Chlamydia treatment failed twice

Chlamydia treatment failed twice DEFAULT

Treatment Failure of Chlamydial Infection in Males and Females in Youth Correctional Facilities

Brief Summary:

Chlamydia is a common infection among youth and can be given from one person to another during sex. Many people who have chlamydia have no signs of infection at all, but can pass the infection to anyone they have sex with. If not treated, chlamydia can lead to serious health problems. This study will look at how well medicines given for chlamydia infection work. The study requires 306 evaluable subjects, chlamydia-positive, males and non-pregnant females, ages 12-21, living in long-term, gender-segregated youth correctional facilities. Participants will be assigned to receive either doxycycline (2 times per day, by mouth, for 7 days) or azithromycin (1 single dose by mouth). Study procedures will include collection of at least 3 urine samples to test for chlamydia. Study visits will occur during initial enrollment in the study, day 28 after starting treatment, and day 67. Participants will be involved in study related procedures for up to 67 days.


Chlamydial InfectionDrug: AzithromycinDrug: DoxycyclinePhase 3

Detailed Description:

Genital chlamydia is a public health concern. The World Health Organization (WHO) estimates that 90 million of all new cases of sexually transmitted diseases (STDs) per year are caused by Chlamydia (C.) trachomatis. In the United States alone, approximately 3 million new cases of chlamydia are reported yearly, and the costs associated with their management and complications exceed $2 billion. Unfortunately, at least 75 percent of females with chlamydia are asymptomatic, and unless the infection is detected through chlamydia testing (screening), their infection may be transmitted to others or lead to complications. The Centers of Disease Control and Prevention (CDC) recommends either azithromycin 1 gram (gm) by mouth (PO) once or doxycycline 100 milligrams (mg) PO twice daily (BID) for 7 days as co-equal therapies for uncomplicated chlamydia. A secondary aim will be to determine demographic predictors of chlamydia treatment failure following azithromycin or doxycycline treatment, and to explore clinical parameters, which distinguish those with persistent infection. The study design of this Phase III trial will address major limitations of prior chlamydia efficacy studies and the findings will reveal both the true efficacy of azithromycin and doxycycline in uncomplicated chlamydia in adolescents and the factors that predict treatment failure. This study is designed primarily to determine the frequency of chlamydia treatment failure following either azithromycin or doxycycline regimens and to evaluate whether the efficacy of the azithromycin regimen is inferior to the doxycycline regimen. Both drugs are Food and Drug Administration (FDA) approved for use in the U.S. The study will enroll 650 males and females age 12-21 years in good health (based on vital signs and provider's clinical evaluation documented in medical records) who are residing in long-term gender-segregated (not co-ed) youth correctional facilities (YCFs) (usual stay >3 weeks) and who are identified as chlamydia-infected would comprise the study population until 306 evaluable subjects are obtained . Only individuals who have a positive chlamydia screening test are enrolled, and those with negative screening tests are excluded. Consenting chlamydia-positive subjects at the enrollment visit (study visit 1) will be enrolled, asked to provide demographic data, to provide a first-void urine sample (not a mid-stream specimen) for repeat chlamydia testing with Gen-Probe (GP) AC2 (for verification of chlamydia), and then randomized to 1 of 2 treatment arms (190 153 subjects per arm): doxycycline 100 mg PO BID for 7 days or azithromycin 1 gm PO single dose. Both therapies are given as directly observed, and side effects are evaluated at the first follow-up visit (day 28 after study drug initiation). If a subject who's GP AC2 from the enrollment treatment visit returns negative for C. trachomatis, they will be categorized as unevaluable and will be removed from the study, then the site investigator will determine whether the subject will complete this treatment or will receive other therapy. Subjects whose GP AC2 at the enrollment treatment visit is positive for C. trachomatis will then be asked to provide a first-void urine sample for repeat chlamydia testing with GP AC2 at 28- and 67-days after study drug initiation [corresponding to the first follow-up visit (study visit 2) and second follow-up visit (study visit 3), respectively].

Study Type : Interventional  (Clinical Trial)
Actual Enrollment :567 participants
Allocation:Randomized
Intervention Model:Parallel Assignment
Masking: None (Open Label)
Primary Purpose: Treatment
Official Title:Randomized Clinical Trial Evaluating Treatment Failure Following Recommended Therapy (Azithromycin Versus Doxycycline) for Genital Chlamydial Infection in Males and Females in Youth Correctional Facilities
Study Start Date :December 2009
Actual Primary Completion Date :May 2014
Actual Study Completion Date :May 2014

Sours: https://www.clinicaltrials.gov/ct2/show/NCT00980148

Yes, Chlamydia Can ‘Come Back’ — Here’s How to Prevent It

How can you be sure you’re experiencing a new bout?

Chlamydia is treated with antibiotics, usually azithromycin or doxycycline.

In order to make sure chlamydia is cured, you need to take the full course of antibiotics as prescribed by your doctor. You need to take every single dose — don’t stop taking the antibiotics until there are none left.

If you’ve taken all your antibiotics but you still have symptoms, contact your doctor or another healthcare professional.

According to the , you’ll need a follow-up test three months after treatment to ensure that the infection is cured.

Why does reoccurrence happen?

There are a few reasons why you might contract chlamydia a second time:

  • The initial infection wasn’t cured because the course of antibiotics wasn’t completed as directed.
  • A sexual partner transmitted chlamydia to you.
  • You used a sex toy that was contaminated with chlamydia.

A 2014 study suggests that chlamydia can live in the gastrointestinal tract and reinfect the genitals, causing chlamydia symptoms to reappear after the genital infection went away.

However, this study only looked at animal models of chlamydia. Research on human participants is needed.

How long does a bout of chlamydia typically last?

The symptoms of chlamydia typically disappear once you finish your antibiotics. This can vary in time, as some chlamydia antibiotic courses are one dose taken on one day, while others last longer.

The recommends waiting seven days after a one-day antibiotic, or until the end of a seven-day antibiotic course, before having partner sex again.

What can you do to relieve your symptoms?

No home remedy for chlamydia can replace antibiotics. Chlamydia is a bacterial infection, so you need to take antibiotics to cure it.

However, there are a few ways you can soothe symptoms while you wait for the antibiotics to get to work. For example:

  • Use pain medications, such as ibuprofen to reduce pain
  • Use a cold pack to soothe inflammation.
  • A herb called goldenseal might reduce inflammation and other symptoms.
  • Use an echinacea supplement aid your immune system.

Remember that these home remedies might soothe the symptoms of chlamydia, but they don’t actually cure chlamydia in itself. The best way to soothe the symptoms is to use antibiotics.

What happens if you don’t seek treatment?

If you take your antibiotics as directed, chlamydia is likely to go away. But if it’s left untreated, it can cause a few complications.

For example, if you have a vulva, you could develop pelvic inflammatory disease (PID). PID is a painful infection that could damage your uterus, cervix, and ovaries.

Untreated chlamydia can also lead to scarred fallopian tubes, which can cause infertility.

If you’re pregnant, untreated chlamydia can be transmitted to the baby during vaginal delivery. Chlamydia can cause eye infections and pneumonia in newborns.

Untreated chlamydia can lead to epididymitis, which is when the epididymis (the tube that holds the testicles in place) becomes inflamed, causing pain.

Chlamydia can also spread to the prostate gland, which can lead to painful sex, lower back pain, and a fever.

Fortunately, treatment for chlamydia is relatively straightforward. And if it’s treated quickly, you’re unlikely to experience any long-term complications.

What exactly causes chlamydia?

A type of bacterium called Chlamydia trachomatis causes chlamydia. This bacterium can take hold in the tissues of your genitals, anus, eyes, or throat.

It’s usually transmitted from one person to another during penetrative vaginal or anal sex or oral sex, although sex without penetration can also transmit it.

Chlamydia can also be transmitted to a baby during vaginal delivery if the person giving birth has an untreated chlamydia infection.

The bottom line

It’s possible to have chlamydia more than once.

To prevent reoccurrence or reinfection, finish your full course of antibiotic treatment, and talk with your sexual partner(s) about getting tested and treated for chlamydia, too.


Sian Ferguson is a freelance writer and editor based in Grahamstown, South Africa. Her writing covers issues relating to social justice, cannabis, and health. You can reach out to her on Twitter.

Sours: https://www.healthline.com/health/healthy-sex/can-chlamydia-come-back
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Chlamydia Can Live in Your Gut And Reinfect You After You’re Cured

chlamydia

Chlamydia is the most commonly reported sexually transmitted diseases in the United States. Thankfully, it’s also curable. But new research suggests that for some people, curing chlamydia doesn’t prevent reinfection, even if they’re not exposed to it again. Apparently the disease can live inside your gut, and reinfect you out of the blue.

