The use of IPT during pregnancy can prevent malaria and the adverse outcomes it causes to pregnant women and their fetuses in SSA. In the current analysis we used a sensitive PCR–based diagnostic method to compare the antimalarial effect of the standard two-dose SP IPTp regimen with monthly SP, alone or in combination with two doses of azithromycin. Compared with the control group, the participants in the monthly SP and AZI-SP groups had relatively 67% and 77% lower prevalence of PCR-diagnosed peripheral P. falciparum malaria parasitaemia at delivery, respectively. In stratified analyses, HIV-negative participants of all gravidities, primigravidae regardless of their HIV-status, and women who did not use a bed net at enrollment had significantly less malaria in both intervention groups than those in the control group.
The study population was drawn from a trial which used broad inclusion criteria, random group allocation and blinding of the outcome assessors 
. The intervention code was linked to the malaria results only after the DNA-based diagnostic tests were completed. The number of participants with available PCR-results, as well as their baseline characteristics, was similar in the three study groups, except for differences in the number of previous pregnancies and the prevalence of malaria parasitaemia. However, similar differences were seen already in the main study 
, and adjusted analyses suggested that these did not bias the results. Confidence interval calculations and hypothesis testing indicated that the probability of type I error was small. We therefore believe that the sample findings were reliable and representative, and that IPTp with monthly SP, with or without azithromycin, was indeed associated with reduced prevalence of PCR-diagnosed peripheral P. falciparum
malaria parasitaemia at delivery in the target population from which the sample was drawn.
A secondary analysis of pooled data from three other clinical trials from Kenya, Malawi and Zambia suggested that compared with the two-dose SP regimen, the monthly regimen had a beneficial effect on malaria prevalence at delivery and birth weight among HIV-positive pregnant women in their first or second pregnancy 
. The previous Malawian study found significantly lower peripheral malaria at delivery in the monthly SP group also among HIV-negative primi- and secundigravidae 
. However, when this data was pooled with the data of HIV-negative participants in the Kenyan study, the analysis failed to document statistical significance 
. None of these earlier studies addressed the efficacy of the monthly SP regimen in a representative sample that contained both HIV-negative women and those with more than one earlier pregnancy 
. Hence, it has thus far been uncertain, whether the monthly SP regimen would best be reserved for some selected risk groups, or it should be given to all pregnant women in malaria endemic areas 
Our results indicate that IPTp with monthly SP can in some conditions reduce the prevalence of peripheral blood P. falciparum
malaria parasitaemia at delivery in the latter, wider target group, as well as among HIV-negative women regardless of their gravidity. This finding is consistent with the results of a recently reported trial from Mali, which found that IPTp with three doses of SP is superior to two doses in reducing malaria prevalence at delivery, LBW and premature delivery among mainly HIV-negative women of different gravidities 
. The sample of the current substudy was not powered to look at more direct health outcomes, but since also PCR-detectable submicroscopic parasitaemias have important consequences to maternal health and birth outcomes, the observed effect is likely to be beneficial both to the mother and the newborn 
(Atupele Kapito-Tembo et al, unpublished results).
Besides the apparently improved efficacy against malaria, monthly dosing would solve some of the problems associated with the two-dose SP IPTp. There are concerns that the dosing interval in the two-dose regimen might be too long even in areas with low levels of SP resistance, and increasing resistance further shortens the duration of the post-treatment prophylactic effect 
. This concerns especially HIV-positive women not taking cotrimoxazole prophylaxis, who have increased susceptibility to malaria in pregnancy and reduced efficacy of antimalarial drugs 
. Also, many pregnant women receive none or only one dose of SP despite making several antenatal care visits during the second and third trimesters 
. One reason for this is the difficulty to determine the appropriate timing of the doses; monthly SP policy would simplify the dosing regimen and thus ensure that more women receive at least two doses of SP 
. Previous results suggest that also monthly SP, alone and in combination with azithromycin, is safe to use during the second and third trimesters of pregnancy 
. All this, together with the ease of single-dose administration which can be done under direct observation at antenatal care visits, and the low price of SP, makes the monthly SP regimen seem suitable for IPTp until an effective, safe and feasible alternative for SP has been identified.
However, before adopting a monthly SP IPTp policy, several issues have to be taken into consideration. The prevalence of mutations in P. falciparum
associated with resistance to SP has increased in Africa, and is very high among pregnant women in some parts of Malawi 
. There have been concerns that IPTp might increase resistance and even cause adverse outcomes, but the results have so far been contradictory 
. Additionally, local conditions such as malaria prevalence and the level of acquired malaria immunity among the target group need to be taken into consideration as they might affect SP IPTp efficacy. In our study area the level of resistance was high already during the enrollment between 2003 and 2007: over 80% of the malaria isolates collected among the LAIS participants at enrollment were found to express quintuple DNA mutations for SP resistance (Steven Meshnick et al, unpublished results).
In our previous analysis we found that the combination regimen of monthly SP and two doses of azithromycin, but not monthly SP alone, was significantly superior to the standard regimen of two SP doses in preventing preterm delivery and LBW 
. Azithromycin is both a modestly active antimalarial and a potent broad-spectrum antimicrobial drug, and thus the improved efficacy of the combination could have been due to either activity 
. However, because the prevalence of microscopic malaria was low, we were earlier unable to adequately separate the antimalarial and other possible effects of azithromycin.
In the current analysis, we found that the monthly SP regimen was associated with statistically significant reductions in PCR-diagnosed malaria prevalence at delivery both alone, as well as in combination with azithromycin, and that there were no major differences between the two intervention regimens in the point-estimates for the effect size. The results thus suggest that the decrease in parasitaemia at delivery was mainly caused by the increased frequency of the SP dosage, while the improvement in birth outcomes might have been due to some other activity of azithromycin, independent of its activity against malaria. A possible explanation for azithromycin’s weak antimalarial effect in our study is the used dosage. Although 1g dose of azithromycin given twice several weeks apart is sufficient to cure many RTIs, it might be suboptimal against P. falciparum
Two limitations of our study are that we analyzed the prevalence of malaria parasitaemia with PCR only at delivery, and only from peripheral blood. It could be argued that the difference in parasitaemia at delivery simply reflects the fact that the last dose in the regimens containing monthly SP was given nearer to delivery than in the two-dose regimen. Thus we cannot rule out that the groups might have had comparable malaria prevalence during pregnancy and that the differences only developed towards delivery. However, it is unlikely that the efficacy of SP would have differed during pregnancy, and therefore the monthly dosing with shorter treatment intervals is likely to have been more effective throughout pregnancy. This is supported by our finding of a comparable effect size in the prevalence of microscopic malaria parasitaemia at 32 gestational weeks in the original sample from which the current study population was drawn 
. Another limitation is that we did not perform analyses with PCR of placental blood and did not collect histological samples of the placenta to diagnose placental malaria. The microscopic results of placental blood showed no difference between the groups 
In conclusion, our results suggest that increasing the frequency of SP administration during pregnancy improves efficacy against malaria at delivery among HIV-negative women as well as a population consisting of both HIV-positive and –negative pregnant women of all gravidities in a setting of relatively low but holoendemic malaria transmission, frequent use of bed nets and high SP resistance. However, further research is needed to study the consequences of the prevention of submicroscopic malaria infections to maternal and child health, as well as the effect of different levels of resistance and malaria prevalence on the IPTp efficacy. We therefore recommend the use of sensitive diagnostic tools for malaria, such as PCR and placental histology, and the determination of the level and change of drug resistance and acquired immunity in future IPTp trials.