Catherine Aude-Garcia

Véronique Collin-Faure

Sylvie Luche

Thierry Rabilloud
email: thierry.rabilloud@cea.fr

Improvements and simplifications in in-gel fluorescent detection of proteins using ruthenium II tris-(bathophenanthroline disulfonate), the poor man's fluorescent detection method

Fluorescent detection of proteins is a popular method of detection allying sensitivity, linearity and compatibility with mass spectrometry. Among the numerous methods described in the literature, staining with ruthenium II tris(bathophenanthroline disulfonate) is particularly cost-effective, but slightly cumbersome owing to difficulties in the preparation of the complex and complexity of staining protocols. We describe here modifications on both aspects that allow to perform a higher contrast staining and offer a more robust method of complex preparation, thereby maximizing the advantages of the method.

Numerous constraints apply to detection methods used in gel-based proteomics. They should be linear over a wide range, homogeneous from protein to protein, and of course sensitive. In addition to these core features, they should also be mass spectrometry-compatible, user-friendly and cost-effective. Among the variety of methods used for protein detection after gel electrophoresis [START_REF] Miller | Protein stains for proteornic applications: Which, when, why?[END_REF], fluorescent methods offer an interesting compromise, especially for detection linearity [START_REF] Patton | A thousand points of light: The application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics[END_REF] and for compatibility with mass spectrometry [START_REF] Chevalier | Different impact of staining procedures using visible stains and fluorescent dyes for large-scale investigation of proteomes by MALDI-TOF mass spectrometry[END_REF]. Among the wide variety of fluorescent detection methods that have been developed to detect proteins after gel electrophoresis, two have emerged as standards in the field, one using epicocconone [START_REF] Mackintosh | A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and twodimensional gel electrophoresis[END_REF] (marketed under the trade name deep purple) and one using an undisclosed fluorescent ruthenium complex and marketed under the trade name Sypro Ruby [START_REF] Berggren | Background-free, high sensitivity staining of proteins in one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex[END_REF][START_REF] Steinberg | Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain[END_REF]. Almost simultaneously with the description of Sypro Ruby staining, another stain using a published ruthenium complex (ruthenium II tris(bathophenanthroline disulfonate) was described [START_REF] Rabilloud | Comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels[END_REF] , with minimal interference with mass spectrometry and much improved cost efficiency compared to commercial formulations. However, the sensitivity of this stain was moderate, and a major improvement was published a few years later [START_REF] Lamanda | Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signalto-background ratio and improved baseline resolution[END_REF]. This improvement resulted, however, in a much longer staining period, extending to almost two days after the end of electrophoresis, thereby decreasing the overall productivity of the proteomics setup. This is why intermediate formulations were developed, claiming for equal sensitivity and much improved speed and simplicity of staining [START_REF] Pluder | One-step procedure for staining of proteins with ruthenium II tris (bathophenanthroline disulfonate)[END_REF]. Furthermore, all the protocols using ruthenium II tris(bathophenanthroline disulfonate) are plagued by difficulties in the preparation of the complex. Indeed this preparation involves both complex formation and reduction from ruthenium III to ruthenium II, as most water-soluble ruthenium salts are ruthenium III salts. In the published protocols, this reduction was achieved either with hypophosphoric acid and sodium hydroxide, or by ascorbic acid and sodium hydroxide. As both redox couples are pH-sensitive, the control of the extent of reduction is difficult to manage in the standard proteomics or biochemistry laboratory, so that preparation of the complex occasionally fails. We therefore decided to revisit both aspects of staining with ruthenium II tris(bathophenanthroline disulfonate), namely complex preparation on the one hand, and staining protocol on the other hand, aiming at more simplicity, robustness and performance. Ammonium formate is a weak but interesting reducing agent [START_REF] Andrade | One-step reduction of chalcones to saturated alcohols by ammonium formate/palladium on carbon: A versatile method[END_REF]. Its solutions are naturally close to neutral pH, thereby needing no pH adjustment, but it is effective only at relatively high temperatures and the overall reducing power is weak. In order to prepare an efficient ruthenium complex, we prepared a solution containing 20 mM potassium pentachloroaquo ruthenate (Alfa Aesar), 60 mM bathophenanthroline disulfonate, disodium salt (Aldrich) and 400 mM ammonium formate (available as a 10 M titrated stock solution from Fluka). Reduction of ruthenium III to ruthenium II and simultaneous complex formation was achieved either by refluxing the solution for three days, or incubating it at 95°C in an oven for three days. In the latter case, because of gas evolution during reduction, it was not possible to tightly close the vessel containing the solution. Thus, a beaker containing water was placed next to the ruthenium solution in the oven set at 95°C, in order to saturate the oven with vapor and limit evaporation in the ruthenium solution. However, some evaporation was frequently encountered, and compensated for at the end of the preparation by water addition to restore the initial volume. Although much longer to perform, we found this method to be much more reliable than those using ascorbate, hypophosphite or borohydride as reducing agents. In addition, the complex solution was found to be stable for over two years in the refrigerator, with no decrease in staining efficiency. This method using ammonium formate also allowed to replace potassium pentachloroaquo ruthenate by simple ruthenium III chloride at equal molar concentrations, and the resulting, even brighter complex was called alternate complex (see figures 1 and 2).

As to the staining protocol itself, we first compared the three published protocols. The initial protocol [START_REF] Rabilloud | Comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels[END_REF] (Figure 1A) was clearly less sensitive than the two improved ones [START_REF] Lamanda | Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signalto-background ratio and improved baseline resolution[END_REF][START_REF] Pluder | One-step procedure for staining of proteins with ruthenium II tris (bathophenanthroline disulfonate)[END_REF] (Figure 1B and1C), but the background was still unsatisfactory with the published protocols, as can be seen in Figure 1. We therefore decided to combine the staining in acidic solution with the destaining step to reduce background. A significant improvement was noted with classical, acetic acid-based solutions (Figure 1 D), but a further improvement in contrast was obtained when the acetic acidethanol solutions described in [START_REF] Lamanda | Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signalto-background ratio and improved baseline resolution[END_REF] and [START_REF] Pluder | One-step procedure for staining of proteins with ruthenium II tris (bathophenanthroline disulfonate)[END_REF] were replaced by a phosphoric acid-ethanol solution, both for staining and destaining (Figure 1 E), resulting in the simple protocol described here. This protocols describes as follows:

Step 1: after electrophoresis, fix the gel for 1 hour in 1% phosphoric acid (v/v, starting from commercial 85% phosphoric acid) and 30% ethanol

