[Lung cancer screening and endoscopic approach of the pulmonary nodule]. / Dépistage du cancer du poumon et approche endoscopique du nodule pulmonaire.

Lung cancer screening corresponds to any investigation carried out to detect the presence of this type of cancer before the appearance of any symptomatology. According to the results of a large multicenter study in the US (NLST), American scientific societies are currently recommending the use of the CT Thoracic Scanner with a low dose of irradiation once a year for lung cancer screening in high-risk populations. European expert groups, for the most part, reserve their recommendations after publication of the results of a major European study (NELSON). As a corollary of screening campaigns, the management of lung nodules is likely to be more and more frequent in the targeted populations. This can be performed in different ways ranging from the less invasive, the CT Scanner follow-up, to the more invasive, the surgical resection. The endoscopic approach of the pulmonary nodule, whose results were so far limited, is actually regaining interest due to the contribution of new technologies in bronchial endoscopy.

Le dépistage du cancer du poumon correspond à toute investigation réalisée dans le but de détecter l'existence de ce type de cancer avant l'apparition de la moindre symptomatologie. Faisant suite à la publication d'une grande étude multicentrique aux USA (NLST), les sociétés savantes américaines recommandent actuellement l'utilisation du CT Scanner Thoracique à faible dose d'irradiation à raison d'une fois par an pour le dépistage du cancer du poumon dans les populations à risque. Les sociétés européennes, pour la plupart, réservent quant à elles leurs recommandations pour après la publication des résultats d'une grande étude européenne (NELSON). Corollaire des campagnes de dépistage, la prise en charge des nodules pulmonaires risque d'être de plus en plus fréquente dans les populations ciblées. Celle-ci peut être réalisée de différentes façons allant de la moins invasive, le suivi par CT Scanner, à la plus invasive, la chirurgie d'exérèse. L'approche endoscopique du nodule pulmonaire, dont les résultats étaient jusqu'à présent limités, connait un regain d'intérêt du fait de l'apport de nouvelles technologies en endoscopie bronchique.

[The effects of hypnosis by virtual reality on tolerance to flexible bronchoscopy]. / Effets de l'hypnose par réalité virtuelle sur la tolérance de la bronchoscopie flexible.

INTRODUCTION: Patients often perceive flexible bronchoscopy as an unpleasant procedure. The aim of this study was to investigate the effect of virtual reality (VR) hypnosis on tolerance to flexible bronchoscopy. MATERIALS AND METHODS: We conducted a prospective, randomized, controlled, monocentric study comparing flexible bronchoscopy with VR-induced hypnosis to the usual procedure. Patient tolerance was evaluated using a visual analogue scale (VAS), the state-trait anxiety inventory (STAI) before and after the procedure and, finally, willingness to repeat the examination under the same conditions (WTR). RESULTS: Among the 70 patients included, 34 were randomized to the VR hypnosis group and 36 to the control group. There was no difference between the 2 groups in terms of modification of the pre-/post-bronchoscopy VAS for anxiety, pain, cough, choking, nausea and overall discomfort, or modification of the STAI score and WTR. Subgroup analysis among patients who were more anxious before the procedure revealed a trend toward reduced anxiety in the VR hypnosis group. CONCLUSION: This study did not observe any effect of VR hypnosis on the tolerance of patients during routine flexible bronchoscopy. However, VR hypnosis may be beneficial in patients with higher anxiety score before bronchoscopy, a hypothesis that needs to be confirmed by further studies with a larger number of subjects.

Intra- and inter-spatial variability of meiofauna in hadal trenches is linked to microbial activity and food availability.

Hadal trenches are depocenters for organic material, and host intensified benthic microbial activity. The enhanced deposition is presumed to be reflected in elevated meiofaunal standing-stock, but available studies are ambiguous. Here, we investigate the distribution of meiofauna along the Atacama Trench axis and adjacent abyssal and bathyal settings in order to relate the meiofauna densities to proxies for food availability. Meiofauna densities peaked at the sediment surface and attenuated steeply with increasing sediment depth. The distribution mirrored the vertical profile of the microbial-driven oxygen consumption rate demonstrating a close linkage between microbial activity and meiofauna density. Meiofaunal standing-stock along the trench axis varied by a factor of two, but were markedly higher than values from the abyssal site at the oceanic plate. Overall, meiofaunal densities poorly correlated with common proxies for food availability such as total organic carbon and phytopigments, but strongly correlated with the microbial benthic O2 consumption rate. We argue that microbial biomass likely represents an important meiofaunal food source for hadal meiofauna. Observations from three trench systems underlying surface water of highly different productivity confirmed elevated meiofaunal densities at the trench axis as compared to abyssal sites on oceanic plates. Food availability appear to drive elevated abundance and variations in meiofauna densities in hadal sediments.

