A novel histological grading scheme for placental malaria applied in areas of high and low malaria...

A novel histological grading scheme for placental malariaapplied in areas of high and low malaria transmission

Atis Muehlenbachs1,2, Michal Fried1,3, Rose McGready4,6,7, Whitney Harrington1,3,Theonest K. Mutabingwa1,5, François Nosten4,6,7, and Patrick E. Duffy1,3,8,*

1Seattle Biomedical Research Institute, Seattle WA, USA 2Department of Pathology, University ofWashington, Seattle WA, USA 3Department of Global Health, University of Washington, SeattleWA, USA 4Shoklo Malaria Research Institute, Mae Sot, Thailand 5National Institute for MedicalResearch, Dar es Salaam, Tanzania 6Mahidol Oxford University Tropical Medicine ResearchProgram, Bangkok, Thailand 7Centre for Tropical Medicine and Vaccinology, Churchill Hospital,Oxford, UK 8National Institute of Allergy and Infectious Diseases, Washington DC, USA

AbstractBACKGROUND—Plasmodium falciparum-infected erythrocytes sequester in the placenta andelicit an inflammatory response that is harmful to both fetus and mother. Histologic measurementsduring placental malaria (PM) might provide surrogate endpoints for interventional trials, butexisting histologic schemes capture limited complexity and are not consistently used among studysites.

METHODS—Using frozen section histology in Tanzania (high transmission area), we establish anovel grading scheme to separately quantify inflammation and pigment deposition during PM(n=102). To generalize this method, formalin-fixed paraffin-embedded placental samples fromKaren women in Thailand (low transmission area) were selected from among those withdocumented antenatal parasitemia near term (n=18).

RESULTS—In the Tanzanian cohort, the inflammation and pigment deposition scores wereindependently associated with birth weight, and the inflammation score was associated withchemokine levels. In the smaller cohort from Thailand, both inflammation and pigment scoreswere associated with birth weight, and the pigment score had an inverse trend with the number ofantenatal clinic visits.

CONCLUSIONS—This semiquantitative pathological grading scheme is simple to implementyet captures information that is associated with outcomes in Asia and Africa, and therefore shouldfacilitate the comparison and standardization of results among clinical trials across areas ofdiffering endemicity.

KeywordsMalaria; Plasmodium falciparum; pregnancy; placenta; histology

*Corresponding Author Contact Information: Patrick Duffy, M.D., Chief, Laboratory of Malaria Immunology and Vaccinology,National Institute of Allergy and Infectious Diseases, Washington DC, USA, Phone:301-443-4605, [email protected] OF INTEREST STATEMENT:All authors declare no conflicts of interest.STATEMENT OF PRIOR PRESENTATION:This work was presented at the American Society of Tropical Medicine and Hygiene and at the United States and Canadian Academyof Pathology annual meetings in 2009 and 2010, respectively.

NIH Public AccessAuthor ManuscriptJ Infect Dis. Author manuscript; available in PMC 2011 November 15.

Published in final edited form as:J Infect Dis. 2010 November 15; 202(10): 1608–1616. doi:10.1086/656723.

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INTRODUCTIONMalaria during pregnancy is associated with morbidity and mortality for pregnant womenand their newborns in tropical areas. During placental malaria (PM), Plasmodiumfalciparum-infected erythrocyes (IE) adhere to chondroitin sulfate A present on thetrophoblast surface (1). Parasite sequestration results in a maternal inflammatory responsethat can be harmful to both the mother and the fetus. Histologic features of PM correlatewith poor clinical outcomes and therefore might be useful as efficacy endpoints forinterventional trials of prophylactic or treatment drugs.

