Previous research has shown that internet education can lead to short-term improvements in clinicians' knowledge, confidence and communication practices. We wished to better understand the duration of these improvements and whether different curriculum delivery strategies differed in affecting these improvements.As previously described, we conducted a randomized control trial comparing four different strategies for delivering an e-curriculum about herbs and other dietary supplements (HDS) to clinicians. The four strategies were delivering the curriculum by: a) email over 10 weeks; b) email within one week; c) web-site over 10 weeks; d) web-site within one week. Participants were surveyed at baseline, immediately after the course and 6–10 months after completing the course (long-term). Long-term outcomes focused on clinicians' knowledge, confidence and communication practices.Of the 780 clinicians who completed the course, 385 (49%) completed the long-term survey. Completers and non-completers of the long-term survey had similar demographics and professional characteristics at baseline. There were statistically significant improvements from baseline to long-term follow-up in knowledge, confidence and communication practices; these improvements did not differ by curriculum delivery strategy. Knowledge scores improved from 67.7 ± 10.3 at baseline to 78.8 ± 12.3 at long-term follow-up (P < 0.001). Confidence scores improved from 53.7 ± 17.8 at baseline to 66.9 ± 12.0 at long term follow-up (P < 0.001); communication scores improved from 2.6 ± 1.9 at baseline to 3.6 ± 2.1 (P < 0.001) at long-term follow-up.This e- curriculum led to significant and sustained improvements in clinicians' expertise about HDS regardless of the delivery strategy. Future studies should compare the impact of required vs. elective courses and self-reported vs. objective measures of behavior change. Herbs and dietary supplements (HDS) are the most commonly used complementary medical therapies purchased in the United States , leadingWe previously reported the short-term outcomes of our randomized controlled trial (RCT) comparing four different strategies of delivering an on-line course about HDS to diverse clinicians . The shoTo answer questions about the duration of these improvements and whether any differences between delivery strategies would emerge over a longer follow-up, we prospectively followed up study participants from the earlier RCT six to ten months after they'd completed the initial study.We conducted a prospective 6 to 10 month follow-up of an RCT comparing four different strategies for delivering an e-curriculum about herbs and dietary supplements to diverse health professionals . BaselinThe intervention and delivery strategies have been described previously ,10. BrieDuring the second week of October 2005 (approximately ten months after the first group and six months after the second group had completed the course) all original enrollees were asked to complete a final course evaluation. The email request contained a link to a web page which included the exact same questions as the immediate outcome survey to assess long-term retention and maintenance of knowledge, confidence, and communication practices among course enrollees. Non-respondents received up to three email requests to complete the survey before the November 30, 2005 deadline.knowledge scores were the percent of the knowledge questions answered correctly . A confidence scale score with a possible range of 19 to 95 was derived from responses to 19 Likert-type questions such as "I feel confident responding to patients' questions about HDS;" it had a Cronbach alpha reliability statistic of 0.96. Respondents who had seen patients within the past 30 days completed the communications practices scale, with a range of scores from 0 to 10; the Cronbach alpha reliability statistic was 0.84 for baseline and 0.92 for the immediate outcome assessments for this scale.The primary study outcomes have also been described previously ,10. BrieChi-square methods were used for evaluation of associations of categorical variables. For continuous outcomes measures, t tests or analysis of variance (ANOVA) were utilized for normally distributed data, and Mann-Whitney U tests or Kruskal-Wallis tests for non-normally distributed variables. For repeated measures outcomes, paired samples t-tests or Wilcoxon signed rank tests were used, depending on data characteristics. Analyses were performed using SPSS 14.0 .This study was approved as "exempt" as an educational research project by the Wake Forest University School of Medicine Institutional Review Board.Of the 780 participants who completed the course, 385 (49%) completed the long-term follow-up survey six to ten months later Table . CompletIn repeated measures analyses, there were significant, sustained improvements in knowledge, confidence, and communication practices compared to baseline among those who completed questionnaires six to ten months after the course Figure . KnowledChanges in knowledge, confidence, and communications practices at the 6 to 10-month follow-up did not differ by curriculum delivery strategy. Nor were there significant differences in improvement by age, gender, profession, baseline HDS use, or having paid for CE/CME credit (data not shown). Improvements in knowledge and confidence were affected by enrollment period  and whether the participant was a trainee or was in practice Table . CommuniIn this long-term follow-up study, the on-line curriculum resulted in significant and sustained improvements in knowledge, confidence, and communication for diverse clinicians regardless of delivery strategy. Outcomes were only related to semester of enrollment and being a trainee versus a practitioner. Those who took the course in the spring had significantly greater improvements in knowledge and confidence scores than those enrolled in the fall. The differences between fall and spring may be because fall completers had substantially more time to forget learned information than their spring counterparts.Similarly, trainees had significantly greater improvements than practitioners in all three outcomes . These differences may be due to two factors. First, trainees had lower baseline scores than practitioners, allowing for greater opportunity for improvement. Secondly, trainees presumably have fewer experiences and habits to unlearn than practitioners.As expected, knowledge scores decreased from initial follow-up to the long-term follow-up. However, even six to ten months after completing the course, knowledge scores were significantly higher than the baseline scores. This suggests significant knowledge retention of the curriculum material. Confidence and communication scores progressively increased from baseline to the long-term follow-up. These observations are consistent with the hypothesis that as individuals had more opportunity to practice the material they had learned, they could reinforce it and feel increasingly more confident and communicate with patients more comfortably.The results of this study are consistent with previous research that demonstrate the effectiveness of online CME courses ,7,9. AltThis long-term follow-up study has several limitations. First, the sample consisted of self selected enrollees who elected to learn more about HDS, which limits generalizability to elective courses; it is possible that outcomes would differ for participants in required courses. Another limitation is the low response rate to the long-term follow-up. This limits the generalizability of the outcomes to those individuals who have a greater willingness to complete surveys even after the completion of the initially planned study. Those who are willing to complete such voluntary questionnaires  may have been more knowledgeable and confident about their ability to do well. This conjecture is supported by the observation that those who completed the long-term follow-up had slightly, but significantly higher knowledge scores than the non-respondents. Also, the study relied on self-reported changes in confidence and communication, which may overestimate actual behavioral changes ; future studies in this field should corroborate self-report with objective measures of clinician behavior. Finally, we did not collect information on the actual costs of delivering the curriculum through each method because study personnel were engaged in both offering and studying the intervention and did not separately allocate research and education efforts. However, it is our impression that bolus-pull delivery is the least expensive to deliver for participants such as those in this study.Despite these limitations, results from this long-term follow-up study have important implications for professional education and future research. Online case-based curriculum with evidence-based resource links results in significant and sustained improvements in knowledge, confidence, and communication. These improvements are substantial and do not appear to depend on the delivery strategy, at least among motivated clinicians. Therefore, educators can choose to offer on-line Continuing Education (CE) courses with confidence. Because the delivery strategy of online curriculum does not affect attainment of learning goals, the most convenient and low-cost delivery method can be utilized. Future studies about one-line CME should focus on whether required curriculum would have similar outcomes as elective courses as well as developing interventions that would further improve clinicians' communication patterns.The author(s) declare that they have no competing interests.TB drafted the manuscript.KK conceived of the project, designed the survey and revised the manuscript.PG reviewed and edited survey questions and revised the manuscript.CW edited survey questions, analyzed the data and revised the manuscript.The pre-publication history for this paper can be accessed here:


