Sunday, May 31, 2015

More on PCR

In the posting below - [link] we gave an overview of the recent Warwick University paper, SE3280, commissioned by Defra in 2012 at a cost of £467,353. We note that sadly, no mention of it or its stunning results have made it into the press.
And considering the indecent haste with which the badger vaccination mischief - [link] of 74 per cent efficacy, bounced around the airwaves, only to be retracted quietly in subsequent months, that is a damned disgrace shame.

 So we will cut and paste from the paper - link] which can be accessed by clicking on Final Report on the previous link. The study set out to explore a non invasive method of identifying diseased badger groups:
. "Controlling disease spread through UK cattle herds is a significant challenge as the European badger (Meles meles) has been highlighted as a wildlife reservoir that may be a significant source of continued re-infection. Determining the disease prevalence and TB status of badger populations is a demanding challenge and currently requires direct interaction with individual animals through expensive and labour intensive trapping and testing regimes. This report describes a robust and reliable non-invasive quantitative polymerase chain reaction (qPCR) assay designed to detect the presence of M. bovis DNA in badger faecal samples."
Those samples were collected from Woodchester Park and matched with cultures and blood assays from cage trapped animals, over a long period of time.
" .. badger faecal samples were obtained from 12 social groups of badgers at Woodchester Park in Gloucestershire (the site of a long-term study on TB in badgers), with a recent history of trapped badgers having positive TB test results. Samples were taken throughout the year in a cross sectional style, contemporary with trapping efforts, while two additional intensive sampling periods were undertaken during spring and autumn. In addition, samples obtained directly from trapped badgers, were directly analysed by qPCR to compare against the culture status as a benchmark. "

The results were given in the paper as follows:
When comparing qPCR on faeces taken from trapped badgers with culture, the qPCR assay exhibited a sensitivity of 100% (95% CI: 30.8-100%) and a specificity of 95.7% (95% CI: 90.3-98.6).
"qPCR results varied by season, with spring and autumn exhibiting 100% and 80% sensitivity respectively against the combined trapped badger diagnostics for the same season. The degree of infection within a social group (trapped badger diagnostics) was strongly correlated with the degree of shedding as determined by faecal qPCR"
By taking samples over a long period of time, the Warwick team ascertained that the best results came from the most highly infected groups and that samples taken in spring gave the most robust results. They explain:
"We determined the optimum sampling strategy to be 20 samples taken over a 2 day period with a few days interval in the spring or early summer. With up to 20 samples from social groups taken across May and June, we had 100% agreement with the suite of other diagnostic tests in terms of identifying positive groups"
The paper explains that this study builds on the rigorous exploration of the contents of badger latrines in Defra project SE 3231 as a non invasive method of ascertaining infective status:
"It is a direct follow on from the rigorous ring trial (Defra project SE3231), during which Warwick University, the AHVLA and VISAVET processed faecal samples from 15 latrines from putative bTB negative badger social groups (Cambridgeshire and Bedfordshire) and 15 latrines from putative positive social groups using real-time PCR (qPCR). All putative negative samples were found to be negative by all labs, two putative positive latrines were positive in all labs and one putative positive latrine was positive in one lab.

The probability of a false positive result for the two latrines detected in common is less than 3􏰄10-9. The methodology has been further optimised such that samples containing a ten-fold lower bacterial cell count can be detected as positive, resulting in considerably increased sensitivity (see Final report SE3231 Fig 11). The test that has been developed is implemented at the social group level, rather than the individual sample."
Also involved in this work, are the Republic of Ireland, which is providing post mortem samples to further quantify qPCR results. This work has found that respiratory shedding can also be identified using faecal samples.
"Other research at Warwick University, with qPCR of faeces and culture performed in parallel on samples taken from badgers in areas in the Republic of Ireland with high levels of TB breakdown in cattle, indicates that faecal shedding is a good proxy for respiratory shedding.

The qPCR test for faeces detected all badgers shown to be also shedding via the tracheal route (n=7). The qPCR and detection of M. bovis in tissue by culture were not significantly different, with a high level of correlation in detection by both methodologies in animals with severe disease progression (Travis et al. manuscript in preparation)."
The aim of the project was to enhance the detection capabilities of qPCR in the field.
"The main aim of this project was to maximise the sensitivity of the qPCR test for applied field detection of M. bovis in badger faeces through optimisation of latrine sampling strategies. A ten-fold increase in the limit of detection has been applied compared with the previous DEFRA project."
Sampling over a long period gave the following results:
The qPCR bacterial load data [] shows that particular social groups are disproportionately responsible for shedding large numbers of M. bovis bacilli into the environment.

The genome equivalents ranged from 1x103 to 4x105 per gram (N.B. 10-100s of genome equivalents are considered to represent 1 cfu)[22]. There is a variation in the cumulative load between social groups, as shown in the bubble plot; a small number of social groups appear to be responsible for most of bacteria shed and therefore potentially represent the greatest risk for onward transmission."
That mention of '1 cfu' reminded us of previous research - [link] which found just how little bacteria is needed to infect a calf.

So, the sampling:
"The state of infection in the social group affects the likelihood of detection: as expected, heavily infected social groups were identified positive with less intensive sampling regimes. The data clearly shows that by sampling in periods of peak badger activity, the chance of detecting a social group as positive with a fixed number of samples increases.

Spring is again clearly shown as the optimal sampling season, with the seven social groups with the highest prevalence of infection detected with 95% probability in 17 or less samples. In autumn 23 or less samples would detect the seven most infected social groups with 95% probability. In spring or autumn, all social groups could be detected positive with 95% certainty within 40 samples"
And the results:
"We have determined an optimal sampling strategy for latrine faecal qPCR testing, which when applied in the field demonstrated a 100% sensitivity, 100% specificity"
"Sampling should occur in the spring or early summer with up to 20 samples taken from each social group across two days with a few days interval."
"The faecal qPCR test has been shown to be robust and reliable with no significant difference observed between results obtained from two centres at the social group level."
And cost?
We would envisage the processing of the initial samples occurring in batches of five samples until either at least one sample was positive or all 20 samples were returned as negative

. To determine a sample as positive, a second and third replicate of that sample must be extracted. A social group will be considered positive if at least one sample is positive on at least one of the confirmatory re- extraction.

This gives a false positive rate of 1%.  The cost for a social group per season would range between £81.30 and £208.20 depending on when a positive sample was detected.

So, in a nutshell, Owen Paterson's qPCR project appears to be able to identify groups of infectious badgers, upspilling zTuberculosis  into the environment. Sensitivity and specificity is 100 per cent and the cost of this non-invasive technology is around £200 per group sampled.

So why no publicity?
Why were Defra giving false information to the secretary of State, about its capabilities?
And why are Defra and assorted fellow travelers so against identifying these highly infected time bombs?

The reason we think is that if a social group of badgers was so identified and APHA failed to act on that information, then as we have said many times, litigation for victims would not be a possibility but a certainty.

Far better to bury this work, and hope it stays buried. Keep killing sentinel tested cattle and ignore the message this canary is offering.

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