MICROBIOLOGY
bottles where lot number/expiry date, destroying this information.
Determination of positivity Another aspect of blood culture bottles that was observed in this study was the relative fluorescence unit (RFU) values. This is the unit of measurement that the analyser algorithm uses to determine positivity. Carbon dioxide produced by microorganisms diffuses across a semi-permeable membrane in the bottle sensor into a water layer. The resultant drop in pH of the sensor causes a colour change, which increases the RFU value of the sensor. Fortunately, a study of blood cultures designated as negative after five days’ incubation in the analyser had been running for several months at the time of writing this report, thereby providing data that may be compared to that of the false-positive bottles. Table 3 shows the minimum, maximum and average RFU values of bottles on insertion into the analyser, removal from the analyser, and the overall RFU increase. In the case of the false-positive bottles, the data for eight bottles were not recorded, thereby giving the number of bottles in this comparison as 52. As can be seen from Table 3, the average
RFU at insertion was slightly higher in the false-positive bottles, while there was less difference in average RFU on removal. Overall, however, the average increase in RFU value of the routine bottles was double that of the false-positive bottles. This can be explained by the fact that the routine bottles were incubated for far longer than the false-positive bottles, as well as the bottle populations being different (1:1 anaerobic:aerobic bottles for the routine bottles, compared to 9:1 anaerobic:aerobic bottles for the false-positive bottles). Nonetheless, there does not seem to be any significant difference between RFU values between the blood culture bottles compared here, thereby ruling out issues with the bottles themselves, confirming the conclusion regarding lot numbers detailed above.
Time to positive Further data were collected on time to positive for each bottle. This showed that the vast majority of bottles (78%; n=45) were positive at 2.2 hours, an apparent ‘benchmark’ time for the analyser algorithm
‘Bottle lot numbers were recorded during this study to establish if there was a link with a certain batch that was causing the problem’
258
may be mistaken for bacterial growth by the analyser algorithm. The bottles in this study period contained volumes of 2–20 mL (average 8 mL). Therefore, it can be seen that, other than in a small number of cases, an excessive amount of blood was not inoculated into bottles, thereby discounting this reason for generating false-positive results during this period.
Blood culture bottles in the BacT/ALERT 3D analyser.
to flag a bottle falsely as positive. This is probably due to the BacT/ALERT 3D having a two-hour initial value algorithm, with first positives being flagged at two hours 10 minutes (two hours plus first reading interval). Other timings of bottles flagging positive, such as 19.2 hours (2%; n=1) and 7.2 hours (2%; n=1) may have been due to other reasons for some bottles being considered falsely as positive during this time period (eg the bottle being overfilled with blood, which is known to be a potential cause of a blood culture bottle being flagged by the analyser as positive when it is, in fact, negative).
Volume of blood As volume of blood inoculated into a bottle is a known risk factor for generating false-positive results, blood volume was also measured in bottles removed from the analyser as false-positives. The manufacturer of the blood culture bottles in use at Nobles Hospital recommends approximately 10 mL blood per bottle as being the optimum amount, striking a balance between sensitivity for detecting organisms that may be present in low numbers and the risk of excess leucocytes and thrombocytes in a high volume of inoculated blood undergoing respiration that
Source of samples In addition to the volume of blood added to bottles, and hence the manner in which they were processed at the service user end, it was also
decided to note the ward of origin in order to rule out a pattern of false-positive bottles coming from a certain area. If a statistically significant number of false-positive bottles were found to originate in a specific ward, it would have suggested practices such as storage and inoculation issues as the cause of the false-positive results. In this period, false-positive bottles were received from 13 wards. The wards with the most false- positive bottles attributed to them were A&E (32%), Ward 6/Medical Assessment Unit (16%) and Ward 17/Intensive Care Unit (11%). However, as these are the wards that send the majority of blood culture bottles, it can be argued that the higher numbers of false-positive bottles from these wards are to be expected.
‘This report has looked into various other potential reasons for the increase in false-positive bottles, but has found no evidence to suggest secondary factors’
Table 3. Relative fluorescence unit (RFU) values of false-positive bottles compared to routine negative bottles removed after five days’ incubation.
False positives (n=52)
RFU value at insertion RFU value on removal Increase in RFU value
Minimum Maximum Average
Minimum Maximum Average
Minimum Maximum Average
2568 3537 3262 3109 3718 3477 56
913 215
Negative after
five days (n=270) 1906 3404 2846 2062 4150 3278 41
1017 450
MAY 2016 THE BIOMEDICAL SCIENTIST
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