Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimens
BackgroundThe current testing approach to diagnose Chlamydia trachomatis (CT) infection relies on nucleic acid amplification tests (NAATs). These tests are highly sensitive, but do not distinguish between active infection and residual bacterial nucleic acid which may remain after resolution of infec...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-08-01
|
| Series: | Frontiers in Reproductive Health |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/frph.2023.1199740/full |
| _version_ | 1797754594074296320 |
|---|---|
| author | Lucia Vojtech Shahrokh Paktinat Tiffany Luu Stella Teichmann Olusegun O. Soge Olusegun O. Soge Robert Suchland Lindley A. Barbee Lindley A. Barbee Christine M. Khosropour |
| author_facet | Lucia Vojtech Shahrokh Paktinat Tiffany Luu Stella Teichmann Olusegun O. Soge Olusegun O. Soge Robert Suchland Lindley A. Barbee Lindley A. Barbee Christine M. Khosropour |
| author_sort | Lucia Vojtech |
| collection | DOAJ |
| description | BackgroundThe current testing approach to diagnose Chlamydia trachomatis (CT) infection relies on nucleic acid amplification tests (NAATs). These tests are highly sensitive, but do not distinguish between active infection and residual bacterial nucleic acid which may remain after resolution of infection, or via cross-contamination. Better methods to assess the viability of CT detected in clinical samples would be useful in determining the relevance of CT detection in a variety of clinical settings. The goal of this study was to test viability PCR (vPCR) as a method to distinguish viable bacteria from non-viable CT.MethodsThe vPCR relies on a propidium monoazide dye (PMAxx), which intercalates into accessible DNA from dead organisms and prevents their detection in a PCR assay for the CT ompA gene. We used digital PCR to quantify absolute genome copy numbers from samples. We validated the vPCR approach using laboratory stocks of CT with known viability. Then, we tested total DNA, viable CT DNA, and culture results from 18 clinical vaginal specimens and 25 rectal clinical specimens, all of which had tested positive by NAAT.ResultsIn laboratory stocks of CT, vPCR using defined ratios of heat-killed to live bacteria tracked closely with expected results. In vaginal clinical specimens, vPCR and total DNA results were correlated, though total DNA genomes outnumbered viable genomes by 2.2–52.6-fold more copies. As expected, vPCR detected more total genomes than culture results. Both vPCR and total DNA correlated with culture results (Spearman correlation R = 0.8425 for total DNA and 0.8056 for vPCR). Ten rectal NAAT positive specimens were negative by total DNA PCR, vPCR, and were negative or inconclusive by culture. Of the 6 rectal specimens that were culture positive, all were total DNA and vPCR positive. vPCR additionally detected viable bacterial DNA in 8 specimens which were NAAT + and culture negative, though levels were very low (mean 1,357 copies/ml)ConclusionsvPCR is a fast and easy method to assess viability in clinical specimens and is more correlated with culture results than total DNA PCR. Inconsistent ratios between total DNA and vPCR results suggest that the amount of dead bacteria varies considerably in clinical specimens. Results from rectal specimens suggest that many NAAT positive specimens do not in fact represent live replicating bacteria, and likely result in significant overuse of unnecessary antibiotics. |
| first_indexed | 2024-03-12T17:35:39Z |
| format | Article |
| id | doaj.art-811572e998364fcc8242143333bc5026 |
| institution | Directory Open Access Journal |
| issn | 2673-3153 |
| language | English |
| last_indexed | 2024-03-12T17:35:39Z |
| publishDate | 2023-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Reproductive Health |
| spelling | doaj.art-811572e998364fcc8242143333bc50262023-08-04T11:46:42ZengFrontiers Media S.A.Frontiers in Reproductive Health2673-31532023-08-01510.3389/frph.2023.11997401199740Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimensLucia Vojtech0Shahrokh Paktinat1Tiffany Luu2Stella Teichmann3Olusegun O. Soge4Olusegun O. Soge5Robert Suchland6Lindley A. Barbee7Lindley A. Barbee8Christine M. Khosropour9Department of Obstetrics and Gynecology, University of Washington, Seattle, WA, United StatesDepartment of Obstetrics and Gynecology, University of Washington, Seattle, WA, United StatesDepartment of Obstetrics and Gynecology, University of Washington, Seattle, WA, United StatesDepartment of Obstetrics and Gynecology, University of Washington, Seattle, WA, United StatesDepartment of Medicine, University of Washington, Seattle, WA, United StatesDepartment of Global Health, University of