Lung recruitment before surfactant administration in extremely preterm neonates with respiratory distress syndrome (IN-REC-SUR-E): a randomised, unblinded, controlled trial.

BACKGROUND: The importance of lung recruitment before surfactant administration has been shown in animal studies. Well designed trials in preterm infants are absent. We aimed to examine whether the application of a recruitment manoeuvre just before surfactant administration, followed by rapid extubation (intubate-recruit-surfactant-extubate [IN-REC-SUR-E]), decreased the need for mechanical ventilation during the first 72 h of life compared with no recruitment manoeuvre (ie, intubate-surfactant-extubate [IN-SUR-E]). METHODS: We did a randomised, unblinded, controlled trial in 35 tertiary neonatal intensive care units in Italy. Spontaneously breathing extremely preterm neonates (24 + 0 to 27 + 6 weeks' gestation) reaching failure criteria for continuous positive airway pressure within the first 24 h of life were randomly assigned (1:1) with a minimisation algorithm to IN-REC-SUR-E or IN-SUR-E using an interactive web-based electronic system, stratified by clinical site and gestational age. The primary outcome was the need for mechanical ventilation in the first 72 h of life. Analyses were done in intention-to-treat and per-protocol populations, with a log-binomial regression model correcting for stratification factors to estimate adjusted relative risk (RR). This study is registered with ClinicalTrials.gov, NCT02482766. FINDINGS: Of 556 infants assessed for eligibility, 218 infants were recruited from Nov 12, 2015, to Sept 23, 2018, and included in the intention-to-treat analysis. The requirement for mechanical ventilation during the first 72 h of life was reduced in the IN-REC-SUR-E group (43 [40%] of 107) compared with the IN-SUR-E group (60 [54%] of 111; adjusted RR 0·75, 95% CI 0·57-0·98; p=0·037), with a number needed to treat of 7·2 (95% CI 3·7-135·0). The addition of the recruitment manoeuvre did not adversely affect the safety outcomes of in-hospital mortality (19 [19%] of 101 in the IN-REC-SUR-E group vs 37 [33%] of 111 in the IN-SUR-E group), pneumothorax (four [4%] of 101 vs seven [6%] of 111), or grade 3 or worse intraventricular haemorrhage (12 [12%] of 101 vs 17 [15%] of 111). INTERPRETATION: A lung recruitment manoeuvre just before surfactant administration improved the efficacy of surfactant treatment in extremely preterm neonates compared with the standard IN-SUR-E technique, without increasing the risk of adverse neonatal outcomes. The reduced need for mechanical ventilation during the first 72 h of life might facilitate implementation of a non-invasive respiratory support strategy. FUNDING: None.

A modified physiologic test for bronchopulmonary dysplasia: a clinical tool for weaning from CPAP and/or oxygen-therapy the premature babies?

BACKGROUND: A physiologic test for estimating BPD rate has been developed by Walsh and collaborators. Actually there are not standard criteria for weaning from CPAP and/or oxygen therapy the premature babies. Aim of this study was to verify if a physiologic test, modified respect to that developed by Walsh and collaborators for estimating BPD rate, can be used as a clinical tool for weaning the premature babies from CPAP and/or oxygen therapy. METHODS: Neonates with BW 500-1250 g and GA ≤ 32 weeks, receiving FiO2 ≤ 0.30 by hood or CPAP, were prospectively studied at 28 days of life and at 36 weeks of postmestrual age. The test was performed in 3 steps: baseline, challenge (FiO2 and CPAP reduction to room air) and post test (room air). Monitoring of transcutaneous CO2 was added to SpO2 and the newborns passing the test were left in room air. RESULTS: Six of 23 tested babies (26%) passed the challenge at 28 days of life, 4 of 10 tested babies (40%) passed the challenge at 36 weeks. Median values of SpO2 were significantly higher in the neonates passing the test, respect to the failing patients. At the same time median values of TcPCO2 were significantly higher in the latter babies. CONCLUSION: TcPCO2 monitoring appeared to be a new useful parameter for failure prediction of weaning. The test represented a clinical guide because the newborns passing it were left in room air.

New Insights on Early Patterns of Respiratory Disease among Extremely Low Gestational Age Newborns.

