Conceived and designed the experiments: ML SRM TK KM PA. Performed the experiments: ML AMR SRM TK KM PA. Analyzed the data: ML YBC PA. Contributed reagents/materials/analysis tools: SRM PA. Wrote the paper: ML SRM PA. Revised the manuscript critically for important intellectual content: AMR YBC TK KM.
Azithromycin and its placebo used in the study were provided free of charge by Pfizer Inc (New York), which had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. The authors have declared that no other competing interests exist.
New regimens for intermittent preventive treatment in pregnancy (IPTp) against malaria are needed as the effectiveness of the standard two-dose sulfadoxine-pyrimethamine (SP) regimen is under threat. Previous trials have shown that IPTp with monthly SP benefits HIV-positive primi- and secundigravidae, but there is no conclusive evidence of the possible benefits of this regimen to HIV-negative women, or to a population comprising of both HIV-positive and –negative women of different gravidities.
This study analyzed 484 samples collected at delivery as part of a randomized, partially placebo controlled clinical trial, conducted in rural Malawi between 2003 and 2007. The study included pregnant women regardless of their gravidity or HIV-infection status. The participants received SP twice (controls), monthly SP, or monthly SP and two doses of azithromycin (AZI-SP). The main outcome was the prevalence of peripheral
Overall prevalence of PCR-diagnosed peripheral
Our results suggest that increasing the frequency of SP administration during pregnancy improves the 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.
ClinicalTrials.gov NCT00131235
Malaria is one of the most important preventable causes of poor maternal health and adverse birth outcomes
At least four trials have tested the efficacy of an IPTp regimen that contains monthly SP-dosing
In our previously reported trial from Malawi we included pregnant women regardless of their gravidity or HIV-infection status
In a subsequent study, we applied a polymerase chain reaction (PCR) – based method to diagnose malaria from peripheral blood samples collected at delivery in the above described trial. With this more sensitive method, we documented an almost five fold higher prevalence of
This study is a secondary analysis of peripheral blood samples collected at delivery for real-time PCR assay targeting
The trial was performed according to Good Clinical Practice guidelines and the ethical standards of Helsinki Declaration. The protocol was approved by the College of Medicine Research and Ethics Committee, University of Malawi, Malawi and the Ethical Committee of Pirkanmaa Hospital District, Finland. Key details of the protocol were published at the clinical trial registry of the National Library of Medicine, Bethesda, Md, USA (
The target population of LAIS comprised pregnant women who came between December 2003 and October 2006 for antenatal care to Lungwena Health Centre, Mangochi district, southern Malawi. Malaria is holoendemic at this rural site
Participants in the control group (“control”) received standard Malawian antenatal care including IPTp with SP (three tablets orally, each containing 500 mg sulfadoxine and 25 mg pyrimethamine), and a placebo to azithromycin, both given twice during pregnancy: at enrollment and between 28th and 34th weeks of gestation. Participants in the first intervention group (“monthly SP”) received otherwise the same treatment, but SP was given monthly from enrollment until 37 gestational weeks. Participants in the second intervention group (“AZI-SP”) received otherwise the same treatment as the monthly SP group, but instead of placebo, they received active azithromycin twice (two tablets orally, each containing 500 mg of azithromycin): at enrollment visit and between 28th and 34th weeks of gestation. All participants received ferrous sulphate (200 mg/day) and folic acid (0.25 mg/day) throughout pregnancy.
Participants diagnosed with malaria at enrollment or at 28–34th week visit received the normal pre-packed study drugs which included SP for all study groups. Laboratory-confirmed malaria at any other point was treated with quinine (two tablets orally three times a day for seven days, each containing 300 mg of quinine).
SP tablets were purchased from Malawi Central Medical Stores that were supplied by Pharmanova, Ipca Laboratories Ltd, F.Hoffmann-La Roche Ltd, and Universal Corporation Kenya Ltd. Both active azithromycin and its placebo were manufactured and donated by Pfizer Inc. We did not perform any pharmacological tests on the study drugs.
A researcher not involved in data collection generated a randomization code list in blocks of nine. Based on this list, an individual drug box was pre-packed for each identification number. The drug box contained appropriate study drugs for each planned study visit in separate opaque drug envelopes labeled with identification number and visit information. Individual slips containing unique identification numbers, but not group allocation, were sealed in individual opaque randomization envelopes. The envelopes were grouped into randomization blocks to ensure similar allocation rates to each group.
At enrollment visit, the research personnel interviewed individuals interested in participating in the study about their socioeconomic status, obstetric history and bed net use, gave pre-test HIV-counseling, and performed an antenatal and laboratory examinations. Testing for HIV was optional. Eligible individuals signed or thumb-printed informed consent and picked one randomization envelope which contained an identification number. A research assistant not involved in outcome assessment gave the corresponding pre-packed study drugs to the participant under direct observation.
