Implementation of helicase-dependent amplification with SYBR Green I for prompt naked-eye detection of bacterial contaminants in platelet products

doi:10.21203/rs.3.rs-2108180/v1

Download PDF

Article

Implementation of helicase-dependent amplification with SYBR Green I for prompt naked-eye detection of bacterial contaminants in platelet products

https://doi.org/10.21203/rs.3.rs-2108180/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 24 Feb, 2023

Read the published version in Scientific Reports →

You are reading this latest preprint version

Platelet transfusions may lead to more significant risks of infection and septic transfusion reactions that can be fatal to the recipient. Platelet products should be screened to limit or detect bacterial contamination before application to patients to minimise any adverse reactions. This study aimed to develop a helicase-dependent amplification (HDA) technique targeting a universal highly conserved bacterial gene, 16S rRNA, in combination with naked-eye detection using SYBR Green I (HDA/SYBR Green I) to detect bacterial contamination in platelet products. Thirty positive samples were obtained from spiked platelet products by five transfusion-relevant bacterial strains and were screened for bacterial contamination by HDA/SYBR Green I. HDA/SYBR Green I showed an enhanced yield of bacterial contaminant detection when performed with medium to late shelf life, Day 2 of storage or later platelet products (96% sensitivity and 100% specificity). The limit of detection of HDA/SYBR Green I was 1 ng, and there was no cross-reaction with other organisms that could likely contaminate platelet products. The developed HDA/SYBR Green I assay is rapid and simplistic and only requires an easy-to-find heat box, available in general blood bank laboratories, for the amplification step. This technique is suitable for further development as an alternative method to detect bacterial contamination in platelet products in the near future.

Platelets

Bacterial contamination

Helicase-dependent amplification

SYBR Green I

Bacterial contamination of blood components is a longstanding concern¹. Receiving bacterial contaminated blood components may lead to transfusion-transmitted fatalities due to bacterial infections. The U.S. Food and Drug Administration (FDA) report from 2012–2016 showed that transfusion-transmitted bacterial infections were the third cause of death from transfusions after transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO)². Even the blood collection process is being improved and is well-controlled to reduce the bacterial contamination of blood components³. However, platelet products present a significant risk due to their storage conditions (20–24°C with agitation), which may support bacteria to multiply and reach clinically harmful levels during their 5-day storage period¹.

Regarding the American Association of Blood Banks (AABB) standard 5.1.5.1, platelet products should be screened to limit or detect bacterial contamination before application to patients to minimise any adverse reactions⁴. Bacterial culture is the gold standard for detecting bacterial contamination from various blood components. However, the traditional culture exhibits some limitations, including an inappropriate practice for bacterial culture in the blood bank working area, time consumption that does not match the short 5-day shelf life of platelets, and high cost-effectiveness of the assay. The BacT/ALERT® culture system is an automated culture-based method for platelet quality control approved by the FDA. Several molecular-based methods have revolutionised the detection of bacterial contamination in platelet products, such as PCR and real-time PCR. However, these techniques are not widely used in routine testing due to a complicated process, high cost-effectiveness, and specific instrument dependence that requires ongoing maintenance^1,5.

The isothermal amplification method is an amplification technique that operates under a constant temperature. The most significant advantage of isothermal amplification is that it does not require a thermocycling machine. Therefore, isothermal amplification requires low energy consumption and can be easily integrated into a simple detection platform, such as in the field or instrumentation-free settings. Helicase-dependent amplification (HDA) is one of the most straightforward approaches for isothermal nucleic acid amplification, mimicking an in vivo process of DNA replication, using a helicase instead of heat to isothermally unwind DNA duplexes. The process occurs under an optimum temperature range from 60 to 65°C and takes 30–120 minutes. HDA has been previously developed as a novel molecular-based tool for rapid pathogen detection^6,7. The aim of this study was to fabricate a simple, rapid, and sensitive screening method for bacterial contaminants in platelet products that can be easily implemented in blood bank service routines. To achieve this objective, HDA amplification targeting 16S rRNA was used with endpoint detection using SYBR Green I (referred to as HDA/SYBR Green I).

