Group B StreptococcusStreptococcus agalactiae

Reproductive Infectious Disease SeminarsSeptember 28, 2004

Natali Aziz, MD, MSReproductive Infectious Disease and Maternal-Fetal Medicine Fellow

Department of Obstetrics, Gynecology and Reproductive Sciences University of California, San Francisco

Microbiology

• Gram-positive cocci• Chains • Encapsulated • Non-motile• Facultative anaerobes• Lactic acid production• Multiple nutritional

requirements • 1-4 mm diameter, grey-

white, flat, mucoid• Selective Broth Media

(SBM) or Lim broth http://medicine.ucsd.edu/NizetLab

Classification• β-hemolysis– RBCs surrounding the colony are

completely lysed – “Hemolysin" toxins

– Orange pigment

– Narrow hemolysis zones

– CAMP factor enhances hemolysis

• Carbohydrate Antigens (C substance)– Lancefield Group B

– Group specific antigen

• Polysaccharide Capsule Serotypes– 150 oligosaccharide subunits with

mono-, di-, tri- side chains

– Ia, Ib, II-VIII

– III and V completely sequenced http://medicine.ucsd.edu/NizetLab

Epidemiology

• Humans (and cattle)• Genitourinary and

gastrointestinal tracts in adults

• Upper respiratory tract in young infants

• Pregnant women, neonates, and non-pregnant adults

Bovine Mastitis

Epidemiology Pregnant Women and Neonates

• Pregnant women: 20-30% rectovaginal colonization • Pregnant women infection 0.3 per 1000

live births • 50-70% infants born to colonized

mothers will become colonized• 1-2% infants develop early-onset

invasive disease (EOID)1

• Heavy colonization associated with PTD• Colonization rates differ by ethnic

groups, geographic locales, and age in pregnant women2

– NY Hispanics, Af AM> Caucasians> Hispanics, Asians

• Serotypes differ by geographic locales in pregnant women

– Japan VI, VIII– Denmark VIII

• Risk factors neonatal EOID: – GBS maternal colonization (x29) – Maternal age < 20 yo– African American race– Heavy colonization (>105 cfu/ml)

– Low anti-GBS capsular Abs– Parity – Intrapartum fever, chorioamnionitis,

GBS bacteriuria, prematurity, prolonged rupture of membranes (>18 hours), PPROM, prior infant with GBS infection

1Prevention of perinatal group B streptococcal disease: a public health perspective. MMWR ;45(RR-7):1-24.2Regan, et al. The epidemiology of GBS colonization in pregnancy. Obstet Gyn 1991;77:604-10.

EpidemiologyNon-pregnant Adults

• Emergence in 1980-90’s – Atlanta: Incidence 2.4, 4.4,

5.9 per 100,000 in 1982, 1990, 19921

– > 7600 adult GBS infections annually2

• ?Virulent strains • ?Chronic diseases / more

immunosuppression• ? Loss of barrier of

protection in chronically colonized site

• 29% serotype V (<Ia, Ib, III)

• Risk factors– African American Race – Increasing age– Institutionalization – Chronic illness – HIV– Alcoholism – Trauma

• Bimodal Distribution – Mid 20’s and mid 60’s

• Mortality 15-38% 1 Farley, et al. A population-based assessment of invasive disease due to group B streptococcus in non-pregnant adults. NEJM 1993;328:446.2 Zangwill, et al. GBS disease in the US, 1990:report from a multi-state active surveillance system. MMWR CDC Surveil Sum 1992;41:25.

Epidemiology

Adult GBS infection: ¾ of invasive disease and 90% of mortality in US

Up To Date: Group B streptococcal infections in non pregnant adults.