At follow-up visits (at four-week intervals until 36 completed gestational weeks and weekly thereafter) the research personnel conducted an antenatal examination. The participants were offered post-test HIV counseling and prevention of mother to child transmission. At the visit during 28th–34th weeks of gestation, a malaria test was conducted. At each visit, the participant took the appropriate pre-packed study drugs under direct observation.
Upon notification of a delivery, a research assistant visited the delivery site. She collected data on delivery, examined the newborn and gave her/him nevirapine or placebo based on maternal HIV status. From participants who delivered at a local health facility, maternal peripheral venous blood was collected as thin and thick blood films, and as dried blood spots on filter paper.
Peripheral blood
Statistical analysis was carried out with Stata 9.2 (StataCorp, College Station, USA). We estimated risk ratio and risk difference for comparison of binary end-points at a single time point. To prevent inflated type I errors due to multiple comparisons, we began hypothesis testing with global null hypotheses of all three groups being identical before doing pair-wise comparisons. We tested the hypotheses with Fisher’s exact test.
The proportion of women with no previous pregnancies was higher in the control and monthly SP groups than in the AZI-SP group, and the proportion with peripheral malaria parasitaemia at enrollment was higher in the control group than in the intervention groups. As sensitivity analyses, we adjusted for these two covariates as categorical variables by generalized linear models and compared the adjusted and main analyses. We performed tests for interaction between the interventions and HIV status (those whose status was known), the number of previous pregnancies (classified as none, and one or more), and bed net use using the likelihood ratio test, and did analyses stratified by the same variables.
Of the 1320 LAIS participants
The mean (standard deviation, SD) number of scheduled SP treatments received was 2.0 (0.2) in the control, 4.2 (0.9) in the monthly SP, and 4.1 (0.8) in the AZI-SP group. Women in the AZI-SP group received a mean (SD) of 2.0 (0.1) azithromycin doses. The mean number (SD) of quinine doses given at non-scheduled outpatient visits was 0.04 (0.23) for the control, 0.05 (0.22) for the monthly SP, and 0.02 (0.15) for the AZI-SP group (P = 0.415). Against the trial protocol some SP-doses were given also at the non-scheduled visits, but the number of these was very small and there were no differences between the groups (4 participants each received one additional dose: 1 in control, 2 in monthly SP and 1 in AZI-SP group).
Characteristic | Sub-characteristic | Control (SP twice)N = 162 | Monthly SPN = 151 | AZI-SPN = 171 | LAIS participants not included in the current analysis (all groups combined)N = 836 |
Mean (SD) age in years | 24 (7) | 24 (6) | 25 (6) | 25 (7) | |
Number (%) of literate participants | 49 (30.3%) | 47 (31.1%) | 65 (38.0%) | 223 (26.7%) | |
Mean (SD) years of schooling completed | 2.5 (2.9) | 2.4 (2.9) | 2.8 (2.9) | 2.0 (2.6) | |
Mean (SD) height in cm | 154.8 (5.9) | 154.4 (5.2) | 155.0 (5.5) | 155.2 (5.5) | |
Mean (SD) BMI as kg/m2 | 21.7 (2.1) | 21.8 (2.3) | 22.1 (2.1) | 21.7 (2.2) | |
Mean (SD) gestational age at enrollment in weeks | 20.3 (3.0) | 19.5 (3.2) | 19.9 (3.0) | 20.2 (3.0) | |
Number (%) of previous pregnancies | |||||
None | 53 (32.7%) | 52 (34.4%) | 34 (19.9%) | 167 (20.0%) | |
One or more | 109 (67.3%) | 99 (65.6%) | 137 (80.1%) | 669 (80.0%) | |
Number (%) of women owning a bed net | 123 (75.9%) | 107 (70.9%) | 130 (76.0%) | 608 (72.7%) | |
Number (%) of women who used bed net the night before enrollment | 104 (64.2%) | 88 (58.3%) | 100 (58.5%) | 505 (60.4%) | |
Maternal HIV status, number (%) | |||||
Positive | 18 (11.1%) | 19 (12.6%) | 23 (13.5%) | 101 (12.1%) | |
Negative | 129 (79.6%) | 119 (78.8%) | 129 (75.4%) | 656 (78.5%) | |
Unknown | 15 (9.3%) | 13 (8.6%) | 19 (11.1%) | 79 (9.5%) | |
Number (%) with microscopic peripheral blood malaria parasitaemia at enrollment | 24 (14.8%) | 13 (8.6%) | 6 (3.5%) | 74 |
|
Mean (SD) blood Hb concentration as g/L | 109 (18) | 110 (16) | 110 (20) | 111 (19) |
Abbreviations: AZI-SP, intervention group that received monthly SP and two doses of azithromycin; SD, standard deviation; BMI, body mass index; Hb, hemoglobin.
