This is the most comprehensive report characterizing resistance associated genetic polymorphisms in P. falciparum field samples collected in southern Pakistan. As such, the results bridge an important knowledge gap of the P. falciparum population in South Asia.
The 93% prevalence of pfcrt 76T, essential for chloroquine resistance, is in line with results from neighbouring countries [16, 31, 32]. This data indicate high levels of in vivo
P. falciparum chloroquine resistance in southern Pakistan. Moreover, the high pfcrt 76T and moderately high (57%) pfmdr1 86Y prevalence also suggests high levels of tolerance/resistance to amodiaquine in the study area.
The high prevalence of the pfdhfr 108N (99%) and 51I + 108N/59R + 108N (92%) in our study indicate that decreased susceptibility to sulphadoxine-pyrimethamine is widespread in Pakistan. However, only seven patients had infections with the triple pfdhfr resistance associated haplotype and only one patient was infected with P. falciparum that had the quintuple pfdhfr + pfdhps haplotype associated with high grade sulphadoxine-pyrimethamine resistance. These results indicate that high grade resistance to sulphadoxine-pyrimethamine is not widespread and suggest that this drug is probably suitable for use with artesunate in southern Pakistan, as recommended by the National Malaria Control Programme. However, the occurrence of triple and quintuple mutant P. falciparum is of concern as widespread use of sulphadoxine-pyrimethamine as a partner to artesunate may rapidly select these haplotypes. Monitoring of pfdhfr and pfdhps resistance associated haplotypes is consequently of importance. Furthermore, artesunate + sulphadoxine-pyrimethamine in vivo efficacy urgently needs to be assessed. This is critical as efficacy studies conducted in Baluchistan (2001-2005) reported 56% treatment failure with sulphadoxine-pyrimethamine monotherapy 
Only one patient had a P. falciparum infection with two copies of pfmdr1, a finding that should be interpreted with caution. Increased pfmdr1 copy number has been associated with an increased risk for treatment failure after mefloquine monotherapy and artesunate-mefloquine therapy . The low prevalence of pfmdr1 amplifications observed in this study suggests that both artesunate-mefloquine and artemether-lumefantrine would be efficacious in southern Pakistan. They may therefore represent potential future treatment alternatives to artesunate + sulphadoxine-pyrimethamine. Furthermore, the observed low prevalence of pfcrt 76K and pfmdr1 86N provides supporting evidence of a probable high artemether-lumefantrine efficacy [20, 24]
This data concur with recent results from a small study (n = 28) conducted in Khyber Pkhtunkhwan Province, Pakistan and previous data from India and Iran . Furthermore, just as in northern India and Iran the pfdhfr A16N51
108I164 and A16
108I164 were the most common double and triple mutants found [35, 36]. These results thus support a selective sweep of chloroquine and sulphadoxine-pyrimethamine resistant P. falciparum from southeast Asia via India to Pakistan and then on to Iran [37–40]
It should be underlined that a majority of patients (74%) presented with symptomatic malaria infection at the Aga Khan University Hospital that represents tertiary level of care. Some of these patients may have received anti-malarial treatment prior to enrolment in this study and the P. falciparum SNP proportions reported may, therefore, not be representative for the parasite population in southern Pakistan. However, the similarity of our results with those reported from neighbouring countries suggests that the results may be generalized.