Over the past five decades, several seroassays have been developed to detect IgA, IgM, and IgG anti-CT serum antibodies. These assays are not recommended for clinical diagnoses of CT infections, and commercial versions of these assays are not approved by regulatory bodies such as the United States Food and Drug Administration. Additionally, use of these assays outside of epidemiologic CT studies is fairly limited, although some argue that seroassays may be useful when evaluating patients with suspected PID[17].

To identify CT seroassays to include in this review, we reviewed English-language publications that either (1) described the development and validation of CT seroassays, (2) compared various types of CT seroassays, or (3) estimated seroprevalence of antibodies to CT and/or the association of seroprevalence of antibodies with adverse reproductive health outcomes in a population. We searched PubMed and Google between October 1, 2021, and August 1, 2022, using the search terms “chlamydia serological assays”, “Chlamydia trachomatis serological assays”, and “Chlamydia trachomatis serology”. We reviewed references from published papers yielded in this search to identify papers that may have been missed in the initial search.

We identified 26 distinct types of validated CT seroassays (further sub-divided into 28 commercially-available versions and 25 non-commercial versions of these assays) that have been reported in 55 publications. A full description of the function, validation, and strengths and weaknesses of these assays is provided in Supplementary Table 1. Because the goal of this review is to focus on CT seroassays that have been used in epidemiologic studies, we provide no further information on the CT seroassays that have been developed and validated but not applied in an epidemiologic context. From the 26 types of assays that we initially identified, 10 have been used or are currently being used in epidemiologic studies to measure seroprevalence of anti-CT antibodies in various populations or the association between seroprevalence and other health outcomes. The remainder of this narrative review focuses on these 10 seroassays (described in detail in Table 1): the microimmunofluorescence assay (MIF), the whole cell inclusion immunofluorescence assay (WIF), the major outer membrane protein ELISA (MOMP ELISA), the Heat Shock Protein 60 ELISA (cHSP60 ELISA), the lipopolysaccharide recombinant ELISA (LPS rELISA), Plasmid Gene Protein 3 ELISA (Pgp3 ELISA), the Luminex MAGPIX multiplex bead array (MBA), the elementary body ELISA (EB ELISA), the mixed peptide ELISA, and the Pgp3 luciferase immunosorbent assay (Pgp3 LISA).

The MIF, WIF, and MBA use immunofluorescence for the detection of antibodies to CT. The MIF was developed by Wang and Grayston in the early 1970s[18] and has historically been the “gold standard” of CT seroassays[19–26]. Many versions use CT elementary bodies, primarily formalin-fixed outer membrane protein A (OmpA) as the antigen on glass slides to detect IgG and IgM antibodies to CT, but other antigen preparations may also be used, including those to detect IgA[23,27,28]. Similar to the MIF, the WIF assay uses fluorescence to detect IgG and IgM antibodies to CT LPS IgG and MOMP, respectively, and in contrast to the MIF, the WIF uses the entire CT inclusion as antigen rather than elementary bodies[29]. The MBA also uses fluorescence to detect IgG antibodies[30,31].

The Pgp3 LISA uses a luciferase immunoprecipitation system[10], where the presence of IgG antibody is detected via luminescence[32]. The remaining assays that we focus on are classified as ELISAs, which use colorimetric substrates and enzyme amplification to detect antibodies[30]. In terms of antibody detection, the MOMP ELISA uses major outer membrane protein, which is encoded by the ompA gene of CT[33] and has been used to detect IgG, IgM, or IgA antibodies. The LPS rELISA[21], EB ELISA[16,34–37], and the mixed peptide ELISA[11] can be used to detect IgG and IgA CT antibodies, while the cHSP60 ELISA[19,20,38,39] and the Pgp3 ELISA[19,26,40] exclusively detect IgG in a manner similar to the MBA.

There is currently no agreed-upon reference standard to evaluate CT seroassays. Most assays included in this review used NAAT at the time serum was drawn as a reference standard for validation. However, using NAAT as a reference standard may not be appropriate when the goal is to estimate history of CT infection. Using NAAT as a comparison indicates whether the assay is sensitive and specific at detecting antibodies during a current infection, but not whether it is a valid assay to detect antibodies from a prior infection. The MIF has also been used as a reference standard for validating several CT seroassays[19,20,22,23,25,26]. However, due to the lower sensitivity of the MIF in comparison to newer CT seroassays such as the MOMP and mixed peptide ELISAs as well as the Pgp3 LISA and MBA, using the MIF alone may no longer be the best choice for a reference standard.

