Multiplexed isothermal assays for the detection of P. falciparum, T. vaginalis, and C. trachomatis in urine

Introduction: : Malaria in pregnancy (MiP) and sexually transmitted infections (STIs) are significant preventable causes of poor pregnancy outcomes. Plasmodium falciparum (Pf), the most virulent of the 5 human malaria species, is estimated to account for 10,000 maternal deaths, 100,000 newborn deaths and 200,000 infant deaths each year. Unlike other human malaria species, Pf can sequester within the placenta, making detection using standard microscopy and antigen point of care (POC) tests difficult due to low peripheral parasitemia. A diagnostic assay with a lower limit of detection (LOD) is needed. Where malaria is prevalent, STI prevalence among pregnant women also leads to adverse outcomes such as stillbirths, low birth rates, and permanent infertility. In some regions like sub-Saharan Africa, this burden is notably high, with Trichomonas vaginalis (TV) and Chlamydia trachomatis (CT) accounting for a large portion of comorbidities. Identifying and distinguishing these infections during prenatal visits will allow for immediate and appropriate treatment and could reduce poor pregnancy outcomes. Here, we present a novel isothermal molecular amplification technique to detect identical multi-repeat sequences (iso-IMRS) from Pf, TV and CT in urine, demonstrating performance comparable to PCR. We also present iso-IMRS multiplexing of Pf with TV and CT. This workflow will allow for earlier and more sensitive detection of MiP, TV, and CT infections in prenatal populations in areas with endemic malaria.

Materials and

Methods: : The IMRS algorithm identifies identical, repetitive sequence substrings from multiple loci within the annotated genome sequence of each pathogen and generates a library of unique repeat sequences that can be used as amplification primers. These repeat sequences are < 30 base pairs apart and primers of opposite orientation (i.e., forward–reverse) were located within an amplifiable region < 3000 base pairs apart to enable (1) strand displacement and (2) isothermal amplification. The primer pairs were evaluated using the NIH’s Basic Local Alignment Search Tool (BLAST) to ensure that they were specific to the pathogen genome. One set of primer pairs will amplify several fragments of DNA of different sizes due to the distribution of primer specific regions throughout the genome, increasing the sensitivity of the assay.
Human urine contains inhibitors that can affect amplification efficiency; therefore, we prepared spiked urine samples for iso-IMRS by performing buffer exchange with desalting chromatography columns. Of note, urine samples were applied to the resin with additional stacking buffer. Processed eluate was collected for amplification.
The 25 μl iso-IMRS reaction mixture comprises 800 U/μL Bst 2.0 polymerase, with 1X isothermal amplification buffer, 2 μM forward primer, and 2 μM reverse primer combined with 1.4 mM dNTPs, 0.4 M Betaine, and 2% molecular biology grade Ficoll-400. 5μL of processed sample was added to each 25 μL reaction. Amplification was carried out at 60°C for 30 min. Amplified products were visualized by lateral flow immunoassays and 12% acrylamide gel electrophoresis.

Results, Conclusions, and Discussions:: We developed and optimized a sample preparation, amplification, and readout strategy for detecting P. falciparum, T. vaginalis, C. trachomatis gDNA in spiked human urine. Along with the development of 3 singleplex assays, we developed 2 multiplex assays — one consisting of P. falciparum and T. vaginalis and the other P. falciparum and C. trachomatis. Notably, sample preparation was completed in only 5 minutes and successful amplification was achieved with only 30 minutes of heating. Following iso-IMRS amplification, amplicons were diluted in buffer and applied to lateral flow strips. Strips were allowed to develop for 15 minutes before reading either by eye or a lateral flow strip reader.
To determine LODs for each pathogen in urine using iso-IMRS, we conducted probit analysis at a 95% hit rate after linear regression. The LODs were P. falciparum: 0.32 copies/µL, T. vaginalis: 0.62 copies/µL, and C. trachomatis: 290.8 copies/µL.
We have confirmed that a novel isothermal nucleic acid detection method called iso-IMRS can sensitively detect P. falciparum, T. vaginalis, and C. trachomatis gDNA in human urine at clinically relevant limits of detection. This approach represents an important step toward bringing advanced nucleic acid-based diagnostics to resource-limited settings. Future work will focus on streamlining sample preparation, consolidating all targets into a single multiplex reaction, and integrating an independent heating element to eliminate the need for auxiliary equipment. We anticipate this assay will improve MiP screening in regions where it currently goes undiagnosed.

Acknowledgements and/or References (Optional)::

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