Women living in different geographical areas experience different levels of malariaexposure, and their level of immunity prior to pregnancy influences disease course duringinfection. In areas of high stable transmission, such as Tanzania, women have significantclinical immunity prior to pregnancy, and PM is often asymptomatic but associated withsevere maternal anemia and fetal growth retardation. In these areas, PM is most frequent andsevere in first-time mothers because women develop specific immunity against placentalforms of IE over successive pregnancies. In areas of low unstable transmission, such as theThai-Burma border, malaria is symptomatic in women of all parities, and associated withhigh rates of fetal loss and maternal death (2). The approach to antenatal care and thesensitivity of the parasite to different antimalarials also differ and could contribute todifferences in outcomes. In Tanzania, insecticide treated bed-nets (ITN) and intermittentpreventive treatment in pregnancy (IPTp) are routinely used as preventive interventions butcase-detection is passive (3), whereas preventive measures are not routine at the Thai-Burmaborder where case-detection is active and prompts early treatment.

Three key histologic features of an untreated PM episode were described by Garnham in1938: parasites, inflammation and pigment deposition, which correspond to three distinctbiologic timelines (4) (Fig 1). Infected erythrocytes accumulate in the intervillous space, andparasitemia changes rapidly over the course of the parasite life cycle and in response totreatment. Maternal inflammatory cells, predominately monocyte-macrophages, alsoaccumulate in the intervillous space, and are estimated to accumulate over the course of daysto weeks in susceptible women and can persist for a brief time following treatment (4).Malaria pigment accumulates in intervillous fibrin, likely originating from degeneratingpigment-laden macrophages, and can persist for months following heavy infection duringgestation, or may become undetectable with adequate treatment and no further reinfection(5,6).

In endemic areas, pathologic features of PM correspond to clinical outcomes. Maternalinflammation is mostly seen in first-time mothers, and is linked to decreased birth weight(7–9) and anemia (9). Pigment present in immune cells or fibrin has been associated withreduced birth weight (8,10). The presence of pigment in women without active parasitemia(past infection) has been associated with decreased BW in some studies (9,11), but not inothers (8,12,13). Differences in outcomes associated with past infections may be related tothe sensitivity of different methods for detecting parasites, especially during very lowparasitemia. Along the Thai-Burma border, an area of low transmission, pathologic changesof PM were rare in women without evidence of recent infection (6).

A pathologic classification scheme for PM was developed by Bulmer in 1993 (14) to reflectthe chronology of the infection. This scheme infers “active” or “acute” infections whenparasites are present in the intervillous spaces with or without pigment in intervillousmonocytes; “chronic” infections when parasites are present in the intervillous spaces alongwith pigment as deposits or in macrophages within fibrin; and “past-chronic” infectionswhen pigment is present in the absence of parasites (Fig 1A). Subsequent modifications and

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additional grading schemes have been developed by Ismail (15) and Rogerson (9) similar inspirit to the Bulmer scheme, as well as by Davison to incorporate other features of placentalinjury including intervillous inflammatory cells (16).

The application of the existing PM grading schemes is limited because chronic PM is abroad category encompassing varying degrees of pathology, scoring criteria vary by studysite, and in general, the degree of inflammation is not independently considered. In contrast,a scheme using two parameters, inflammation and fibrosis, is widely used in monitoringprogression and treatment of chronic viral hepatitis (17). We have developed a twoparameter semi-quantitative grading scheme that scores the degrees of inflammation andpigment deposition during PM. The proposed scheme is simple to perform, corresponds topregnancy outcomes in two continents, and may improve the comparison andstandardization of results among clinical trials across areas of differing endemicity.

METHODSStudy sites

Placental samples from study populations living in two areas of different malaria endemicitywere analyzed. Informed consent was obtained at both sites. Women living in the hightransmission area around Muheza, Tanzania, were enrolled in the Mother Offspring MalariaStudies Project during their delivery at the Muheza Designated District Hospital. Thesewomen received antenatal care according to Tanzanian national guidelines, including mostlypassive malaria case detection and one or two presumptive antimalarial treatments. Womenfrom refugee and migrant populations living in an area of sporadic transmission along theThai-Burma received weekly antenatal care with active malaria screening at one of fiveclinics operated by the Shoklo Malaria Research Unit. Antenatal care was provided free ofcharge, and a subset of women enrolled in clinical trials. The demographics of the cohortsare presented in Table 1.