Despite the lack of a reference standard for CT seroassays, the seroassays we reviewed provided published estimates of “sensitivity” and “specificity”. Although this terminology may not be accurate (i.e., true sensitivity would be a measure of how many individuals were seropositive of all those infected, which is unknown), these published values permit comparisons across assays that used the same reference (e.g., NAAT). Here, we present these values as positive percent agreement (PPA) and negative percent agreement (NPA), which represent the percent of people with current infection who are seropositive and percent without current infection who are seronegative, respectively. In Table 1 we report the mean and range of these percentages for each assay in the detection of IgG when NAAT was used as the reference standard (except where otherwise noted, see footnotes). The mean PPA across the assays ranged from 52.4% to 100%. The mean NPA ranged from 5.9% to 100%. A detailed description of the populations included in these validation studies and the raw sensitivity and specificity values for assays with multiple validation studies where we present means and ranges are provided in Supplementary Table 1.

The assays with mean PPA >80% were the WIF, LPS rELISA, MBA, mixed peptide ELISA, and the Pgp3 LISA, with the highest PPA (~93% or higher) noted for the WIF, MBA, and the Pgp3 LISA. A mean NPA of 80% or higher was noted for all seroassays except the WIF, LPS rELISA, and the MBA. The EB ELISA, mixed peptide ELISA, and the Pgp3 LISA all reported a mean NPA >98%. However, these results for the WIF, Pgp3 ELISA, MBA, mixed peptide ELISA, and the Pgp3 LISA are each based on only one validation study. Additionally, the validation study for the WIF tested IgG, IgM, and IgA together while the validation studies for the other assay types only tested IgG. Although comparing the performance of different assays is difficult due to inconsistent reference standards used, there have been improvements in these agreements between NAAT results and these assays in recent years. Most notably, the mixed peptide ELISA[11] (composite reference standard of commercial seroassays) and the Pgp3 LISA (NAAT as reference)[10] both have a PPA >85% and NPA >98%.

Table 1 details how CT seroassays have been applied in epidemiologic research. The assays with commercially-available versions have been used in epidemiologic studies considerably more often than laboratory-developed assays. The MIF has been used to estimate CT seroprevalence among select populations in the Netherlands[41–43], Japan[44,45], and Jamaica[46], and an in-house version of the WIF examined CT seroprevalence among select populations in Finland[47]. Laboratory-developed EB ELISAs[16] have been used to measure CT prevalence from key populations in the US[37], and have been used to examine the association between CT seropositivity and gastroschisis[36], pregnancy outcomes[37], and tubal factor infertility (TFI)[48]. The MOMP ELISA has been applied to explore the correlation between seroprevalence of anti-CT antibodies and subfertility or infertility in females in the Netherlands[43], Rwanda[49], Samoa[50], and Iran[51s]. The one commercial version of the cHSP60 ELISA was used among females who were subfertile, infertile, had TFI, or had a male partner who was infertile[41,50,52s,53s]. The Pgp3 ELISA has measured seroprevalence in population-based samples of women in the United Kingdom[54s] and the U.S.[55s] and to predict the CT-attributable population fraction of TFI by race in U.S. females[56s]. The Pgp3 LISA was applied to estimate seroprevalence of antibodies to CT in adults in Northern China[10]. The single commercial version of the LPS rELISA was applied in Germany to measure the association between CT seropositivity and infertility in males[21]. The mixed peptide ELISA is currently being used to estimate the lifetime prevalence of CT in U.S. men and characterize factors associated with recent versus past infection[57s].

There are substantial differences between these assays regarding their ease of implementation and reproducibility. In general, MIFs are harder to implement and less reproducible. The MIF requires subjective microscopic interpretation which makes it more labor-intensive than ELISAs, and this subjective interpretation can impact reproducibility[26]. Across all the assays, the availability of commercial versions generally allows for easier use and greater reliability. When no commercial version is available, labs must recreate the assays themselves based on the protocols of other research groups. The MIF, WIF, EB ELISA, MOMP ELISA, cHSP60 ELISA, LPS rELISA, and the MBA all have at least one commercial version available, while the mixed peptide ELISA, Pgp3 ELISA, and the Pgp3 LISA do not. In summary, the use of ELISAs and newer techniques like LISA and MBA may be less labor-intensive and more reproducible than older assays[58s].