High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) nerve fibers are present in the central nucleus of amygdala (CeA), a brain region implicated in the control of fear-related behavior. This study evaluated PACAPergic modulation of fear responses at the CeA in male Sprague-Dawley rats. PACAP (50–100 pmol) microinfusion via intra-CeA cannulae produced increases in immobility and time the rats spent withdrawn into a corner opposite to the electrified probe compared to controls in the shock-probe fear/defensive burying test. Shock-probe burying and exploration, numbers of shocks received, locomotion distance, and velocity were all reduced by intra-CeA PACAP injection. Further, intra-CeA PACAP effects were manifested only when the animals were challenged by shock, as intra-CeA PACAP injections did not cause significant changes in the behaviors of unshocked rats. Thus, intra-CeA administration of PACAP produces a distinct reorganization of stress-coping behaviors from active (burying) to passive modes, such as withdrawal and immobility. These findings are potentially significant toward enhancing our understanding of the involvement of PACAP and the CeA in the neural basis of fear and anxiety. Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of thesecretin/glucagon/vasoactive intestinal peptide superfamily (Arimura and Shioda ), is a pThe influence of PACAP on brain function has also been investigated in learning and memory studies. For example, icv injection of PACAP facilitated the learning, as well as retrieval, of the passive avoidance response (Telegdy and Kokavszky ). This fThe dense innervation of the amygdala by PACAP nerve fibers clearly indicates that this peptide can exert a strong, but largely unknown, influence on amygdaloid function. The present study, therefore, was designed to explore PACAP's contribution to the regulation of fear behavior, specifically at the level of the central nucleus of amygdala, using the shock-probe fear test. This methodwas originally developed as the defensive burying paradigm by Treit andcoworkers (Treit and Pinel ). Findinn = 58). Animals were pair-housed until cannulaimplantation surgery and then single-housed in polycarbonate cages andmaintained on a 12:12-hour light/dark cycle (lights on at 0700 hours), with food andwater available ad libitum. Rats were handled daily for a week and habituated to the environmental conditions in the testing room. All procedures were carried out in accordance with the University of South Florida Institutional Animal Careand Use Committee guidelines regarding the care and use of experimentalanimals. After an initial 1-week acclimation and handling period, rats wereanesthetized with Ketamine (90 mg/kg) and Xylazine (10 mg/kg) and a 24-gauge stainless steel guide cannula  was unilaterally implanted into the right central nucleus of amygdala under stereotaxic control through a burr hole in theskull. Cannulae were secured to the skull with three stainless steel anchorscrews and cranioplastic cement and temporarily occluded with a dummy cannula.Following surgery, ketoprofen (5 mg/kg) was injected subcutaneously to minimizepost-surgical pain and inflammation. Unilateral cannulation of the rightamygdala , weighing 210–240 upon arrival, were used for the study  was chon et al. ) and uniFor four consecutive days before behavioral experimentation, rats were given mock injections by attaching the guide cannula to an empty injection connector tubing for 2 minutes in their home cage and then the tubing was disconnected and the rats were exposed to the test chamber without the shock probe for 20 minutes. During the pretest session (the day before the experiment) individual rats were given amock injection, and exposed to the test chamber in presence of an unelectrifiedshock probe for 20 minutes. Animal behavior was recorded onto digital video files at these pretest sessions.μL over a 30-second period. The internal cannula remained inserted for 1 minute post injection to prevent backflow and to allow for diffusion of the peptide. The internal cannula was then withdrawn and the animal was placed immediately into theshock-probe fear test chamber.Rats were randomly assigned into either the control or experimental groups prior to behavioral testing and infused with artificial cerebrospinal fluid (aCSF control) or PACAP  using a BAS bee syringe pump system  connected to a 31-gauge internal cannula  with 2.5 mm protrusion below the end of the guide cannula to reach the target region. PACAP was diluted in sterile aCSF containing 0.05% bovine serum albumin  and administered at a dose of 50 or 100 pmol into CeA in a volume of 0.2 The shock-probe fear test apparatus consisted of a 46.6 × 28 × 26 cm Plexiglas chamber, evenly covered with 5 cm of Tek-fresh odor-absorbent bedding material . The shock-probe (8 cm long and 0.8 cm in diameter) was inserted through a hole on one wall of the chamber, 2 cm above the bedding material and helically wrapped with two copper wires through which electric current could be administered. The probe was not electrified until the spontaneously moving rat touched it with its forepaws, at which point the animal received a brief, 2 mA shock from the shock source , remotely activated by an investigator using a footswitch. The 20-minute test began once the rat received its first shock and the probe remained electrified for the remainder of this period. To determine whether intra-CeA infusion of PACAP, without shocks, would produce alterations in behaviors, a group of rats was subjected to intra-CeA aCSF or PACAP injections and 20-minute exposure to the test chamber in the presence of an unelectrified probe. Animal behavior in the test chamber was recorded onto digital video tape and then saved as MPEG2 digital video files for subsequent observation, scoring, and automated analysis.μm thickness were pretreated with 1% sodium borohydride in distilled water followed by .5% hydrogen peroxide in phosphate-buffered saline and then preincubated in 10% normal horse serum. Sections were incubated for 3 days at 4°C in rabbit anti-PACAP serum  diluted at 1:10,000 followed by sequential incubations in biotinylated donkey anti-rabbit IgG  and the ABC elite kit . Immunoreactivity was visualized with diaminobenzidine (DAB) as chromogen. A total of fifteen animals with missed cannula placements were excluded from statistical analysis. Ten additional animals were excluded for other problems such as bleeding, necrosis, or inadequate spread of synthetic PACAP immunoreactivity.Immediately after behavioral testing, animals were deeply anesthetizedwith Nembutal  and perfused transcardially with heparinizedsaline followed by a solution containing 2% paraformaldehyde and 2.5% acroleinin .1 M phosphate buffer. Standard Nissl staining by cresyl violet andimmunolabeling for PACAP were used to evaluate the injection sites. Tissuepreparation for immunohistochemistry was performed according to a previouslydescribed method , an event-recorder software: (a) locomotion parameters: locomotion distance, defined as the total distance moved in the arena during the test period and mean velocity of locomotion, (b) probe exploration, including a stretched/attend-like posture oriented toward the probe or directly touching or sniffing the probe, (c) immobility, defined as crouching, sitting, or standing still on at least three feet, with the body motionless except for small and slow, lateral scanning movements of the head, (d) zonal preference, defined as time spent in the zone either away from the probe or near the probe, generated by dividing the length of the test chamber into two equal halves, (e) burying parameters: latency to bury, defined as the time between the first shock and the first burying event, duration of time spent on burying the probe such as spraying bedding materials toward or over the probe, thefrequency of burying events and the height of bedding material over the probe atthe end of session, (f) numbers of contact-induced shocks, (g) rearing time andnumbers of rearing events, (h) grooming time and numbers of grooming events. Therats' reactivity to shock was scored according to a four-point scale  where “nd Treit ; Treit nd Treit ). Mean sP < .05 was considered to be statistically significant.All data were expressed as means ± SEM and analyzed by ANOVA, followed by post hoc analysis using the student-newman-keuls multiple comparisons test . A probability level of P = .911 and P = .854, resp.).As indicators of baseline behavior, measures of exploration of the unelectrifiedprobe were evaluated from recordings made during pretest sessions  = 11.05; P = .001] and the duration of probe exploration  in shocked animals  = 6.49, P < .05), away zone time;  = 6.52, P < .05)] in rats tested with the electrified shock probe  = 11.46, P < .001] and movement velocity  were significantly reduced by intra-CeA PACAP injection in shocked groups during the 20-minute test session  = 10.49, P = .001] and total time spent on immobility behavior  were significantly increased by intra-CeA PACAP-injection relative to aCSF-injected controls  = 6.55, P < .05], total duration of burying , bury events , and the height of bedding over the probe  as compared to aCSF controls. Probe burying was significantly delayed in PACAP-injectedrats compared to aCSF controls  = 5.12, P < .05]  andvelocity of movement were reduced in intra-CeA PACAP-injected animals testedwith the electrified shock probe. In the 4-point shock-reactivity scale, , producIt has been recognized that the CeA serves as an output nucleus of the amygdala. Its efferent fibers project to the hypothalamus and brainstem areas such as the periaqueductal gray, parabrachial and caudal pontine reticular nuclei and the nucleus of the solitary tract, which are poised to mediate fear-related behaviors, including immobility and autonomic responses . The prInteractions between PACAP and other neuropeptides/neurotransmitters, such as CRH, are quite likely to occur. Based on earlier reports, we hypothesize that the effects of PACAP on fear-related behaviors may be mediated through interaction between PACAP and CRH neurons at hypothalamic, as well as extrahypothalamic, sites , a key structure in the organization ofgoal-oriented behaviors (Haddon and Killcross ). The prIt is important to further note that PACAP injection alone, in the presence of an unelectrified probe, did not have an effect on measures of locomotion, immobility, frequency, and duration of probe exploration and zonal preference as compared to the corresponding aCSF- injected controls. Thus, the potentiation of fear-related behaviors by intra-CeA PACAP injection occurred only in shocked rats. This finding provides strong support for the notion that PACAP is active in modifying CeA functions only when the animal is challenged by an aversive stimulus.In summary, the present study revealssubstantial effects of PACAP microinjection into the CeA on the expression ofbehavioral coping strategies in response to a fear-provoking stimulus. In theshock-probe fear test (defensive burying paradigm), intra-CeA PACAP at 50 or100 pmol doses induced a remarkable shift from active (burying) to passive coping strategies. Infusion of PACAP into CeA resulted in nospecific alterations in locomotion or probe exploration responses when animalswere tested with an unelectrified probe, indicating that PACAP's effects weremanifested only when the animal was challenged by aversive stimuli (shock).Thus, in addition to delineating the PACAPergic modulation of amygdalaphysiology and the neurobiology of fear, these studies may also have importantimplications toward understanding the role of PACAP in the neural basis ofanxiety disorders.