Turnover of CD4+ and CD8+ T lymphocytes in HIV-1 infection as measured by Ki-67 antigen.

We investigated CD4+ and CD8+ T cell turnover in both healthy and HIV-1-infected adults by measuring the nuclear antigen Ki-67 specific for cell proliferation. The mean growth fraction, corresponding to the expression of Ki-67, was 1.1% for CD4(+) T cells and 1.0% in CD8(+) T cells in healthy adults, and 6.5 and 4.3% in HIV-1-infected individuals, respectively. Analysis of CD45RA+ and CD45RO+ T cell subsets revealed a selective expansion of the CD8+ CD45RO+ subset in HIV-1-positive individuals. On the basis of the growth fraction, we derived the potential doubling time and the daily turnover of CD4+ and CD8+ T cells. In HIV-1-infected individuals, the mean potential doubling time of T cells was five times shorter than that of healthy adults. The mean daily turnover of CD4+ and CD8+ T cells in HIV-1-infected individuals was increased 2- and 6-fold, respectively, with more than 40-fold interindividual variation. In patients with <200 CD4+ counts, CD4+ turnover dropped markedly, whereas CD8+ turnover remained elevated. The large variations in CD4+ T cell turnover might be relevant to individual differences in disease progression.

Critical role of cytosolic phospholipase A2{alpha} in bronchial mucus hypersecretion in CFTR-deficient mice.

Cystic fibrosis (CF) is due to mutations in the CF transmembrane conductance regulator gene CFTR. CF is characterised by mucus dehydration, chronic bacterial infection and inflammation, and increased levels of cytosolic phospholipase A2α (cPLA2α) products in airways. We aimed to examine the role of cPLA2α in the modulation of mucus production and inflammation in CFTR-deficient mice and epithelial cells. Mucus production was assessed using histological analyses, immuno-histochemistry and MUC5AC ELISA. cPLA2α activation was measured using an enzymatic assay and lung inflammation determined by histological analyses and polymorphonuclear neutrophil counts in bronchoalveolar lavages. In lungs from Cftr(-/-) mice, lipopolysaccharide induced mucus overproduction and MUC5AC expression associated with an increased cPLA2α activity. Mucus overproduction was mimicked by instillation of the cPLA2α product arachidonic acid, and abolished by either a cPLA2α null mutation or pharmacological inhibition. An increased cPLA2α activity was observed in bronchial explants from CF patients. CFTR silencing induced cPLA2α activation and MUC5AC expression in bronchial human epithelial cells. This expression was enhanced by arachidonic acid and reduced by cPLA2α inhibition. However, inhibition of CFTR chloride transport function had no effect on MUC5AC expression. Reduction of CFTR expression increased cPLA2α activity. This led to an enhanced mucus production in airway epithelia independent of CFTR chloride transport function. cPLA2α represents a suitable new target for therapeutic intervention in CF.

The value of endoscopic ultrasound after bronchoscopy to diagnose thoracic sarcoidosis.