When designing studies and interpreting results, researchers should consider what is currently known about antibody isotypes and the timing of antibody development.[14–16,59s,60s].The majority of people (61–90%) appear to experience IgG seroconversion within 3 months of a positive NAAT result, though a small proportion will develop antibodies between 3 months and several years after an initial positive NAAT.[14–16] This wide range of estimates of the timing of seroconversion is likely due to several factors, including host genotype[61s,62s], number of previous infections[15,61s], and uncertainty about when an individual actually acquired a CT infection versus when they first tested positive. Another unknown is antibody persistence, which may vary based on the anatomic site of infection. Among children likely exposed to ocular CT, anti-CT IgG levels have been shown to remain stable for 3 years[12]. Öhman and colleagues found that the proportion of people with IgG antibodies from urogenital CT declined from 65.5% to 34.5% 3–10 years after baseline[14], and Alexiou and colleagues found that only 42% of women who were IgG seropositive at the time of a positive NAAT for a urogenital CT infection were still positive 6 years later[60s]. For IgA, one study found that anti-CT IgA seroprevalence declined from 73% to 61% within 6 months of a positive NAAT[16]. Another found that 32% of female participants who were positive for IgA at the time of a positive NAAT no longer had detectable IgA 20–400 days post-NAAT[13].

Investigators using CT seroassays to estimate CT prevalence should take care to understand the timing of serum collection relative to when an individual may have been exposed and/or infected with CT. Due to the variability in timing of seroconversion across individuals, if serum is drawn from participants too proximal to when they acquired an infection, they may not have developed antibodies yet (even if they test positive for CT by NAAT)[14–16]. Likewise, if serum is drawn from participants several years after their infection, it is possible that no serum antibodies would be detected. In both of these situations, there is a high likelihood of underestimating CT seroprevalence, since participants would be misclassified as never being infected with CT when they truly were.

Additionally, the ability to establish timing of infection is a desired attribute when using these seroassays in epidemiologic studies. Although none of the assays can reliably estimate timing of infection, the mixed peptide ELISA is the only assay that we focus on with the purported ability to distinguish between past and recent infection[11]. Rahman and coauthors suggest that this may be possible by testing serum for IgG1 and IgG3, with the presence of IgG3 indicating a recent infection due to fairly quick seroconversion and seroreversion[34].

When selecting a CT seroassay for use in epidemiologic research, assays that have cross-reactivity with other Chlamydia species should be avoided if possible. This is especially relevant when working with populations that may have been exposed to C. pneumoniae or other Chlamydia species. This cross-reactivity is due to the highly conserved genomes of members of the Chlamydia genus[63s]. The MIF, WIF[26], LPS rELISA[21], and some versions of the MOMP ELISA are cross-reactive with Chlamydia pneumoniae as shown in Table 1[20]. Other versions of the MOMP ELISA and the cHSP60 ELISA, the Pgp3 ELISA, and the Pgp3 LISA have little cross-reactivity with C. pneumoniae, but they are cross-reactive with other Chlamydia species that cause zoonotic diseases such as C. psittaci[20]. Notably, the mixed peptide ELISA[34] and MBA[64s] have little to no cross-reactivity with other human and veterinary Chlamydia species, including species such as C. suis and C. avium[65s] in addition to C. pneumoniae and C. psittaci[11]. Assays developed after 2008 tend to be less cross-reactive with other Chlamydia species compared to assays developed earlier, and thus recent epidemiologic studies have used these newer assays.

An additional challenge in using these seroassays to measure CT seroprevalence is that they do not provide information about the anatomic site of infection. Most studies of CT seroprevalence and implementation of CT seroassays have focused on urogenital or ocular CT infections, but there is growing recognition that rectal CT infections are common in both males and females[66s–69s]. Researchers attempting to distinguish between past infections at different anatomic sites may choose to pair serology data with sexual history data to understand which anatomic sites may have exposed prior to drawing conclusions about infections at the urogenital site. This may be of particular relevance to populations where ocular CT infections are endemic. In these populations, a positive CT serology result may not necessarily indicate a CT infection in the genital tract, and studies that aim to examine the association between CT and adverse reproductive health outcomes in trachoma-endemic areas should interpret their results with this limitation in mind[70s].