Clinical Presentation of Early Syphilis and Genomic Sequences of Treponema pallidum Strains in Patient Specimens and Isolates Obtained by Rabbit Inoculation

Abstract

Background

The global resurgence of syphilis necessitates vaccine development.

Methods

We collected ulcer exudates and blood from 17 participants with primary syphilis (PS) and skin biopsies and blood from 51 patients with secondary syphilis (SS) in Guangzhou, China, for Treponema pallidum subsp pallidum (TPA) quantitative polymerase chain reaction, whole genome sequencing (WGS), and isolation of TPA in rabbits.

Results

TPA DNA was detected in 15 of 17 ulcer exudates and 3 of 17 blood PS specimens. TPA DNA was detected in 50 of 51 SS skin biopsies and 27 of 51 blood specimens. TPA was isolated from 47 rabbits with success rates of 71% (12/17) and 69% (35/51), respectively, from ulcer exudates and SS bloods. We obtained paired genomic sequences from 24 clinical samples and corresponding rabbit isolates. Six SS14- and 2 Nichols-clade genome pairs contained rare discordances. Forty-one of the 51 unique TPA genomes clustered within SS14 subgroups largely from East Asia, while 10 fell into Nichols C and E subgroups.

Conclusions

Our TPA detection rate was high from PS ulcer exudates and SS skin biopsies and over 50% from SS blood, with TPA isolation in more than two-thirds of samples. Our results support the use of WGS from rabbit isolates to inform vaccine development.

Graphical Abstract

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During the past 20 years, syphilis, caused by Treponema pallidum subspecies pallidum (TPA), has resurged as a global public health problem [1]. In China, 57 196 new syphilis cases (12.7% of all cases) were reported in Guangdong Province in 2021 [2].

Whole genome sequencing (WGS) of TPA genomes from geographically diverse regions is essential for development of a syphilis vaccine with global efficacy. At present, most TPA genomic sequences have originated from Europe, North America, and Australia [3–8]; fewer Asian strains have been sequenced [3, 9]. Most clinical specimens used for WGS have been primary syphilis (PS) ulcer swabs, samples that typically contain high TPA burdens. Skin biopsies and whole blood (WB) from patients with secondary syphilis (SS) are other potential sources of spirochete genomes but have considerably lower TPA burdens than ulcer exudates [10–12]. Rabbit infectivity testing (RIT) allows strain recovery from samples with low TPA burdens [13], but it is unclear whether the genomes of treponemes obtained by RIT accurately reflect those in the corresponding clinical specimens.

We recruited patients with PS and SS from Guangzhou, China, as part of a parent project for syphilis vaccine development [14]. We collected ulcer exudates and WB from patients with PS and skin biopsy and WB from patients with SS for quantitative polymerase chain reaction (qPCR), WGS, and RIT. In addition to characterizing the clinical presentation of early syphilis in our patient population, we aimed to (i) compare TPA qPCR and RIT results between specimen types, (ii) compare TPA WGS results from paired clinical samples and TPA rabbit isolates, and (iii) phylogenetically analyze TPA strains to aid in the design and development of syphilis vaccines.

MATERIALS AND METHODS

Ethics Statements

This research was approved by the Medical Ethics Committee Dermatology Hospital of Southern Medical University (SMU; GDDHLS-20181202) and the Institutional Review Board (IRB) of the University of North Carolina at Chapel Hill (UNC; IRB protocol 19-0311) [14]. Rabbit experimentation was approved by the Ethics Committee of Dermatology Hospital of SMU (GDDHLS-20181202,12/12/2018) and South China Agricultural University (SCAU) Experimental Animal Ethics Committee (2020C004, 07/05/2020). Rabbits were housed under approved conditions at SCAU.

Participant Recruitment, Diagnosis, and Management

Patients visiting the sexually transmitted infections (STIs) department of the Dermatology Hospital of SMU from December 2019 to March 2022 were recruited (Figure 1). Individuals aged ≥18 years diagnosed with PS or SS were eligible for enrollment. Diagnosis of PS was based on the presence of 1 or more anogenital ulcers with a positive darkfield microscopy (DFM) and/or reactive nontreponemal (toluidine red unheated serum test [TRUST]) and treponemal (T pallidum particle agglutination [TPPA]) tests [15, 16]. Diagnosis of SS was based on characteristic skin or mucosal lesions plus reactive TRUST and TPPA tests [15, 16].