Malaria Diagnosis and Sample SelectionIn the Tanzanian cohort, PM was detected by microscopy of Giemsa-stained thick and thinsmears of blood extracted from placental tissue by mechanical grinding. Placental parasitedensity was quantified as percent IE. The primary analysis is among PM-positive women inorder to identify features that are specifically linked to poor outcomes within this group. Inthe cohort from the Thai-Burma border, malaria episodes were detected by peripheral bloodsmear at weekly antenatal clinic visits and at delivery, with additional examination ofmother peripheral blood and placental blood collected by incision at delivery. Samples wereselected from among those with peripheral blood parasitemia at least 2 weeks prior todelivery. Only women with recent infection were included to avoid examining negativehistopathology samples (6), and to facilitate comparison with the Tanzanian cohort.

HistologyParaffin-embedded blocks were previously generated from tissue samples collected on theThai-Burma border (6), from which Giemsa-stained slides were made (Fig 2A–B). InTanzania, placental tissue was placed in polyvinyl alcohol, snap frozen in liquid nitrogen,and stored at −80°C. Sections were made on a cryostat, air-dried, methanol-fixed andGiemsa-stained for 12 minutes (Fig 2C–E). The proposed grading scheme is detailed belowin the results section. Placental malaria episodes were also classified as acute or chronic(14), with chronic PM defined by the presence of pigment in fibrin (greater than 1 in 50 highpower fields).

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Biomarker analysisQuantitative PCR was performed as previously described (18). Briefly, total RNA wasextracted from frozen placental cryosections using RNeasy mini kits (Qiagen) and realtimePCR was performed using SYBR Green Master Mix, an ABI Prism 7500 system (AppliedBiosystems) and intron-spanning primers for CXCL13 and for KRT7 (a gene expressed bythe trophoblast).

Statistical analysisAnalyses were performed using Statview and SAS (SAS Institute). Continuous andcategorical variables were analyzed by Student’s t-test and Fisher's exact test. Bar graphs arepresented as mean (standard error). Percent infected erythrocytes were log transformed priorto analysis. Logistic regression and analysis of variance (ANOVA) were used formultivariate analyses.

RESULTSThe Inflammation Score

The inflammation categories are qualitative and are intended to reflect distinct biologicentities (Fig 3A). “Minimal inflammation” (I) describes cases with no appreciableintervillous inflammation. Pigmented monocytes are rare, and the intervillous space whitecell density is not increased above the level of peripheral white blood cells expected intransit. “Inflammation present” (II) describes cases with mononuclear cells sequestering inthe intervillous space, particularly pigment-laden macrophages. This is a broad categorydescribing the intermediate stage between no inflammation and massive intervillositis.Massive intervillositis (III) is a distinct entity where the intervillous space contains sheets ofdensely packed mononuclear cells (20–22). It was reported in 6.3% of PM cases in southernTanzania (8), in 7 of 102 (6.9%) PM cases in MOMS Project in northeastern Tanzania, andin 1.7% (3/175) of women who had malaria during pregnancy on the Thai-Burma border (6).Massive intervillositis is a rare finding in the absence of malaria, where it is associated withrecurrent miscarriage (20).

The Pigment Deposition ScoreThe pigment deposition score is semiquantitative and can be rapidly assessed by quantifyingthe percentage of high power fields that are positive for pigment in fibrin within intervillousspaces (Fig 3B). The scoring method excludes pigment in erythrocytes or monocytes.Malaria pigment is golden brown after Giemsa stain, and can either be identified on paraffinor frozen sections. The use of a 60× objective is recommended, because pigment is presentin fine granules, and at least 40 fields within the intervillous space are counted. The totalfield count excludes stromal tissue within the decidua, basal plate and stem villi. Althoughthe total amount of fibrin may be variable, the total field count includes fields withinintervillous spaces that do not have fibrin.