Apparently doctors have known that chlamydia can reappear in cured patients for about 80 years, but they’ve been stumped as to how exactly it happens. This study points out that, in many animals, chlamydia has been found to live in the gastrointestinal tract. “Thus, if gastrointestinal infection occurs in most hosts,” the authors write, “then it is very likely that gastrointestinal infection occurs in humans as well.”

The study in question doesn’t actually test this theory on any human beings. Instead it looks at data in animal models about reinfection, and the failure of certain drugs to treat chlamydia when it lives in the gut. From there, they propose that women who are infected with chlamydia could see the same kind of issues: the drugs they’re given might cure the disease genitally, but not gastrointestinally, leaving the bug to live inside waiting for the right time to strike.

Jason Koebler at Motherboad says that earlier studies suggest that women are more likely to see these spontaneously reoccurring infections.

Two things would explain that—treatment failures that could occur because of antibiotic resistance, or reinfection. Rank says that, though treatment failure in chlamydia is rising, in mice studies, antibiotics were much less effective on GI chlamydia than on genital chlamydia. Rank suggests that women suffer self-reinfection at a higher rate than men for the same reason that they are more likely to have urinary tract infections. He says that alternative antibiotics or closer monitoring might be necessary to ensure a patient is cured.

For those who have been treated for chlamydia, it’s probably not time to freak out just yet. Reemergence is rare, and when chlamydia does come back, it’s still treatable. But if they do wind up with a repeat case, it might not be time to blame your partner for cheating just yet. Their guts could be playing tricks on you.

Recommended Videos

Sours: https://www.smithsonianmag.com/smart-news/chlamydia-can-live-your-gut-and-reinfect-you-after-youre-cured-180949688/
Sexual Health - Chlamydia (Female)

Doxycycline vs Azithromycin: Think Twice About the 2020 CDC Guideline Update on Treatment of Gonorrhea and Chlamydia

cdc gonorrhea chlamydia doxycycline

When the new Centers for Disease Control and Prevention (CDC) recommendations1 regarding the treatment of uncomplicated gonorrhea (and indirectly chlamydia) debuted like a slice of antibiotic resistance doom, it felt like another “gift” had arrived from 2020. Intramuscular (IM) ceftriaxone dosing has increased from 250 mg to 500 mg (or 1 g for weight ≥150 kg). Empiric chlamydia coverage switched from a single dose of 1 g of azithromycin to doxycycline 100 mg PO BID for 7 days. Being deferential to CDC expertise, many providers accepted them uncritically. Compliance rates with a switch from a 1-time to a 7-day regimen are not addressed, especially worrisome for a condition that can be minimally or asymptomatic. 

Case

A young woman presents with new and concerning discharge after an unprotected encounter. Her pregnancy test is negative. After agreement for empiric treatment, the patient then refuses empiric treatment when told about the new guidelines (2 injections and 14 chances for esophagitis). Patient specifically asks for the old regime or will just leave against medical advice.

Why a higher dose of ceftriaxone for gonorrhea?

It is important to note that the evidence of ceftriaxone, cefixime, and azithromycin resistance for gonorrhea is substantial.2 Observational data from across the United States and world demonstrate worsening resistance patterns. Many of our pharmacy colleagues are working on obtaining 500 mg/2 mL ceftriaxone for injection vials, so it can be given in single injection (or two for morbidly obese patients). While this guideline may be existentially troubling, this change is practically feasible and should become standard of care.

Read more about the Trick of the Trade on administering IV instead of IM ceftriaxone for gonorrohea.

Why no mention of the single-dose azithromycin option for chlamydia?

The evidence basis for the change to doxycycline for treatment of chlamydia co-infection coverage is substantially weaker. It is also decidedly mute on the risks of partial or non-compliance with treatment. The question then becomes: How profound is the treatment effect and how does it balance against its risks?

The guideline states, as evidence for the doxycycline switch:

“A recent investigation comparing children who received twice-yearly azithromycin with children who received placebo found that the gut’s resistome, a reservoir of antimicrobial resistance genes in the body, had increased determinants of macrolide and nonmacrolide resistance, including beta-lactam antibiotics, among children receiving azithromycin (10).3 A higher proportion of macrolide resistance in nasopharyngeal Streptococcus pneumoniae was demonstrated in communities receiving mass administration of oral azithromycin (11).4 Azithromycin resistance has been demonstrated in another STI, Mycoplasma genitalium, and sexually transmissible enteric pathogens (e.g., Shigella and Campylobacter) (12–14)5-7. In addition, evidence supports increasing concern for the efficacy of azithromycin to treat chlamydial infections, especially rectal infections (15,16)8,9.”

Citations 10 and 11 speak in generalities of resistance patterns, with citation 11 being a secondary analysis of a mass azithromycin treatment trial of young children in Niger. Citations 12-14 discuss rates of coinfection treatment failure – an important consideration, but only secondarily relevant. That leaves 2 citations (15 and 16)– one a meta-analysis and one a small poster that isn’t even available online related to known anorectal chlamydia.

That really leaves the meta-analysis8 to answer our question: how best do we protect the reproductive health of our patients in the setting of diagnostic uncertainty?

The meta-analysis

The meta-analysis is somewhat messy with substantial heterogeneity in many relevant subgroups.8 A single study comprises the majority of the evidence that shows doxycycline superiority in non-gonococcal urethritis.10 It was from 2011 and revealed that while doxycycline may be better for chlamydia treatment, azithromycin was better for coinfection treatment (such as shigella or mycoplasma). And to top the whole thing, the doxycycline superiority line reads:

“We found a pooled efficacy difference in favor of doxycycline of 1.5%… to 2.6%.”

In men with symptomatic urethritis, the superiority of doxycycline increases to 7% (an NNT of 14). If you ignore the heterogeneity and pool everyone, we arrive with an overall NNT for doxycycline over azithromycin of 38 (fixed effects model size was a 2.6% advantage). If the above study10 was removed, the pooled difference would have been non-significant with an NNT of at least 50.

Having thought perhaps they just didn’t include all the evidence, a secondary literature review was undertaken. A few small case studies11 and older observational studies12,13 were found, which showed a potential treatment failure rate of azithromycin of up to 8%, but comparable rates with doxycycline.12 That’s it. There is also genuine concern that use of azithromycin may induce resistance not only for itself but other antibiotic classes3,4 but this concern is based on fecal biome sampling from toddlers and requires a couple of steps to be relevant to our question. Doxycycline, an essential medication in its own right, for treatment of tick-borne disease, ascending genital tract infections, COPD exacerbation and MRSA, also requires our stewardship.

Medication compliance questions

Given patient non-compliance with filling and completing ED prescriptions approach rates of 20%,14,15 the recommendation for a 7-day course of doxycycline for chlamydia over single-dose azithromycin is fraught with peril. Additionally, consider that the patient may be relatively asymptomatic, placing them even more at risk for medication non-compliance for the 7-day course of doxycycline. Contrast this with the risks of pelvic inflammatory disease and infertility if untreated.

Conclusion

Given the sparse, heterogenous literature, we should have strong reservations about recommending doxycycline for patients for whom chlamydia has not been excluded. New gonorrhea treatment recommendations should be followed and efforts made to stock appropriate concentrations of ceftriaxone. A single-dose of azithromycin may be a reasonable alternative for your patient for non-gonococcal disease, after considering and discussing the risks and benefits. Pregnant patients require close follow up but should also continue to receive azithromycin.

If you are prescribing doxycycline, remember:

  • Each pill should be taken with 6-8 oz of liquid, water preferred. 
  • If taken with food, it decreases the risk of dyspepsia.
  • One should sit upright for 30 minutes following each pill, especially those with history of GERD.
  • If substantially sunexposed, sunscreen or full skin coverage should be recommended to prevent photosensitive reactions (which can be mild to quite severe).

If you are prescribing azithromycin, remember:

  • Azithromycin can cause clinically significant increases of QTc even with a single dose, but typically only to those with multiple risk factors.16 Consider ECG if patient on QTc prolonging medications and/or coexisting electrolyte derangements discovered.
  • The risk of treatment failure for chlamydia and other non-gonococcal coinfections is real. For men with symptomatic urethritis, that risk is substantially higher.
  • Have a shared decision discussion about doxycycline versus azithromycin.
  • While all patients should receive verbal and written follow-up instructions, close follow up should be emphasized, given that you are essentially contravening a CDC guideline.

Patient case resolution

You explain to your patient that the new guidelines should be followed for gonorrhea, and so she receives 500 mg of IM ceftriaxone. While the new guideline for doxycycline MAY be slightly more effective for the treatment of chlamydia, using shared decision making, she receives the old regimen (single-dose azithromycin). You verbally emphasize and document in the discharge instructions the importance they follow up with either their PCP, gynecologist, or the local sexually transmitted infection clinic for a recheck, if their symptoms don’t resolve within 7 days.


