• View in gallery

    Map of the four study sites in Mali. The size of the red dots indicates the relative approximate number of inhabitants of Bamako, Bougoula, Djoliba, Samako, and Kolle. Samako and Kolle were pooled as a single site because of geographic proximity.

  • View in gallery

    UPGMA dendrogram displaying the relationship between the Plasmodium falciparum haplotypes (N = 75). The isolates are colored according to geographic location in the dendrogram. Red: Djoliba; blue: Samako/Kolle; green: Bamako; and gold: Bougoula.

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Analyzing Deoxyribose Nucleic Acid from Malaria Rapid Diagnostic Tests to Study Plasmodium falciparum Genetic Diversity in Mali

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  • UMR MD3 Infections Parasitaires Transmission Pharmacologie et Thérapeutique (IP-TPT), Aix-Marseille University, Marseilles, France; Parasitology Laboratory, Timone Hospital, Marseilles, France; Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, UMI 3189, Faculty of Medicine and Dentistry, University of Sciences, Techniques and Technology, Bamako, Mali; University of Pavia, Pavia, Italy

We evaluated the use of positive malaria rapid diagnostic tests (mRDTs) to determine genetic diversity of Plasmodium falciparum in Mali. Genetic diversity was assessed via multiple loci variable number of tandem repeats analysis (MLVA). We performed DNA extraction from 104 positive and 30 negative used mRDTs that had been stored at ambient temperature for up to 14 months. Extracted DNA was analyzed via quantitative polymerase chain reaction (qPCR), and MLVA genotyping was then assessed on positive qPCR samples. Eighty-three of the positive mRDTs (83/104, 79.8%) and none of the negative mRDTs were confirmed P. falciparum positive via qPCR. We achieved complete genotyping of 90.4% (75/83) of the qPCR-positive samples. Genotyping revealed high genetic diversity among P. falciparum populations in Mali and an absence of population clustering. We show that mRDTs are useful to monitor P. falciparum genetic diversity and thereby can provide essential data to guide malaria control programs.

Genetic diversity analysis of Plasmodium falciparum in humans and parasite population dynamics are useful to monitor malaria control and elimination strategies. Indeed, several multiple loci variable number of tandem repeats analysis (MLVA) studies using microsatellite markers have shown that P. falciparum population genetics correlated with malaria transmission intensity in Africa and provided epidemiologically relevant information.15 Assessing malaria parasite genetic diversity over time can also help to evaluate malaria control programs. For instance, a P. falciparum microsatellite-based survey in Djibouti has revealed a significant decline in genetic diversity between 1998 and 2009, which was compatible with pre-elimination goals.5

Plasmodium falciparum genetic diversity has been well described in humans throughout the world15; however, this type of study is difficult to manage in the field. Indeed, collection, transportation, and storage of large amounts of blood samples remain difficult in remote areas with a tropical climate. Sampling of dried blood spots on filter papers partially circumvents these technical difficulties, thereby enabling detection and genotyping of P. falciparum from archived specimens.1,4,5 Nevertheless, patient consent is required as long as specific blood sampling is required for the study.

Malaria rapid diagnostic tests (mRDTs) detect the presence of circulating P. falciparum-specific antigens, such as histidine-rich protein 2 (PfHRP2) and lactate dehydrogenase. They are highly recommended by the World Health Organization for systematic malaria diagnosis before artemisinin-based combination therapy of uncomplicated malaria cases.6 As mRDTs are already used for case management, they are widely available in malaria-endemic countries and therefore represent a potential source of P. falciparum DNA for large population studies. Furthermore, patient consent is not required for such investigations, as mRDTs are passively collected during patient care and would normally been discarded once interpreted. Previous studies have shown that used mRDT nitrocellulose strips harbor P. falciparum DNA,710 and thereby enable the detection of antimalarial drug-resistant genes via the single nucleotide polymorphism genotyping.8 This study aimed to assess whether used mRDTs stored at room temperature provide sufficient quality DNA to conduct MLVA genotyping of P. falciparum in Mali.

