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Longitudinal Assessment of Infecting Serovars of Chlamydia trachomatis in Seattle Public Health Clinics: 1988–1996

SUCHLAND, ROBERT J. BS*; ECKERT, LINDA O. MD; HAWES, STEPHEN E. PhD; STAMM, WALTER E. MD*

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Sexually Transmitted Diseases: April 2003 - Volume 30 - Issue 4 - p 357-361
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IT IS ESTIMATED that 3 million cases of chlamydial urogenital infections occur annually in the United States, at an estimated cost of $2 billion. 1 The number of reported cases in women has increased dramatically since chlamydial infections became a reportable disease in 1986. In 1999, over 500,000 cases were reported, making Chlamydia trachomatis disease the most common reportable disease in the United States. 1 Most epidemiologic studies of C trachomatis to date have focused on identifying demographic and behavioral risk factors, consistently demonstrating young age and race/ethnicity as significant risk factors. 2

Other studies have evaluated relationships between the infecting C trachomatis serovar and clinical manifestations, demographic factors, and chlamydial inclusion forming units (IFUs) in culture. 3–5 However, large studies evaluating changes in the infecting chlamydial serovars over time in a given population have been infrequent. Such studies could improve our understanding of the potential importance of antigenic variation and acquired immunity in the basic reproductive rates and spread of chlamydial infections.

Chlamydial cultures have been routinely performed for men and women attending the Seattle–King County Public Health Clinics over many years, with use of standardized methods of specimen collection, transport, and culture. Serotyping has also been routinely done, with a standardized methodology. In this study, we used multivariate analyses to explore the relationships between age, gender, and race/ethnicity with serovar, and we analyzed temporal variations of infecting serovars over a 9-year period of time.

Methods

Patient Population

The study population consisted of 7110 women and 4344 men who had their first culture-documented episode of C trachomatis genital infection recorded at any of the Seattle–King County Health Department Sexually Transmitted Disease (STD) clinics over the 9 years (1988–1996) studied. Information on chlamydia tests that were positive in other clinics or before 1988 were not analyzed in this study. Gender, age, race, and collection date were recorded for every patient on a standardized interview sheet. Recurrent episodes of infection were excluded from the analysis.

Specimen Collection and Isolation of C trachomatis

The specimen collection and culture isolation techniques we utilized have previously been described in detail. 3 In brief, urethral specimens were collected from men with small Dacron swabs (Hardwood, Guilford, ME) inserted 1 to 2 cm into the urethra, and female cervical specimens were collected with a larger Dacron swab after cleansing of the cervix with a cotton pledget. Swabs were stored in 1500 μl of transport media at 4 °C and transported within 24 hours to the laboratory. Each specimen was inoculated onto cycloheximide-treated McCoy cells, and chlamydial inclusions were detected with a genus-specific fluorescein-conjugated monoclonal antibody. Specimens producing inclusions were stored at −70 °C for serotyping.

Serotyping

A low-passage microtiter plate culture method was used for rapid immunotyping, with use of a panel of 17 subspecies- and serovar-specific monoclonal antibodies on mature fixed inclusions, as previously described in detail. 3,6 Monoclonal antibodies were run individually or in non-cross-reactive pools to prevent ambiguous cross-reactivity. The wells were then reacted with an FITC conjugate (Sigma, St. Louis, MO) and read with an epifluorescence microscope. The final reaction patterns were compared with prototype strain reactions to determine serovar.

Data Collection and Statistical Analysis

Each patient's age, gender, race, and culture site were provided on the laboratory requisition form accompanying the cultures. These data were entered into the laboratory computer database, as were the culture results and serotyping results as they became available. Univariate comparisons were based on the Pearson chi-square statistic for categorical variables and on the Student t statistic for continuous variables. Because serovars may shift with time, year of infection was included in the multiple regression models, as well as gender, race, and age.

Results

Table 1 summarizes the 11,454 positive cultures by serovar, patient gender, race, age, and year of infection. Consistent with other study findings, serovar E was the most prevalent (32%), followed by F (18%) and D (13%). 7,8 Serovar I was the least common (0.3%); 2.6% of patients had a mixed infection with more than one serovar. In this urban clinic population, 7110 (62%) of the infections detected were found in females and 4344 (38%) in males. Serovar type was associated with race (especially with types E, G, and H;P < 0.001) and year of infection (P < 0.001) and was not associated with gender (P = 0.06). Because of potential interrelationships between all of these factors, multivariate regression analyses were performed.