The categories are determined as follows: I (<10%), II (10–40%) and III (>40% of fieldspositive). The cut off values of 10% and 40% per high power field corresponded to the 50th

and 90th percentiles respectively for both multiparous Tanzanian PM+ women and for theselected cohort from Thailand (all parities). Among first-time mothers with PM fromTanzania, these values corresponded to the 20th and 75th percentiles respectively.

A null (“0”) category could be considered for women if pigment is absent, particularly if thepurpose of the study is to detect past infections during drug trials. In the MOMS Project,pigment was absent in 10% of PM-positive women, and these women had a trend towardsincreased birth weight and multiparity (data not shown). We elected to include these cases in

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category I for this study because the criteria for pigment exclusion would require a greateramount of tissue examined and would be problematic in formalin-fixed tissues with even ascant amount of formalin pigment deposition. In Tanzania, among PM-negative first-timemothers, histologic evidence of past infection was not associated with decreased birthweight (n=58 of 104; p=0.462).

The Tanzanian CohortOf 984 singleton deliveries in Muheza, Tanzania, 12% (124/984) had PM, as determined byplacental blood smear. Tissue was available for frozen section histology on 82% (102/124).Based on the traditional histologic grading scheme, chronic PM was associated with adecrease in mean birth weight compared to acute PM that approached significance (0.21 kg,p-value 0.057). When the cohort was stratified by inflammation and pigment depositionscores using the new scheme (Fig 4), both scores were associated with birth weight. Pigmentand inflammation scores were strongly related (p-value 0.005), but nevertheless wereindependently associated with birth weight by ANOVA (p=0.016 and 0.005, respectively;n=101). Inflammation and pigment scores of III were independently associated with lowbirth weight by logistic regression analysis (p=0.003 and 0.017, respectively; n=101).

Primiparous women were analyzed as a subgroup because first-time mothers are at risk forpoor outcomes (Fig 5). Based on the traditional grading scheme, chronic PM was notassociated with a significant decrease in mean birth weight compared to acute PM (0.22 kg,p-value 0.46). Pigment and inflammation scores were related (p-value 0.042, n=47), andbirth weight was inversely associated both with inflammation and with pigment scores,however by multivariate analysis neither pigment nor inflammation was independentlyassociated with birth weight. Placental parasitemia was inversely associated with pigmentdeposition. Placental mRNA levels of CXCL13, a PM biomarker, were 200-fold higher inplacental tissue with inflammation score III versus score I, and 16-fold higher with pigmentscores of III versus I.

The Thai-Burma CohortSamples collected during a histopathologic study at the Thai-Burma border from 1995–1997(6) were reviewed. Of the 175 placental samples from women with malaria episodes, 18women had P. falciparum within the 2 weeks prior to delivery (Range 0–10 days, median 2days) and were included for analysis. Inflammation and pigment scores I through III wereobserved. A single sample demonstrated both pigment and inflammation scores of III, andno inflammation was identified in 10 and no pigment was identified in 5 of 18 samples.Inflammation and pigment were not significantly related in this small sample size (p-value0.28).

We conducted preliminary analyses of this small sample size to examine trends inrelationships between scores and clinical outcomes. Pigment deposition together withinflammation by histology was associated with decreased birth weight (Fig 6). Estimatedgestational age (EGA) did not significantly differ by inflammation or pigment category,although EGA did decrease with increasing pigment. The number of antenatal clinic visits(where women are screened and promptly treated for malaria) was inversely related withpigment, but this was not significant. CXCL13 was not examined in these samples becauseof formalin fixation.

Comparison across study sitesA formal analysis across study sites is limited by differences in study design, malariadiagnosis, and histologic technique. Samples from the Thai-Burma border were compared tothose from Tanzanian first-time mothers, the group that has the least immunity and is most

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susceptible to PM. Pigment deposition was significantly lower in the Thai-Burma cohort (p-value = 0.002), but the presence or degree of inflammation did not significantly differ (p-value = 0.267, n= 18 Karen versus 47 Tanzanian). The parasite densities in the placentawere not examined across sites due to different modalities of tissue processing and malariadiagnosis.

