Open Access

Evaluation of the intra- and inter-specific genetic variability of Plasmodium lactate dehydrogenase

  • Arthur M Talman1, 2,
  • Linda Duval1,
  • Eric Legrand3,
  • Véronique Hubert4,
  • Seiha Yen1,
  • David Bell5,
  • Jacques Le Bras4,
  • Frédéric Ariey1Email author and
  • Sandrine Houze4
Malaria Journal20076:140

DOI: 10.1186/1475-2875-6-140

Received: 20 March 2007

Accepted: 25 October 2007

Published: 25 October 2007

Abstract

Background

Malaria diagnosis is vital to efficient control programmes and the recent advent of malaria rapid diagnostic tests (RDTs) provides a reliable and simple diagnostic method. However a characterization of the efficiency of these tests and the proteins they detect is needed to maximize RDT sensitivity.

Methods

Plasmodial lactate dehydrogenase (pLDH) gene of wild isolates of the four human species of Plasmodium from a variety of malaria endemic settings were sequenced and analysed.

Results

No variation in nucleotide was found within Plasmodium falciparum, synonymous mutations were found for Plasmodium malariae and Plasmodium. vivax; and three different types of amino acid sequence were found for Plasmodium ovale. Conserved and variable regions were identified within each species.

Conclusion

The results indicate that antigen variability is unlikely to explain variability in performance of RDTs detecting pLDH from cases of P. falciparum, P. vivax or P. malariae malaria, but may contribute to poor detection of P. ovale.

Background

Rapid and reliable diagnosis is one of the key factors in promoting malaria control. The gold standard for malaria diagnosis remains the examination of Giemsa-stained smears by light microscopy. Whilst this standard has a good sensitivity and specificity and allows species and stage differentiation, it does require the expertise of a trained and experienced microscopist, is time-consuming (30 minutes per diagnostic) and requires equipment not always available or maintainable in remote areas. The 1990's have seen the advent of a new rapid diagnostic method, the immunochromatography-based malaria Rapid Diagnostic Tests (RDTs). These assays are fast (revealed in 15 minutes) and, for the most part, very simple to use. Moreover with the change of therapeutic practice towards relatively expensive artemisinin-based combination therapies [1], a good diagnostic has become essential to limit inappropriate treatment and the development of resistance. Although the use of RDTs has spread, their reliability is still questioned in numerous studies [2, 3]. These assays detect one or several antigens, the most common are: histidine-rich protein-2 (HRP-2), aldolase and lactate dehydrogenase (pLDH).

Lactate dehydrogenase is an enzyme that catalyzes the inter-conversion of lactate into pyruvate. This enzyme is essential for energy production in Plasmodium [4]. The level of pLDH in the blood has been directly linked to the level of parasitaemia [5].

The genetic diversity of HRP2 has been examined and partly linked to RDT detection sensitivity [6], the genetic variability has also been assessed in aldolase, it has been ruled out as a possible cue for variation RDT sensitivity [7]. Here is the first study of Plasmodium LDH genetic variability as a possible cause of variation in sensitivity of RDTs.

Methods

A total of eight Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium. ovale, Plasmodium malariae, Plasmodium yoeli, Plasmodium chabaudi, Plasmodium berghei and Plasmodium. reichnowi), including the four human pathogens, from numerous origins (Figure 1) were examined with a nested-PCR assay amplifying a 543 bp fragment: corresponding to the 57 to 237 amino acid position of the reference P. falciparum LDH coding sequence (pf13_0141). All field samples analysed were diagnosed by microscopic examination and confirmed by PCR [8] and conserved from previous studies and approved at the time by respective National Ethics Committees. Two sets of PCR and nested primers were designed for this study based on the sequences available on GenBank (Table 1) one set use for P. vivax and P. falciparum, and the other for P. ovale and P. malariae.
https://static-content.springer.com/image/art%3A10.1186%2F1475-2875-6-140/MediaObjects/12936_2007_Article_445_Fig1_HTML.jpg
Figure 1

Worldwide distribution of the isolate sequenced in the study, grouped by species. One dot corresponds to one isolate, in red the malaria endemic area.