TABLE 1
TABLE 1:
Prevalence of Chlamydia trachomatis Genital Infections in Seattle, 1988–1996

Because age is the singly most significant factor associated with serovar type, we wanted to further investigate what factors were associated with age. Table 2 utilizes a multivariate regression model to examine associations of gender, race, year of infection, and serovar type with age. A white male infected with serovar type E in 1988 is the reference (serovar type E is the most prevalent and thus was the referent serovar type). The age at intercept for this reference was 24.26 years. A negative regression coefficient implies younger age than the reference at time of infection. A positive coefficient implies an older age, while controlling for the other factors in the model. As expected, females found to have positive C trachomatis cultures were 3.75 years younger than males (P < 0.001). The percentage of infections in females increased over time. Infected African Americans were 0.55 years younger than whites, while Asian and Hispanic patients were older (1.16 years and 0.99 years, respectively) than whites (all differences:P < 0.001).

TABLE 2
TABLE 2:
Multivariate Regression of Factors Associated With Age*

Chlamydia-infected patients became younger as the study progressed. Each year, the mean age of patients presenting to the clinics with C trachomatis infection declined by 0.09 years in this multivariate analysis (P < 0.001). The big change in age distribution (especially among males) was the increase in the percentage of positive infections in those aged 10 to 15 years (1.6% to 3.5% for males [P < 0.001], and 10.8% to 12.9% for females [P > 0.05]).

We found significant differences between age and serovar type after we controlled for other factors in the model. Mixed infections (P < 0.001) were associated with younger age (with serovar E as the reference). Those infected with serovars D, D-, F, G, H, J, and K all had higher mean ages than those infected with serovar E. Serovars G and K were the “oldest” relative to E (2.26 and 1.56 years older than E, respectively;P < 0.001 for both).

In a multivariate logistical regression analysis of serovar type by race, using white race as the reference and adjusting for age, gender, and year of infection, we identified racial differences in the prevalence of infecting serovar. Compared with whites, African Americans were significantly more likely to be infected with serovar typed D- and Ia and less likely to be infected with serovar types D, E, F, G, and K (all P values <0.05.) In Asian patients, serovars type G, H, and K occurred more frequently, while Ia and J were less common than in whites. In Hispanic patients, serovar types E and Ia were isolated more frequently, while serovars D, F, and J were detected less frequently than in whites. No differences in serovar distribution between Native Americans and whites were detected, but this was the ethnic group least well represented in our study population, accounting for only 3.9% of total chlamydial infections.

Figure 1 presents serovar distributions over the 9-year time period in this study. In this population, the major serovar groups were generally quite stable over time; those serovar types that did not significantly change over time represented 75.3% of all serovar types seen during the study period. However, in part because of the large sample size, statistically significant variation in the overall distribution of serovar types occurred between 1988 and 1996. Greater fluctuation over time occurred in some of the minor serovars such as I and K. Figure 2 presents those serovars that had a significant percentage change over the 9-year period, with years 1988 to 1990 as the baseline. Serovars B and E did not have significant changes over time (P = 0.06 for both).

Fig. 1
Fig. 1:
Serovar types over time.
Fig. 2
Fig. 2:
Percentage of change in serovar types over time (with 1988–1990 as baseline).

Similarly, the percentage of chlamydial infections that were due to serovar F varied significantly over time but by 1996 had stabilized to near baseline. Serovar type G increased significantly from baseline, nearly doubling from 1.8% of infections early in the study to 3.0% by the mid-1990s. Because of the large numbers in the study, the absolute change in serovar distribution over time might account for a relatively small proportion of all infections. However, the change within that serovar type could be marked. For example, we found 24 infections with serovar I in 1988 to 1990 and only two in 1994 to 1996. While infections with serovar I are a very small portion of the 11,454 infections, this represents a dramatic decrease in infections with serovar I.

Discussion

The strengths of this investigation included the large number of patients studied, the consistency of laboratory methodology over time, and the use of multivariate analyses to adjust for potential confounders. Our data are consistent with the serovar distributions previously reported from different parts of the world. 7,8 This similarity in relative distribution of serovars over time and over wide geographic regions is striking and overshadows smaller changes in rare serovars.