The aim was to assess the value of magnetic resonance mammography (MRM) in the detection of recurrent breast cancer on the prior lumpectomy site in patients with previous conservative surgery and radiotherapy.Between April 1999 and July 2003, 93 consecutive patients with breast cancer treated with conservative surgery and radiotherapy underwent MRM, when a malignant lesion on the site of lumpectomy was suspected by ultrasound and/or mammography. MRM scans were evaluated by morphological and dynamic characteristics. MRM diagnosis was compared with histology or with a 36-month imaging follow-up. Enhancing areas independent of the prior lumpectomy site, incidentally detected during the MRM, were also evaluated.MRM findings were compared with histology in 29 patients and with a 36-month follow-up in 64 patients. MRM showed 90% sensitivity, 91.6% specificity, 56.3% positive predictive value and 98.7% negative predictive value for detection of recurrence on the surgical scar. MRM detected 13 lesions remote from the scar. The overall sensitivity, specificity, positive predictive value and negative predictive value of MRM for detection of breast malignancy were 93.8%, 90%, 62.5% and 98.8%, respectively.MRM is a sensitive method to differentiate recurrence from post-treatment changes at the prior lumpectomy site after conservative surgery and radiation therapy. The high negative predictive value of this technique can avoid unnecessary biopsies or surgical treatments. Recurrence of breast cancer lesions on the surgical scar after conservative surgery and radiation therapy have been reported to occur in at least 1–2% of cases per year . DetectiSeveral recent studies have shown the important role of breast magnetic resonance imaging (MRI) imaging for detection of recurrent lesions in patients treated with conservative surgery (quadrantectomy) and radiation therapy -5. MagneTo the best of our knowledge, no specific data are available on the accuracy of MRM to differentiate recurrent lesions from normal changes of the surgical scar at the site of prior lumpectomy when a multifactorial MRM evaluation protocol is used to classify enhancing lesions.This study was designed to determine whether MRM, performed with a multifactorial evaluation of enhancing lesions, improves the accuracy of diagnosis of recurrence on the surgical scar in patients who underwent quadrantectomy and local radiation therapy. Furthermore, the overall accuracy of contrast-enhanced MRM for the detection of suspicious enhancing lesions, even when not closely related to the surgical scar, has also been assessed in the series reported.The study cohort comprised 93 female patients who underwent breast MRI examination between April 1999 and July 2003 for suspected recurrence on the site of conservative surgery (quadrantectomy) for breast cancer, at least 6 months after the end of radiation therapy.All patients underwent a yearly breast evaluation, performed by the breast radiologist with mammography, associated or not with an ultrasound examination, according to the characteristics of the breast tissue density, and underwent a clinical examination. The clinical examination routinely performed by the breast radiologists after the evaluation of mammographic scans, matched in the same report of mammography, and was also used to decide the level of suspicion.Recurrence was suspected either by ultrasound or by mammography, or by both examinations. When a malignant lesion on the surgical scar was suspected (ultrasound and/or mammographic Breast Imaging Reporting and Data System (BI-RADS) III or BI-RADS IV), the patient underwent a contrast-enhanced breast magnetic resonance mammography (MRM), after signing a proper informed consent and after exclusion of contraindications to exposure to magnetic fields.2. The slice thickness was chosen between 2 mm and 3 mm, depending on the breast size, in order to maintain each sequence time within 90 s.All MRM examinations were performed on a Signa Horizon LX , with a field strength of 1 T. The entire breast was scanned with a two-channel, phased-array, bilateral dedicated coil and the following parameters: Repetition Time = 7 ms, Echo Time = 1.6 ms, flip angle = 10°, TI = 25 ms, receiver bandwidth = 32 kHz, Number of Excitations = 1, matrix = 320 × 320, Field of View = 36 × 18 cmCoronal T1-weighted, fat-suppressed  FSPGR 3D sequences were acquired once before and five times after intravenous contrast injection . The acquisition of dynamic images started 10 s after the contrast injection.To determine the contrast medium uptake, baseline images were subtracted from images obtained after contrast medium injection. All dynamic images were sent to a diagnostic workstation  where the maximum intensity projection and the multiplanar reformation were obtained.One senior radiologist, aware of the time of surgery and the location of the primary breast cancer, and of the mammographic and ultrasonographic findings, reviewed each MRM examination on a diagnostic workstation.Each lesion was retrospectively reviewed and classified by the reader according to a multifactorial evaluation protocol for enhancing lesions connected or not to the surgical scar. Morphological features and time–signal intensity curves of enhancing lesions were classified according to the Fischer multifactorial evaluation  to diffeThe morphological features evaluated and graded were form, margins and enhanced pattern. Form was considered suspicious for malignancy when it was branching or spiculated, whereas it was considered benign when it was rounded. Margins were considered suspicious for malignancy if indistinct, while they were considered benign if well defined. The enhancing pattern was suspicious for malignancy if it was inhomogeneous or ring shaped, whereas it was considered benign if it was homogeneous. Lesions were accordingly graded on a one-point scale for form and for margins , and on a two-point scale for enhancing patterns .According to the same classification of Fischer and colleagues , the dynUsing this multifactorial evaluation protocol, the overall maximum score achievable is 8: a total score lower than 3 points indicated benign lesions, whereas a total score higher than 4 points indicated malignant lesions.The American College of Radiology recently introduced a classification for lesions detected by MRM (adapted from the mammographic BI-RADS classification)  in five The Fischer score was accordingly converted into the MRM-BI-RADS score of the American College of Radiology, as follows: Fischer 0, 1 = BI-RADS I; Fischer 2 = BI-RADS II; Fischer 3 = BI-RADS III; Fischer 4 and Fischer 5 = BI-RADS IV; Fischer 6, Fischer 7 and Fischer 8 = BI-RADS V.MRI findings were compared either with histological findings, if the patient underwent biopsy or surgery, or with a 36-month follow-up with the usual imaging modalities (ultrasound and/or mammography), if the patient did not undergo any biopsy or surgery.True-positive, true-negative, false-positive and false-negative cases were recorded as follows. True-positive cases were patients with histological signs of disease within 3 months after the MRM indicating the lesion as suspicious for recurrence. True-negative cases were patients with negative findings at MRM who had negative findings at ultrasonography and/or mammography during the following 36-month follow-up. False-positive cases were patients who underwent biopsy or surgery because MRM indicated an enhancing lesion as suspicious for malignancy but histological findings and follow-up examinations were negative for malignancies. False-negative cases were patients who underwent biopsy or surgery within 3 months after a negative MRM, because of clinical, ultrasound or mammographic findings suspicious for recurrence. These patients were considered false negative at MRM if histological examination demonstrated the presence of cancer.The sensitivity, specificity, positive predictive value and negative predictive value and the accuracy of MRM in the detection of recurrent disease on the surgical scar were calculated. The same values, with 95% confidence intervals relating to the sensitivity and specificity, were calculated for the lesions detected in areas not related to the scar, in order to define the overall accuracy of MRM in the detection of new breast cancer lesions.Ninety-three patients  were evaluated in the present study: 40 patients had conservative surgery on the right breast, and 53 patients had conservative surgery on the left breast. Follow-up evaluation after surgery included mammography for all 93 patients and ultrasound for 74 patients. Recurrence was suspected by mammography in 27 patients , by ultrasound in 58 patients , and by both examinations in eight patients .The mean time of follow-up after MRM, performed with mammography, ultrasonography or both examinations, was 36 months . The mean age of patients at the time of new diagnosis was 52 years.MRM findings were confirmed by histological findings in 29 patients and by follow-up imaging modalities (ultrasound and/or mammography) in 64 patients.The 93 lesions studied by MRM on the surgical scar, evaluated according to the Fischer criteria, included 59 lesions graded as Fischer 0, two lesions graded as Fischer 1, eight lesions graded as Fischer 2, eight lesions graded as Fischer 3, 12 lesions graded as Fischer 4, no lesions graded as Fischer 5, four lesions graded as Fischer 6, and no lesions graded as Fischer 7 and Fischer 8. The same lesions subsequently classified into the magnetic resonance BI-RADS classification were: 61 lesions graded as BI-RADS I, eight lesions graded as BI-RADS II, eight lesions graded as BI-RADS III, 12 lesions graded as BI-RADS IV, and four lesions graded as BI-RADS V , by mammography alone in two cases (both graded as mammographic BI-RADS IV), and by both examinations in two cases . Histological proof of malignancy was obtained with a vacuum-assisted breast biopsy in four patients, with a tru-cut biopsy in three cases, with fine-needle aspiration cytology in one case, and with fine-needle aspiration cytology + tru-cut in one case. Four out of nine patients underwent a complete mastectomy, 2/9 underwent a second conservative treatment (quadrantectomy), 1/9 died without undergoing further surgery , and the remaining 2/9 underwent surgery in other institutions – we received the pathological report without a detailed description of the type of surgery. Histological types of the true-positive cases were 8/9 infiltrating ductal carcinoma and 1/9 cribriform infiltrating carcinoma. The mean dimension of lesions was 1.96 cm . Six out of these nine lesions were graded as Fischer 4, and the other three lesions were graded as Fischer 6 , by mammography in 24 cases , and by both examinations in five cases . Fifty-nine of these 76 cases were graded as Fischer 0, two lesions were graded as Fischer 1, seven lesions were graded as Fischer 2, and eight lesions were graded as Fischer 3. Among lesions graded as Fischer 3 (probably benign), three cases underwent biopsy while five cases underwent a 6-month MRI and a 12-month MRI as the patients refused to undergo a biopsy.Seven false-positive cases were detected on the surgical scar based on histological findings. Malignancy was suspected by ultrasound alone in five