Representative bacterial strains

Five strains of bacteria, including Staphylococcus aureus (ATCC 29523), Staphylococcus epidermidis, Bacillus cereus, Escherichia coli (ATCC 25922), and Serratia marcescens, were used as representative strains. All of these strains have been previously reported as common transfusion-relevant bacterial strains of platelet products^1,8,9. The ATCC bacterial strains were kindly provided by Assoc. Pitak Santanirand, the Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. S. epidermidis, B. cereus, and S. marcescens were clinical isolates obtained from leftover specimens from the Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, and were confirmed at the species level by conventional biochemical tests and PCR sequencing. The bacteria were subcultured on tryptic soy agar (TSA) at 37°C for 24 hrs before use. Bacterial genomic DNA was extracted by the boiling method. A single 24 hr colony of each strain was resuspended in 1 ml of sterile distilled water and boiled at 95°C for 10 min, followed by a 5-min centrifugation at 12,000 rpm. The supernatant was used as a DNA template for HDA/SYBR Green I optimisation.

Primer Design And Screening

An HDA/SYBR Green I assay was developed targeting the highly conserved bacterial 16S rRNA gene, the most widely used marker gene employed to identify bacteria and differentiate between closely related bacterial species. HDA primers were designed from the conserved fragments of 16S rDNA sequences predicted to cover 275,057 eubacterial species derived from the Ribosomal Database Project of a previous study¹⁰ using the Primer3 program (version 0.4.0; http://bioinfo.ut.ee/primer3-0.4.0/). This selected 16S rRNA gene fragment is dominant among Firmicutes, Gemmatimonadetes and Proteobacteria, which are the predominant phyla in all microbiomes¹⁰. These HDA primer candidates contained 20–32 bases, as recommended by the IsoAmp®II Universal tHDA kit (New England BioLabs, USA). The primer melting temperature ranged from 60 to 74°C, and the GC% was within the interval of 35–60% ⁶. The OligoAnalyser 3.1 program (https://eu.idtdna.com/calc/analyser) and BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi) were used to assess the possibility of secondary structure formation and the specificity of the primers, respectively. Four forward and reverse primers were designed in this study. Each forward and reverse primer candidate was paired and preliminarily screened for its ability to amplify specific targets with an IsoAmp®II Universal tHDA kit as recommended by the manufacturer. The most suitable primer pair was selected and finally used in our fabricated HDA/SYBR assay as follows: 16S rRNA forward (5’AGTCCCRYAACGAGCGCAACCC 3’) and 16S rRNA reverse (5’TTGACGTCRTCCCCRCCTTCC 3’), generating an HDA product size of 104 bp.

Hda/sybr Green I Optimisation

The optimised HDA/SYBR Green I assay was evaluated using genomic DNA of S. aureus (ATCC 29523), S. epidermidis, B. cereus, E. coli (ATCC 25922), and S. marcescens as positive controls to obtain the most appropriate conditions for HDA amplification and endpoint SYBR Green I detection. During HDA/SYBR Green I assay development, the amount of DNA template (1–20 ng), primer concentration (50–200 nM), MgSO₄ concentration (3-4.5 mM), NaCl concentration (20–50 mM), incubation temperature (63–69°C) and incubation period (15–90 minutes) were optimised until the most appropriate conditions were obtained. Sterile distilled water served as a negative control. Amplified 16S rRNA was directly detected by the naked-eye in natural light by adding 1 µL of 100-500x SYBR to a final volume of 50 µL of HDA products and immediately observing a colour change of the solution. The solution changed from light orange to bright green in the presence of the HDA product, indicating that bacterial DNA was detected. Conversely, the solution remained light orange in the absence of the HDA product, suggesting that no bacterial DNA was detected.

In every optimisation step, the HDA products were examined by SYBR Green I and compared with agarose gel electrophoresis. The condition giving clear readout results when observed with SYBR Green I, without nonspecific bands upon agarose gel electrophoresis, was chosen (data not shown).

Spiked Platelet Product Samples For The Performance Test

Spiked platelet samples were established to mimic natural bacterial-contaminated platelet products. This study used sterile leukocyte-poor platelet concentrates (LPPC) supplemented with a platelet additive solution (PAS). All the platelet concentrates were due to the expiration of the shelf life (after Day 5 of storage) and were collected from the Regional Blood Centre IV, Thai Red Cross Society. The discarded platelet products were checked for purity by plating out onto blood agar and observed for any growth colony until 7 days of aerobic and anaerobic incubations. The study was approved by the Research Ethics Committee, National Blood Centre, Thai Red Cross Society, Bangkok, Thailand (COA.NBC 15/2019).

