malaria is the major cause of morbidity and mortality 
 in children under five years in sub - saharan africa . in areas 
 of stable malaria transmission , like equatorial guinea , 2540% of the 
 patients in clinics and 2050% of hospital admissions are due to malaria . the 
 falciparum malaria is the cause of at least 20% of death in fewer than five 
 years old children . in sub - saharan africa , the mortality in children under 
 five years old during the period between 1990 and 1998 was doubled compared 
 with the period from 1982 to 1989 . one of the most important reasons of this 
 increase in the mortality rates is the spread of antimalarial drug resistance in africa . the prevalence of malaria infection in children less 
 than five years is 22% and 48% in malabo 
  ( island region ) and in bata ( continental region ) . the more frequent species in malabo 
  are plasmodium 
 falciparum ( 92% ) , p. malariae ( 3% ) , p. 
 ovale ( 1% ) ,   and mixed infection ( 4% ) ; in bata are plasmodium 
 falciparum ( 97% ) , p. malariae ( 2% ) ,   and mix 
 infection ( 1% ) ( unpublished data ) . in equatorial guinea , as per the 
 national malaria program , chloroquine ( cq ) is the first line of treatment , 
 whereas quinine is for treatment of severe malaria . due to the initiative of 
 medical care international development ( mcdi ) , the equatorial guinea ministry 
 of health has started to use the combination of as + sp as a first line treatment 
 of uncomplicated falciparum malaria in children and pregnant women since 2004 in bioko island . in different in vivo studies carried out in 
 equatorial guinea ( bioko island ) by the institute of health carlos iii ( madrid , 
 spain ) , in collaboration with the ministry of health of this country from 1992 
 to 1999 , it was concluded that resistance to cloroquine ( cq ) was over 40% in 
 children under five , and resistance to sulphadoxine - pyrimethamine ( sp ) was 
 around 25% in the same age group . in the continental area of the country , 
 studies undertaken during 2001   gave the same results as in the island . during the years 2002 and 2003 , the sensitivity of plasmodiumfalciparum to as + sp combination was 
 evaluated in equatorial 
  guinea 
  ( unpublished study ) ,   and an adequate 
 clinical response of 95% was achieved . as a result of the problem of drug resistance 
 developed in p. falciparum , the word health organization ( who ) 
 recommended to improve access of population to a quick and efficacy treatment 
 to decrease the burden of malaria . the current recommendation is the use of a combination therapy following the 
 experience of treatments in other diseases , such as tuberculosis , leprosy ,   and 
 hiv infection . a combination therapy would be helpful in simultaneous use of 
 two or more blood schizontocidal drugs with independent modes of action and 
 different biochemical targets in the parasite 
 . present study was undertaken jointly with the malaria 
 national control program for evaluation of as + sp and aq + sp combinations in 
 treatment of uncomplicated falciparum malaria . aq + sp combination is presently 
 recommended by the who , if the efficacy is acceptable . it is less expensive than a 
 combination with artesunate and can be used as a first line of treatment keeping 
 in view the economic resources of the country rather than introducing directly 
 a combination with artesunate . the second one   as + sp is being used in the island ,   and its efficacy needs to be monitored for its 
 further use in the malaria national control program . for any in vivo study , it is necessary to differentiate 
 recrudescence or reinfection , when the country is having high incidence of 
 malaria and with a high transmission rate . for this reason , polymorphic markers 
 as msp-1 and msp-2 or microsatellite markers are useful for this 
 purpose . in this study , we evaluated the efficacy of the as + sp 
 and aq + sp combinations as   a first - line treatment for uncomplicated malaria in 
 equatorial guinea , and the outcome of infection and treatment was determined by 
 molecular analysis to present a real scenario on use of these two combinations . it is divided in two regions , the continental area , called rio muni between cameroon and gabon , 
 and the island region ( bioko , annobn and corisco valley ) . bioko , the biggest island , is placed 40  km from cameroon 
  coast . population 
 estimation is around 484  000 habitants in an area of 28  051  km between both regions 
 . malabo is the capital of the 
 country and is placed in the island of bioko . there is a 
 tropical climate with a rainy season from may to october and a dry period from 
 december to march . it is a mesoendemic transmission area with a plasmodium 
 index of 21.7% in 2005 . bata is the most important city in the continental region and has also a 
 tropical climate with two dry seasons ( december  march , june it is a mesohyperendemic 
 area with a plasmodium index between 41% and 75% in children under five years 