We identified no major differences in serovar type in relation to gender. Prior studies have reported gender differences in serovar type, 9 but these studies utilized univariate analyses only. Our univariate analysis did show that D, F, and G were more common in men than in women, and these serovars are also found more often in older patients. Because the men in our study were older than the women, gender differences in serovar were no longer significant after adjustment for age.

It was of interest that our population became younger as the study progressed. This may be due to a number of factors. For females, the age of sexual debut is gradually decreasing. 10,11 In addition, more emphasis is placed now on initiating chlamydial screening at an earlier age than it was a decade ago. As data about the high prevalence of chlamydia infections in teenagers becomes public knowledge, more teenagers may be presenting to these public health clinics for screening at a younger age. Additionally, as patients age, healthcare–seeking behaviors may change, and women may seek health care in facilities other than public health clinics.

We found that African American patients with chlamydia were younger than white patients, while Asian and Hispanic patients were older than white patients. These age differences may be a result of behavioral factors such as age of sexual debut, or they may reflect healthcare–seeking behavior.

Using multivariate analyses, we found associations between serovar and age. After adjusting for other factors in the model, we noted that those with infections due to B, Ia, and mixed serovar types were younger, while those with infections due to serovars D, F, H, and K were older. Those with serovar G tended to be the oldest of all. C class serovars might be more common in older patients because immunity against the more prevalent B serovars developed earlier in life. This hypothesis is consistent with previous work demonstrating lower IFUs in C class serovars and C class serovars in older patients. 3

Association of race with serovar was also demonstrated in our multivariate analyses. Serovar Ia was common in African American patients but rarely seen in Asian patients. These data may indicate that there is little mixing of sex partners between these two population groups in our geographic area. In addition, the data are consistent with the possibility of core groups in which one or a few serovars are transmitted frequently within the core population.

Mixed infections were also more common in younger patients. This is likely consistent with a greater likelihood of having multiple partners at a young age, leading to a mixed infection. It is also possible that a longer duration of infection is also seen in younger women, increasing the likelihood of mixed infections. Further, immunity to certain strains may be acquired with exposure, decreasing the likelihood of mixed infections in older patients. Underlying differences in the immune response by racial group could also contribute to racial differences in predominant serovars. For example, the amount of interferon gamma in cervical secretions, a measure of Th1 response, was lower in African American women than in white women in one study. 12 It is also possible that acquired immunity to certain serovars may be more prevalent among different races.

Despite the large database, shortcomings of the analysis should also be mentioned. These data concern presumed first infections only and may not be generalizable to subsequent infections. We also do not know whether infections may have been diagnosed and treated outside of our clinics, potentially causing misclassification of patients as having single episodes of infection. In addition, this is not a true population-based study and may be biased by differences in healthcare–seeking behavior that we were not able to assess. Different racial groups and groups of patients of specific ages may be more likely to present to the clinics sampled for this study. Although these same clinics were studied over the 9-year period, changes in the clinic populations over time may have occurred that could have influenced the outcomes measured.

The extent to which acquired immunity causes changes in serovar distribution over time is unclear. Predominant serovar types in a population may shift as acquired immunity to selected strains becomes more common. This has been demonstrated with gonococcal infections. 13 Perhaps the major change of interest in our study was the significant temporal increase over the 9-year period of the intermediate class serovar G. Interestingly, two recent seroepidemiologic studies suggest that the relatively rare serovar G is a risk factor for the development of squamous cell carcinoma (SCC). One study found more antibodies to the GFK serotype pool in women who developed SCC than in controls, 14 and the other study reported a serological correlation between past C trachomatis infection, particularly that with serovar G, and the development of SCC. 15

Other studies have associated serotype G with symptomatic infections and upper genital tract infections. 16,17 Serovar G has continued to increase in percentage in our population over the period from 1997 to 2001 (unpublished data). Although serovar G is a minor serovar, representing only 2.3% of all our infections between 1988 and 1996, a steady increase in its frequency could have important public health ramifications.

Despite shifts in the prevalence of some of the minor serovar types, the major Chlamydia serovar types in our study population remained surprisingly stable over the 9-year study period. This temporal stability of major infecting serovar types would seem to support continued efforts toward developing a chlamydia vaccine.

References

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