In areas where P. falciparum and P. vivax co-exist, parasite-specific diagnosis and choice of effective treatment is crucial to prevent the emergence and spread of resistance. In malaria-endemic areas, the occurrence of P. falciparum/P. vivax co-infection is frequent and it is, therefore, common for P. vivax to have been exposed to treatment with SP.
In Ethiopia, not only are there wide interregional differences in the endemicity and transmission of malaria, but there is also a significant lack of information on the effectiveness of anti-malarial drugs. In 1999, the CQ treatment failure rate in the first two weeks still stood at 88% in the centre of the country . In 2005, SP therapeutic failure within two to four weeks of follow-up was 36% and 72% respectively. In this same year, however, treatment with AL yielded an appropriate clinical and parasitological response of 99% .
With regard to the prevalence of species, a decrease of P. falciparum mono-infection and an increase of P. vivax mono-infection was observed in contrast to other results obtained by other studies previously conducted in Ethiopia [[6, 7, 43, 44]]. The predominant species was P. falciparum, which appeared in approximately 53% of cases, followed by P. vivax with a prevalence higher than 30%, and a small proportion cases of co-infection by both species. In addition, two cases of infection by Plasmodium ovale were detected. This finding is rather singular; in that P. ovale has never been previously described in Ethiopia, and might be accounted for by the migration process from the western side of the continent to this country, or people returning after having gone to work in other areas of Africa with prevalence of P. ovale. In the future, more studies about the prevalence of malaria species should be performed in Ethiopia, to know the factors are influencing in these changes of prevalence.
As with other studies in which high rates of therapeutic failure of CQ and SP have been detected, our results likewise show a high prevalence of these mutations linked to resistance in P. falciparum, particularly T76 in pfcrt (responsible for resistance to CQ) and the triple mutation in pfdhfr (responsible for treatment failure in SP) (see Table 3 and Figure 2). Furthermore, the high rate of the pfdhps double mutation, G437/E540, responsible for conferring a high degree of resistance to sulphadoxine and for therapeutic failure to SP in the presence of the triple mutation in pfdhfr, has resulted in the pfdhfr/pfdhps quintuple mutation being present in over 80% of cases. This corresponds to the low SP-treatment efficacy rates registered in different regions of Ethiopia  and confirms the need for AL to be made available countrywide as the first-line treatment.
The pfcrt T76, pfmdr1 Y86 and pfmdr1 Y1246 mutations are very useful molecular markers of CQ resistance in areas where resistance rates are low to mild [45, 46]. This study showed high to moderate prevalence of pfcrt and pfmdr1 mutations, respectively, to be consistent with low CQ efficacy in Ethiopia .
In the case of P. vivax, there was a high prevalence of the pvdhfr R58 and pvdhfr N117 mutations. Furthermore, the pvdhfr double mutation, N117/R58, appeared in 52 out of 57 cases infected by P. vivax (taking the cases of P. falciparum co-infection into account), and 42 out of 47 cases infected by this species alone. The pvdhps G553 mutation always appeared in combination with pvdhfr double mutation R58/N117, resulting pvdhfr/pvdhps R58/N117/G553 mutation in two cases of P. vivax mono-infection. This finding could explain a possible SP treatment failure in these patients, as has been associated in other studies [36, 37]. Yet pvdhfr T117, L57, pvdhfr triple or pvdhfr/pvdhps quadruple mutations were not in evidence, in the last case neither in P. vivax mono-infection nor in P. vivax-P. falciparum co-infection. Although these multiple mutations seem to be necessary for in vivo resistance [[17, 18, 43]], this finding nevertheless suggests widespread SP use, since the pvdhfr mutations arise first under drug pressure [[17, 18, 43]].
It should be taken into account that, when it comes to combined P. falciparum/P. vivax infection, patients should be treated with a blood schizonticide (for both species) and a tissue schizonticide (for hepatic hypnozoites of P. vivax). In the past, SP was used as treatment for P. falciparum. As a result, P. vivax came into contact with the same treatment when it was present in mixed infections, and mutations in different genes linked to SP resistance have thus appeared in this species. The mutations in the pvdhps gene would be equally accounted for, i.e., here, only the G553 mutation, implicated in resistance to sulphadoxine and corresponding to the G581 mutation in P. falciparum, displayed a very low prevalence and appeared in the presence of the pvdhfr double mutation, N117/R58. In contrast, the G383 mutation, which corresponds to G437 in P. falciparum, was observed in neither case. This strengthens the hypothesis that the asymmetric selection process of mutations observed in P. falciparum is also applicable to P. vivax .
Samples co-infected by these two species showed mutations in either pfdhfr/pfdhps or pvdhfr, which would explain why treatment for both these species of Plasmodium could fail in such patients.
In view of the genotype results obtained, it would be advisable for a study of therapeutic efficacy to be conducted in this area, to ascertain whether the genotypic are the same as the phenotypic data, i.e., whether the parity mutation-resistance is produced in vivo.
Moreover, it would be advisable for future research to verify whether there were mutations in genes (such as atp6) related with resistance to artemisinin derivatives, the use of which as an anti-malarial drug has spread in Africa. Artemisinin-based combination therapy (ACT) (for example, using Coartem®) has come into use in Ethiopia, though it is proving difficult to implement in rural areas due to the continued use of drugs, such as SP and CQ, in combination with other medication, a practice that continues to favour the expansion of resistance.