresponds to Duffy-negative genotype, although the 12-bp fragment is not captured. Simulated identification was given for each isolate for representation purposes. The negative control was for no human blood.

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Plasmodium vivax Infections Detected in a Large Number of Febrile Duffy-Negative Africans in Dschang, Cameroon

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  • 1 Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy;
  • | 2 Evangelical University of Cameroon, Bandjoun, Cameroon;
  • | 3 Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland;
  • | 4 Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy;
  • | 5 Botswana-University of Pennsylvania Partnership, Gaborone, Botswana;
  • | 6 Division of Infectious Diseases, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania;
  • | 7 Department of Biomedical Sciences, Faculty of Medicine, University of Botswana, Gaborone, Botswana;
  • | 8 Mbangue District Hospital, Douala, Cameroon;
  • | 9 Dschang District Hospital, Dschang, Cameroon;
  • | 10 Faculty of Medicine and Pharmaceutical Sciences, University of Dschang, Dschang, Cameroon

ABSTRACT

The Duffy blood group is a critical receptor for Plasmodium vivax (P. vivax) invasion of red blood cells, and consequently, P. vivax infections were considered rare in sub-Saharan Africa where the prevalence of Duffy-negativity is high. However, recently, P. vivax infections have been found in Duffy-negative Africans throughout the malaria transmission area of sub-Saharan Africa, raising important questions concerning the molecular composition of these P. vivax clones and the red blood cell receptors that facilitate their invasion. Here, we describe an unusually high number of P. vivax infections in febrile Duffy-negative Africans in Dschang, Cameroon (177 of 500 outpatients), as compared with Santchou (two of 400 outpatients) and Kyé-ossi (two of 101 outpatients), in other areas in Cameroon. In the discussion, we speculate on the possible reasons why Dschang might account for the unusually large numbers of P. vivax infections in Duffy-negative individuals living there.

INTRODUCTION

Individuals living in sub-Saharan Africa were considered resistant to Plasmodium vivax (P. vivax) because of the absence of the Duffy blood group antigen which is a critical receptor for red blood cell invasion by P. vivax.1,2 More recently, P. vivax infections in Duffy-negative Africans, first described in Kenya,3 have been documented in all regions of sub-Saharan Africa.4 Duffy-negative Africans infected with P. vivax often have extremely low parasitemia, and the diagnosis depends on PCR.4 In some countries (e.g., Madagascar, Ethiopia, Sudan, and Mauritania), Duffy-positive and -negative Africans live side-by-side, but Duffy-negative Africans infected with P. vivax still have lower parasitemia and less fever,48 although anemia may be a problem.9 This difference indicates that P. vivax causes a distinct disease pattern in Duffy-positive versus Duffy-negative individuals.

Recent studies in sub-Saharan Africa reported varying prevalence of P. vivax, but the variability in the blood sample collection makes direct comparisons of the prevalence of P. vivax between studies difficult. Some were surveyed in their homes,10 and others were seen in a hospital or clinic.11 Some had fever (including the present study), and others were asymptomatic and had no fever.9,1214 Some were collected during the rainy season when transmission occurs and others during the dry season when transmission depends on other factors or no transmission occurs.

Although P. vivax infections have been clearly documented in Duffy-negative Africans, our knowledge of the molecular composition of these parasites and the receptors they use for invasion is incomplete. Plasmodium vivax infections appeared first in higher apes in Africa and then infected Duffy-positive Africans in close contact to the apes.15 Subsequently, P. vivax was selected for the loss of the Duffy blood group, the receptor for P. vivax. However, the relationship between the current P. vivax clones circulating in Africa and the clones that first infected Africans is not known. To better understand the factors that impact transmission of P. vivax, we compared the number of P. vivax infections in febrile Duffy-negative Africans living in three distinct areas in Cameroon, Dschang, at an altitude of 1,400 m and Santchou and Kyé-Ossi at lower altitudes. Previous studies of febrile patients in the hospital in Dschang during the dry season reported 27 P. vivax–positive individuals in 484 total patients (5.6%).11 However, in this study, we report that the number of P. vivax infections during the rainy season was extremely high, with 177 P. vivax–infected individuals in a total of 500 patients (35.2%). During the same period, we observed only small numbers of P. vivax infections in Santchou (two of 400, 0.5%) and in Kyé-Ossi (two of 101, 2%). We speculate in the discussion on possible reasons why Dschang might account for the unusually large numbers of P. vivax infections.

METHODS

Ethical clearance.

