Objective
The objective of the study was to determine whether single-nucleotide polymorphisms (SNPs) that influence the maternal innate immune response are associated with puerperal group A streptococcal sepsis.
Study Design
Subjects with confirmed puerperal group A streptococal infection were prospectively identified in 2 tertiary care hospitals over 18 years. Controls were racially matched subjects with term, uncomplicated deliveries. Thirty-eight polymorphisms associated with the innate immune response to bacterial infection were analyzed. Allele and genotype frequencies for subjects and controls were compared.
Results
Forty-eight women with puerperal group A streptococcal infection were identified. DNA was obtained for 28 subjects and 54 controls. Allele frequencies were significantly different between subjects and controls for polymorphisms in Toll-like receptor (TLR) 9-1486 ( P = .03) and heat shock protein (HSP) 70-2 1267 ( P = .003). Genotype frequencies were significantly different between subjects and controls for TLR9-1486 ( P = .025), HSP70-2 1267 ( P = .02), and interleukin (IL)-1β-511 ( P = .016).
Conclusion
Puerperal group A streptococcal sepsis may be associated with innate immune response gene polymorphisms in TLR9, HSP70-2, and IL1β.
Group A β-hemolytic streptococcus, also known as Streptococcus pyogenes , is a Gram-positive extracellular bacterial pathogen. It commonly colonizes the throat and skin and is responsible for bacterial pharyngitis, impetigo, postpartum infection, and streptococcal toxic shock syndrome. Group A streptococci also play an important role in poststreptococcal syndromes, which include acute rheumatic fever, acute glomerulonephritis, and reactive arthritis. Since the mid-1980s, an unexplained resurgence of this pathogen has been observed in the form of rheumatic fever, streptococcal toxic shock syndrome, and invasive group A streptococcal skin and soft tissue infections. This resurgence has been demonstrated in obstetrics in the form of puerperal group A streptococcal infections.
Group A streptococcal puerperal infection is an uncommon but potentially fatal disease. In 2002, a population-based estimate of the incidence of invasive postpartum group A streptococcal infections was projected to be 220 cases occurring annually nationwide. The clinical severity of infection varies widely, from mild endometritis that resolves with antibiotics to invasive disease that necessitates intensive care and/or surgery. Pathogenesis in invasive group A streptococcal infections has been linked to bacterial virulence factors. However, these virulence factors do not adequately predict the acquisition and clinical severity of infection. Many women are exposed to group A streptococcus, but only a subset develop clinical disease.
Recently it has been proposed that an individual’s response to infection may be influenced by genetic variations within the immune system. The innate immune system is responsible for early detection and control of infection. Evidence is mounting that an individual’s genetic makeup plays an important role in the susceptibility to sepsis and influences its severity and outcomes. There are currently no studies that investigate the association of innate immune response gene polymorphisms and puerperal group A streptococcal sepsis. We hypothesized that these polymorphisms alter susceptibility to group A streptococcal puerperal infection.
Materials and Methods
Subjects
This is a case-control analysis of the association of innate immune response gene polymorphisms and puerperal group A streptococcal sepsis. The study group is illustrated in the Figure . Forty-eight cases of puerperal group A streptococcal sepsis were prospectively identified from 1991 to 2009 at 2 tertiary care centers in Salt Lake City, UT, via voluntary reporting by physicians and surveillance of microbiology culture logs. Twenty-eight women consented for study participation; we were unable to contact 14 women and 6 declined to participate. Case characteristics and medical/obstetric histories were obtained via chart abstraction and telephone interviews.
Cases demonstrated clinical evidence of endometritis and/or had febrile illness during the postpartum period. They also had at least 1 positive culture for group A streptococcus from a normally sterile body site. Cases were further stratified as having either severe or mild disease. Severe disease was considered to be present in patients with at least 1 of the following previously published criteria for severe (ie, disseminated or invasive) disease: (1) disease requiring surgical exploration or debridement, (2) admission to the intensive care unit, (3) group A streptococcal toxic shock syndrome, or (4) hospitalization for 14 days or longer. Controls were racially matched healthy subjects with a history of term, uncomplicated deliveries and no history of puerperal infection.