Between October 2013 and January 2015, we randomly selected a total of 134 used mRDTs (104 PfHRP2 positive and 30 PfHRP2 negative) from four sites in Mali (Figure 1, Table 1). Blood samples were collected via finger prick (5–10 μL) and absorbed onto mRDTs (SD BIOLINE Malaria Ag P.f® and P.f/Pan®, Standard Diagnostics, Kyonggi, Republic of Korea) by Malaria Research and Training Center (MRTC) clinicians during the systematic testing of symptomatic febrile patients. Used mRDT samples were stored and transported at ambient temperature to the MRTC Central Laboratory in Bamako, Mali. The samples were then randomly selected and air transported to Marseilles, France, where P. falciparum DNA was extracted from January 2015 to August 2015.

Figure 1.
Figure 1.

Map of the four study sites in Mali. The size of the red dots indicates the relative approximate number of inhabitants of Bamako, Bougoula, Djoliba, Samako, and Kolle. Samako and Kolle were pooled as a single site because of geographic proximity.

Citation: The American Society of Tropical Medicine and Hygiene 94, 6; 10.4269/ajtmh.15-0832

Table 1

Epidemiological and sampling characteristics as well as the qPCR and MLVA results of the 104 mRDT-positive samples collected from four sites in Mali

Study sitesDjolibaKolle (K) Samako (S)BamakoBougoulaTotal
Sampling periodOctober 2013 to January 2014November 2014 (K)December 2014January 2015 
December 2014 (K)January 2015  
January 2015 (K) (S)   
Endemicity*HyperendemicHyperendemicHypoendemicHyperendemic 
Patients sampled, no.34272815104
Positive PCR for Plasmodium falciparum, n (%)29 (85.3)24 (88.9)20 (71.4)10 (66.7)83 (79.8)
Complete genotype (N = 8 loci), n (%)28 (96.5)21 (87.5)18 (90.0)8 (80.0)75 (90.4)
Genetic diversity (mean ± SD)0.76 ± 0.180.75 ± 0.160.78 ± 0.170.69 ± 0.360.75 ± 0.22

MLVA = multiple loci variable number of tandem repeats analysis; mRDTs = malaria Rapid Diagnostic Tests; qPCR = quantitative polymerase chain reaction; SD = standard deviation.

Data from various Malian official reports and studies reviewed in a published thesis in French. (Doumbo O, 1992. Epidémiologie du paludisme au Mali, étude de la chloroquinorésistance, essai de stratégie de contrôle basée sur l'utilisation de rideaux imprégnés de permethrine associée au traitement systématique des accès fébriles. Thèse de Doctorat. Sciences Biologiques, Montpellier II, France.)

Malaria endemicity levels based on P. falciparum prevalence among children aged 2–10 years (PfPR2–10), according to the World Health Organization classification: hypoendemic, 0–10%; hyperendemic, 50–75%.