Five strains of bacteria, including S. aureus (ATCC 29523), S. epidermidis, B. cereus, E. coli (ATCC 25922), and S. marcescens, were grown overnight in tryptic soy broth (TSB) and adjusted to 0.5 McFarland standard (10⁸ CFU/ml). Serial 10-fold dilutions were prepared in TSB to achieve a concentration of 10² CFU/ml. Then, 3 ml of each bacterial strain was injected into a separate bag containing 297 ml of sterile platelet products, achieving a final bacteria concentration of 1 CFU/ml. The freshly prepared spiked sample was named Day 0 (representing the collection day). The platelet samples were maintained under standard platelet storage conditions at 20–24°C under agitation until 5 days and named Day 1 to Day 5, respectively. During storage, platelet products were sampled daily from every bag (Day 0 to Day 5) inoculated with each bacterial strain. In total, 30 contaminated samples were obtained and set as the positive samples. Twelve sterile samples were set as the negative samples. One millilitre of platelet product was aliquoted daily, and 10-fold dilutions were prepared in TSB and plated on TSA. Colony counting was performed after 24 hrs of incubation to determine the bacterial concentration of each strain in the platelet product on each storage day. All experiments were performed in triplicate. Bacterial DNA was extracted from another 1 ml of daily aliquoted platelet products using the QIAamp DNA Microbiome Kit (Qiagen, Germany) as recommended by the manufacturer. The DNA was stored at -20°C until use.

Bact/alert® Culture System Protocol

A 10 ml aliquoted sample was taken from Day 0 to Day 5 platelet products and inoculated into BacT/ALERT® BPA culture bottles (BioMérieux, France). The samples were incubated with the automated BacT/Alert system for 7 days, and CO₂ production indicating bacterial growth was automatically measured via a fluorescent signal. All positive bottles were subcultured, and bacterial colonies were reidentified for confirmation by the matrix-assisted laser desorption/ionization (MALDI-TOF) Microflex series (Bruker Diagnostics, Germany).

Hda/sybr Green I Assay Protocol

DNA samples derived from Day 0 to Day 5 of platelet products containing 5 individual bacterial strains were used in the HDA/SYBR Green I assay protocol as positive contaminated samples. In contrast, sterile distilled water and sterile platelet product were used as negative samples. The HDA reaction was performed with the IsoAmp®II Universal tHDA kit (New England BioLabs, USA) as recommended by the manufacturer. Each HDA reaction consisted of 5 µM 16S rRNA gene forward and reverse primers, 5 µl 10X annealing buffer II, 2 µl MgSO₄, 4 µl NaCl, 3.5 µl IsoAmp dNTPs solution, 3.5 µl IsoAmp enzyme mix, 10 ng of DNA and sterile distilled water up to 50 µl. The HDA reaction mixture was incubated at 65°C for 60 minutes. After amplification, each reaction was evaluated by SYBR green I (HDA/SYBR Green I assay) and 2% agarose gel electrophoresis. One microlitre of 400x SYBR Green I (Takara Bio, Japan) was added to the 25 µl HDA products, and a colour change of the solution in natural light was immediately observed by the naked-eye. All experiments were performed in triplicate. HDA/SYBR Green I was blindly interpreted by two different investigators to eliminate bias.

Analytical Limit Of Detection

An analytical limit of detection was established to determine the lowest concentration of DNA enabling detection by the HDA/SYBR Green I assay. Genomic colony DNA of S. aureus (ATCC 29523), S. epidermidis, B. cereus, E. coli (ATCC 25922), and S. marcescens was 10-fold serially diluted from 10 to 0.000001 ng. Each DNA template was amplified by HDA using sterile distilled water and sterile platelet product as negative controls. The HDA products were analysed by SYBR Green I and 2% agarose gel electrophoresis.

Cross-reaction Testing

The specificity of HDA/SYBR Green I was evaluated with DNA extracted from microbial pathogens other than bacteria. These pathogens included the common fungi Candida albicans, Cryptococcus neoformans, Aspergillus spp., Rhizopus spp. (kindly provided with no patient data links by the Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand), and bloodborne hepatitis B virus (kindly provided with no patient data links by Prof. Yong Poovorawan, the Centre of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Human genomic DNA was extracted from a sterile discarded platelet product and included in cross-reaction testing. A total of 10 ng of individual DNA templates was used for HDA amplification. The HDA product was evaluated by SYBR Green I and 2% agarose gel electrophoresis. Sterile distilled water was used as a negative control.