The Ethics Review and Consultancy Committee (ERCC) of the Cameroon Bioethics Initiative (CAMBIN) gave approval for this study, by delivering the ethical clearance CBI/427/ERCC/CAMBIN.

Inclusion criteria.

Febrile outpatients of all ages coming for consultation in the selected healthcare facilities of Santchou, Dschang, and Ambam health districts.

Exclusion criteria.

Febrile outpatients who withdrew during the data/sample collection and patients whose questionnaire did not answer at least 70% of the questions. Those who did not give their consent to participate in the study were treated for their illness even if they did not consent. Febrile outpatients transferred from another healthcare facility and patients coming for the second time during the data/sample collection period were excluded.

Recruitment process.

Participants were recruited at the reception of the selected healthcare facilities after detecting a fever above 37.5°C (auxiliary temperature) or reporting a fever history the day before. These patients were informed about the study, its objectives, and all the requirements (blood collection and answering a questionnaire) to obtain their written consent by signing the informed consent form. The languages used were principally French and English, but in cases where the participants did not know these languages, a translator was available for Bamiléké language, especially Yemba, and for the Southern language, especially Ntumu. Those are the main local languages of the study areas. The informed consent form was signed in two copies, one of which was given to the participant. They were also made aware of the possibility to withdraw from the study at any time while still receiving therapy for their illness. The patients younger than 21 years (Cameroonian legal age) were enrolled after the authorization of their parents or guardians. All the consenting patients fitting with the criteria were enrolled in the study.

Study areas.

A cross-sectional descriptive study was carried out in the rainy season in Santchou (August–December 2016) and Kyé-Ossi (May–September 2017) and Dschang (May–August 2017) (Figure 1). The study sites were Santchou health district, Dschang health district, and Ambam health district in Kyé-Ossi. Dschang and Santchou are characterized by a strong seasonal transmission pattern, whereas Kyé-Ossi (Ambam) has a perennial transmission pattern.16

Figure 1.
Figure 1.

Location of the Plasmodium vivax studies in Cameroon in the present paper. (A) Plasmodium vivax samples were collected in febrile patients in the district hospitals in Santchou, Dschang, and Kyé-Ossi in Cameroon. (B) Table shows the altitude, temperature, rainfall, and malaria transmission in Santchou, Dschang, and Kyé-Ossi.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 3; 10.4269/ajtmh.20-1255

Santchou health district is a small health district consisting of five health areas with 10 health centers. The most important activities are farming and trade. Data and samples were collected in two healthcare facilities (Santchou District Hospital and the Santchou Catholic Health Center). Santchou (5.28°N, 9.97°E)17 is located at an altitude of 750 m18 above sea level at 22 km from Dschang. The average temperature is 23.8°C, and rainfall is 2,208 mm in a year.19

Dschang health district is found in the west region of Cameroon. It is bounded to the south by Santchou and Bandja health districts, Penka-Michel to the east, Batcham to the northeast, and the southwest and northwest regions to the west. Dschang (5.44°N, 10.05°E)17 is located at an average altitude of 1,400 m18 above sea level. In a year, the temperature averages 21.7°C and the rainfall is 2090 mm/year.19 Data and samples were collected in the reference healthcare facility (Dschang district hospital).

Ambam health district includes Kyé-Ossi which was the study area in the south region. Kyé-Ossi (2.17°N, 11.33°E)17 is a joint and exchange point between Cameroon, Equatorial Guinea, and Gabon. It is located at an altitude of 556 m,20 with an average temperature of 24.6°C and a rainfall of 1797 mm a year.19 The population is mostly made of traders and very few civil servants; farmers are found in the remote areas. Data and samples were collected in the medical center of Kyé-Ossi.

Data and sample collection.

Authorizations were taken from different health districts and healthcare facility administrators before any data or samples were collected. After obtaining the consent of the participants, trained field surveyors administered a questionnaire for about 5–10 minutes.

Blood samples were collected using filter paper (Whatman), Little Chalfont, Buckinghamshire, United Kingdom in the normal patients’ routine in the healthcare facility. For all patients needing to do complementary tests before the final diagnosis, their blood was collected from the sample they donated to the hospital laboratory. For healthcare facilities not having laboratories, blood was collected through a finger prick. The dried blood spots collected were kept in individual plastic bags and stored in boxes containing silica gel bags at room temperature. The samples from Santchou were collected on Whatman 003 paper and put into individual bags with one silica gel per bag. Samples from Dschang and Kyé-ossi were collected on Whatman 1 paper with silica gel in each box.

DNA extraction.