Study participants received standard care including physical examination and treatment with 2.4 million units (MU) of intramuscular benzathine penicillin G weekly for 2 consecutive weeks (total of 4.8 MU) in accordance with Chinese Center for Disease Control and Prevention treatment guidelines [16]. Patients were asked to return within 90 days for a follow-up examination and repeat TRUST titers; TPPA testing was repeated if the initial test was negative.

Data and Specimen Collection

A case report form was used to collect demographic and clinical data, including sexual orientation, sexual and medical histories, results of physical examination, and laboratory findings [14]. For patients with PS, we recorded the number and location of ulcers. For patients with SS, we recorded the type and location of rash and other mucocutaneous manifestations. For rashes, we estimated body surface area of involvement (BSA) score, a disease outcome predictor used for other skin diseases [17].

From PS participants, we collected ulcer exudates for DFM, DNA extraction, and RIT (Figure 1). The first swab was used for DFM; the residual of the first swab plus a second swab were placed into 1 mL of TpCM-2 medium [18] for RIT [13]. The third and fourth swabs were placed in tubes containing DNA/RNA shield (Zymo Research, Irvine, California; R1100–250) for DNA extraction. WB specimens were also collected from participants with PS. From participants with SS, two 4-mm punch skin biopsies were obtained from rashes and stored in DNA/RNA shield. WB specimens were also collected from SS participants for DNA extraction and RIT (Figure 1).

Assessment of T pallidum Burdens by qPCR

DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions; 100 μL of nuclease-free water was used to elute the DNA, followed by −80°C storage. TaqMan PCR for TPA polA was performed as described by Chen et al [19] using 1.25 μL of 10 μM primers, and measured on real-time PCR instruments (Bio-Rad CFX96, Hercules, California).

Rabbit Infectivity Testing

RIT was performed as previously described [13]. Adult New Zealand White rabbits (males, 3 months of age, 2.5–3 kg) were prescreened to confirm nonreactive syphilis serologies. Freshly obtained genital ulcer exudate from participants, diluted with 1 mL of TpCM-2 medium or 1 mL of freshly collected WB, was injected into each testis. The serologic status of the rabbits was monitored weekly by TPPA beginning with the second month after inoculation. Seroconverted rabbits were euthanized, and their testes were aseptically removed for TPA isolation. When the testicular extract from the seroconverted rabbit was negative by DFM, the extract was serially passaged once or twice to “amplify” the isolate for WGS and banking. Seronegative rabbits were euthanized after 3 months followed by passage of testicular extract to a second rabbit to confirm lack of infection.

WGS and Bioinformatic Analysis

Whole genome sequencing was carried out on ulcer exudates from individuals with PS, skin biopsy specimens from patients with SS, and paired rabbit-passaged isolates (Figure 1) using custom 120-nucleotide RNA oligonucleotide baits obtained from Agilent Technologies (Santa Clara, California). TPA enrichment was performed using the Sure Select XT Low Input kit at UNC and the Sure Select XTHS2 kit at SMU, following the previously established protocol [9]. The resulting TPA-enriched libraries were sequenced using the Illumina MiSeq platform at UNC or the NovaSeq platform at SMU, generating paired-end, 150-bp reads. The raw sequencing data, with residual human reads removed, have been deposited at the Sequence Read Archive database (PRJNA815321). Fifty-one TPA genomes from China included in this manuscript were reported in a separate study [14].

The sequencing reads and publicly available data were processed using a conservative bioinformatic pipeline with minor modifications [9], available at https://github.com/IDEELResearch/tpallidum_genomics. Only sequences with at least 80% of their genome covered 3× were retained for variant calling. Variant calling was performed with GATK HaplotypeCaller [20] with joint genotyping using GenotypeVCFs module and hard filtering by VariantFiltration.