Test Performance Analyses

The sensitivity and specificity of HDA/SYBR Green I were evaluated with the FDA-recommended BacT/Alert system. Sensitivity (a true positive rate) was computed from sensitivity (%) = TP/(TP + FN) x 100, where ‘TP’ is the number of positive samples, and ‘FN’ is the number of false negatives when spiked samples were used. Specificity (a true negative rate) was computed from specificity (%) = TN/(TN + FP) x 100, where ‘TN’ is the number of negative samples and ‘FP’ is the number of false-positives when nonspiked samples (sterile distilled water and sterile platelet product) were used. To further evaluate the reliability of HDA/SYBR in detecting bacterial contamination in platelet products, the percent agreement between the two methods (HDA/SYBR Green I and BacT/Alert® system) was calculated. The Kappa result was interpreted as follows: values ≤ 0 indicated no agreement, 0.01–0.20 indicated slight agreement, 0.21–0.40 indicated fair agreement, 0.41–0.60 indicated moderate agreement, 0.61–0.80 indicated substantial agreement, and 0.81-1.00 indicated almost perfect agreement.

Establishment of spiked platelet product samples

The average bacterial concentration in the platelet products from Day 0 to Day 5 of storage is shown in Table 1. A visible colony of every bacterial strain, except S. epidermidis, was initially observed in platelet products collected from Day 1 of storage, with concentrations ranging from 8.8x10² to 1.9x10³ CFU/ml. In contrast, the colony of S. epidermidis was slower on Day 2 of storage, with a concentration of 2.2x10³ CFU/ml. The number of bacterial cells in platelet products increased during a more extended storage period in a time-dependent manner. The maximum bacterial concentration was 1.2x10¹⁰ CFU/ml, which was found on Day 5 of storage when inoculated with S. marcescens.

Table 1

Detection of bacterial contaminants in platelet products using HDA/SYBR Green I assay compared with colony count during storage periods.
Bacterial strains	Storage periods of platelet products
Bacterial strains	Day 0	Day 1	Day 2	Day 3	Day 4	Day 5
S. aureus (ATCC29523)	0/0^a No visible colony	0/0 (1.7 x10³ CFU/ml) ^b	3/3 (1.3x10⁸ CFU/ml)	3/3 (5.0x10⁸ CFU/ml)	3/3 (6.6x10⁸ CFU/ml)	3/3 (1.4x10⁹ CFU/ml)
S. epidermidis	0/0 No visible colony	0/0 (0 CFU/ml)	2/3 (2.2x10³ CFU/ml)	3/3 (8.4x10⁵ CFU/ml)	3/3 (9.5x10⁶ CFU/ml)	3/3 (1.2x10⁸ CFU/ml)
B. cereus	0/0 No visible colony	3/3 (1.9 x10³ CFU/ml)	3/3 (2.0x10⁸ CFU/ml)	3/3 (4.1x10⁸ CFU/ml)	3/3 (6.9x10⁸ CFU/ml)	3/3 (7.8x10⁸ CFU/ml)
E. coli (ATCC25922)	0/0 No visible colony	0/0 (1.8 x10³ CFU/ml)	3/3 (1.1x10⁸ CFU/ml)	3/3 (2.4x10⁸ CFU/ml)	3/3 (2.2x10⁸ CFU/ml)	3/3 (2.9x10⁹ CFU/ml)
S. marcescens	0/0 No visible colony	0/0 (8.8 x10² CFU/ml)	3/3 (7.4x10⁴ CFU/ml)	3/3 (2.6x10⁹ CFU/ml)	3/3 (1.1x10¹⁰ CFU/ml)	3/3 (1.2x10¹⁰ CFU/ml)
^a Number of a positive HAD/SYBR reactions / replicates
^b Bacterial concentration acquired by colony count assay

Bact/alert® Culture System Detected Bacterial Contamination In Platelet Products

The BacT/ALERT® system could detect bacterial contamination in all storage periods of spiked platelet products, starting from the collection day (Day 0), except for platelets inoculated by B. cereus, which started to be detected on Day 1 of storage. The average processing time of the BacT/ALERT® system from sample incubation until a positive signal was detected ranged from 10 hrs and 54 mins to 18 hrs and 15 mins (Supplement Table 1).

Detection Of Bacterial Contamination By Hda/sybr Green I

After complete optimisation of HDA/SYBR Green I, the appropriate assay conditions were achieved. When evaluated with genomic DNA derived from a colony of 5 representative bacterial strains, SYBR Green I stayed orange in a negative control tube due to the lack of amplification of the HDA product. In contrast, SYBR Green I switched to shining green in the positive control tubes, implying that DNA products were amplified by HDA with 16S rRNA gene-specific primers (Fig. 1). HDA/SYBR Green I concordantly exhibited positive and negative results for agarose gel electrophoresis. The presence of a bottom primer-dimer band on an agarose gel in Fig. 1 (Lanes 1, 2, 7 and 8) did not cause a false-positive or background on SYBR Green I detection or interfere with the assay. When testing with Day 0 to Day 5 of stored (contaminated samples) and sterile platelet products (sterile samples) in triplicate, HDA/SYBR Green I could correctly detect positive and negative samples, as described in Table 1. Platelets inoculated with B. cereus were first detected on Day 1 of storage (bacterial concentration was 1.9x10³ CFU/ml), whereas the rest of the contaminants started to be detected on Day 2 of storage (bacterial concentration ranged from 7.4x10⁴ to 2.0x10⁸ CFU/ml). The average test time of HDA/SYBR Green I, from the reaction began until the sample readout, was 1 hr and 40 mins.