A clinicoradiological presentation of thoracic sarcoidosis requires histopathology in order to establish the diagnosis. Flexible bronchoscopy has a reasonable diagnostic yield and is the procedure of first choice for diagnosis. Endoscopic ultrasound (endoscopic ultrasound-guided fine needle aspiration/endobronchial ultrasound-guided transbronchial needle aspiration) can help in the diagnosis of sarcoidosis. An implementation strategy of endoscopic ultrasound for the diagnosis of sarcoidosis following negative flexible bronchoscopy results was examined prospectively in 15 clinics. A total of 137 patients (92 males; median age 43 yrs) were included, and sarcoidosis was found in 115 (84%). Alternative diagnoses were tuberculosis, lymphangitis carcinomatosa, pneumoconiosis and alveolitis. All patients were sent for flexible bronchoscopy, which was performed in 121 (88%), resulting in a definite diagnosis in 57 (42%). A total of 80 patients were sent for endoscopic ultrasound, which could be performed in 72 (90%), yielding a definite diagnosis in 47 (59%). Endoscopic ultrasound following negative flexible bronchoscopy avoided a surgical procedure in 47 out of 80 patients. The sensitivity of flexible bronchoscopy for sarcoidosis was 45% (95% confidence interval 35-54%), but 62% (50-72%) if biopsy specimens were taken. The sensitivity of endoscopic ultrasound following negative flexible bronchoscopy results was 71% (58-82%). With this strategy, 97 out of 115 (84% (76-90%)) of proven sarcoidosis was diagnosed using endoscopy. This large prospective implementation study (trial number NCT00888212; ClinicalTrials.gov) shows that endoscopic ultrasound is valuable for diagnosing sarcoidosis after negative flexible bronchoscopy results.

Validation of the Wii Balance Board to assess balance modifications induced by increased respiratory loads in healthy subjects.

BACKGROUND: There is a link between breathing and balance and posture. When the inspiratory loads are increased by pathologies, there is a decrease of postural control. The increase of the inspiratory load on respiratory muscles is a common feature in various chronic pulmonary pathologies. Consequently, the balance of those patients is likely affected. RESEARCH QUESTION: The aim of this study is to validate the use of the Nintendo Wii Balance Board (WBB) to assess balance modifications induced by increased respiratory loads in healthy subjects. METHODS: Thirty-seven healthy young participants (25 ± 4 years old, 17 women) participated in this study. Five different conditions were tested: without anything (control), throughout a mouthpiece, and throughout three inspiratory threshold loads (ITL) at 10% (low), 40% (mid) and 60% (high) of the maximal inspiratory pressure. Each trial lasted for 60 s. Nine parameters were extracted based on center of pressure displacement based on a previously-validated method. ANOVA tests were used to compare the different conditions followed by Bonferroni's corrections. RESULTS: Highly statistically significant differences (all p < 0.01) and large effect sizes (all ω2 > 0.24) were obtained for all parameters between the different loads and the mouthpiece condition. There is a linear relationship between the load and balance perturbation. SIGNIFICANCE: In this study, we demonstrated the validity of the WBB to detect the effect of the inspiratory load on balance in young healthy subjects. Further studies are needed to determine if such a kind of evaluation can be used in clinics with patients suffering from chronic respiratory disease.

Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice.

This report examines the effect of recombinant murine (rm) IL-10 on antigen-induced cellular recruitment into the airways of sensitized Balb/c mice. The intranasal instillation of 10 micrograms ovalbumin induced an early (6-24 h) increase in the number of neutrophils, and a late rise (24-96 h) in that of eosinophils in the bronchoalveolar lavage (BAL) fluid and bronchial tissue. A single intranasal instillation of 0.01-0.1 microgram of rmIL-10, administered concurrently with ovalbumin, but not 1 or 3 h thereafter, dose-dependently inhibited both airway neutrophilia and eosinophilia. This phenomenon was suppressed by treating the sensitized mice with 1 mg/mouse of a neutralizing anti-IL-10 mAb, which increased significantly ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These results suggest that antigen stimulation may trigger the in vivo generation of IL-10, which, in turn, participates in the leukocyte infiltration into the airways. rmIL-10 also reduced TNF-alpha release in the BAL fluid observed 1 and 3 h after antigen challenge. Furthermore, the intranasal instillation of an anti-TNF-alpha antiserum to sensitized mice markedly reduced ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These findings indicate that leukocyte infiltration into the airways of antigen-challenged mice is regulated by IL-10. Furthermore, inhibition of TNF-alpha production by rmIL-10 suggests that allergic airway inflammation and TNF-alpha formation are parallel events in this model.

Self-assembly of influenza hemagglutinin: studies of ectodomain aggregation by in situ atomic force microscopy.