The mRDT nitrocellulose strips (cut into five identical pieces) were incubated for 48 hours at ambient temperature in 800 μL of lysis buffer (bioMérieux, Marcy l'Etoile, France). The extracted DNA was eluted in 100 μL of elution buffer using a NucliSENS EasyMAG instrument (bioMérieux).11 Screening for P. falciparum was performed via quantitative polymerase chain reaction (qPCR) using a LightCycler 480 PCR system (Roche Diagnostics, Meylan, France) with specific primers targeting the 18S rRNA gene.12 Each experimental run included both a negative (no template) and a positive (P. falciparum 18S rRNA plasmid) control. Standard curves were generated with serial 10-fold dilutions of the plasmid to allow for species-specific quantification of parasite density (number of parasites/μL of blood). We assumed that each genome of P. falciparum has five copies of the 18S rRNA gene (as observed in the 3D7 genome).13 The qPCR-positive samples were genotyped using eight polymorphic microsatellite markers specific for P. falciparum (Poly α, TA109, TA1, TA81, TA42, ARA2, PfPK2, and Pfg377).1 Microsatellite amplification was performed applying a semi-nested PCR strategy using fluorescent end-labeled primers.1 PCR products were analyzed using an ABI 3130XL capillary sequencer (Applied Biosystems, Foster City, CA). To differentiate allele peaks from stutter peak artifacts, we scored multiple alleles per locus if minor peaks were > 33% of the height of the major peak, corresponding to the predominant allele.1 We also discarded peaks with a fluorescence intensity < 100 units. Genetic diversity metrics were assessed using Arlequin v3.5 (Excoffier & Lischer 2010) software based on complete genotypes. For a haploid organism, genetic diversity was defined as a measure of the probability to randomly draw a pair of different alleles from an allelic pool. Potential values ranged from 0 (no diversity, 100% similarity between alleles) to one (maximal diversity, 100% of the alleles are different). Because of the frequent occurrence of multiclonal infections, we only considered major peaks when determining genotypes for genetic diversity and haplotype analysis.15 To analyze the relationship between P. falciparum haplotypes, we computed a dendrogram using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method (BioNumerics v7.1 Software, Applied Maths, Ghent, Belgium).

Eighty-three of the positive mRDTs (83/104, 79.8%) and none of the negative mRDTs were confirmed P. falciparum-positive via qPCR (Table 1). Among the 83 qPCR-positive samples, 75 (90.4%) were successfully genotyped for all test loci (Table 1). The eight remaining samples displayed one (N = 3), two (N = 2), three (N = 1), and four (N = 2) negative loci of the eight loci tested (see Table 3 for complete microsatellite data). Mean PCR cycle threshold (Ct) values were 31.94 ± 3.15 for the 75 samples that were completely genotyped (estimated parasitemia ranging from 11 to 512,000 parasites/μL) versus 37.52 ± 1.92 for the eight incompletely genotyped samples (estimated parasitemia ranging from 0.01 to 3,620 parasites/μL) (Table 2).

Table 2

Quantification of samples via qPCR cycle threshold and estimated parasitemia based on the number of positive loci

 Complete genotypes (8 positive loci; N = 75)Incomplete genotypes (< 8 positive loci; N = 8)
PCR cycle threshold (mean ± SD)31.94 ± 3.1537.52 ± 1.92
Estimated parasitemia* (parasites/μL blood) (mean ± SD)43,067 ± 82,558749 ± 1,280
Range of estimated values of parasitemia* (parasites/μL blood)11–512,0000.01–3,620

qPCR = quantitative polymerase chain reaction; SD = standard deviation.

Estimation of parasite density: ([copy number of the gene in 1 μL of DNA] × [100/25]). We assumed that each genome of Plasmodium falciparum has five copies of the 18S rRNA gene,13 that 5 μL of blood was spotted onto malaria rapid diagnostic tests, and that extracted DNA was eluted into 100 μL.