Performance Of Contaminated Bacterial Detection By Hda/sybr Green I

The performance of HDA/SYBR Green I to detect bacterial contamination in platelet products was validated with the BacT/ALERT® culture system. HDA/SYBR Green I could not detect bacterial contamination in freshly prepared Day 0 of storage samples and exhibited a very low sensitivity when tested with Day 1 of storage samples. The sensitivity and specificity of HDA/SYBR Green I when tested with Day 1 of storage samples was 3.33% and 100%, respectively, with slight agreement with a standard method. However, when tested with samples from Day 2 of storage or later, HDA/SYBR Green I showed markedly increased sensitivity of 98.89–100% and specificity of 100% with almost perfect agreement (Table 2).

Table 2

Performance of HDA/SYBR Green I assay during storage periods.
Storage periods of platelet products	Sensitivity	Specificity	PPV	NPV	Cohen kappa's coefficient
Day 0	0.00% (95% CI: 0.00-4.02%)	100% (95% CI: 90.26–100%)	- -	28.57% (95% CI: 28.57–28.57%)	0.000 (95% CI: 0.000–0.000)
Day 1	3.33% (95% CI: 0.69–9.43%)	100% (95% CI: 90.26–100%)	100% -	29.27% (95% CI: 28.48–30.07%)	0.019 (95% CI: 0.003–0.042)
Day 2	98.89% (95% CI: 93.96–99.97%)	100% (95% CI: 90.26–100%)	100% -	97.30% (95% CI: 88.68–99.61%)	0.981 (95% CI: 0.943-1.000)
Day 3	100% (95% CI: 95.98–100%)	100% (95% CI: 90.26–100%)	100% -	100% -	1.000 (95% CI: 1.000–1.000)
Day 4	100% (95% CI: 95.98–100%)	100% (95% CI: 95.98–100%)	100% -	100% -	1.000 (95% CI: 1.000–1.000)
Day 5	100% (95% CI: 95.98–100%)	100% (95% CI: 95.98–100%)	100% -	100% -	1.000 (95% CI: 1.000–1.000)

Lod And Cross-reactivity Of Hda/sybr Green I

The LOD of the HDA/SYBR Green I assay was 1 ng. When the HDA product was detected by agarose gel electrophoresis, the LOD was as low as 0.1 ng. HDA/SYBR Green I did not cross-react with C. albicans, C. neoformans, Aspergillus spp., Rhizopus spp., hepatitis B virus, or human genomic DNA. Figures 2 and 3 show the LOD and cross-reactivity testing of HDA/SYBR Green I, respectively.

A transfusion with bacterial contaminated platelets can lead to transfusion-transmitted diseases such as transfusion-transmitted bacterial infection (TTBI) and septic transfusion reactions (STRs), which are significant sources of morbidity and mortality. Between 1 in 1000 to 2000 platelet units are bacterially contaminated. Thus, a strategy to relieve the risk of bacterial contamination in platelet products is challenging. Different strategies have been proposed, including preventing bacterial contamination during platelet collection, pathogen reduction/inactivation technologies and testing methods for platelet bacterial contamination¹¹. In 2004, AABB standard 5.1.5.1 instructed that the blood bank or transfusion service should have methods to limit and detect bacterial contamination in platelet products before applying to the patients to minimise any adverse reactions⁴.