The study was approved by the institutional review board and written informed consent was obtained from all subjects prior to participation.
Deoxyribonucleic acid (DNA) extraction
DNA was extracted from either saliva or blood samples collected at the time of study enrollment. Saliva samples were obtained from cases with a standard collection kit, and DNA was then extracted using the PureGene DNA purification system (Qiagen, Valencia, CA). DNA from controls was extracted from peripheral blood leukocytes using the same techniques.
Selection of polymorphisms for genotyping
Thirty-eight polymorphisms in genes involved in the innate immune response were selected based on hypothesized causal pathways and associations with bacterial infection, sepsis, and septic shock in the infectious disease and critical care literature. The polymorphisms included in our analysis are shown in Table 1 .
| Gene | Symbol | Polymorphism | RS number |
|---|---|---|---|
| Tumor necrosis factor α | TNFα | –308(G/A) | rs1800629 |
| Interleukin 1 α | IL1α | –889(C/T) | rs1800587 |
| Interleukin 1 receptor antagonist | IL1ra | VNTR | |
| Interleukin 1 β | IL1β | –511(A/G) | rs16944 |
| Interleukin 6 | IL6 | –174(G/C) | rs1800795 |
| –596(G/A) | rs1800797 | ||
| Interleukin 8 | IL8 | –251(T/A) | rs4073 |
| Interleukin 10 | IL10 | –592(C/A) | rs1800872 |
| –819(C/T) | rs1800871 | ||
| –1082(G/A) | rs1800896 | ||
| Interleukin 13 | IL13 | Arg130gln(T/C) | rs20541 |
| Mannose-binding lectin | MBL2 | –550(G/C) | rs11003125 |
| –221(G/C) | rs7096206 | ||
| arg52cys(T/C) | rs5030737 | ||
| gly54asp(A/G) | rs1800450 | ||
| gly57glu(A/G) | rs1800451 | ||
| Toll-like receptor 1 | TLR1 | 1805(T/G) | rs5743618 |
| –7202(A/G) | rs5743551 | ||
| Toll-like receptor 2 | TLR2 | arg753gln | rs5743708 |
| –16933(T/A) | rs4696480 | ||
| Toll-like receptor 4 | TLR4 | asp299gly | rs4986790 |
| thr399ile | rs4986791 | ||
| Toll-like receptor 6 | TLR6 | 745(C/T) | rs5743810 |
| Toll-like receptor 9 | TLR9 | –1486(C/T) | rs187084 |
| Matrix metalloproteinase 16 | MMP16 | 39811(A/G) | rs2664349 |
| 43827(C/T) | rs2664325 | ||
| CD14 | CD14 | –260(C/T) | rs2569190 |
| –159(C/T) | rs2569191 | ||
| High-mobility group box 1 | HMGB1 | 982(C/T) | rs1060348 |
| Interleukin 1 receptor-associated kinase 1 | IRAK-1 | 1595(T/C) | rs1059703 |
| Myosin light chain kinase | MYLK | A/G | rs820336 |
| G/C | rs33264 | ||
| A/G | rs820325 | ||
| Heat shock protein 70-2 | HSP 70-2 | 1267(G/A) | rs740598 |
| Plasminogin activator inhibitor 1 | PAI-1 | 11053(G/T) | rs7242 |
| Factor V Leiden | F5 | arg506gln(A/G) | rs6025 |
| Angiotensin-converting enzyme | ACE | Intron 16 I/D | rs4646994 |
| FC-gamma-RIIa | FCgammaRIIa | his131arg | rs1801274 |
Genotyping
The Taqman single-nucleotide polymorphism (SNP) allele discrimination assay was used for SNP genotyping (Applied Biosystems, Inc, Foster City, CA). Samples were amplified per the manufacturer’s instructions in an ABI 9700 thermocycler (Applied Biosystems). Fluorescence signal was determined via endpoint analysis on an ABI 7900 real-time polymerase chain reaction (PCR) instrument (Applied Biosystems). Genotypes were determined using Applied Biosystems’ automated Taqman genotyping software (SDS, version 2.3).