DISCUSSIONThe clinical and epidemiologic features of PM vary widely with malaria transmission levels,as do control measures. We developed a 2-parameter grading scheme measuringinflammation and pigment deposition that is simple to use, and captures more informationthan earlier schemes. We find that the criteria for histologic classification are applicable inan African setting with high malaria transmission and an Asian setting with lowtransmission. Further, the scores are significantly related with pregnancy outcomes in arelatively large Tanzanian cohort, and have similar trends in a small study of Karen womenin Thailand. Based on these preliminary findings, we propose that this scheme be furtherevaluated in clinical trials, where it may facilitate comparison and standardization of resultsbetween sites.

As a result of differences in exposure, immunity, and treatment, malaria episodes arecomplex. Chronic PM can result from several factors, including low host immunity and lackof effective antenatal control measures. Women can be inoculated multiple times duringpregnancy, be treated or not treated adequately, and experience reinfections andrecrudescences (23,24) (Fig 1B). For example, in a recently published treatment study alongthe Thai-Burma border 253 women had a median number of 2 (range of 1 to 11) episodes ofmalaria (P.falciparum and P.vivax) detected and treated in pregnancy (25).

In this study, pigment deposition and inflammation were associated with decreased birthweight at two distant sites. In Tanzania, both pigment deposition and inflammation wereindependently associated with low birth weight in the overall cohort but not the primiparoussubgroup. Inflammation by histology was strongly associated with CXCL13, a proposedbiomarker of inflammatory PM (22), and has been linked to maternal peripheral blood levelsof IL10 (27), suggesting utility for these or other markers to monitor placental inflammationprior to delivery.

On the Thai-Burma border, the relationship between pathologic features of malaria andoutcome differs from areas of high stable transmission. In one study where all episodes ofperipheral parasitemia were detected by active screening during the pregnancy and treated,antenatal P. falciparum or P. vivax infections were both associated with birth weightreduction (26), although the impact was greater with P. falciparum than with P. vivax. Noplacental pigment was observed in 33% (16/49) of women with documented P. falciparuminfections during pregnancy (6). However, pathological changes were more likely to beobserved when malaria was diagnosed in the month before delivery, and more pigment (inimmune cells and fibrin) was observed when malaria was diagnosed in the week beforedelivery (6).

This is the first study to document conserved histopathologic features in pregnancy malariabetween Africa and Asia. Although a formal analysis across study sites is precluded bydifferences in study design and histologic technique, samples from Tanzanian first-timemothers were compared to selected samples representing the small number of Karen womenwho experienced P. falciparum malaria within the last 2 weeks of pregnancy. Thistimeframe was chosen in order to maximize the likelihood of finding histologic changes,including the presence of pigment (6). The histologic degree of placental inflammation did

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not significantly differ between these two groups, however there was significantly decreasedpigment deposition in the Karen women. This likely reflects the increased inoculation rateand limited treatment and antenatal care efficacy in Tanzania versus the effect of earlydetection and prompt treatment on the Thai-Burma border, since pigment deposition wasinversely related to frequency of ANC visits among Karen women. These differences mightalso reflect the differing biologic timelines that regulate inflammation and pigmentdeposition during PM: inflammation changes more dynamically in response to parasitemia,whereas pigment persists over a longer period of time (Fig 1). Interpretation of theassociations of inflammation and pigment with birth weight would have been improved ifgestational age data were available for all the study sites, and would have allowed theassociation to be examined in growth-restricted term infants.

