Amanda R. Highet

An infectious aetiology of sudden infant death syndrome

Due attention has been given to infectious agents and immune responses to infection in sudden infant death syndrome (SIDS). It has been acknowledged that the pathological, epidemiological and genotypic findings in SIDS infants suggest an infectious aetiology possibly being potentiated by immunoregulatory polymorphisms, however, the cause of SIDS is a mystery and remains open to debate. Consistent pathological findings are seen which display similarities to the pathogenesis of toxaemic shock and/or sepsis. The major risk factors for SIDS parallel those for increased colonization and serious bacterial infections and the natural variation in the incidence of SIDS cases is typical of an infectious disease. The roles played by viral infection, immunoregulatory genes and suspected bacterial species are discussed herein.

Gut microbiome in sudden infant death syndrome (SIDS) differs from that in healthy comparison babies and offers an explanation for the risk factor of prone position

The role of bacteria in the causation of sudden infant death syndrome (SIDS) is gaining acceptance. Mainstream research favouring respiratory compromise has failed to provide a plausible pathogenetic mechanism despite many years of investigation and thousands of research papers. Bacterial colonisation of the colon of the human infant is influenced by many factors including age, mode of delivery, diet, environment, and antibiotic exposure. The gut microbiome influences development of the immune system. The gut microflora could be important in protection against the bacteria and/or their toxins purportedly involved in SIDS pathogenesis. The aim was to perform a preliminary investigation of the gut microflora in sudden infant death syndrome (SIDS) compared with live comparison babies. The intestinal contents from 52 SIDS, and 102 faecal samples from age-matched live comparison infants were screened by PCR to target 16s RNA genes of Clostridium innocuum, Cl. Perfringens, Cl. difficile, Bacteroides thetaiotaomicron and Staphylococcus aureus. Gut colonisation of the babies with these bacteria was analysed in relation to age, gender and type of feeding; and for SIDS babies sleeping position. Cl. difficile, Cl. innocuum and B. thetaiotaomicron were significantly associated with SIDS with 25%, 46% and 30% of cases PCR positive for these respective bacteria compared with only 6%, 23% and 8.8% respectively in the comparison group. SIDS babies had dual colonisation by both Cl. perfringens and Cl. difficile significantly more often than comparison babies and also with triple colonisation by Cl. perfringens, Cl. difficile and Cl. innocuum. SIDS babies were more often colonised by S. aureus than comparison babies. In addition, SIDS babies found prone were significantly more likely to be colonised by S. aureus than for other positions recorded (OR = ∞; CI = 2·04 - ∞). No significant differences between breast and bottle-fed SIDS babies was observed in regard to each clostridial bacterium, or S. aureus, however Cl. innocuum was found to be significantly associated with formula feeding in the comparison cohort. Comparison of breast and formula feeding of SIDS babies with live comparison babies revealed significant differences with regards to some of the clostridial bacteria. Age-specific differences in gut bacterial microbiome were observed in both SIDS and comparison healthy babies. This study gives an insight into differences in the gut bacterial microbiome of SIDS babies compared with healthy babies. These differences could be important in contributing to a babys susceptibility to infection and therefore to SIDS. The association of S. aureus colonisation with prone sleep position supports the hypothesis that prone sleep position could increase the risk of ingestion/inhalation of bacteria contaminating the sleeping surface and could account for the increased risk of SIDS in babies who are put to sleep prone. The study provides impetus for broader studies into the gut microbiome of babies and could lead to effective approaches to SIDS prevention.

Staphylococcal enterotoxin genes are common in Staphylococcus aureus intestinal flora in Sudden Infant Death Syndrome (SIDS) and live comparison infants

Pathological and epidemiological findings in sudden infant death syndrome (SIDS) suggest an infectious aetiology with indications of involvement of staphylococcal enterotoxins (SEs). While SEA, SEB and SEC have been found in the sera and tissues of SIDS cases, little is known about the role of intestinal Staphylococcus aureus or the roles of later-described toxins SEE, SEG, SEH, SEI and SEJ in SIDS. We used a molecular-based approach to define whether the intestinal tract could be a source of SEs to support the staphylococcal toxic shock hypothesis for SIDS. Intestinal contents from 57 SIDS infants and faeces from 79 age- and gender-matched live comparison infants were cultured and tested for S. aureus and sea-b-c-e-g-h-j and TSST using PCR. High proportions of infants in both groups carried toxigenic and nontoxigenic S. aureus. Significantly greater proportions of SIDS compared with comparison babies were positive for S. aureus (68.4% vs. 40.5%) and for SE genes (43.8% vs. 21.5%), suggesting a possible role in SIDS. The results indicate that colonization by S. aureus with SE genes is common in infants; however, their detection is unlikely to be a strong predictive tool for SIDS. Other factors (including immune response) may reveal a specific susceptibility to SEs in SIDS infants.