Adult Clinical Manifestations • Pregnant Women Infections

– Urinary tract infections • Asymptomatic bacteriuria

• Cystitis

• Pyelonephritis

– Chorioamnionitis• Heavy 2nd trimester colonization

– Puerperal sepsis

(<12 hours post delivery)

– Endometritis(> 24 hours after delivery)

– Meningitis, endocarditis, abdominal abscess, necrotizing fasciitis

• Non-pregnant Adult Infections– Skin and soft tissue (15-40%)

– Bacteremia (30-40%)

– UTIs (5-15%)

– Pneumonia (6-12%)

– Bone and joint(2-15%)

– Cardiac (2-9%)• Endocarditis on native valves, L-

sided

– CNS (4%)

– Toxic shock-like syndrome

Treatment: PCN G; vancomycin if allergic; gentamicin for synergy in endocarditis

Neonatal Clinical Manifestations

• Acquired in-utero or at delivery • Serotypes III, Ia, >II, V account

for 85% of infections in infants • Early-onset disease:

– Up to 7th day of life– Usually within 12-24 hours of

life– Serotypes I and II – Bacteremia– Sepsis– Pneumonia– Meningitis (10-15%)

• Late-onset disease: – 7-89 days old – 50% attributable to vertical transmission– Serotype III – Meningitis (35%)/seizures– Pneumonia, septic arthritis,

osteomyelitis, cellulitis, adenitis, and others

– Current Treatment• Amp/Gent PCN G once dx confirmed

– PCN at high dose as has higher MIC; more selective

– Resistance to erythromycin, clindamycin, TMP-SMX, and gentamicin

• IVIG and GBS hyperimmune globulin

GBS Neonatal Infection Incidence Trends1

1Prevention of perinatal group B streptococcal disease: revised guidelines from CDC. MMWR 2002;51(RR-11):1-24.

Incidence of EOID 1990: 1.8 per 1000 live births1999: 0.5 per 1000 live births2000: 0.6 per 1000 live births

Guidelines for Prevention of Perinatal GBS Disease

• Schrag, et al 2002

– Antenatal screening strategy prevented 55% more cases of EOID than risk-based approach

– Prevention of Perinatal GBS Disease: Revised Guidelines form CDC. MMWR 2002;51 (RR-11):1-24.

Guidelines for Prevention of Perinatal GBS Disease

Prevention of Perinatal GBS Disease: Revised Guidelines form CDC. MMWR 2002;51 (RR-11):1-24.

Intrapartum Antibiotic Prophylaxis

Prevention of Perinatal GBS Disease: Revised Guidelines form CDC. MMWR 2002;51 (RR-11):1-24.

PathogenesisNeonatal Disease

• Vaginal colonization in pregnant women– Adherence to epithelial cells and resisting

mucosal immune defenses• Fetus aspirates infected fluid as organism

ascends into the amniotic cavity by penetration of placental membranes or at time of delivery with ROM

• Bacteria enter the fetal lung through aspiration of infected amniotic fluid.   

• Pneumonia with lung epithelial and endothelial cell injury are characteristic of early onset disease,

– cytotoxic properties of ß-hemolysin and the influx of host neutrophils. 

• GBS invade alveolar epithelial and pulmonary endothelial cells within membrane-bound vacuoles

• Newborn infants, particularly premature infants, have fewer alveolar macrophages than adults and exhibit poor neutrophil chemotaxis. 

• GBS are inefficiently phagocytosed in the absence of opsonization by specific antibody or complement, diminished in neonatal serum. 

• Polysaccharide capsule of GBS has a marked inhibitory effect on phagocytic clearance by preventing complement deposition on the bacterial surface.

• Components of GBS surface protein C may both retard opsonization and decrease killing of GBS taken up by neutrophils

• Cell wall-associated components of circulating GBS induce a sepsis syndrome characterized by severe systemic hypotension, pulmonary hypertension, hypoxemia and acidosis. 

• Effects of a host inflammatory response mediated by release of tumor necrosis factor (TNFalpha), interleukins, prostaglandins and thromboxane. 

• Bloodstream dissemination allows GBS to reach multiple body sites, and invasion of brain microvascular endothelial cells may be the first step in production of meningitis.

Pathogenesis

Doran, Kelly S. & Nizet, Victor. Molecular pathogenesis of neonatal group B streptococcal infection: no longer in its infancy. Molecular Microbiology  2004;54:23-31.

Fig. 1. Stages in the molecular and cellular pathogenesis of neonatal group B Streptococcal (GBS) infection.