This study was not powered to analyze the result of HIV co-infection (n=4, Tanzaniancohort). Since HIV and malaria both affect birth weight and pregnancy outcome, HIV couldalter the reported findings. Several studies have found an increased risk of placental malariawith HIV co-infection in endemic areas (28,29), and that PM episodes are more likely tohave greater parasitemia and increased pigment deposition by histology (28). Co-infectedwomen exhibit altered humoral immune responses to placental IE (30) and immune cellsisolated from the intervillous spaces of co-infected women exhibit decreased IL-12production (31) and contain an expanded CD16+ monocyte subset (32). In areas of lowtransmission, such as the Thai-Burma border where HIV prevalence is low (33), the effect ofHIV co-infection on PM episodes has not been determined.

Here, we demonstrated that the new grading scheme is applicable on both frozen andformalin-fixed paraffin-embedded (FFPE) sections. A direct comparison of frozen versusFFPE histology to detect the features of PM was not performed and would require bothmethods to be performed on the same sample population. FFPE tissue preserves fine detailsof cellular morphology and erythrocytes remain intact, whereas in frozen tissue cellulararchitecture is disrupted and erythrocytes lyse. The formation of formalin pigment or acidhematin during prolonged storage or during processing of FFPE tissue can obscure malarialhemozoin, often precluding analysis; formalin pigment formation is reduced with smallertissue samples, generous amounts of buffered formalin, and prompt passage through gradedalcohols following fixation. Frozen tissue is handled minimally, decreasing the concern ofwashing out intervillous contents. Frozen section histology is not routinely available in mostrural tropical areas, however the infrastructural costs are significantly less than those forFFPE histology. Frozen sections have several advantages: no formalin pigment artifact,reduced laboratory infrastructure, and preserved tissue that yields high quality RNA (22,23),antigens (22), protein and DNA for use in molecular analyses.

In the Tanzanian cohort, parasitemia was diagnosed by placental blood smear, andparasitemia per se is not included in the proposed grading scheme. Placental blood smearsmay not be available for all studies, with diagnosis and quantification of parasitemia relyingon histologic sections or other ancillary tests. As described by Ewing (34), and in ourexperience (AM, MF), the identification of low level parasitemia (<1%) by histology ischallenging due to routine dehydration and sectioning of erythrocytes and can be precludedby intraerythrocyte formalin pigment formation. Parasitemias above 5% may be readilydetected and parasitemia quantified in histologic sections.

In this paper, PM refers strictly to the placental sequestration of P falciparum IE because itis not known whether all P. falciparum episodes during pregnancy have a placentalphenotype, and there is no evidence at this time that P. vivax sequesters in the placenta. Themechanisms by which malaria episodes (both P. falciparum and vivax) in the first or secondtrimester lead to poor birth outcomes are not known, particularly in the absence of malaria-

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specific histologic features at term. Pathologic studies of miscarriages due to both species ofmalaria, or longitudinal studies that incorporate ultrasound measurements and biomarkers ofinfection would be of great interest.

In conclusion, we provide a semi-quantitative pathological grading scheme is simple toimplement, specifically documents inflammation, and is associated with outcomes. Futureclinical trials may benefit from this scheme for evaluation of histologic endpoints and forcomparison across study sites.

AcknowledgmentsBillie Davidson performed initial histology on the cohort of Karen women. Ronald S. Veazey coordinated thetransfer of samples. Corinne Fligner contributed to the pathological analyses. Wonjong Moon contributed todemographic analyses of the Tanzanian cohort. This work was supported by a University of Washington HouseStaff Association grant and the Benjamin H Kean fellowship from the American Society of Tropical Medicine andHygiene (to AM). The Shoklo Malaria Research Unit is part of the Wellcome-Mahidol University- Oxford TropicalMedicine Research Program funded by the Wellcome Trust of Great Britain. Part of this work was supported byPREMA-EU (Contract no ICA4-CT-2001-10012). The Mother Offspring Malaria Studies Project was supported bygrants from the Bill and Melinda Gates Foundation (29202) and National Institutes of Health (R01AI52059) toPED.