Table 3

Microsatellite data of the 83 genotyped samples

SiteSamplePoly αTA109PfPK2ARA2Pfg377TA42TA81TA1
KolleT10166163157 183   168171 7682  104107 192  125128 168180  
KolleT11157   171   180  67   104  207  143  180   
KolleT12160   168   165  76   107  207  125  174   
KolleT13157136  180   180  64   104  192  137  180   
KolleT15157   192   168  73   104  192  131  180168  
KolleT16157   183   165  70   104  192  131  171   
KolleT17154   183195  180  67   104  192  131137 168189  
KolleT18169   183171  165183 6470  104  183  131  183180  
KolleT19148166  183   171168174827067 107  207192 125131 189174183 
KolleT129163   180   177  67   107  192  134  171   
KolleT130163   180   168      107  192  125  165   
KolleT131163   213   177  82   107  192  131  174   
SamakoT20    168   180171     107  192  128      
SamakoT21163   168183  174168 7067  107  192  131125 180   
SamakoT22163157  180   174  82   107  207  128  174   
SamakoT23169172157 168180  171  7064  104107 192  134143 168177189 
SamakoT24    180198  174      104  192  128  189186  
SamakoT25151160  183   174171 6779  104  192  128131 165168  
SamakoT26157   168   174171 6479  107  192  128125 183180  
SamakoT28154157  186201  177174 73   104  192  137  168171  
SamakoT124157142166175168183180198168171 70646782107  192207 125131 165171189198
SamakoT125109   168182  1681741808273  104113 192  131137 171189  
SamakoT126157   168   177  70   107  192  131  180   
SamakoT127157172  189180168 165195 7364  107  207192183125  183165  
DjolibaT29178   180   177  73   107  192  131  177   
DjolibaT30169   168   174  79   104  207  131  165   
DjolibaT31154   186   189  73   107  192  128  174   
DjolibaT32169154  171   186168 76   107  192  137125 180   
DjolibaT33160157  168180  171165 76   104107 192  134128 177165  
DjolibaT34163   183   192  94   107  207  131  174   
DjolibaT35172184  168174  171195 70   107  192189 128  174   
DjolibaT36157   180183  171165 70   104  207222 131137 171183  
DjolibaT37163   204   171  85   107  207  122  174   
DjolibaT38163184  195183171 174177 73   107  192  128  171   
DjolibaT39115160  159   168171 70   104  192  125170 174   
DjolibaT40154166  153   171  7673  113  207  131  171   
DjolibaT41178   183   168  73   107  192  125128 174   
DjolibaT42154   207171  168  946473 107  192  131  183171  
DjolibaT43169160  180   180165 79   107  207  125  171   
DjolibaT44175   171   165  79   104  192  125  183   
DjolibaT45184   189   174  70   101  192  125  174   
DjolibaT46157   183      73      207  140  174   
DjolibaT47148   180   171  73   110  192  125  168   
DjolibaT48160   171   165171 7970  101107 195  143134 174   
DjolibaT50157160163 183171186204168171174766770 104101107192  131128134165174168177
DjolibaT51166   171   174  70   107  192  140  165   
DjolibaT52157   183168  1831771717667  107  192  125  168   
DjolibaT54154   180   198162 76   104  192207 128  171   
DjolibaT55160154  168   168177 76   107104 192  125128134171168  
DjolibaT56169   162   186177 70   107  192207 128  189177  
DjolibaT57157   168   171  73   107  192  116128 165   
DjolibaT58160178  177189  174  7064  107  192207 125  168174  
DjolibaT59142   183189  168  7667  107  192  125  177183  
BougoulaT133160   183   186  76   107  192  116  171   
BougoulaT134157   201                128125 132   
BougoulaT135151154  180183  192  8573  107  192  125  171   
BougoulaT137181   171   171  82   107113 192204207128  168   
BougoulaT138166154  168183  168180 767082 10792 192  125  171   
BougoulaT139157166  177168  168  7670  107  192  128  165174  
BougoulaT140151   171168180 171  70   107  192207 134131 177180174 
BougoulaT141160157  165192  168165 82   107  192  131134128174   
BougoulaT144151   189   174  70   104  192  122  183   
BougoulaT146    168   186  64   107  192  128134 171   
BamakoT1169   177   174  82   104  192  134128 171   
BamakoT3154178  189   174  70   104  207  128  201   
BamakoT6166   165   168  7082  104  192  131  186   
BamakoT7139   180   165  70   110  207  128  168   
BamakoT9154   189   168  67   98  192  134128 174   
BamakoT155    180   171  76   107  192  140  171   
BamakoT156157   171   168  7073  107  192  134  186   
BamakoT157157   168   171  79   104  192  131  165   
BamakoT159169   168   174  76   107  207  131  180   
BamakoT160139   171   165  7073  110  207  125131 174165  
BamakoT162172   183186  171162 6473  101107 192  125134 177   
BamakoT163172   183   171  76   104  192  128  177   
BamakoT171178   168   165  70   11095 192  128  174   
BamakoT172157   171180  174  6773  104110 192  131  168180  
BamakoT174166   183   171  70   107  192  128  186   
BamakoT176163   183   171  76   10489 192  134  192   
BamakoT177154   183   177            131      
BamakoT178154157  168   177  73   10792 192  125  180   
BamakoT179160   180   168  79   10792 192  128  177   
BamakoT180157169148 180168183 177171180737685 107  192  125  171174  