References

  1. St. Cyr S, Barbee L, Workowski KA, et al. Update to CDC’s Treatment Guidelines for Gonococcal Infection, 2020. MMWR Morb Mortal Wkly Rep 2020;69:1911–1916. DOI: http://dx.doi.org/10.15585/mmwr.mm6950a6external icon
  2. https://www.cdc.gov/std/treatment-guidelines/toe/GCEvidenceTables2020.xlsx [Download file link]
  3. Doan T, Worden L, Hinterwirth A, et al. Macrolide and nonmacrolide resistance with mass azithromycin distribution. N Engl J Med 2020;383:1941–50. PMID 33176084
  4. Doan T, Arzika AM, Hinterwirth A, et al.; MORDOR Study Group. Macrolide resistance in MORDOR I—a cluster-randomized trial in Niger. N Engl J Med 2019;380:2271–3. PMID 31167060
  5. Bachmann LH, Kirkcaldy RD, Geisler WM, et al. Prevalence of Mycoplasma genitalium infection, antimicrobial resistance mutations and symptom resolution following treatment of urethritis. Clin Infect Dis 2020;ciaa293. Epub March 18, 2020. PMID 32185385
  6. Yousfi K, Gaudreau C, Pilon PA, et al. Genetic mechanisms behind the spread of reduced susceptibility to azithromycin in Shigella strains isolated from men who have sex with men in Québec, Canada. Antimicrob Agents Chemother 2019;63:e01679–18. PMID 30455248
  7. Gaudreau C, Pilon PA, Sylvestre JL, Boucher F, Bekal S. Multidrug-resistant Campylobacter coli in men who have sex with men, Quebec, Canada, 2015. Emerg Infect Dis 2016;22:1661–3. PMID 27533504
  8. Kong FY, Tabrizi SN, Law M, et al. Azithromycin versus doxycycline for the treatment of genital chlamydia infection: a meta-analysis of randomized controlled trials. Clin Infect Dis 2014;59:193–205. PMID 24729507
  9. Dombrowski JC, Wierzbicki MR, Newman L, et al. A randomized trial of azithromycin vs. doxycycline for the treatment of rectal chlamydia in men who have sex with men. Presented at the National STD Prevention Conference, Atlanta, GA: September 14–24, 2020.
  10. Schwebke JR, Rompalo A, Taylor S, et al. Re-evaluating the treatment of nongonococcal urethritis: emphasizing emerging pathogens randomized clinical trial. Clin Infect Dis. 2011 Jan 15;52(2):163-70. PMID 21288838
  11. Bhengraj AR, Vardhan H, Srivastava P, Salhan S, Mittal A. Decreased susceptibility to azithromycin and doxycycline in clinical isolates of Chlamydia trachomatis obtained from recurrently infected female patients in India. Chemotherapy. 2010;56(5):371-7. PMID 20938174
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Kory London, MD

Director of Clinical Operations, Jefferson Methodist Hospital ED
Associate Director of Quality Assurance and Practice Improvement
Assistant Professor of Emergency Medicine
Sidney Kimmel Medical College
Thomas Jefferson University

Kory London, MD
Sours: https://www.aliem.com/doxycycline-vs-azithromycin-cdc-guideline-gonorrhea-chlamydia/

Twice chlamydia treatment failed

Urogenital chlamydia trachomatis treatment failure with azithromycin: A meta-analysis

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Sours: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804326/
Sexual Health - Chlamydia (Female)

A cohort study of Chlamydia trachomatis treatment failure in women: a study protocol

  • Study protocol
  • Open Access
  • Published:
  • Jane S Hocking1,8,
  • Lenka A Vodstrcil1,2,
  • Wilhelmina M Huston3,
  • Peter Timms3,
  • Marcus Y Chen1,4,
  • Karen Worthington4,
  • Ruthy McIver5 &
  • Sepehr N Tabrizi2,6,7

BMC Infectious Diseasesvolume 13, Article number: 379 (2013) Cite this article

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Abstract

Background

Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection in the developed world and diagnosis rates have increased dramatically over the last decade. Repeat infections of chlamydia are very common and may represent re-infection from an untreated partner or treatment failure. The aim of this cohort study is to estimate the proportion of women infected with chlamydia who experience treatment failure after treatment with 1 gram azithromycin.

Methods/design

This cohort study will follow women diagnosed with chlamydia for up to 56 days post treatment. Women will provide weekly genital specimens for further assay. The primary outcome is the proportion of women who are classified as having treatment failure 28, 42 or 56 days after recruitment. Comprehensive sexual behavior data collection and the detection of Y chromosome DNA and high discriminatory chlamydial genotyping will be used to differentiate between chlamydia re-infection and treatment failure. Azithromycin levels in high-vaginal specimens will be measured using a validated liquid chromatography – tandem mass spectrometry method to assess whether poor azithromycin absorption could be a cause of treatment failure. Chlamydia culture and minimal inhibitory concentrations will be performed to further characterize the chlamydia infections.

Discussion

Distinguishing between treatment failure and re-infection is important in order to refine treatment recommendations and focus infection control mechanisms. If a large proportion of repeat chlamydia infections are due to antibiotic treatment failure, then international recommendations on chlamydia treatment may need to be re-evaluated. If most are re-infections, then strategies to expedite partner treatment are necessary.

Peer Review reports

Background

Over 100 million men and women worldwide are infected with chlamydia at any point in time [1]. It is the most commonly reported bacterial sexually transmitted infection (STI) in developed countries, with over 1.4 million cases reported in the United States in 2011 [2]. Left untreated, chlamydia can ascend from the endocervix to the upper genital tract in women and cause pelvic inflammatory disease (PID) which can increase the risk of developing fallopian tube scarring, potentially leading to ectopic pregnancy, tubal infertility and chronic pelvic pain [3–5]. In addition, genital infection in pregnant women increases the risk of preterm delivery, can be passed on to the baby during vaginal delivery and may result in eye and lung infections in the new born [3, 6]. Infection with genital chlamydia can also increase the risk of HIV acquisition in both men and women, and cause epididymo-orchitis in men [4, 7].

Repeat chlamydia infections are common following treatment. Among women who tested positive in an Australian cohort of 1116 young women who were treated at recruitment, 18% tested positive again at 3 months (95% CI: 8%, 34%) [8]. In the UK, a prospective cohort of 16 to 24 year old women treated for chlamydia in general practice, reported a repeat infection rate of 29.9% per year (95% CI: 19.7%, 45.4%) [9]. Another cohort of adolescent women in the US reported a repeat infection rate of 34% per year [10] and a recent systematic review of chlamydia repeat infection following treatment found that the overall median proportion testing positive again for chlamydia was 13.9% [11].

Repeat infections are generally considered to be re-infections through exposure to an infected partner. However, emerging evidence suggests that treatment failure following azithromycin may account for a substantial proportion and this has led to considerable debate in the medical and scientific literature [12–16]. Among female participants in a partner treatment trial who reported no sexual intercourse after treatment, 22 of 289 (8%; 95% CI: 5%, 11%) had persistent infection suggestive of treatment failure at follow up [17]. Similarly, the treatment failure rate in a cohort of adolescent females was 7.9% (95% CI: 4%, 10.1%) [10]. Both of these studies attempted to differentiate between re-infection and treatment failure using sexual behavior questionnaires. Batteiger et al. (2010) included genotyping as a further tool to identify re-infections, but these studies were based on self-report of sexual behavior which may have been unreliable.

The recommended first line treatment for uncomplicated genital chlamydia infection in most developed countries is a 1 gram dose of the macrolide antibiotic, azithromycin [18–20]. Although doxycycline 100 mg twice daily for 7 days is a second line treatment for uncomplicated chlamydia, it is not widely used because there are concerns about compliance given the longer duration of treatment [21]. A meta-analysis of chlamydia treatment reported a 97% cure rate for azithromycin and 98% for doxycycline [22]. Of the 12 trials included in the meta-analysis, 11 used culture or immunoassay rather than the more sensitive nucleic acid amplification tests (NAAT) to determine microbial cure at study end [22]. Given the use of culture rather than NAATs, it is possible that the treatment efficacies in these trials were over-estimated [12, 13, 23].

Governments throughout the developed world are pushing for increased chlamydia testing and the Australian government has invested in a large chlamydia screening randomized controlled trial [24]. It is imperative, therefore, to rigorously investigate the adequacy of currently recommended treatment for chlamydia. We describe here a cohort study, the Australian Chlamydia Treatment Study (ACTS), that aims to measure the proportion of chlamydia infected women who fail treatment when treated with 1 gram azithromycin using advanced microbiological techniques to differentiate between re-infection and treatment failure.

Research aim

The primary aim is to estimate the proportion of women infected with chlamydia who fail treatment with 1 gram azithromycin, the most widely recommended first line treatment for genital chlamydia infection [18–20]. The secondary aim is to determine the role of C. trachomatis organism load, azithromycin tissue absorption of azithromycin and antimicrobial resistance in chlamydia treatment failure.