Step 2: stain overnight with 1µM ruthenium complex in 1% phosphoric acid-30% ethanol

Step 3: destain for 4-6 hours in 1% phosphoric acid-30% ethanol. Shorter destaining (1-2 hours) did not afford sufficient background reduction, while longer destaining (e.g. overnight) reduced the sensitivity. The times indicated here are therefore a good compromise

Step 4: rinse in water (one rinse, 5-15 minutes) prior to imaging. This step is just intended to avoid contaminating the UV table with phosphoric acid and ethanol from the liquid film coming along with the gels Further tests showed that the performances were maintained by using the complex prepared from RuCl3 in place of the complex prepared from K2RuCl5 (Figure 1F), and that the performances were marginally inferior to those obtained with Sypro Ruby (Figure 1G). However, fluorescent staining with such methods was clearly not as sensitive as a silver staining (Figure 1H)

We then evaluated the different staining methods in the context of two-dimensional gels, where the homogeneity of the stain and interference from carrier ampholytes can be evaluated, and the results are described in Figure 2 and in Supplementary Figure 1 with raw images (i.e. without color inversion and gray level conversion) . Here again, it can be seen that the improved protocol (Figure 2 panels A-D) is faster and/or more sensitive (because of better contrast) than those published previously [START_REF] Lamanda | Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signalto-background ratio and improved baseline resolution[END_REF][START_REF] Pluder | One-step procedure for staining of proteins with ruthenium II tris (bathophenanthroline disulfonate)[END_REF] (Figure 2 panels E-I). We also found during this step of the study that 1% phosphoric acid offers the best signal to noise ratio (Figure 2 panels B andD). Higher concentrations of acid increased the background and thus decreased sensitivity (Figure 2 panels A andC). We also tried to reduce further the phosphoric acid concentration below 1%, but the stain became more erratic and less reproducible. It is also noteworthy that gels stained by the method of Pluder et al. showed a very high background (Figure 2G), in contrast to what is described in the original publication [START_REF] Pluder | One-step procedure for staining of proteins with ruthenium II tris (bathophenanthroline disulfonate)[END_REF]. However, it shall be mentioned that the original publication uses a laser excitation at 532 nm, i.e. quite far from the excitation maxima of the ruthenium complex (280 and 470 nm), whereas excitation was performed at 312 nm in the present study.

Finally, we evaluated the new staining method in a real proteomic-type experiment based on two-dimensional gels, and the results are shown on Figure 3 and in Supplementary Figure 2 with raw images (i.e. without color inversion and gray level conversion). The detection protocol as a whole, i.e. staining and imaging, was found to be very reproducible, due to its low number of steps and to flexibility in each step duration. As a result, the median rsd (relative standard deviation) in a set of four parallel gels was found to be 15-16%, to be compared to the 20-25% previously described for several detection methods including colloidal Coomassie Blue [START_REF] Luche | Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines[END_REF]. We could stain up to four gels per dish, provided that 250ml of solution was present per gel (160x200x1.5 mm) at each step.

Compared to the early protocols [START_REF] Rabilloud | Comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels[END_REF][START_REF] Lamanda | Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signalto-background ratio and improved baseline resolution[END_REF], the new staining protocol uses far fewer steps, thereby minimizing the risk of external contamination of the protein spots, e.g. by keratins. Avoidance of acetic acid also decreases the risk of artefactual acetylation.

In conclusion, the modified protocol described in this technical brief offers an improved detection method using the ruthenium bathophenanthroline complex, being faster and simpler, and offering better contrast than previously published protocols using the same fluorescent agent. Furthermore, an improved preparation of the fluorescent complex that can be made in all biochemical laboratories without any special glassware is also described. This protocol offers the benefits of the metalbased fluorescent complex detection, i.e. sensitivity, robustness, resistance to fading, at a minimal cost. The proteins were loaded on top of 12% acrylamide gels ( 80 x 50 x 1mm) run in Tris-glycine buffer. After electrophoresis, the proteins were detected by fluorescence, and imaged on a UV table (312nm emission) with a digital camera (canon A95) equipped with a UV filter and a yellow filter to cut the light emission of the table. Photography for 4 seconds at f-stop 8.

A: staining by 200 nM complex in water, no destaining [START_REF] Rabilloud | Comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels[END_REF]. B: staining by 1 µM complex in water, destaining in 10% acetic acid+40% ethanol [START_REF] Lamanda | Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signalto-background ratio and improved baseline resolution[END_REF]. C: staining by 1 µM complex in 10% acetic acid+30% ethanol, no destaining [START_REF] Pluder | One-step procedure for staining of proteins with ruthenium II tris (bathophenanthroline disulfonate)[END_REF]. D: staining by 1 µM complex in 10% acetic acid + 30 % ethanol, destaining in the same solution. E: staining by 1 µM complex in 2% phosphoric acid + 30 % ethanol, destaining in the same solution F: staining by 1µM alternate complex (from RuCl3) in 2% phosphoric acid + 30 % ethanol, destaining in the same solution G: staining with Sypro Ruby [START_REF] Berggren | Background-free, high sensitivity staining of proteins in one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex[END_REF] H: silver staining (fast silver nitrate staining) [START_REF] Chevallet | Silver staining of proteins in polyacrylamide gels[END_REF] The strips were transferred on top of 10% acrylamide gels ( 80 x 50 x 1mm) run in Tris-taurine buffer. [START_REF] Tastet | A versatile electrophoresis system for the analysis of high-and low-molecular-weight proteins[END_REF] After electrophoresis, the proteins were detected by fluorescence, and imaged on a UV table (312nm emission) with a digital camera (canon A95) equipped with a UV filter and a yellow filter to cut the light emission of the table. Photography for 4 seconds at f-stop 8, unless otherwise indicated.