Comparison of paired TPA genomes (direct sequencing from clinical samples vs rabbit isolates) was conducted manually using a VCF file obtained from joint genotyping, applying strict filtering parameters (depth ≥8×, genotype quality ≥20, and variant allele frequency [VAF] ≥0.85). Variant genes (eg, tprK) were considered heterogeneous if the proportion of reads supporting less frequent alleles was >15% (ie, VAF between 0.15 and 0.85). To generate a multiple alignment using the MAFFT (v7.520) [21], we combined 51 Nichols- and SS14-clade consensus sequences generated in this study, 109 published TPA genomes, and 2 outgroup genomes (T pallidum subsp endemicum Bosnia A and T pallidum subsp pertenue Samoa D). The resulting alignment was then inputted into Gubbins (v3.2.1) [22], which processed the alignment with default parameters for predicting and masking homologous recombination regions. A phylogenetic tree was constructed under the GTRGAMMA model with 1000 bootstrap replicates by Gubbins (v3.2.1), which then was visualized using the ggtree package in R software [23]. The genotypic resistance of macrolide was analyzed by competitive mapping as previously described by Beale et al [4, 24].

Statistical Analysis

Descriptive statistics were used to assess participant characteristics. All data were analyzed using SPSS 22.0 (IBM SPSS Statistics).

RESULTS

Sociodemographic Data, Clinical Characteristics, and Laboratory Findings

From December 2019 to March 2022, 154 individuals were screened, 68 of whom (17 PS and 51 SS) consented to enrollment (Figure 1 and Table 1); 7 participants were excluded due to errors in the informed consent process. Among enrolled participants, the median age was 27 (interquartile range [IQR], 23–33) years, 84% were male, and 33% were men who have sex with men or men who have sex with men and women. All but 1 participant was HIV negative, 9% had a history of prior syphilis, and 22% had a history of other STIs.

Sixteen of the 17 participants with PS were male, with the majority (59%) presenting with solitary ulcers (Table 2 and Figure 2A). Spirochetes were visualized by DFM in 12 of the 17 (71%) patients with exudative lesions. The TRUST and TPPA tests were positive in 7 (41%) and 15 (88%) patients with PS, respectively. TPA DNA was detected in 15 of 17 (88%) ulcer exudates and 3 of 17 (18%) PS blood specimens. TPA polA copy numbers ranged from 1.43 to 2250 copies/μL in ulcer exudates and 0.288 to 37.99 copies/μL in whole blood (Supplementary Table 1). All 12 DFM-positive ulcer specimens were positive by TPA PCR (Supplementary Table 1). TPA DNA was detected in ulcer exudates and WB in 2 PS cases with negative TRUST and TPPA results (Supplementary Table 1).

Forty-two of the 51 (82%) SS participants presented with a rash only; 8 (16%) presented with rash and other manifestations (Table 2 and Figure 2BitalicD). BSA scores were <3% in 15 participants (30%), 3%–10% in 10 (19%), and >10% in 26 (51%). All SS participants had reactive TRUST and TPPA tests. TPA DNA was detected in 50 of 51 (98%) skin biopsies and 27 of 51 (53%) WB specimens. TPA polA copy numbers ranged from 0.610 to 10 200 copies/μL in skin biopsies and 0.110 to 3850 copies/μL in blood (Supplementary Table 2).

Isolation of T pallidum by Rabbit Inoculation

We inoculated New Zealand White rabbits with 17 ulcer exudates from patients with PS and 51 WB specimens from patients with SS (Figure 1). Fifty rabbits (74%) developed reactive TPPA tests, which included 14 of 17 (82%) rabbits inoculated with ulcer exudates and 36 of 51 (71%) inoculated with blood (Table 3). TPA strains were isolated from 47 of the 50 rabbits that TPPA seroconverted—12 of 17 (71%) from ulcer exudates and 35 of 51 (69%) from SS blood samples. Thirty strains were isolated from the first inoculation—11 of 30 from ulcer exudates and 19 of 30 from SS WB. Eleven strains were isolated following a second passage (1/11 from ulcer exudates and 10/11 from SS WB). Six strains isolated from SS WB required a third passage. One of 2 rabbits inoculated with PCR-negative exudates and 15 of 24 rabbits inoculated with PCR-negative blood samples yielded isolates. Conversely, RIT was negative in 2 of 15 PCR-positive ulcer exudates and 7 of 27 PCR-positive blood specimens. TPA polA copy numbers in the 2 PCR-positive, RIT-negative ulcer exudates were comparable to those in PCR-positive, RIT-positive samples (Supplementary Table 1). In contrast, polA copy numbers were at the low end in 5 of the 7 PCR-positive, RIT-negative blood samples (Supplementary Table 2).