A variable number tandem repeat (VNTR) within the interleukin (IL)-1 receptor antagonist (IL1ra) gene and an insertion/deletion polymorphism within the angiotensin-converting enzyme (ACE) gene that have been associated with septic shock were also genotyped. Both the VNTR and the insertion/deletion polymorphism were amplified under typical PCR conditions using an ABI 9700 thermocycler (Applied Biosystems). One primer for each polymorphism was labeled on the 5′ end with the fluorescent tag 6-FAMM to allow detection of the genotype. After amplification, samples were analyzed on an ABI 3730XL capillary electrophoresis instrument (Applied Biosystems). Genotypes were determined using Applied Biosystems’ GeneMapper version 3.5.1 software.
Statistics
Demographic and clinical characteristics of cases and controls were compared using the χ 2 or Fisher’s exact test for categorical variables and the Wilcoxon rank sum test for continuous variables. Differences in allele and genotype frequencies between cases and controls were tested for each polymorphism with the use of the χ 2 or Fisher’s exact tests, as appropriate. Tests for Hardy-Weinberg equilibrium (χ 2 or Fisher’s exact) were performed on control subjects for each polymorphism. Because this was an exploratory study, no adjustments were made for multiple comparisons, and all comparisons are reported. P < .05 was considered to be statistically significant. All calculations were performed using SAS software (SAS Institute, Inc, Cary, NC).
Results
The demographic and clinical characteristics of puerperal group A streptococcal cases included and excluded from this study based on consent for participation and DNA analysis are shown in Table 2 . Subjects who declined participation had a longer length of hospitalization ( P = .04) and were more likely to have had surgical debridement ( P = .02). The majority of enrolled subjects occurred following multiparous (64.3%), term (89.3%), and vaginal deliveries (85.7%). Thirty-nine percent of enrolled subjects qualified as having severe disease; 32.1% required surgical exploration and/or debridement and 17.9% had a hysterectomy. There were no maternal deaths.
| Characteristic | Included | Excluded | P value |
|---|---|---|---|
| Subjects, n (%) | 28 (58.3) | 20 (41.7) | |
| Age, mean ± SD, y | 29.3 ± 6.6 | 26.5 ± 5.5 | .16 |
| Parity, n (%) | |||
| Primiparous | 10 (35.7) | 10 (50) | .32 |
| Multiparous | 18 (64.3) | 10 (50) | |
| Gestational age at delivery, n (%) | |||
| Term | 25 (89.3) | 19 (95) | .63 |
| Preterm (<37 wks) | 13 (10.7) | 1 (5) | |
| Mode of delivery, n (%) | |||
| Vaginal | 24 (85.7) | 16 (80) | .70 |
| Cesarean | 4 (14.3) | 4 (20) | |
| Severity of disease, n (%) a | |||
| Mild | 17 (60.7) | 9 (45) | .28 |
| Severe | 11 (39.3) | 11 (55) | |
| Symptoms onset, mean ± SD (days postpartum) | 5.1 ± 8.3 | 4.3 ± 3.5 | .51 |
| Length of hospitalization, mean ± SD (d) | 14.1 ± 24.3 | 19.2 ± 24.2 | .04 |
| Surgical debridement, n (%) | 9 (32.1) | 13 (65) | .02 |
| Hysterectomy, n (%) | 5 (17.9) | 5 (25) | .72 |
| Maternal death, n | 0 | 0 |
a Severe disease was considered to be present in patients with at least 1 of the following criteria: (1) disease requiring surgical exploration or debridement, (2) admission to the intensive care unit, (3) group A streptococcal toxic shock syndrome, or (4) hospitalization for 14 days or longer.
The demographic and clinical characteristics of cases and controls are shown in Table 3 . Cases and controls were similar with regard to age, race/ethnicity, parity, and mode of delivery.
