Assessment of epidermal growth factor receptor -LRB- EGFR -RRB- expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray Metastatic colorectal carcinomas -LRB- CRC -RRB- resistant to chemotherapy may benefit from targeting monoclonal therapy cetuximab when they express the epidermal growth factor receptor -LRB- EGFR -RRB- . Because of its clinical implications , we studied EGFR expression by immunohistochemistry on tissue sections of primary CRC -LRB- n = 32 -RRB- and their related metastases -LRB- n = 53 -RRB- . A tissue microarray -LRB- TMA -RRB- was generated from the same paraffin blocks to determine whether this technique could be used for EGFR screening in CRC . On tissue sections , 84 % of the primary CRC and 94 % of the metastases were EGFR-positive . When matched , they showed a concordant EGFR-positive status in 78 % of the cases . Moreover , staining intensity and extent of EGFR-positive cells in the primary CRC correlated with those observed in the synchronous metastases . On TMA , 65 % of the primary CRC , 66 % of the metastases , and 43 % of the matched primary CRC metastases were EGFR-positive . There was no concordant EGFR status between the primary and the metastatic sites . A strong discrepancy of EGFR status was noted between TMA and tissue sections . In conclusion , EGFR expression measured in tissue sections from primary CRC and their related metastases was found to be similar and frequent , but it was significantly underestimated by the TMA technique . Introduction Epidermal growth factor receptor -LRB- EGFR -RRB- , also called HER1 , is a member of the transmembrane tyrosine kinase receptor family . In normal and malignant cells , the activation of EGFR receptor cascades has multiple consequences , such as cell growth , differentiation , and proliferation -LSB- 10 -RSB- . The EGFR signaling pathway may also promote malignant transformation , angiogenesis , and metastatic dissemination -LSB- 10 -RSB- . To block the activation of this receptor , targeted therapies have been developed , representing a new and promising strategy for cancer management -LSB- 15 -RSB- . Thus , antibodies directed against EGFR have recently been integrated into the treatment of metastatic colorectal carcinomas -LRB- CRC -RRB- resistant to chemotherapy -LSB- 3 , 23 -RSB- . Such a strategy requires an EGFR immunohistochemical assessment by the pathologist to ensure that the targeted receptor is present in the tumor . Cancer patients included in these protocols are not always treated in the same institute for their primary disease and for their metastatic disease . Moreover , metastases are not always available for histology . It is thus possible that EGFR expression be evaluated in only one of the tumor locations -LRB- i.e. , primary or metastatic -RRB- . However , few and controversial data are available concerning the EGFR status in its primary site and the related metastatic sites -LSB- 6 , 11 , 13 , 24 -RSB- . The first goal of our study was to analyze whether EFGR expression in colorectal primary tumors and their metastatic sites was similar or not . To answer this question , formalin-fixed and paraffin-embedded primary CRC from 32 patients and their related synchronous metastases -LRB- n = 45 -RRB- were examined using a standardized EGFR immunohistochemical procedure . Additionally , metachronous metastases -LRB- n = 8 -RRB- from four out of 32 patients were also processed for EGFR immunohistochemistry . The second goal of this study was to evaluate EGFR expression on a tissue microarray -LRB- TMA -RRB- generated from the same paraffin blocks and to compare these results with those obtained by using the whole tissue sections . This comparison was carried out to determine whether the increasingly used TMA technology is a reliable tool for high-throughput EGFR screening in CRC -LSB- 17 , 26 -RSB- . Materials and methods Patient characteristics Clinical and histopathological characteristics of the patients are reported in Table 1 . Thirty-two patients , having undergone surgical resection of both the primary tumor and the corresponding metastatic sites in our institution between 2000 and 2004 , were selected from a pathological database of colorectal cancer cases . There were 15 men -LRB- 47 % -RRB- and 17 women -LRB- 53 % -RRB- . Median age at surgery was 60 years -LRB- ranging from 45 to 81 years -RRB- . Twenty-one patients -LRB- 66 % -RRB- had colon cancer and 11 -LRB- 34 % -RRB- had rectal cancer . The most common histological type was adenocarcinoma -LRB- 91 % -RRB- , the remaining three cases -LRB- 9 % -RRB- being mucinous carcinomas . According to the latest TNM classification -LSB- 28 -RSB- , all the patients had stage-IV disease because of synchronous metastases at the time of diagnosis . Among them , four patients received neoadjuvant radiotherapy -LRB- n = 2 -RRB- or radiochemotherapy -LRB- n = 2 -RRB- for rectal carcinomas and six others received neoadjuvant chemotherapy before surgical resection of their metastases . Eighty-five paraffin-embedded tumor samples from CRC -LRB- n = 32 -RRB- and liver , pulmonary , and ovarian metastases -LRB- n = 51 , 1 , and 1 , respectively -RRB- were available . Synchronous metastases -LRB- n = 45 -RRB- were unique in 23 patients and multiple -LRB- n = 22 -RRB- in nine patients , consisting of two , three , or four locations in the liver -LRB- Table 1 -RRB- . Metachronous metastases -LRB- n = 8 -RRB- from four patients were also collected and assessed for EGFR immunohistochemistry . They consisted of five multiple liver metastases for one patient , one single liver metastasis for two patients , and one pulmonary metastasis for one patient . Synchronous and metachronous metastases were obtained by metastasectomy . All resected samples were received fresh , then immediately fixed in 10 % pH neutral formalin for 48 h and embedded in paraffin before processing . Table 1Clinical data and EGFR status among primary CRC and their related metastasesClinical dataEGFR statusa on tissue sectionsCase no.SexAge -LRB- years -RRB- pTNMLocationNeoadjuvant treatmentPrimary siteSynchronous metastases -LRB- location number -RRB- Metachronous metastases -LRB- location number -RRB- 1234123451M60T3N1M1C + + + + +2 M60T3N2M1RRCT + + + +3 M66T2N0M1R + + + +4 M49T3N2M1RCT + + + + + + + +5 M70T3N1M1C + + +6 F74T3N1M1RRCT + + +7 F59T3N2M1RCT + + + − 8M71T3N2M1C + + +9 F55T3N2M1R − + − 10F61T4N1M1CCT + + +11 F68T3N1M1CCT + + +12 M75T3N1M1R + +13 M72T3N1M1C + +14 M54T3N0M1C + +15 F55T4N1M1C + +16 F57T4N2M1C + +17 F52T3N1M1R + +18 M53T3N0M1C + +19 M62T4N2M1C + +20 F77T3N2M1C + +21 F49T3N1M1C + +22 F51T3N2M1C + +23 M62T3N2M1RRT + +24 F65T3N1M1CCT + +25 M56T4N1M1CCT + +26 F47T3N1M1RRT + +27 F59T3N1M1C + − 28F45T3N2M1C + − 29F81T3N1M1R − +30 M58T2N0M1C − +31 M69T3N1M1C − +32 F68T3N2M1C − + Liver metastases in all cases except for a metachronous pulmonary metastasis and a synchronous ovarian metastasis in patients 7 and 15 , respectivelyC colon , R rectum , RCT radio-chemotherapy , CT chemotherapy before surgical resection of metastasis , RT radiotherapyaPositive if > 1 % of tumor cells expressed EGFR EGFR immunohistochemistry EGFR immunohistochemistry was carried out on freshly sectioned tissue slides which has been shown to be critical for optimal EGFR immunoreactivity -LSB- 1 -RSB- . Immunohistochemistry of the tumors was performed by using the Dako autostainer and the EGFR pharmDx ™ kit -LRB- K1494 , Dako , Glostrup , Denmark -RRB- , according to the manufacturer 's instructions . Briefly , 4-μm-thick sections were mounted on silanized slides and allowed to dry overnight at 37 °C . After deparaffinization and rehydratation , slides were incubated with 3 % hydrogen peroxide for 5 min . After washing with the supplied buffer , tissue sections were covered for 5 min with a proteinase K solution . The slides were then incubated for 30 min with the primary mouse anti-EGFR monoclonal antibody -LRB- clone 2-18C9 -RRB- , which binds to a formalin-resistant epitope near the ligand-binding site on the extracellular domain of EGFR . After two rinses in buffer , the slides were incubated with a horseradish peroxidase-labeled polymer coupled to secondary antibodies for 30 min . Tissue staining was visualized with diaminobenzidine as substrate-chromogen solution . Slides were counterstained with hematoxylin , dehydrated , and mounted . Negative control sections were processed without the primary antibody but with an irrelevant murine IgG1 supplied with the kit . Negative and positive control cell slides -LRB- CAMA-1 and HT29 cell lines , respectively -RRB- , provided with the EGFR pharmDx ™ kit , were also used to ensure that each assay run was performed appropriately and according to protocol