BACKGROUND: The analysis of early patterns of lung disease among preterm infants may help to identify predictors of pulmonary deterioration. OBJECTIVES: To analyze FIO2 requirement in the first 14 days of life among preterm infants and to find predictors of bronchopulmonary dysplasia (BPD). METHODS: Retrospective cohort study. SETTING: 3 Italian level III NICUs. POPULATION: infants born between 240/7 and 276/7 weeks' gestational age (GA) who survived to 14 days. A consecutive sample of 588 infants was analyzed. Daily mode FIO2 in the first 2 weeks of life were analyzed according to the criteria defined by Laughon et al. [Pediatrics 2009;123:1124-1131], who found 3 early respiratory patterns: consistently low FIO2 (LowFIO2), pulmonary deterioration (PD), and early persistent pulmonary deterioration (EPPD). Factors associated with pulmonary deterioration were studied by univariate and multivariate analysis. RESULTS: Forty percent of infants had low FIO2, 18% had pulmonary deterioation, 21% had early persistent pulmonary deterioration, and 21% had a previously unreported pattern (pulmonary improvement, PI). The prevalence of BPD was 7% in the LowFIO2 group, 28% in the PI group, 44% in the PD group, and 62% in the EPPD group (p = 0.000). Infants with lung deterioration were more frequently males (OR = 2.019, CI: 1.319-3.090, p = 0.001), had lower GA (OR = 0.945, CI: 0.915-0.975, p = 0.000), higher incidence of severe respiratory distress syndrome (OR = 2.956, CI: 1.430-6.112, p = 0.003), and lack of postnatal caffeine (OR = 0.167, CI: 0.052-0.541, p = 0.003). CONCLUSIONS: We report 4 distinct patterns of early respiratory disease associated with significantly different prevalence of BPD and discuss risk factors for lung deterioration.

Efficacy of a new technique - INtubate-RECruit-SURfactant-Extubate - "IN-REC-SUR-E" - in preterm neonates with respiratory distress syndrome: study protocol for a randomized controlled trial.

BACKGROUND: Although beneficial in clinical practice, the INtubate-SURfactant-Extubate (IN-SUR-E) method is not successful in all preterm neonates with respiratory distress syndrome, with a reported failure rate ranging from 19 to 69 %. One of the possible mechanisms responsible for the unsuccessful IN-SUR-E method, requiring subsequent re-intubation and mechanical ventilation, is the inability of the preterm lung to achieve and maintain an "optimal" functional residual capacity. The importance of lung recruitment before surfactant administration has been demonstrated in animal studies showing that recruitment leads to a more homogeneous surfactant distribution within the lungs. Therefore, the aim of this study is to compare the application of a recruitment maneuver using the high-frequency oscillatory ventilation (HFOV) modality just before the surfactant administration followed by rapid extubation (INtubate-RECruit-SURfactant-Extubate: IN-REC-SUR-E) with IN-SUR-E alone in spontaneously breathing preterm infants requiring nasal continuous positive airway pressure (nCPAP) as initial respiratory support and reaching pre-defined CPAP failure criteria. METHODS/DESIGN: In this study, 206 spontaneously breathing infants born at 24(+0)-27(+6) weeks' gestation and failing nCPAP during the first 24 h of life, will be randomized to receive an HFOV recruitment maneuver (IN-REC-SUR-E) or no recruitment maneuver (IN-SUR-E) just prior to surfactant administration followed by prompt extubation. The primary outcome is the need for mechanical ventilation within the first 3 days of life. Infants in both groups will be considered to have reached the primary outcome when they are not extubated within 30 min after surfactant administration or when they meet the nCPAP failure criteria after extubation. DISCUSSION: From all available data no definitive evidence exists about a positive effect of recruitment before surfactant instillation, but a rationale exists for testing the following hypothesis: a lung recruitment maneuver performed with a step-by-step Continuous Distending Pressure increase during High-Frequency Oscillatory Ventilation (and not with a sustained inflation) could have a positive effects in terms of improved surfactant distribution and consequent its major efficacy in preterm newborns with respiratory distress syndrome. This represents our challenge. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02482766 . Registered on 1 June 2015.

Changes in ventilator strategies and outcomes in preterm infants.