Novel hypothesis for unexplained sudden unexpected death in infancy (SUDI)

Objective: Two recent retrospective studies independently reported typically pathogenic bacteria in normally sterile sites of infants succumbing to sudden unexpected death in infancy (SUDI). These findings suggested a proportion of unexplained SUDI might be triggered by bacteraemia. The objective was to assess these observations in the context of the pathology and epidemiology of sudden infant death syndrome (SIDS) in relation to the role of infection and inflammation as triggers of these deaths. Design: A review of the literature to identify potential risk factors for unexplained infant deaths and proposal of a theoretical model for SUDI. Results: Pathologic and epidemiological evidence suggests a hypothesis based on three factors: bacterial translocation, pathogen pattern recognition insufficiency and prenatal exposure to infection. Conclusion: We propose that sterile site infections in which common toxigenic bacteria are identified indicate a brief bacteraemic episode prior to death. This might reflect an ineffective innate response to invasive pathogens that results in reduced clearance of the bacteria. Thymomegaly observed consistently among infants diagnosed under the category of SIDS might have its origins in prenatal life, perhaps generated via in utero infection or exposure to microbial antigens which results in thymocyte priming. There is consistent evidence for an infectious aetiology in many unexplained SUDI. Future directions for research are suggested.

The frequency of molecular detection of virulence genes encoding cytolysin A, high-pathogenicity island and cytolethal distending toxin of Escherichia coli in cases of sudden infant death syndrome does not differ from that in other infant deaths and healthy infants

Consistent pathological findings in sudden infant death syndrome (SIDS) are seen which display similarities to the pathogenesis of toxaemic shock and/or sepsis. A key candidate infectious agent that is possibly involved is Escherichia coli, given its universal early colonization of the intestinal tract of infants and an increased frequency of toxigenic and mouse-lethal isolates from SIDS compared with comparison infants. An explanation for these findings has yet to be identified. Using PCR, we screened E. coli isolates from 145 SIDS and 101 dead control and healthy infants for three new candidate pathogenicity-related genes: clyA (cytolysin A), irp2 [high-pathogenicity island (HPI)-specific gene] and cdt (cytolethal distending toxin). The results failed to show a positive correlation with SIDS, instead proving that clyA and irp2 genes were common to the infant intestinal E. coli. Interestingly we observed a high rate of carriage of these two potentially pathogenic genes in E. coli from healthy infants in the absence of diarrhoeal disease, and we report that in a number of cases, the detection of HPI-specific genes was predictable by serotype. Despite the lack of associations defined so far, there remains the likelihood that genetic determinants influence the interactions between E. coli and the host, so these factors may be part of the multi-factorial aspect of SIDS.

Hypoxia induced HIF-1/HIF-2 activity alters trophoblast transcriptional regulation and promotes invasion.

Reduced or absent cytotrophoblast invasion of the maternal uterine spiral arterioles is a common clinical finding in studies of pregnancies complicated by preeclampsia, suggesting that the mechanisms mediating invasion of these cells is perturbed. The placenta initially develops in a low oxygen environment of 1-2% oxygen until after the 10th week of pregnancy. During this time oxygen concentration exerts a major influence over trophoblast activity and, hypoxia inducible factors are proposed to be one of many key regulators of first trimester trophoblast behaviour. We used a global gene expression microarray approach to identify signalling pathways and hypoxia-responsive genes of interest involved in invasion of the first trimester trophoblast cell line HTR8/SVneo under hypoxic conditions where HIF-1 was active. Additionally, first trimester placental samples from different gestational age groups were labelled with anti HIF-1α and HIF-2α to evaluate whether HIFs are differentially expressed and localised across two periods of placental development: (1) early first trimester characterised by hypoxia (6-8 weeks) and (2) late first trimester after initiation of maternal blood flow into the placenta (10-12 weeks). Invasion of HTR8/SVneo was assessed in real-time and was significantly increased in 1% compared with 5% and 21% oxygen and did not differ between 5% and 21% oxygen treatments. Eighty-eight genes were differentially expressed between cells cultured in 1% oxygen (where HIF-1α protein was localised to the nucleus) and 5% oxygen (where HIF-1α was mainly cytoplasmic). 65% of the genes were predicted to contain HIF-1α:HIF-1β transcription factor binding sites. While HIF-2α staining intensity in trophoblasts of late first trimester placenta was higher than early first trimester (+57%) the percentage of positively stained trophoblast nuclei did not differ between the two time points. There was no difference in the expression level of any of the hypoxia responsive genes of interest, IGFBP3, P4HA1, P4HA2, ANGPTL4 and MMP1 between early and late first trimester placenta. While HIF-1α and its downstream targets are clearly induced in HTR8/SVneo during in vitro hypoxic conditions, it appears that hypoxia inducible factors and genes are not altered throughout the first 7-12 weeks of placental development, during which the onset of maternal blood flow to the intervillous space takes place.