specifications . Furthermore , perineurium and normal mucosae on primary CRC and hepatocytes surrounding the metastases were considered as positive internal controls on tumor slides . Tissue microarray Tissue blocks appearing to have enough material upon gross inspection were initially selected and hematoxylin -- eosin-stained sections were evaluated by two observers -LRB- F. Bibeau and F. Boissière-Michot -RRB- for the presence of carcinoma . The areas to be used for the construction of the TMAs were marked on the slide and the donor block . Particular attention was made to select invasive cells near the tumor front . The tissues corresponding to selected areas were sampled using a manual arraying instrument -LRB- Manual Tissue Arrayer 1 , Beecher Instruments , Sun Prairie , WI , USA -RRB- . Two TMAs were constructed using 0.6-mm tissue cores . The sampling consisted of three malignant cores from different areas of the tumor from a single case of invasive colorectal cancer , placed at specified coordinates . When possible , normal mucosa was also sampled as internal control . After the arraying was completed , TMA blocks were sectioned at a thickness of 4 μm . One section was stained with hematoxylin -- eosin , and EGFR immunostaining using the immunohistochemical system kit EGFR pharmDx ™ was performed on the adjacent section . Among the 32 cases sampled , 31 primary CRC , 29 metastatic sites , and 28 matched sites were assessable with this technology . EGFR immunohistochemical staining scoring system EGFR assessment was realized according to the EGFR pharmDx ™ scoring guidelines . EGFR expression was evaluated on sections including the deepest region of tumor invasion as this region was shown to contain the greatest density of EGFR-positive cells -LSB- 6 -RSB- . Results were reported as positive when a complete or incomplete circumferential membrane staining was observed in at least 1 % of the tumor cells . Staining was defined as immunostaining of tumor cell membranes above background level and scored as follows : 1 + = weak , 2 + = moderate , and 3 + = strong . The absence of membrane staining or cytoplasmic staining was reported as negative . The percentage of stained cells was assessed as follows : 1 -- 10 , 10 -- 50 , and > 50 % . Slides were scored by two pathologists -LRB- F. Bibeau and J.C. Sabourin -RRB- , who were blinded to the patients ' characteristics . In the case of disagreement -LRB- three out of 91 specimens -RRB- , the EGFR status was determined by consensus after simultaneous dual reexamination . In addition to these standardized criteria , a semiquantitative analysis was performed taking into account both the staining intensity and the percentage of positive tumor cells . Statistical analysis The cut-off value for negative/positive expression of EGFR was 1 % of tumor cells displaying a membranous staining , as specified by the EGFR pharmDx ™ scoring guidelines . Data were summarized by frequency and percentage for categorical variables and by means , standard deviations , median , and range for continuous variables . For TMA analysis , the percentage of positive cells was calculated from the mean of triple cores . The correlation between the percentage of positive cells observed on primary tumors and matched metastases was evaluated with the Spearman correlation coefficient . Associations between categorical variables were examined using McNemar 's chi2 test . A P value of less than 0.05 was considered statistically significant . Due to the small number of patients with metachronous metastases , only the correlation between the matched primary CRC and the synchronous metastatic sites was made . Results EGFR expression of primary CRC and their related metastases on tissue sections EGFR reactivity was not homogeneous throughout the tumors . EGFR immunostaining was mainly observed in the deepest region of the primary CRC or at the periphery of metastases , especially in the liver . Of note , EGFR immunoreactivity was often greater in isolated tumor cells and in small clusters of tumor cells . Eighty-four percent of primary CRC -LRB- 27 out of 32 -RRB- and 94 % of the synchronous metastases -LRB- 30 out of 32 -RRB- displayed EGFR reactivity in more than 1 % of the tumor cells -LRB- Table 1 -RRB- . Overall , all patients displayed an EGFR-positive status considering EGFR-expression in the primary tumor and/or the metastatic sites . EGFR expression was positive at both sites in most of the cases -LRB- 25 out of 32 ; 78 % -RRB- and rarely in only one site , i.e. , primitive -LRB- two out of 32 ; 6 % -RRB- or metastastic -LRB- five out of 32 ; 16 % -RRB- . However , the discordant cases -LRB- i.e. , positive primary sites with negative metastasis and conversely -RRB- were not statistically significant -LRB- P = 0.453 -RRB- . Nine -LRB- 28 % -RRB- , 10 -LRB- 31 % -RRB- , and eight -LRB- 25 % -RRB- of the primary CRC and seven -LRB- 22 % -RRB- , 14 -LRB- 44 % -RRB- , and nine -LRB- 28 % -RRB- of the synchronous metastases displayed a weak , moderate , and strong positivity , respectively . According to the extent of EGFR immunoreactivity , five -LRB- 16 % -RRB- primary CRC had no reactivity , 12 -LRB- 37.5 % -RRB- had < 10 % reactive cells , eight -LRB- 25 % -RRB- had > 10 -- 50 % positive cells , and seven -LRB- 22 % -RRB- had > 50 % labeled cells . In the synchronous metastases , two cases -LRB- 6 % -RRB- were negative , 13 -LRB- 41 % -RRB- had < 10 % reactive cells , 11 -LRB- 34 % -RRB- had > 10 -- 50 % positive cells , and six -LRB- 19 % -RRB- had > 50 % labeled cells . Interestingly , the percentage of EGFR-positive tumor cells in the primary site was significantly correlated with that observed in the synchronous metastases -LRB- r2 = 0.616 , P = 0.0002 -RRB- . This correlation was also demonstrated when combining EGFR scoring -LRB- labeling intensity -RRB- with the percentage of EGFR-positive tumor cells -LRB- r2 = 0.559 , P = 0.0009 -RRB- . One example of the pattern of EGFR labeling in both sites is shown in Fig. 1 . EGFR expression was positive among the multiple synchronous liver metastases in eight out of nine patients -LRB- i.e. , 21 out of 22 samples , Table 1 -RRB- . The related primary site of the discordant case -LRB- two synchronous metastases : one was EGFR-positive , the other was EGFR-negative -RRB- displayed an EGFR-negative status -LRB- case number 9 , Table 1 -RRB- . Fig. 1Example of EGFR expression in tissue sections in a primary colorectal carcinoma -LRB- a -RRB- and the matched metastasis -LRB- b -RRB- . Strong membranous staining of numerous cells in both sites -LRB- immunoperoxidase ; bar 50 μm -RRB- Regarding the analysis of EGFR expression in metachronous metastases , we observed an analogous EGFR-positive status between all the available liver metastases -LRB- i.e. , five metastases for one patient , one metastasis for two patients -RRB- and their related primary sites and synchronous metastases -LRB- case numbers 4 , 10 , and 11 , Table 1 -RRB- . The only metachronous pulmonary metastasis available for analysis did not show any EGFR immunoreactivity , whereas both the corresponding hepatic synchronous metastases and the primary site expressed EGFR -LRB- case number 7 , Table 1 -RRB- . EGFR expression of primary CRC and their related metastases on TMA On the first TMA block , the EGFR status was found to be positive in 18 out of the 31 -LRB- 58 % -RRB- assessable primary tumors and 18 out of 29 metastases -LRB- 62 % -RRB- -LRB- Table 2 -RRB- . Only nine out of 28 -LRB- 32 % -RRB- assessable matched tumors were positive for both the primary and the metastatic sites , whereas four out of 28 -LRB- 14 % -RRB- were negative . Fifteen out of 28 -LRB- 54 % -RRB- cases were discordant -LRB- i.e. , seven positive primary and negative metastases , and eight , conversely -RRB- . Table 2Comparison of EGFR expression in primary CRC and their synchronous metastatic sites on whole tissue sections and TMA Primary siteMetastasesMatched sitesTissue sections27/32 -LRB- 84 % -RRB- 30/32 -LRB- 94 % -RRB- 25/32 -LRB- 78 % -RRB- First TMA18/31 -LRB- 58 % -RRB- 18/29 -LRB- 62 % -RRB- 9/28 -LRB- 32 % -RRB- Second TMA14/31 -LRB- 45 % -RRB- 16/29 -LRB- 55 % -RRB- 8/28 -LRB- 29 % -RRB- Both TMAs20/31 -LRB- 65 % -RRB- 19/29 -LRB- 66 % -RRB- 12/28 -LRB- 43 % -RRB- Data are expressed as the number of positive cases/total number of specimens On the second TMA block , 14 out of 31 -LRB- 45 % -RRB- primary tumors and 16 out of 29 -LRB- 55 % -RRB- metastases were EGFR-positive -LRB- Table 2 -RRB- . Eight out of 28 -LRB- 29 % -RRB- assessable matched tumors were positive for both sites , whereas nine -LRB- 32 % -RRB- were negative . Eleven out of 28 cases -LRB- 39 % -RRB- were discordant -LRB- i.e. , four positive primary sites and negative metastases , and seven , conversely -RRB- . When combining the two TMAs -LRB- six tissue cores -RRB- , the number of positive cases increased , to reach 20 out of 31 of the primary tumors -LRB- 65 % -RRB- and 19 out of 29 of the metastases -LRB- 66 % -RRB- -LRB- Table 2 -RRB- . Twelve out of 28 -LRB- 43 % -RRB- assessable matched tumors were positive for both sites , whereas three -LRB- 11 % -RRB- were negative . Nevertheless , 13 cases -LRB- 46 % -RRB- remained discordant -LRB- i.e. , six positive primary and negative metastases , and seven , conversely -RRB- . Moreover , using the TMA technology and whatever the number of cores -LRB- three or six -RRB- , we failed to demonstrate any correlation of EGFR expression between primitive sites and matched metastases . Comparison of EGFR expression between tissue sections and TMA The EGFR status on the triple core-tissue array was significantly underestimated at both sites when compared with tissue sections -LRB- P = 0.011 for both primitive and metastatic sites for the first TMA ; P = 0.0005 and P = 0.0023 for primitive and metastatic sites , respectively , for the second TMA -RRB- . When combining both TMAs -LRB- six tissue cores -RRB- , although the number of positive cases increased , we still observed a significant discrepancy between both techniques for primitive sites -LRB- P = 0.034 -RRB- and metastases -LRB- P = 0.0001 -RRB- . Discussion EGFR is a 170-kDa transmembrane cell surface receptor encoded by the human HER1 gene , which promotes malignant cell proliferation and cancer progression -LSB- 15 -RSB- . Interfering with EGFR cell signaling by targeting strategies represents a novel approach to the treatment of solid tumors -LSB- 15 -RSB- . Among them , cetuximab -LRB- Erbitux ® -RRB- , a chimeric monoclonal human -- mouse antibody , is used for the treatment of EGFR-expressing metastatic CRC that have progressed following chemotherapy -LSB- 3 , 23 -RSB- . To identify patients eligible for this treatment , an immunohistochemical evaluation of EGFR expression is required . Although numerous studies have demonstrated EGFR expression in 70 -- 97 % of CRC -LSB- 4 , 6 , 29 -RSB- , only few and controversial data are available on the EGFR status in distant metastases -LSB- 6 , 11 , 13 , 24 -RSB- . Consequently , knowledge of EGFR metastastic status could be of potential value for therapeutic decisions , particularly if it differs from the primary tumors . To answer the question as to whether EGFR expression is similar in different tumor sites , we compared the EGFR status of a set of primary CRC and their corresponding synchronous distant metastases , using a standardized immunohistochemistry test -LRB- EGFR pharmDx ™ , Dako -RRB- . We also analyzed EGFR expression in metachronous metastases , when available , to study possible changes of EGFR expression during the metastatic process of CRC . On whole tissue sections , our results demonstrated that EGFR expression was frequent and similar in both the primary and the metastatic sites . In our study , immunoreactivity was observed in 84 and 94 % of the primary tumors and the metastases , respectively -LRB- Table 1 -RRB- . The 32 matched primary and synchronous metastatic CRC expressed a concordant EGFR status in 78 % of the cases , all immunopositive , and the discordant cases were not statistically significant -LRB- P = 0.453 -RRB- . Our data are in agreement with three recent studies that showed a high frequency and concordance of EGFR expression in matched primary tumors and distant metastatic lesions of CRC -LSB- 8 , 11 , 13 -RSB- . To our knowledge , only one study reported no correlation of EGFR expression on primary CRC and related metastases -LSB- 24 -RSB- . In addition to the concordant EGFR status between primary and metastatic locations , our study showed that both the staining intensity and the percentage of EGFR-positive cells in the primary site were significantly correlated with those observed in synchronous metastases -LRB- Fig. 1 -RRB- . Our work also emphasized the EGFR-positive status among multiple synchronous and metachronous metastatic sites in most of the patients -LRB- Table 1 -RRB- . Furthermore , we noticed an analogous EGFR-positive status between all metachronous metastases but one , their related synchronous metastases , and their primary CRC -LRB- Table 1 -RRB- . Such data , to our knowledge , have never been reported in clinical samples and seem to agree with the preclinical results , suggesting that EGFR expression is required for the tumor to acquire metastatic potential -LSB- 19 , 22 , 27 -RSB- . Moreover , in our cohort , all the patients displayed an EGFR-positive status . This would mean that most of the patients are potentially eligible for therapy with EGFR-targeted monoclonal antibodies , given the results of the immunohistochemistry . The clinical relevance of EGFR immunohistochemical detection is today a matter of debate because EGFR-negative patients might respond to cetuximab and EGFR-positive patients might not -LSB- 2 , 3 , 5 , 23 -RSB- . One reason proposed to explain this lack of correlation is a possible difference in the EGFR status between the primary tumor and the metastatic sites -LSB- 24 -RSB- . Our results do not support this hypothesis . The lack of response in EGFR-positive patients could more likely be explained by alterations in the critical downstream signals , activated via other receptors or other pathways . It is also plausible that the lack of correlation between EGFR expression and antitumor activity could be due to the potential for cetuximab to induce antibody-dependent cell-mediated cytotoxicity and to the absence of discrimination between the high - and low-affinity receptors with the commonly used antibodies -LSB- 2 , 14 , 16 -RSB- . Moreover , negative immunohistochemical results may be linked to the heterogeneity of EGFR expression and could , in part , represent false negative cases that might respond to EGFR-targeted therapy . The second goal of our study was to perform the same analysis on TMA generated with the same paraffin blocks used for EGFR assessment on the whole tissue sections . TMA has been employed to study the molecular profile of different cancer types and to test hypotheses regarding colorectal carcinogenesis and prognosis -LSB- 9 , 12 , 20 -RSB- . However , EGFR expression in primary CRC and their related metastases has rarely been studied with this approach and the few published results are controversial -LSB- 8 , 25 -RSB- . In our work , TMA was carried on multiple -LRB- three -RRB- cores , selected from the deepest region of tumor invasion . Nevertheless , the results obtained were systematically lower than those observed on the whole tissue sections . Even when combining the two TMAs , EGFR positivity only reached 65 and 66 % in primary CRC and their related metastases , respectively -LRB- Table 2 -RRB- . In contrast to what we observed on tissue sections , we failed to demonstrate a correlation of EGFR expression between both sites using the TMA technology . The discordant results between the two technologies may be explained by cases containing rare stained cells -LRB- i.e. , < 10 % -RRB- or small invasive clusters , which may be not selected when TMA is performed even using three or six cores . These small tumor clusters , also called tumor `` budding , '' belong to a phenomenon which might be involved in metastatic spreading and seem to represent a poor prognostic factor -LSB- 18 , 21 , 30 -RSB- . Such data are of importance because TMA has been recognized as a useful and powerful tool for screening immunohistochemical markers for prognostic purposes and for carcinogenesis studies , as well . In conclusion , we demonstrated that EGFR expression on whole tissue sections is frequent and similar in primary CRC and their related metastases . Our results also indicated that the EGFR status of the primary is concordant with those of metastases , whatever the number of sites and the time of occurrence , i.e. , synchronous or metachronous . This may have clinical implications because patients are not always treated in the same institute for their primary disease and for their metastatic disease . In our experience , TMA does not represent an appropriate technique for EGFR screening in CRC because it significantly underestimates EGFR expression and does not provide data similar to those obtained with whole tissue sections . Undoubtedly , this technology is a reliable tool for the high-throughput assessment of homogeneously distributed proteins -LSB- 7 -RSB- , but it appears to generate unreliable information with very heterogeneous markers such as EGFR .

