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Effect of Cervical Cytologic Status on the Association between Human Papillomavirus Type 16 DNA Load and the Risk of Cervical Intraepithelial Neoplasia Grade 3

  1. Long Fu Xi1,2,
  2. Nancy B. Kiviat1,
  3. Denise A. Galloway3,
  4. Xiao-Hua Zhou4,
  5. Jesse Ho1 and
  6. Laura A. Koutsky2
  1. 1Department of Pathology, School of Medicine, University of Washington, Seattle, Washington
  2. 2Department of Epidemiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington
  3. 3Fred Hutchinson Cancer Research Center, Seattle, Washington
  4. 4Department of Biostatistics, School of Public Health and Community Medicine, University of Washington, Seattle, Washington
  1. Reprints or correspondence: Dr. Long Fu Xi, Dept. of Pathology, University of Washington, 1914 N. 34th St., Ste. 300, Seattle, WA 98103 (longfu{at}u.washington.edu).
 
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Abstract

Background. Although correlations between cervical cytologic status and both human papillomavirus (HPV) load and histopathologic status are recognized, it is largely undetermined whether the association between the HPV DNA load and the risk of cervical intraepithelial neoplasia grade 3 (CIN-3) differs on the basis of cervical cytologic findings.

Methods. Study subjects were 821 women enrolled in the ASCUS-LSIL Triage Study who tested positive for HPV-16 at entry. Women were followed semiannually for 2 years. The baseline HPV-16 load was measured by real-time polymerase chain reaction.

Results. CIN-3 was confirmed in 286 (34.8%) of 821 women during the 2-year follow-up period. The adjusted odds ratio (OR) of the 2-year cumulative risk of CIN-3 was 1.46 (95% confidence interval [CI], 1.29–1.64) per 1 log10 increase in virus load. The ORs varied from 1.66 (95% CI, 1.16–2.37) for women with normal cytologic findings at enrollment to 0.86 (95% CI, 0.61–1.20) for those with high-grade squamous intraepithelial lesions. Among women with normal cytologic findings at enrollment, the area under the receiver operating characteristic curve for detection of CIN-3 on the basis of the virus load was 0.70 (95% CI, 0.61–0.78).

Conclusion. The HPV-16 DNA load was associated with the risk of developing CIN-3, but the associations varied with cytologic findings at the time of virus load measurement. The clinical usefulness of measuring the HPV-16 load as a means to detect CIN-3 was minimal, even in women with normal cytologic findings.

Quantitative analyses of human papillomavirus (HPV) DNA not only indicate whether HPV infection is present but also reflect the HPV DNA load. Risk associations between high-grade cervical intraepithelial neoplasia (CIN) and elevated virus load have been found in some studies [1–18] but not in other studies [19–24]. Identification of factors that affect the association between the HPV DNA load and the risk of CIN may help clarify these discrepant findings and improve the understanding of the etiologic role of the HPV DNA load in CIN.

Cervical cytologic abnormalities are thought to be manifestations of active HPV infections. HPV DNA load has been shown to be significantly correlated with concurrent presence [14, 25–29] and future development [30, 31] of cytologic abnormalities. A study by Sherman et al. [32] has demonstrated that, among women with CIN grade 3 (CIN-3), the amount of HPV DNA was substantially affected by the numbers of atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) cells present in exfoliative cervical samples. Given the close correlation between findings of cytologic and histopathologic testing of cervical specimens, it is likely that the association between the virus load and the risk of high-grade CIN may differ on the basis of cytomorphologic findings of Papanicolaou (Pap) smears.

Although a few studies have attempted to assess the risk of high-grade CIN associated with HPV DNA load in women with normal results of cytologic tests separately from those with ASCUS or LSIL [4, 30, 33], their findings are limited by semiquantitative measurement of additive virus loads for different HPV types or by insufficient statistical power. Complete understanding of the effects of cervical cytologic status on the association between the virus load and the risk of CIN would be clinically important, because testing for HPV has now been recommended as an adjunct to Pap testing for screening of cervical cancer [34–37].

To determine whether the HPV-16 DNA load can predict the risk of CIN-3, we performed a longitudinal evaluation, stratified by findings of cytologic testing, of the association between baseline HPV-16 DNA load and 2-year cumulative risk of CIN-3 among women in the ASCUS-LSIL Triage Study (ALTS).