Variant interleukin 1 receptor antagonist gene alleles in sudden infant death syndrome

Objective To investigate if carriage of interleukin 1 (IL-1) receptor antagonist gene variants are associated with sudden infant death syndrome (SIDS) in a large cohort of case–control demographically matched infants. Design 118 SIDS and 233 control infants, who were matched to each SIDS infant by date of birth, sex, birth weight (±500 g), gestational age and ethnicity, were genotyped for an IL-1RN 89 bp tandem repeat polymorphism and analysed for significant associations. Results No significant difference in genotype frequencies was observed between low and normal birthweight infants and year of birth (1987–1994, when the SIDS incidence was higher). In infants born between 1987 and 1994, an association was observed with SIDS and allele 2 where 18% of SIDS infants carried the 2/2 genotype compared with 9% of controls (χ2 p=0.026, OR 2.46). Allele 3 was found at a low frequency, but was significantly more common in SIDS infants (3.1%) compared with controls (0.9%, Fishers exact p=0.04, OR 3.76). Conclusion The higher prevalence of IL-1RN allele 2, which predisposes to poor outcomes from infection, in SIDS infants born between 1987 and 1994 (ie, prior to the dramatic decrease in SIDS incidence) suggests that the high incidence during this period could point to infection playing a role in aetiology. An association of IL-1RN allele 3 with SIDS was also found, but should be interpreted with caution due to the low frequency of this variant. The consequence of allele 3 carriage is currently unknown in the absence of functionality studies for this isoform.

Distribution of interleukin-1 receptor antagonist genotypes in Sudden Unexpected Death in Infancy (SUDI); unexplained SUDI have a higher frequency of allele 2

Abstract Aims. This investigation was designed to explore the role of IL-1RN genotype in unexplained infant deaths (including sudden infant death syndrome (SIDS)), non-infectious infant deaths, and infectious infant deaths, and to investigate whether IL-1RN genotype is related to the finding of organisms in normally sterile sites in infant deaths. Methods. IL-1RN 89bp variable number of tandem repeat polymorphism genotype was determined using polymerase chain reaction for 49 cases of unexplained sudden unexpected death in infancy (uSUDI), 13 cases of infectious sudden unexpected death in infancy, 10 cases of non-infectious sudden unexpected death in infancy, and 103 live control infants. IL-1RN genotype was then compared with the presence of bacteria in normally sterile sites in infant deaths. Results. An association was found between the homozygous A2 allele and uSUDI (P = 0.007; 95% confidence interval 1.41-17.67) where carriage of the 2/2 genotype was 4.85 times more likely to increase risk of uSUDI compared with the predominant 1/1 genotype. Conclusions. The role of infection in uSUDI and SIDS may be via an immune response pathway where IL-1RN A2 affects interleukin (IL)-1 regulation. These results are consistent with previous research where polymorphic genotypes conferring more severe proinflammatory responses are found more frequently in uSUDI/SIDS infants than in controls.

Maternal and perinatal risk factors for SIDS: a novel analysis utilizing pregnancy outcome data

A number of maternal and perinatal factors to increase an infant’s risk of sudden infant death syndrome (SIDS) have been found in past investigations. We analysed data for potential SIDS risk factors including the presence of complications or conditions considered as detrimental to the infant’s or mother’s health. The data for 118 SIDS cases and 227 matched controls were obtained from a state pregnancy outcome unit. SIDS was found to be significantly more common in cases where the infant’s mother was not in a relationship (i.e. divorced, separated or never married) (p = 0.005), if the infant was not the first born (p = 0.0001) and when the mother resided in a socioeconomically disadvantaged area (p = 0.03). Conclusion: Overall, this SIDS cohort appears to display classical SIDS associations, and our findings are consistent with those from other regions. This novel epidemiological tool opens the way for a national Australia-wide study using pregnancy outcome data collected by the individual states and could be helpful in assessing maternal and fetal risk factors for other paediatric medical conditions.

CD14 (C-260T) polymorphism is not associated with sudden infant death syndrome (SIDS) in a large South Australian cohort

Similarities have been drawn between models of endotoxic shock and gross and microscopic pathology observed in sudden infant death syndrome (SIDS) cases. Polymorphisms in genes that influence the expression of endotoxin receptors could affect the outcome of toxaemia, and could, therefore, play a role in SIDS. The CD14 gene promoter contains a single nucleotide polymorphism that affects the level of CD14 gene expression. The TT genotype of the CD14 (C-260T) polymorphism causes a significantly higher density of CD14 receptor expression on monocytes which makes the individual more sensitive to endotoxin than those with the wild-type (CC). This investigation was designed to determine whether SIDS infants have a higher frequency of the CD14 (C-260T) polymorphism compared with non-SIDS controls. One hundred and sixteen SIDS and 228 control infants were genotyped using PCR followed by restriction fragment length analysis of amplified product. Carriage of the TT or CT genotypes did not significantly differ between SIDS and control infants (P = 0.218 and 0.081, respectively). The frequencies observed in the control group were consistent with Hardy—Weinberg equilibrium and did not differ significantly from the published frequencies in Caucasian Australians. These results suggest that CD14 (C-260T) polymorphism is unlikely to be implicated in SIDS.