O O B I I I O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

epidermal growth factor receptor
colorectal carcinoma
metastases
tissue microarray


Associations

between

mild-to-moderate

anaemia

in

pregnancy

and

helminth

malaria

and

HIV

infection

in

Entebbe

Uganda

Summary

Introduction

Anaemia

in

pregnancy

contributes

to

maternal

deaths

and

may

also

contribute

to

adverse

birth

outcomes

including

intrauterine

growth

retardation

and

prematurity

and

hence

to

perinatal

morbidity

and

mortality

-LRB-

WHO

-RRB-

Hookworm

is

an

important

cause

of

anaemia

in

developing

countries

but

lack

of

consensus

regarding

the

risks

and

benefits

of

treating

helminths

in

pregnancy

has

until

recently

led

to

a

tendency

to

exclude

pregnant

and

even

breastfeeding

women

from

deworming

programmes

In

a

call

was

made

for

research

leading

to

improved

estimates

of

hookworm

infection

in

women

of

child-bearing

age

and

for

evaluation

of

interventions

that

might

be

beneficial

-LRB-

WHO

-RRB-

but

to

our

knowledge

only

one

placebo-controlled

trial

of

the

treatment

of

hookworm

in

pregnancy

has

been

reported

-LRB-

Torlesse

and

Hodges

-RRB-

More

recently

an

Informal

Consultation

held

by

the

WHO

gave

consideration

to

the

possible

effects

of

schistosomiasis

in

pregnancy

and

it

was

suggested

that

anaemia

might

be

among

them

-LRB-

Allen

et

al.

WHO

-RRB-

Although

praziquantel

had

been

widely

avoided

in

pregnant

and

lactating

women

it

was

noted

that

there

was

no

evidence

from

studies

in

animals

from

case

reports

or

from

mass

treatment

campaigns

in

humans

of

any

major

adverse

effects

Treatment

of

schistosomiasis

during

pregnancy

was

therefore

advocated

by

the

Consultation

Committee

We

have

undertaken

a

trial

designed

to

examine

the

effects

of

maternal

helminths

and

of

deworming

during

pregnancy

on

the

response

to

immunisation

and

susceptibility

to

infection

and

disease

in

infancy

-LSB-

-RSB-

-LRB-

Elliott

et

al.