 old . ( unpublished study from a national 
 prevalence survey 2006 , ministry of health and instituto de salud carlos iii . ) in 
 december 2005 , an in vivo study was carried out in the two regional 
 hospitals of malabo 
  and bata . the cases that were included in 
 the assessment came from the paediatric external consultation . it 
 was a two - armed prospective evaluation of the clinical and parasitological 
 responses to different antimalarial combinations , as as + sp and aq + sp . the 
 minimal sample size was different depending on the combinations . in order to 
 have a confidence level of 95% and a precision of 10% and as the proportion of 
 treatment failure with as + sp was already known , a minimum of 50 patients had to 
 be included . with aq + sp , there was no evidence of treatment failure , so the 
 minimum sample size had to be 96 for having a precision of 10% with a 
 confidence level of 95% . the inclusion criteria were age between 
 6 to 59 months , no signs of severe malnutrition , a slide confirmed 
 monoespecific infection of p. 
  falciparum , parasite density between 2000 and 200  000 asexual 
 parasites / microlitre , axillary temperature > 37.5c , easy access to the 
 hospital , absence of history of hypersensitivity reactions to any of the drugs 
 being evaluated and informed consent provided by parents or caregivers . any danger signs of severe malaria such as 
 inability to drink or breastfeed , vomiting , recent history of convulsions , 
 lethargy , and inability to sit or stand up were considered as exclusion 
 criteria . they were evaluated 
 clinically and parasitologically during 14 days ; treatment was given during 
 the first three days under direct observation . a 
 record form , which included : age , sex , address , name of the caregiver , contact 
 telephone ( if available ) , was maintained . during the followup , parameters such 
 as temperature ( during the six days ) , doses of drug ( days 0 , 1 , and 2 ) , 
 parasitaemia ( days 0 , 1 , 2 , 3 , 7 , and 14 or whatever day that children was bringing 
 to the hospital ) , haematocrit ( days 0 and 14 ) and filter paper ( days 0 , 14 ,   or any 
 day in case of treatment failure for analysis of the molecular markers ) were 
 recorded . finger prick blood samples that were collected in whatman paper and in a thick - and - thin smear stained 
 with giemsa for microscopy exam were undertaken . parasitaemia 
 was quantified by a standard approximation method , that is ,   number of asexual 
 parasites seen per 200 white blood cells in a high - power examination of a thick 
 blood film . a positive smear was defined as the presence of at least one 
 asexual parasite seen after examining 100 thick smear fields . quality control 
 of blood smears was done by rereading 10% of slides selected randomly . 055585 . expired 11/2008 ) , sulphadoxine 500  mg + pyrimethamine 25  mg ( ida . amodiaquine was given as 10  mg / kg of body weight per 
 day for three days . artesunate given as 4  mg / kg of body weight per day for three 
 days and sulphadoxine - pyrimethamine given as sulphadoxine 500  mg + pyrimethamine 
 25  mg / kg of body weight in a single dose   for the first day . in case of treatment 
 failure , quinine at 10  mg / kg of body weight every 8 hours for seven days was 
 administered . the criteria to determine the 
 treatment failure were the following : early treatment failure ( etf ) was 
 considered if development of severe malaria or danger signs during days 1 , 2 , or 
 3 in presence of parasitaemia , or parasitaemia on day 2 is higher than day 0 
 irrespective of axillary temperature , or parasitaemia on day 3 with axillary 
 temperature   > 37.5c , or parasitaemia on day 3 more than 25% of day 0 . late clinical failure ( lcf ) was considered by 
 development of severe malaria or danger signs after day 3 with parasitaemia 
 without previous criteria of etf , or parasitaemia and temperature > 37.5c on any day between 4 
 and 14 without previous criteria of etf . late parasitological failure ( lpf ) was 
 determined based on the presence of parasitaemia between days 7 and 14 with 
 temperature > 37.5c , 
 without previous criteria of etf or lcf . adequate clinical and parasitological response 
 was determined ( acpr ) by the absence of parasitaemia on day 14 , irrespective of 
 axillary temperature , without meeting the criteria of etf , lcf , and lpf . some of the cases were considered as withdrawal , when 
 caregivers of children decided not to continue with the study despite all 
 efforts and lost to follow up ; children could not be found at hospital , in the 
 community , or in the study area . we cut a circle of paper ( 40  mm   ) with blood , and dna was extracted by phenol / chloroform 
 method . this dna was used for the different molecular assays : ( a ) semi - nested - multiplex pcr for the diagnosis of malaria to confirm the plasmodium species ( b ) study of the msp-1 and msp-2 genes of p. 