Methods/design

Study design and setting

This is a cohort study of women who test positive for genital chlamydia at one of two large, publically funded sexual health centres in Melbourne and Sydney, Australia. Participants will be followed up for up to 56 days post treatment and will provide weekly genital specimens for further assay.

Duration of study

Recruitment commenced in October of 2012 and is expected to continue until October 2014, with results reported late 2015.

Participant eligibility

Women who are diagnosed with genital chlamydia and who meet eligibility criteria will be invited to participate in the study. The study is limited to women because of their increased risk of serious chlamydia related complications.

The inclusion criteria are:

  • Female;

  • Positive test for genital chlamydia using Nucleic Acid Amplification Test polymerase chain reaction [PCR] (performed on specimens collected from women recruited from Sydney Sexual Health Centre) or strand-displacement assay (performed on specimens collection from women recruited from Melbourne Sexual Health Centre);

  • Age ≥16 years;

  • Adequate English and comprehension skills to give informed consent;

  • Able to attend their recruitment clinic for follow up and specimen collection at day 7;

  • Resides in a jurisdiction serviced by one of the two clinics and plan to stay in the jurisdiction for the next 8 weeks.

The exclusion criteria are:

  • Current infection detected as a part of a routine test for re-infection [18];

  • Concomitant infection with another bacterial STI;

  • Concurrent PID;

  • Self report of antibiotic use in the last 2 weeks;

  • Current commercial sex work;

  • Women who do not wish to receive study packs by post;

  • Women who do not have a mobile phone;

  • HIV positive status;

  • Concurrent medication likely to significantly interact with azithromycin (e.g. cyclosporine, digoxin);

  • Known macrolide allergy.

Recruitment

Women who have tested positive for chlamydia will be seen by a research nurse at a participating clinic at point of treatment. The research nurse will explain the study to them, assess eligibility and take informed consent. The research nurse will collect specimens for further testing (Table 1) and provide treatment with a single dose of 1 gram azithromycin. The nurse will observe the participant as she takes her treatment to ensure compliance. This is classified as the baseline visit (Day 0). All clinic visits, pathology costs and treatment for participants and their partners will be provided free of charge.

Full size table

Primary outcome

The primary outcome is the proportion of women who are classified as having chlamydia treatment failure following detection of chlamydia on a self-collected high-vaginal specimen using a polymerase chain reaction (PCR – see below) Specimens will be collected for testing on days 28, 42 or 56 after recruitment (referred to as ‘test of cure’ specimens for the purpose of this study) according to the algorithm in Figure 1. This algorithm follows a previously described method [10] with the addition of the detection of Y chromosome DNA and performing high discriminatory chlamydial genotyping. Y chromosome DNA will be used to validate sexual behaviour data because it can be detected in the vagina for up to 14 days after unprotected intercourse with a male partner [25, 26]. Specimens will be collected from participants every 7 days for Y chromosome detection and genotyping will be undertaken on any chlamydia positive specimens collected on days 28, 42 or 56 after recruitment. Chlamydial genotype fingerprinting will be performed to determine any differences in chlamydia strain/serovar between a participant’s baseline specimen and any repeat positive specimens detected at day 28, 42 or 56 [27]. If a woman has the same serovar present at follow up, further testing by multilocus sequence typing (MLST) targeting ompA and five house-keeping genes will be undertaken [28, 29]. Detection of same MLST type without detection of Y chromosome DNA or any reports of unprotected sex will be classified as treatment failure. If the woman has a different serovar, it will be classified as a re-infection. A repeat positive chlamydia diagnosis will also be classified as a re-infection if the woman’s repeat specimen has the same MLST type and Y chromosome is detected or she reports unprotected sexual contact.

Algorithm for classifying treatment failure.

Full size image

Study endpoint

A study participant will reach the study endpoint when she is diagnosed with treatment failure, re-infection or treatment success. The study endpoint will be measured by a test of cure PCR conducted after 28, 42 or 56 days follow up according to Figure 2.

Classification of study endpoint.

Full size image

Further classification of treatment failure

Treatment failure will be further classified as:

Poor azithromycin absorption: There is no vomiting or diarrhoea reported after treatment but low levels of azithromycin concentration are found in the vaginal cells [30, 31] and the MIC for the initial chlamydia culture is within reported range.

Persistent chlamydia not responding to azithromycin: Azithromycin absorption shows adequate levels of azithromycin in the vaginal cells, the minimal inhibitory concentration (MIC) for the initial chlamydia culture is within reported range and the subsequent chlamydia culture is negative [13, 23, 32, 33].

Reduced antimicrobial susceptibility and resistance: Some viable infectious chlamydia is present when cultured with antibiotic series compared with cultures with no antibiotic series. The MIC for initial chlamydia culture is elevated compared with reported range [13, 15, 32, 34, 35]. Evidence of 23S rRNA gene mutation may or may not be present.

Specimen collection and testing

Participants will be asked to return to the clinic for follow up testing at day 7 so that the research nurse can collect further specimens. Thereafter, weekly high-vaginal specimens will be self-collected at home and mailed in a postage-paid envelope to the study centre. Participants will receive weekly short message service (SMS) prompts reminding them to return specimens and questionnaires and will be telephoned if they test chlamydia positive again throughout the study. These women will be requested to attend the clinic for treatment and further specimen collection. Table 1 outlines the number of specimens collected and the tests to be undertaken at each stage of the study.

Chlamydia polymerase chain reaction (PCR)

Test of cure swab specimens collected on day 28, 42 and day 56 will be placed in Cobas® PCR Media (Roche Diagnostics) and will be tested in batches on COBAS 4800 CT/NG (Roche Diagnostics). The remaining eluted DNA from COBAS 4800 will be stored at −80°C until required for further assay. For specimens collected at other time points, swabs collected will be rotated for 30 seconds in 1 ml of phosphate buffered saline (PBS) and stored at −80°C until further testing required. Specimens will be subsequently extracted with MagNA Pure 96 (Roche Diagnostics) using 200 μl of the cellular suspension in conjunction with the total nucleic acid isolation kit. Eluted nucleic acid of 100 μl will be tested as required.

Genotyping & quantification of organism load

Chlamydial fingerprinting will be conducted on specimens that are PCR positive at baseline and at test of cure. We will determine the organism load, identify the chlamydia serovar(s) (genovar) of each infection through a series of quantitative PCR assays to establish whether or not the genotype of the chlamydia detected in those women who have a repeat positive is the same as the type present at baseline [27]. Initial qPCR primers and probes have been designed to predict antigenic differences in major outer membrane protein (MOMP) to determine the serovar as previously described [27, 36–38]. The primary chlamydia group-specific multiplex quantitative PCR will target conserved regions of the ompA gene specific to all chlamydia serovar groups, including the B group (serovars B, E, D, L1, and L2), C group (serovars A, C, H, I, J, K, and L3) or intermediate group (serovars F and G) serovars. This assay will enable quantification of organism load and will be used to direct serovar-specific PCRs to determine serovars present in the specimen, including possibility of mixed serovars [27, 39].

Chlamydia organism load in each specimen will be quantified by comparing the crossing-threshold of each specimen to the crossing-threshold of a standard curve constructed by amplifying different known copy numbers of the ompA gene. The beta globin gene, present at one haploid copy per nucleated cells [40], will also be quantified using qPCR to assess specimen adequacy as well as to measure sampling variability between participants and specimens by correlation with the number of eukaryotic cells collected. The quantity of chlamydia will be divided by the number of eukaryotic cells and expressed as the number of organisms present per 100 eukaryotic cells [40].

Specimens from participants where the same serovar has been detected at baseline and follow up will undergo further discriminatory confirmation of relatedness using sequencing of ompA gene, as well as five house-keeping genes (hctB, CT058, CT144, CT172, and CT682 [pbpB]) utilizing a multilocus sequence typing (MLST) approach [28, 29, 41, 42]. This will include the amplification and sequencing [29, 43, 44] followed by determining MLST type using C. trachomatis MLST database (mlstdb.bmc.uu.se).

DNA sequencing to identify mutation in 23S rRNA gene

Each specimen will be subjected to DNA sequencing (as described above) of PCR-amplified region of the 23S rRNA gene flanking the positions 2057 and 2611. Presence of changes in three locations, A2057G, A2059G and T2611C (E. coli numbering), will be determined as they are associated with increased macrolide resistance [45]. The data arising from this experiment will be correlated with MICs as described below.

Detection of Y chromosome

A separate swab will be collected each week and placed in 400 μl of PBS for Y-chromosome detection as evidence of unprotected sexual exposure. Real-time PCR directed at amplification of a region of the Y chromosome targeting SRY (sex determining region Y) will be conducted [25, 26].