-RRB-

Anaemia

is

among

the

secondary

outcomes

of

the

trial

The

trial

is

ongoing

In

view

of

current

interest

in

the

role

of

helminths

in

anaemia

during

pregnancy

we

have

examined

associations

between

anaemia

and

helminths

and

other

major

infections

-LRB-

malaria

and

HIV

-RRB-

among

pregnant

women

at

enrolment

into

the

trial

Materials

and

methods

Study

population

and

procedures

The

study

area

comprises

Entebbe

Municipality

and

the

adjacent

subcounty

of

Katabi

This

area

supports

semi-urban

rural

and

fishing

communities

residing

on

the

Entebbe

peninsula

in

Lake

Victoria

Uganda

Women

were

recruited

at

the

antenatal

clinic

at

Entebbe

Hospital

between

April

and

November

Women

were

assessed

for

screening

at

their

first

antenatal

visit

thus

initial

screening

could

take

place

in

any

trimester

of

pregnancy

They

were

eligible

for

screening

if

they

were

well

resident

in

the

study

area

planning

to

deliver

their

baby

at

the

hospital

willing

to

participate

and

willing

to

know

their

HIV

status

On

the

screening

day

after

giving

written

informed

consent

eligible

women

were

interviewed

regarding

sociodemographic

characteristics

and

risk

factors

for

helminth

infection

malaria

and

HIV

and

were

examined

by

a

midwife

A

blood

sample

was

obtained

for

investigations

including

haemoglobin

-LRB-

Hb

-RRB-

estimation

examination

for

microfilariae

-LRB-

mf

-RRB-

and

malaria

parasites

syphilis

and

HIV

serology

Screened

women

were

asked

to

return

for

enrolment

within

month

with

a

stool

sample

They

were

excluded

from

enrolment

if

they

had

a

Hb

level

g/dl

clinically

apparent

severe

liver

disease

diarrhoea

with

blood

in

the

stool

an

abnormal

pregnancy

a

history

of

adverse

reaction

to

anthelminthic

drugs

or

had

already

participated

in

the

study

during

an

earlier

pregnancy

Women

were

enrolled

in

the

trial

when

they

returned

with

a

stool

sample

if

they

were

in

the

second

or

third

trimester

and

full

eligibility

was

confirmed

All

women

received

routine

antenatal

care

including

haematinics

and

intermittent

presumptive

treatment

for

malaria

using

sulfadoxine/pyrimethamine

Women

were

treated

for

syphilis

and

provided

with

nevirapine

for

prevention

of

mother-to-child

HIV

transmission

if

indicated

Women

who

were

excluded

from

the

study

on

grounds

of

severe

anaemia

were

treated

with

albendazole

and

haematinics

and

referred

for

transfusion

if

required

Haemoglobin

estimation

and

definition

of

anaemia

Hb

was

estimated

at

the

antenatal

clinic

using

a

colorimetric

haemoglobinometer

-LRB-

DHT

haemoglobin

meter

Developing

Health

Technology

Barton

Mills

UK

-RRB-

with

same-day

results

The

same

sample

was

then

sent

to

the

Medical

Research

Council/Uganda

Virus

Research

Institute

-LRB-

MRC/UVRI

-RRB-

laboratories

for

analysis

by

a

Coulter

analyser

-LRB-

Beckman

Coulter

AC-T

diff

CP

Beckman

Coulter

Nyon

Switzerland

-RRB-

Quality

control

for

the

Coulter

analyser

was

provided

through

the

United

Kingdom

National

External

Quality

Assessment

Schemes

with

consistently

good

results

Initial

evaluation

suggested

that

the

haemoglobinometer

results

reliably

matched

the

Coulter

analyser

results

-LRB-

intraclass

correlation

coefficient

-LRB-

ICC

-RRB-

CI

-RRB-

and

the

immediately

available

haemoglobinometer

results

were

used

to

determine

enrolment

Evaluation

of

results

to

March

showed

less

reliability

-LRB-

ICC

CI

-RRB-

and

it

was

noted

that

women

had

been

enrolled

with

haemoglobinometer

results

g/dl

but

Coulter

analyser

values

below

this

cut-off

thereafter

Coulter

analyser

results

were

used

for

enrolment

Coulter

analyser

results

have

been

used

in

this

analysis

In

accordance

with

WHO

criteria

anaemia

was

defined

as

Hb

g/dl

i.e.

g/dl

above

the

standard

cut-off

of

g/dl

to

allow

for

the

altitude

in

Entebbe

-LRB-

m

a.s.l.

-RRB-

-LRB-

-RRB-

-LRB-

WHO

-RRB-

Parasitology

Examination

of

stool

samples

was

performed

as

described

previously

-LRB-

Bukusuba

et

al.

-RRB-

using

the

Kato

Katz

method

-LRB-

Katz

et

al.

-RRB-

and

charcoal

culture

for

Strongyloides

-LRB-

Friend

-RRB-

Two

Kato

Katz

slides

were

prepared

from

each

sample

each

examined

within

min

for

hookworm

or

the

following

day

for

other

parasites

Blood

was

examined

for

Mansonella

by

a

modified

Knott

's

method

-LRB-

Melrose

et

al.

-RRB-

Intensity

of

infection

was

assessed

by

egg

counts

in

stool

and

mf

counts

in

blood

Intensities

were

categorised

as

follows

hookworm

light

eggs

per

gram

of

stool

-LRB-

epg

-RRB-

moderate

epg

high

epg

-LRB-

WHO

-RRB-

Schistosoma

mansoni

light

epg

moderate

epg

high

epg

-LRB-

WHO

-RRB-

Trichuris

trichiura

light

epg

moderate

epg

high

epg

-LRB-

WHO

-RRB-

No

standard

categories

are

available

for

Mansonella

intensity

therefore

arbitrary

categories

were

defined

to

obtain

approximately

equal

numbers

of

participants

in

each

category

light

mf/ml

moderate

mf/ml

high

mf/ml

HIV

serology

HIV

serology

was

performed

using

a

rapid

test

algorithm

with

same-day

results

Testing

kits

provided

by

the

Ministry

of

Health

varied

with

availability

Most

commonly

Determine

-LRB-

Abbott

Laboratories

Abbott

Japan

Co.

Ltd.

Tokyo

Japan

-RRB-

was

used

for

screening

with

positive

results

confirmed

by

Unigold

-LRB-

Trinity

Biotech

plc

Bray

Ireland

-RRB-

Samples

with

differing

results

were

referred

for

analysis

by

non-rapid

ELISA

tests

or

were

examined

using

a

tie-breaker

rapid

test

usually

Statpack

-LRB-

Chembio

Diagnostic

Systems

Medford

NY

USA

-RRB-

A

proportion

of

samples

including

all

those

with

differing

results

by

rapid

test

were

re-examined

at

the

MRC/UVRI

laboratories

for

quality

control

with

high

agreement

of

the

results

Demographic

information

and

potential

risk

factors

for

anaemia

Socioeconomic

data

were

summarised

by

developing

two

indices

based

on

the

variables

that

appeared

to

describe

socioeconomic

status

most

usefully

These

were

woman

's

socioeconomic

index

comprised

of

education

personal

income

and

occupation

and

household

socioeconomic

index

comprised

of

building

materials

number

of

rooms

and

items

collectively

owned

The

relationship

between

potential

confounding

factors

and

anaemia

and

helminths

malaria

and

HIV

were

considered

and

a

diagram

describing

hypothesised

relationships

was

developed

-LRB-

Figure

-RRB-

Age

socioeconomic

status

and

tribe

were

considered

to

be

possible

confounders

with

gravidity

of

potential

importance

particularly

for

malaria

to

which

primigravidae

are

known

to

be

particularly

susceptible

Data

management

and

statistical

analysis

Data

were

entered

using

Microsoft

Access

-LRB-

Microsoft

Corp.