Human and parasite DNA was extracted from dried blood spots using the PureLink genomic DNA extraction mini kit (Invitrogen Life technologies, 2012, Carlsbad, CA) according to the manufacturer’s instruction.

Parasite detection.

Parasite detection was performed in Italy at the Istituto Superiore di Sanità, malaria research unit laboratory. The extracted DNA was used for nested PCR (N-PCR) and parasite DNA sequencing. We did not attempt to identify P. vivax microscopically.

Nested PCR.

The PCR analysis of the four Plasmodium species (Plasmodium falciparum [P. falciparum], P. vivax, Plasmodium ovale [P. ovale], Plasmodium malariae [P. malariae]) was performed by a N-PCR of a specific 18S ribosomal RNA rss rRNA gene fragments as previously described.21 Positive control DNAs representing each of the four Plasmodium species assessed (laboratory strains for P. falciparum 3D7 and isolates from sick patients for other species) and two negative controls (for the outer and N-PCR, respectively) were used to ensure the efficiency of the amplification.

Plasmodium vivax DNA sequencing.

Samples were amplified using a protocol previously described.22 The target was specifically P. vivax Merozoite Surface Protein 1 (PvMSP1). Positive results were obtained by amplification through outer and N-PCR with primers PvMSP1. The amplified samples were sent to Eurofins Genomics (Ebersberg, Germany) for sequencing. Multiple sequence alignment analysis was performed using Clustal Omega (EMBL-EBI, Hinxton, Cambridgeshire, UK). Six sequenced isolates were compared with MSP1 from P. vivax P01 (Papua Indonesia) and Salvador I reference sequences from PlasmoDB. DNA to protein conversion was carried out using the ExPASy tool.23

Duffy blood group genotyping.

Duffy blood group genotyping was performed at the University of Botswana–University of Pennsylvania Joint molecular laboratory in Gaborone, Botswana. The extracted DNA was used for PCR–restriction fragment length polymorphism targeting the gene DARC (−33T > C) rs2814778 and treated with endonuclease StyI according to the protocol described previously.12 The fragment sizes for Duffy negative are 82, 65, and 64 base pairs (bp) (12 bp is not seen) and for Duffy positive are 82, 77, and 64 bp.

RESULTS

Plasmodium vivax has been found in multiple areas of Cameroon, all in Duffy blood group–negative people.11,2426 We studied febrile patients in three areas of Cameroon: Dschang and Santchou with seasonal transmission in the west region and Kyé-Ossi with year-round transmission in Ambam health district in the south region (Figure 1). Dschang, compared with the other two districts, had an unusually high frequency of P. vivax (Table 1). As expected, the infection of P. vivax was high during the rainy season in Dschang compared with the dry season in the previous study11 (Table 2). Not only was it high but also was more frequent than P. falciparum (Table 1), the universal infection in sub-Saharan Africa. In the other two areas, low frequencies of P. vivax were observed (Table 1), as described previously in other areas of Cameroon.2426

Table 1

Plasmodium spp. in three areas of Cameroon

Study siteNumber of patient samplesPf*Pv*Po*Pm*CoinfectionsDuffy blood group (negative)Pv (% in malaria cases)Plasmodium falciparum (% of malaria cases)Number of malaria cases
Pf-PvPf-PoPf-PmPv-Pm
Santchou400170120121022 (1%)174 (98%)177
Dschang500881390336012177177 (66%)125 (46%)269
Kyé-Ossi10129203002022 (6%)31 (86%)36

Pf = Plasmodium falciparum; Pv = Plasmodium vivax; Pm = Plasmodium malariae; Po = Plasmodium ovale.

Only Pv cases were Duffy blood group typed.

Table 2

Plasmodium vivax and Plasmodium falciparum in the dry and the rainy seasons in Dschang

SeasonTotal number of patientsNested PCR analysis
PfPv
Infected patientsPercentageInfected patientsPercentage
Dry 2013*484449.1275.6
Rainy 20175001252517735.2

Pf = Plasmodium falciparum; Pv = Plasmodium vivax.

Reference (Russo et al.11).

Plasmodium spp. infections were determined by N-PCR analysis using the 18S ribosomal RNA gene for P. vivax, P. falciparum, P. malariae, or P. ovale.21 Plasmodium vivax and P. falciparum were identified by the fragment size of 120 and 205 base pairs, respectively. Representative patient samples that are positive by nested PCR for both P. vivax (Supplemental Figure S1A) and P. falciparum are shown (Supplemental Figure S1B).