Table 1

PCR and nested-PCR primers used in the study

PCR primers

Primer sequence 5' to 3'

Fv1

ATGATYGGAGGMGTWATGGC

Fv2

GCCTTCATYCTYTTMGTYTC

Mo1

ATGATWGGAGGTGTTATGGC

Mo2

TGTGTCCRTATTGDCCTTC

Nested Primers

Fv1n

AATGTKATGGCWTATTCMAATTGC

Fv2n

AACRASAGGWGTACCACC

Mo1n

TAGGMGATGTTGTTATGTTYG

Mo2n

ATTTCRATAATAGCAGCAGC

Forty PCR cycle were undertaken using 94°C for 30 s, 55°C for 60 s and 72°C for 75 s; the same cycle was used for the nested-PCR but only repeated 35 times. Positive and negative controls were included in all amplification assays. The amplified products were purified using a Quiaquick PCR purification kit (QIAGEN, Valencia, CA) according to the manufacturer's recommendations, and sequenced using Big Dye Terminator kit v1.1 (Applied Biosystems, Foster City, CA) in an AbiPrism 3130 sequencing machine (Applied Biosystems, Foster City, CA).

Results

No variability was observed in P. falciparum (n = 49) with a homology of 100% between all newly sequenced sequences (named F). A single reference sequence on GenBank (corresponding to the FCC1/HN strain) exhibited a different amino acid sequence (named F1). For P. vivax (n = 10), four different types of sequence were found, the mutations observed were all synonymous (named V); no geographic pattern was identified. P. malariae (n = 17) exhibited three different type of sequences, one for African and American isolates and the other two for the south-east Asian isolate and reference strain respectively. Those variations resulted in the same amino acid sequence (named M).

P. ovale (n = 13) exhibited three different types of nucleotide sequences, leading to three different types of amino acid sequences (named O1, O2 and O3). P. berghei and P. yoeli sequences exhibited synonymous mutations (named Y). P. chabaudi exhibited a nucleotide sequence (named C).P. reichnowi and P. falciparum sequences exhibited synonymous mutations.

Interestingly a comparison of the sequences of different species reveals the existence of conserved regions and other very variable ones; this inter-specific variation is exhibited in Figure 2. Table 2 gives details of the analysed isolates.
https://static-content.springer.com/image/art%3A10.1186%2F1475-2875-6-140/MediaObjects/12936_2007_Article_445_Fig2_HTML.jpg
Figure 2

Schematic representation of the 181 amino acid sequence variation (each different marks correspond to one amino acid change). F, F1, M, V, O1, O2, O, Y and C correspond to the sequence identified in the study (see result). The conserved regions of the Plasmodium pLDH gene for all species are highlighted in green.

Table 2

Result of the sequence analysis for the isolates tested in this paper.

ID Code

Year

species

Origin

Seq

AA

5353A

2005

PF

South Africa

F

F

5353B

2005

PF

South Africa

F

F

5421

2005

PF

Benin

F

F

5445

2005

PF

Brazil

F

F

4899

2004

PF

Burkina Faso

F

F

CAMBF

2001

PF

Cambodia

F

F

5203

2005

PF

Cameroon

F

F

5848

2005

PF

Cap Verde

F

F

5265

2005

PF

Republic of Central Africa

F

F

3414

2002

PF

Colombia

F

F

4682

2004

PF

Comoros

F

F

5405

2005

PF

Congo

F

F

4919

2004

PF

Ivory Cost

F

F

5600

2005

PF

Dominican Republic

F

F

1628

1999

PF

Ecuador

F

F

5648

2005

PF

Gabon

F

F

5083

2004

PF

Gambia

F

F

5094

2004

PF

Ghana

F

F

5898

2005

PF

Guinea

F

F

5339

2005

PF

Equatorial Guinea

F

F

FguyF

2003

PF

French Guiana

F

F

5555

2005

PF

Haiti

F

F

5745

2005

PF

India

F

F

2038

2000

PF

Kenya

F

F

4548

2004

PF

Liberia

F

F

4609

2004

PF

Madagascar

F

F

2686

2001

PF

Malaysia

F

F

5296

2005

PF

Malawi

F

F

5173

2004

PF

Mali

F

F

5793

2005

PF

Mali

F

F

4807

2004

PF

Mauritania

F

F

4629

2004

PF

Mozambique

F

F

5323

2005

PF

Namibia

F

F

5822

2005

PF

Niger

F

F

4582

2004

PF

Nigeria

F

F

5846

2005

PF

Pakistan

F

F

1317

1998

PF

Papua New Guinea

F

F

5225

2005

PF

Sao Tome

F

F

4838A

2004

PF

Senegal

F

F

4512

2004

PF

Sierra Leone

F

F

4764

2004

PF

Sir Lanka

F

F

4562

2004

PF

Sudan

F

F

5224

2005

PF

Tanzania

F

F

5647

2005

PF

Chad

F

F

604

1997

PF

Thailand

F

F

4751A

2004

PF

Togo

F

F

4751B

2004

PF

Togo

F

F

542

1997

PF

Yemen

F

F

5197

2005

PF

Congo Democratic Republic

F

F

ID Code

Year

species

Origin

Seq

AA

Plasmodium malariae

CAMBM

2001

PM

Cambodia

M2

M

3413

2002

PM

Cameroon

M3

M

4739

2004

PM

Cameroon

M3

M

5990

2006

PM

Cameroon

M3

M

1909

1999

PM

Republic of Central Africa

M3

M

3670

2002

PM

Comoros

M3

M

4014

2003

PM

Comoros

M3

M

1548

1999

PM

Congo

M3

M

2667

2001

PM

Ivory Cost

M3

M

5041

2004

PM

Ivory Cost

M3

M

4568

2004

PM

French Guiana

M3

M

4774

2004

PM

Madagascar

M3

M

516

1997

PM

Senegal

M3

M

1018

1998

PM

Togo

M3

M

2389

2000

PM

Congo Democratic Republic

M3

M

Plasmodium ovale

5894

2005

PO

Angola

O2

O2

CAMBO

2001

PO

Cambodia

O2

O2

3044

2001

PO

Republic of Central Africa

O2

O2

5979

2006

PO

Ivory Cost

O2

O2

3149

2002

PO

Gabon

O2

O2

4646

2004

PO

Guinea

O2

O2

3740

2002

PO

Congo Democratic Republic

O2

O2

4419

2003

PO

Cameroon

O3

O3

5401

2005

PO

Madagascar

O3

O3

2132

2000

PO

Mali

O3

O3

5994

2006

PO

Mali

O3

O3

2668

2001

PO

Rwanda

O3

O3

3043

2001

PO

Zimbabwe

O3

O3

Plasmodium vivax

3019

2001

PV

French Guiana

V1

V

1977

2000

PV

India

V1

V

1866

1999

PV

Nicaragua

V1

V

800

1997

PV

Thailand

V1

V

2642

2001

PV

Madagascar

V2

V

5315

2005

PV

Chine

V3

V

CAMBV

2001

PV

Cambodia

V4

V

5753

2005

PV

Comoros

V4

V

1173

1998

PV

Laos

V4

V

ID Code

 

species

Origin

Seq

AA

Reference strains

3D7

 

PF

pf13_0141

F

F

FCC1/HN

 

PF

dq825436

F1

F

EMBL

 

PM

ay486059

M1

M

EMBL

 

PO

ay486058

O1

O1

EMBL

 

PV

ay486060

V1

V

YOELII

 

PY

xm_719008

Y

Y

CHABAUDI

 

PC

xm_740087

C

C

BERGHEI

 

PB

ay437808

B

Y

REICHNOWII

 

PR

ab122147

R

F

Seq = Nucleotide sequence, AA = aminoacid sequence

Discussion

Here is described, for the first time, the sequence variability of pLDH in the four human's species of malaria and four animal Plasmodium species and analysed them together with published sequences. The results indicate the existence of both variable and conserved regions in plasmodial lactate dehydrogenase.

The intra-specific geographic conservation of pLDH suggests that genetic variability may not be linked to disparities in sensitivities or specificities observed in the detection of P. falciparum [3] with anti-pan LDH antibodies. The falciparum-specific epitope detected by RDTs is probably situated in the inter-specific variable regions we have identified; whilst the pan-malarial epitope is more likely situated in a conserved region. However, Moody et al. [2] reported that one pan-specific monoclonal antibody used in a pLDH RDT has a lower affinity to P. malariae and P. ovale antigens, the attribution of this to a sequence divergence must not be neglected and should be further investigated.

Conclusion

The WHO states: "Rapid diagnostic tests (RDTs) offer the potential to provide accurate diagnosis to all at risk populations (...) The success of RDTs in malaria control will depend on good quality planning and implementation" [9]. Moreover a rapid diagnostic test needs to be reliable globally, to detect an antigen that mirrors accurately blood parasitaemia; therefore part of a good quality assurance is to monitor such factors.

As part of this quality assurance, we have identified that an intra-specific genetic variability is not a significant factor in the variation of efficiency observed in rapid diagnostic tests in the detection of P. falciparum, P vivax and P. malariae, although it may explain the poor sensitivity to P. ovale [7]. Similar findings of low variability have been demonstrated for plasmodial aldolase another target antigen of MRDTs [10] despite a bad sensitivity in the dectection of P. ovale infection [11] in contrast to HRP2, a target antigen of P. falciparum with high variability affecting MRDT sensitivity. In this regard, pLDH offers advantages as a target antigen for diagnosis. The identification of pan-specific and species-specific regions may help in development of more sensitive and specific monoclonal antibodies for MRDTs.

Declarations

Acknowledgements

This Work has been supported by a WHO grant and is part of the Modipop Project (Institut Pasteur de Paris).

Authors’ Affiliations

(1)
Unité d'Epidémiologie Moléculaire, Institut Pasteur in Cambodia
(2)
Division of Cell and Molecular Biology, Imperial College London
(3)
Centre National de Référence sur la Chimiorésistance du Paludisme dans la région Antilles-Guyane, Institut Pasteur de la Guyane
(4)
Centre National de Référence du Paludisme, AP-HP, Hôpital Bichat-Claude Bernard
(5)
Malaria, and other Vector-borne and Parasitic Diseases, World Health Organization-Regional Office for the Western Pacific

References

  1. Snow RW, Eckert E, Teklehaimanot A: Estimating the needs for artesunate-based combination therapy for malaria case-management in Africa. Trends Parasitol. 2003, 19: 363-369. 10.1016/S1471-4922(03)00168-5.View ArticlePubMedGoogle Scholar
  2. Moody A, Hunt-Cooke A, Gabbett E, Chiodini P: Performance of the OptiMAL® malaria antigen capture dipstick for malaria diagnosis and treatment monitoring at the Hospital for Tropical Diseases, London. Br J Haematol. 2000, 109: 891-894. 10.1046/j.1365-2141.2000.01974.x.View ArticlePubMedGoogle Scholar
  3. Murray CK, Bell D, Gasser RA, Wongsrichanalai C: Rapid diagnostic testing for malaria. Trop Med Int Health. 2003, 8: 876-883. 10.1046/j.1365-3156.2003.01115.x.View ArticlePubMedGoogle Scholar
  4. Brown WM, Yowell CA, Hoard A, Vander Jagt TA, Hunsaker LA, Deck LM, Royer RE, Piper RC, Dame JB, Makler MT, VanderJagt DL: Comparative structural analysis and kinetic properties of lactate dehydrogenases from the four species of human malarial parasites. Biochem. 2004, 43: 6219-6229. 10.1021/bi049892w.View ArticleGoogle Scholar
  5. Piper R, Lebras J, Wentworth L, Hunt-Cooke A, Houze S, Chiodini P, Makler M: Immunocapture diagnostic assays for malaria using Plasmodium lactate dehydrogenase (pLDH). Am J Trop Med Hyg. 1999, 60: 109-114.PubMedGoogle Scholar
  6. Baker J, McCarthy J, Gatton M, Kyle DE, Belizario V, Luchavez J, Bell D, Cheng Q: Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests. J Infect Dis. 2005, 192: 870-877. 10.1086/432010.View ArticlePubMedGoogle Scholar
  7. Moody A: Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev. 2002, 15: 66-78. 10.1128/CMR.15.1.66-78.2002.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Snounou G: Detection and Identification of the four malaria parasite species Infecting humans by PCR amplification. Methods Mol Biol. 1996, 50: 263-291.PubMedGoogle Scholar
  9. WHO Meeting Report: Informal Consultation on Field Trials and Quality Assurance on Malaria Rapid Diagnostic Tests, WORLD HEALTH ORGANIZATION Regional Office for the Western Pacific MALARIA RAPID DIAGNOSIS. 2003, [http://www.who.int/malaria/rdt.html]Google Scholar
  10. Lee N, Baker J, Bell D, McCarthy J, Cheng Q: Assessing the genetic diversity of the aldolase genes of Plasmodium falciparum and Plasmodium vivax and its potential effect on the performance of Aldolase-detecting Rapid Diagnostic Tests (RDTs). J Clin Microbiol. 2006, 44: 4547-9. 10.1128/JCM.01611-06.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Bigaillon C, Fontan E, Cavallo JD, Hernandez E: Ineffectiveness of the Binax NOW Malaria test for diagnosisof Plasmodium ovale Malaria. J Clin Microbiol. 2005, 3: 1011-10.1128/JCM.43.2.1011.2005.View ArticleGoogle Scholar

Copyright

© Talman et al; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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