Pathogenesis

• Maximal cell adherence at acidic pH of vaginal mucosa• Selective fibronectin adherence mediated by RogB and FbsA • Rib protein expressed by invasive isolates • Traverses chorionic, not amniotic, cells by generation of oxygen radicals

and PGE2– Suspected association with membrane rupture and preterm delivery

• Infant lung damage: intracellular invasion, direct cytolytic injury, and damage induced by the inflammatory response

• Loss of pulmonary and blood-brain barrier integrity loss due to β-hemolysin/cytolysin promoting IL-8 release

• Effects can be neutralized by major surfactant phospholipid constituent • CAMP factor triggers cell lysis by creating discrete pores • Conserved GBS surface proteins (ScpB, Sip, BSP)

Virulence FactorGBS Surface Polysaccharide Capsule

• Antiphagocytic properties • Capsule-deficient mutants

diminished virulence in animal models

• Sialic acid residues on capsule inhibit the binding of opsonically-active C3 component of complement to the cell surface– blocking activation of the

alternative pathway

• Transplacental passage of type-specific anticapsular IgG antibody from mother to infant is an important protective factor against invasive disease

http://medicine.ucsd.edu/NizetLab

Virulence FactorGBS β-hemolysin

• Cytotoxic to pulmonary epithelial and endothelial cells– Pulmonary injury and alveolar protein exudate in early-onset

pneumonia

• Activity is blocked by surfactant phospholipid– Increased risk of premature, surfactant-deficient neonates for severe

pneumonia

• Induces cytokine release and nitric oxide production in macrophages– Stimulate elements of the sepsis cascade

Virulence FactorC5a-peptidase

• Cleaves and inactivates the complement-derived neutrophil chemoattractant C5a

• C5a-peptidase-deficient mutants are more rapidly cleared from the lungs of infected animals when compared to the isogenic wild-type strain

Immune Mechanismand Pregnancy

• Smith, et al 2001 – Purpose:

• To evaluate functional capacity of granulocytes and monocytes from pregnant and non-pregnant women in relation to GBS colonization status

– Methods: • Engulfment of fluorescent GBS by peripheral phagocytes in GBS

colonized and uncolonized women measured by flow cytometry– Conclusions:

• Monocytes from pregnant, GBS colonized women engulfed more GBS and released significantly more superoxide into the extracellular milieu than did granulocytes from the same women.

• Unlikely effective defense mechanism against intracellular bacteria• Components of innate immune system may function suboptimally,

contributing to the colonization process by GBS

Smith JM, et al. Differences in innate immunologic response to group B streptococcus between colonized and noncolonized women. Infect Dis Obstet Gynecol 2001;9:125-32.

Immune Mechanismand Pregnancy

• Campbell, et al 2000– Objective:

• To investigate relationship between serum concentration of GBS capsular polysaccharide-specific IgG, colonization status, race or ethnicity, and age in pregnant women

– Results/Conclusion: • Women aged <20 yo had lowest median

serum concentrations of CPS IgG

• Colonization with GBS may elicit a systemic immune response, with increased prevalence of CPS IgG with increasing age.

• Low IgG levels in teenagers may account for increased risk of GBS disease in neonates born to these mothers.

Figure 4. Distribution of group B streptococcal capsular polysaccharide–specific immunoglobulin G (Ig) G concentrations in delivery sera of 294 colonized women categorized by age. Distribution is illustrated as the percentage of each age group with less than 0.5 (white bars), 0.5–1.0 (dotted bars), or more than 1.0 (black bars) mcg/mL of specific IgG.

Campbell JR, et al. GBS colonization and serotype-specific immunity in pregnant women at delivery. Obstet Gyn 2000;96:498-503.

Diagnosis and Screening

• Culture– Gold Standard – Selective broth medium (SMB or

Lim Broth)

• PCR (Berger, et al 2000)

– Comparable in sensitivity to culture in extremely controlled laboratory environment

– Limited studied of clinical practice

• Immunoassay– Enzyme and Optical– Poor sensitivity – Antigen detection may assist with

diagnosis of CSF infection Bergeron MG, et al. Rapid detection of GBS in pregnant women at delivery. NEJM 2000;343:175.

