Tag Design
NIBSC product
2.8. Application of Methods Developed to Real and Modelled Clinical Samples
Several sample types, including real clinical samples and infection models were prepared for sequencing in parallel. This allowed the sample processing developed in 2.6.4 to be tested for utility on several sample types simultaneously. All samples were sequenced in a single sequencing run on the HiSeq. A summary of all samples can be found in Table 2-12
2.8.1. Negative Samples
An extraction negative control was set up by starting with 1ml of PBS in place of sample and the entire sample processing including reverse transcription and ɸ29 MDA was performed, and the products sequenced. The second negative control was water which was used in a reverse transcription and ɸ29 MDA reaction.
2.8.2. Further Blood Models
Four further bacteraemia models were created to model more challenging diagnosis. The f irst was a mixture of S. Pyogenes (clinical isolate 15M153838) and Influenza virus (NIBSC control media Influenza A H3N2 07/298) to model cases of secondary bacteraemia following viral
infection. The second model was a Shigella, which many methods (including MALDI-TOF) cannot differentiate from E. coli. A model using non Typhi invasive Salmonella was used, as whole genome sequencing was available from the Salmonella Reference Unit at PHE Colindale, and this provided an opportunity to compare straight from sample methods to culture based sequence library preparation. The final model was a P.aeruginosa, which was highly resistant, including to carbapenems.
Dilutions of bacteria were prepared as described in 2.3.4.2 and single cells of S.Pyogenes, Salmonella sp and P.aeruginosa were inoculated into 1 ml horse blood. Ten cells of S. sonnei were inoculated into 1 ml horse blood, due to poor survival rates shown in 2.6.1. For the
Influenza in the mixed model, 20 µl of a 1:100 dilutions of the control material was used, to aim at 25 viral copies, as described in 2.5.4.1.
2.8.3. Tissue Model
Post mortem none human primate tissue (rhesus macaque) was obtained from PHE Porton Down. Roughly 1cm3 tissue samples were collected and were stored in RNAlater (Sigma) until use. The first tissue model was to simulate endocarditis, by inoculating heart tissue with H. influenzae
92 (clinical isolate 15M154514) dilutions of an overnight culture were prepared as described in
2.3.4.2 and five cells were inoculated into the tissue. The second model was to simulate viral
hepatitis, using Hepatitis A virus control media, the aim was to inoculate 25 viral particles, and so 65 µl of the control media was added (as in 2.5.4.1) to the tissue. The third tissue was a model of a mixed infection using Bacteroides vulgatus (14M180862), Streptococcus oralis (15M150586), Streptococcus mitis (15M150586) and Streptococcus anginosus (15M152569). Five cells of each bacterium were mixed into a total volume of 50 µl and inoculated into the brain tissue. The final model was a viral kidney infection, for this CMV was used, 20 µl of 1:100 control media was inoculated into the tissue, this value was aiming at ~25 viral particles based on previous work using VZV virus in 2.5.4.1
Samples were inoculated using, extra-long 10 µl pipette tips (VWR®) and incubated at 37oC for one hour. The tissue samples were first ground using Dounce tissue grinder set (Sigma-Aldrich), with the addition of 500 µl sterile PBS. 50 U of collagenase (Sigma) was added and the sample was incubated on a shaker at 37oC. 250 µl of 2% 2-mercaptoethanol (Sigma) was then added and the sample incubated a further hour at room temperature on a shaker. The sample was then
centrifuge at 6000 xg for 5 minutes and the supernatant discarded. The sample was then washed three times using 3 ml PBS and centrifuged at 6000 xg. The final pellet was resuspended in 5ml of PBS. This was then vacuum filtered using a 100µm filter (Millipore). The filter was discarded and the filtered sample was used as the input for the previously developed sample process.
2.8.4. Urines
Urine samples were sent from Addenbrookes hospital for deep sequencing analysis as part of an on-going project looking at genotyping BK virus directly from samples. Samples from
symptomatic patients were included in the project and four of these urines were randomly chosen and 50 µl used for processing in this thesis.
2.8.5. CSF
CSF samples with unknown aetiology from symptomatic patients were collected as part of a PHE historic study. 20 µl of four of these CSF were processed, however due to the uncertain age and handling of these samples some adaptations of the sample processing was made. DNAse and RNAse stages weren’t carried out, to be certain of the recovery of pathogen DNA which may have been degraded.
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2.8.6. STI swabs
Four blinded samples were used from sexually transmitted bacteria reference unit (STBRU) at PHE Colindale full sample details are in Table 2-11. These were samples which had been referred to the reference laboratory for diagnostic confirmation. The samples consisted of swabs which had been placed in buffer and vortexed to deposit material into liquid. After completion of STBRU processing 40 µl of sample was processed as shown in Figure 2-4
Swab type Buffer type
Reference lab results
1 RECTAL BD VIPER C. trachomatis + ([email protected])
LGV + ([email protected]/23.63)
2 VAGINAL BD VIPER C. trachomatis + ([email protected])
LGV -
3 VAGINAL COBAS N. gonorrhoeae + (Ct @29.14/29.14)
4 VAGINAL COBAS N. gonorrhoeae + (Ct @32.64/30.36)
Table 2-11 Deta ils of STBRU samples processed i n this thesis, including swab site, buffer a nd STBRU results LGV=
Lymphogra nuloma venereum
2.8.7. Treponema pallidum Samples
In collaboration with St Marys Hospital six samples with presumed T. pallidum infections were tested, two sets of matched samples from patients positive for Treponema pallidum (whole blood and ulcer samples) and two further blood samples from an additional two patients. Samples were retried from the Imperial College London Communicable Disease Research Tissue Bank. Ulcer samples were shipped in RNA later solution (Sigma).
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Sample number
Sample type Input Input amount
1 Control Negative extract 1 ml PBS
2 Control Negative amplification 10 µl water
3 Bacteraemia model Streptococcus Pyogenes
(15M153838) Influenza A H3N2 (07/298) Single cell 20 µl 1:100 dilution of control media (~25 VP)
4 Bacteraemia model Shigella sonnei
(15M005139)
Ten cells
5 Bacteraemia model Salmonella Sp.
(14M180208)
Single cell
6 Bacteraemia model Pseudomonas aurginosa
(13M155577)
Single cell
7 Endocarditis model Haemophilus influenzae
(15M154514)
Five cells
8 Viral Hepatitis HAV RNA (13/102) 65 µl control media
(~25 VP)
9 Mixed brain abscess Bacteroides vulgatus
(14M180862) Streptococcus oralis (15M150586) Streptococcus mitis (15M150586) Streptococcus anginosus (15M152569)
Five cells of each
10 Viral kidney infection human cytomegalovirus