cases , by mammography alone in one case (graded as BI-RADS IV), and by both examinations in one case (graded as mammographic BI-RADS III and ultrasound BI-RADS IV). Six of these false-positive cases were graded at MRM as Fischer 4 Figure  and one We found only one false-negative case at MRM. The ultrasonographic detection of a hypoechoic inhomogeneous lesion of 2 cm in correspondence with the surgical scar (ultrasound BI-RADS IV) gave indication to perform MRM. The subsequent MRM scan depicted the lesion as rounded, with regular shape and homogeneous contrast enhancement. The time–intensity curve showed a moderate initial increase and a steady increase in later sequences Figure . The lesThirteen lesions remote from the surgical scar of prior lumpectomy were detected in seven patients. These lesions were classified as: one Fischer 2, four Fischer 3, three Fischer 4, two Fischer 5, two Fischer 6, and one Fischer 8. The same lesions according to the BI-RADS classes were: 0 cases graded as BI-RADS I, one case graded as BI-RADS II, four cases graded as BI-RADS III, five cases graded as BI-RADS IV, and three cases graded as BI-RADS V.Six true-positive cases were identified, one case graded as Fischer 4, two cases graded as Fischer 5, two cases graded as Fischer 6, and one case graded as Fischer 8. Among these patients, 3/6 had no malignancy on the surgical scar: one of them underwent a controlateral quadrantectomy; the remaining two patients, having enhancing lesions in a different quadrant, underwent a total mastectomy. Three out of six patients had second cancer foci distant from the scar as well as on the scar, confirmed by histology as multifocal carcinomas: in two cases the enhancing lesions were on the same quadrant and the patients underwent a second quadrantectomy, and in one case the lesion was in another quadrant and the patient underwent a total mastectomy  were, respectively, 93.8% , 90% , 98.8%, 62.5% and 90.6%.The long-term survival rate among women who undergo breast-conserving surgery is the same as that among women who undergo radical mastectomy . Breast-The diagnostic evaluation of the treated breast is unfortunately still a challenge because post-treatment changes of breast tissue can show great variability, hiding or mimicking recurrent lesions ,14. ChanAt our institution the usual follow-up of breast cancer patients treated with breast-conserving surgery and irradiation is based on annual imaging evaluation performed with mammography, completed by clinical examination and/or ultrasound examination, according to the density of the breast.Local recurrence is the development of a tumor in the ipsilateral (treated) breast that occurs after treatment of the initial breast cancer . CompariFor these reasons, MRM is considered a useful additional examination in patients with suspicion of recurrence ,8. ProgrIn the present study, the multifactorial evaluation protocol proposed by Fischer and colleagues  was usedThe dynamic enhancement pattern, combined with morphology, on contrast-enhanced MRI of breast masses, allows reproducible lesion characterization , and it The sole false-negative lesion was an infiltrating mucinous carcinoma. The lesion showed well-defined margins, regular shape and homogeneous contrast enhancement, with a moderate initial increase and a steady increase in later sequences (graded as Fischer 2 and BI-RADS II). Other cases of infiltrating mucinous breast cancer not detected by MRM have been described ,28. The MRM in the present study found seven false-positive cases, although all examinations were performed at least 6 months after treatment. Other studies have demonstrated difficulties in differentiating a recurrent tumor from postoperative changes within 6 months after surgery  and withThe high sensitivity in detection of multifocality, in particular for lesions located far from the scar, has influenced the type of repeat surgery (quadrantectomy versus mastectomy). These results are comparable with other studies  performeOf particular relevance is the very high negative predictive value of MRM (98.7%), which indicates a very low likelihood of new malignancy if MRM defines the lesion as benign. These results suggest that lesions graded by MRM as Fischer I–II (BI-RADS I–II) can be safely monitored with the usual yearly follow-up. A repeat MRM examination after 6 months is recommended for lesions graded as Fischer III (BI-RADS III), if there is no clinical suspicion of recurrence before 6 months. For lesions graded higher than Fischer IV (BI-RADS IV–V), further cytological or histological evaluation is mandatory.in situ ductal carcinoma in our series. Ductal carcinoma in situ is considered one of the most common causes of false-negative results at MRM [in situ, and this does probably justify the 93.8% overall sensitivity with only one false-negative case. Another limitation was that imaging examinations were evaluated by a single reviewer. We therefore did not assess the interobserver variability in the use of this multifactorial protocol.One limitation of this study is the lack of s at MRM , and thiThis series demonstrates the high sensitivity and high specificity of MRM in confirming or excluding recurrence at the prior lumpectomy site, after conservative surgery and radiation therapy, when recurrence was already suspected either by mammography  or by ultrasonography. MRM has shown an overall high negative predictive value (98.8%) in the detection of breast cancer, including lesions not related to the surgical scar.Despite the high accuracy of MRM in detecting recurrence on the site of lumpectomy, its cost and low availability limit its use for the routine follow-up of treated patients. In some conditions, however, as in the presence of radiodense breasts and/or structural post-treatment changes, MRM represents an important diagnostic modality in support of the other traditional imaging modalities, and it can be considered conclusive when showing negative findings.BI-RADS = Breast Imaging Reporting and Data System; MRI = magnetic resonance imaging; MRM = magnetic resonance mammography.The authors declare that they have no competing interests.LP contributed to the study concept and design, manuscript drafting and editing, approval for important intellectual concepts, and literature research. GV contributed to the clinical study, data acquisition, literature research, and manuscript drafting and editing. SR contributed to the clinical study, data acquisition, statistical analysis, literature research, and manuscript drafting and editing. LB contributed to the clinical study, data acquisition, literature research and manuscript drafting. DO contributed to the clinical study, data acquisition, data analysis and interpretation, literature research, and manuscript drafting and editing. EC contributed to the clinical study, data acquisition, literature research and manuscript drafting. MB contributed to the study concept and design, manuscript drafting and editing, approval for important intellectual concepts, data analysis and interpretation. All authors approved the final version of the submitted manuscript.


To determine the prevalence of abnormal findings on brain magnetic resonance (MR) examinations in adult participants of brain docking in order to assess its usefulness.We analyzed screening brain MR examinations for 1113 adults  performed during 6-year period from April 1998 to March 2004. All participants voluntarily sought a brain MR examination at their own expense. All subjects were studied using the same 1.0-T MR scanner, on axial T1-weighted spin echo (SE) images, proton-density-weighted and T2-weighted fast SE images, and intracranial MR angiography (MRA). All abnormal findings were classified into three basic categories: (1) findings with no referral necessary; (2) findings not requiring further evaluation, but which needed to be reported to the referring physician; (3) findings requiring further evaluation.Participants with abnormal MR findings requiring further evaluation accounted for 1.3 %, but five of seven suspected intracranial aneurysms were not confirmed by other imaging modalities . No malignant tumors or other life-threatening pathology was detected, and only three participants (0.27 %) with abnormalities underwent surgical treatment. No participant groups were identified from our data as being high risk for MR abnormal findings requiring further evaluation.Brain-docking participants had a variety of abnormalities on brain MR examinations, but only a small percentage of these findings required further evaluation. The usefulness of the brain docking with MRI and MRA has yet to be proven, and at this time we cannot approve this screening procedure. This unique Japanese practice, which may be performed as a part of an annual medical check-up, usually consists of brain magnetic resonance (MR) imaging and MR angiography (MRA) in addition to routine physical and laboratory examinations. It has been believed that brain docking may be beneficial for early diagnosis of some brain disorders, since it is well established that unexpected abnormalities are sometimes detected on brain MR examinations, usually in the setting of an investigation for a reason unrelated to the abnoramlity -4.A screening examination is an examination on individuals who are at risk for a particular disease or condition, but who lack any signs or symptoms of the disease or condition, to determine if the disease or condition is present. It should be done only when clinical studies have demonstrated that screening examinations may do more good than harm . The extIn the current study, we analyzed the results of screening brain MR examinations in order to assess its usefulness. We also attempted to identify participant groups which are high risk for intracranial abnormalities on brain MR examinations, as limiting the screened population to those who are high risk for target disease would be better for cost effectiveness.We included 1113 consecutive adult participants  on whom brain MR examinations were performed during 6-year period from April 1998 to March 2004 as a part of an annual medical check-up in our hospital. All participants voluntarily sought a brain MR examination at their own expense. They usually believed that they were neurologically healthy at the time of the medical check-up, although some participants had a past history of brain infarction, bleeding, trauma or tumor. Some of them had recurrent headache or vertigo/dizziness, but they did not feel that immediate medical advice was required.2 (kg/m2), was used as the index of relative weight. Self-reported data on cigarette smoking were used to classify subjects as nonsmokers, current smokers of 1 to 20 cigarettes per day (moderate smokers), and those smoking more than 20 cigarettes per day (heavy smokers). Regular alcohol consumption was recorded as grams of average absolute ethanol per day, and categorized as follows; nondrinkers, low alcohol intake , and high alcohol intakes . Individuals were considered to have systemic hypertension if their blood pressure readings had repeatedly exceeded 140 mmHg systolic or 90 mmHg diastolic, if they were currently taking antihypertensive therapy, or if they had a past medical history of systemic hypertension. Diabetes mellitus was diagnosed by a fasting serum glucose concentration of > 140 mg/dl or under current treatment for diabetes. High serum cholesterol level was diagnosed by a fasting serum total cholesterol level of > 250 mg/dl, or having a past medical history of high serum cholesterol level.All participants underwent a clinical interview, physical and neurological examinations by a well-experienced neurologist. The presence of recurrent or chronic headache and vertigo/dizziness was always questioned by the neurologist. Since vertigo and dizziness were not always clearly differentiated, these two symptoms were combined in our analyses. Participants' demographic data consisted of age and sex. The body-mass-index (BMI), calculated as weight/heightBrain stroke was defined as a history of physician-diagnosed symptomatic infarction and hemorrhage. Transient ischemic attack (TIA) was not considered a brain stroke. Cardiac diseases were defined as a history of congestive heart failure, myocardial infarction, angina pectoris, left ventricular hypertrophy, or atrial fibrillation; or electrocardiographic evidence of past myocardial infarction, left ventricular hypertrophy, or atrial fibrillation.