1 Introduction WHO, 1991 WHO, 1994 Torlesse and Hodges, 2000, 2001 Allen et al., 2002; WHO, 2002 Elliott et al., 2007 2 Materials and methods 2.1 Study population and procedures The study area comprises Entebbe Municipality and the adjacent subcounty of Katabi. This area supports semi-urban, rural and fishing communities residing on the Entebbe peninsula in Lake Victoria, Uganda. Women were recruited at the antenatal clinic at Entebbe Hospital between April 2003 and November 2005. Women were assessed for screening at their first antenatal visit, thus initial screening could take place in any trimester of pregnancy. They were eligible for screening if they were well, resident in the study area, planning to deliver their baby at the hospital, willing to participate and willing to know their HIV status. On the screening day, after giving written informed consent, eligible women were interviewed regarding sociodemographic characteristics and risk factors for helminth infection, malaria and HIV and were examined by a midwife. A blood sample was obtained for investigations including haemoglobin (Hb) estimation, examination for microfilariae (mf) and malaria parasites, syphilis and HIV serology. Screened women were asked to return for enrolment within 1 month with a stool sample. They were excluded from enrolment if they had a Hb level <8 g/dl, clinically apparent severe liver disease, diarrhoea with blood in the stool, an abnormal pregnancy, a history of adverse reaction to anthelminthic drugs or had already participated in the study during an earlier pregnancy. Women were enrolled in the trial when they returned with a stool sample if they were in the second or third trimester and full eligibility was confirmed. All women received routine antenatal care including haematinics and intermittent presumptive treatment for malaria using sulfadoxine/pyrimethamine. Women were treated for syphilis and provided with nevirapine for prevention of mother-to-child HIV transmission, if indicated. Women who were excluded from the study on grounds of severe anaemia were treated with albendazole and haematinics and referred for transfusion if required. 2.2 Haemoglobin estimation and definition of anaemia Hb was estimated at the antenatal clinic using a colorimetric haemoglobinometer (DHT haemoglobin meter; Developing Health Technology, Barton Mills, UK) with same-day results. The same sample was then sent to the Medical Research Council/Uganda Virus Research Institute (MRC/UVRI) laboratories for analysis by a Coulter analyser (Beckman Coulter AC-T 5 diff CP; Beckman Coulter, Nyon, Switzerland). Quality control for the Coulter analyser was provided through the United Kingdom National External Quality Assessment Schemes, with consistently good results. Initial evaluation suggested that the haemoglobinometer results reliably matched the Coulter analyser results (intraclass correlation coefficient (ICC) = 0.81, 95% CI 0.74–0.88) and the immediately available haemoglobinometer results were used to determine enrolment. Evaluation of results to March 2005 showed less reliability (ICC = 0.54, 95% CI 0.51–0.58) and it was noted that 37 women had been enrolled with haemoglobinometer results >8 g/dl but Coulter analyser values below this cut-off; thereafter, Coulter analyser results were used for enrolment. Coulter analyser results have been used in this analysis. http://www.sph.emory.edu/∼cdckms/hbadj2.html WHO, 1999a 2.3 Parasitology Bukusuba et al., 2004 Katz et al., 1972 Strongyloides Friend, 1996 . Mansonella Melrose et al., 2000 WHO, 1994 Schistosoma mansoni WHO, 1999b Trichuris trichiura WHO, 1999b Mansonella 2.4 HIV serology HIV serology was performed using a rapid test algorithm with same-day results. Testing kits, provided by the Ministry of Health, varied with availability. Most commonly, Determine (Abbott Laboratories, Abbott Japan Co. Ltd., Tokyo, Japan) was used for screening, with positive results confirmed by Unigold (Trinity Biotech plc, Bray, Ireland). Samples with differing results were referred for analysis by non-rapid ELISA tests or were examined using a ‘tie-breaker’ rapid test, usually Statpack (Chembio Diagnostic Systems, Medford, NY, USA). A proportion of samples, including all those with differing results by rapid test, were re-examined at the MRC/UVRI laboratories for quality control, with high agreement of the results. 