We have used in situ tapping mode atomic force microscopy (AFM) to study the structural morphology of two fragments of the influenza hemagglutinin protein bound to supported bilayers. The two proteins that we studied are the bromelain-cleaved hemagglutinin (BHA), corresponding to the full ectodomain of the hemagglutinin protein, and FHA2, the 127 amino acid N-terminal fragment of the HA2 subunit of the hemagglutinin protein. While BHA is water soluble at neutral pH and is known to bind to membranes via specific interactions with a viral receptor, FHA2 can only be solubilized in water with an appropriate detergent. Furthermore, FHA2 is known to readily bind to membranes at neutral pH in the absence of a receptor. Our in situ AFM studies demonstrated that, when bound to supported bilayers at neutral pH, both these proteins are self-assembled as single trimeric molecules. In situ acidification resulted in further lateral association of the FHA2 without a large perturbation of the bilayer. In contrast, BHA remained largely unaffected by acidification, except in areas of exposed mica where it is aggregated. Remarkably, these results are consistent with previous observations that FHA2 promotes membrane fusion while BHA only induces liposome leakage at low pH. The results presented here are the first example of in situ imaging of the ectodomain of a viral envelope protein allowing characterization of the real-time self-assembly of a membrane fusion protein.

[Manual abdominal compression for the detection of expiratory flow limitation]. / La compression abdominale manuelle dans la détection de la limitation du débit expiratoire.

INTRODUCTION: Expiratory flow limitation (EFL) is a characteristic feature of chronic obstructive pulmonary disease (COPD) and leads to dynamic hyperinflation (DH) which is a major source of dyspnoea, particularly during exercise. STATE OF THE ART: A new technique for the detection of EFL, based on manual compression of the abdomen (MCA), was assessed both in normal subjects and patients with COPD. MCA was always associated with a moderate increase in pleural pressure and allowed the detection of EFL in a reproducible manner, in both the seated and supine postures. The technique was well tolerated. It was also a reliable method for the detection of EFL during exercise since EFL detection was effectively associated with the development of DH. Finally, MCA was also compared to NEP in patients with obstructive sleep apnoea syndrome (OSAS) and in these patients, MCA invariably increased expiratory flow whereas the NEP method produced flow limitation in some cases because of upper airway collapse. PERSPECTIVES: EFL detection with MCA may be clinically useful since EFL is a determinant of dyspnoea, affects ventilatory response to exercise as well as maximum exercise capacity. CONCLUSIONS: MCA is a reliable technique for the detection of EFL in different positions, during resting breathing or exercise, requiring neither special equipment nor patient cooperation.

[Techniques and strategy of pathological sampling in the diagnostic and therapeutic management of lung cancer]. / Techniques et stratégie de prise en charge des prélèvements anatomopathologiques dans le cadre de l'approche diagnostique et thérapeutique du cancer bronchique.

Histopathology is key to the diagnosis and staging of lung cancer. This analysis requires tissue sampling from primary and/or metastatic lesions. The choice of sampling technique is intended to optimize diagnostic yield while avoiding unnecessarily invasive procedures. Recent developments in targeted therapy require increasingly precise histological and molecular characterization of the tumor. Therefore, pathologists must be economical with tissue samples to ensure that they have the opportunity to perform all the analyses required. More than ever, good communication between clinician, endoscopist or surgeon, and pathologist is essential. This is necessary to ensure that all participants in the process of lung cancer diagnosis collaborate to ensure that the appropriate number and type of biopsies are performed with the appropriate tissue sampling treatment. This will allow performance of all the necessary analyses leading to a more precise characterization of the tumor, and thus the optimal treatment for patients with lung cancer.

Drug interactions between voriconazole, darunavir/ritonavir and tenofovir/emtricitabine in an HIV-infected patient treated for Aspergillus candidus lung abscess.

We report an unusual case of pulmonary aspergillosis in a patient with AIDS and we demonstrated the drug-drug interactions between voriconazole, darunavir/ritonavir and tenofovir/emtricitabine. The combination darunavir-ritonavir-voriconazole should be avoided.

Toxicity, efficacy, plasma drug concentrations and protease mutations in patients with advanced HIV infection treated with ritonavir plus saquinavir. Swiss HIV Cohort Study.