A: staining by 1 µM complex in 2% phosphoric acid + 30 % ethanol, destaining in the same solution B: staining by 1 µM complex in 1% phosphoric acid + 30 % ethanol, destaining in the same solution C: staining by 1 µM alternate complex in 2% phosphoric acid + 30 % ethanol, destaining in the same solution D: staining by 1 µM alternate complex in 1% phosphoric acid + 30 % ethanol, destaining in the same solution E: staining by the method of Lamanda et al [START_REF] Lamanda | Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signalto-background ratio and improved baseline resolution[END_REF] F: same as E, but photography for 2 seconds G: staining by the method of Pluder et al. [START_REF] Pluder | One-step procedure for staining of proteins with ruthenium II tris (bathophenanthroline disulfonate)[END_REF] H: same as G, but photography for 2 seconds I: same as G, but photography for 1 second Figure 3: reproducibility analysis in a proteomics context Total protein extracts from the M1 myeloblastoid cell line (ATCC TIB 192), induced or not with dexamethasone for 3 days, were analyzed by 2D electrophoresis. 300 µg were loaded on the first dimension gels (immobilized pH 4-8 gradients). The second dimension was 10% acrylamide gels, operating in the Tris Taurine system [START_REF] Tastet | A versatile electrophoresis system for the analysis of high-and low-molecular-weight proteins[END_REF]. Staining by 1µM complex in 1% phosphoric acid + 30 % ethanol, destaining in the same solution, as described in the main text. Imaging on a UV table (312nm emission) with a digital camera (canon A95) equipped with a UV filter and a yellow filter to cut the light emission of the table. Photography for 4 seconds at f-stop 8. The images were then analyzed by the Delta2D software (v 3.6, Decodon, Germany). For each group (control and treated cells) ca. 1600 spots were detected, and the median rsd (relative standard deviation) was calculated. The control group had a median rsd of 15.1 % and the treated group had a median rsd of 15.9 %. Two gels are shown for each group, while four were made and included per group for the computerized analysis.

A, B: control cells, fluorescence detection 300 µg/gel. C: control cells, 120 µg loaded, silver staining D, E: dexamethasone-treated cells, fluorescence detection 300 µg/gel. F: dexamethasone-treated cells, 120 µg loaded, silver staining

Figure 1 :

 1 Figure 1: sensitivity evaluation with 1D SDS PAGE

Figure 2 :

 2 Figure 2: sensitivity evaluation with 2D PAGE


Keywords: theexperienceofHIV-positive womeninBurkinaFaso BurkinaFaso, Africa, infantfeeding, HIV, localcultures, women'sstrategies, socialchange, PaulCezanneUniversityofAix-Marseille, InstitutdeRecherchespourle

In low-resource areas of West Africa where infant feeding patterns are dominated by prolongedbreastfeeding, theprevention ofmother-to-childHIV transmissionrequires new feedingpractices:formulafeedingorexclusivebreastfeedinglimitedtosixmonthsfollowed by rapid weaning. Both patterns are innovations for the majority of women in all social categoriesatthelocallevel.Asinnovations,thesepracticesareappliedonlyundercertain conditionsmetbymothers;theyalsohavesocialconsequences.Twoethnographicstudies conducted in 1998-2000 and in 2003-2007 explored women's perceptions about "good infantfeeding"withimplicationsfor"goodmothering,"andthesocialrelationshipsthatare involvedininfantfeedingmanagementinasettingshapedbypatrilinearorganization.These studiesalsoshowthecontradictionsthatHIV-positivewomenfaceintwolocalsub-cultures ofbreastfeeding:theoneinvolvingthebaby'sfather,thefamilyandneighborhood,andthe other involving health services and PLHIV support organizations. Women must rely on a rangeofstrategiestofacedifficultiesrelatedtothelackofeconomicorsocialautonomyor supportfromthechild'sfather,theriskofstigma,socialnormsregardingbreastfeedingand contradictorydiscoursesamonghealthworkers.HIV-positivemothers'experiencesbringto lightseveralkeyfeaturesoflocalinfantfeedingcultures,includingthechangesthatoccurred over the last ten years regarding the fathers' role, the impact of infant feeding conceptualization in biomedical institutions and the promotion of a model of infant care based on the dual mother-and-child relationship. These dimensions are considered in relationtogeneralsocialtrendsinaWest-Africansocietyregardingwomen'sautonomy,the roleofthecoupleinthehouseholdandthemedicalizationofinfantfeeding.

Facingcompetingculturesofbreastfeeding:theexperienceofHIV-positivewomenin BurkinaFaso

1.Introduction

ItiscommonlyassumedthatinallsocietiestheHIVpandemichasrevealedandtransformed socialpracticesandtheirsymbolicunderlyinglogics.Itsdimensionofrevelationisrelatedto the link between HIV modes of transmission and the circulation of biological fluids among individuals:HIVmanagementgivespublicexposuretopracticesthatareusuallyintimateor hidden.ItalsoappearswhenepidemiologicaldiscrepanciesinHIVprevalenceratesamong populations reveal underlying structural inequities. Its dimension of transformation is related,amongotheraspects,totheextentoftheepidemic'ssocialandculturalimpactand thepublichealthmeasurestofightit,withconsequencesatmicro-andmacro-sociallevels. Which significations might these notions hold regarding infant feeding practices in West Africa: were these practices or related norms, perceptions, social roles and relationships revealedortransformedbyHIV?

Despitehighnumbers(severalhundredthousandinfantsbecomingHIV-positiveeveryyear inAfricaduetotransmissionthroughbreastfeeding), 3 itwasonlyattheendofthe1990s 4 that this issue became a worldwide public concern beyond specialists' circles, when UN agencies published a policy (WHO/UNAIDS/UNICEF 1998). Until then, several factors had hinderedthedefinitionofpreventivestrategies,includingthelackofeasilyavailableandlow cost technical measures and the difficulty in implementing measures that could fit in with varied social, economic, educational and material environments, when all HIV-positive women had turned to replacement feeding in developed countries. Other factors included concernsaboutthespreadofformulafeedinginresource-poorsettings(seechapterXXby Thairu) and the conceptual and organizational difficulty for health agencies that promoted breastfeedingasahealthybehaviorwhenconsideringHIVtransmission(Desclaux2000).This situation of "secrecy and ignorance" about HIV transmission through breastfeeding and consequences regarding pediatric AIDS, while other aspects of the pandemic such as HIV sexual transmission and clinical aspects of AIDS in adults were fully acknowledged at the publiclevel,hadaparticulareffectonthecourseofHIV/AIDSpublicpoliciesregardingthis modeoftransmission,thatfirstpreventedany"revelation"or"transformation."

