From the Cold Chain to Point-of Care Tests for Chlamydia and Gonorrhea: Personal Reflections

Both authors have closely collaborated on point-of-care (POC) tests for sexually transmitted infections (STIs) in the last decade. However, when Charlotte Gaydos entered the chlamydia/gonorrhea laboratory at the Johns Hopkins Division of Infectious Diseases in 1987, there was a required cold chain to transport clinician obtained cervical or urethral swabs for culture of chlamydia in McCoy cells. Candle jar transport was required for gonorrhea specimens to culture on chocolate and Thayer Martin agar plates. In retrospect, we were in the dark ages and have come a long way since. Improvements came quickly. Although labor-intensive, chlamydia elementary bodies could be detected by direct fluorescent antibody staining of smears. Next, enzyme immunoassay tests were commercialized (e.g., SyvaR company); although they lacked sensitivity, enzyme immunoassay obviated the requirement for a cold chain. Culture was still considered the criterion standard for chlamydia testing. Similarly, gonorrhea culture and biochemical confirmatory tests continued to be used because there were other similar nonpathogenic strains such as Neisseria cinerea. Culture was being supplanted by more rapid methods. In 1986, a new strain of chlamydia, Taiwan acute respiratory agent (TWAR), was reported that caused respiratory infections.1 First grown from a trachoma study in Taiwan from an eye swab, TWAR (soon renamed Chlamydia pneumoniae) was the causative agent of respiratory infections in college students in Seattle, Washington, as well as in Finish military groups.2 Drs. Thomas Quinn and Hugh Taylor, trachoma specialists, soon obtained a live strain for our laboratory. Shortly thereafter, cynomolgus monkeys were inoculated with the organism in the eye, nose, and pharynx. Around that time, an Infectious Disease Fellow, Dr. Steve Holland, ran into the laboratory to tell us about a new diagnostic test called polymerase chain reaction (PCR) invented by Kary Mullis—there was now a machine that could perform the necessary temperature changes automatically for annealing of primers, extension, and amplification of target DNA for 40 cycles. Amazingly, the target DNA from the organism was “amplified” from a few copies to millions of copies, “making the needle in the hay stack into the hay stack,” some quipped. Tom Quinn immediately bought that Perkin Elmer machine. Diagnostic detection underwent a revolution. Polymerase chain reaction was born for diagnostics. We used it to test various chlamydia isolates, as well as the specimens from the monkeys. Drs. Quinn and Charlotte Gaydos later decided C. pneumoniae was a great project for a doctoral thesis based on sequencing the C. pneumoniae gene.3 That is another story. We started to perform PCR for Chlamydia trachomatis, C. pneumoniae, and Chlamydia psittaci to differentiate them.4,5 We showed that we could differentiate chlamydia species by enzyme restriction analysis of the PCR product. The Roche company soon conducted a PCR clinical trial to identify C. trachomatis. Our laboratory participated.6 The assay was commercialized, and gonorrhea was added to the assay. We had now moved from culture to identifying both pathogens by PCR. Urine and vaginal swab sample types were validated for use with PCR. Culture was no longer the criterion standard. Other companies followed with commercialization of amplification assays similar to PCR, but slightly different. The term “nucleic acid amplification tests” (NAATs) was coined by Dr. Julius Schachter to refer to any amplification assay of DNA or RNA. Roche's AMPLICOR, Becton Dickenson's ProbeTech, GenProbe's (Hologic) transcription-mediated amplification (TMA), and Abbott's ligase amplification (LCR) came along quickly.7–10 An outstanding innovation by many companies was to validate tests with patient self-collected, diagnostic specimens, namely, male and female urine and vaginal swabs for women. Studies showed that self-collected specimens provided results as accurate as PCR of clinician-collected cervical swabs and male urethral swabs. The Food and Drug Administration (FDA) astounded some in approving these types of specimens. The stage was set for more changes to STI diagnostic assays. Interest developed in having a rapid NAAT that could provide quicker results in the clinics at the POC. In 2007, we received a grant from the National Institutes of Biomedical Imaging and Bioengineering to work with companies who wanted to develop POC tests for the diagnosis of STIs. We were able to fund many companies with small research grants, while conducting needs assessments surveys with key opinion leaders, STI clinic personnel, and other clinicians, about the desirable characteristics of POC tests.11 Not surprisingly, we found sensitivity, specificity, turnaround times, and costs to be important variables in assessing the utility of POC tests for chlamydia, gonorrhea, and HIV. We also instituted a study for women and men to request an STI test via the Internet and mail self-collected samples from home to our research laboratory for NAAT testing, which we named “iwantthekit.”12 This was a nice fit with the FDA's clearance for self-collected specimens. The Cepheid Company was successful in developing the first FDA-cleared PCR test for chlamydia and gonorrhea, which could be performed near the patient in 90 minutes using patient self-collected specimens.13 The faster turnaround time was much shorter than the 2 to 4 days observed with commercial laboratory NAAT assays and has been used in emergency department settings.14 Specimens could be self-collected in a pharmacy, and participants were willing to wait until results were ready.15 This ushered in a new era of technologic innovation and bioengineering to produce devices that could be used at POC. Now in our fourth 5-year cycle, our National Institutes of Biomedical Imaging and Bioengineering research grants mirrored the progress being made in POC diagnostics. We were able to give small grants to many companies to develop POC tests that met the desired characteristics for acceptability and performance, especially turnaround times. By 2021, 2 tests, Atlas Genetics (now binx) and Click (now Visby), were both FDA cleared as 30-minute Clinical Laboratory Improved Amendments–waived POC tests.16–18 A whirlwind of progress over 3 decades has changed diagnostic expectations for all infectious diseases. Unfortunately, the retail cost of these POC tests has not yet met expectations. Financial sustainability may be a barricade to adoption, but progress is being made.19 In conclusion, the revolution in STI diagnostic testing from reference laboratory to POC described previously was monitored and encouraged by Dr. Julius Schachter, our prior Journal editor, and many of these accomplishments were documented in the STD Journal. After the COVID-19 epidemic, POC tests and even self-tests have achieved phenomenal diagnostic adoption. We expect POC tests for STIs and infectious diseases to continue to move forward in giant leaps.20