Performance of automated real-time PCR-based method for bacteria detection

doi:10.21203/rs.3.rs-8392071/v1

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Performance of automated real-time PCR-based method for bacteria detection

https://doi.org/10.21203/rs.3.rs-8392071/v1

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In the field of bacterial disease diagnosis, a rapid detection of the causative pathogen(s) may help initiation of appropriate antimicrobial treatment. Culture based methods are considered as standard methods, though they are time consuming. A more rapid assay for bacterial identification would have several benefits compared to culture methods. Nucleic acid amplification-based methods are significantly faster and are increasingly being utilized to detect specific pathogens from various sample types. Here, we present a proof of concept for a fully automated real-time PCR-based identification method for specific bacteria. We were able to detect 87% of culture positive samples from various sample types with a turnaround time of approximately two hours. Such an approach might provide a relatively easy and cost-effective method for indicating bacteria presence in a patient sample.

Nucleic Acid

Pseudomonas

Staphylococcus

Streptococcus

Wounds

RT-PCR

Time is a crucial factor for patient care both in clinical and economical aspects. Bacterial infections may be life-threatening and thus require immediate diagnostics and treatment. Simultaneously, a major goal in health care is shortening the time a patient is hospitalized. Optimizing the treatment and minimizing the time a patient spends in hospital have become more important as expenses of health care rise and the numbers of qualified staff decreases.

Mucosal and dermal barriers efficiently sustain homeostasis and sterility from environmental microbes and as a result, clinical samples, such as deep tissue swabs, peritoneal or synovial fluids are normally sterile. However, after injury, operation or infection, normally sterile tissue may become infected with mono- or multiflora. In these instances, rapid detection of bacteria is important for initiation of appropriate antibacterial treatment. Traditionally, this has been done by culture-based methods which take 1–2 days with subsequent antibiotic sensitivity assays taking further several days for slow growing bacteria. The development of PCR methodology has enabled rapid detection of bacterial genomes. Recently, multiplexed assays to detect pathogens from a single sample have been developed and are in use in clinical laboratories worldwide^2–5.

One challenge with both culture-based and traditional PCR methods is the need for well-trained laboratory personnel. For this, commercial providers have launched a variety of automated PCR systems which significantly simplify the PCR workflow by removing the need for separate extraction of nucleic acids and PCR set-up and analysis. In addition, such automated platforms may include an open channel for Laboratory Developed Tests (LDTs). Fully automated platforms with multiplex assay capability hold an exciting potential to be used for rapid screening of bacterial presence in normally sterile body fluids. However, at present there is no assay which could be used to rapidly analyze samples for possible bacterial presence without the need for specialized trained professionals

The performance of pan-pathogen PCR assays has been evaluated by many groups. There are studies of the performance of 16S rDNA amplification as a tool for diagnosis in microbial infections^6–9. Similarly, culture-free methods have been developed for fungi¹⁰. These methods are based on extraction of nucleic acids followed by nucleic acid amplification and 16S rDNA sequencing. The performance of these assays as measured by their capability to identify pathogens in culture negative samples has varied from 4.3% ⁶ to 42.9 % ⁷. A pointed out above, these methods require highly skilled personnel and specific instrumentation and therefore may represent a challenge for microbiology laboratories in routine screening for bacterial infections.

We describe here a technical proof of concept for a novel real-time PCR based screening method which allows a shorter Turn Around Time (TAT) using the fully automated Laboratory Developed Test (LDT) mode function (Fig. 1). As model organisms we used Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes which are common causes of bacterial infections^11,12,13. We also demonstrate that DNA from these bacteria can be detected in clinical samples using the fully automated LDT mode.

Samples were processed using primary tubes on the fully automated assay; the complete workflow with automatic nucleic acid extraction, real-time PCR and analysis of the results, including reporting to the Laboratory Information System (LIS) via LIS connectivity.

