Chlamydia trachomatis is a Gram-negative, obligate intracellular pathogen that causes the most common bacterial sexually transmitted infections (STIs) (Woodhall et al., 2018; Murray and McKay, 2021), with an estimated ~130 million new cases worldwide each year, thereby remaining a major global health challenge, particularly in underdeveloped regions (World Health Organization, 2016). C. trachomatis infection in women can cause pelvic inflammatory disease, including salpingitis, tubo-ovarian abscesses, endometritis, pelvic peritonitis, and cervical carcinoma (Cooksey et al., 2010; Di Pietro et al., 2019). Also, chlamydial infections during pregnancy can lead to adverse outcomes, such as premature rupture of membranes, stillbirth, miscarriages, and preterm labor with low birth weight (Adachi et al., 2016). Maternal transmission to neonate during birth may also cause infant pneumonia, otitis media, and inclusion conjunctivitis (Wise et al., 2015). Chlamydial infection in men can lead to orchitis, epididymitis, and urethritis (Dukers-Muijrers et al., 2016; Rowley et al., 2019). Furthermore, C. trachomatis infection is related to an increased risk of human immunodeficiency virus (HIV) infection and transmission (Khosropour et al., 2021). In addition, ocular infection with C. trachomatis can bring about trachoma, which is the leading cause of blindness worldwide (Pourabbas et al., 2018; Khosropour et al., 2021). Owing to the majority of patients being asymptomatic, making early diagnosis is difficult (Park et al., 2021). Developing a rapid and sensitive point-of-care (POC) testing for accurate screening of C. trachomatis infection is critical for earlier treatment to prevent transmission.

Traditional laboratory-based diagnosis of urogenital C. trachomatis infection was based on cell cultivation (Meyer, 2016). Isolating pathogens from cell cultures was considered as the initial gold standard for diagnosis of C. trachomatis (Meyer, 2016; Kelly et al., 2017). However, the sensitivity was low (ranging from 60 to 80%) and requires stringent conditions in specimen collection, transport, and cultivation (Peng et al., 2020). In addition, cell culture is labor-intensive and time-consuming (48–72 h). Hence, it is rarely performed in routine clinical diagnostic application. Nucleic acid amplification technologies (NAATs), including polymerase chain reaction (PCR) and ligase chain reaction, have been considered as a major breakthrough and have been regarded as the new gold standard for the detection of C. trachomatis infection for high sensitivity, specificity, and automation (Gaydos et al., 2013; Safarkar et al., 2017). Nevertheless, NAATs are often unaffordable and inaccessible in resource-limited regions due to requiring skilled technicians and expensive analytical instruments. Therefore, devising a cheap, rapid, simple, and accurate assay is critical to making an early diagnosis and follow-up antibiotic use during the initial stage of C. trachomatis infection.

Loop-mediated isothermal amplification (LAMP), an innovative nucleic acid isothermal amplification technique, was considered as an attractive alternative to traditional NAATs, which has potential applications as a POC testing for its simplicity, rapidity, and easy-to-operate (Notomi et al., 2000; Sahoo et al., 2016; Shirato, 2019). More importantly, it has been widely applied to diagnosis of various pathogens, such as SARS-CoV-2, Neisseria gonorrhoeae, and HIV (Rudolph et al., 2015; Chen et al., 2021a,b). LAMP amplicons can be analyzed with electrophoresis, turbidity, fluorescent dye, and colorimetric indicators (Li et al., 2017; Shirato, 2019). Nevertheless, all of these techniques require special facilities and reagents. To conveniently detect the LAMP products, gold nanoparticle-based lateral flow biosensor (LFB) is used as a paper-based and POC testing platform, which is currently of enormous potential for clinical applications due to its rapidness, visualization, simplicity, cost-saving, and portability of detection of molecular markers (Anfossi et al., 2019; Ye et al., 2020).

In this study, LAMP integrated with gold nanoparticle-based LFB (LAMP-LFB) was first developed for visual, rapid, specific, sensitive, portable, identification of C. trachomatis by targeting the ompA gene (Molano et al., 2018; Somboonna et al., 2018). The principle of C. trachomatis-LAMP-LFB is illustrated in Figure 1, and its feasibility was confirmed with clinical samples. The assay was completed with a high accuracy within 60 min. Therefore, the LAMP-LFB method can be regarded as a valuable POC assay for the detection of C. trachomatis infection, particularly in resource-scarce regions.