Denominator is 835 because of one missing malaria result.
The overall prevalence of PCR-diagnosed peripheral
PCR-diagnosed malaria at delivery | Number with outcome/total number with known outcome data | Comparison between monthly SP and control group | Comparison between AZI-SP and control group | |||||
Control | Monthly SP | AZI-SP | P-value | Risk ratio (95% CI) | P-value | Risk ratio (95% CI) | P-value | |
Unadjusted | 33/162 (20.4%) | 10/151 (6.6%) | 8/171 (4.7%) | <0.001 | 0.33 (0.17 to 0.64) | <0.001 | 0.23 (0.11 to 0.48) | <0.001 |
Adjusted for the number of previous pregnancies | 0.31 (0.16 to 0.61) | 0.001 | 0.25 (0.12 to 0.52) | <0.001 | ||||
Adjusted for microscopic malaria at enrollment | 0.33 (0.17 to 0.65) | 0.001 | 0.24 (0.11 to 0.51) | <0.001 |
Abbreviations: AZI-SP, intervention group that received monthly SP and two doses of azithromycin; CI, confidence interval.
Sensitivity analyses, adjusting either for the number of previous pregnancies or microscopic malaria parasitaemia at enrollment, gave essentially identical results to those of the unadjusted analysis (
In a stratified analysis including only HIV-negative participants, those in the monthly SP group had a RR (95% CI) of 0.26 (0.12 to 0.57, P<0.001) and those in the AZI-SP group 0.24 (0.11 to 0.53, P<0.001) for PCR-diagnosed malaria at delivery, when compared with the controls (
Stratified analysis of malaria at delivery based on | Number of participants with malaria (%) | Comparison between monthly SP and control group | Comparison between AZI-SP and control group | ||||||
All groups | Control | Monthly SP | AZI-SP | P-Value | Risk ratio (95% CI) | P-Value | Risk ratio (95% CI) | P- Value | |
|
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Positive | 3/60 (5.0%) | 2/18 (11.1%) | 1/19 (5.3%) | 0/23 (0.0%) | 0.189 | 0.47 (0.05 to 4.78) | 0.604 | 0 | 0.187 |
Negative | 43/377 (11.4%) | 29/129 (22.5%) | 7/119 (5.9%) | 7/129 (5.4%) | <0.001 | 0.26 (0.12 to 0.57) | <0.001 | 0.24 (0.11 to 0.53) | <0.001 |
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None | 23/139 (16.6%) | 18/53 (34.0%) | 3/52 (5.8%) | 2/34 (5.9%) | <0.001 | 0.17 (0.05 to 0.54) | <0.001 | 0.17 (0.04 to 0.70) | 0.003 |
One or more | 28/345 (8.1%) | 15/109 (13.8%) | 7/99 (7.1%) | 6/137 (4.4%) | 0.028 | 0.51 (0.22 to 1.21) | 0.175 | 0.32 (0.13 to 0.79) | 0.011 |
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No | 32/192 (16.7%) | 21/58 (36.2%) | 6/63 (9.5%) | 5/71 (7.0%) | <0.001 | 0.26 (0.11 to 0.61) | <0.001 | 0.19 (0.08 to 0.48) | <0.001 |
Yes | 19/292 (6.5%) | 12/104 (11.5%) | 4/88 (4.6%) | 3/100 (3.0%) | 0.044 | 0.39 (0.13 to 1.18) | 0.115 | 0.26 (0.08 to 0.89) | 0.029 |
Abbreviations: AZI-SP, intervention group that received monthly SP and two doses of azithromycin; CI, confidence interval.
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
The study population was drawn from a trial which used broad inclusion criteria, random group allocation and blinding of the outcome assessors
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
Our results indicate that IPTp with monthly SP can in some conditions reduce the prevalence of peripheral blood
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
However, before adopting a monthly SP IPTp policy, several issues have to be taken into consideration. The prevalence of mutations in
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
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
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
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.
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We thank the study participants and the people of Lungwena; the staff at the Lungwena Training Health Centre, Malindi and Mangochi Hospitals and our research nurses and assistants for their positive attitude, support and help in all stages of the study; B Mbewe for supervising part of the data collection; E Molyneux, S A White, G Kafulafula for monitoring the study as the data safety and monitoring board members; J Kumwenda for making the site monitoring visit; L Csonka for designing the data entry program; S Taylor for his assistance in the development of the PCR methodology.