Redmond

WA

USA

-RRB-

and

analysed

using

STATA

version

-LRB-

Stata

Corp.

College

Station

TX

USA

-RRB-

The

initial

analysis

was

based

on

a

binary

variable

for

outcome

-LRB-

anaemic/not

anaemic

-RRB-

and

categorical

variables

for

exposure

with

binary

variables

for

exposures

to

helminths

malaria

and

HIV

infection

-LRB-

infected/not

infected

-RRB-

Effects

of

helminth

infection

were

then

investigated

in

more

detail

by

examining

categories

of

infection

intensity

-LRB-

light

moderate

or

heavy

-RRB-

For

these

analyses

cross-tabulations

were

made

between

selected

risk

factors

and

the

presence

of

anaemia

Logistic

regression

was

used

to

estimate

unadjusted

and

adjusted

odds

ratios

-LRB-

OR

-RRB-

Likelihood

ratio

tests

were

used

to

determine

P-values

Continuous

variables

for

anaemia

-LRB-

Hb

level

-RRB-

and

infection

intensity

-LRB-

egg

or

parasite

count

-RRB-

were

then

used

to

examine

effects

of

intensity

in

more

detail

among

infected

participants

using

linear

regression

Adjustment

was

made

for

the

same

potential

confounders

in

logistic

and

linear

regression

models

Results

A

total

of

women

registered

at

the

antenatal

clinic

during

the

recruitment

period

of

whom

were

assessed

for

inclusion

in

the

study

and

were

considered

eligible

and

screened

The

commonest

reasons

for

ineligibility

were

residence

outside

the

study

area

-LRB-

-RRB-

unwillingness

to

have

an

HIV

test

-LRB-

-RRB-

unwillingness

to

join

the

study

-LRB-

-RRB-

and

enrolment

during

an

earlier

pregnancy

-LRB-

-RRB-

Of

the

screened

were

enrolled

of

these

were

subsequently

excluded

because

they

had

been

enrolled

during

a

previous

pregnancy

Of

the

women

screened

but

not

enrolled

the

majority

-LRB-

-RRB-

failed

to

return

for

enrolment

and

only

brought

a

stool

sample

Since

intestinal

helminth

infection

detected

by

stool

analysis

was

a

focus

of

interest

this

analysis

was

confined

to

the

women

who

were

enrolled

in

the

trial

and

for

whom

all

or

almost

all

relevant

data

were

available

Characteristics

of

the

study

women

Maternal

age

ranged

from

years

to

years

-LRB-

mean

years

-RRB-

The

majority

were

Baganda

-LRB-

-RRB-

the

predominant

tribe

of

the

district

Most

-LRB-

-RRB-

were

married

with

single

widows

and

divorced

or

separated

Education

varied

from

-LRB-

-RRB-

to

primary

-LRB-

-RRB-

secondary

-LRB-

-RRB-

and

tertiary

-LRB-

-RRB-

and

most

women

were

poor

-LRB-

with

a

personal

income

of

less

than

per

month

-RRB-

Primigravidae

comprised

of

the

women

studied

Prevalence

of

infections

and

anaemia

Complete

data

were

obtained

for

Kato

Katz

assays

from

women

for

Strongyloides

assays

from

women

for

Mansonella

from

women

and

for

malaria

from

women

The

prevalence

of

hookworm

was

Mansonella

perstans

S.

mansoni

Strongyloides

stercoralis

T.

trichiura

Ascaris

lumbricoides

Trichostrongylus

sp.