The BacT/ALERT^® culture system is the primary automated culture-based method commonly used worldwide and in major transfusion service centres in Thailand. The assay has been approved by the FDA and is regarded as the gold standard for bacterial contaminant screening in platelet products after collection. In our experimental setting, the BacT/ALERT^® culture system effectively detected bacterial contamination in collection day (Day 0) and early storage (Day 1) platelet samples inoculated with bacterial strains (e.g., S. aureus, S. epidermidis, B. cereus, E. coli, and S. marcescens) with an incubation time of 10 to 18 hrs. A previous study showed that the level of detection of the BacT/ALERT^® culture system against those relative bacteria was as low as 1–10 CFU/ml⁵. The BacT/ALERT^® system has been shown to reduce the incidence of TTBI in screening programs for bacterial contamination of platelet products¹². However, this current automatic culture system may not effectively detect biofilm-producing organisms¹³. Moreover, due to high cost-effectiveness, implementing the BacT/ALERT^® system may be limited in many transfusion service routines in Thailand. Currently, molecular biology techniques, such as real-time PCR, are being developed to detect bacterial contamination in platelet products targeting highly conserved 16S rRNA and 23S rRNA genes^8,14. The analytical sensitivities ranged from 5 to 50 CFU/ml. However, the ideal screening test should have a high diagnostic sensitivity and specificity and be affordable, reliable, and rapid. Real-time PCR relies on a specific thermocycler, which is not yet widely used in transfusion service routines to detect bacterial platelet contamination. Isothermal-based amplification is a molecular method that depends on a constant temperature during the entire amplification process. Among putative isothermal amplification techniques, HDA is one of the most straightforward methods⁷. Similar to the traditional PCR primer design, the HDA primer is simple and easy to tailor. HDA has been successfully developed as a novel molecular-based method for the rapid and sensitive detection of bacteria (e.g., E. coli, S. aureus, Chlamydia trachomatis and Neisseria gonorrhoeae) and viruses (e.g., human papillomavirus 16 and 18)⁶. The optimum HDA temperature ranges from 60 to 65°C; thus, the entire reaction can be constantly operated by a simple heat block that is generally used in blood bank laboratories.

Here, HDA was chosen to amplify bacterial target DNA, the 16S rRNA gene, to establish specific instrument-free amplification that depends on a simple heat generating box available in general transfusion service centres. The 16S rRNA gene has been a mainstay of molecular-based bacterial investigation for decades and can be implemented in aerobic, anaerobic, facultative, and fastidious bacteria. The copy numbers of 16S rRNA are diverse among bacterial phyla. They were high in Firmicutes, Gammaproteobacteria and Fusobacteria, with averages greater than five copies per genome. In contrast, most bacterial phyla contain at least a single 16S rRNA copy¹⁵. Target selection and primer design are critical in assay development. Therefore, the target sequence and initial primer selection were first verified in silico before the candidate set of primers was experimentally screened with reference strains. However, our 16S rRNA primers were designed in silico to cover 275,057 eubacterial species, including the commonly contaminating bacteria, and the detection of some species may eventually fail¹⁰.

The HDA/SYBR Green I assay provided high sensitivity and specificity and was rapid for detecting bacterial contamination in platelet products by the naked-eye. The developed assay was conducted under a constant temperature of 65°C, and the test time was approximately 1 hr and 40 min from sampling to readout. In our study, the early sampling with a small sample volume (1 ml) might have caused false negative results of the HDA/SYBR Green I assay compared with the BacT/ALERT^® system, which was processed with a larger sample volume (10 ml). In a previous study, a high-volume extraction (2.4 ml of sample volume) of total DNA by magnetic bead technology was shown to enhance the sensitivity of the 23S rRNA real-time reverse transcription PCR assay for detecting bacterial contamination of platelet products¹⁴. HDA/SYBR Green I showed poor sensitivity in detecting bacterial contamination in samples collected from early shelf life, Day 0 and Day 1. However, the assay showed an enhanced yield of detection when performed with medium to late shelf life, Day 2 of storage or later platelet products, with a sensitivity ranging from 98.89–100%. The sample collection at an early stage of platelet shelf life can cause a sampling error, owing to a low bacterial contamination number at the start time of storage, mainly when slow-growing bacteria are implicated in the contamination. Previously, the risk of sampling errors continuously decreased during storage; the detection rate of bacterial contamination changed from 14.9% for sampling on Day 1 to 100% for sampling on Day 7¹⁶. The bacterial load in platelet products has been suggested to be associated with storage times. One approach to improving the sensitivity of contaminated platelet screening could be to collect samples later in platelet storage (e.g., Day 3 to Day 5 of storage)⁹. Another factor that might interfere with the efficacy of the HDA assay during the amplification process is the presence of human genomic DNA in clinical samples, presumably through binding HDA enzymes. Doseeva et al. showed that more than 200 ng of human genomic DNA per HDA assay significantly interfered with the specific amplification¹⁷. Removal of background human genomic DNA originating from platelet cells may increase either sensitivity or specificity of the HDA assay.

