Introduction
Necrotizing enterocolitis (NEC) remains a major cause of mortality and morbidity in premature infants. Risk factors for NEC include prematurity, intestinal hypoxia/ischaemia, formula feeding and colonization of the intestine by pathogenic organisms. These factors may promote an inflammatory cascade as a common final pathway [1]. Physiologically, the intestinal mucosal barrier prevents penetration of bacteria, toxins and antigens, and B and T lymphocytes are essential components of this mucosal immune system. Immaturity of the intestinal mucosal barrier may play an important role in the pathogenesis of NEC in premature infants [2].
There has been only one published report of intestinal symptoms observed in neonates born to HIV-positive mothers; the two infants presented with hypoperistalsis and intestinal pseudo-obstruction [3]. Poor mobility of the intestine, leading to bacterial overgrowth, may increase the risk of NEC. In addition, the complex immunological changes, involving cytokine production and T cell function, observed in uninfected infants born to HIV-infected mothers [4,5] also support the hypothesis of a possible link between HIV vertical exposure and NEC.
This case-control study was designed to test the hypothesis that being born prematurely to an HIV-positive mother is an independent risk factor for developing NEC.
Methods
The study was performed on premature infants born before a postmenstrual age (PMA) of 37 weeks in our institution, from January 1995 to July 2003.
Identification and management of necrotizing enterocolitis
In this population of premature infants, all occurrences of frank red blood in the stools were identified. In these infants, NEC was diagnosed using a modified Bell's classification [6]. Only 'proven' (stage IIB) and 'advanced' (stages IIIA and IIIB) NEC was included in the study. This meant that pneumatosis intestinalis was observed by abdominal radiography in addition to the detection of red blood in the stools. Under the clinic's usual procedures, abdominal radiographs were analysed by both the neonatology team and a paediatric radiologist to ensure correct interpretation. A paediatric surgeon was consulted if this was considered necessary. Age at onset, modified Bell's stage and treatment were recorded. In order to study all NEC cases occurring in our premature infants until home discharge, information was collected for each infant for the entire clinical course, including hospital stays in any other institution as many of our infants are referred to other hospitals before being discharged home.
The routine care of infants with NEC did not change during this period. When the diagnosis of NEC was suspected, feeding was immediately stopped, total parenteral nutrition was initiated, a sepsis workup was performed and intravenous antibiotics were started. If the baby deteriorated clinically, intubation was performed and mechanical ventilation was provided. Continuous gastric aspiration was started if there was abdominal distension. Serial abdominal radiographs were performed during the acute phase of NEC. Surgery was indicated when free air was detected in the abdomen, when pneumatosis was extensive and/or was suspected to reach the small intestine, or when deterioration continued despite optimal medical management. It consisted of exploratory laparotomy and ileostomy in a normal intestinal zone above the lesions. Bowel resection was not considered at this stage.
Controls
For each case of NEC, two matched controls were retrospectively chosen. Matching was based on PMA at birth (same week), intrauterine growth [classified as adequate or growth restriction (< 10th centile of Lubchenco's growth chart [7])] and chronological order of admission. Following one case of NEC, the next two patients born at the same week of PMA and with or without intrauterine growth restriction were chosen. Since NEC can occur after a few weeks of life, control infants were only selected if they survived until home discharge.
Perinatal characteristics of cases and controls
Perinatal characteristics were recorded, with special attention to those identified as risk factors for NEC.
Cocaine and morphine abuse were determined from the maternal history. Prenatal corticosteroids were considered to have been used for fetal lung maturation when they were initiated more than 12 h and less than 15 days before birth. Abnormal umbilical artery velocity during fetal life was defined as absent or reverse end-diastolic velocities [8]. Fetal heart rate was considered abnormal when any one of the following was observed: late fetal heart rate decelerations, poor variability or persistent tachycardia.
CRIB scores [9] were determined. PMA was estimated from the date of the last menstrual period and routinely confirmed by ultrasound examination in the first trimester. In the case of discrepancy of more than 6 days, PMA was based on the ultrasound determination. Additional data (not listed) were respiratory outcome and cerebral complications detected by ultrasound.