 falciparum through nested - pcr . in order to distinguish recrudescence from 
 reinfection cases , we studied the three different allelic families of msp-1 
 gene : mad20 , ro33 and k1 ,   and msp-2 gene . recrudescence was defined as 
 the same population of parasites appeared on day 7 or day 14 as in day 0 . reinfection 
 was defined as a new population of parasites detected on day 14 compared to day 
 0 . after the pcr , we analysed the result in the bioanalyzer 2100 expert 
 ( agilent technologies ) , this method has a very high resolution and we can 
 distinguish amplification fragments with a little difference in the basepairs . baseline 
 characteristics , means ,   and percentages were compared in study subjects of both areas 
 by student t - test and by chi - square test respectively . the non - parametric 
 alternative for t - test and   mann - whitney test was used to compare the baseline 
 parasite density . therapeutic efficacy was calculated by dividing the number of 
 acpr cases by the number of patients evaluated on 14th day . log - rank test for equality of 
 survivor functions was calculated in order to compare time to parasitological 
 clearance . between 
 august and december 2005 , 206 children were randomised from the external 
 consultation of malabo and bata regional hospitals 
  in order to 
 evaluate the efficacy of two antimalarial combinations : aq + sp and as + sp . 
 baseline characteristics of children are shown in table 1 . as expected , no 
 differences were found between groups , except in the weight . the enrolled children were given one of the two 
 combinations in a random way : 120 were treated with aq + sp and 86 received as + sp . 
 in the first group 95% ( 114/120 ) finished the study and 91.9% ( 79/86 ) in the 
 second group . there 
 were 13 children who lost to follow up in aq + sp group . the percentage of lost 
 to follow up was 5% ( 6/120 ) and in the as + sp arm was 8.1% ( 7/86 ) . in general 
 children who lost to follow up were fewer ( 6.3% ; 13/206 ) than 10% of the children as per who 
 recommendations . with 
 aq + sp , 4 cases of lpf were registered and the proportion of treatment failure 
 was 3.5% ( 4/114 ) . with as + sp , 2 cases of lpf 2.5% ( 2/79 ) were 
 registered , the cure rate was 97.5% ( c.i . all of the 
 failure cases received rescue treatment with oral quinine and cured ( see table 2 ) . after the molecular correction , the acpr changed from 96.5% to 97.3% with 
 aq + sp combination . both combinations were well tolerated , no deaths were 
 notified and no severe reactions were recorded , except 3 patients ( 2.5% ) had 
 complaints of itching in aq + sp treatment group , they were given antihistamine . four 
 patients ( 3.3% ) in aq + sp and 1 patient ( 1.2% ) in as + sp treatment groups vomited , 
 which could not be confirmed whether this was the consequence of malaria or the 
 treatment effect . the parasite 
 clearance ( figure 2 ) was faster 
 in the group who received the combination with artesunate ( log - rank test 
 p < .001 ) . in as + sp treatment group , 77.2% ( 61/79 ) and in aq + sp treatment 
 group , 94.7% ( 108/114 ) presented parasitemia on day 1 . on day 2 , 3.8% ( 3/79 ) of 
 as + sp and 55.3% ( 63/114 ) of aq + sp treatment groups were parasitemic . by day 3 , after receiving 
 treatment , gametocyte density was found to be higher in as + sp patients than 
 aq + sp . with the first combination , by day 2 , 12% of children had gametocytes on 