®, Shimadzu, Kyoto, Japan), including axial T1-weighted spin echo , and proton-density-weighted and T2-weighted fast SE  images. The matrix was 256 * 192 and section thickness was 5 mm with a gap of 2.5 mm for all sequences. MR angiography (MRA) was also performed using time-of-flight (TOF) technique , and 16 projections of the MRA of the circle of Willis were created by a maximum-intensity projection (MIP) algorithm around the head-to-foot axis and right-to-left axis. Contrast-enhanced T1-weighted images were not obtained in any cases.All participants were studied on the same 1.0-T MR scanner (Magnex All MR images were interpreted by a board-certified diagnostic radiologist (YT) with 17-years of experience as a general radiologist, and all MR abnormalities were classified into three basic categories: (1) findings with no referral necessary; (2) findings not requiring further evaluation, but which needed to be reported to the referring physician; (3) findings requiring further evaluation. A well-experienced neurosurgeon also separately interpreted all images. When there was disagreement in image interpretation and neutral consensus could not be reached, the radiologist made the final decision. For MRA image interpretation, only the MIP images were evaluated, and the source images were not used.White-matter signal abnormalities on MR images were considered present if visible as high intensity on proton-density and T2-weighted images, without prominent low intensity on T1-weighted images. According to Fazekas scale , periven®, GE-Yokogawa, Tokyo, Japan) with an intravenous bolus injection (2.5 ml/sec) of contrast material , and surface rendering (SR) images were constructed from 40 or 50 axial images  using an Advantage Workstation ® .When an intracranial aneurysm was suspected on MRA, a three-dimensional computed tomography (3D-CT) examination with contrast material or digital subtraction angiography (DSA) was recommended to confirm the diagnosis. 3D-CT was performed using a single-detector helical CT scanner . For statistical analyses, Student t test and Fisher exact test were used. P values less than 0.05 were considered significant. We set the screening cost for MR examination at US$200, although we know that the cost is much higher in US and EU countries.Nine of 1113 participants had a past history of intracranial disease or trauma . Of 1113 participants, 939 (84.4 %) were categorized was having no abnormal findings or findings with no referral necessary. Abnormal MR findings were demonstrated in 15.6 % of the participants and were classified as follows: 159 (14.3 %) with findings not requiring further evaluation, but needed to be reported to the referring physician; 15 (1.3 %) with findings requiring further evaluation Table . No maliOn MRA, seven intracranial aneurysms and one middle cerebral artery stenosis in eight participants were suspected Table , and allIn our screened population, three participants (0.27%) with abnormalities  underwent surgical treatment.Demographic and clinical data of the participants were summarized in Table The cost for a screening brain MR examination was approximately US$200 in Japan, thus the estimated cost for the identification of one participant with a finding requiring further evaluation (1.3 %) was US$ 14840. When the cases with unconfirmed intracranial aneurysms were excluded, there were nine participants who had findings requiring further evaluation, and the cost for the identification of one participant with a finding requiring further evaluation (0.8 %) rose to US$ 24733.In the current study, a variety of abnormal findings was discovered on screening brain MR examinations, but most of them were not serious. The prevalence of the abnormal findings requiring further evaluation in our study was only 1.3 %, and no malignant tumors or other life-threatening pathology was detected in any participants.Some studies have attempted to determine the prevalence of abnormal findings in screening brain MR examinations, although most of these prior reports have focused on some specific abnormalities, such as silent infarctions , brain tIt was quite difficult to determine the cost effectiveness of brain docking, since a variety of intracranial diseases were discovered. It would be better to calculate quality-adjusted life-years (QALYs) gained by this screening procedure but this was also difficult for the same reason. From our study, however, since no malignant tumors or other life-threatening diseases (except for an aneurysm) were discovered in any participants, the cost-effectiveness of this screening procedure would largely depend on the prevalence of intracranial aneurysms. However, the wisdom of searching for and treating small asymptomatic intracranial aneurysms has been widely questioned.Yoshimoto et al.  reportedNot only MRA but also brain MRI may miss some conditions, and a diagnosis of normal might be inaccurate. However, it was less likely that the field strength of the MR unit and the quality of hardware and software would significantly affect the accuracy of detecting clinically important intracranial abnormalities on MRI, since findings less conspicuous than those detected in our study were unlikely to be clinically important. The lack of clinical follow-up, which may result in the underestimation of false negative findings, was also a shortcoming of our study. Currently, the false negative rate of brain docking is unknown.Another important concern was that the diagnoses of uncertain or unimportant findings may require additional invasive testing or elaborate follow-up, which may place the persons at risk for unexpected health consequences and lead them to unwarranted health expenditures, and will result in increased patient anxiety . In factFrom the view point of cost-effectiveness, it is better to limit the screened population who are at high risk for the target diseases. However, we were not able to identify any groups which were high risk for having intracranial MR abnormalities requiring further evaluation.Brain-docking participants had a variety of abnormalities on brain MR examinations, but the percentage of these findings requiring further evaluation was small. The usefulness of the brain docking with MRI and MRA has yet to be proven, and at this time we cannot approve this screening procedure.The author(s) declare that they have no competing interests.YT participated in the design of the study and performed the statistical analysis. KE and ATT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


XRCC1  and XPD (Lys751Gln) modify individual breast cancer risk, with emphasis on tobacco smoking.It has been suggested that individuals with reduced DNA repair capacities might have increased susceptibility to environmentally induced cancer. In this study, we evaluated if polymorphisms in DNA repair genes The study population consisted of 483 incident breast cancer cases and 482 population controls of Finnish Caucasian origin. The genotypes were determined by PCR-RFLP-based methods. Odds ratio (OR) and confidence intervals (CIs) were calculated by unconditional logistic regression analyses.XRCC1-399 Gln allele  or XPD-751 Gln/Gln genotype  compared to smoking women not carrying these genotypes. The risks were found to be confined to women smoking at least five pack-years; the respective ORs were 4.14 (95% CI 1.66–10.3) and 4.41 (95% CI 1.62–12.0). Moreover, a significant trend of increasing risk with increasing number of the putative at-risk genotypes (p for trend 0.042) was seen. Women with at least two at-risk genotypes had an OR of 1.54 (95% CI 1.00–2.41) compared to women with no at-risk genotypes. Even higher estimates were seen for ever actively smoking women with at least two at-risk genotypes.No statistically significant overall effect in the breast cancer risk was seen for any of the studied polymorphisms. However, a significant increase in breast cancer risk was seen among ever smoking women if they carried at least one XRCC1 and XPD polymorphisms in breast cancer susceptibility, but suggest that they may modify the risk especially among smoking women.Our results do not indicate a major role for  Breast cancer is the most common female cancer among western societies and its incidence increases constantly. Hormonal factors like early age at menarche, later age at menopause, later age at first full term pregnancy, and hormone replacement therapy are known to be the main risk factors for sporadic breast cancer ,2. Also,Unrepaired or misrepaired DNA damage can lead to gene mutations, chromosomal alterations, and genomic instability, known to have a role in cancer initiation. Accordingly, individuals with reduced DNA repair capacities might have an altered cancer risk -11. DuriXRCC1 (X-ray repair cross-complementing group 1) is known to participate in base excision repair (BER) of small lesions such as oxidized or reduced bases, fragmented or nonbulky adducts, and lesions caused by methylating agents . The XRCXRCC1 gene [XRCC1 Arg280His polymorphism lies in between the DNA polymerase β and PARP binding areas [280His variant allele has been suggested to confer increased mutagen sensitivity [Three gene polymorphisms resulting in non-conservative amino acid substitutions  have been identified in the CC1 gene . Two of CC1 gene . The XRCng areas . The 280sitivity  and breasitivity -22.XRCC1 Arg399Gln polymorphism is located in the area coding for a PARP binding site. PARP is a zinc-finger containing enzyme that detects DNA strand breaks [XRCC1-399 Gln variant allele have been shown to have higher levels of DNA adducts [399Gln variant allele and breast cancer risk has been seen in some studies [The d breaks . Carrier adducts  and to b adducts  and toba adducts -27. A po studies ,22,28,29 studies ,29-36.XPD (XP complementation group D) gene encodes a helicase involved in transcription and in the NER pathway by unwinding of double helix at the site of deleterious DNA lesions [XPD gene. Two of these SNPs lead to amino acid change, Asp312Asn in exon 10 and Lys751Gln in exon 23, and are in strong linkage disequilibrium