Over the next 11 years, 5 strategies proposed in 1998 were implemented, promoting particularinfantfeedingpracticesamongHIV-positivewomen-exclusivebreastfeedingwith 3 ThefirstestimatesofglobalfiguresforAfricabyUNagenciesremainedunpublisheduntilthe2000s. 4 This was 17 years after the identification of HIV and 8 years after the quantification of HIV transmission throughbreastfeedinginAfrica. 5 NewrecommendationshavebeenpublishedinNovember2009that,usingantiretroviraltreatments,propose that norms regarding infant feeding pattern for all HIV-positive mothers be defined at the national level, choosingbetweenexclusiveprolongedbreastfeedingorreplacementfeeding(WHO2009). early weaning 6 or replacement feeding-that fit in with local norms and habits among generalpopulations.Thedecisiontoimplementoneofthesetwofeedingpatternswas"left" to women, and should have resulted from an interaction with health workers during counseling sessions before the infant's birth. In this era of "HIV particularism," health administrationswererequestedtosetverticalprogramsforPreventionofMother-to-Child Transmission of HIV (PMTCT), responsible for making prevention available and sustainable forallwomenatanindividuallevel.Incountrieswherepreventiveinfantfeedingoptionsdid not fit in with local habits, HIV-positive women were supposed to adopt "new" patterns recommendedbythehealthsystem.Theirexperiencesatthisintersectionbetweentwosets of norms would depend on the nature and extent of divergences between locally-and medically-defined feeding patterns, the adaptability of each pattern to the local environment,themeaningsconveyedby"new"feedingpatternsatthelocallevelandthe socialconsequencesofadoptingparticularfeedingpatterns.

Thiseraof"HIVparticularism"mayend,replacedbyaneraof"normalization"whenrecent WHO recommendations will be implemented, since they request HIV-positive mothers to use preventive antiretroviral treatment and apply an infant feeding pattern defined at the national level. HIV-positive women's experience regarding infant feeding choice and implementation in the 2000s may remain unique. Though public health studies mostly assessed them through successes or failures in achieving safe practices, these experiences maybeconsideredasexperimentsofasocialinnovation,revealingthecapacityforchange andunderlyingdeepculturaltrendsinsocialcontexts.WasthisinnovationendorsedinWest Africa? What do HIV-positive women's experiences reveal about the local and medical culturesofinfantfeeding?HastheHIVepidemicjeopardizedlocalculturesofinfantfeeding andbroughtaboutchanges,oppositionorresistanceinpractices,meaningsordiscourses?

Burkina Faso is a particularly relevant site to study these issues. There, prolonged breastfeedingisappliedbymostwomenandishardlyever"exclusive." 7 TheHIVprevalence ratewas8.5%amongadultsatthebeginningofthe2000s,whichmeantthattheepidemic was"generalized"beyondvulnerablegroupsandeveryfamilyhadhadorwasrelatedtoa personlivingwithHIVoran"AIDScase." 8 InBurkinaFaso,WHOrecommendationsproposed that a very high percentage of women known to be HIV-positive adopt infant feeding practices that differed from local habits; the recommendations also outlined that feeding patternsshouldbedefinedbasedoneachindividualwoman'sdecision.

Transformations in infant feeding were experienced by HIV-positive women at the intersection of two "sub-cultures"-defined as systems of values, beliefs and behaviors associatedwithasocialgroup:(1)thelocalcultureofinfantfeedingrelatedtoinfantcare provision and perceptions of motherhood in a multiethnic, mostly patrilinear society with 6 Categories for breastfeeding practices, which have different rates for HIV transmission, include: exclusive breastfeeding, predominant breastfeeding, complementary feeding, breastfeeding, and bottle-feeding (WHO 2008,p.4).

7 Some 85% of children are still breastfed at the age of 20-23 months; 7% are exclusively breastfed at 0-7 months;50%getcomplementaryfoodat6-9months(DHS2003 epidemic,in1998-1999(DesclauxandTaverne2000).Thisenabledustoidentifythemain trends in perceptions and practices, changes over a five to eight year time span and variations between pilot sites hosting PMTCT programs receiving external support and "ordinary"sitesfromthenationalprogram.Italsoenabledustoidentifycommonsituations across countries and local cultural particularities regarding counseling (Desclaux & Alfieri 2009), interactions between mothers and health workers (Desclaux et al. 2006), roles of fathersandfeasibilityandacceptabilityofpreventivestrategies(Desclaux&Alfieri2008).

2.TheculturallandscapeofinfantfeedinginBurkinaFasointheearly2000s

In the early 2000s, feeding patterns most resembled those described in the 1980s in neighboringMali(Dettwyler1987 Developed Country," 11 most women do agricultural work and handicrafts or have small businessintheinformalsector,oftenasindividualentrepreneurs.Thissituationallowsthem to keep infants "au dos" (keeping them on their backs), while doing their work and breastfeeding at any time; situations of incapacity to breastfeed in the workplace are infrequent.

Infantsarebreastfeduntilthemeanageoftwoyears:itmaybemuchlater,sometimesupto fiveyears,orlessifthemotherbecomespregnantagain.Waterandherbalteasaregivenas early as the first days of life, considered as cleansing or preventive liquids rather than as nutritive;thisincludeslustralwaterproducedbyusingwatertorinseKoranicverseswritten on a slate and retaining the water as an "indigenous vaccine." Mothers start giving small pieces of various foods and millet gruel when the infant is three to four months or when teethappear.Thisgruelmaybeenrichedwithsugarandsalt,apieceofsmokedfishoran egg, as advised by Mother-and-Child Health Services or elder women. Concentrated or powdered milk is also often added, and this gruel is used until the infant is six or seven months old, progressively becoming the predominant food. Fruits such as bananas or orangesarealsogivensquashed.Infantsareweanedratherrapidlywhentheygainacertain levelofautonomyandmayreceivefoodfromthefamilydish.However,weaningremainsan importantstageinlocalperceptionsofinfantdevelopmentandisnotconsideredbeforethe childlookshealthyandstrongenough.

Infant feeding practices are firstly a matter of inter-generational transmission between women at the household level. The influence of "health education" in Mother-and-Child HealthServicesisalsoperceptibleinwomen'swordswhentheyexplaintheadvantagesof breastfeedinginnutritionaltermsandwhentheystatethatbreastfeedingis"thebestway tocareforone'sbaby,"shiftingtonormativestatements.Theseperceptionsareshapedby thediscoursesofbreastfeedingpromotion,whichdevelopedinWestAfricaduringthe1990s (Desclaux 2000). Though Burkina Faso was never affected by the extension of formula feeding, programs were developed by UNICEF and the Ministry of Health to promote breastfeeding(ProgrammeHôpitauxAmisdesBébés,Programmenationaldepromotionde l'allaitement maternel) with the help of a national association (APAIB: Association pour la promotion de l'Alimentation Infantile au Burkina Faso) and an international nongovernmental organization based in Ouagadougou (IBFAN-FAN: International Baby Food Action Network-French speaking African Network). APAIB brought together mostly health workerswhowerealsoexperiencedmothersandworkedtosetupatechnicaldiscourseand "best practices" regarding breastfeeding, focusing on feeding positions, duration, mastitis prevention and treatment and exclusive breastfeeding, among other topics. However, besidesthisattemptto"professionalize"infantfeedingcare,breastfeedingremainsmostlya matteroffolkhealingandhouseholdcare.Exclusivebreastfeedingwasmainlyadoptedby women who were influenced by health workers-often as friends or relatives-or NGO members.