 the thick film , compared with the 7.5% with aq + sp . by day 3 , the 7.8% with 
 as + sp and the 6% with aq + sp had gametocytes . by day 14 , haematocrit at enrolment showed that   37% 
 of the children had moderate anaemia with haematocrit 2429 , 49% had mild 
 anaemia with haematocrit 3034 ,   and 14% were nonanaemic . after treatment the 
 moderate anaemia decreased from 37% to 23% ,   and nonanaemic cases increased from 
 14% to 22% . molecular correction was done to distinguish recrudescent from reinfection in 6 
 treatment failure cases : 4 cases from bata , 3 in the aq + sp combination , 
 and 1 in the as + sp , and 2 in malabo 
  with two 
 combinations ( aq + sp and as + sp ) . table 3 shows the number of alleles of msp-1 allelic family , and msp-2 detected in the six treatment 
 failure cases . we could confirm the resistant 
 cases from bata by molecular analysis ; because the parasite genotypes detected 
 by pcr on day 14 were similar with those on day 0 . however , in the aq + sp treatment 
 failure cases from malabo , 
 one of them was reinfection because a different set of genotypes appeared on 
 day 14 as shown in table 3 . there is a need to give a thought about any monotherapy as a 
 first - line treatment in case of patients report with uncomplicated falciparum malaria . keeping this in view , this study would 
 be helpful in national malaria control program of equatorial guinea to 
 introduce a new combination therapy for malaria treatment . two combinations were assessed , the combinations as + sp , where 
 the as is an efficacious drug and reduces the parasitaemia faster than others , 
 and the second combination , aq + sp , is also an effective combination but with the 
 risk to develop resistant in due course of time , but more accessible to the 
 population when compared with artemisinin derivatives . the study demonstrated that both combinations are safe and 
 efficacious for the treatment of nonsevere falciparum malaria in equatorial guinea . a study carried out in tanzania 
  in an area of high malaria 
 transmission compared the efficacy and safety of aq and sp in monotherapy and 
 aq + sp as a combination . the study demonstrated that aq + sp combination was safe , 
 and the combination of the two drugs had higher efficacy ( 96.2% ) than 
 monotherapy . 
 in a similar study carried out in gambian children , concluded that as + sp 
 combination was safe and the efficacy was 98.4% . a study carried out in guinea 
  showed an efficacy of 99% with this combination . these results are similar to those of the present study with cure rates of 
 97.5% ( 77/79 ) in 
 as + sp combination and 97.3% ( 111/114 ) in aq + sp . the 
 situation in equatorial 
  guinea 
  is similar with other african 
 countries . the cure - rates with the two combination drugs , aq + sp and as + sp ,   are 
 over the 95% , which is the limit that who suggested for a combination to be 
 used as a first line of treatment . in general terms , drugs were well tolerated , with a low 
 percentage of adverse effects . in the group that received aq + sp , a small number 
 of children required retreatment after vomiting after treatment . severe 
 reactions with amodiaquine as neutropenia have been described also in other 
 publications . in one study , it was shown that the risk was associated with amodiaquine used as a weekly 
 prophylaxis , not like a treatment . 