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Subjects, Materials, and Methods

Study subjects and design. The ASCUS-LSIL Triage Study was a randomized multicenter clinical trial designed to evaluate strategies for management of women with equivocal or mildly abnormal results of cervical cytologic tests. A detailed description of the ALTS design and study population is presented elsewhere [38, 39]. Briefly, between January 1997 and December 1998, a total of 5060 women were enrolled an average of 2 months after a referral Pap smear revealed ASCUS or LSIL. Participants were randomly assigned to undergo immediate colposcopy, to undergo HPV triage, or to receive conservative management. These arms differed only with respect to which women were referred for colposcopy at enrollment: all women in the immediate colposcopy arm were referred for colposcopy-directed biopsy of visible lesions, women in the conservative management arm were referred only if cytologic testing resulted in a diagnosis of high-grade SIL (HSIL), and women in the HPV triage group were referred only if they tested positive for oncogenic HPV types or if cytologic analysis revealed HSIL. All women, regardless of the study arm, were scheduled for follow-up cytologic analyses and HPV tests at 6-month intervals over a 2-year period. If HSIL was found during follow-up, women were referred a second time for colposcopy and biopsy. At the end of the follow-up period, participants were required to undergo a series of exit procedures, including cytologic analysis, HPV testing, and colposcopic examination with biopsy of any visible lesions. Women who received a diagnosis of CIN grade 2 or higher were promptly treated, usually by a loop electrosurgical excision procedure (LEEP).

An ALTS participant was eligible for the present study if her enrollment cervical sample was positive for HPV-16 DNA by polymerase chain reaction (PCR)-based reverse-line blot. The ALTS protocol was approved by the institutional review boards at the National Cancer Institute and each of the 4 clinical centers involved in the trial. The protocol for this study was approved by the institutional review board at the University of Washington. Of 846 eligible women, 7 were excluded because their enrollment samples were later confirmed to be negative for HPV-16 DNA. We additionally excluded 12 women whose enrollment samples were unavailable for virus load testing and 6 women who had an unsatisfactory cytologic diagnosis at enrollment, leaving 821 in the analysis.

Clinical end point. Pathologists at the clinical centers made the initial diagnosis for ALTS subjects on the basis of cervical cytologic and histologic findings, and diagnoses were reviewed by a panel of expert pathologists for quality control and safety monitoring. Histologic diagnosis was made on the basis of analysis of tissues obtained by biopsy, endocervical curettage, and/or LEEP. The most severe diagnosis was used if >1 tissue block was obtained and examined at a single visit. We used the 2-year cumulative diagnosis of CIN-3 as the clinical end point in order to overcome potential study arm-related missing and/or delayed diagnoses of CIN-3 at enrollment [40]. For women with >1 CIN-3 diagnosis during the study period, only the first diagnosis was counted. Cervical cytologic and histologic data used in the present study were from women whose diagnosis was made by the panel of expert pathologists. The results did not change appreciably when diagnoses made by the clinical center pathologists were used as clinical outcomes (data not shown).

Quantification of HPV-16 DNA load. A multiplex real-time PCR was performed on all HPV-16-positive enrollment samples for quantification of HPV-16 DNA. The assay was set up in a reaction volume of 25 µL with the TaqMan Universal PCR Master Mix kit (Applied Biosystems). Sequences of primers and the probe for the HPV-16 E7 gene were as follows: forward primer: nucleotide positions 700–720, 5′-CCG-G-A-C-A-G-A-G-C-C-C-A-T-T-A-C-AAT; reverse primer: nucleotide positions 782–762, 5′-ACG-T-G-T-G-T-G-C-T-T-T-G-T-A-C-G-CAC; and florescence-labeled probe: nucleotide positions 733–760, 5′-FAM-TGT-T-G-C-A-A-G-T-G-T-G-A-C-T-C-T-A-C-G-C-T-T-C-GGT-TAMRA. The primers and probe for the human β-actin gene were commercially available (Applied Biosystems). Amplification was performed using Applied Biosystems 7900 HT sequence detection system, with a cycling program of holding at 50°C for 2 min and at 95°C for 10 min, followed by a 2-step cycle of 10 s at 95°C and 1 min at 60°C for 40 cycles.

Two log-phase 5-point standard curves were implemented in each set of the assay, one for HPV-16 and the other for cellular DNA. The number of HPV-16 E7 copies was normalized according to the input amount of cellular DNA (β-actin). Each sample was assayed in triplicate, and a mean of 3 measurements was used for analysis. As assessed by 1-way analysis of variance with random effects, the estimated reliability of the mean value of the triplicate measurements was 0.981, with an intraclass correlation of 0.947 (95% confidence interval [CI], 0.941–0.954). The virus load was expressed as the mean of log10 HPV-16 E7 copy number per nanogram of cellular DNA.