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22. Ordi J, et al. Massive chronic intervillositis of the placenta associated with malaria infection. Am JSurg Pathol. 1998; 22:1006–1011. [PubMed: 9706981]

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24. Mutabingwa TK, et al. Randomized trial of artesunate+amodiaquine, sulfadoxine−pyrimethamine+amodiaquine, chlorproguanal-dapsone and sp for malaria in pregnancy in tanzania. PLoS One.2009; 4:e5138. [PubMed: 19352498]

25. McGready R, et al. A randomised controlled trial of artemether-lumefantrine versus artesunate foruncomplicated plasmodium falciparum treatment in pregnancy. PLoS Med. 2008; 5:e253.[PubMed: 19265453]

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https://www.researchgate.net/publication/38045500_Contribution_to_the_Pathological_Anatomy_of_Malaria_Fever?el=1_x_8&enrichId=rgreq-fadd967aae318013e0e0dc3184272b81-XXX&enrichSource=Y292ZXJQYWdlOzQ3MzY5NTA5O0FTOjEwMTc1ODI2OTM5NDk0NEAxNDAxMjcyNDcwNDMy

https://www.researchgate.net/publication/38045500_Contribution_to_the_Pathological_Anatomy_of_Malaria_Fever?el=1_x_8&enrichId=rgreq-fadd967aae318013e0e0dc3184272b81-XXX&enrichSource=Y292ZXJQYWdlOzQ3MzY5NTA5O0FTOjEwMTc1ODI2OTM5NDk0NEAxNDAxMjcyNDcwNDMy

https://www.researchgate.net/publication/12216364_Chronic_histiocytic_intervillositis_A_placental_lesion_associated_with_recurrent_reproductive_loss?el=1_x_8&enrichId=rgreq-fadd967aae318013e0e0dc3184272b81-XXX&enrichSource=Y292ZXJQYWdlOzQ3MzY5NTA5O0FTOjEwMTc1ODI2OTM5NDk0NEAxNDAxMjcyNDcwNDMy

https://www.researchgate.net/publication/12216364_Chronic_histiocytic_intervillositis_A_placental_lesion_associated_with_recurrent_reproductive_loss?el=1_x_8&enrichId=rgreq-fadd967aae318013e0e0dc3184272b81-XXX&enrichSource=Y292ZXJQYWdlOzQ3MzY5NTA5O0FTOjEwMTc1ODI2OTM5NDk0NEAxNDAxMjcyNDcwNDMy

https://www.researchgate.net/publication/15696465_Batts_KP_Ludwig_J_Chronic_hepatitis_An_update_on_terminology_and_reporting?el=1_x_8&enrichId=rgreq-fadd967aae318013e0e0dc3184272b81-XXX&enrichSource=Y292ZXJQYWdlOzQ3MzY5NTA5O0FTOjEwMTc1ODI2OTM5NDk0NEAxNDAxMjcyNDcwNDMy

https://www.researchgate.net/publication/15696465_Batts_KP_Ludwig_J_Chronic_hepatitis_An_update_on_terminology_and_reporting?el=1_x_8&enrichId=rgreq-fadd967aae318013e0e0dc3184272b81-XXX&enrichSource=Y292ZXJQYWdlOzQ3MzY5NTA5O0FTOjEwMTc1ODI2OTM5NDk0NEAxNDAxMjcyNDcwNDMy

Figure 1.Schematics of A) a hypothetical single PM episode in a susceptible woman, and B)recrudescenses and reinfections in a single woman that can lead to varying degrees ofparasitemia, inflammation and pigment, especially when the drugs for IPT or treatment arenot effective.

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Figure 2.Formalin-fixed paraffin embedded placental tissue section from PM cases on the Thai-Burma border. A) Remote infection, with no histologic alteration. B) P. falciparum atdelivery, with inflammatory infiltrate (asterisk) and pigment deposition in fibrin (arrow)..Fresh-frozen Giemsa-stained sections from PM cases in Tanzania. The features of placentalmalaria are readily identified: C) Parasites; D) Inflammation; and E) Pigment deposition infibrin. 400× magnification.