11 AccordingtothisUnitedNationscategorization,thesecountries(49in2009)meetthefollowingcriteria:low income,humanresourcesweaknessandeconomicvulnerability.AccordingtotheGPI,BurkinaFasoranked164 forGrossNationalIncomepercapitawith480USDandranked177fortheHumanDevelopmentIndexin2008 (WorldBank,seeUNICEF2007).

Moreover, the social uses of breastfeeding promotion went beyond the limitations of the healthsystem:agrowingnumberofstakeholdersfelttheyhadsomethingtosayregarding breastfeeding,whenevertheirdiscoursewaslimitedtoanormativeassertionwhendirecting mothers to breastfeed; this was the case for political and religious authorities locally involved in pro-breastfeeding activities, particularly during Breastfeeding World Week (Desclaux 2000). Overall, the promotion of breastfeeding has seldom introduced new concepts, 12 techniques or practices at the community level, but it has introduced notions thatconvergewithlocalperceptionsinfavorofbreastfeedingandhasgivenbreastfeedinga moral content and a strong normative value. These notions put forward biological (nutritional) and social (pro "interaction between mother and infant") advantages and turned breastfeeding into a symbol of "a mother's love"-a notion rooted in a Western culturalunderstandingofindividualrelationshipsinnuclearfamilies.Womencombinethese notions with body perceptions that are based on local ethnophysiology and the actual inscribingofmother-childrelationshipintothefamily,asdefinedbykinshipstructures. 13 Infantfeedingpracticeshaveundergonerecentchangesinotheraspects.Forinstance,two decadesago,itwasstillcommontoencounteraninfantbreastfedbyawomanwhowasnot his/herbiologicalmother.Aclassificatorymotherinthelocalkinshipsystem,whetheritbea biological grand-mother or a mother's sister, or any woman, might replace the mother by chance if the infant was found crying from hunger or fatigue, or in case of the mother's unavailabilityforeithershortorprolongedperiodsduetoillness,absenceordisqualification byanethnomedicaldiagnosisofinsanity.Thesepracticesseemtohavedisappearedinurban settings,exceptforafewhoursafterbirthwhenamother'ssistermayfeedtheinfantuntil themothergetsenoughmilktodoitherself;replacementbreastfeedingisnowlimitedto situationsofreplacementmothering-suchasfostering.Thisevolutionseemstoresultfrom theinfluenceofhealthworkersthatdiscouragedbreastfeedingbywomenwhowerenotthe biologicalmother,evenbeforetheHIVpandemicoutbreak-whichreinforcedthistrend.

3.TheHIVoutbreak:introducing"choice"ininfantfeeding

The UN agencies' recommendations regarding prevention of HIV transmission through breastfeeding were first applied in Burkina Faso within pilot projects either supported by NGOs or implemented in conjunction with clinical trials. It took a few years to adapt a strategybeyondthesesitesinanenvironmentthatlackednutritionalsecurityandadequate sanitation. In 2000, the National PMTCT program adopted the international recommendations that promoted "informed choice" by women. After undergoing a collectiveand(atleastone)individualcounselingsessionduringantenatalvisitsafterherHIV statuswasdisclosed,awomanshouldopteitherforexclusivebreastfeedingwithearlyand rapid weaning between four and six months, or for replacement feeding if feasible, accessible,acceptable,sustainableandsecure. 14 12 Forinstancethosementionedinfootnote4. 13 FormoreinformationontheseaspectsseeDesclaux&Taverne2000.

14 Inbothcases,psychosocialandnutritionalcounselingshouldbeprovidedregularlyinpost-natalclinicsand thewomanshouldgethelpinnon-governmentalorganizationsandassociationssupportingpeoplelivingwith HIV. In addition, other measures were progressively set up to consider mothers' care for HIV, based on the scaling-upofHIVcareandantiretroviraltreatmentavailability.

In practice, many women had the feeling that what was labeled as "choice" by health workers was instead a decision made under constraints, since the cost of formula and its preparation prevented many of them from considering this option. The second main constraint was the social risk due to HIV stigma related to formula feeding, since, as one mother said about people in Bobo-Dioulasso: "They say that all mothers who do not breastfeed are HIV-positive." The ability to "have a choice" depended essentially on the context:anactual"choice"occurredinservicessupportedbyanNGOoraresearchproject that could provide long-term material and psychological support to women; in other facilities,womenwerecompelledtoturntoexclusivebreastfeeding,exceptthosewhocould get economic support from the child's father. Besides these structural-economic and social-factors,women'schangesininfantfeedingpatternswerealsoinfluencedbytheway "counseling for infant feeding" was interpreted by health workers. Some of them decided which option would be the best for women, based on their social and economic status. In other health services however, where formula and equipment were provided, women reporteddiscussionswithcounselorsontheadvantagesandinconveniencesofeachfeeding option.Interviewsshowthatinmostcases,womenwouldpreferreplacementfeeding,since any risk of HIV transmission seems unbearable for a mother to consciously take for her infant. In the "balance of risks," compared to HIV infection, the seriousness of diarrhea or malnutrition related to formula feeding was considered insignificant, since these diseases arecommoninBurkinaFasoandcurable. 15 Observations of counseling sessions and narratives of infant feeding decisions by women showedthatfatherswereoftenconsultedbeforethedecision,whichmightinvolvewomen's HIV status disclosure. Health workers encouraged this disclosure but it was not always planned and might be unavoidable when a father's suspicion was aroused by unusual discussion about infant feeding, normally a banal infant care practice, or when the father hadsomeknowledgeaboutHIVtransmissionthroughbreastfeedingandpersonalreasonsto suspectthewoman'sHIV-positivestatus.Inanycase,despitethehealthsystem'seffortto support a rational decision based on the respective advantages and risks of every feeding option, this "choice under constraints" of an infant feeding option involved relational aspectswithastrongemotionalcontentforwomenwhofrequentlyhadlimitedtimetoplan fortheirinfantfeedingdecisionaftertheywereinformedabouttheirownHIVstatus. Regarding practices, some women managed to implement formula feeding, mostly in pilot sites and when living with a certain level of social and economic autonomy or education. Manymothersfaceddifficultiesforimplementationintheirhouseholdduetotheeconomic and social constraints mentioned above, particularly in cases of shortages of formula providedbythenationalprogram.Contrarytowhatsomehealthworkershadanticipated, exclusivebreastfeedingwasnoteasy,duetothediscrepanciesbetweenthisfeedingpattern and usual ones, for instance, a mother applying a different one from the one used with a previous child. Among the women we met, many made a "default choice" to breastfeed exclusively, then practiced mixed feeding (giving liquids in addition to breastfeeding). This 15 However,breastfeedingwassometimeschosenforitscontraceptiveeffectsbywomenwhocouldnotaccess other contraceptive means. The rate of contraceptive coverage in Burkina Faso is low; moreover, access and information about contraception among persons living with HIV, as a component of HIV care, are poorly managedinmanycountries:itisaconcernofUNagencies. situation 16 resultedfromtheirinabilitytoconstantlyavoidtheintroductionofliquidsorfood otherthanbreastmilkforsixmonths.However,womensetupvariousstrategiestocomply with recommendations and to hide their unusual "choice" on matters previously managed byfamilyandcommunity"know-how."