HPV-16 E7 DNA was undetectable by real-time PCR in 61 samples that were initially positive by PCR-based reverse-line blot. The negative test results were not explained by the presence of potential inhibitors or the lack of sufficient sample inputs, because the amounts of cellular DNA in samples with and samples without detectable HPV-16 E7 DNA were similar (data not shown). Because the samples with negative results might have contained levels of DNA that were below the lower limit of detection, a load of 1 copy per nanogram of cellular DNA (equal to a log10-transformed value of 0) was assigned to each of these samples. Similar results were obtained when these samples were excluded from the analysis. For simplicity, these results are not presented.

Statistical analysis. HPV-16 DNA load was treated as a continuous variable. The normality of the distribution of the log10-transformed virus load was assessed by a Q-Q plot. The 2-year cumulative risk of CIN-3 associated with the baseline HPV-16 DNA load was evaluated using unconditional logistic regression [41]. The effects of cervical cytologic findings at enrollment on the risk association were assessed by stratified analyses while controlling for age at enrollment, current use of hormonal contraceptives, lifetime number of male sex partners, and study arm.

A receiver operating characteristic (ROC) analysis was used to evaluate whether testing for baseline HPV-16 DNA load would provide added value to identification of women with 2-year prevalent CIN-3 [42]. The ROC curves, stratified by cytologic findings, were constructed by computing the true-positive rate against the false-positive rate for various virus loads. We used R to implement the algorithms proposed by Delong et al. [43]. The accuracy of the test for discriminating women with from those without CIN-3 was assessed by the area under the ROC curve, computed using maximum likelihood estimates to fit a smooth curve to all data points. These areas represent the probabilities that the CIN-3 status of randomly drawn pairs of women with and women without CIN-3 can be accurately classified by the test. An area of 1 represents a perfect test (100% sensitivity and 100% specificity), and an area of 0.5 represents a worthless test.

A linear trend of increasing baseline HPV-16 DNA load with increasing severity of concurrent cervical cytologic findings was tested by assigning scores to cytologic diagnoses and treating this scored factor as a continuous variable. The Student t test or 1-way analysis of variance, whichever was appropriate, was used to compare HPV-16 DNA loads on the basis of demographic characteristics, sexual behavior, study arm, HPV-16 variants, and coinfection with other types of HPV. All statistical tests were 2-sided and had an α of 0.05.

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Results

The mean log10 number of HPV-16 E7 copies per nanogram of cellular DNA was 2.78 (95% CI, 2.69–2.87) for 821 women in whom HPV-16 infection was initially detected at enrollment. As shown in figure 1, the points for the observed values against values from a normal distribution were clustered around a straight line, suggesting that the distribution of these log10-transformed values was approximately normal. Of these 821 women, 503 had a referral cytologic diagnosis of ASCUS, and the rest had a referral cytologic diagnosis of LSIL. Abnormal cervical cytologic findings at enrollment were diagnosed in 665 women (81.0%), including 240 (29.2%) with ASCUS, 271 (33.0%) with LSIL, and 154 (18.8%) with HSIL. The baseline HPV-16 DNA load increased significantly with increasing severity of concurrent cytologic abnormalities in women with a referral diagnosis of either ASCUS or LSIL (P<.001, by the test for trend, for both) (table 1). The trend remained similar when the analysis was restricted to women who did not have a diagnosis of 2-year prevalent CIN-2–3 (data not shown).

Figure 1
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Figure 1

Normal Q-Q plot of the log10-transformed human papillomavirus type 16 (HPV-16) DNA load for women with baseline infection. Observed values are plotted against values expected from a normal distribution. If the sample is from a normal distribution, the points cluster around a straight line.

Figure 2
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Figure 2

Receiver operating characteristic (ROC) curves of quantitative analysis of baseline human papillomavirus type 16 (HPV-16) DNA load for detection of 2-year prevalent cervical intraepithelial neoplasia grade 3. The area under the ROC curve was 0.63 (95% confidence interval [CI], 0.59–0.67) for the entire group of HPV-16-positive women, 0.70 (95% CI, 0.61–0.78) for those with normal cytologic findings at enrollment, 0.62 (95% CI, 0.55–0.69) for those with atypical squamous cells of undetermined significance (ASCUS), 0.60 (95% CI, 0.55–0.65) for those with low-grade squamous intraepithelial lesion (LSIL), and 0.46 (95% CI, 0.38–0.54) for those with high-grade squamous intraepithelial lesion (HSIL).

Table 1
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Table 1

Baseline human papillomavirus type 16 (HPV-16) DNA load, by referral and enrollment cervical cytologic findings.