Our study has proven a concept to combine isothermal-based amplification, HDA, with naked-eye detection using SYBR Green I to detect bacterial contamination in platelet products. HDA/SYBR Green I is less laborious and expensive (equipment, reagents) than real-time PCR and the BacT/ALERT^® system. The total test time of HDA/SYBR Green I is faster than that of culture-based assays, and can be implemented for routine contaminant bacterial screening in platelet products before providing them to a patient. The ideal test to detect bacterial contamination in platelet products should be highly sensitive (1 CFU/ml), specific, rapid (less than 2 hrs), inexpensive, reliable, and simple¹⁸. HDA/SYBR Green I first detected bacteria on Day 1 of storage (1.9x10³ CFU/ml) as the earliest detection, whereas the rest of the contaminants started to be detected on Day 2 of storage (7.4x10⁴ to 2.0x10⁸ CFU/ml). The analytical sensitivity of HDA/SYBR Green I was similar to that obtained from real-time PCR for S. epidermidis (2.9x10⁴ CFU/ml) and E. coli (2.2x10⁴ CFU/ml) in a previous study¹⁴. Earlier published surveillance data show that platelet units with bacterial concentrations of < 10⁵ CFU/ml are much less likely to cause severe STRs than units with higher bacterial concentrations¹⁹. HDA/SYBR Green I potentially detected bacterial concentrations in platelet products at the minimum clinical significance cut-off of < 10⁵ CFU/ml. Nevertheless, the sensitivity of our HDA/SYBR Green I assay requires further improvement.

In conclusion, the HDA/SYBR Green I assay in combination with late sampling can enhance the possibility of detecting bacteria in platelet products and can be easily adopted in a blood bank service for the routine screening of bacterial contaminants. Our developed HDA/SYBR Green I assay is rapid and simplistic and only requires an easy-to-find heat box, which is available in general blood bank laboratories, for the amplification step. This technique is suitable for further development as an alternative method to detect bacterial contamination in platelet products in the future.

Acknowledgements

This research was financially supported by the 90th Anniversary Chulalongkorn University Fund and the Chulalongkorn University Ratchadaphiseksomphot Endowment Fund (CU- GR_63_83_37_10). The authors would like to acknowledge the Prof. Yong Poovorawan, the Centre of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, for providing hepatitis B virus DNA, and Assoc. Pitak Santanirand, the Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, for providing bacterial ATCC and clinical strains DNA.

Author contributions statement

Panan Ratthawongjirakul (P.R.) created the conceptualisation, conducted the experiments, supervision and did funding acquisition and project administration. Warangkana Yamket (W.Y.) conducted all the experiments and did the first draft manuscript. Panuwat Sathianpitayakul (P.S.) performed BacT/ALERT^® culture and reidentified bacteria using MALDI-TOF. Pitak Santanirand (P.S.) was an essentially intellectual contributor, conducted the experiments and analysed the data. P.R. and W.Y. analysed the statistical data and wrote the manuscript. All the authors read and approved the final manuscript.

Additional information

The authors declare that they have no conflicts of interest related to the publication of this article.

Data availability

The datasets analysed during the current study are available in the DNA Data Bank of Japan (DDBJ) repository, LC730901, LC730902, LC730903, LC730904, and LC730905 for analysed sequences of 16S rRNA of S. aureus, S. epidermidis, B. cereus, E. coli, and P. aeruginosa, respectively, generated in the study.