BACKGROUND: Although life-saving, intubation and mechanical ventilation can lead to complications including bronchopulmonary dysplasia (BPD). In order to reduce the incidence of BPD, non-invasive ventilation (NIV) is increasingly used. OBJECTIVE: The aim of our study was to describe changes in ventilator strategies and outcomes between 2006 and 2010 in the Italian Neonatal Network (INN). DESIGN: Multicentre cohort study. SETTINGS: 31 tertiary level neonatal units participating in INN in 2006 and 2010. PATIENTS: 2465 preterm infants 23-30 weeks gestational age (GA) without congenital anomalies. MAIN OUTCOMES MEASURES: Death, BPD and other variables defined according to Vermont Oxford Network. Logistic regressions, adjusting for confounders and clustering for hospitals, were used. RESULTS: Similar numbers of infants were studied between 2006 and 2010 (1234 in 2006 and 1231 in 2010). The baseline risk of populations studied (GA, birth weight and Vermont Oxford Network Risk-Adjustment score) did not change. After adjusting for confounding variables, infants receiving invasive mechanical ventilation decreased (OR=0.72, 95% CI 0.58 to 0.89) while NIV increased (OR=1.75, 95% CI 1.39 to 2.21); intubation in delivery room decreased (OR=0.64, 95% CI 0.51 to 0.79). Considering outcomes, there was a significant reduction in mortality (OR=0.73, 95% CI 0.55 to 0.96) and in the combined outcome mortality or BPD (OR=0.76, 95% CI 0.62 to 0.94). CONCLUSIONS: Despite a stable baseline risk, from 2006 to 2010, we observed a lower level of invasiveness, a reduction of mechanical ventilation and an increase of NIV use, and this was accompanied by a decrease in risk-adjusted mortality and BPD.

Clinical prediction models for bronchopulmonary dysplasia: a systematic review and external validation study.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth. Very different models using clinical parameters at an early postnatal age to predict BPD have been developed with little extensive quantitative validation. The objective of this study is to review and validate clinical prediction models for BPD. METHODS: We searched the main electronic databases and abstracts from annual meetings. The STROBE instrument was used to assess the methodological quality. External validation of the retrieved models was performed using an individual patient dataset of 3229 patients at risk for BPD. Receiver operating characteristic curves were used to assess discrimination for each model by calculating the area under the curve (AUC). Calibration was assessed for the best discriminating models by visually comparing predicted and observed BPD probabilities. RESULTS: We identified 26 clinical prediction models for BPD. Although the STROBE instrument judged the quality from moderate to excellent, only four models utilised external validation and none presented calibration of the predictive value. For 19 prediction models with variables matched to our dataset, the AUCs ranged from 0.50 to 0.76 for the outcome BPD. Only two of the five best discriminating models showed good calibration. CONCLUSIONS: External validation demonstrates that, except for two promising models, most existing clinical prediction models are poor to moderate predictors for BPD. To improve the predictive accuracy and identify preterm infants for future intervention studies aiming to reduce the risk of BPD, additional variables are required. Subsequently, that model should be externally validated using a proper impact analysis before its clinical implementation.

Calcium signaling-related proteins are associated with broncho-pulmonary dysplasia progression.

Broncho-pulmonary dysplasia (BPD) is a chronic pulmonary disorder that follows premature birth. It is preceded by respiratory distress syndrome (RDS), characterized by acute respiratory failure due to deficiency of surfactant at birth. Clinical characteristics of infants affected by BPD have widely changed in the last decades: they are extraordinarly immature, with impaired alveolar and vascular lung development. To build up new therapeutic strategies for BPD babies, it is necessary to understand the pathogenic mechanisms, which are complicated by environmental risk factors and genetic predisposition. Therefore, the aim of this study was to highlight protein changes in the broncho-alveolar lavage fluid (BALF), thus providing an appropriate picture on what is happening in the locus of injury. We analyzed BALF samples from preterm babies, born at different stages of lung development. We confirmed that gestational age is relevant for BPD progression, but we also detected few de-regulated proteins in the younger babies; we discovered less abundant calcium signaling-related proteins, consistent with BPD severity, comparing severe to mild BPD babies with matched gestational age. In conclusion, this study suggests a subset of proteins to be investigated to better treat BPD babies and facilitate the definition of potential drug targets for novel therapies. BIOLOGICAL SIGNIFICANCE: Pulmonary biomarkers are needed to predict the clinical course of lung disease, status, progression and response to treatment. A key aspect in biomarker discovery is uncovering molecules that appear early during disease initiation, when the natural history of the disease can be modified. Using a proteomic-based approach we compared broncho-alveolar lavage fluid (BALF) protein profile from preterm neonates at different postmenstrual ages, to have a molecular description of broncho-pulmonary dysplasia (BPD) progression. BALF provided a snapshot of local molecular changes, which are relevant for early diagnosis, assessment and characterization of lung disorders. We showed that even if the studied patients had similar clinical phenotype (they all developed severe BPD and they were all cured in the same way in terms of mechanical ventilation, surfactant administration, antenatal steroid treatment and ibuprofen treatment for patent ductus arteriosus), however their BALF protein profiling displayed significant differences in a subset of proteins, which could be exploited to facilitate the development of novel effective therapies, distinct for age and severity of the disease.