Hymenolepis

nana

and

Loa

loa

Ova

of

Fasciola

hepatica

and

Dicrocoelium

dendriticum

were

found

in

samples

from

one

and

five

women

respectively

but

infection

was

not

confirmed

in

follow-up

samples

and

eggs

probably

originated

from

liver

or

offal

in

the

women

's

diet

detection

of

ova

of

these

two

species

was

not

considered

to

indicate

infection

The

prevalence

of

asymptomatic

Plasmodium

falciparum

malaria

parasitaemia

was

and

HIV

infection

The

prevalence

of

anaemia

-LRB-

Hb

g/dl

-RRB-

was

Six

women

were

enrolled

in

error

with

Hb

g/dl

by

both

methods

-LRB-

Coulter

analyser

results

g/dl

-RRB-

These

as

well

as

the

women

enrolled

prior

to

March

with

haemoglobinometer

results

above

but

Coulter

analyser

results

below

g/dl

have

been

retained

in

this

analysis

Associations

between

characteristics

of

pregnant

women

and

anaemia

in

pregnancy

Relationships

between

characteristics

of

the

participating

women

and

anaemia

are

shown

in

Table

The

prevalence

of

anaemia

declined

with

age

Anaemia

was

associated

with

tribe

with

Basoga

most

likely

to

be

anaemic

Anaemia

showed

no

association

with

women

's

socioeconomic

index

but

low

household

socioeconomic

status

was

associated

with

anaemia

Primigravidae

were

more

likely

to

be

anaemic

than

multigravidae

Relationship

between

infections

and

anaemia

in

pregnancy

Relationships

between

infections

and

anaemia

are

shown

in

Table

of

the

helminths

showed

an

association

with

anaemia

when

considered

using

categorical

variables

for

the

presence

or

absence

of

infection

Hookworm

showed

a

weak

positive

association

but

this

was

reduced

in

the

adjusted

model

Malaria

parasitaemia

and

HIV

infection

were

strongly

associated

with

anaemia

for

malaria

the

effect

was

unchanged

and

for

HIV

it

increased

after

adjusting

for

potential

confounding

factors

Malaria

was

also

associated

with

HIV

infection

-LRB-

OR

CI

P

-RRB-

the

association

between

HIV

and

anaemia

was

reduced

slightly

but

not

explained

when

malaria

was

added

to

the

model

-LRB-

adjusted

OR

-LRB-

AOR

-RRB-

adjusted

for

age

tribe

socioeconomic

index

gravidity

and

malaria

CI

P

-RRB-

The

likelihood

of

being

anaemic

was

particularly

high

among

women

with

both

HIV

infection

and

malaria

compared

with

women

with

neither

infection

-LRB-

AOR

CI

P

-RRB-

Comparing

those

with

HIV

only

to

those

with

neither

gave

an

AOR

of

-LRB-

CI

P

-RRB-

and

comparing

those

with

malaria

only

to

those

with

neither

gave

an

AOR

of

-LRB-

CI

P

-RRB-

Hookworm

and

Mansonella

showed

positive

associations

with

malaria

parasitaemia

and

hookworm

showed

a

negative

association

with

HIV

infection

but

adjusting

for

these

infections

had

minimal

effect

on

the

observed

associations

between

helminths

and

anaemia

-LRB-

data

not

shown

-RRB-

Attributable

fractions

for

anaemia

were

for

hookworm

for

malaria

and

for

HIV

infection

Relationship

between

anaemia

and

infection

intensity

Associations

between

infections

and

anaemia

were

further

explored

by

examining

infection

intensity

-LRB-

Table

-RRB-

The

prevalence

of

anaemia

increased

with

each

category

of

hookworm

infection

intensity

However

this

trend

was

greatly

weakened

showing

no

evidence

of

association

after

adjusting

for

age

tribe

socioeconomic

index

gravidity

malaria

and

HIV

On

the

other

hand

there

was

a

small

negative

association

between

hookworm

egg

count

and

Hb

in

hookworm-infected

women

-LRB-

adjusted

regression

coefficient

CI

to

P

-RRB-

i.e.

women

with

the

highest

hookworm

intensity

-LRB-

approximately

epg

-RRB-

had

on

average

a

Hb

level

g/dl

lower

than

those

with

the

lowest

detectable

egg

counts

-LRB-

epg

-RRB-

Anaemia

prevalence

was

higher

in

women

with

heavy

S.

mansoni

infection

than

in

those

with

lower

intensity

infection

or

no

infection

but

the

number

of

such

women

was

small

and

the

effect

was

not

statistically

significant

Infections

with

Trichuris

were

light

in

all

but

six

of

the

infected

women

therefore

associations

between

anaemia

and

moderate-to-heavy

Trichuris

infections

could

not

be

examined

There

was

no

evidence

of

an

association

between

Hb

and

egg

count

for

women

infected

with

S.

mansoni

or

Trichuris

There

was

a

negative

association

between

malaria

parasite

count

-LRB-

parasites

per

white

blood

cells

-RRB-

and

Hb

but

this

was

reduced

in

the

adjusted

model

-LRB-

crude

regression

coefficient

CI

to

P

adjusted

regression

coefficient

CI

to

P

-RRB-

Hb

was

not

significantly

associated

with

T-cell

count

among

HIV-positive

women

-LRB-

adjusted

regression

coefficient

CI

to

P

-RRB-

Anaemia

in

women

excluded

from

enrolment

Hb

g/dl

was

an

exclusion

criterion

Among

the

women

who

were

screened

but

never

enrolled

Hb

g/dl

was

the

reason

given

in

cases

but

Coulter

analyser

Hb

was

g/dl

for

a

further

women

excluded

for

other

reasons

The

prevalence

of

anaemia

was

-LRB-

-RRB-

among

those

excluded

compared

with

-LRB-

-RRB-

among

those

enrolled

-LRB-

P

-RRB-

giving

an

overall

prevalence

of

among

all

women

screened

Since

stool

results

were

only

available

for

of

the

excluded

women

associations

with

intestinal

helminths

could

not

be

analysed

Of

the

women

excluded

and

with

Coulter

Hb

g/dl

two

had

stool

results

both

had

hookworm

infections

-LRB-

one

light

and

one

moderate

intensity

-RRB-

and

neither

had

schistosomiasis

A

sensitivity

analysis

was

performed

assuming

that

all

women

excluded

and

with

Coulter

Hb

g/dl

had

hookworm

As

expected

a

slightly

stronger

association

was

obtained

but

this

was

again

reduced

after

adjusting

for

confounding

factors

-LRB-

crude

OR

CI

P

AOR

adjusted

for

age

tribe

socioeconomic

indices

and

gravidity

CI

P

-RRB-

Associations

with

malaria

and

HIV

were

similar

in

the

excluded

group

to

those

in

the

enrolled

group

with

crude

ORs

of

-LRB-

CI

P

-RRB-

and

-LRB-

CI

P

-RRB-

respectively

Discussion

This

study

suggests

that

among

pregnant

women

in

Entebbe

Uganda

malaria

and

HIV

are

more

important

infectious

causes

of

anaemia

than

helminths

No

association

was

observed

between

mild-to-moderate

anaemia

and

any

species

of

helminth

and

a

weak

association

between

anaemia

and

increasing

intensity

of

hookworm

infection

was

reduced

after

adjusting

for

confounding

factors

Anaemia

was

slightly

more

common

among

women

heavily

infected

with

S.

mansoni

but

the

number

of

such

women

was

small

In

keeping

with

the

exclusion

of

women

with

Hb

g/dl

excluded

women

were

more

likely

to

be

anaemic

than

enrolled

women

but

this

had

minimal

impact

on

the

overall

estimate

of

prevalence

of

anaemia

-LRB-

-RRB-

Given

the

recognised

ability

of

hookworm

to

cause

anaemia

-LRB-

Bondevik

et

al.

Hotez

et

al.

Shulman

et

al.

-RRB-

we

conducted

a

sensitivity

analysis

to

estimate

the

effect

of

hookworm

if

all

women

with

Hb

g/dl

and

no

stool

result

had

hookworm

The

result

was

in

keeping

with

a

possible

effect

of

hookworm

but

after

adjusting

for

confounding

factors

the

effect

was

small

Associations

between

anaemia

and

malaria

and

between

anaemia

and

HIV

infection

were

similar

in

enrolled

and

excluded

women

Our

investigations

for

intestinal

helminths

used

only

one

stool

sample

from

each

woman

meaning

that

a

proportion

of

women

with

low-intensity

infections

will

have

been

misclassified

as

uninfected

and

that

estimates

of

intensity

will

have

been

imprecise

-LRB-

Hall

Utzinger

et

al.

-RRB-

Recent

comparable

studies

have

the

same

limitation

-LRB-

Ajanga

et

al.

Bondevik

et

al.

Dreyfuss

et

al.

Larocque

et

al.

-RRB-

In

our

study

this

may

again

be

important

for

hookworm

where

there

was

a

small

increase

in

anaemia

with

infection

intensity

but

may

not

explain

the

lack

of

association

for

schistosomiasis

where

there

was

no

suggestion

of

an

effect

of

light-to-moderate

infections

Our

method

of

investigation

for

Mansonella

and

intensity

does

not

suffer

from

this

limitation

repeat

examinations

among

women

showed

agreement

for

the

binary

variable

-LRB-

infected/uninfected

-RRB-

with

an

ICC

for

mf/ml

of

-LRB-

CI

-RRB-

-LRB-

unpublished

data

-RRB-

Not

all

pregnant

women

in

Entebbe

attend

the

district

hospital

antenatal

clinic

but

a

community

survey

undertaken

in

the

study

area

showed

an

increase

in

the

proportion

choosing

this

clinic

during

the

recruitment

period

to

approximately

and

most

of

the

personal

and

socioeconomic

characteristics

of

women

choosing

or

not

choosing

to

attend

the

district

hospital

clinic

were

similar

-LRB-

unpublished

data

-RRB-

Thus

our

results

are

likely

to

be

reasonably

representative

of

pregnant

women

in

this

area

Recent

studies

from

different

settings

give

similar

results

in

relation

to

the

effects

of

hookworm

and

S.

mansoni

on

anaemia

in

pregnancy

In

Peru

-LRB-

where

hookworm

and

Trichuris

are

predominant

-RRB-

in

Tanzania

-LRB-

S.

mansoni

and

hookworm

-RRB-

in

the

Democratic

Republic

of

Congo

-LRB-

Ascaris

and

hookworm

-RRB-

and

in

Java

-LRB-

Trichuris

and

hookworm

-RRB-

no

association

was

observed

between

anaemia

and

infection

with

any

single

species

-LRB-

Ajanga

et

al.