Culture

Endocervical specimens collected by speculum examination will be used for chlamydia culture. The research nurse will place the swab immediately into culture medium, store at −80°C, and courier to the laboratory on dry ice for processing. Chlamydia isolates will be cultured on HEp-2 to propagate each isolate prior to progressing to MICs. Specimens will be prepared by vortexing before addition to 20 hour HEp-2 cultures in antibiotic free DMEM supplemented with 10% serum. Cultures will be incubated at 37°C, 5% CO2 for 48 hours and viable infectious chlamydia elementary bodies will be harvested by mild sonication. A serial dilution of the specimen cultured onto coverslips in 24 well plates with 20 hr growth 2 × 105 HEp-2 cells to determine the viable infectious yield [46].

Minimum inhibitory concentration (MIC)

MIC will be conducted by culturing chlamydia on 20 hr HEp-2 cells. HEp-2 cells will be used for all MIC determinations as they are the most relevant immortalised cell line (cervical), are widely used, and have previously been shown to have consistent performance in MIC assays when compared to other cell lines [35]. Low passage clinical isolates prepared above will be inoculated onto the HEp-2 cells at 2500 and 5000 IFU per well. Cultures will be centrifuged for 1 hr (1200 × g) to ensure equal infection rates. These inoculums will be used as they have previously been reported to be an appropriate dose to predict the MCC (minimum chlamydicidal concentration) and are adequate numbers to accurately assess the inclusions formed [35]. The antibiotic will be titrated into the infected cultures at 4 hours after addition of the chlamydia using a twofold dilution series from 140 g/ml to 0.008 g/ml (in fresh media with cycloheximide 1 g/ml). Antibiotic will be freshly prepared as per the manufacturer’s instructions for each experiment. The numbers of correctly formed inclusions will be determined by methanol fixing the coverslips at 30 hours and stained to allow clear visualisation and counting of the inclusions on the microscope (Chlamydia Cel, Vital diagnostics). The MIC will be defined as the concentration at which no typical inclusions can be seen on the entire coverslip by microscopy examination.

Azithromycin absorption

High-vaginal specimens collected by the research nurse at the day 7 visit will be preserved in 1 ml of 100% methanol and stored at −80°C prior to analysis. Azithromycin levels are thought to remain well above the reported MIC for chlamydia for between 10 to 14 days post-treatment with 1 g dose [30, 31]. Azithromycin levels in high-vaginal material (cells and mucus) will be measured using a validated liquid chromatography – tandem mass spectrometry method (LC-MS/MS) [47–50]. An azithromycin standard curve will be prepared in high-vaginal specimens from a separate individual not exposed to azithromycin, and azithromycin concentrations detected by LC-MS/MS will be normalised to lipid concentrations [50]. Azithromycin concentrations in high-vaginal specimens will be normalised to membrane lipid concentrations and then calculated using the standard curve.

Immunological markers

Blood specimens will be taken at the recruitment visit and from all women who return a positive test of cure test. Serum will be analyzed for chlamydia antibodies titres against peptide and protein antigens using previously described methods [51, 52].

Chlamydia test results and management

Women will be treated with 1 gram azithromycin according to site protocol at the time of recruitment. Those who have a positive test of cure by PCR at day 28, 42 or 56 will be asked to return to the clinic for further treatment of doxycycline 100 mg twice daily for ten days. All laboratory assay results will be forwarded to the study centre and entered into the study database.

Partner notification

At the time of recruitment, the research nurse will explain the importance of treating sexual partners and avoiding sexual contact for 7 days. Multiple strategies to support partner treatment will be explored. Participants will be given a business card that contains the web address for Let Them Know (http://www.letthemknow.org.au/), a partner notification service for sexual contacts of chlamydia and other STIs. Let them know allows individuals to send a message to a partner, either anonymously or named, by SMS or email. If preferred, women may direct their partners to call the research nurse for information and a referral to appropriate clinical care. Women may also consent to have their sexual partner(s) contacted for a telephone consultation and, if appropriate, prescribed azithromycin treatment for partner or mail delivery. This method has been used in several studies at Melbourne Sexual Health Centre and has been very effective at maximizing partner treatment [53, 54].

Data collection

Participants will be asked to complete questionnaires at the time of recruitment and each week until they reach study endpoint. The following data will be collected:

  • Age, height, weight

  • Genital symptoms

  • Reason for attending clinic for their initial test

  • Current medications including contraceptives and concurrent antibiotics

  • Date of last menses

  • Sexual practice data including number of sexual partners, type of sexual partners (casual, occasional, once off or regular), type of sexual contact (vaginal or anal sex), condom use; and

  • Whether partners have been notified and treated

Data analysis

Sample size estimates

To detect a treatment failure risk of 8% with a 95% CI of 5.5%, 10.5%, 450 women are required. If the risk of treatment failure is observed to be 3% (as reported by the earlier meta-analysis [22]), then a sample size of 450 generates a 95% CI of (1.7%, 5.0%). To account for a loss to follow up of 15% based on previous studies [8], 520 women will be recruited.

Statistical analysis

The population risk of treatment failure will be estimated as the observed proportion of women in the sample with treatment failure, with 95% CI calculated assuming an underlying binomial distribution. Multinomial logistic regression will be used to explore the association between the three-level outcome variable (treatment failure, treatment success, re-infection) and risk factors such as the impact of organism load, azithromycin absorption and MIC. One advantage of a multinominal model over two separate logistic regression models (one comparing the prevalence of treatment failure with treatment success, the other comparing the prevalence of re-infection with treatment success) is that the question of whether treatment failure and re-infection share common risk factors can be addressed formally by comparing estimated odds ratios generated by the same model. Linear regression analyses will investigate trends in chlamydia organism load over time and explore any associations with symptoms, chlamydia serovar and participant age. Both linear and multinomial regression models will be implemented with fixed and random regression effects to accommodate the longitudinal and repeated measures nature of the data. All analyses will be conducted in Stata 12.0.

Loss to follow up

The provision of reimbursement payments throughout the study is paramount to maximize retention. Women will be reimbursed with vouchers totalling up to $100 to cover their transport and time costs during the study. This will be broken down into smaller payments, $25 at their first follow up visit, $10 for their day 14 specimen, $10 for their day 21 specimen, $25 for their day 28 specimen and then $30 upon completion of the study (day 56 specimen or earlier if repeat positive). Reimbursements will reflect how many specimens and questionnaires are returned.

Discussion

Distinguishing between chlamydia re-infection and treatment failure is important to focus treatment recommendations and infection control mechanisms. For example, if most repeat infections in this study are found to be re-infections, then strategies to expedite partner treatment are necessary. If many repeat infections are due to antibiotic treatment failure, then international recommendations on chlamydia treatment need to be re-evaluated.

Previous studies that have reported an azithromycin treatment failure rate of 2-3% utilized test of cure by chlamydia culture which, in the last decade, has been replaced by more sensitive PCR testing [22]. Chlamydia treatment studies that have utilized PCR testing have reported much higher treatment failure rates of up to 8%; however these studies were not designed to reliably distinguish between re-infections and treatment failure [10, 17]. Our study is one of the first to use both robust molecular microbiological testing and validated behavioural data so that treatment failure rates can be more accurately estimated. We hypothesize that the treatment failure rate will be closer to 8%.

If our hypothesis is correct and 8%, rather than 2-3%, fail chlamydia treatment with azithromycin, then nearly 3,000 women in Australia and 70,000 women in the USA were inadequately treated for chlamydia in 2011 [2, 55]. Treatment failure will lead to persistent infection with a longer duration of infection and increased risk of complications as well as continued transmission in the population. Further, as chlamydia culture has been rarely undertaken over the last decade, there is little monitoring of chlamydia antibiotic susceptibility, and the role of antimicrobial resistance in treatment failure is unknown [35]. The ability to differentiate between re-infection and treatment failure is a strength of this study. Techniques used will include measuring azithromycin absorption in genital cells, chlamydia culture, genotyping, DNA sequencing and antimicrobial sensitivity determinations of the chlamydia isolates obtained. The results from this study will inform discussions about chlamydia treatment failure and help to establish whether 1 gram azithromycin, the most widely recommended chlamydia treatment internationally, is appropriate. If treatment failure is confirmed to be higher than previously estimated, then further chlamydia treatment trials to suggest changes to international treatment guidelines will be indicated.

Ethics approval

Ethical approval for this study was granted by the Alfred Hospital Ethics Committee and the Southern Eastern Sydney Local Health District Human Research Ethics Committee (Southern Sector).

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Abstract

In vitro susceptibility testing and genotyping were done on urogenital isolates of Chlamydia trachomatis from 3 patients, 2 of whom showed evidence of clinical treatment failure with azithromycin and one of whom was the wife of a patient. All 3 isolates demonstrated multidrug resistance to doxycycline, azithromycin, and ofloxacin at concentrations >4.0 μg/mL. Recurrent disease due to relapsing infection with the same resistant isolate was documented on the basis of identical genotypes of both organisms. This first report of clinically significant multidrug-resistant C. trachomatis causing relapsing or persistent infection may portend an emerging problem to clinicians and public health officials.