Rapid Screening: Clinical Experience

• Objectives– To compare the efficacy of

the rapid optical immunoassay (OIA) and real-time polymerase-chain reaction (PCR) screening techniques to the standard enrichment broth culture for the detection of GBS colonization in pregnant women during labor.

– To evaluate real-time PCR screening technique in an intrapartum setting.

– To reexamine prior OIA performance studies conducted at Stanford/Lucile Packard Hospital

• Methods– Double vaginal swabs collected from 315

patients at time of presentation for delivery – OIA performed with one swab within 2 h

of collection per our current stat protocol. – PCR performed on second swab-- placed

in buffer, 50 µl of which was used for the real-time PCR protocol, performed on a Cepheid Smartcycler once every 24 h.

– Another 500 µl of buffer and swab were incubated in Lim broth for culture detection of GBS.

– For 75% of samples yielding discrepant PCR and culture results, another larger volume extraction and subsequent amplification were done using remaining cell eluate.

– Sensitivity/specificity/confidence intervals calculated.

Rapid Screening: Clinical Experience

SENS SPEC PPV NPV

OIA 0.071 0.981 0.444 0.830

PCR 0.625 0.973 0.833 0.923

Alternate PCR

0.821 0.973 0.868 0.962

GBS vaginal colonization rate: 17.8%

Aziz N, et al. Comparison of OIA and PCR for GBS rapid intrapartum screening. Manuscript in preparation.

Rapid Screening: Clinical Experience

• Conclusions– Rapid OIA technique is not a reliable method for GBS

screening in an intrapartum setting. – Although real-time PCR technique is a more reliable

method than the rapid OIA technique for GBS screening in an intrapartum setting, it was not as sensitive as Bergeron findings in intrapartum setting.

– Real-time PCR technique may have a useful role in the management of women whose GBS status is unknown at time of presentation for delivery.

– Laboratory technique may significantly impact OIA results.– “Research Method” tends to yield better results than “Routine

Laboratory” application for both techniques.

Immunoprophylaxis• Maternal antibody against type-

specific capsular polysaccharide protective

– May not be sufficient and infants < 32 wks do not benefit from placental

transport of maternal IgG

• Limitations with current guidelines

– Administration of unnecessary antibiotics

– Late to care and unknown status

– RF strategy not good enough

– Antibiotic adverse effects

– Increased E coli Amp-resistance in infants <1500gm

– No change in late onset disease

– Invasive (IV)

• Targets for vaccine – Group B carbohydrate Ab’s not protective

(not virulent factor)– Capsular polysaccharide confers

specificity except two cases – Others: Sialic acid, protein antigens, Beta

C protein, Alpha C protein• Vaccine Formulations

– Polysaccharide (CPS)• Phase I study promising for serotype

III, even in pregnant patients • Studies for Ia and II variable results

– Polysaccharide-protein conjugate • Animal studies more immunogenic

with coupling – Potential GBS protein vaccines

• Difficulty finding conservation among serotypes

Immunoprophylaxis Challenges• Proving vaccine efficacy is going to be very difficult.

– To demonstrate an 80% reduction in risk, from 0.5 per 1000 to 0.2 per 1000 births with an alpha of 0.05 and beta of 0.8, will require enrollment of 125,000 women who deliver babies during the follow-up period.

– Will likely need an even larger number, since some women may not end up (or stay) pregnant– Will require large and expensive trial.

• Organisms such as pneumococcus and GBS have polysaccharide antigens that are poor immunogens.

– Coupling them to haptens like pertussis or tetanus toxoids often works for individual polysaccharides,

– However, when combining into polyvalent vaccines, the responses are often inconsistent.– Constructing a vaccine to all of the common serotypes (now at least Ia, Ib, II, III, and V) is a

challenging task. – Antigen drift in the population of colonizing organisms may make today's vaccine obsolete

tomorrow, as those serotypes disappear and type VIII and/or others become predominant.• Women whose infants are at greatest risk are characterized by heavy GBS

colonization and the absence of effective opsonizing antibody. – The latter is clearly not a consequence of a lack of exposure to the antigens -- they're heavily

colonized, after all; likely that these women may have some immunogenetic impediment to development of protective antibodies, and it is not clear that they would do that better after exposure to a vaccine than they do after natural exposure.