with each other [XPD-751 Gln variant allele has been associated with increased DNA adduct levels [The nucleotide excision repair (NER) pathway repairs a wide variety of DNA damage, including cross-links, oxidative damage and bulky adducts (such as polycyclic aromatic hydrocarbon (PAH)-DNA adducts). The  lesions . Severalch other . The XPDt levels ,40 and st levels ,38, but t levels . It has t levels  and squat levels , and rect levels . Contrast levels ,40,45-47XRCC1 Arg280His, XRCC1 Arg399Gln, and XPD Lys751Gln polymorphisms in breast cancer susceptibility in our Finnish Caucasian study population. We especially aimed at examining the genotype effects in relation to smoking, which is known to cause DNA damage repaired by DNA repair enzymes of the BER and NER pathways. The role of alcohol was also evaluated in this context.In this study, we evaluated the role of Breast cancer cases had been referred by a physician to the Kuopio University Hospital (Finland) during the recruiting period from 1990 to 1995 because of a suspect breast sign or symptom. Women were asked to participate in the study and were interviewed by a trained study nurse before any diagnostic procedures. The study was approved by the Joint Committee of the University of Kuopio and Kuopio University Hospital. Participation was based on written consent.A total of 516 out of 1,919 women were eventually diagnosed with histologically confirmed breast cancer. The details of the study population and recruitment have been described earlier . The conHealthy population controls living in the same area as the cases were a randomly selected group of subjects drawn from the Finnish National Population register. They were initially contacted by a letter explaining the study protocol, and subsequently called up and invited to the hospital to be interviewed. In all, 514 controls were interviewed in parallel with the cases, all of whom agreed to participate in a genetic study. The cooperation rate for the controls was 72%.Blood samples were collected at the time of the interview. Lymphocyte DNA was available for 483 breast cancer patients and 488 controls. Six subjects among controls were excluded, four because of earlier breast cancer diagnosis and two because of non-Finnish origin. The final case group included 483 cases  and 482 controls , all of Finnish Caucasian origin.Detailed data on smoking habits included the amount of cigarettes with or without filter/day, years of smoking, time since quitting smoking, and time (in years) of passive smoking at home and/or at work.XRCC1 Arg280His genotype determination, a 282 base pair (bp) fragment was amplified using the primers described earlier [RsaI, the 280Arg wild-type allele was revealed by two 141 bp fragments, while an intact 282 bp fragment indicated the presence of the 280His variant allele.Lymphocyte DNA (100 ng) extracted by standard methods was used as a template in PCR-based restriction fragment length polymorphism (RFLP) assays. For the  earlier . After dXRCC1 Arg399Gln genotype analysis, a 615 bp fragment amplified using the primers described in [MspI restriction enzyme; the XRCC1-399 Arg wild-type allele was revealed by the presence of 221 bp and 374 bp fragments while the 399Gln variant allele remained intact (615 bp).In the ribed in  was digeXPD Lys751Gln genotypes were determined by amplifying a 324 bp fragment with the primers described in [PstI; the amplicon from the 751Lys wild-type allele was cut into 220 bp and 104 bp fragments while the amplicon from the 751Gln variant allele was cut into 157 bp, 104 bp and 63 bp fragments.The ribed in . The resXRCC1 Arg280His genotype could not be determined for three cases and three controls, the Arg399Gln genotype for four cases and four controls, and the XPD Lys751Gln genotype for two cases and two controls.The genotype analyses were performed unaware of the case-control status. Two positive controls with known genotype and two negative controls were used within each PCR amplification batch, and two independent researchers interpreted the gel images to ensure the validity of genotyping for each polymorphism. The PCR for samples with divergent results was repeated and an additional 10% of all samples were reanalysed for each polymorphism for quality control. No discrepancies were found in the replicate tests. The Association between genotypes and risk of breast cancer were evaluated by unconditional logistic regression to calculate multivariate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) using SPSS version 11.5 . Calendar age, age at menarche, age at first full term pregnancy, number of children, history of benign breast diseases, first degree  family history of breast cancer, waist-to-hip ratio and use of alcohol and smoking were used as adjusting variables in all analyses. Subjects with missing values in any of the adjusting variables were excluded from the analysis. When the adjusted ORs differed significantly from the unadjusted ORs, both are shown.Women who had smoked daily for longer than three months were considered as smokers. Never smokers were categorized as those who had never been exposed to either passive or active smoking, and as those exposed to passive smoking only. Ever smokers were further categorized according to the approximate median smoking years (15 years), daily consumption (10 cigarettes) and pack-years of smoking (5 pack-years) in the control population. Pack-years of cigarettes were calculated as the number of packs (20 cigarettes/pack) smoked per day multiplied by years of smoking.XRCC1-280 Arg/Arg and XRCC1-399 Arg/Arg genotypes were used as reference categories for all separate analyses. As the frequency of the XRCC1-280 His variant allele was low, the ORs were calculated for the combined XRCC1-280 His allele containing genotypes in all analyses. Similarly, in the stratified analyses, the XRCC1-399 Gln allele containing genotypes were combined to increase statistical power. In contrast, a recessive model was used for the XPD Lys751Gln polymorphism based on the observed genotype distributions and some earlier publications [XPD-751 Lys/Gln genotype was combined with the wild-type Lys/Lys genotype to serve as a reference category. Possible gene environment interactions were assessed using stratified analyses and interaction terms. Variables of interest were smoking and use of alcohol. Tests for interactions were assessed by the likelihood ratio test to compare goodness of fit of the model with the interaction term, to the reduced model including the main effect variable (genotype) and the main adjusting variables. All reported p-values are two-sided. No attempt was made to adjust for multiple comparisons.Based on the prior knowledge of the functional significance or epidemiological evidence, the ications ,51. Therp = 0.10), or in smoking years .Characteristics of the study population are shown in Table XRCC1-280 His, XRCC1-399 Gln, and XPD-751 Gln variant alleles in controls were 0.08, 0.27, and 0.43, respectively. All the genotype distributions in the control population conformed to Hardy-Weinberg equilibrium . No statistically significant differences were seen in the frequency of these genotypes between cases and controls  compared to those with the Arg/Arg genotype. No increase in risk was seen for lower stage (I or II) breast cancer . A similar tendency of increased risk for advanced stage cancer was seen for subjects with the XRCC1-280 Arg/His or His/His genotype  compared to subjects with the Arg/Arg genotype. Similarly, women with the XRCC1-399 Gln/Gln genotype presented with a significantly increased risk for grade II and grade III tumours  when compared to those with the Arg/Arg genotype, while no increase was seen for grade I tumours . Moreover, women who carried the XRCC1-399 Gln/Gln genotype and were diagnosed with early stage (I or II) breast cancer had tumours of higher grade (II or III) marginally  more often than those with the Arg/Arg genotype, 86.1% (31/36) versus 72.5 % (137/189), respectively.When subjects were studied by the stage of the disease at the time of diagnosis, a significant increase for advanced stage (III or IV) breast cancer was seen for women with the XRCC1-399 genotypes and breast cancer risk was studied according to smoking habits, increased breast cancer risk with dose-response was seen among women who had ever smoked actively and carried either one  or two  XRCC1-399 Gln variant alleles compared to those carrying the Arg/Arg genotype (p for interaction between smoking habits and XRCC1-399 genotype 0.025) .When the association between XRCC1-399 Gln allele , or the XPD-751 Gln/Gln genotype  compared to similarly smoking women without these genotypes  or by smoking years . The ORs were 5.32 (95% CI 1.97–14.4) for women smoking ≥10 cigarettes/day and carrying at least one XRCC1-399 Gln allele, and 4.03 (95% CI 1.40–11.6) for women who had smoked ≥15 years and carried at least one XRCC1-399 Gln allele, compared to women with similar smoking habits but with the XRCC1-399 Arg/Arg genotype. For the carriers of the XPD-751 Gln/Gln genotype, a similar increase in the risk of breast cancer was seen for women smoking ≥10 cigarettes/day  while no statistically significant increase was seen by smoking years , compared to women smoking the same amount, but not carrying the homozygous variant XPD-751 Gln/Gln genotype.When ever smoking women were further stratified by pack-years smoked , the increase in risk was seen to be confined to those who had smoked over five pack-years and carried at least one XPD-751 Gln/Gln genotype were at 3.18-fold (95% CI 1.34–7.57) increased risk of breast cancer compared to similarly drinking women carrying the other genotypes (p for interaction 0.026). No interaction was found between XRCC1-280 or XRCC1-399 genotypes and current use of alcohol (data not shown).When stratified by current use of alcohol, women who reported using alcohol weekly to daily and carried the XRCC1-280, XRCC1-399, and XPD-751 genotypes was studied, a statistically significant increase in the risk of breast cancer was seen for subjects carrying two at-risk genotypes of these genes  compared to subjects with the wild-type genotypes for all three polymorphic sites . A trend of increasing risk with increasing number of at-risk genotypes was seen (p for trend 0.042). However, this estimate did not reach statistical significance , possibly due to the low number of subjects with all the three at-risk genotypes (four cases and one control). When the combined effects were studied among ever active smokers, women who carried any two at-risk genotypes were at remarkably increased risk of breast cancer; the adjusted OR was 10.7 (95% CI 3.62–31.6) compared to those without these genotypes . When the combined effect was calculated for the number of at risk alleles , a similar increase in the risk was seen; subjects with three at-risk alleles had an OR of 1.72  among all women, and OR 4.62  among ever actively smoking women compared to women with no at-risk alleles. Only four cases and one control carried simultaneously four at-risk alleles, and none more than four (of the six).When the joint effect of the XRCC1 Arg280His, XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms in relation to breast cancer risk in a Finnish study