Genital infection with C. trachomatis is considered to be one of the most common bacterial STIs worldwide (Ramadhani et al., 2019; La Rosa et al., 2021). A distinguishing feature of C. trachomatis infection is that the patients are asymptomatic at the early stage of infection and usually delay or do not seek medical treatment, especially in less-developed regions, which brings about the continued transmission (Tadele et al., 2019; Caven and Carabeo, 2020). Hence, a rapid, accurate, cost-saving, and easy-to-operate assay for early diagnosis of C. trachomatis is critical for addressing ever-increasing C. trachomatis infection transmission rates. Here, a novel C. trachomatis-LAMP-LFB assay, which combined LAMP amplification with gold nanoparticle-based biosensor, was devised and applied successfully to the visual and rapid testing of C. trachomatis in clinical samples.

Traditional diagnostic techniques, including cell culture, enzyme immunoassay, and direct fluorescence assay, were used to diagnose C. trachomatis infection (Kelly et al., 2017; Peng et al., 2020). However, all of these tools cannot meet fully the requirement of early and easy-to-operate diagnosis in clinical application. Cell culture was regarded as the initial gold standard for C. trachomatis detection, but C. trachomatis require fastidious and demanding cultivation conditions, and the procedure of cell culture is time-consuming and labor-intensive (Herbst De Cortina et al., 2016). Therefore, it is now seldom utilized in clinical laboratory. Compared with cell cultivation, enzyme immunoassay and direct fluorescence assay are relatively simple and rapid, but are not recommended as routine diagnostic methods due to low diagnostic accuracy (Peng et al., 2020). Nucleic acid amplification tests are more sensitive and have an excellent specificity, which is recommended as the new gold diagnostic method for C. trachomatis detection (Herbst De Cortina et al., 2016; Safarkar et al., 2017). However, their use is confined due to requiring complicated laboratory facilities and trained technicians in resource-impoverished regions. In this study, the C. trachomatis-LAMP-LFB method only requires simple instruments, such as water bath, heating block, or even a thermos cup that can maintain a constant temperature (67°C). More importantly, the detection results can be visually read out with LFB. The whole diagnosis procedure, including genomic DNA releasing (~15 min), LAMP amplification (35 min), and result visual interpretation (<2 min), can be completed within 60 min.

In this study, LAMP was applied to amplify the target gene of C. trachomatis, which is a novel nucleic acid amplification technique devised by Notomi et al., usually 100-fold higher sensitivity than traditional PCR (Notomi et al., 2000; Avendaño and Patarroyo, 2020; Chaouch, 2021). A set of four or six primers spanning six or eight different regions of the target gene makes the amplification results highly specific. The primer set contains two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB) (Panno et al., 2020). Typically, four primers (F3, B3, FIP, and BIP) are enough to amplify a target fragment and improve its efficiency and specificity, while the two loop primers are added to the LAMP amplification system (Augustine et al., 2020; García-Bernalt Diego et al., 2021). In addition, a Bst DNA polymerase with chain displacement capability is critical for LAMP amplification at a fixed temperature (Notomi et al., 2000). Here, a set of C. trachomatis-LAMP primers were successfully designed for targeting 8 regions of ompA gene from 14 C. trachomatis serological variants (serovar A, B, C, D, E, F, G, H, I, J, K, L1, L2, L3). The specificity analysis confirmed that the C. trachomatis-LAMP-LFB assay can correctly identify the target pathogens and have no cross-reactions with no-C. trachomatis templates (Table 2; Supplementary Figure 2). Furthermore, the LoD of C. trachomatis-LAMP-LFB assay is as low as 50 copies/ml. To further confirm the feasibility of the assay in clinical application, 87 genomic DNA samples isolated from suspected C. trachomatis-infected patients were tested simultaneously with C. trachomatis-LAMP-LFB and qPCR. The data indicated that our assay is strengthened in detecting C. trachomatis-infected genital secretion samples (Table 3; Supplementar Table 1). To further verify its feasibility, much more low copy number of clinical samples should be collected and tested in the subsequent study. The lower detection rate of qPCR may be attributed to low copy number of the C. trachomatis DNA templates, and the qPCR detection was performed with a commercial real-time TaqMan PCR Kit (Da An Gene Co., Ltd. China). The concentrations of C. trachomatis < 500 copies/ml will be regarded as a negative outcome according to the illustrations of the manufacturer. In this study, three “negative” clinical specimens (C. trachomatis concentrations ranging from 300 to 500 copies/ml) presented positive results using C. trachomatis-LAMP-LFB, which further verified that the sensitivity of our assay is higher than the traditional PCR technique.