The allele length (in base pairs) for each Plasmodium falciparum tested loci (Poly α, TA109, TA1, TA81, TA42, ARA2, PfPK2, and Pfg377) is provided. The empty cells of the table correspond to negative loci.

Genetic diversity was high (mean value of 0.75) and did not differ between the urban site of Bamako and the rural sites of Samako/Kolle (which were pooled together because of geographical proximity) and Djoliba (Table 1). The dendrogram based on the analysis of the 75 complete haplotypes (Figure 2) demonstrates that each haplotype was unique, which is consistent with the high level of isolate genetic diversity. The absence of major haplotype clustering between and within study sites indicates a lack of local clonal expansion of P. falciparum.

Figure 2.
Figure 2.

UPGMA dendrogram displaying the relationship between the Plasmodium falciparum haplotypes (N = 75). The isolates are colored according to geographic location in the dendrogram. Red: Djoliba; blue: Samako/Kolle; green: Bamako; and gold: Bougoula.

Citation: The American Society of Tropical Medicine and Hygiene 94, 6; 10.4269/ajtmh.15-0832

Our findings show that mRDTs are a very accessible, convenient, and efficient tool to detect and genotype P. falciparum from patient blood samples. As mRDTs are already widely used for malaria case management, such samples can be collected and stored in large-scale in malaria-endemic countries. Systematic collection of used mRDTs can be considered for genetic diversity studies without specific population sampling procedures or patient consent requirements. Malaria RDTs are also suitable for field conditions, as they can be transported and stored at ambient temperature, even for extended periods. Indeed, after a 14-month storage period at ambient temperature, DNA quality and quantity were sufficient to perform complete genotyping of 90.4% of the qPCR-positive samples. In addition, mRDTs can be transported in simple packages; this feature is critical as it is increasingly difficult to ship biological samples because of the expansion of international biological risk.

Of 104 mRDT-positive samples, 21 tested negative via qPCR. This discrepancy may be due to false-positive mRDT results associated with the persistence of PfHRP2 antigenemia following malaria treatment, as previously observed.10,14 Malaria RDT-positive samples that yielded negative qPCR results could also be interpreted as false-negative qPCR results because of the presence of PCR inhibitors or DNA extraction failure. Insufficient parasitic material may also yield a false-negative result, as the blood volume spotted onto mRDTs is low (5–10 μL) compared with the blood volume usually spotted on filter papers (50 μL). However, previous studies have reported the absence of PCR inhibitors in samples obtained from mRDTs.7,9 Furthermore, the estimated qPCR sensitivity from mRDT samples is much higher than that of mRDT antigen capture and field microscopy (both estimated to 100 parasites/μL).15 Indeed, the presence of a single parasite in a 5–10 μL blood sample spotted onto an mRDT should be sufficient to yield a positive qPCR result.9

Eight of 83 samples (9.6%) were not successfully genotyped for all test loci despite a positive qPCR result. This outcome may be due to low parasite density, as indicated by the higher Ct values (mean Ct of 37) compared with the successfully genotyped samples (mean Ct of 32). This result may also be due to unsuccessful primer recognition because of high polymorphism or local DNA alterations of the target sequence. Nevertheless, 90.4% of samples were completely genotyped for all eight test loci, which provided sufficient data to examine P. falciparum genetic diversity.