Washington, Seattle, WA, United StatesDepartment of Medicine, University of Washington, Seattle, WA, United StatesDepartment of Medicine, University of Washington, Seattle, WA, United StatesPublic Health – Seattle and King County, HIV/STD Program, Seattle, WA, United StatesDepartment of Epidemiology, University of Washington, Seattle, WA, United StatesBackgroundThe current testing approach to diagnose Chlamydia trachomatis (CT) infection relies on nucleic acid amplification tests (NAATs). These tests are highly sensitive, but do not distinguish between active infection and residual bacterial nucleic acid which may remain after resolution of infection, or via cross-contamination. Better methods to assess the viability of CT detected in clinical samples would be useful in determining the relevance of CT detection in a variety of clinical settings. The goal of this study was to test viability PCR (vPCR) as a method to distinguish viable bacteria from non-viable CT.MethodsThe vPCR relies on a propidium monoazide dye (PMAxx), which intercalates into accessible DNA from dead organisms and prevents their detection in a PCR assay for the CT ompA gene. We used digital PCR to quantify absolute genome copy numbers from samples. We validated the vPCR approach using laboratory stocks of CT with known viability. Then, we tested total DNA, viable CT DNA, and culture results from 18 clinical vaginal specimens and 25 rectal clinical specimens, all of which had tested positive by NAAT.ResultsIn laboratory stocks of CT, vPCR using defined ratios of heat-killed to live bacteria tracked closely with expected results. In vaginal clinical specimens, vPCR and total DNA results were correlated, though total DNA genomes outnumbered viable genomes by 2.2–52.6-fold more copies. As expected, vPCR detected more total genomes than culture results. Both vPCR and total DNA correlated with culture results (Spearman correlation R = 0.8425 for total DNA and 0.8056 for vPCR). Ten rectal NAAT positive specimens were negative by total DNA PCR, vPCR, and were negative or inconclusive by culture. Of the 6 rectal specimens that were culture positive, all were total DNA and vPCR positive. vPCR additionally detected viable bacterial DNA in 8 specimens which were NAAT + and culture negative, though levels were very low (mean 1,357 copies/ml)ConclusionsvPCR is a fast and easy method to assess viability in clinical specimens and is more correlated with culture results than total DNA PCR. Inconsistent ratios between total DNA and vPCR results suggest that the amount of dead bacteria varies considerably in clinical specimens. Results from rectal specimens suggest that many NAAT positive specimens do not in fact represent live replicating bacteria, and likely result in significant overuse of unnecessary antibiotics.https://www.frontiersin.org/articles/10.3389/frph.2023.1199740/fullChlamydiaPCRsexually transmitted infectionnucleic acidsNAATDNA extraction |
| spellingShingle | Lucia Vojtech Shahrokh Paktinat Tiffany Luu Stella Teichmann Olusegun O. Soge Olusegun O. Soge Robert Suchland Lindley A. Barbee Lindley A. Barbee Christine M. Khosropour Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimens Frontiers in Reproductive Health Chlamydia PCR sexually transmitted infection nucleic acids NAAT DNA extraction |
| title | Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimens |
| title_full | Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimens |
| title_fullStr | Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimens |
| title_full_unstemmed | Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimens |
| title_short | Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimens |
| title_sort | use of viability pcr for detection of live chlamydia trachomatis in clinical specimens |
| topic | Chlamydia PCR sexually transmitted infection nucleic acids NAAT DNA extraction |
| url | https://www.frontiersin.org/articles/10.3389/frph.2023.1199740/full |
| work_keys_str_mv | AT luciavojtech useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT shahrokhpaktinat useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT tiffanyluu useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT stellateichmann useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT olusegunosoge useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT olusegunosoge useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT robertsuchland useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT lindleyabarbee useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT lindleyabarbee useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens AT christinemkhosropour useofviabilitypcrfordetectionoflivechlamydiatrachomatisinclinicalspecimens |