Qiasymphony platform

Lumbar puncture was performed immediately prior to craniotomy for tumor debulking. After sterile field preparation, the thecal sac was cannulated between the L3 and L5 intervertebral spaces using a 0.61‐mm gauge lumbar puncture needle, and 10 ml of CSF was removed. After collection, CSF, whole blood, and urine samples were immediately placed on ice and then rapidly transferred to a pre‐chilled centrifuge for processing. For urine samples, 0.5 M EDTA was added within an hour of collection. Samples were centrifuged at 1,500 g at 4°C for 10 min. Supernatant was removed and further centrifuged at 20,000 g for 10 min and aliquoted into 2 ml microtubes for storage at −80°C (Sarstedt, Germany). Tumor tissue DNA was extracted and isolated as described previously (Mouliere et al, 2018b (link)). Fluids were extracted using the QIAsymphony platform (Qiagen, Germany). Up to 10 ml of plasma, 10 ml of urine and 8 ml of CSF were used per sample. DNA from cancer plasma, urine, and CSF samples was eluted in 90 μl and further concentrated down to 30 μl using a Speed‐Vac concentrator (Eppendorf, Germany).

Blood from those who provided written consent was used to prepare a dried blood spot (DBS) on filter paper at the same time when a malaria RDT was performed. In the second round of testing, an additional PfLDH RDT (CareStart Malaria pLDH Pf/Pan, Cat No G0121) was performed only in cases where the initial PfHRP2 test (SD Bioline Malaria Ag Pf/Pan, Cat No 05FK60; used in both rounds) was negative. If the second RDT was positive, the sample was flagged for additional testing to detect pfhrp2/3 deletions. All patients with a confirmed malaria infection received anti-malarial treatment free of charge per the national treatment guideline.
Only DBS from persons with P. falciparum infection confirmed with a positive RDT result were retained for further molecular testing. Each DBS was assigned a unique identification number and stored in a separate resealable plastic bag with silica gel desiccant until DNA extraction. The unique identifier was recorded along with the date of sample collection, the age and sex of the participants and, in the second round, the RDT test results. The collected samples along with the corresponding logs were shipped to the Asia–Pacific Regional Centre of the WorldWide Antimalarial Resistance Network in Bangkok, Thailand. DNA extraction from the DBS was performed using the semi-automated QIASymphony® platform and Qiagen DNA Mini Kits.

We differentiated A–K from L1–L3 CT biovares by multiplex PCR and subsequent hybridization with specific probes in urethral or rectal exudate samples.
Gene extraction and purification was performed using the QIASymphony platform (QIAGEN, Venlo, The Netherlands) with the DSP DNA Pathogen kit. CT DNA detection was performed using the Allplex™ II STI-7 Detection kit (Seegene Inc, South Korea) on the AriaMX Real-time PCR platform, and the subsequent genotypic characterization using the STD Direct Flow Chip amplification assay (Vitro, Granada, Spain), in all cases following the manufacturer’s instructions.

PBMCs were purified from EDTA blood using Lymphoprep (Stemcell Technologies, Cambridge, UK) and nucleic acids were extracted as previously described with the QIAsymphony platform and the DSP virus/pathogen Mini kit (Qiagen, 22 (link)). For RNA analysis total nucleic acids were treated with DNaseI by adding 0.5µL DNaseI (2000U/ml, NEB) and 1µL 10x DNaseI buffer (NEB) to 10µL of extract followed by incubation for 10min at 37°C and 10min inactivation of the enzyme at 72°C.

RNA testing involved elution from the DBS by lysing a 6 mm spot for 2 h at 56°C with 20 µl of proteinase K and 300 µl of ATL lysis buffer (Qiagen products: 19133 and 19076). The entire eluate was extracted on the Qiagen Qiasymphony platform using the Qiasymphony DSP Virus/Pathogen mini kit (Qiagen product: 937036) and ‘cell‐free V6/7 DSP default IC’ protocol. Bacteriophage MS2 was added as the internal control.

Thick and thin blood films were stained with Giemsa and
Pf-infected red cells counted against 500 leukocytes and 1,000 red blood cells, respectively. To detect lower parasite densities, a highly sensitive
Pf -specific PCR assay based on
18 (link) was performed.
A sensitive high qPCR assay was used for detection where 500 µl of whole venous blood was used to extract DNA using an automated DNA extraction and purification method (QIAsymphony platform, Qiagen, Germany) according to the manufacturer’s instructions. DNA was eluted in 100 µl of DNAse free water/elution buffer from which 13.5 µl was used to amplify the 18S ribosomal RNA gene by qPCR (we used Applied Biosystems’ TaqMan™Universal PCR Master Mix (cat no 4318157) which already contains the DNA polymerase (AmpliTaq Gold™DNA Polymerase)) in triplicates in a hydrolysis probe assay using primers and probes previously described. The PCR cycling conditions were carried as described using Applied Biosystems 7500 real-time PCR system. Non-template control was used as a negative control (in triplicate wells) with parasite quantification against known cultured parasite standards comprising of six serial dilutions of extracted DNA also run-in triplicate.