population. As the products of these genes act in BER and NER pathways, and as some evidence exists on the association of these polymorphisms with smoking-related cancers [XPD-751 Gln/Gln genotype and breast cancer risk in smoking women [In this study, we examined the role of  cancers ,42,43,52ng women .Arg280His and Arg399Gln in the coding region of the XRCC1 gene were recently predicted to be 'possibly damaging' to XRCC1 function based on the conservation of the sequences in mammalian orthologues [XRCC1-399 Gln allele was somewhat higher among the present cases compared to controls, leading to a tendency of increased breast cancer risk. A similar effect has been reported in studies among Korean [The two polymorphisms hologues . In agreg Korean , Indian g Korean , and Afrg Korean  women. Ng Korean , in agreg Korean -33. Moreg Korean , French g Korean , Canadiag Korean , Turkishg Korean  and Danig Korean  women.XRCC1 Arg399Gln polymorphisms, the Arg280His polymorphism did not significantly modify breast cancer risk in the present study. Similarly, no association was seen for Indian women [XRCC1-280 Arg/His genotype [280His allele is low (0.08) among Caucasians, including Finns. Consequently, even though having almost twice the size of the French study, the power of our study to detect an OR of 1.5 at a 0.05 significance level was only 67% for the XRCC1-280 His allele containing genotypes. As the XRCC1-194 Trp variant allele is even less frequent among Finns (approximately 0.03) [In contrast to the an women  or for Uan women . On the genotype . One realy 0.03)  comparedly 0.03) , we deciXPD Lys751Gln polymorphism has been suggested to be the most important functional polymorphism in the gene due to major change in the electronic configuration of the respective amino acid in an important interaction domain of the protein [XPD Lys751Gln genotypes. This was in agreement with the other five studies on the XPD Lys751Gln polymorphism and breast cancer risk including one in Finnish [XPD-751 Gln allele and breast cancer risk was seen in a recent study among American women [XPD Asp312Asn polymorphism was recently shown to be associated with breast cancer risk in a German population [XPD Asp312Asn polymorphism as it has been shown to be strictly linked with the Lys751Gln polymorphism [The  protein . However Finnish , one in  Finnish , one in  Finnish , and two Finnish ,40. In can women . Moreovepulation . We decimorphism .XRCC1-399 Gln allele posed an elevated risk for more advanced stage breast cancer. A similar tendency of increased risk for more advanced stage breast cancer was also seen for the Arg280His polymorphism. It can be hypothesized that defective DNA repair leads to more aggressive and, therefore, more advanced tumours at the time of diagnosis. This was also supported by the association of the XRCC1-399 Gln allele with higher grade tumours. However, as earlier studies on breast cancer have not evaluated the genotype effects by the stage of the disease or tumour grade, these findings remain to be confirmed in future studies.When the present study subjects were stratified by stage of disease, the XRCC1 Arg399Gln (p = 0.025) or XPD Lys751Gln (p = 0.011) genotypes. Subjects with the variant Gln/Gln genotypes were at increased risk of developing breast cancer if they had ever smoked. Furthermore, a gene-dosage effect was seen for the XRCC1 Arg399Gln genotype; the increased risk was higher for subjects carrying two XRCC1-399 Gln alleles. In contrast, the effect of the XPD-751 Gln allele seemed to be recessive. When ever smokers were further stratified by the amount of smoking, the risk estimates remained statistically significant among those who reported having smoked more than five pack-years.Smoking alone did not significantly affect breast cancer risk in the present study. This is in agreement with the majority of epidemiological studies on smoking and breast cancer risk, as well as with a recent report of the Collaborative Group on Hormonal Factors in Breast cancer, which concluded that cigarette smoking has little or no effect on the risk of developing breast cancer . There aXRCC1 Arg399Gln and/or XPD Lys751Gln genotypes, smoking, and breast cancer risk are biologically plausible. Cigarette smoke is a rich source of chemical carcinogens and ROS [XRCC1-399 Gln/Gln genotype has been found to be related to increased sister chromatid exchange frequencies among smokers [399Gln allele who have smoked ≥10 cigarettes/day [XRCC1-399 Gln allele containing genotypes compared to cells homozygous for the Arg allele [XRCC1-399 Gln allele is associated with increased risk of smoking-dependent cancers. In agreement with this, a higher risk of lung cancer has been reported for the carriers of the XRCC1 Gln/Gln genotype [XRCC1-399 Gln allele was significantly associated with detectable PAH-adducts only in never smokers [XRCC1-399 polymorphism and smoking, but in contrast to our study, the highest risk was seen for subjects who were homozygous for the XRCC1-399 Arg allele [XRCC1-399 genotypes [The associations found between the  smokers . Similarttes/day , and higg allele . It can genotype , althouggenotype ,55. Brea smokers . They spg allele . Unfortuenotypes ,30,31,36XPD-751 Gln allele has been associated with increased DNA adduct levels in never smokers [XPD-751 Gln/Gln genotype and increased lung cancer risk [XPD-751 Gln/Gln genotype [In addition to ROS, tobacco smoke includes chemical carcinogens, such as polycyclic aromatic hydrocarbons, aromatic amines and tobacco-specific nitrosamines, that can produce bulky DNA adducts, which are repaired through the NER pathway. There is evidence that tobacco smoke constituents can reach breast tissue , and hig smokers  and in t smokers , as well smokers ,38. A recer risk . This wacer risk . In accogenotype . SimilarXPD-751 Gln/Gln genotype and used alcohol weekly or daily. No earlier studies on the XPD genotype and breast cancer have reported an association with the use of alcohol. However, a 2.59-fold (95% CI 1.25–5.34) risk was seen for squamous cell carcinoma of head and neck among current drinkers carrying the XPD-751 Gln/Gln genotype compared to those drinking the same amount but with the other genotypes [Alcohol is a known risk factor for breast cancer; increased estrogen and androgen levels have been observed in alcohol consuming women . Alcoholenotypes .XRCC1-399 and XPD-751 genotypes while the XRCC1-280 genotype was a minor contributor, possibly partly due to the very low frequency of the XRCC1-280His variant alleles. When the combined effect was studied according to the number of at-risk alleles, subjects with any three variant alleles were found to be at increased risk. There was no subject who simultaneously carried more than four at-risk alleles in these three polymorphic sites.When the gene-gene interactions were studied, clear combined effects were seen, especially among ever smoking women; smoking women with any two at-risk genotypes were at remarkably increased risk of breast cancer  compared to smoking women without these genotypes. These effects were mainly due to It should be noted that even if our study involved a reasonable number of study subjects, the numbers in some subgroup analyses were small, and the statistical power to detect significant point-estimates is, therefore, low. Furthermore, due to multiple comparisons performed in the study, the possibility of chance findings should be borne in mind. However, in addition to the plausibility of the findings, the dose-response seen by the number of at-risk alleles or genotypes and amounts smoked support the causality of the findings.In case-control studies, the possibility of selection and recall bias should be considered. However, the detailed data about smoking habits and other life style factors were collected prior to diagnosis, and the exposure to passive smoking, quite often disregarded, was taken into account. It is also unlikely that selection would be related to an individual's genotype. As the statistical analyses were performed within exposure groups and not between them, the possibility of biased estimates is further reduced.RAI, ASE-1 and ERCC1, in the same chromosomal region 19q13.2-3 define a high-risk haplotype that poses women under 55 years to a significantly increased breast cancer risk [XRCC1 or XPD genotypes but the effects were not studied by smoking habits. In our study, no difference in the risk estimates was seen by age.Even though the present findings are biologically plausible, the possibility of the association with another gene polymorphism in the same region should be considered. Accordingly, it was recently reported that three markers, cer risk . In thatXRCC1 Arg194Trp polymorphism, association has been seen with increased mutagen sensitivity [OGG1, encoding an enzyme important in the BER pathway, as well as numerous other genes encoding for possibly important repair enzymes in the NER pathway, namely XPA, XPC, XPF and XPG.Finally, it should be noted that not all the known polymorphisms in the BER and NER pathways were analysed in this study. For the sitivity , risk ofsitivity , and it XRCC1 and XPD genotypes and breast cancer risk in Finnish smoking women, especially when enzymes in both DNA repair pathways were defective. This finding is of particular interest as smoking, being the main preventable risk factor for cancer, is constantly increasing among female adolescents in many countries, including Finland. However, it should be borne in mind that the two studied DNA repair genes account for only a small part of the known genetic variations in these pathways and, therefore, most probably do not explain the whole picture, but should be considered in the context of other possible genes. These results, together with our earlier results concerning polymorphisms in estrogen metabolizing enzymes [We found a statistically significant association between the  enzymes , supportBER = base excision repair; bp = base pair; CI = confidence interval; NER, nucleotide excision repair; OR = odds ratio; PARP = poly(ADP-ribose)polymerase; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism; ROS = reactive oxygen species.The authors declare that they have no competing interests.KM was responsible for supervising the laboratory work, for performing statistical analyses, and for manuscript preparation. VK participated in the design and coordination of the study, helped to draft the manuscript and acts as the principal clinician in the study group. PS, PS and LH carried out the genotyping analyses and drafted the manuscript. ME participated in the design and coordination of the study and was clinical coordinator of collection of study patient material. VMK performed histopathological analyses of all cases, and helped to draft the manuscript. MU has been involved in the planning of the study, collection of clinical data, in particular with respect to the genetic part of the study, and he has commented on the manuscript in the writing phase. AH participated in the design and coordination of the molecular epidemiology part of the study, was responsible for acquisition of funding for the molecular epidemiology part, and was involved in the preparation of the manuscript. All authors read and approved the final manuscript.