Our study demonstrates high genetic diversity among P. falciparum populations in Mali (mean genetic diversity = 0.75), which indicates a high intensity of malaria transmission in the country. These results are compatible with local malaria epidemiology, as Mali has yet to reach the malaria pre-elimination stage. Microsatellite studies performed in other malaria-endemic countries in Africa have also reported high genetic diversity of P. falciparum, ranging from 0.72 to 0.8.14

These techniques of DNA extraction and genotyping from used mRDTs have already been transferred to the MRTC in Mali and will serve to monitor genetic diversity of P. falciparum in the context of malaria control. This approach will also facilitate the monitoring of drug resistance via genotyping of resistance genes. Using mRDTs, future large-scale P. falciparum genetic studies would be relatively cost-effective and easy to carry out, as they circumvent specific blood sampling, storage, and transportation. This technique thus represents a breakthrough in the capacity to bolster field malaria epidemiological studies.

ACKNOWLEDGMENTS

We would like to express our gratitude to the study population and volunteers as well as the MRTC field and laboratory staff. We would also like to acknowledge APPLIED MATHS for use of the BioNumerics software platform.

  • 1.

    Anderson TJ, Haubold B, Williams JT, Estrada-Franco JG, Richardson L, Mollinedo R, Bockarie M, Mokili J, Mharakurwa S, French N, Whitworth J, Velez ID, Brockman AH, Nosten F, Ferreira MU, Day KP, 2000. Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol Biol Evol 17: 14671482.

    • Search Google Scholar
    • Export Citation
  • 2.

    Bogreau H, Renaud F, Bouchiba H, Durand P, Assi SB, Henry MC, Garnotel E, Pradines B, Fusai T, Wade B, Adehossi E, Parola P, Kamil MA, Puijalon O, Rogier C, 2006. Genetic diversity and structure of African Plasmodium falciparum populations in urban and rural areas. Am J Trop Med Hyg 74: 953959.

    • Search Google Scholar
    • Export Citation
  • 3.

    Mobegi VA, Loua KM, Ahouidi AD, Satoguina J, Nwakanma DC, Amambua-Ngwa A, Conway DJ, 2012. Population genetic structure of Plasmodium falciparum across a region of diverse endemicity in west Africa. Malar J 11: 223.

    • Search Google Scholar
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  • 4.

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Author Notes

* Address correspondence to Renaud Piarroux, Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire de La Timone, 264 rue Saint Pierre, 13385 Marseilles Cedex 5, France. E-mail: renaud.piarroux@ap-hm.fr

Financial support: Sample collection in Mali was supported by EDTCP1-WANETAM and WANECAM grants. Sample genotyping was supported by the Parasitology and Mycology Laboratory, La Timone Hospital, Marseilles, France.

Authors' addresses: Cécile Nabet, Coralie L'Ollivier, and Renaud Piarroux, UMR MD3 IP-TPT, Aix Marseille University, Marseille, France, and Parasitology and Mycology Laboratory, Assistance Publique des Hôpitaux de Marseille (APHM), Marseille, France, E-mails: cecilenabet7@gmail.com, coralie.lollivier@ap-hm.fr, and renaud.piarroux@ap-hm.fr. Safiatou Doumbo, Issaka Sagara, Amadou Tapily, Abdoulaye Djimde, and Ogobara K. Doumbo, Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, Faculty of Medicine and Dentistry, University of Sciences, Techniques and Technologies, Bamako, Mali, E-mails: sdoumbo@icermali.org, isagara@icermali.org, atapily@icermali.org, adjimde@icermali.org, and okd@icermali.org. Fakhri Jeddi, Parasitology and Mycology Laboratory, APHM, Marseille, France, E-mail: fakhri.jeddi@ap-hm.fr. Tommaso Manciulli, UMR MD3 IP-TPT, Aix-Marseille University, Marseille, France, and University of Pavia, Faculty of Medicine and Surgery, Pavia, Italy, E-mail: tommaso.manciulli01@universitadipavia.it.

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