2.5 Demographic information and potential risk factors for anaemia Figure 1 2.6 Data management and statistical analysis P 3 Results A total of 15 035 women registered at the antenatal clinic during the recruitment period, of whom 11 783 were assessed for inclusion in the study and 3163 were considered eligible and screened. The commonest reasons for ineligibility were residence outside the study area (6243), unwillingness to have an HIV test (1186), unwillingness to join the study (874) and enrolment during an earlier pregnancy (115). Of the 3163 screened, 2515 were enrolled; 8 of these were subsequently excluded because they had been enrolled during a previous pregnancy. Of the 648 women screened but not enrolled, the majority (596) failed to return for enrolment and only 15 brought a stool sample. Since intestinal helminth infection detected by stool analysis was a focus of interest, this analysis was confined to the 2507 women who were enrolled in the trial and for whom all or almost all relevant data were available. 3.1 Characteristics of the study women Maternal age ranged from 14 years to 47 years (mean 23.6 years). The majority were Baganda (49.1%), the predominant tribe of the district. Most (83.8%) were married, with 13.4% single, 0.6% widows and 2.3% divorced or separated. Education varied from none (3.9%), to primary (50.5%), secondary (37.3%) and tertiary (8.4%) and most women were poor (85.1% with a personal income of less than £10 per month). Primigravidae comprised 27.7% of the women studied. 3.2 Prevalence of infections and anaemia Strongyloides Mansonella Mansonella perstans S. mansoni Strongyloides stercoralis T. trichiura Ascaris lumbricoides Trichostrongylus Hymenolepis nana Loa loa Fasciola hepatica Dicrocoelium dendriticum Plasmodium falciparum The prevalence of anaemia (Hb < 11.2 g/dl) was 39.7%. Six women were enrolled in error with Hb < 8 g/dl by both methods (Coulter analyser results 5.6–7.9 g/dl). These, as well as the 37 women enrolled prior to March 2005 with haemoglobinometer results above but Coulter analyser results below 8 g/dl, have been retained in this analysis. 3.3 Associations between characteristics of pregnant women and anaemia in pregnancy Table 1 3.4 Relationship between infections and anaemia in pregnancy Table 2 P P P P P Mansonella Attributable fractions for anaemia were 3.1% for hookworm, 12.3% for malaria and 10.2% for HIV infection. 3.5 Relationship between anaemia and infection intensity Table 3 10 P S. mansoni Trichuris Trichuris S. mansoni Trichuris 10 P P 10 P 3.6 Anaemia in women excluded from enrolment P P P P P 4 Discussion S. mansoni Bondevik et al., 2000; Hotez et al., 2004; Shulman et al., 1996 Hall, 1981; Utzinger et al., 2001 Ajanga et al., 2006; Bondevik et al., 2000; Dreyfuss et al., 2000; Larocque et al., 2005 Mansonella Not all pregnant women in Entebbe attend the district hospital antenatal clinic, but a community survey undertaken in the study area showed an increase in the proportion choosing this clinic during the recruitment period, to approximately 80%, and most of the personal and socioeconomic characteristics of women choosing, or not choosing, to attend the district hospital clinic were similar (unpublished data). Thus, our results are likely to be reasonably representative of pregnant women in this area. S. mansoni Trichuris S. mansoni Ascaris Trichuris Ajanga et al., 2006; Kalenga et al., 2003; Larocque et al., 2005; Nurdia et al., 2001 S. mansoni S. mansoni S. mansoni Dreyfuss et al., 2000 Bondevik et al., 2000 Bondevik et al., 2000; Dreyfuss et al., 2000 Bondevik et al., 2000; Olsen et al., 1998 Torlesse and Hodges, 2001 Mansonella Trichuris Strongyloides Dreyfuss et al., 2000; Larocque et al., 2005; Nurdia et al., 2001 Larocque et al. (2005) Trichuris Trichuris Ramdath et al., 1995 Nurdia et al. (2001) Trichuris The strong effects of malaria and HIV contrast with the weak effect of hookworm and the lack of effects of other helminths in this study. Shulman and Dorman, 2003 Belperio and Rhew, 2004 Dairo et al., 2005; McIntyre, 2003 Shulman and Dorman, 2003; ter Kuile et al., 2004 Christian et al., 2004 Authors’ contributions AME designed the study; JN and CA carried out interviews and recruited participants; MO and HM carried out clinical assessments; NO and DK carried out laboratory assessments; LM, PW and LAM analysed and interpreted the data; LM, PW, LAM and AME drafted the manuscript. All authors reviewed and approved the final manuscript. LM and AME are guarantors of the paper. Funding Wellcome Trust Career Post fellowship held by Dr Elliott, grant number 064693; PMTCT programme, Ministry of Health, Uganda. Conflicts of interest None declared. Ethical approval The Science and Ethics Committee, Uganda Virus Research Institute, the Uganda National Council for Science & Technology, and the London School of Hygiene & Tropical Medicine.