Variability of anti-staphylococcal antibodies in healthy volunteers and pre-cardiac surgery patients

The incidence of wound infection following cardiac surgery is approximately 1–4 %,
of which approximately one fifth is due to methicillin-resistant Staphylococcus aureus (MRSA) (Allen et al. 2014]). Post-operative MRSA infection is associated with a mortality ranging from 10 to
14 % (Ridderstolpe et al. 2001]). In addition, there is a considerable cost both economically and in terms of patient
suffering that result from sternal wound breakdown, staphylococcal septicaemia and
the associated increased length of hospital stay (Coskun et al. 2005]). Kanafani demonstrated that whilst certain pre-operative characteristics were associated
with an increased risk of post-operative staphylococcal sepsis, the majority of those
that developed such complications had no such risk factors (Kanafani et al. 2009]). These patients had no measure made of their underlying immunity to Staphylococcus. Similarly, in one of the largest retrospective studies of cardiac surgical patients
(n??330,000), there were again relationships drawn between pre-operative risk factors
such as body mass index (BMI), diabetes, chronic renal insufficiency, increasing age
and immunosuppressive therapy and development of infective complications (Fowler et
al. 2005]). In this study, again, no measure had been made of the patients’ own underlying
ability to resist infection.

The humoral immune system provides a level of protection to patients against a number
of infective/inflammatory conditions and indeed our own institution has been involved
in demonstrating links between low levels of pre-operative antibody against endotoxin
and outcome following cardiac surgery (Hamilton-Davies et al. 1997]; Bennett-Guerrero et al. 1997]). It is likely that everyone has a varying level of antibodies to common infective
organisms, thus maintaining a degree of circulating immunity to these organisms, and
that these antibodies are functional, i.e. have neutralising or opsonizing functions.

It has been shown that the alpha-toxin fragments secreted by Staphylococcus can impair gut mucosal integrity and thus further worsen the potential for sepsis
by enabling Gram-negative organism/endotoxin translocation from the gut lumen (Kwak
et al. 2012]).

Anti-staphylococcal antibodies to a variety of epitopes can be measured by enzyme-linked
immunosorbent assay (ELISA) (PhPlate AB, Stockholm, Sweden) (Colque-Navarro et al.
1998]), and the levels of these antibodies are likely to vary between individuals. It is
well established that there is considerable variation in antibody levels in healthy
volunteers and in infected patients (Dryla et al. 2005]). This is similar to the variation in levels of endogenous endotoxin antibodies,
which are predictive of post-operative complications (Down et al. 2004]).

We are particularly interested in those patients who develop serious S. aureus infections (deep-seated wound infection, serious bacteraemia, endocarditis) and examining
whether there is a relationship with the patient’s own immune state. We set out to
determine if these antibodies were measurable in individuals (healthy volunteers and
pre-operative cardiac surgical patients) and whether there was measurable variation
in antibody levels in these two groups. We planned to study the younger volunteers
as they were likely to have an active, healthy immune system as compared to the patient
group who may be expected to exhibit a degree of immunosenescence and have lower levels
of circulating antibodies.

We chose to assay for antibodies to alpha-toxin (AT), an extracellular polypeptide,
and to teichoic acid (TA), a major surface antigen of the staphylococcal organism;
both are present in almost all strains of S. aureus. A high proportion (86–95 %) of the S. aureus isolates in clinical infections produce an anti-alpha-toxin antibody response (Mollby
1983]) (Fig. 1). In cases of serious staphylococcal infection, the levels of alpha-toxin have been
demonstrated to be very high, suggesting that the antigen is highly immunogenic (Soderquist
BC-N et al. 1993]). Teichoic acid is particularly expressed in case of long-standing staphylococcal
infection, for example, deep-seated wound infection or endocarditis (Colque-Navarro
et al. 1998]). These two antibody types are likely to be reliably expressed in those patients
that we are most interested in studying; those that develop deep-seated staphylococcal
wound infections following cardiac surgery.

Fig. 1. Categorised antibody response (positive, weakly positive, negative) to alpha-toxin
and teichoic acid domains in healthy volunteers (HV), n?=?25, and in pre-operative cardiac surgical patients (PS), n?=?25. Fisher’s exact test (Freeman-Halton extension) used to test variability of
response between groups

The IgG class of antibody is likely to be a more reliable indicator due to the reliability
in appearance and longevity of response. Peak levels of the IgM class may be missed
due to its more transient nature (Barclay 1995]).

This investigation formed the first part of an ongoing investigation into peri-operative
staphylococcal antibody levels and clinical outcome. As this was a pilot study and
we were uncertain as to the degree of variation in antibody levels within and between
the groups, we did not perform power calculations. The results of this investigation
will help with the prediction of sample sizes in our future studies.

Hypotheses

There is a measurable intra-population and inter-population variability in anti-staphylococcal
antibodies amongst different populations of individuals.