Feeding guidelines were fairly uniform during the study period. Indeed, enteral feeding was started as early as day 2 or 3 in premature infants with PMA < 32 weeks; after initial stabilization, infants were given human milk by orogastric infusion over 2 h, eight times a day. Volumes were slowly increased by 10-15 ml/kg, every 24 to 48 h, according to clinical tolerance. Whenever available, milk from the infant's own HIV-negative mother was used. When milk from HIV-negative mothers was not available, and in all infants born to HIV-positive mothers, banked human milk was used. In infants born between 32 and 36 weeks of PMA, enteral feeding was usually started using preterm infant formula with volume increases of 20 ml/kg every 24 h.
The standard care of HIV-positive women and newborn infants was oral zidovudine (3′-azido-3′deoxythymidine), which was started between 14 and 34 weeks of pregnancy and continued until the onset of labour for the mother and after delivery to the newborn infant in order to prevent maternal-infant HIV transmission [10]. Zidovudine was used alone or in combination with other antiretroviral drugs. Combinations consisted of two nucleoside reverse transcriptase inhibitors (NRTI: zidovudine and usually didanosine or lamivudine) and protease inhibitors (indinavir, ritonavir or nelfinavir, or a combination of ritonavir and lopinavir). An injection of zidovudine was also administered during labour.
Newborn infants with PMA ≤ 34 weeks were initially given zidovudine intravenously. This was changed to enteral administration when enteral feeding was tolerated. Newborn infants with PMA > 34 weeks were given zidovudine enterally right from birth. The total duration of treatment was 6 weeks. Before 1999, the zidovudine dosage regimens were 1.5 mg/kg and 2 mg/kg every 6 h for intravenous and enteral administration, respectively. Since 1999, the same dose is given every 12 h, because of the excessive plasma levels recorded with the previous schedule, in agreement with published data [11,12]. Alternative treatments were considered if the HIV was resistant to zidovudine.
Diagnosis of infantile HIV infection was based on two techniques. HIV DNA polymerase chain reaction was performed on peripheral blood mononuclear cells obtained from samples taken at birth and at 1, 3 and 6 months of age. Antibody testing was performed at birth, and at 6, 12 and 18 months of age. HIV infection was excluded when HIV DNA was not detected by 6 months of age, and when antibody tests became negative at 12 or 18 months of age.
Data analysis
Univariate analysis was used to assess differences between cases and controls, focusing on previously reported NEC risk factors. Student's t test or the non-parametric Mann-Whitney U test was used to compare numerical data for the two groups, as appropriate. The χ2 test or the exact Fisher test was used to analyse categorical variables. Multivariate logistic regression was performed to estimate the independent effects of each selected risk factor. All variables found to be associated with NEC at a limit of 0.1 on univariate analysis were included in the final model. Odds ratios, adjusted for all other factors, were calculated. All analyses were performed with Statview 5.0 statistical software package (SAS Institute, Cary, North Carolina, USA).
Results
Of approximately 30 000 infants born in our centre between January 1995 and July 2003, 4009 (13.3%) were born before a PMA of 37 weeks and were admitted to the neonatal unit. In 1917 of these 4009 infants, PMA was < 32 weeks. During the same period, 631 infants of HIV-positive mothers who had received perinatal care were born in our institution; 78 of these infants were born before a PMA of 37 weeks and were admitted to the neonatal unit. In 42 of these 78, PMA was < 32 weeks (Table 1). Relevant data recorded at birth and during the neonatal course are listed in Tables 2 and 3.
Necrotizing enterocolitis
Of the 4009 premature infants, 79 (2%) developed NEC and 62 of these were from the group of 1917 infants born with a PMA of < 32 weeks. During the study period, the incidence of NEC was 1.8% (72/3931) in preterm infants born to HIV-negative mothers and 8.9% (7/78) in the premature infants born to HIV-positive mothers.