Plasmodium vivax was further confirmed by the sequence of a section of PvMSP1 in six samples comparing with the known P. vivax sequences from P01 and Sal I sequence from PlasmoDB by multiple sequence alignment. P01 and Salvador I MSP1 sequences have sequence variations in the selected regions (Supplemental Figure S2A). Five of the isolates showed sequence similarities to P01 PvMSP1 by genomic (Figure 2A) and protein sequences (Figure 2B). Although five sequences have close similarity to P01 sequence, all five isolates had unique mutations, suggesting different clones of P. vivax in Dschang. One isolate had close similarity to the Salvador I MSP1 sequence, again with unique mutations (Supplemental Figure S2B).

Figure 2.
Figure 2.

Sequencing of the PvMSP1 gene in Plasmodium vivax isolated in Dschang. To further confirm the specificity of P. vivax, six samples that were positive for P. vivax by N-PCR were selected. Sequencing was performed on these six samples for the MSP1 gene. (A) Multiple sequence alignment for five isolates shows high similarity with the reference MSP1 sequence from P01 (Papua Indonesia). (B) The DNA sequence of the five isolates were translated into protein sequence, and alignment was compared with the reference protein sequence for MSP1 sequence from P01 (Papua Indonesia). The schematic diagram is the full length PvMSP1 gene and shows the region in which the DNA sequencing was obtained. aa = amino acid; SP = signal peptide; GPI = glycosylphosphatidylinositol.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 3; 10.4269/ajtmh.20-1255

Duffy blood group genotyping was performed on the samples that were positive for P. vivax and was determined by restriction fragment length polymorphism using Sty I enzyme. Representative patient samples are shown (Figure 3) for Duffy-negative genotyping characterized by bands 82-, 65-, 64-, and 12-bp digestion products (Figure 3) compared with 82-, 77-, and 64-bp fragments (Supplemental Figure S3) for Duffy-positive. However, the 12-bp product was not captured for Duffy-negatives because of small fragment size (Figure 3). All the P. vivax–infected samples were in Duffy blood group–negative Cameroonians (Table 1).

Figure 3.
Figure 3.

Determination of the Duffy genotype in Plasmodium vivax–infected people in Dschang. Duffy genotyping was determined using PCR product in the Duffy promoter region which was digested by the Sty I restriction enzyme. The expected fragment size of 82, 65, 64, and 12 bp of the digested product corresponds to Duffy-negative genotype, although the 12-bp fragment is not captured. Simulated identification was given for each isolate for representation purposes. The negative control was for no human blood.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 3; 10.4269/ajtmh.20-1255

DISCUSSION

The most striking observation reported here was the unusually high frequency of P. vivax infections among febrile Duffy-negative Africans in Dschang (35%) as compared with Santchou (0.5%) and Kyé-ossi (2%). By contrast, the frequencies of P. falciparum infections were relatively similar in the three regions (25%, 43%, and 30%, respectively). There are a variety of differences in the three areas that could affect P. vivax transmission, but these would also be predicted to impact P. falciparum transmission. For example, Dschang is at an altitude of 1,400 m where P. falciparum is less effectively transmitted.27 The lower transmission relates to the lower temperature (below 16°C) at which P. falciparum develops poorly in mosquitoes.28 Moreover, there is little evidence that P. falciparum infections can suppress P. vivax.29

It would seem that at least three possibilities exist for the high frequency of P. vivax in Dschang in Duffy-negative Africans. First, efficient P. vivax transmission could be due to an unknown novel mosquito vector present in Dschang but not yet identified. Consistent with this idea, no P. vivax was detected in mosquitoes caught in Dschang houses 10 years before.18 These data suggest that P. vivax incidence may have changed in Dschang in the last 10 years or that P. vivax may have transmitted through a vector that feeds outside the houses. Second, new mutations in P. vivax ligands arising only in Dschang may increase P. vivax infections. Analysis of P. vivax from Dschang could address this possibility. Third, the most interesting but completely speculative idea is the possibility that P. vivax–like parasites from higher apes have undergone introgression into human P. vivax in Dschang. Three invasion ligands (PvRBP2d: PVX_101585, PvRBP2e: PVP01_0700500, and PvRBP3: PVX_101495) are pseudogenes in P. vivax but are genes in P. vivax–like parasites in higher apes.30 Thus, sequencing P. vivax from Dschang could address this possibility directly. However, both Dschang and Santchou are far from the sites where P. vivax–infected apes live. But it is possible that the higher altitude in Dschang may play a role in the proposed introgression into human P. vivax in Dschang. Perhaps, relevant to this possibility is the observation that in Botswana at an altitude of 1,000 m where P. falciparum has almost been eliminated and P. vivax is more common.14,31 Clearly, we must await the sequence of P. vivax in Dschang to answer these important questions.