2.1. Bacterial dilution series and culture-based quantification

Bacterial cultures were incubated overnight at 37°C in standard CO₂ atmosphere. A ~0.5 McFarland suspensions of each bacterium were prepared in 0.9% NaCl solution. A 1:10 serial dilution series were done in Universal Transport Medium (UTM, Copan). Concentration of suspensions were verified by plating on Blood agar (ThermoFisher, Finland). Colony Forming Units in ml (log cfu/ml) were calculated after overnight growth at 37°C (Figure 2). Primary concentrations of bacterial strains are presented in Table 1. Serial dilutions of bacterial suspensions were then used for assay development.

Assay development – primers and probes

Commercially available research use only (RUO) microbial DNA qPCR assays (ready-to-use mixes of primers and probes) for S. aureus, P. aeruginosa and S. pyogenes, (reference numbers BPID00314AR, BPID00288AR, BPID00332AR, respectively, Catalogue number 330025 GeneGlobe, QIAGEN) were used for assay development. Sensitivity of each specific assay (single-plex) was first evaluated using the 10-fold pure culture dilution series (Fig. 2). To amplify the three target microorganisms simultaneously, a multiplex assay was created (subsequently referred to as 3-plex). The performance of the assays was evaluated by comparing Cycle time (Ct) values and dilution factors (Fig. 2). Additionally, cross reactivity tests were performed using the 1-plex assays. For each PCR reaction, a volume of 0,65 µl of the commercial RUO qPCR assay was used. The assays were diluted with microbial free water (QIAGEN) and loaded into the NeuMoDx LDT Primer/Probe Strip (ref 100400) well for a total volume of 6,5 µl using manufacturers NeuMoDx LDT Master Mix, DNA (ref 210100).

2.2. Assay development - Automated sample processing conditions

Samples were processed using the fully automated System. The system performs the complete workflow automatically, including nucleic acid extraction, real-time PCR, analysis of the results and reporting the results to the LIS system. A modified Assay Definition File (ADF) template “TM DNA Qual” was used. For a complete run protocol see the modified ADF template displayed in Table 2. Specific reagents and materials for nucleic acid extraction and PCR were purchased from QIAGEN and used according to instructions by the manufacturer.

2.3. Clinical samples

A total of 97 clinical swab samples from daily routine were analyzed with the NeuMoDx 3-plex LDT method. Samples were chosen according to the preliminary culture results. A swab sample was placed in phosphate buffered saline solution (Vacuette, BD, Finland) and pre-treated by vortexing for 20 seconds.

For decades, clinical laboratories have identified bacteria using culture-based methods. As the demand for shorter TAT for microbiological results is increasing the traditional microbiological methods are unable to respond to the current clinical needs.

A challenge with 16S rDNA and PCR screening methods is the need for specialized laboratory personnel¹⁴For this, we measured the capability of LDT 3-plex assay to identify bacteria in culture suspension. The LDT 3-plex assay performed without difficulties with pure culture suspensions (Table 3). Additionally, the LDT assay gave mainly comparable results with culture method (See Appendix). In duplicate analyses, a total of 86 samples gave positive results in at least one pair (87%) (see Appendix 1). These samples were then cultured to verify the growth. Samples that were negative in the 3-plex assay contained mixed aerobic flora (n = 5) or contained only a limited amount of target (n = 2) which may have affected LDT 3-plex results. Additionally, three samples did not contain target microbes as confirmed by culture and can be considered true negatives.

Each specific LDT single-plex LDT assay detected the respective bacterial strain from pure culture samples (Fig. 3), performed in triplicates. No cross reactivity was found for the specific assays i.e., only specific assays gave positive results. The serial dilution series showed that P. aeruginosa and S. pyogenes were able to be detected until the 6th dilution step and S. aureus until the 5th dilution step. Negative samples showed no amplification, while the internal control was detected in all samples.

The performance of method in samples derived from patients (in duplicates) was good (Table 3). Samples that contained high counts (+++) of target pathogen were also positive with the 3-plex assay (n = 42). However, there were three wound samples containing S. aureus that remained negative (in triplicate analysis). Two of these negative samples contained mixed flora. Additionally, four samples that gave both positive and negative result when samples were analyzed in duplicate. Also, there were three wound samples and one eye swab sample containing S. aureus (lower amount according plate count method) which were negative with the 3-plex LDT.