The characteristics of the 79 cases of NEC and the 158 controls are shown in Tables 1 and 2. Comparison of the main prenatal characteristics of the two groups showed significant differences (Table 1). Multiple pregnancies and abnormalities of both umbilical artery velocity and fetal heart rate were more frequent in the cases than in the controls. Prenatal corticosteroid use, as defined in the Methods, was less frequent in the NEC group. This difference, however, was no longer observed when all cases of prenatal corticosteroid use were considered (any number of injections, any time before delivery). Finally, HIV-positive status of the mothers was more frequent in the NEC group, as 7 of 79 NEC cases and 2 of 158 controls were born to HIV-positive mothers (P = 0.01).
Comparison of neonatal characteristics (Table 2) and outcome (Table 3) of cases and controls did not reveal any significant difference, including the incidence of patent ductus arteriosus and the use of indometacin. Plasma haemoglobin and lactate levels on the day of admission were also similar. In the NEC group, the median age at NEC onset was 20 days (range, 1-78). Surgery was performed during the acute phase in 27 (34%). Four patients required delayed surgery, owing to intestinal stenosis. NEC-related mortality rate was 10%.
HIV-positive mothers and their infants
Nine of the mothers were HIV-positive (seven in the NEC group and two in the control group; Table 4). Severity of NEC in these infants is indicated by the need for surgery in five of the seven.
Of the seven HIV-positive mothers whose infants developed NEC, six had received antiretroviral drugs during pregnancy (Table 4). In two, this treatment consisted of zidovudine alone. In four, it consisted of combination therapy with two NRTI (zidovudine and didanosine or lamivudine) and one protease inhibitor. The treatments were given during the third trimester, and one zidovudine injection was given during labour. The last HIV-positve mother was not treated during the third trimester but received zidovudine during labour. The seven infants also received zidovudine: only intravenous in three,intravenous followed by enteral administration in three and only enterally in one. Treatment was immediately stopped in six of the seven infants.
The two HIV-positive mothers whose infants were included in the control group received combination therapy during the third trimester and zidovudine injection during labour. The two infants received zidovudine, which was stopped after one injection in one because of HIV resistance to zidovudine. This infant subsequently received one dose of nevirapine.
None of the nine infants of HIV-positive mothers was infected.
Multivariate analysis
Perinatal characteristics that were independently and significantly associated with an increased risk of NEC were identified using multivariable logistic regression (Table 5). Among these characteristics, HIV-positive mother status had the highest odds ratio (6.63; 95% confidence interval, 1.26-34.8; P = 0.02).
Discussion
This case-control study suggests a possible association between maternal HIV-positive status and development of NEC in premature infants. This association, which might be of clinical relevance, has not been previously reported.
The limitations of the study comprise inclusion of a fairly small number of cases and the inability to demonstrate a causal relationship because of the retrospective design. However, the following elements support the validity of our observations. NEC diagnosis was carefully established, based on objective criteria and selection of only proven or advanced stage disease [6]. Correct identification of NEC in infants of HIV-positive mothers was further supported by the requirement for surgery in five of the seven, although these numbers are too small to allow comparisons of NEC severity in relation to the maternal HIV status. Lack of bias in selection of the control group is supported by the similar incidence of HIV-positive mothers in this group (1.3%) and in our overall population of premature infants (78 HIV-positive mothers for 4009 premature infants, or 1.9%). In addition, we assumed 100% identification of HIV infected mothers based on the almost universal HIV testing during pregnancy in France.
NEC, which is a gastrointestinal disease resulting in significant morbidity and mortality, predominantly affects premature infants weighing < 1500 g at birth. Our NEC incidence was similar to that of a recent report [13].