Among women with normal cytologic findings at enrollment who did not have CIN-2–3 during the study period, an increased baseline HPV-16 DNA load was associated with an age of <25 years (P=.007), current use of hormonal contraceptives (P=.006), and ≥6 lifetime number of male sex partners (P=.03). There were no appreciable differences in HPV-16 DNA load with respect to race, smoking status, number of Pap tests in the past 5 years, presence of HPV-16 variants, coinfection with other HPV types, and study arm (table 2).

Table 2
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Table 2

Baseline human papillomavirus type 16 (HPV-16) DNA load, by demographic and clinical characteristics, of women with normal cytologic findings at enrollment who did not develop cervical intraepithelial neoplasia grade 2 or 3 during the 2-year study period.

CIN-3 was histologically confirmed in 286 (34.8%) of 821 women during the 2-year study period, including 30 (19.2%) of 156 who had had normal cytologic findings at enrollment, 86 (35.8%) of 240 who had received a diagnosis of ASCUS, 76 (28.0%) of 271 who had received a diagnosis of LSIL, and 94 (61.0%) of 154 who had received a diagnosis of HSIL. The odds ratio (OR) of the 2-year cumulative risk of CIN-3 was 1.46 (95% CI, 1.29–1.64) per 1 log10 increase in virus load, after adjustment for age at enrollment, current use of hormonal contraceptives, lifetime number of male sex partners, and study arm (table 3). The increase in 2-year cumulative risk was statistically significant for women with normal cytologic findings (adjusted OR, 1.66; 95% CI, 1.16–2.37), ASCUS (adjusted OR, 1.51; 95% CI, 1.17–1.94), or LSIL at enrollment (adjusted OR, 1.34; 95% CI, 1.07–1.69), although the magnitude of the risk was less substantial in the latter group. There was no appreciable association of 2-year cumulative risk with the baseline HPV-16 DNA load among women with HSIL at enrollment.

Table 3
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Table 3

Risk of 2-year prevalent cervical intraepithelial neoplasia grade 3 (CIN-3) associated with baseline human papillomavirus type 16 (HPV-16) DNA load, by baseline cytologic finding.

Approximately 23% of 821 women exited the trial before their last scheduled visit. The mean number of visits was 4.37, 4.31, 4.52, and 4.44 for women with normal cytologic findings, ASCUS, LSIL, and HSIL, respectively, at baseline. The log10-transformed HPV-16 DNA load was similar between women who completed the last visit and those who did not (2.73 vs. 2.79 HPV-16 E7 copies per nanogram of cellular DNA; P=.54). Additional adjustment for the number of follow-up visits did not considerably change the overall risk estimate (adjusted OR, 1.47; 95% CI, 1.30–1.66) or the risk estimates stratified by cytologic findings (adjusted OR, 1.68 [95% CI, 1.17–2.43] for women with normal cytologic findings, 1.55 [95% CI, 1.19–2.00] for those with ASCUS, 1.35 [95% CI, 1.07–1.71] for those with LSIL, and 0.89 [95% CI, 0.62–1.27] for those HSIL). The risk estimates remained similar when the analysis was restricted to women who attended the last scheduled visit (data not shown).

A total of 232 women tested positive for HPV-16 alone at enrollment. The mean (±SD) of the log10-transformed virus loads were 3.13±1.04 and 2.82±1.45 HPV-16 E7 copies per nanogram of cellular DNA for women with and women without CIN-3, respectively, who tested positive for HPV-16 alone; and the log10-transformed virus loads were 3.20±1.05 and 2.47±1.41 HPV-16 E7 copies per nanogram of cellular DNA for women with and women without CIN-3, respectively, who tested positive for multiple types of HPV. The increased risk of CIN-3, although not statistically significant, remained associated with an elevated virus load in women who tested positive for HPV-16 alone (adjusted OR, 1.19; 95% CI, 0.96–1.47). The impact of cervical cytologic findings on the risk association was similar between women with and those without coinfection with other HPV types; that is, the association was evident in women with normal cytologic findings or ASCUS (although it was not statistically significant in those positive for HPV-16 alone) but not in women with HSIL (data not shown). Additional adjustment for coinfection with other types did not appreciably alter the risk estimates (data not shown).

Although an elevated HPV-16 DNA load was significantly associated with the risk of CIN-3 for all women except those with HSIL, the clinical value of testing for virus load at a single point in time to discriminate women with from women without CIN-3 was minimal. As shown in figure 2, the area under the ROC curve for detecting 2-year prevalent CIN-3 by testing for baseline HPV-16 DNA load was 0.63 (95% CI, 0.59–0.67) for HPV-16-positive women overall. