Sin ttulo de diapositiva

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Analysis of single-nucleotide polymorphisms on
resistance genes of P. falciparum isolates from
two endemic regions of Colombia.

• Maestre A, Montoya P, Restrepo E, Tobón A,
  Carmona J.
• GRUPO MALARIA, Universidad de Antioquia
• Medellín-Colombia

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The Malaria problem in Colombia

• 140.000 cases in 2005
• P. vivax 59
• P. falciparum 39
• Mixed infection 2

High risk Moderate risk Low risk
World Malaria Report, 2005
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P. falciparum MALARIA CASES IN COLOMBIA
2005 56.288 reports 73.021 cases
Major sub-report!!
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Frequency of treatment failure to the
antimalarials used in Colombia for falciparum
malaria.
AQ
MQ
CQ
SP
AS
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pfcrt allelic variants
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pfmdr-1 allelic variants
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Objectives

• To assess and compare the frequency of mutations
  in the genes pfcrt and pfmdr in recent clinical
  isolates from Turbo, El Bagre and Tumaco,
  Colombia.

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Metodology

• Patients 300 patients from the Municipalities of
  Turbo, el Bagre and Tumaco.
• P. falciparum infection Giemsa stained blood
  smear.
• Efficacy studies Treatment response according to
  WHO, 1998 Early Treatment Failure, Late
  Treatment Failure, Adequate Clinical Response
• Genotyping of resistance genes
• PCR-RFLP as described by Djimde A. et al. A
  molecular marker for chloroquine-resistant
  falciparum malaria. N Engl J Med
  2001344(4)257-63.
• Copy number for Pfmdr-1 by real time PCR

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pfcrt protein
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pfcrt protein
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Study population
Turbo PAI 57.7
Zaragoza PAI 109 El Bagre PAI 67.7 (Bajo Cauca)
Tumaco PAI 33.4
PAI gt 10 High risk
Boletín de actividades de la DSSA- SIVIGILA. 2005
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METHODOLOGY
P. falciparum patients confirmed by microscopy
Whole blood
Filter paper blood spot
In vitro culture
In vitro sensibility
Real time PCR gene copy number
PCR -RFLP
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Distribution of mutations in pfcrt, codons 72 ,
74, 75 and 76 in 149 isolates according to
origin of the sample.
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Overall distribution of haplotypes of pfcrt,
codons 72, 74, 75 and 76 (percentage) in the
regions studied
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pfmdr1 codons 86 1246
Mutant Alele
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pfmdr1 mutations and treatment response to
chloroquine or amodiaquine
N17
N33
Codon 86 was either wild or wild/mutant in all
patients studied
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pfmdr1 mutations and treatment response to
mefloquine
N91
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Pfmdr1 copy number




(mean copy number)



Sample number
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Conclusions

• pfcrt
• We confirmed the presence of pfcrt T76 in all
  isolates studied, indicating complete selection
  of this polymorphism in the regions.
• The most common pfcrt haplotype was CMNT,
• The SMET haplotype was observed in 9 of the
  isolates, the later has been reported in Brazil,
  but not in Peru.
• The CMET mutant haplotype reported by us in 9 of
  the isolates, has never been reported in South
  America.
• We detected mixed mutant and wild-type alleles in
  single isolates in 12 of the samples,
  independent of the time of collection (either
  from day 0 or failure day).

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Conclusions

• pfmdr-1
• Mutations of pfmdr- 1 1246 are high in the
  regions studied, while codon 86 is wild. No
  significant association could be observed between
  a particular allele and the treatment response to
  the antimalarials tested.
• Change in copy number of pfmdr1 has no
  relationship with in vitro resistance to any
  antimalarial assayed. Therefore trascriptional
  control in this gene should be monitored.

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Conclusions
pfcrt 72m 15 74m 0 75m 23 76m 100 Pfmdr
86m 0 1246m 97
pfcrt 72m 21 74m 0 75m 17 76m 100 Pfmdr
86m 5.5 1246m 98
pfcrt 72m 21 74m 0 75m 17 76m 100 Pfmdr
86m 0 1246m 28
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Acknowledgments

• COLCIENCIAS grant Contract RC156- 2002
• CODI, Universidad de Antioquia
• Grupo Malaria, Universidad de Antioquia