Comparison of TPA Genome Sequences From Paired Clinical Samples and Rabbit Isolates

Our genomic dataset consisted of 75 genomes from 51 of the 68 participants (Figure 1). These included (i) 24 paired TPA genomic sequences—5 from PS ulcer exudates and TPA strains recovered from the exudates (n = 10) and 19 from SS skin biopsies and TPA strains recovered from the corresponding patient blood samples (n = 38) (Table 4); (ii) 23 unpaired genomes from rabbit isolates (7 ulcer exudates and 16 SS blood samples); and (iii) 4 unpaired genomes from SS skin biopsies (see Supplementary Table 3 for complete details).

Before proceeding to phylogenetic analysis (see below), we first compared the 24 paired genomes to ascertain whether WGS from rabbit-passaged isolates accurately reflected those obtained directly from clinical specimens. Twenty-two of the 24 paired genomes were identified as SS14 lineage, while the remaining 2 were Nichols lineage (Table 4). Within the SS14-lineage group, we observed 5 discordant variants at 3 specific genomic sites when we applied the rigorous variant filtering criteria described in the Methods (Supplementary Table 4). Two mutations are in genes (tp0179 and tp0618) encoding hypothetical proteins. The third, found in rabbit isolates from 3 patients, occurred in the gene encoding an outer membrane factor (TP0967) for an efflux pump [25]. Our structural model predicts that this mutation adds 3 glycine residues to a polyglycine tract (G152 to G161) in extracellular loop 1 (ECL1) adjacent to an aspartic acid residue (D162) in β-strand 2 (Supplementary Figure 1); this insertion would not be expected to affect antigenicity or function. With manual inspection, we found 3 additional discrepant heterogenous variants (including 1 single-nucleotide polymorphism and 2 indels) in tprK (Supplementary Table 5). Within the 2 paired Nichols lineage genomes, we identified a total of 28 discrepant variations, all located exclusively within tprK (Supplementary Table 6). Two of these discordant variants were found in 1 of the paired genomes, while the remaining 26 discordant variants were present in the other (Supplementary Table 6). Due to the complexity of the tprK locus and the limitations of short-read sequencing, we did not go deeper to uncover additional possible tprK variants. Overall, the results clearly showed that rabbit passage has minimal effects on TPA genomic stability.

Phylogenetic Diversity of TPA Strains From Guangzhou

We constructed a phylogenetic tree to assess the diversity of the newly sequenced TPA strains (Figure 3). For the paired genomes, we used the sequences from the clinical samples; when clinical samples were unavailable, we utilized genomes from isolates. The final analyzed dataset consisted of 51 unique TPA genomes from 51 participants—28 from clinical samples (24 paired, 4 unpaired) and 23 from unpaired rabbit isolates described above (Figure 1). We also included 109 previously published TPA genomes, including 28 TPA strains from Japan and 22 others available from China up to the present date. Additionally, our analysis included outgroup genomes from Treponema pallidum subsp endemicum Bosnia A and Treponema pallidum subsp pertenue Samoa D (Figure 3). The 51 newly sequenced Chinese strains fell into 3 subgroups: 41 clustered within an SS14 subgroup largely comprised of genomes from East Asia, 8 were classified into the Nichols C subgroup, and 2 were assigned to the Nichols E subgroup.

TPA strains harboring mutations in the 23S rRNA gene at positions A2058G or A2059G are associated with genotypic resistance to macrolides [24, 29]. Forty-seven of the newly sequenced Chinese TPA strains exhibited genotypic macrolide resistance, predominantly through the A2058G allele mutation, while 4 strains harbored genotypic macrolide resistance with A2059G allele mutation (Figure 3).

DISCUSSION

We enrolled participants who presented with a wide range of clinical manifestations of early syphilis, allowing for multiple specimen types for TPA molecular detection and WGS. In addition, we utilized RIT, a traditional, highly specialized syphilis research tool, for a purpose of direct relevance to vaccine development—obtaining isolates for WGS from blood. The TPA genome sequences from our site with accompanying clinical data have provided new insights into the syphilis epidemic in Guangzhou, paving the way for molecular epidemiological studies that can synergize with future vaccine strategies.