Chlamydia trachomatis, a nonmotile, gram-negative obligate intracellular bacterium, is primarily a human pathogen that causes inclusion conjunctivitis, lymphogranuloma venereum, and urogenital tract disease. Genital tract infection with C. trachomatis is asymptomatic in 50%–80% of men and women [1, 2]. In men, symptomatic C. trachomatis infection may manifest as urethritis or epididymitis, whereas in women it often presents as cervicitis, urethritis, salpingitis, or endometritis. Asymptomatic or “silent,” chronic infection in women has been recently recognized as a significant cause of infertility [3]. C. trachomatis infection is the most commonly reported infectious disease in the United States. This may be in part because of its well-known ability to cause asymptomatic infection, thus creating a reservoir that facilitates widespread transmission among multiple partners [4–7]. Until recently, asymptomatic infection and the lack of a simple and sensitive screening test have been barriers to the accurate detection of C. trachomatis infection. With the development of nucleic acid amplification technology, efforts to define the epidemiology of C. trachomatis infection have been renewed.

A well-documented feature of chlamydial infection has been its high rate of recurrence among sexually active populations [8]. However, determining whether the high rate of recurrent disease is due to reinfection or to persistent infection with the same organism has been difficult [9]. Immunity to chlamydial infections is type specific; thus, once an initial infection is resolved, reinfection is believed to be the result of exposure to chlamydial strains that differ in type from the initial infecting strain [10]. In contrast, persistent infections are those in which Chlamydia has entered a metabolically quiescent and noninfectious state; such infections have been demonstrated in both mouse models and cell culture [11–13]. Unless the interval between infections is too short to mount an immune response, persistent infection can presumably be distinguished from reinfection by demonstrating that the chlamydial strains from both the initial and subsequently detected infections have identical major outer membrane protein (MOMP) gene sequences. However, chlamydial genotyping data that aid distinction between reinfection and persistent infection have been limited in reported studies of recurrent infections. Chlamydia-specific DNA and antigens have been found in the upper genital tracts of infertile women, but attempts to culture C. trachomatis from these specimens have generally been unsuccessful, suggesting that persistent infection may not be uncommon [13–15].

Of particular concern in this era of increasing antibiotic resistance is whether persistent infection is a consequence of increasing resistance to standard antimicrobial agents. Although C trachomatis has been historically sensitive to the tetracyclines, macrolides, and fluoroquinolones, recent reports have noted increasing in vitro resistance [16]. However, although in vitro antimicrobial resistance to tetracycline and erythromycin has been described [17], the clinical significance of these findings is unknown.

We describe 2 patients with C. trachomatis infections that persisted after standard treatment and that demonstrated multidrug resistance. To our knowledge, these are the first reported cases of clinically significant C. trachomatis infection resistant to ofloxacin and azithromycin. In addition, for one of the patients, we documented recurrent disease due to relapse with the same resistant isolate, on the basis of genotyping of both organisms. We also describe the wife of one of the patients, who was infected with the same multidrug-resistant strain. We believe these cases may signify an emerging problem with resistant C. trachomatis infections, which could have far-reaching implications for subsequent patient management.

Materials and Methods

Case reports

Patient 1 was a 17-year-old pregnant woman from Wyoming who was seen on 30 May 1997 for her first prenatal visit at 30 weeks of pregnancy. Routine screening for C. trachomatis was positive by ligase chain reaction (LCR) testing of urine. She was given erythromycin on 6 June 1997, but her treatment was changed to amoxicillin, 500 mg 3 times daily, on 7 June 1997 because of gastrointestinal intolerance, and she completed the recommended 7-day course of therapy. Results of the LCR test of urine were positive on 20 June 1997. She denied being sexually active since becoming pregnant and affirmed compliance with treatment. On 3 July 1997 she was given 1 g of azithromycin. Repeat LCR tests of urine were positive on 18 and 28 July 1997. She delivered a normal infant on 23 August 1997, at which time a cervical swab specimen was tested by culture and found to be positive for C. trachomatis. Further clinical information on the mother and child were not available. Because of the persistence of her chlamydial infection, her isolate was sent to the Centers for Disease Control and Prevention (CDC) for susceptibility testing (table 1).

Table 1

Antibiotic susceptibilities of Chlamydia trachomatis isolates from 3 patients and of positive and negative control isolates.

Table 1

Antibiotic susceptibilities of Chlamydia trachomatis isolates from 3 patients and of positive and negative control isolates.

Patient 2 was a 29-year-old heterosexual man from Atlanta who had recurrent episodes of C. trachomatis urethritis that were documented by DNA probe on 13 October 1995, 6 March 1996, 6 October 1996, and 2 and 30 April 1997. Before April 1997, he had clinically responded to standard doxycycline therapy (100 mg orally twice daily for 7 days), with resolution of symptoms. His wife (patient 3) was treated with doxycycline at each episode of his infection, but specimens collected from her were negative by DNA probe throughout this time period (October 1995 to April 1997). On 2 April 1997, he received 1 g of azithromycin for symptomatic urethritis, but he returned with persistent symptoms on 30 April 1997. On 12 May 1997, he described intermittent dysuria and discharge but had no objective evidence of urethritis. Urine and urethral specimens collected on 12 May 1997 were positive for C. trachomatis by polymerase chain reaction (PCR). Human immunodeficiency virus (HIV) antibody tests, serologic testing for syphilis, and culture for Neisseria gonorrhoeae were all negative. He was reevaluated on 21 May 1997 for persistent dysuria, and a urethral Gram's stain revealed urethritis (21 May 1997). Urine and urethral specimens collected on 21 May 1997 were tested at the CDC by PCR and culture of C. trachomatis and were assayed for antimicrobial susceptibility. C. trachomatis culture and PCR results were positive, and the isolate showed resistance to doxycycline, azithromycin, and ofloxacin (table 1). Patient 2 was treated with azithromycin on 21 May 1997, with subsequent resolution of his symptoms. Patient 3 was empirically treated with azithromycin on the same day (21 May 1997) by another clinician on the basis of known contact exposure to her husband but had no C. trachomatis diagnostic testing done at that time. Follow-up urine specimens from both patients and the urethral swab from patient 2 were negative for C. trachomatis by PCR on 23 June 1997. Six months later, patient 3 noted increasing vaginal discharge and sought medical attention in January 1998. At this time, both she and her husband (patient 2) denied any history of marital infidelity during the previous 6 months. Pelvic examination revealed cervicitis, and culture of cervical specimens was positive for C. trachomatis. HIV testing, serologic testing for syphilis, and culture of cervical specimens for herpes simplex virus and N. gonorrhoeae were negative. Also at this time, her husband (patient 2) complained of slight dysuria. A urethral swab specimen from him was also culture positive for C. trachomatis. Genotypes of both isolates and antibiotic susceptibilities were determined at the CDC. Both patients were treated with azithromycin; subsequent PCR testing of urine was negative 4 weeks and 3 months later. Further clinical information and laboratory evaluation of these patients were not available.

PCR

A commercially available PCR test (Amplicor PCR; Roche Diagnostics, Indianapolis) was used to detect C trachomatis in urine and urethral and cervical swab specimens submitted to the CDC. The PCR test was done according to the manufacturer's protocol.

Antibiotic susceptibility testing

Antimicrobial susceptibility testing with doxycycline, azithromycin, and ofloxacin was done on C. trachomatis strains isolated by culture in BGMK cells from urethral or cervical swab specimens that had been transported in M4 transport medium (MicroTest, Snellville, GA). In vitro antimicrobial susceptibility testing was done as described elsewhere [18–20] with minor modifications [16]. Briefly, susceptibility testing was done in cell culture with BGMK cells grown to 90% confluence in 48-well microtiter plates (Costar, Cambridge, MA). Each well was inoculated with 300 μL of the test isolate, diluted in tissue culture medium (Iscove's modified Dulbecco's medium; Life Technologies GIBCO BRL, Gaithersburg, MD) to yield ∼10,000 inclusion-forming units (ifu)/well (or ∼40 ifu/×400 field). This inoculum resulted in infection of ∼20% of the host cells in the monolayer. Microtiter plates were centrifuged at 1750 g for 1 h, after which the supernatants were aspirated. Antimicrobial agents were obtained as standard powders for in vitro susceptibility testing and were reconstituted according to the manufacturers' instructions: doxycycline and azithromycin from Pfizer Laboratories (Groton, CT) and ofloxacin from Ortho Pharmaceuticals (Raritan, NJ). Antimicrobial agents were prepared by 2-fold dilution in Iscove's modified Dulbecco's medium containing 1.8 μg/mL cycloheximide, 584 mg/L l-glutamine, and 10% fetal calf serum and then were added to each well to give a final concentration range of 0.008–4.0 μg/mL. Plates were incubated at 35°C in 5% CO2 for 48–72 h. After incubation, wells were fixed with methanol and stained with a Chlamydia genus—specific monoclonal antibody reagent (Pathfinder; Kallestad Diagnostics, Austin, TX) for identification of the inclusions to determine the MIC and MIC90 of the antimicrobial agents for these isolates. The minimum chlamydicidal concentration (MCC) of antimicrobial agents for these isolates was determined after a subsequent passage of the contents of duplicate unstained wells to a fresh monolayer in antibiotic-free medium. Because we have observed previously that the earliest and most sensitive measure of an inhibitory effect of antimicrobials on C. trachomatis is a dramatic alteration of the morphology and size of the inclusions, the MIC of each agent was defined as the concentration of antibiotic at which no inclusions of typical morphology were identified on direct fluorescent antibody staining after incubation in cell culture [16]. The MIC90 was defined as the concentration of antibiotics at which 90% of typical inclusions were inhibited after incubation in cell culture. The MCC was defined as the lowest concentration of drug that permitted no inclusions to be formed on passage in an antibiotic-free medium.