OBJECTIVE: To assess the safety, efficacy and plasma drug levels of the combination of ritonavir plus saquinavir for the treatment of advanced HIV infection. DESIGN: Multicentre pilot study. PATIENTS: Eighteen protease inhibitor-naive patients, with intolerance or contraindication to reverse transcriptase inhibitors, a median CD4 cell count of 12 x 10(6)/l (range, 1-50 x 10(6)/l), and a median HIV viraemia of 5.25 log10 copies/ml (range, 4.00-6.13 log10 copies/ml). METHODS: Patients received 600 mg twice daily of both ritonavir and saquinavir. Viraemia was measured at baseline and at weeks 5, 9 and 13. Response was defined as a drop of viraemia of more than 1 log10 at week 5. Plasma drug levels were determined after at least 3 weeks of combined treatment: samples were collected before and 1, 2, and 4 h after the morning ingestion of both drugs. The protease gene was sequenced at baseline and under treatment. RESULTS: Among the 16 patients evaluable at week 5, 11 were responders, and among these patients, six remained responders at week 13 (two with undetectable viraemia). Study discontinuations were due to side-effects (n = 4), patient choice (n = 3), protocol violation (n = 1) and death (n = 1). Responders had higher drug levels than non-responders (P < 0.01 for saquinavir, P = 0.04 for ritonavir). In two non-responders, development of multiple new mutations at positions 10, 20, 48, 82, 84 and 90 was observed after 5-13 weeks. CONCLUSION: The response to ritonavir plus saquinavir in advanced HIV infection is unpredictable. A minority of patients respond with disappearance of HIV viraemia. In other patients, rapid cumulative emergence of protease mutations conferring resistance to treatment cannot always be prevented by good compliance and relatively high plasma drug levels.

Long-term hydroxyurea in combination with didanosine and stavudine for the treatment of HIV-1 infection. Swiss HIV Cohort Study.

OBJECTIVE AND METHODS: In 1998 we reported on a randomized comparison between stavudine plus didanosine plus placebo versus stavudine plus didanosine plus hydroxyurea (HU), in patients with a CD4 count of 200-500 x 10(6) cells/l. After 3 months, the HU group had a higher proportion of patients with viral load < 200 x 10 cells/l. At the end of the 3 months blinded period, patients in the placebo group had the option to add HU if their viral load remained > 200 x 10(6) cells/l. We report results after 24 months. RESULTS: Seventy-two patients were randomized to the HU arm, and a further 30 elected to add HU after 12 weeks. Twenty-four months after the start of the trial, only 25% of the 72 patients originally randomized to HU, and 20% of the 30 who added HU after week 12, were still taking it. The reasons for stopping HU were: lack of efficacy (45%), adverse events (37%) and patient or physician preference (18%). Side effects were more frequent in the didanosine/stavudine/HU group than in the didanosine/stavudine group: neuropathy (35 versus 15%, P< 0.02), fatigue (22 versus 7%, P< 0.01), and nausea or vomiting (26 versus 9%, P< 0.01). Of those who had discontinued HU, 73% were taking three drugs including a protease inhibitor. Patients who had started HU were compared with similar patients who had started protease inhibitors in the Swiss cohort. The probability of stopping HU was higher than the probability of stopping nelfinavir or indinavir, and similar to the probability of stopping ritonavir. CONCLUSION: HU increased the antiviral effect of stavudine plus didanosine. However, side effects were more frequent, and after 24 months the majority of patients had switched to protease inhibitor regimens.

A placebo-controlled trial of didanosine plus stavudine, with and without hydroxyurea, for HIV infection. The Swiss HIV Cohort Study.

OBJECTIVE: To explore the short-term effects on surrogate markers for HIV progression of didanosine (ddl) plus stavudine (d4T), with or without hydroxyurea. DESIGN: Randomized, double-blinded, prospective study. SETTING: Swiss HIV Cohort Study. PATIENTS: A total of 144 patients (75% antiretroviral-naive) were studied (mean baseline HIV-1 RNA, 4.53 log10 copies/ml; mean CD4 cell count, 370 x 10(6)/l). INTERVENTION: Patients received ddl (200 mg twice daily) plus d4T (40 mg twice daily), with additional hydroxyurea (500 mg twice daily) or placebo. MAIN OUTCOME MEASURES: The primary endpoint was a reduction of viraemia below 200 copies/ml after 12 weeks. At that time, patients who did not reach the primary endpoint were withdrawn in the hydroxyurea arm, whereas patients in the placebo group had the option of adding hydroxyurea to ddl and d4T. All patients were followed until week 24. RESULTS: After 12 weeks, 54% of the patients randomized to hydroxyurea had viraemia below 200 copies/ml, compared with 28% on placebo (P < 0.001). Using an ultrasensitive assay with a limit of detection of 20 copies/ml, 19% of patients receiving hydroxyurea had viraemia levels below 20 copies/ml, compared with 8% on placebo (P = 0.05). Mean decrease in HIV-1 RNA was 2.3 and 1.7 log10 copies/ml for hydroxyurea and placebo groups, respectively (P = 0.001). Hydroxyurea was found to induce lymphopenia (-124 x 10(6)/l). Increase in CD4 cell counts was +28 x 10(6)/l during hydroxyurea treatment compared with +107 x 10(6)/l on placebo (P = 0.001). CONCLUSIONS: Hydroxyurea improved the antiviral activity of d4T and ddl over a 12-week period, but was associated with a smaller increase in CD4 cell counts due to hydroxyurea-induced lymphopenia.

