2.2. MARCO CONCEPTUAL
3.2.1. DETERMINACIÓN DE VOLUMEN DE PROBETAS
3.2.1.2. CÁLCULOS PARA LA CONCENTRACIÓN DE SAL METÁLICA EN LA
To evaluate further the humoral immune response in hydrocephalus shunt infections, protein antigen was substituted for polysaccharide B in the ELISA test. ii.) Methods
Preparation of antigen
The method is based on that described by Dryden et al (1992). Three organisms, F ll, F743 and F544 were used. These were inoculated onto blood agar plates and incubated at 37®C overnight. Three colonies were taken and inoculated into 5 glass universal bottles containing 20ml of BHI and incubated on an orbital shaker overnight at 37°C.
Organisms were harvested by centrifugation at 2000rpmy+4®C/20 mins and the supernatant discarded. The deposits were washed twice in distilled water and the pellets resuspended in 0.5ml distilled water and sonicated for 5 mins in 5 sec cycles (20Khz, 47W) in an ice-water bath. The sonicate was then spun in a microcentifuge at 12800 rpm for 10 minutes and the supernatant fluid stored at -20°C for use.
The test method used was that described for polysaccharide B with the exception that only 50 microlitre volumes were used throughout. The same ELISA system as that used for polysaccharide B was used for the determination of antibody to cell proteins of coagulase negative staphylococci.
in the same way as described for the polysaccharide B study. The same sera were used to assess any difference in the immune response between polysaccharide and protein antigens. Patients were divided into groups as described before for polysaccharide B.
Sera from shunted patients were again divided into three categories according to whether they had proven infections, suspected infection or no infection. The criteria for these categories have already been described and the absorbances for each are given in Tables A 19 to A33 (Appendix III).
iii.) Results
Positive ELISA results were seen in all but one case (19) with proven VA shunt infection. In this case, the mean IgM value was 0.032 and the mean IgG was 0.016, both extremely low values and it may be that in this case this constitutes a false negative result. In one other (Case 15), ELISA was IgG positive only and in another two (38,42), was IgM positive only.
The ASET was found to be significantly raised for age in all those with a proven VA shunt infection and in twelve, the CRP was raised also. Eight of these had immune complex nephritis. In the four outstanding, the CRP was thought to be raised due to surgical trauma in three and a urinary tract infection in one.
In all cases of proven VP shunt infection, a positive ELISA IgM and IgG result was recorded. The ASET was found to be raised in one (24) and this child had a St. epidermidis bacteraemia. In two others (Cases 3 & 30), a bacteraemia was also found. In the former, St. haemolyticus was isolated and in the latter, a Micrococcus sp.
In neither case was the ASET found to be raised and this was due to the presence of high titres of specific antibody to the infecting organism as previously described.
In all cases of proven infection, the infected shunt was removed and a new shunt inserted. All patients remain well.
for both IgM and IgG antibody .In the two remaining (Case 21,28), one was IgM positive only (28) and the other IgG positive only .
The ASET was found to be raised in four (17,20,2832) and in two (Cases 20 & 28), St. epidermidis was isolated from blood cultures and the shunt proximal catheter respectively. These patients remain well after shunt removal.
In the two cases outstanding, blood cultures and CSF samples have thus far been culture negative. The unreliability of culture results in shunt infection has been discussed before (Bayston et al 1991).
In the three where the ASET was not significantly raised but at the top of the normal range for age, all had symptoms which could be associated with shunt infection. Cultures of CSF and blood have been negative so far and these children remain under surveillance.
It is possible that ELISA results obtained for these patients constitute false positives but the absorbance values obtained for both polysaccharide B and whole cell protein for IgM and IgG were not low values. Other sera from known positives and negatives tested in the same batch gave expected results and it is therefore unlikely that the results obtained are due to technical error.
The CRP was found to be raised in only one case (Case 14) and this was due to a urinary tract infection for which the child was treated.
In those with VP shunts in this group, all were ELISA IgM and IgG positive except one which was IgM positive only (35). The CRP was raised in five of these (Cases 9,12,22,27,35). In the two remaining cases (Case 7 & 37), antibiotic treatment had been commenced for a urinary tract infection in one (7), but no explanation for a negative result could be found for the other (37). It may be that the patient was receiving antibiotics but there was no evidence to support this in the notes.
The ASET was also found to be raised in two cases (Case 9 & 27), and in each case the shunt was removed. In the former, St. epidermidis was isolated from blood cultures, but CSF and shunt were culture negative. In the latter, all cultures
were negative.
Shunt culture has been shown previously to provide the most reliable microbiological results (Bayston et al 1991). In the two cases discussed here, only the proximal catheter was cultured and this may explain the negative results obtained.
In the fifteen patients with implants who were not infected, one VA shunt patient (Case 44) was found to be IgG positive only whilst two VP shunt cases (2„41,) were positive for IgG and IgM respectively. In the fifty control sera tested from patients without shunts, two were ELISA positive; in both (Case 16, 37), this was to IgM only. These were results which were not expected and constitute false positives.
STATISTICAL ANALYSIS
As with ELISA Polysaccharide B, a Kruskal-Wallis one-way analysis of variance was used to determine whether there were significant differences between the groups.
Data were plotted (Fig 6 & 7) to illustrate where observed differences were occurring. The non-parametric test of Kruskal-Wallis was then applied to reach a value for probability (p) which was found to be <0.0005 suggesting a significant difference between all groups tested. The groups on the graphs were as described previously for polysaccharide B. The analysis was performed for IgM and IgG separately.
To measure the effect of using each of the three antigens in this test, multiple regression analysis was carried out The values for the three antigens were used in the same model for multiple regression as polysaccharide B and the values obtained for coefficients and confidence intervals have been stated earlier.
The results showed a good agreement between antigens and extracts used in the ELISA test.
Fig 6