Resistant and susceptible controls, consisting of C. trachomatis laboratory strains with previously characterized susceptibilities (one fully susceptible [N-8685] and one resistant to doxycycline, azithromycin, and ofloxacin [CDC-TU-486]), were included in each assay. N-8685 is a serovar D strain that was originally obtained from the University of Washington. N-8685 is routinely used as a susceptible control strain in our laboratory because it exhibits MIC and MCC activities against doxycycline, azithromycin, ofloxacin, and clindamycin that are representative of ∼40 laboratory strains and clinical isolates in a collection at the CDC of strains of the 3 most prevalent serovars (D, E, and F). Some of these isolates were in the asymptomatic infection group previously described by our laboratory in a study of >40 C. trachomatis isolates that were evaluated for susceptibilities to commonly used antibiotics, including doxycycline, azithromycin, and ofloxacin [16]. The resistant control strain CDC-TU-486 is a clinical isolate from an 18-year-old woman with cervicitis who was seen at a teen clinic in Atlanta in 1996. She was treated with azithromycin but then was lost to follow-up, so we have no knowledge of whether symptoms resolved or treatment failed.

Each assay was repeated with a lower inoculum size (5000 ifu/well or 10 ifu/×400 field) and with 6.0-mL glass vials containing a 12-mm coverslip used in place of 48-well tissue culture cluster plates to confirm results. Patient isolates determined to be resistant and negative controls without identifiers were sent to the laboratory of R. Jones (Indiana University School of Medicine, Indianapolis) for independent confirmation of the antimicrobial susceptibility results.

Genotyping

C. trachomatis DNA samples were prepared from 500 μL of the culture transport medium from endocervical (patients 1 and 3) or urethral (patient 2) swab specimens by centrifugation for 10 min at 12,000 g followed by resuspension of the pellet in a lysis buffer containing 10 mM Tris-HCl at pH 8.3, 0.05% Triton X, and proteinase K (final concentration, 100 μg/mL) for 1 h. Proteinase K was then inactivated by incubation at 95°C for 30 min. Processed samples were amplified by a nested PCR assay developed in our laboratory at the CDC. The PCR assay used primers specific to the C. trachomatis MOMP gene (omp1). For the primary amplification, 25 μL of the lysate was added to 75 μL of a reaction mix with a final concentration of 10 mM Tris-HCl at pH 8.3, 50 mM KCl, 200 mM of each dNTP, 25 pmol of each primer (CT90UF: 5′-GGACATCTTGTCTGGCTTTAACT-3′ and CT220DR: 5′-GCGCTCAAGTAGACCGATATAGTA-3′), and 2.5 U of Taq polymerase (Perkin Elmer, Foster City, CA). Amplification was done in a thermocycler (Perkin Elmer 9600) under the following conditions: 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 52°C for 1 min, and 72°C for 1 min and a final extension at 72°C for 5 min. For the nested PCR reaction, 5 μL of the first-round PCR product was added to 95 μL of a reaction mix prepared as described above except with the substitution of the primer pair (CT40UF: 5′-ATAGCGAGCACAAAGAGAGC-3′ and CT80DR: 5′-CCAGAAACACGGATAGTGTTATTA-3′). Amplification was done as described above. PCR products were analyzed on a 1.2% agarose gel and were stained with ethidium bromide to confirm amplification and DNA fragment size. Amplified products were purified with a kit (PCR purification kit; Qiagen, Chatsworth, CA). Sequencing was done on an automated sequencer by use of a dRhodamine Terminator Cycle Sequencing kit (model 377; Perkin Elmer Biosystems, Foster City, CA), according to the manufacturer's instructions. Edited sequences were aligned and analyzed with the GCG software package (Genetic Computer Group, Madison, WI). Genotypes were determined by comparison of our sequences with reference C. trachomatis omp1 sequences in the GenBank database.

Serologic testing

Serum samples were tested by microimmunofluorescence (MIF) testing as described elsewhere [21], with commercially available C. trachomatis antigen pools representing D-K and the L1–3 serovars (Washington Research Foundation, Seattle). To label specifically bound anti-chlamydial antibody in the test serum, we used fluorescein isothiocyanate-conjugated goat anti-human IgG monoclonal antibody. Positive and negative reference control sera were included in each assay, and each test was repeated to confirm the result.

Results

The MIC and MCC activities of multiple antibiotics for the chlamydial isolates from our 3 patients are described in table 1. The MIC and MCC activities for all drugs tested were similar for both the high (10,000 ifu/well) and low (5000 ifu/well) inoculum sizes. In all but 1 case (patient 1, azithromycin MIC), MIC and MCC activities of all drugs tested were markedly higher for patient isolates than for a susceptible control C. trachomatis strain. Doxycycline MICs were >4.0 μg/mL for isolates from patient 2 and 3 but only 0.015 μg/mL for the susceptible control strain. The azithromycin MICs ranged from 0.5 μg/mL to >4.0 μg/mL for patient isolates, compared with 0.5 μg/mL for the susceptible control strain. All isolates were also resistant to ofloxacin, with MICs of 2.0 and >4.0 μg/mL for patient isolates and 0.5 μg/mL for the control strain. MIC and MCC activities of isolates from patients 2 and 3 were tested blindly by an independent laboratory and found to be similar to our results.

The clinical isolate from patient 1 was determined to be C. trachomatis subtype (or serovar) E on the basis of the deduced amino acid sequence of the MOMP. Additional clinical material could not be obtained from patient 1. All clinical isolates from patient 2 and his wife (patient 3) were determined to be subtype F. The omp1 genotypes of the isolates from patients 2 and 3 were found to be identical. Furthermore, genotypes of the organisms obtained from patient 2 at initial diagnosis and during his relapse in January 1998 were identical. Interestingly, at the time of relapse, MIF testing of serum samples revealed a titer of anti-chlamydia IgG of ⩽1 : 16 for patient 2 and 1 : 256 for his wife. Measurement of IgM yielded negative results for both.

Discussion

These 3 patients, 2 of whom failed to respond clinically to antibiotic treatment, represent the first well-documented cases of infection with multidrug-resistant C. trachomatis isolates. In addition to alerting us to the likelihood of emerging resistance of C. trachomatis, clinical findings from patient 2 in particular suggest that C. trachomatis infection may remain in a latent state, evident by the negative intervening PCR testing, and then relapse months later, causing symptomatic disease.

The characteristics of antibiotic resistance of C. trachomatis differ significantly from those of other bacteria in several ways. First, because chlamydiae are intracellular pathogens, antimicrobial susceptibility must be determined by their ability to proliferate within a host cell in the presence of varying concentrations of antibiotic. Second, unlike the case for most bacteria, when C. trachomatis organisms are found to be resistant to typically effective antibiotics such as tetracycline, the resistance is not absolute. In fact, C. trachomatis displays what is known as “heterotypic resistance” in vitro; that is, the chlamydial population contains both susceptible and resistant organisms. Thus, although it is possible that all organisms within a population may be capable of expressing resistance, only a small proportion do so at any one time. The marked differences that were observed between the MIC90 and MIC for isolates from patients 2 and 3 illustrate this concept, as does the difference between the MIC and MCC for patient 1 (table 1). Removal of the antibiotic from the medium during testing for the MCC allows the small percentage of organisms that were resistant to the first exposure to antibiotic (MIC) to then multiply and form inclusions. In our laboratory, heterotypic resistance exhibited by some C. trachomatis strains would have been missed unless both MIC and MCC testing were done (CDC, unpublished data). Similar to our findings, Jones et al. [17] reported a tetracycline-resistant isolate of C. trachomatis for which only ∼1% of the population demonstrated resistance. More recently, Lefevre et al. [22] reported an infection with tetracycline-resistant C. trachomatis in a woman who was found to have asymptomatic inflammation on cervical cytologic testing. Their antimicrobial susceptibility studies suggested that only a small proportion (<1%) of organisms were resistant. Although resistant isolates do form inclusions at high concentrations of tetracycline, there are far fewer inclusions formed than at the lower concentrations, suggesting that only a small proportion of organisms within the population express resistance. Furthermore, in strains that exhibit heterotypic resistance, we see many aberrant inclusions, and the proportion of atypical to typical inclusions gradually increases along with a decrease in the overall number of inclusions until all inclusions are aberrant or absent; this may explain the large difference in isolates from patients 2 and 3 between the MIC90 (some inclusions are still typical) and the MIC (no inclusions, or all inclusions are atypical). We believe this to be a result of the fact that the resistance exhibited by individual organisms within the chlamydial population is heterogenous (defined as heterotypic resistance).