Survey of neonatal respiratory care and surfactant administration in very preterm infants in the Italian Neonatal Network.

INTRODUCTION: Variation of respiratory care is described between centers around the world. The Italian Neonatal Network (INN), as a national group of the Vermont-Oxford Network (VON) allows to perform a wide analysis of respiratory care in very low birth weight infants. METHODS: We analyzed the dataset of infants enrolled in the INN in 2009 and 2010 and, for surfactant administration only, from 2006 to 2010 from 83 participating centers. All definitions are those of the (VON). A questionnaire analysis was also performed with a questionnaire on centers practices. RESULTS: We report data for 8297 infants. Data on ventilator practices and outcomes are outlined. Variation for both practices and outcome is found. Trend in surfactant administration is also analyzed. CONCLUSIONS. The great variation across hospitals in all the surveyed techniques points to the possibility of implementing potentially better practices with the aim of reducing unwanted variation. These data also show the power of large neonatal networks in identifying areas for potential improvement.

Nasal continuous positive airway pressure with heliox in preterm infants with respiratory distress syndrome.

OBJECTIVE: To assess the therapeutic effects of breathing a low-density helium and oxygen mixture (heliox, 80% helium and 20% oxygen) in premature infants with respiratory distress syndrome (RDS) treated with nasal continuous positive airway pressure (NCPAP). METHODS: Infants born between 28 and 32 weeks of gestational age with radiologic findings and clinical symptoms of RDS and requiring respiratory support with NCPAP within the first hour of life were included. These infants were randomly assigned to receive either standard medical air (control group) or a 4:1 helium and oxygen mixture (heliox group) during the first 12 hours of enrollment, followed by medical air until NCPAP was no longer needed. RESULTS: From February 2008 to September 2010, 51 newborn infants were randomly assigned to two groups, 24 in the control group and 27 in the heliox group. NCPAP with heliox significantly decreased the risk of mechanical ventilation in comparison with NCPAP with medical air (14.8% vs 45.8%). CONCLUSIONS: Heliox increases the effectiveness of NCPAP in the treatment of RDS in premature infants.

Elective high-frequency oscillatory versus conventional ventilation in preterm infants: a systematic review and meta-analysis of individual patients' data.

BACKGROUND: Population and study design heterogeneity has confounded previous meta-analyses, leading to uncertainty about effectiveness and safety of elective high-frequency oscillatory ventilation (HFOV) in preterm infants. We assessed effectiveness of elective HFOV versus conventional ventilation in this group. METHODS: We did a systematic review and meta-analysis of individual patients' data from 3229 participants in ten randomised controlled trials, with the primary outcomes of death or bronchopulmonary dysplasia at 36 weeks' postmenstrual age, death or severe adverse neurological event, or any of these outcomes. FINDINGS: For infants ventilated with HFOV, the relative risk of death or bronchopulmonary dysplasia at 36 weeks' postmenstrual age was 0.95 (95% CI 0.88-1.03), of death or severe adverse neurological event 1.00 (0.88-1.13), or any of these outcomes 0.98 (0.91-1.05). No subgroup of infants (eg, gestational age, birthweight for gestation, initial lung disease severity, or exposure to antenatal corticosteroids) benefited more or less from HFOV. Ventilator type or ventilation strategy did not change the overall treatment effect. INTERPRETATION: HFOV seems equally effective to conventional ventilation in preterm infants. Our results do not support selection of preterm infants for HFOV on the basis of gestational age, birthweight for gestation, initial lung disease severity, or exposure to antenatal corticosteroids. FUNDING: Nestlé Belgium, Belgian Red Cross, and Dräger International.