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Figure 3.Schematic demonstrating placental villi (v) for grading the histologic features of PM. A)Categories of maternal inflammation in the intervillous spaces (ivs): I- minimal; II-present;III- massive. B) Cut off values for categorizing malarial pigment deposition withinintervillous fibrin (f). (% 60× high power fields).

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Figure 4.Inflammation and pigment scores in relation to birth weight in Tanzanian women of allparities. * p<0.05; ** p<0.01.

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Figure 5.Inflammation and pigment scores examined in Tanzanian first-time mothers. Relationshipbetween A) birth weight and pigment score, B) birth weight and inflammation score, C)placental parasitemia and pigment score, and D) placental CXCL13 transcript levels (foldover cytokeratin 7 expression) and inflammation score. * p<0.05; ** p<0.01.

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Figure 6.Inflammation and pigment scores in Karen women who had been diagnosed with P.falciparum malaria within 2 weeks of delivery. Relationships between: A) birth weight andpigment score; B) birth weight and inflammation score; C) estimated gestational age (EGA)and pigment score; and D) number of antenatal clinic (ANC) visits and pigment score. *p<0.05; ** p<0.01.

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Table 1

Pregnancy Malaria Epidemiology, Therapy, and Antenatal care in the Tanzania and Thailand cohorts.

Africa, Tanzania, 2002–2005 Asia, Thailand, 1995–2002

Malaria incidenceduring pregnancy

34% of women reportedtreatment for acute malaria

~5–30% with episodes duringpregnancy (varies by site);< 1 P.falciparum infectionper woman/year<1.5 P.vivax infection perwoman/year

Cases of pregnancymalaria due to non-falciparum species

< 1% ~60% P.vivax

Case-detectionmethod duringpregnancy

Passive (screening availableat ANC)

Active (weekly visits) andPassive (24 hour malariascreening available)

Placental malariaincidence at delivery

12.6% all women,19.4% of first-time mothers

< 1%

Women ReceivingAntenatal Care (ANC)

36% with documented ANCattendance83% with reported IPTpexposure

>90% attend ANC in refugeecamp

Average Number(Range) of AntenatalVisits

4 (1–12), wheredocumented by ANC card

Weekly ANC>50% in first trimesterAverage 15–20 visits

Preventive Treatment SP-IPTp None as high levels of MDR-Pf resistant strains. SP notused in Thailand > 30 years.

Women ReportingIPTp Usage

83% N/A

Average Number(Range) of IPTp doses

1 (0–3), where documentedby ANC card

N/A

Drugs used to treatmalaria duringpregnancy

ACT, quinine Quinine, mefloquine,artesunate monotherapy,ACT

Efficacy of availabledrugs

Limited:High level resistance to firstline therapy (SP) 2002–2008;Currently efficacious:ACT (artemether-lumefantrine), quinineSevere malaria=IV quinine

Limited:Quinine and mefloquinemonotherapy, 1995–2002Currently efficacious:1st trimester = QC72nd&3rd trimester =ACTSevere malaria=IVartesunate

Bednet usage 62%, any net19%, treated net

~90% treated net

Screening foranaemia: Hb or HCT

At most ANC visits Every 2nd week

Nutritionalsupplements

Ferrous and folic acid atmost ANC visits

At each visit:Prophylactic: ferrous 200 mgdaily and folic acid 5mg/wkTreatment: ferrous 400mgBID and folic acid 5mg daily

Antihelminthic policy All women: stool and urinetesting

Anaemic women stooltesting

Prevalence HIV inpregnant women

5–7%, from (35). <0.5%, from (33)

SP: Sulfadoxine pyrimethamine.


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IPTp: intermittent preventive treatment in pregnancy.

ACT: Artemisinin-based combination therapy currently (2009) artesunate+clindamycin 7 days.

QC7: Quinine and Clindamycin for 7 days.


A novel histological grading scheme for placental malaria applied in areas of high and low malaria...

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