We observed several interesting clinical findings. Forty-one percent of our participants with PS presented with multiple ulcers in the anogenital area, although single ulcers are typical [30, 31]; more than half of all participants with PS had negative TRUST titers, underscoring the importance of direct detection for diagnosis. We found the sensitivity of DFM for PS to be 71%, within the reported range of 70%–100% [12, 32, 33]. Among our participants with SS, we noted that more than half had >10% BSA scores, indicative of widespread TPA dissemination. Rashes on the palms and soles are often indicative of a secondary syphilis diagnosis, reported in 40%–80% of cases in the literature [34, 35]; less than half of our participants, however, had these typical findings.

We found that the sensitivity of polA PCR was 88% for chancres and 18% for WB from PS participants, compared to 98% for skin biopsies and 53% for blood from SS participants. The sensitivity of PCR for SS blood in the current study (53%) is similar to the value (47%) we reported previously [10]. The limited sensitivity of molecular assays for detection of TPA in blood of early syphilis patients is well recognized [12, 36]. Although our PCR sensitivity for WB was higher than in some reports [12, 36, 37], the overall low detection rates are likely explained by the low concentration of treponemes circulating in blood and the small volumes of specimens used for PCR testing [38]. The high PCR positivity for SS skin biopsies supports its use for diagnosis if it can be performed.

RIT has long been regarded as the gold standard for detection of small numbers of live treponemes in clinical specimens [13, 39]. While a variety of specimens have been successfully inoculated into rabbits over the years [13, 39], studies of acquired syphilis in the antibiotic era have largely focused on cerebrospinal fluid to identify patients with asymptomatic neurosyphilis [40, 41]. We isolated TPA in some rabbits inoculated with PCR-negative blood, emphasizing the extraordinary sensitivity of RIT. The differences in sensitivity between RIT and PCR may reflect the volume of whole blood used for RIT versus TPA qPCR. On the other hand, we also noted that specimens with detectable TPA DNA were not uniformly RIT-positive. Treponemal burdens at or near the minimal infectious inoculum of individual TPA strains may partially explain these discordant results [42], while the presence of TPA DNA does not always reflect viable treponemes [43]. Moreover, clinical strains often do not readily adapt to rabbits [44]. Indeed, as shown herein, amplification of the initial inoculum by serial rabbit passage is often required. We also noted that organisms may not be recoverable from rabbits that became infected based on conversion of their TPPA tests.

A critical question in the use of RIT for TPA enrichment before WGS is whether rabbit passage induces genomic alterations in the parental strain harbored by the inoculated clinical sample. We addressed this issue by performing the first large-scale comparison of genomic sequences from matched clinical samples and rabbit isolates. The results showed that rabbit passage has minimal effects on TPA genomic stability, although our analysis excluded some regions of the genome that could not be confidently resolved using short-read sequencing. The genomes of all 22 recovered SS14 clade strains were essentially identical to their clinical counterparts, while variation in the 2 Nichols-clade isolates was confined to their tprK loci. Evidence from WGS for recombination in TPA strains [45, 46] implies that coinfection must occasionally occur, presumably in genital ulcers where high treponemal burdens would maximize the probability for genetic exchange. However, in all 24 paired cases, genomes in clinical samples and rabbit isolates clearly derived from the same TPA strain.

The newly sequenced TPA genomes were found to cluster within the Nichols C and E subgroups and an SS14 subgroup largely comprised of genomes from East Asia. Interestingly, although 3 previously reported Chinese SS14 clade strains were found to cluster in the SS14-Omega subgroup [9], the vast majority of Chinese TPA strains published to date belonged to the SS14-Omega East Asian subgroup [26, 27, 47]. Furthermore, the Chinese TPA strains in both the SS14 and Nichols lineages showed a high degree of relatedness to publicly available Japanese TPA strains. Notably, genotypic resistance to macrolides was observed in all newly sequenced strains, indicating that azithromycin should not be considered as an alternative drug for treating syphilis patients in China.