4.HIV-positivemothers'strategiesinthecommunity

Thestrategiessetupbywomentoavoidthesocialconsequencesofdiscrepanciesbetween preventivepatternsandthelocalsub-cultureofinfantfeedingareparticularlynoticeablefor threeaspects:avoidanceofHIVstigma,implementationof"proper"breastfeedingandthe relationship with the father. Some mothers invented infant feeding patterns that had not been proposed during counseling but that allowed them to minimize the social risks, the economiccostandthebiologicalrisksofHIVtransmissionwhilepreservinginfanthealth.

Since infant breastfeeding is a common public practice when infants are carried on their mothers'backsuntiltheycanwalkalone,orevenlaterwhentheyaretired,commentson avoidanceofbreastfeedingpracticemaycomefromthefamilyandkinship,neighborhood, mothers'friendsorwomenencounteredinpublicplaceslikethemarket,thefieldorother work places. The use of formula or early weaning may be equivalent to public disclosure aboutamother'sHIV-infectionandarouseinterpretationsaboutthewoman'sabilitytobea "good mother." Some mothers would combine options on a sequential mode, applying breastfeedingforafewdaysorweeksafterbirthtobeacknowledgedasa"goodmother" and to avoid being labeled as HIV-positive then turning to formula feeding to prevent transmission. This use of breastfeeding as "complementary feeding" was also adopted by mothers who maintained or re-started breastfeeding instead of replacement feeding if formula became unavailable or was provided in quantities perceived as insufficient; it also allowed women to avoid suspicion of being HIV-positive. In both cases, the use of breastfeeding for social reasons was not an easy experience for women who feared HIV transmission,sincetheyfelt,asonemotherstated:"EverytimeIfeedmybabyItellmyselfI may be transmitting the virus." Their strategy to reduce the overall proportion of breastfeeding in infant feeding was their interpretation, which was not considered by the PMTCTprogram. HIVstigmawasespeciallydifficulttoavoidforpoorerwomenwhoseformulawasprovided by the national program or in pilot sites. In their case, their supposed ability to meet this cost was questioned by the household or neighbors; women had to provide believable reasons for using expensive food instead of free mother's milk. Some of them evoked medical reasons that protected them from being considered HIV-positive: inadequate lactation, a breast pathology such as mastitis, breast cancer, etc., which some health workershelpedtoputintoconvincingwords. Formanymothersthegreatestdifficultywasavoiding"herbalteas"consideredessentialby elder women who have authority on infant care in the household. On that matter as on others,themother'sabilitytoignoreorcontradictelderwomen,whoseexpertiseshouldbe acknowledged by all young women, depends on her relative status in the household and 16 MixedfeedingcarriesthehighestlevelofriskforHIVtransmission.

relationshipwiththeinfant'sfather,sinceanelderwomancouldeasilygetayoungmother rejected.Inthatcasetoo,somewomenmanagedsocialandbiologicalrisksinadiachronic way: they would start with non-exclusive breastfeeding by letting their mothers-in-law performritualbathingontheinfantdespitethepossibilitythatthisincludesfeedingliquids, thenturntoexclusivebreastfeedingfortheremainingmonths.

Water is also often given to infants by female relatives or neighbors, for instance as a "greeting present" for a mother and her infant when they visit a household. One way to comply with preventive recommendations according to mothers' emic perceptions is to requestthatonlybottledwaterbegiventotheirinfants,sincetheyconsideritpurerthan other kinds of water. 17 Lastly, the ultimate strategy to avoid confrontation, which was adopted by many women to varying degrees, was to remain secluded with their infants, protecting them from any social contact that could jeopardize the exclusiveness of breastfeeding or lead to criticism and questioning. For this reason, some women avoided being helped by young family housekeeping assistants who usually take care of infants. Others would adopt a less extreme strategy, for instance by returning to their parents' household for a few months where they could more easily limit social contact for their infants. Regarding fathers' attitudes towards mothers, our study shows a slight change between 1999and2007.Ininitialinvestigations,mothersgenerallyconsideredfathersasobstaclesto changes in infant feeding practices, since they were reluctant about women's unusual feedingpracticesexceptincasesofamedicallydefinedreason.Theyplayedanactiverolein feedingchoicesfortheirinfantsonlywhenmothersweretooilltodoso(orweredead)and whenfamilysupportfromwomenwaslacking.Atthattime,fewwomendisclosedtheirHIVstatustotheirpartners:inourfirststudyonlyone-thirdhadtoldthemsixmonthsafterthe infant's birth. Most women feared rejection, when being the first one in the couple to be diagnosedwithHIVoftenmeantherbeingconsideredastheonewhohadbroughtHIVto thecouple.Inrecentinterviews,womendescribemostfathersassupportiveorindifferent, whichisofteninterpretedbymothersastoleranceabouttheirHIV-statusandperpetuation of ascribed social roles that give mothers full responsibility for infant care. Fewer women thaninourpreviousstudy(aboutone-third)maintainedsecrecyabouttheirHIV-statusasa strategy to avoid fathers' adverse reactions. In most cases, they used many precautions whencommunicatingwiththem:carefullychoosingthetimetodiscussthismatter;relying onsignificantotherssuchasafather'srelativetoshowsupportortoactasanintermediary; requesting health workers to disclose their HIV-status for them; or using means in the interaction that allowed denial in case of violent reaction-such as using "half words," speaking of HIV-positive persons in general before coming to their own case or using euphemisticwords.Theseaspectsarenotsecondarymattersregardinginfantfeeding,since rejectionofamotherbyherhusbandmaycompletelydisableherfromprovidingproperand safecaretoherinfant. Fathers' attitudes about infant feeding varied, depending on their perceptions of feeding optionsandtheirtrustinpreventivestrategieswhenconsideringthecostforthehousehold.