 in a study carried out in madagascar , using combination of aq + sp like our work , 
 no severe reaction was described using aq in a 3-day treatment schedule . clearance of parasites was faster in case of combination 
 with artemisinin derivatives , due to the mode of action of artemisine , which 
 reduces parasitaemia by a factor of 10 in each cycle . in uganda , was 
 described the difference in parasitemia clearance using a combination with and 
 without artemisinin derivatives . by day two , in the first case just 3% were 
 positive compared to the second group , where 48% were positive . these results coincide with our study , where by day two , 3.8% were parasitaemic 
 compared with the 55.3% of children who received aq + sp on the same day . due to faster 
 parasite clearance , clinical improvement and disappearance of symptoms were 
 faster in children treated with as + sp . because of this rapid effect , use of 
 artemisinin derivatives in a combination would be appropriate in terms of 
 decreasing the possible development of parasite resistance to the drug by rapid 
 clearance thus they are not in contact with the drug for a long time . contrary to other studies , we have not seen any 
 difference in gametocytes carriage . from this study , we could not say that using 
 of artemisinin - based combination therapies may reflect in decrease of malaria 
 transmission in the area . one study in tanzania , where different drugs in 
 monotherapy and combinations were compared , demonstrated that   aq + sp 
 gametocytes on day 14 were   25.7% in children , which was double than using 
 the combination with as , where just 11.9% of children had gametocytes . it is necessary to assess other therapies 
 without this drug due to the following reasons . first , sp is the only safe drug 
 actually to be used as intermittent preventive treatment for pregnant women . following who recommendations , we need to protect the future development of 
 resistance to this drug . and the second reason is that sp has a resistance 
 around 18% in monotherapy ( unpublished data , 2002 ) . drugs that have been used for a long time as monotherapy and have 
 already demonstrated resistance have the risk to develop resistance in 
 combination in due course of time . from the results of this study , we may suggest that in 
 all in vivo studies a molecular assay for discriminating recrudescence 
 from reinfection should be done . the molecular correction is the only method 
 that allows us to give a real data about the level of resistance detected in an 
 endemic area . this way , it is possible to analyse different markers like msp-1 and   msp-2 genes of p. falciparum , or other polymorphic markers 
 as microsatellites . we need to continue the epidemiological surveillance 
 and monitoring resistance in order to give enough information to the national malaria 
 program of equatorial guinea for revising malaria treatment policy in the 
 country . this should be done as per the recommendation of who before the drug resistance 
 reaches at levels not allowed to be used . the conclusions of this study are that   both the combinations are safe and efficacious 
 for their use as a first - line treatment in case of nonsevere falciparum malaria . to choose one of 
 them will depend on economic resources of equatorial guinea in the moment to 
 change malaria policy of the country ; the molecular correction gives 
 complementary information to strengthen the findings of in vivo studies carried 
 out in areas with a high level of transmission .
objectives . the objectives of the study were ( i ) to evaluate the efficacy of combination drugs , such as artesunate + sulphadoxine - pyrimethamine ( as  +  sp ) and amodiaquine + sulphadoxine - pyripethamine ( aq  +  sp ) in treatment of uncomplicated falciparum malaria ( ii ) to differentiate recrudescence from reinfection by analysing msp-1 and msp-2 genes of plasmodium falciparum in treatment failure cases . 
 methods . 
 we carried out an in vivo study in the year 2005 in 206 children between 6 to 59 months age groups . 
 of the 206 , 120 received aq  +  sp , and 86 received as  +  sp . a clinical and parasitological followup during 14 days was undertaken . 
 finger - prick blood sample from each patient was taken on whatman filter paper ( no . 
 3 ) on days 0 , 7 , 14 and also the day when the parasite and symptoms reappeared for pcr analysis . 
 results . 
 late treatment failure was observed in 3.5% ( 4/114 ) with aq  +  sp , and 2.5% ( 2/79 ) with as  +  sp . the success rate was 96.5% with aq  +  sp and 97.5% with as 
 +  sp . 
 no deaths and severe reactions were recorded . out of the 6 treatment failure cases , 
 one was reinfection as observed by pcr analysis of msp-1 and msp-2 genes on day 14 . 
 discussion . 
 both the combinations found to be efficacious and safe and could be used as a first - line treatment for uncomplicated falciparum malaria in equatorial guinea .