The FilmArray GI panel seems to be a promising tool for the simple and rapid detection and preliminary identification of multiple pathogens in contaminated river water at levels close to infectious dose. Further studies are warranted in order to investigate the performance
(sensitivity & specificity) and applicability of the device under field conditions and with military relevant pathogens and matrixes, including water/food-borne viruses.
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7 Appendix
Initial experimental work and method optimization
We looked at a range of different factors that could be adjusted to try and optimise the
methodology and reduce the time taken to analyse each sample. Firstly we looked at the limits of detection (LoD) with and without culture steps and then we looked at how we could try to modify and combine the NMKL methods and EPA method 1623 thus allowing us only one filtration step. Finally we looked at how to reduce enrichment time without compromising sensitivity.
7.1 Isolation of bacterial genomic DNA and preparation of DNA stock solutions
DNA was isolated from 1mL bacterial culture. QIAamp DNA mini kit (Qiagen cat nr: 51304) according to the manufacturer’s instructions. DNA concentration (ng/µL) was measured with Qubit fluorimeter (Invitrogen) and GU/ µL was calculated with the following formula based on genome size:
GU
µL =ng
µL 𝑥𝑥 6,0221415x1023 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐/𝑔𝑔 genome size (bp)𝑥𝑥660 𝑔𝑔/𝑚𝑚𝑐𝑐𝑚𝑚
𝑏𝑏𝑐𝑐 𝑥𝑥 109
Genome size Yersinia enterocolitica: ~4.6 Mb Genome size Campylobacter jejuni: ~1.7 Mb
7.2 Quantitative determination of Yersinia and Campylobacter genome copies (GU) by qPCR
In order to be able to quantify and evaluate the serial dilution effect on the concentration (GU/mL) of Campylobacter jejuni and Yersinia enterocolitica in our samples qPCR assays were established using the primers and protocols as specified in Nord Val 017 and NMKL 163.
These real-time PCR analyses were not carried out in the main study where the focus was on culture and Film-Array. DNA was extracted from 1 mL liquid sample using QIAamp DNA mini kit (art.nr 51306) and analysed in triplicate using Probe Master I (Roche), primers and
conditions as shown in Table 7.2. All analyses were run on the Light cycler 480 (Roche) real-time PCR instrument. In addition, serial dilutions of the spiked water sample was cultured to estimate the concentration (cfu mL -1) at T=0, prior to filtration and subsequent enrichment and culturing steps.
Table 7.1 The primers used for the PCR detection of Campylobacter jejuni and Yersinia enterocolitica.
Primers (Invitrogen) and probes (TIBmolbiol) (end
concentration) PCR conditions
Campylobacter
jejuni Campy primer 1: 5‘CTG CTT AAC ACA AGT TGA GTA GG 3‘ (440 nM)
enterocolitica YE-R primer:5‘CCC AGT AAT CCA TAA AGG CTA ACA TAT 3‘
YE-F primer: 5‘ATG ATA ACT GGG GAG TAA TAG GTT CG 3‘
Range of quantification: Yersinia: 3*106 GU/µL – 3*100 GU/ µL and Campylobacter: 2,2*107 GU/µL – 2,2*10-1 GU/ µL.
7.3 Optimisation of filtration and extraction of bacteria from filter The initial trials looking at using one single water filter for both bacteriological and
parasitological analyses showed we had to rethink this strategy. The bacteriological NMKL methods describe adding the filter to the enrichment media. The parasitological washing and centrifugation method (EPA 2005) resulted in considerably lower GU/mL for the bacteria than adding half the filter directly to the enrichment medium. We therefore chose to carry out two separate filtrations: one for the parasitological and direct FilmArray analysis and one, in which the filter was cut into two pieces, for the bacteriological analyses in the further work carried out.
Figure 7.1 Detection of Campylobacter and Yersinia with qPCR in mixed sample after enrichment (T16) and two different filter washing methods. EPA method:
Parasitological washing in PBS and centrifugation method. NMKL Method 2: filter cut in half and each half placed directly into the specific culture mediums for each of the bacteria investigated. DNA extraction was carried out and GU/mL calculated after the qPCR.
7.4 Optimisation of enrichment time
The NMKL methods indicate an optimal enrichment time of 48 hours for the Campylobacter and 21 hours for the Yersinia. We wanted to establish what the optimal enrichment time would be for a sample containing a mixture of two bacterial species (Yersinia enterocolitica and Campylobacter jejuni). The initial culturing studies showed that 4 hours was insufficient and 24 hours more than sufficient for detecting Yersinia and Campylobacter in the water samples. We therefore investigated whether 16 hours would also be sufficient given that this would allow us to carry out filtration and set up the sample for enrichment in the afternoon and then carry out DNA extraction and PCR analysis the following morning. All the samples had a CT value <25 after the 16 hour enrichment step. We therefore opted to use 16h enrichment and not 21h or 48h as described in the NMKL methods.
Measured GU/mL (t16)
Start concentration 10 3 cfu/mL (estimated)
Yersinia Campylobacter 1x108
1x107 1x106 1x105 1x104 1x103 1x102 1x101 1x100
EPA (2005) filter washing
method NMKL filter washing
method
7.5 Pilot studies with parasites and FilmArray
There was initial concern that FilmArray might not be able to detect the Cryptosporidium species used in the Accuspike kit. We therefore carried out an additional analysis with a faecal sample from a calf with confirmed cryptosporidiosis (courtesy of the Norwegian Veterinary Institute). FilmArray detected Cryptosporidium in the clinical sample from the animal. In addition to this FilmArray also detected Rotavirus. The laboratory that had donated the clinical sample was informed of the additional findings. This highlights one of the benefits of using such a panel to screen for a range of gastrointestinal pathogens. It also highlights that the detection of one or more of the agents on the panel may not necessarily confirm detection of a human pathogen. Positive findings should always be corroborated further.