There were several limitations to our study. One is the small sample size from a single clinical site in China. Nevertheless, we believe our population is representative of patients presenting to a provincial urban hospital in a large city with a broad catchment area. We did not compare genomes from paired PS ulcer exudates and blood samples; however, such a comparison would be challenging and expensive given the low TPA copy numbers and rates of spirochetemia in patients with PS. Moreover, there is no obvious reason why this would yield a different conclusion from our comparison of genomes from skin biopsies and blood isolates from patients with SS. Last, our short-read sequencing and analysis approach prevents us from fully resolving challenging but important loci such as tprK. However, our conservative variant calling approach allowed us to focus on high-confidence discordant loci.

Our study serves as a reminder of the remarkable diversity of PS and SS clinical manifestations. We showed that DFM, which is no longer performed in most STI clinics, remains a valuable tool for rapid diagnosis of exudative mucocutaneous lesions. We also confirmed previously reported high qPCR sensitivities for ulcer exudates and skin biopsies. Although exclusively a research tool, RIT provided a powerful means to obtain TPA strains for WGS from blood—a compartment critical to the systemic disease process but heretofore inaccessible to genomic investigation. The greater availability of WGS will open the door to genome-based studies of local sexual networks that can assist in the development of novel epidemiologic strategies for syphilis control in hyperendemic areas [48]. TPA outer membrane proteins are widely considered the prime candidates for syphilis vaccine design [25]. Identification of immunologically relevant outer membrane protein variants through WGS of TPA strains from clinical specimens and RIT passaged isolates will facilitate development of a globally efficacious syphilis vaccine.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Acknowledgments. The authors thank all study participants and research staff in Guangzhou and Chapel Hill. They also thank Myron Cohen for his support and guidance, Andreea Waltmann for her contribution to the development of genomic sequencing methods, and Fredrick Nindo for his contribution to the development of data analysis pipelines.

Author contributions. L. Y., A. C. S., J. C. S., K. L. H., M. J. C., J. D. R., H. Z., M. A. M., and B. Y. conceptualized the study. H. Z. and J. B. P. led the genomic sequencing efforts. L. Y. and B. Y. led the clinical sites in enrollment, participant data, and specimen collection. L. Y., J. D. T., H. Z., B. Y., K. L. H., M. J. C., J. C. S., J. D. R., and J. B. P. supervised the clinical and/or research laboratory sites. F. Z. provided critical administrative support for all facets of the project. A. C. S., L. Y., J. S. C., and J. D. T. developed the case report forms. X. Z., R. C., L. W., and W. K. participated in subject enrollment and data and specimen collection. X. Z., P. Z., F. Z., A. C. S., and J. S. C. conducted data curation and formal analysis of the clinical data. W. C., Y. J., and H. Z. conducted the rabbit infectivity testing. W. C. and C. M. H. performed DNA extraction and polA qPCR assays. F. A., W. C., H. Z., C. M. H., and J. B. P. contributed to genome sequencing and phylogenetic analyses. X. Z., P. Z., M. J. C., A. C. S., J. C. S., K. L. H., E. B. B., and J. D. R. provided input into data analysis. L. Y., X. Z., W. C., A. C. S., E. B. B., J. D. R., and J. S. C. prepared the tables and figures. A. C. S., J. S. C., F. A., and J. B. P. verified the underlying data. L. Y., X. Z., W. C., A. C. S., E. B. B., and J. D. R. prepared the original and/or revised manuscripts. All authors reviewed the results and approved the final versions of the manuscript.

Financial support. This work was supported by the National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIAID) (grant number U19AI144177 to J. D. R. and M. A. M.). This work also was supported, in part, by the Bill & Melinda Gates Foundation (grant number INV-036560 to A. C. S.); strategic research dollars from Connecticut Children's; NIAID (grant number: T32 T32AI007151 to F. F. A.); and the National Nature Science Foundation of China (grant number 82220108006 to B. Y., 82302579 to W. C., and 82072321 to W. K.). Under the grant conditions of the Bill & Melinda Gates Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.

Data availability

The de-identified participant REDCap database will be made publicly available at the time of publication. Requests to share the informed consent forms and database can be made by emailing Drs Justin Radolf (jradolf@uchc.edu) or Yang Bin (yangbin101@hotmail.com). Raw sequencing data from this study with residual human reads removed are available through the Sequence Read Archive (BioProject PRJNA815321). Data supporting the findings of this study are available within the manuscript and Supplementary Materials.

References

Author notes