Kalenga

et

al.

Larocque

et

al.

Nurdia

et

al.

-RRB-

In

Peru

there

was

an

association

between

anaemia

and

higher

hookworm

intensities

and

in

Java

there

was

a

negative

association

between

serum

ferritin

and

hookworm

suggesting

an

effect

of

hookworm

on

iron

status

but

not

anaemia

in

Tanzania

in

an

area

of

higher

S.

mansoni

prevalence

and

intensity

a

strong

association

between

anaemia

and

heavy

S.

mansoni

infection

was

observed

Infection

intensity

thus

appears

to

be

important

with

light-to-moderate

hookworm

or

S.

mansoni

infections

having

relatively

weak

effects

on

Hb

levels

However

a

second

important

factor

is

the

underlying

nutritional

status

of

the

women

In

two

studies

in

Nepal

among

populations

perhaps

poorer

and

less

well

nourished

than

ours

-LRB-

as

indicated

by

items

owned

anthropometry

and

vitamin

A

status

-LRB-

Dreyfuss

et

al.

-RRB-

and

with

a

traditionally

vegetarian

diet

-LRB-

Bondevik

et

al.

-RRB-

-RRB-

anaemia

showed

a

significant

association

with

hookworm

infection

-LRB-

Bondevik

et

al.

Dreyfuss

et

al.

-RRB-

The

effects

of

hookworm

infection

are

partially

mediated

by

iron

deficiency

-LRB-

Bondevik

et

al.

Olsen

et

al.

-RRB-

and

in

the

trial

conducted

in

Sierra

Leone

iron-folate

supplements

had

a

greater

benefit

for

anaemia

in

pregnancy

than

treatment

with

albendazole

-LRB-

Torlesse

and

Hodges

-RRB-

We

found

no

suggestion

of

an

association

between

anaemia

and

any

other

helminth

species

that

was

common

in

our

environment

-LRB-

Mansonella

Trichuris

or

Strongyloides

-RRB-

This

again

is

in

agreement

with

other

studies

-LRB-

Dreyfuss

et

al.

Larocque

et

al.

Nurdia

et

al.

-RRB-

Larocque

et

al.

-LRB-

-RRB-

noted

a

stronger

effect

of

moderate-to-heavy

hookworm

infection

when

combined

with

moderate-to-heavy

Trichuris

infection

Although

there

was

broad

overlap

between

the

confidence

intervals

for

these

effects

in

their

analysis

such

an

effect

is

plausible

given

evidence

in

children

that

heavy

Trichuris

infections

-LRB-

epg

-RRB-

can

be

associated

with

anaemia

-LRB-

Ramdath

et

al.

-RRB-

In

our

study

as

in

that

reported

by

Nurdia

et

al.

-LRB-

-RRB-

no

such

heavy

Trichuris

infections

were

observed

The

strong

effects

of

malaria

and

HIV

contrast

with

the

weak

effect

of

hookworm

and

the

lack

of

effects

of

other

helminths

in

this

study

Of

note

women

were

screened

for

this

study

when

they

were

apparently

healthy

thus

both

malaria

and

HIV

infection

were

largely

asymptomatic

The

effects

of

these

infections

are

not

mediated

by

iron

deficiency

and

may

override

any

benefit

of

good

nutrition

The

importance

of

malaria

as

a

cause

of

anaemia

in

pregnancy

is

well

established

-LRB-

Shulman

and

Dorman

-RRB-

HIV

infection

is

also

a

recognised

cause

of

anaemia

-LRB-

Belperio

and

Rhew

-RRB-

and

anaemia

may

be

one

mechanism

by

which

it

causes

adverse

birth

outcomes

-LRB-

Dairo

et

al.

McIntyre

-RRB-

There

are

compelling

reasons

for

preventing

and

treating

malaria

and

HIV

during

pregnancy

-LRB-

Shulman

and

Dorman

ter

Kuile

et

al.

-RRB-

Our

results

highlight

that

anaemia

is

among

them

On

the

other

hand

our

results

as

well

as

recent

literature

suggest

that

associations

between

helminth

infections

and

anaemia

in

pregnancy

are

weaker

with

regional

variations

that

may

be

based

on

nutrition

and

intensity

of

helminth

infection

These

findings

are

relevant

when

estimating

the

relative

disease

burden

of

helminths

and

other

infections

and

the

relative

value

of

possible

interventions

in

pregnancy

Globally

the

majority

of

helminth

infections

are

of

low

intensity

so

in

some

settings

the

benefit

of

routine

deworming

during

pregnancy

in

relation

to

anaemia

may

be

modest

The

effects

of

deworming

during

pregnancy

on

other

parameters

including

birth

outcome

birth

weight

and

long-term

effects

on

health

in

infancy

and

childhood

also

need

to

be

considered

-LRB-

Christian

et

al.

-RRB-

The

forthcoming

results

of

our

ongoing

trial

of

deworming

in

pregnancy

are

expected

to

contribute

further

to

this

debate

Authors

contributions

AME

designed

the

study

JN

and

CA

carried

out

interviews

and

recruited

participants

MO

and

HM

carried

out

clinical

assessments

NO

and

DK

carried

out

laboratory

assessments

LM

PW

and

LAM

analysed

and

interpreted

the

data

LM

PW

LAM

and

AME

drafted

the

manuscript

All

authors

reviewed

and

approved

the

final

manuscript

LM

and

AME

are

guarantors

of

the

paper

Funding

Wellcome

Trust

Career

Post

fellowship

held

by

Dr

Elliott

grant

number

PMTCT

programme

Ministry

of

Health

Uganda

Conflicts

of

interest

declared

Ethical

approval

The

Science

and

Ethics

Committee

Uganda

Virus

Research

Institute

the

Uganda

National

Council

for

Science

Technology

and

the

London

School

of

Hygiene

Tropical

Medicine

O O O B O B O B O B O B O O O O B O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O B O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O B O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O B O O B O O O O O O B O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O B O O O O B O O O O O B O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O B O O O O O O O O O B O O O O B O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O B O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O B O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O

anaemia helminth pregnancy hiv malaria uganda