Thereisnoepidemiologicalevidencefortherelativesafetyofbottledwatercomparedtowaterfromother sourcesregardingtheriskofHIVtransmissionwhenbreastfeedingisnolongerexclusive.

The father's own experience with his HIV status, whether denial, ignorance or awareness, andhisrelationshiptothemotherwilldeterminewhethershemusthandlethingsaloneor whether he will help her with child care after the disclosure. In some cases she will implementastrategythatalsomeetstheneedsofherotherchildren.Recentinvestigations showed that more fathers supported women in their preventive decisions, which made it easierformotherstohandlequestionsfromtheirfamilies.Thiswasespeciallythecasewhen fathers knew that they were HIV-positive themselves. In some cases, fathers shared the decisionaboutinfantfeeding;theywouldoftenbemorecautiousthanwomeninavoiding anyexposuretoHIVwhateverthebenefitsofbreastfeedingandadvicefromhealthworkers onthattopicmaybe.

5.HIV-positivemothers'strategiesinhealthservices

Contrarytowhatmightbeexpected,women'sinterviewsshowedthattheirsituationswere nottotallysecuretoimplementpreventiveinfantfeedingoptionsinhealthservicessettings. The first reason is related to the institutional organization of the management of infant feeding and HIV issues. Since relevant programs-the program for the promotion of breastfeedingandtheAIDSprogram-arebothvertically-designed,anddespitethepolitical willto"integrate"thematthelocallevelduringthe2000s,theirimplementationinhealth services never underwent consistent integration. As a result, the particular needs of HIVpositive women, for instance regarding preventive feeding patterns, were still treated as contradictingandendangeringthepromotionofbreastfeeding.HIV-positivemothersfound this to be especially true when they were in Mother-and Child-Health services and confronted by posters claiming that "Breast is Best," even though PMTCT programs were housed in the same services. Women would also receive contradictory messages, for instance between Mother-and-Child Health workers unaware of a mother's HIV-positive status encouraging her to breastfeed as soon and as long as possible, when other health workers assigned to the PMTCT program advised her to do early weaning or replacement feeding.Inthiscase,silencewasthemostfrequentlyusedstrategy,whatevertheuneasiness generatedbythecontradictionsbetweenhealthworkers'messages. Women experienced the risk of being labeled as HIV-positive, which has not totally disappearedinhealthservices.Althoughstigmaandrejectionarenowlesscommon,some healthworkersstillavoidrelationshipswithHIV-positivepersonsfearingtheywouldnotact properly or may breach confidentiality about their HIV status. In maternity wards where healthworkersconsideredpromotionofbreastfeedingastheirpriority,somewomenwere compelledtorevealtheirHIVstatustodefendtheirwithdrawalofcolostrumortheirrefusal toplacetheinfantonthebreastimmediatelyafterbirth.Womensometimesusedstrategies thatcouldbedangerousforthemselvesortheirbabies,asinthecaseofonewomanwho only gave water to her baby for three days pretending that she was breastfeeding him, fearing both HIV transmission if she breastfed and being labeled as HIV-positive if she neededformula. HIV-positive women also create strategies to work around follow-up by health workers. In terms of counseling, follow-up for nutritional purposes might be fairly prescriptive or comprehensive,dependingonthehealthworkers'knowledgeandexperienceregardingHIV andcounseling.Healthworkers'attitudeswerealsodifferentamongthosewhohadstrong opinionsinfavorofbreastfeedingandadviseditinanysituationandotherswhoconsidered respectiveHIV-levelsofriskandthoughtthatformulawasfeasibleinthelocalcontext.There were also differences in discourses between health workers who were "optimistic" about women's ability to stick to the option she had chosen and those who were "realistic" and considered that each choice might entail challenges in an evolving context. In many cases womenwerecriticizedforbeingunabletoapplypreventivepatternsthatwerebeyondtheir scope, since this depended on the unpredictable attitudes of persons such as husbands or elder women who had authority in the household. Thus, the easiest strategy to avoid this undeservedtreatmentwaslyingtohealthworkersaboutthefeedingpatternusedorhowit was implemented. Some health workers would respond by using their own strategies to verify the mothers' statements, observing the infant's body language in proximity of the breastofamotherwhopretendednottobreastfeedorbypinchingthemother'snippleto seeifshewasstillbreastfeedingaftertheprescribedweaningdate. As a result of HIV stigmatization, women who feared being identified as HIV-positive also had to set strategies to avoid being seen in HIV-related places such as associations for peoplelivingwithAIDS,pretendingtogotoMother-and-ChildHealthServicesorelsewhere. Yet, women who were supported by an NGO or PLHIV group or by counselors from a research team managed to comply more easily with their chosen feeding option. Material means,freeprovisionofsubstitutesandequipmentandthedeliveryofmoreindividualized informationarenottheonlyfactorsinvolved.Inthe"spaceofconfidentiality"ofasupport group,theyarenotcompelled,asinhealthservicesandathome,toavoidcertainsubjectsor justify their actions. They appreciate the opportunity to talk freely about various psychological and social aspects of HIV infection related to managing relationships with significantothers,sincetheseaspectsareimportantforthefeasibilityofpreventiveoptions. TheyalsofindmorecoherentandcontextualizeddiscoursesregardinginfantfeedingandHIV than in some health services, where partially different and non-articulated discourses for HIV-positive and HIV-negative mothers are confusing. Yet, many women did not take full advantageofthesepossibilitieswhentheywereunabletojustifytotheirhusbandswhythey were leaving the household for an indisputable medical reason, as is the case for going to healthservices.

6.Conclusion:TheevolvingcultureofinfantfeedingintheageofHIV

Women's experiences and the strategies they must set up to apply preventive feeding patterns reveal several aspects of local cultures that are not exclusively related to either healthservicesorthecommunity. HIV-positive women who are literate and have a certain level of social and economic autonomy may assert their decision and manage implementing any infant feeding option, including formula feeding, whatever criticisms may arise in either health services or households; this is also the case for women who share the management of HIV with their husbands.Thisobservation,whichstressestheimportanceofpersonalautonomyorsupport inthecouple,isconsistentwithobservationsmadeaboutHIVpreventioninothersettings (Buskens et al. 2007; Leshabari et al. 2007; Hofmann et al. 2009) or for other kinds of challengesinwomen'slives,whatevertheculturalsetting.