For visual and rapid presentation of C. trachomatis-LAMP results, gold nanoparticle-based LFB was used in this assay system. LFB is a paper-based device that has been extensively used in the diagnosis of infectious diseases, foodborne pathogens, cancer biomarkers, and cardiovascular disease, owing to its low-cost, rapid detection with good robustness, specificity, sensitivity, and easy-to-operate (Hsieh et al., 2017; Huang et al., 2020; Wang et al., 2021). Here, the LFB strips were immobilized with anti-FAM and BSA-biotin on TL and CL, respectively. For positive outcomes, the FAM/biotin-labeled C. trachomatis-LAMP amplicons were arrested by anti-FAM at the TL and the streptavidin-DPNs were captured by biotin-BSA at the CL, respectively. For negative results, only the streptavidin-DPNs were arrested by biotin-BSA at the CL. Although the MG regents and real-time turbidity were able to test the C. trachomatis-LAMP results in this study, the former is ambiguous when LAMP amplicon concentrations were low (Figure 6C), and the latter technique requires expensive apparatus. The LFB method is simple and cost-effective (~US$2.0 for each strip). Hence, the overall cost of each C. trachomatis-LAMP-LFB test, including DNA template extraction (~US$1.0), LAMP reaction (~US$3.0), and LFB readout (~US$2.0), was calculated at US$6.0. In previous studies, LAMP has already been used to identify C. trachomatis. Choopara et al. (2017) and Somboonna and Choopara (2019) combined LAMP with hydroxynaphthol blue for visual color detection of C. trachomatis by the naked eye, but the results of this assay were ambiguous when LAMP product concentrations were low. Dean et al. (2021) have also utilized the LAMP assay for testing C. trachomatis, and the C. trachomatis-LAMP amplification products were examined with colorimetric chemistry, which must rely on optical detection instrumentation. In this study, we first combined LAMP amplification with LFB for the identification of C. trachomatis, which is more convenient and rapid than other methods.

The C. trachomatis-LAMP-LFB also has some shortcomings. First, there is a risk of carryover contamination; the LAMP reaction tube must be taken off for LFB identification. Spraying a 10–15% sodium hypochlorite solution and 70% ethanol after LFB detection is an effective way of avoiding nucleic acid contamination in laboratory. In this study, no false-positive outcomes were observed in non-C. trachomatis templates. It is indicated that the cross-contamination can be effectively controlled in our laboratory. Secondly, our assay can be used for qualitative detection of C. trachomatis, but not measurement of the concentrations of C. trachomatis in sample; the quantitative determination of LAMP-LFB could be considered for further study in the future. Thirdly, the C. trachomatis-LAMP-LFB system just detected the ompA gene from 14 C. trachomatis serological variants (serovar A-K, L1, L2, L3) and neglected the internal control gene. For better fitting the clinical application, the C. trachomatis-LAMP amplification system should add a set of LAMP primers based on the internal control gene (human gene) for avoiding false-negative results. The LFB has designed three lines, including CL, internal control line, and TL, which can effectively avoid false-positive results.

In conclusion, we combined a LAMP reaction with LFB readout platform for successfully devising a novel C. trachomatis-LAMP-LFB for the rapid, visual, cost-effective, specific, and sensitive identification of C. trachomatis agent in clinical samples. The LoD of our assay was 50 copies/ml and had no cross-reaction with non-C. trachomatis pathogens. The whole assay process can be completed within 60 min without any special instrument. Therefore, the C. trachomatis-LAMP-LFB shows great potential as POC testing for C. trachomatis screening and detection in clinical settings, especially in resource-starved regions.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The study was approved by the Human Ethics Committee of Hangzhou Women's Hospital (Approval No. [2021]-K (2)-8) and complied with the Declaration of Helsinki. Before clinical samples/isolates were obtained, all personal patient identifiers were removed. Patients' informed consent was waived by the committee.

XC and SD designed and conceived this study and drafted and revised the manuscript. XC, QZ, YT, RW, XW, JL, RL, SW, and SD collected clinical samples and performed the experiments. XC, QZ, and SD analyzed the data. All authors read and approved the final manuscript.

This study was funded by the Program of Scientific and Technological Project in Guizhou Province (Grant No. Qian Ke He [2020]4Y184), the Scientific and Technological in Guiyang City (Grant No. Zhu Ke He [2020]-10-5), and the Public Welfare Technology Research Program in Zhejiang Province (Grant No. LGF21H190001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.