Jones et al. [17] noted that the heterotypic resistance shown by isolates of C. trachomatis was apparent only with large inocula (15.0 × 103 ifu/well). This is because with inocula of <5 × 103 ifu, the number of inclusion-forming units that remain viable after exposure of heterotypically resistant strains to the antibiotic is so small that they can easily be missed during visualization under a microscope for MIC determinations. We used inocula of both 5 × 103 and 10 × 103 ifu in our study and found no difference in MIC and MCC activities for any strains tested. However, the susceptible control strain failed to show any resistance at either inoculum size; thus, it is not the inoculum size itself that produces the apparent resistance.

The mechanism responsible for heterotypic resistance in C. trachomatis is not known. It is possible that the multidrug resistance that we observed is phenotypic in nature rather than genotypic, because the molecular targets of azithromycin, doxycycline, and ofloxacin are quite different, and it is unlikely that a single or limited number of gene mutation(s) could be responsible for simultaneous resistance to these diverse agents. We suspect that, rather than being direct resistance, heterotypic resistance may be a by-product of some undefined alteration of the growth rate or life cycle, resulting in a longer phase or intermediate stage that is more refractive to antimicrobial agents. Alternatively, heterotypic resistance may be mediated by some kind of mechanism that excludes the drug from the chlamydial cell or inclusion (e.g., an efflux pump). Future studies are needed to test these hypotheses.

It is possible that the phenomenon of heterotypic resistance is not new but remains largely undetected, because test-of-cure is not routinely done for chlamydial infections and a clinician is not likely to suspect persistence because the rate of “recurrent” infections due to reexposure is so high. Whereas in vitro antimicrobial resistance of C. trachomatis has been recognized since as early as 1980, its clinical significance has been unclear, because patients have responded to the antimicrobial agent nevertheless [17, 23]. Jones et al. [17] reported 5 C. trachomatis isolates that exhibited resistance to tetracycline, doxycycline, erythromycin, and clindamycin but were sensitive to ofloxacin and ciprofloxacin. Of the 5 patients, 3 had negative follow-up cultures (2 after treatment with minocycline, 1 with doxycycline) and 2 were lost to follow-up. Additionally, in vitro C. trachomatis resistance from female genital tract isolates has been described since the early 1990s. Recently, however, we have observed a higher level of resistance, with many typical inclusions seen at first exposure to high concentrations of drug (MIC) instead of only after passage of the strain in antibiotic-free medium (MCC) (authors' unpublished data). It is only recently, however, that the technology for molecular typing of chlamydial strains has evolved to the point that we can distinguish those recurrent infections that are most likely to be persistent. Further study is needed to support or refute the notion that heterotypic resistance of C. trachomatis is emerging and is related to increases in clinical treatment failures.

The recovery of C. trachomatis isolates demonstrating high-level resistance to multiple antimicrobial agents, including doxycycline, azithromycin, and ofloxacin, along with demonstrated clinical failure of antibiotic treatment, with questionable organism eradication in our 2 cases, may portend an emerging problem. In France, a tetracycline-resistant isolate was recovered from a woman who had persistent infection after doxycycline treatment [22]. However, susceptibility testing to azithromycin or ofloxacin was not reported. The extent that failed treatment causes persistent infection with antibiotic-resistant organisms is unknown. Certainly, the technical expertise and time required to determine antimicrobial susceptibilities for C. trachomatis hinder efforts to understand the extent of this problem. Furthermore, routine test-of-cure is not the standard of care. Further efforts are needed to determine the scope of this problem. Although routine testing for cure following treatment of C. trachomatis infection and more aggressive workup of recurrent C. trachomatis disease, including testing isolates for susceptibility, are not practical in most clinical settings, these measures and the possibility of resistance should be considered when treatment with standard therapy has failed.

Recurrent chlamydial genital tract infection is a common and well-documented phenomenon. Its etiology is likely multifactorial, including treatment noncompliance, repeated exposure leading to reinfection, and only partially protective immunity following infection. Recrudescence of a latent C. trachomatis infection may also be a cause of recurrent disease, as has been demonstrated recently. Dean et al. [12] identified 7 women who had multiple positive chlamydial cultures from episodes of recurrent infection with the same serovar over several years. omp1 genotyping suggested long-term persistence of the women's original strains. Latent infection in other chlamydial diseases, such as trachoma and pneumonia, has also been shown to occur [24–27]. In addition, the phenomenon of persistent C. pneumoniae infection has been reported elsewhere, with increased azithromycin MICs and MCCs for the clinical isolate despite patient recovery [28].

It is unclear why a multiresistant C. trachomatis infection appeared to resolve, at least as indicated by the very sensitive PCR assay of urine, in patient 2. It may be that the phenomenon of heterotypic resistance to antibiotics seen in cell culture occurs in vivo, so that only a small percentage of infecting organisms are resistant. Perhaps the majority of the infectious organisms are susceptible to the antibiotic, and the remaining resistant organisms are sufficiently few to be either undetectable by the diagnostic test or, in most but not all cases, eradicated by the host's immune response. In either case, it is plausible that a small number of residual organisms remain in a nonreplicative form until certain conditions restore the organism to its infectious form. Although this scenario is speculative in humans, studies with a mouse model have shown reactivation with viable C. trachomatis following apparent clearance of primary infection when the animals were given an immunosuppressive agent [11]. Additionally, in vitro evidence suggests that low doses of interferon-γ can induce C. trachomatis organisms into a nonreplicative state; interestingly, the organisms can revert back to infectious, viable forms when the interferon-γ is removed [29]. Furthermore, and most relevant to our report, a number of studies have reported that various antimicrobial agents, including penicillin, chlortetracycline, erythromycin, and sulfonamides, can also induce persistence of chlamydial infection in vitro (reviewed in [13]). In these studies, the chlamydiae produced aberrant noninfectious forms in the presence of the antibiotic that reverted to typical and infectious forms once the antibiotic was removed from the culture medium. The variety of different molecular targets of these antimicrobial agents suggests that persistence can be generated by more than one mechanism. The host's immune response also may affect clearance of C. trachomatis infection [30]. However, a much greater understanding of the elements of persistent C. trachomatis infection and the resultant immune response is necessary [13, 31]. The negative MIF serology of patient 2 is a surprising finding, although difficult to interpret. MIF serology, although the most sensitive of the serologic tests for chlamydia, has not been widely studied in men [31, 32]. Among women, positive MIF antibody titers have been shown to correlate with chronic upper genital tract infection [32–34]. In a study of 387 men attending a sexually transmitted disease clinic, 91.3% of men whose culture of urethral specimens was positive were also positive for IgG by MIF, and 80.4% of culture-negative men were positive for IgG [35]. IgM seroreactivity was much less common in both groups of men. It may be that patient 2 was unable to mount an effective immune response; however, subsequent serologic testing would be useful to delineate any future immune response.

There are no data regarding management of clinically resistant C. trachomatis infection. In vitro data suggest that resistance to ofloxacin imparts resistance to other fluoroquinolones, such as ciprofloxacin. Although many of the newer quinolones, including trovafloxacin, sparfloxacin, grepafloxacin, and tosufloxacin, have equal or greater MICs for C. trachomatis, they need to be tested against an ofloxacin-resistant strain [36, 37]. Perhaps a prolonged course of therapy with a standard agent such as doxycycline or azithromycin would be effective against resistant C. trachomatis disease, because such therapy has been efficacious against C. pneumoniae infection in cases of relapse [38].

In conclusion, we present the first 2 cases of clinically significant multidrug-resistant C. trachomatis infection causing relapsing or persistent infection. The additional case we present is the wife of one of these patients, who also had a multidrug-resistant infection but in whom we could not document persistent infection. These cases may suggest an emerging problem to clinicians and public health officials. We believe that increased surveillance for both in vitro resistance and treatment failure should be implemented in select settings to determine the role of heterotypic resistance in transmission and maintenance of C. trachomatis infections.

Acknowledgments

We thank Robert Ellis (Powell, Wyoming) for his generous contributions to this study, Barbara Van der Pol and Bob Jones (Indiana University School of Medicine) for performing susceptibility testing on our clinical isolates and controls, and Teresa Brown (Chlamydia Laboratory, CDC) for her contributions to the manuscript.

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