Early mannan detection in bronchoalveolar lavage fluid with preemptive treatment reduces the incidence of invasive Candida infections in preterm infants.

BACKGROUND: Candida colonization is an important predictor for development of invasive fungal infection (IFI). We investigated whether early detection of Candida mannan (Mn) in bronchoalveolar lavage fluid (BALF) reduces IFI among preterm infants. METHODS: We conducted an observational study of infants with gestational age of < or =28 weeks, where a group undergoing Candida surveillance cultures (pre-Mn detection group) was compared with a group defined after the initiation of routine use of Candida Mn detection in BALF (Mn detection group). Antifungal treatment was started based on positive microbiologic (surveillance culture or Mn-antigen assay) results. RESULTS: No significant differences were detected when the groups were compared for several predictors of IFI. IFI was observed for 12 (23%) of 51 infants in the pre-Mn detection group, and for 0 (0%) of 29 infants in the Mn detection group (P = 0.003). Surveillance cultures in the pre-Mn detection group became positive at 15.0 +/- 7.2 days after birth, whereas the mean age at time of positive Mn antigen results in the Mn detection group was 4.3 +/- 3.1 days (P < 0.0001). Among 16 infants positive for surveillance cultures, 12 (75%) developed IFI (P < 0.0001). CONCLUSIONS: This study suggests that Candida Mn detection in BALF may be useful for earlier identification and preemptive therapy targeting preterm infants at high risk of IFI.

[Respiratory failure in "late preterm" infants: a retrospective cohort study]. / Insufficienza respiratoria nei neonati "late preterm": uno studio retrospettivo di coorte.

OBJECTIVE: To evaluate the incidence and characteristics of the respiratory failure in late preterm infants. STUDY DESIGN: Retrospective data analysis in years 2006-2007 in late preterm infants (GA 34(+0)-36(+6) weeks) with respiratory failure, admitted at a tertiary level NICU. RESULT: Data from 1011 late preterm infants, which accounted for 7% of all deliveries and 65% of preterm births were analyzed; 29% (292/1011) required intensive care and 13% (136/1011) presented respiratory failure (16% of all ventilated infants in the period). In late preterms with respiratory failure 23% (32/136) were treated with prenatal steroids 46% (62/136) with non -invasive ventilation (nasal continuous positive airways pressure = nCPAP) while 41% (56/136) were intubated and received exogenous surfactant. Mean days of ventilation were 5.3 +/- 6.5 (0.5-55); 3.7% (5/136) developed bronchopulmonary dysplasia defined as oxygen-dependency at 36 postconceptional age and mortality was 1.5% (2/136). CONCLUSION: Respiratory failure incidence and characteristics in late preterms suggest their peculiarity and relevance in neonatal intensive care.

The role of Candida surveillance cultures for identification of a preterm subpopulation at highest risk for invasive fungal infection.

Candida surveillance cultures were obtained from 51 neonatal intensive care unit patients. Sixteen infants showing positive cultures developed subsequent Candida infection, whereas it did not occur for the 35 infants with negative cultures. Fifteen of 16 infants (<1000 g) had <27 weeks of gestational age. Antifungal treatment was started at the time colonization was detected; only 1 infant died of Candida infection.

Mannose-binding lectin polymorphisms and pulmonary outcome in premature neonates: a pilot study.