Pediatricians and general practitioners: a comparison of the management of children with febrile illness.

To assess the relative competence of pediatricians and general practitioners in managing febrile illnesses in childhood, a prospective study was undertaken of 259 children less than 10 years of age seen consecutively at the emergency room of a children's hospital and a general hospital. Both groups of patients were similar in demographic characteristics, age, sex, presenting complaints, and height and duration of fever. Of the 148 patients entered in the study at children's hospital and the 111 at general hospital, 90% and 94%, respectively, were interviewed by telephone within two weeks of their emergency room visit to determine outcomes based on duration of the acute illness episode and further physician contacts or admissions to hospital. Although no significant differences in the measured outcomes of febrile illnesses seen by pediatricians or general practitioners were found, a trend in favor of those cared for by pediatricians was discernible with respect to two key measures: unresolved symptoms at one week (8.3% vs 12.5%) and subsequent hospitalization (0.8% vs 3.0%). The overall frequency of laboratory use and antibiotic prescriptions was the same in both groups; however, significant differences were noted in the type of laboratory test used: general practitioners ordered three times more roentgenograms than pediatricians and one fifth the number of microbiologic tests. Larger prospective studies are needed to test how these findings may be generalized in view of their importance for quality assurance in the primary care of children.

Parental fever phobia and its correlates.

Parents of 202 young febrile children were surveyed about their knowledge, attitudes, and fears concerning fever and its treatment. Forty-eight percent of the parents considered temperatures less than 38.0 degrees C to be "fevers", 43% felt that temperatures less than 40.0 degrees C could be dangerous to a child, 21% favored treatment for fevers less than 38.0 degrees C, and 15% believed that, left untreated, temperature could rise to 42.0 degrees C or higher. Fifty-three percent advocated waking a febrile child at night to administer antipyretic therapy. Young age of the child was associated with a preference for use of acetaminophen over aspirin and, unexpectedly, with a higher parental threshold for consideration of fever. The higher their child's temperature at the time they were questioned, the higher the minimum temperature that parents considered a cause for concern. Surprisingly, higher socioeconomic status was not associated with a lesser degree of fever phobia. In fact, parents of higher socioeconomic status were more concerned about the risks of brain damage or seizures as sequelae of fever than were parents of lower socioeconomic status. It is concluded that undue fear and overly aggressive treatment of fever are epidemic among parents of infants and young children, even among the highly educated and well-to-do. Considerable effort will be required on the part of pediatricians and other child health workers to reeducate these parents about the definition, consequences, and appropriate treatment of fever.

Effect of formula supplementation in the hospital on the duration of breast-feeding: a controlled clinical trial.

To avoid methodologic pitfalls in previous observational studies linking formula supplementation in the hospital to early discontinuation of breast-feeding, a controlled clinical trial of restricted supplementation was conducted. In a pretrial sample of 621 newborns, a comparison of two "well-baby" nurseries found no differences in either hospital supplementation practices or the proportion of infants still being breast-fed at 4 or 9 weeks postpartum. Restriction of supplementation in one of the nurseries for the trial period (n = 781) did not result in higher breast-feeding rates at 4 or 9 weeks. There was, however, a slightly greater mean percent of birth weight lost in the restricted group (6.0% v 5.1%; P less than .001). In examining the control group for evidence of an "observational" association, it was found that infants still breast-feeding at 4 or 9 weeks were far more likely to have been unsupplemented than those no longer being breast-fed. It thus appears that formula supplementation in the hospital is a marker, rather than a cause, of breast-feeding difficulty.