The pathogenesis of NEC involves several factors, including enteral feeding, mesenteric ischaemia and bacterial colonization. These factors are believed to stimulate proinflammatory mediators, resulting in bowel necrosis [1]. Our multivariate analysis identified significant differences between the infants with NEC and the control group. Multiple pregnancy was more frequent in infants who subsequently developed NEC, as previously reported [14]. We confirmed that abnormal umbilical artery velocity, which is associated with decreased perfusion of the intestine, was more frequent in fetuses who subsequently developed NEC after birth [8]. The more frequent abnormal fetal heart rate before postnatal occurrence of NEC highlights the role of perinatal asphyxia and ischaemic mesenteric injury in the pathogenesis of this disease. Prenatal corticosteroid administration may have a protective effect against the risk of NEC [15], although this factor was not statistically significant in our multivariate analysis. On the contrary, prenatal corticosteroids have been reported to increase the risk of NEC [13,16], possibly because of frequent exposure to multiple courses of corticosteroids that has occurred over recent years compared with the previous decade [13].
Our study suggests an association between maternal HIV-positive status and the development of NEC, as 8.9% of mothers in the NEC group were HIV positive, compared with 1.3% in the control group. On multivariate analysis, maternal HIV-positive status was an independent risk factor of NEC with an odds ratio of 6.6 (95% confidence interval, 1.26-34.8).
Two mechanisms can be proposed to explain the hypothesis of an increased risk of NEC in premature infants exposed to HIV: the actual HIV exposure during fetal life and the use of zidovudine.
The risk of NEC might be increased by exposure to HIV during fetal life. The absence of any HIV infection at follow-up of the infants does not rule out this hypothesis, as significant haematological and immunological abnormalities, including cytokine production, have been reported in HIV-uninfected infants of HIV-positive mothers [4,5]. These deficiencies may make these infants more prone to infections, and possibly to developing NEC. Abnormalities of interleukin-12 regulation constitute a possible link between HIV exposure and the occurrence of NEC. Impaired interleukin-12 production has been demonstrated at birth in uninfected infants born to HIV-positive mothers and may persist after birth [17]. Decreases in both production [18] and mRNA expression [19] of interleukin-12 in gut mucosa may contribute to the pathogenesis of NEC, whereas increased interleukin-12 levels appear to protect against pan-necrosis in infants with acute NEC [20].
The risk of NEC might be increased by the use of zidovudine. In newborn infants, the commonest adverse effect of zidovudine is reversible macrocytic anaemia [21], which was not observed at the time of admission in our infants. NEC has not been reported before. Zidovudine might increase the risk of NEC by increasing feeding osmolarity [22], through the use of high osmolarity (3600 mOsm/kg) zidovudine syrup. However, the occurrence of three of our seven cases of NEC while using only the intravenous route makes this mechanism very unlikely. Another possibility is that ZDV-induced mitochondrial dysfunction could be responsible for the development of NEC. Several adverse effects of NRTI on a large number of organs are caused by drug-induced mitochondrial dysfunction, which results from the effect of NRTI on mitochondrial DNA polymerases [23-25]. Transplacental zidovudine exposure can also result in mitochondrial damage [26-28]. In newborn infants, mitochondrial toxicity of zidovudine may account for the occurrence of transient lactic acadaemia [29]. Neuman et al. [3] hypothesized that mitochondrial toxicity of zidovudine could also account for the occurrence of hypoperistalsis and intestinal pseudo-obstruction. The low level of mitochondrial DNA at birth and the common observation of gastrointestinal symptoms in adults presenting with defects of mitochondrial DNA [24,30] support this hypothesis. We speculate that zidovudine -induced transient enteric mitochondrial dysfunction may also contribute to the development of NEC.
In conclusion, our case-control study suggests an association between perinatal HIV exposure and an increased risk of NEC in premature infants. Because of the limitations of the study, no definite conclusions can be drawn. Other studies would help to establish whether the suspected association really exists. A causal relationship simply remains a hypothesis at the present time. Possible mechanisms include immunological alterations in uninfected infants born to HIV-positive mothers, or transient mitochondrial dysfunction induced by NRTI, a mild adverse effect in comparison with the enormous benefits resulting from the use of zidovudine during human pregnancy and in newborn infants. An awareness of a possible association between perinatal HIV exposure and an increased risk of NEC in premature infants might help to improve the management of these infants.
Acknowledgements
We gratefully acknowledge the contributions of Stephane Blanche for helpful comments on the article and Anthony Saul for stylistic review of the manuscript.
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