The advantage of fully automated PCR instruments would open new possibilities to switch from culture-based methods to PCR screening and detection with minimal human intervention. The time to result with nucleic acid-based methods is significantly faster compared to culture methods (Fig. 1). Furthermore, these methods can also serve as a screening method for bacteria, notably also for slow-growing or non–culturable species^15,16,17.

In our catchment area, our clinical laboratory analyzes around 60 000 bacterial samples annually and the average TAT is approximately 3 days for aerobic bacteria and approximately 5 days for anaerobic bacteria. For indicating the presence of bacteria in a sterile sample, the laborious and time-consuming culture methods could be replaced or complemented with nucleotide-based technologies such as PCR or 16S rDNA screening 2–9.

Data generated with the fully automated LDT 3-plex assay and specific bacterial LDT assays demonstrated good analytical performance. As compared to the culture-based methods, it is important to acknowledge, that PCR-based methodologies may have limitations. PCR methods detect also DNA from non-living bacteria. If nucleic acids of bacteria are present in normally sterile tissues but culture remains negative, it is an indication of possibility that a bacteria may have caused an infection, but for example, an empiric antibacterial treatment has terminated bacterial growth. Wu et al. (2022)¹⁸ presented future possibilities in microfluidics to detect viable Escherichia coli bacteria in urine samples.

The use of PCR-based screening methods designed for bacteria using pan 16S primers and probes could also detect unculturable pathogens with unknown clinical relevance. Obviously, this poses a challenge for probe design and likely and RNA/DNA sequencing might be more suitable approach in this sense. PCR based indication of bacterial DNA combined with other technologies such as NGS might hold a great promise for understanding and combining data about human genetics and pathogen presence.

In conclusion this study proves that the newly developed LDT performs well in various clinical samples and provides a significantly shorter TAT by combining an assay to a single-phase detection from sample to result method. Further evaluation is needed to understand further possible benefits of this screening assay, for example for post-operative patients and other patient groups with infections present in normally sterile tissues. Further development of this assay could be performed to include additional clinically relevant or difficult to culture bacteria^15,16,17. While the platform presented in this work is discontinued, the technical feasibility of such an approach is evident and future efforts to set up a 2-step presence of 16s RNA and bacteria-specific probes should be continued. There are currently other similar platforms available commercially, namely Abbott Alinity m YOU-CREATE and Roche Cobas Omni Utility Channel. We continue pursuing toward this automated analysis assay with one of these platforms.

Author contributions

Vesa Mäki-Koivisto: writing the initial report. Vesa Mäki-Koivisto, Elina Aho-Laukkanen, and Ilkka S. Junttila reviewed the article, Vesa Mäki-Koivisto, study design, laboratory work and data analysis. Ilkka S. Junttila supervision and support.

Funding

Vesa Mäki-Koivisto is supported by Competitive State Research Financing of the Expert Responsibility Area of POHDE. Ilkka S. Junttila is supported by Nordlab Laboratories (grant: X3710-KT0011), Competitive State Research Financing of the Expert Responsibility Area of Fimlab Laboratories (grant: X51409) / Competitive State Research Financing of the Expert Responsibility Area of POHDE and Tampere Tuberculosis Foundation. NeuMoDx reagents and consumables were provided free of charge by QIAGEN for this study.

Conflict of interest:

Vesa Mäki-Koivisto, Elina Aho-Laukkanen and Ilkka S. Junttila none.

Data availability:

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request..

Acknowledgements: Authors would like to thank Qiagen for excellent technical support in designing and performing assays.

Ethics declaration: not applicable – no patient data was included in this study.

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Table 1 to 3 are available in the Supplementary Files section.

No competing interests reported.

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You are reading this latest preprint version

Performance of automated real-time PCR-based method for bacteria detection

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Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Bacterial dilution series and culture-based quantification

2.2. Assay development - Automated sample processing conditions

2.3. Clinical samples

3. Results

4. Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1