Theirexperiencesalsoshowthatmostwomenmayapplypreventiverecommendationsfully and with ease only when they are alone with their infants. They often are compelled to "maintain distance" from the actors of infant feeding in order to avoid the risk of being labeled as HIV-positive or as a "bad mother." This explains situations of psychological distress among some women who bear these constraints alone in addition to planned isolation.Italsorevealstowhatextentinfantfeedingisasocialbehavior,involvingseveral protagonists, many of whom feel they have the legitimacy to define what is good for the infant. On this issue, the HIV pandemic stresses the discrepancy between community and healthservices,sincetheinternationalmedicalcultureconsidersthemotherastheinfant's sole caretaker who can make "individual choices," and often blames her when she is the victim, 18 whichisaconsequenceofindividualizingsocialproblems(seechapterXXbyLee). Thedifficultieswomenfacewhentryingtoapplyanalternativetobreastfeedingortolimit its duration to four to six months reveal not only the normative strength of breastfeeding promotionandpopularperceptions,butalsotheirrationales.Inhealthservicesaswellasin the community, biological considerations and precise categorization have little weight comparedtothesocialandsymboliclogicsindefenseofbreastfeeding-whateveritsmode ofimplementationaccordingtonutritionists'categories-whenafullrangeofactorsunder variousideologicalorsocialguidancehaveaddedan"HIV-freelabel"valueforthemotherto its long-standing perceived benefits. This strange ambiguity turns maintaining a mode of HIV-transmission into the sign signaling the absence of contamination and thus challenges communication about health. For this aspect, the HIV pandemic revealed, and maybe in some areas such as Burkina Faso paradoxically reinforced, the symbolic value of breastfeeding.ThethreatthatformulafeedingpracticeswouldspilloverbeyondHIV-positive mothers,oftenevokedbychildspecialists,neverbecameanempiricalconcernsinceformula isasignofmothers'HIVinfectionandthusahighlystigmatizedbehavior. The mothers' experiences stress the symbolic construction, perceptible in both health discoursesandinstitutionalorganization,ofanoppositionbetweenbreastfeedingandHIVtwo heterogeneous categories. Some health workers still seem to believe, as one public health official said in the late 1990s: "The problem is not breastfeeding, it is HIV-positive mothers." 19 In this context, narrated experiences may be interpreted as the result of the embodiment of global categories with profound symbolic content (considering "good" breastfeeding as opposed to "bad" HIV) for women who may both breastfeed and be infectedbyHIV.Asforconceptualizationofbreastfeedingwhich,eveninhealthsettings,is still considered under a monothetic category when characterization on a biological basis requiresatleastfourcategories, 20 thissymbolicopposition,whichseemshardlyquestioned byevidence,appearsasafundamentalconceptinmedicalculture. The range of strategies applied by women to reduce social and biological risks for their infantsandthemselvesiswide.Forinstance,theywouldoftenuseseveralfeedingoptions 18 Theidiom"blamingthevictim"coinedinthemid-1970sbythesociologistWilliamRyanwasappliedinthe 1990s by Paul Farmer in the field of AIDS and summarizes a major theoretical interpretation trend of social processesrelatedtotheAIDSpandemic(Farmer1992).

19 Abidjan,personalcommunication,author'stranslation("Leproblèmec'estpasl'allaitement,c'estlesfemmes séropositives."). 20 Seefootnote4 one after another when medical recommendations request that one is chosen "for good" andappliedwithoutanyvariation.Womenwouldalsoadaptthefeedingpatternaccording to changing social contexts, which is not supported by PMTCT. They would set "risk reduction"whenPMTCTclaims"riskwithdrawal,"whentheyeitherusedmixedfeedingor were ready to re-start breastfeeding in case of a formula shortage. These hybrid patterns, whichshowedwomen's capacityand limits inadaptingtotheir context, are notvaluedby the health system since they do not correspond to the strictly defined patterns that were validatedbyexperimentalclinicaltrials. 21 There,women'sexperiencesrevealtherigidityof medical concepts, which lack the sensitivity to contexts that are empirically developed by women. Further studies in political science are necessary to understand how this lack of acknowledgementis,onthisparticulartopic,relatedtosocialandgenderstatusdifferences between women in Burkina Faso-who are poorly represented as a community force capableofsocial"transformation"-andscientistsattheinternationallevel. Shareddecisionmakingwithfatherswasadvisedbyhealthworkers,whoacknowledgethe need for fathers' psychological and material involvement to support mothers made vulnerablebytheriskofHIVstigma;itmayalsoresultfromdefinedgenderrolesinspiredby western orientated patterns, which underlie UN agency recommendations for HIV counseling, implemented by heath workers in PMTCT programs. Before, mainly women providedcare,assistedbygrandmothersandelderwomen.Now,becausetheyareafraidof apossiblebreachinconfidentialityandrejectionfromtheextendedfamily,decisionmaking isincreasinglylimitedtocouples,whichsubsequentlymeansgreaterinvolvementfrommen. This focus on the couple, which also emphasizes sharing responsibilities equally between menandwomenandpromotingrightsofindividualsamongPLHIVwhatevertheirgenderas ameansofprotectionagainstrejection,isalsoenhancedinsomePLHIVorganizationsand publicallydemonstratedbytheirleaders. Focusingontheparentalcoupleornuclearfamilyandonthemother-childinteractionrather thanonextendedfamilyarepreventivediscoursesthatconveysocialmodelsfromwestern or global cultures. Whether instrumental, aimed at avoiding HIV-labeling by the extended familyorintertwinedwithmedicalmediaandmessages,thesemodelshavespreadwiththe globalized AIDS culture conveyed particularly by NGOs and PLHIV associations. More than the "therapeutic citizenship" that has not yet developed in Africa in the field of PMTCT (Blystad & Moland 2009), contrary to other fields (Nguyen et al. 2007), this individualizing andnuclearpatterninfamilysocialorganization,whichalsocorrespondstoashiftingender roles, may be the social transformation brought about by the HIV pandemic through the preventionofHIVbreastfeedingtransmission.

Acknowledgements

Wewouldliketothankwomenandcaregiversthatdevotedtimetothisresearch.Wealso thank Kesho Bora Group, Centre Muraz, Association Espoir pour Demain (Bobo-Dioulasso), Centre Medical avec Antenne chirurgicale de Pissy, Vie Positive, Médecins sans Frontières (Ouagadougou),andtheteamsinhealthfacilitiesthatmadethisresearchpossible. 21 Fromanepidemiologicalperspective,theoverallreductionofmilkintakeshouldreduceHIVtransmission.