OBJECTIVE: Mannose-binding lectin (MBL2) is a collectin molecule able to activate the complement system and the subsequent inflammatory mechanisms. Several MBL2 genetic variants have been described, including the six variants studied in this report, which are those analyzed in most detail in the medical literature. DESIGN: The present study analyzes the prevalence of MBL2 gene variants in preterm newborns and associates individual genotypes with pulmonary outcome variables. All polymorphisms were analyzed by means of a commercially available reverse dot-blot kit. SETTING: Tertiary neonatal intensive care unit. PATIENTS AND PARTICIPANTS: Seventy-five consecutive preterm newborns. MEASUREMENTS AND RESULTS: Two variants were particularly analyzed: -550G > C and R52C. The first one is known to be associated with lower protein synthesis when included in specific haplotypes. The homozygous and heterozygous -550G > C mutations were significantly associated with protective effects regarding different lung outcome variables, including shorter duration of mechanical ventilation, hours of continuous positive airway pressure and lower number of hemotransfusions. In contrast, the heterozygous R52C mutation was associated with unfavorable outcome, including higher bronchopulmonary dysplasia prevalence. Multivariate logistic regression analysis showed that these associations were independent of gestational age and birth weight. In addition, four groups of patients were defined on the basis of haplotype combinations. Those known to be associated with low serum MBL2 levels were linked to a better outcome in terms of factors such as hours of mechanical ventilation, continuous positive airway pressure, number of hemotransfusions and bronchopulmonary disease development. CONCLUSIONS: The four haplotype combination groups may have a potential diagnostic use as opposite risk factors for lung disease of prematurity.

Epithelial lining fluid free IGF-I-to-PAPP-A ratio is associated with bronchopulmonary dysplasia in preterm infants.

Preterm newborns developing retinopathy of prematurity (ROP) and bronchopulmonary dysplasia (BPD) show persistently low levels of insulin-like growth factor-I (IGF-I) in sera. They also present higher free IGF-I concentrations in epithelial lining fluids (ELFs) and lung tissues. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase that dissociates three binding proteins from the active form of IGF-I, namely free IGF-I. The present study analyzes the ELF concentrations of free IGF-I, PAPP-A, and their ratios in preterm newborns developing or not BPD, defined as O(2) dependence at 36 wk postmenstrual age. Bronchoalveolar lavage fluids of 41 infants (34 without and 7 with BPD) were analyzed on the 2nd and 4th day after birth. Infants developing BPD showed increased ELF free IGF-I and decreased PAPP-A concentrations on both days 2 and 4 compared with newborns without BPD. A nonsignificant trend between these 2 days was observed for free IGF-I (increasing) and PAPP-A (decreasing). On the same days, the free IGF-I-to-PAPP-A ratio was always significantly higher in patients developing BPD. These differences were more significant than those of IGF-I or PAPP-A when individually evaluated. A multivariate analysis confirmed the significance for free IGF-I on day 4, whereas the ratio was confirmed on both days 2 and 4. The same ratio was significantly correlated with some indexes of disease severity, such as hours of oxygen administration, days of hospitalization, and ROP severity scores. Finally, the ratio between ELF free IGF-I and PAPP-A appears to be a useful marker for lung injury of premature newborns.

Serum levels of seven cytokines in premature ventilated newborns: correlations with old and new forms of bronchopulmonary dysplasia.

OBJECTIVE: In addition to the previous classification of chronic lung disease (CLD) O2 dependency at 36 weeks of postmenstrual age, a new definition of CLD has recently been proposed: new bronchopulmonary-dysplasia (BPD). This uses total duration of O2 supplementation and positive pressure requirements to delineate three degrees of severity (mild, moderate, and severe) according to the respiratory status at 36 weeks postmenstrual age. We analyzed the balance of serum proinflammatory and profibrotic/angiogenic cytokine concentrations in relation to CLD and the new BPD definition. DESIGN AND SETTING: Descriptive study in a third-level neonatal ICU. PATIENTS: Thirty-one preterm neonates with a gestational age of 24-29 weeks were studied to evaluate their serum cytokine concentration; they were previously enrolled in a randomized clinical trial to compare the effects of high-frequency oscillatory ventilation vs. intermittent mandatory ventilation in terms of pulmonary mechanics and lung cytokines. Serum samples were collected on days 1, 3, and 5 after birth until extubation to detect the levels of three proinflammatory cytokines plus four profibrotic/angiogenic cytokines, and correlations were examined to old CLD and new BPD. Ventilation treatments were distributed homogeneously between the groups and did not interfere with the results presented here. RESULTS AND CONCLUSIONS: Old CLD development, mainly corresponding to the moderate/severe forms of new BPD, was associated with increased proinflammatory and profibrotic/angiogenic cytokines, while mild forms of new BPD were characterized only by increases in profibrotic/angiogenic cytokines, suggesting a different balance of two pathogenic mechanisms in different phases of the disease.