In addition to Duffy blood group, there are other critical receptors for infection by P. vivax such as the transferrin receptor. A mutation in the transferrin receptor that blocks P. vivax infection without affecting transferrin uptake demonstrated the critical role of the transferrin receptor for P. vivax invasion.32 However, it is unknown whether this mutation, if occurred, would have been selected, as it may have a negative effect on transferrin uptake and survival over time.

Much of the new findings of P. vivax and its sequestration in the bone marrow33,34 may affect the biology of P. vivax, but the primary factor is the Duffy-negative blood group. This mutation in the expression of Duffy blood group determines the low level of infection in Africa, even in populations where Duffy-positives and Duffy-negatives live side-by-side, such as Madagascar.5 Many possibilities exist and must await a deeper study of P. vivax in Dschang.

Supplemental figures and table

ACKNOWLEDGMENTS

G. B. D. D. was supported by a PhD student mobility grant 2017, Sapienza University of Rome (Disp no. 280/2017, Prot. No. 0006599, January 31, 2017). We thank the non-profit associations Mingha Africa Onlus (Rome, Italy) and Projet Intégré pour la Promotion de l’Auto Développement (PIPAD, Dschang, Cameroon) for their logistic support. G. M. P. was supported by the Penn Center for AIDS Research (grant # P30 AI045008). K. G. and L. H. M. were supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Our special thanks to Armand Tiotsia Tsapi, Tsi Kien-Atsu, Raoul Nicolas Takou, and Aboubakar Yombo A. Kimbere for their support in data and sample collection and Raoul Sebastien Ngako for his help in data management and analysis. We thank Susan K. Pierce, NIH, for valuable suggestions and critical reading of the manuscript. We thank Google Maps for the map presented in Figure 1.

REFERENCES

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    • Search Google Scholar
    • Export Citation
  • 2.

    Miller LH, Mason SJ, Dvorak JA, McGinniss MH, Rothman IK, 1975. Erythrocyte receptors for (Plasmodium knowlesi) malaria: duffy blood group determinants. Science 189: 561563.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ryan JR et al. 2006. Evidence for transmission of Plasmodium vivax among a duffy antigen negative population in Western Kenya. Am J Trop Med Hyg 75: 575581.

    • Search Google Scholar
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Author Notes

Address correspondence to Louis H. Miller, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Rockville, MD 20852, E-mail: lmiller@niaid.nih.gov or Gianluca Russo, Department of Public Health and Infectious Diseases, Sapienza University of Rome, Piazzale Aldo Moro n.5, 00185 Rome, Italy, E-mail: gianluca.russo@uniroma1.it.

Authors’ addresses: Ghyslaine Bruna Djeunang Dongho and Gianluca Russo, Department of Public Health and Infectious Diseases, Sapienza University of Rome, Roma, Italy, E-mails: bruna.djeunang@uniroma1.it and gianluca.russo@uniroma1.it. Karthigayan Gunalan and Louis H. Miller, LMVR, NIAID, NIH, Rockville, MD, E-mails: karthigayan.gunalan@nih.gov and lmiller@niaid.nih.gov. Mariangela L’Episcopia and Michela Menegon, Department of Infectious, Parasitic and Immuno-mediated Diseases (MIPI), Istituto Superiore di Sanita, Roma, Italy, E-mails: mlepiscopia@gmail.com and michela.menegon@iss.it. Giacomo Maria Paganotti, Department of Medicine, Botswana-UPenn Partnership Program, Gaborone, Botswana, E-mail: paganottig@bup.org.bw. Rose Efeutmecheh Sangong, Mbangue District Hospital, Douala, Cameroon, and National AIDS Control Committee, Bamenda, Cameroon, E-mail: sangongrose@yahoo.fr. Bouting Mayaka Georges, Dschang District Hospital, Dschang, Cameroon, E-mail: boutingmayaka@yahoo.fr. Joseph Fondop and Martin Sanou Sobze, Faculty of Medicine and Pharmaceutical Sciences, University of Dschang, Dschang, Cameroon, E-mails: docfondopj@yahoo.fr and martinsobze@hotmail.com. Carlo Severini, Department of Infectious Disease, Istituto Superiore di Sanita, Roma, Italy, E-mail: carlo.severini@iss.it.

These authors contributed equally to this work.

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