Jerrold, H. L., Matthew, D. N. & Jay, H. H. Bacterial contamination of platelets for transfusion: strategies for prevention. Critical. Care. 22, 271. https://doi.org/10.1186/s13054-018-2212-9 (2018).
Fatalities reported to FDA Following blood collection and transfusion annual summary for FY2016 (Food and Drug Administration, 2016).
Ibáñez-Cervantes, G., Bello-López, J. M., Fernández-Sánchez, V., Domínguez-Mendoza, C. A. & Acevedo-Alfaro, L. I. Prevalence of bacterial contamination in platelet concentrates at the National Center of Blood Transfusion (Mexico). Transfus. Clin. Biol. 24, 56-61. https://doi.org/10.1016/j.tracli.2017.03.003 (2017).
Further guidance on methods to detect bacterial contamination of platelet components: AABB Association Bulletin No. 03-12. (American Association of Blood Banks, 2003).
Störmer, M. & Vollmer, T. Diagnostic methods for platelet bacteria screening: current status and developments. Transfus. Med. Hemother. 41, 19–27. https://doi.org/10.1159/000357651 (2014).
Barreda-García, S., Miranda-Castro, R., de-Los-Santos-Álvarez, N., Miranda-Ordieres, A. J. & Lobo-Castañón, M. J. Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection. Anal. Bioanal. Chem. 410, 679-693. https://doi.org/10.1007/s00216-017-0620-3 (2018).
Zhao, Y., Chen, F., Li, Q., Wang, L. & Fan C. Isothermal amplification of nucleic acids. Chem. Rev. 115, 12491−12545. https://doi.org/10.1021/acs.chemrev.5b00428 (2015).
Viana, J. D. et al. Detection of bacterial contamination in platelet concentrates from Brazilian donors by molecular amplification of the ribosomal 16S gene. Transfus. Med. 28, 420–426. https://doi.org/10.1111/tme.12561 (2018).
Sireis, W. et al. Extension of platelet shelf life from 4 to 5 days by implementation of a new screening strategy in Germany. Vox. Sang. 101, 191–199. https://doi.org/10.1111/j.1423-0410.2011.01485.x (2011).
Wang, Y. & Qian, P.Y. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS One. 9, 4(10):e7401. https://doi:10.1371/journal.pone.0007401 (2009).
Standards for Blood Banks and Transfusion Services: 31^th Edition. (American Association of Blood Banks, 2018).
Heaton, W. A., Good, C. E., Galloway-Haskins, R., Yomtovian, R. A & Jacobs, M. R. Evaluation of a rapid colorimetric assay for detection of bacterial contamination in apheresis and pooled random-donor platelet units. Transfusion. 54, 1634–1641. https://doi.org/10.1111/trf.12603 (2014).
Greco-Stewart, V. S. et al. Serratia marcescens strains implicated in adverse transfusion reactions form biofilms in platelet concentrates and demonstrate reduced detection by automated culture. Vox. Sang. 102, 212–220. https://doi.org/10.1111/j.1423-0410.2011.01550.x (2012).
Störmer, M., Kleesiek, K. & Dreier, J. High-volume extraction of nucleic acids by magnetic bead technology for ultrasensitive detection of bacteria in blood components. Clin. Chem. 53, 104–110. https://doi.org/10.1373/clinchem.2006.070987 (2007).
Veˇtrovsky, T. & Baldrian, P. The variability of the 16S rRNA Gene in bacterial genomes and its consequences for bacterial community analyses. PLoS ONE. 8(2):e57923. https://doi.org/10.1371/journal.pone.0057923 (2013).
Palavecino, E. L., Yomtovian, R. A. & Jacobs, M. R. Bacterial contamination of platelets. Transfus. Apher. Sci. 42, 71-82. https://doi.org/10.1016/j.transci.2009.10.009 (2010).
Doseeva, V. et al. Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. Diagn. Microbiol. Infect. Dis. 71, 354-365. https://doi.org/10.1016/j.diagmicrobio.2011.08.021. (2011).
McDonald, C. P. Transfusion risk reduction: testing for bacteria. ISBT. Sci. Ser. 8, 73–79. (2013).
Jacobs, M. R., Good, C. E., Lazarus, H. M. & Yomtovian, R. A. Relationship between bacterial load, species virulence, and transfusion reaction with transfusion of bacterially contaminated platelets. Clin. Infect. Dis. 46, 1214-1220. https://doi.org/10.1086/529143 (2008).

No competing interests reported.

supplementfileshouldbePDF.docx

Download PDF

Journal Publication

published 24 Feb, 2023

Read the published version in Scientific Reports →

Editorial decision: Major revision
28 Dec, 2022
Reviews received at journal
27 Dec, 2022
Reviewers agreed at journal
20 Dec, 2022
Reviews received at journal
30 Nov, 2022
Reviewers agreed at journal
27 Nov, 2022
Reviewers invited by journal
27 Oct, 2022
Editor assigned by journal
19 Oct, 2022
Editor invited by journal
10 Oct, 2022
Submission checks completed at journal
10 Oct, 2022
First submitted to journal
27 Sep, 2022

You are reading this latest preprint version

Implementation of helicase-dependent amplification with SYBR Green I for prompt naked-eye detection of bacterial contaminants in platelet products

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Representative bacterial strains

Primer Design And Screening

Hda/sybr Green I Optimisation

Spiked Platelet Product Samples For The Performance Test

Bact/alert® Culture System Protocol

Hda/sybr Green I Assay Protocol

Analytical Limit Of Detection

Cross-reaction Testing

Test Performance Analyses

Results

Establishment of spiked platelet product samples

Bact/alert® Culture System Detected Bacterial Contamination In Platelet Products

Detection Of Bacterial Contamination By Hda/sybr Green I

Performance Of Contaminated Bacterial Detection By Hda/sybr Green I

Lod And Cross-reactivity Of Hda/sybr Green I

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1