Falciparum malaria is an important cause of maternal, perinatal and neonatal morbidity in high transmission settings in Sub-Saharan Africa. Intermittent preventive treatment with sulphadoxine-pyrimethamine (SP-IPT) has proven efficacious in reducing the burden of pregnancy-associated malaria but increasing levels of parasite resistance mean that the benefits of national SP-IPT programmes may soon be seriously undermined in much of the region. Hence, there is an urgent need to develop alternative drug regimens for IPT in pregnancy. This paper reviews published safety and efficacy data on various antimalarials and proposes several candidate combination regimens for assessment in phase II/III clinical trials. Falciparum malaria is an important cause of maternal anaemia, intra-uterine growth retardation, intrauterine death, stillbirth, premature delivery, low birth weight (LBW), perinatal and neonatal morbidity and mortality -5 and povs mefloquine[vs SP plus artesunate[vs SP plus azithromycin[In areas of high or moderate transmission, most malaria infections in pregnant women are asymptomatic and infected women do not present for treatment. In such areas, the World Health Organization recommends a combination of interventions to prevent malaria in pregnancy including insecticide-treated bednets (ITNs), intermittent preventive treatment in pregnancy (IPTp) and effective case management and treatment,17. A smefloquine; SP alonrtesunate; and SP thromycin respectithromycin, but conthromycin,49 and wthromycin, are likThe future of SP for intermittent preventive treatment of malaria in pregnancy is tenuous, particularly in East and Southern Africa, and there are insufficient, reliable data on the safety and efficacy of alternative antimalarials for the prevention and treatment of malaria in pregnancy. Hence, ©, Malorone©, Coartem©, Lapdap©, truncated forms and associated terms  were used where it was felt appropriate. Manufacturers' information sheets, guidelines and reports from the World Health Organization (WHO), Medicines for Malaria Venture (MMV), Gates Malaria Partnership (GMP) and other leading organizations in the field were also scrutinized.A two-stage, systematic search was carried out in PubMed and EMBASE using combinations of the keywords: pregnancy, malaria, antenatal, prophylaxis, intermittent, prevention, clinical trial. Alternative spellings and truncated forms were also used. ArticleMefloquine hydrochloride is a 4-quinolinemethanol derivative with a chemical structure related to that of quinine. In healthy, non-pregnant human volunteers, peak plasma concentrations of around 250 μg/L are observed 6–24 h following a single 250 mg oral administration. MefloquMefloquine has been used alone for prophylaxis in the pre-conception period and in all trimesters of pregnancy-39,41,60Mefloquine, at standard prophylactic doses of 250 mg/week appears safe and effective when taken before conception and during all trimesters of pregnancy,39,41. IHowever, an observational, non-comparative study among seventy-two, female US soldiers stationed in Somalia reported a risk of spontaneous abortion of 16.7% (12/72) among women who took a standard dose mefloquine prophylaxis in the first trimester. One molThere is good evidence that mefloquine is safe and effective in the treatment of falciparum malaria in pregnancy. A low dose mefloquine treatment regimen  given to 33 women in the second or third trimester in Zaria, Nigeria was well tolerated, safe and effective with no adverse maternal or foetal outcomes reported. In a laIn contrast, a retrospective study in Thailand reported a borderline statistical association between mefloquine treatment during the second and third trimester of pregnancy and excess odds of stillbirth compared to women treated either with quinine or other antimalarials. Among 2Several studies were identified which looked at the safety and efficacy of mefloquine in combination with other antimalarials. All were conducted among women in the second or third trimester. A randomized, controlled trial with 40 subjects in Nigeria compared artemether (3.2 mg/kg intramuscularly once at entry) plus low dose mefloquine  versus artemether alone (3.2 mg/kg at entry followed by 1.6 mg/kg/day for 4 days) for the treatment of multidrug-resistant falciparum malaria. No seriA randomized, open-label clinical trial among 108 second and third trimester pregnant women in northwest Thailand compared a three-day mefloquine-artesunate regimen  versus seven days treatment with quinine  for multidrug-resistant falciparum malaria. A numbeA phase III equivalence trial comparing intermittent SP and mefloquine alone for the prevention of malaria-related morbidity in pregnancy is currently underway in Benin.Azithromycin is a macrolide antibiotic derived from erythromycin which has an elimination half-life of 68 h in healthy human volunteers and is excreted largely unchanged in the bile following hepatic clearance. In a prAzithromycin has been used extensively to treat sexually-transmitted and other bacterial infections during pregnancy in total treatment doses of up to 1–2 g and is considered a safe alternative for the treatment of chlamydia, mycoplasma and group B streptococci in pregnant women who are allergic to beta-lactam antibiotics -73.P. falciparum in vitro[Azithromycin is not yet licensed for use as an antimalarial agent but has shown promising activity against  in vitro,75, in t in vitro and in r in vitro-79. A ph in vitro. No seri in vitro. The effTo date, no clinical trials have been conducted among pregnant women but a phase III clinical trial of intermittent SP plus azithromycin in pregnancy versus intermittent SP alone is currently underway in Malawi.The elimination half-life of proguanil is 12 – 21 h in healthy adult volunteers and around 40–60% of the drug is eliminated by urinary excretion. Proguanil is metabolized by a cytochrome P450 isoenzyme to an active metabolite (cycloguanil), responsible for antimalarial activity. Late prProguanil has been used extensively for malaria chemoprophylaxis in travellers, including pregnant women and children and is considered one of the safest antimalarials currently available,42,58,82© (GlaxoSmithKline). Atovaquone has an elimination half life of 2–3 days in adult volunteers and around 95% is excreted unchanged in the bile[Proguanil has also been used in fixed combination with atovaquone (100 mg/250 mg respectively) in the commercially available preparation Malarone the bile. A rando the bile. In an o the bile. In a sm the bile. Median A randomized open-label clinical trial among 81 second and third trimester pregnant women in Thailand compared quinine and artesunate-atovaquone-proguanil (AAP) for the treatment of uncomplicated falciparum malaria and found that women treated with AAP had shorter fever and parasite clearance times and lower treatment failure rates (5% vs. 37%) compared to those in the quinine group. The AAP®) has a similar mode of action and pharmacokinetic profile to proguanil but has a slightly longer elimination half-life (around 32 h)[© (GlaxoSmithKline). Despite evidence from a number of clinical trials of the safety and efficacy of this combination in the treatment of uncomplicated malaria in children[Chlorproguanil (Lapudrineund 32 h). The dru children,87,88 an children, a recen children conclude children and in M children. A combi children and a mu children.Artemisia annua), a herb which has been used in traditional Chinese medicine for over 2000 years[Artemisinin compounds  are derived from sweet wormwood  have been reported,95. TherFollowing two expert consultations in 2002, WHO recommended that artemisinins should not be used in the first trimester except in life-threatening situations where other antimalarials were not suitable, should be used with caution in the second and third trimesters and that further research be conducted to establish the pharmacokinetics of artemisinins during pregnancy, mechanism of toxicity and the relevance of animal toxicity data to humans. Pharmac©/Riamet©), which is available at subsidized cost to malaria-endemic countries following a special pricing agreement established in 2001 between WHO and the manufacturer, Novartis[The only fixed-dose artemisinin combination therapy (ACT) currently available is artemether-lumefantrine  was developed through an international public-private partnership between Holleykin Pharmaceutical, China; Sigma-Tau Industries, Italy; the University of Oxford, UK; and the Medicines for Malaria Venture (MMV)[Dihydroartemisinin is a derivative of artemisinin and the principal active metabolite of artesunate and artemether with an elimination half-life of 40–60 minutes. Piperaqnd others. Dihydroure (MMV). DP has ure (MMV),105-107.ure (MMV) and the © (Sanofi-Aventis). Clinical trials in Ghana[A variety of new ACTs are currently being evaluated in phase II and III clinical trials in children and non-pregnant adults. Amodiaquine plus artesunate is being developed in fixed dose co-formulation as Coarsucam in Ghana and Tanz in Ghana have rep in Ghana. Given u in Ghana,49, it i in Ghana-113.The efficacy of dihydroartemisinin-mefloquine (DHA-MQ) compared to DHA alone and chloroquine alone for the treatment of uncomplicated falciparum malaria was recently described in a small study among 75 children aged 2–13 years conducted in Nigeria but no pPyronaridine, a Mannich-base schizontocide structurally related to the aminoacridine drug quinacrine, which exerts its antimalarial action by forming complexes with haematin, is beinChlorproguanil-dapsone-artesunate (CDA), artesunate-atovaquone-proguanil (AAP) and artesunate-SP are other promising ACTs under development, as described above.P. falciparum-infected erythrocytes following their adherence to placental chondroitin sulphate A (CSA)[The pharmacokinetics, dosing regimen, efficacy and safety profile of antimalarials currently used to treat and prevent falciparum malaria in adults and children are well-described. This is not the case in pregnancy where the limited data available in each of these areas makes it difficult to predict which drugs are likely to be the best candidates for treating and preventing infection. Many antimalarials are contra-indicated in pregnancy , whilst the safety of several promising candidates remains unclear. In malae A (CSA) and hyale A (CSA), which ee A (CSA). Pregnane A (CSA)-123, whie A (CSA),8. Physie A (CSA). For exae A (CSA),101. Fore A (CSA),55. Simie A (CSA). Given te A (CSA), it seemHow then to decide which antimalarials are likely to be effective for IPT in pregnancy and which combinations should be prioritized for investigation in clinical trials Table ? If the ©)[Based on these criteria, dihydroartemisinin-piperaquine appears an appropriate candidate for IPTp and should be considered for phase II/III clinical trials in second and third trimester pregnant women. The combination is relatively inexpensive ; combineArtemether-lumefantrine may also be a suitable candidate: a relatively inexpensive, fixed co-formulation is available and the partner drugs have different molecular targets. The 'prophylactic effect' conferred by lumefantrine may not be sufficient however, given its relatively short half-life compared to sulphadoxine, pyrimethamine, mefloquine or piperaquine.Mefloquine in combination with artemether or artesunate has the advantage of a similar elimination profile to dihydroartemisnin-piperaquine and the safety of such combinations in the treatment of pregnancy-associated malaria is reasonably well-established. Indeed, the extensive data available on mefloquine use in pregnancy compared to piperaquine suggest that dihydroartemisinin-mefloquine could enter phase III trials ahead of dihydroartemisinin-piperaquine. Cost and the lack of fixed dose co-formulations  are disadvantages. Combining mefloquine with the relatively long acting azithromycin is another option and likely to have additional health benefits by reducing maternal and perinatal infections, which may offset cost concerns. Sexually-transmitted infections (STIs) are common in women of childbearing age in Africa and may go undiagnosed and untreated during pregnancy. MonthlyPatterns of SP resistance vary across sub-Saharan Africa so that new artemisinin-containing combinations with SP may be appropriate in West Africa, given that these would fulfil many of the criteria above. Such combinations are, however, unlikely to succeed in East and Southern Africa, given prevailing levels of resistance.Plasmodium vivax infection also needs to be taken into consideration[P. vivax infection[Are different types of drug combination needed in different areas of malaria transmission? Evidence for the effectiveness of IPTp comes primarily from studies conducted in areas of intense perennial transmission , but there may be a case for evaluating IPTp in areas of low transmission . The World Health Organization recommends that IPTp be an integral part of national malaria control programmes in high/medium perennial or seasonal transmission areas but caution that there is insufficient evidence for introducing IPTp in low transmission settings where the principal focus should be on effective case management and insecticide-treated bednets. In areaideration. In addiinfection,131.AV participated in the conception and design of the review, coordinated the literature review and drafted the manuscript.LV participated in the conception and design of the review, conducted the literature search, obtained full text articles and helped draft the manuscript.JC participated in the design of the review and helped draft the manuscript.BG participated in the conception and design of the review, provided additional information e.g. conference proceedings and helped draft the manuscript.DC conceived the study, participated in its design and coordination and helped draft the manuscript.All authors have read and approved the final manuscript.The author(s) declare that they have no competing interests.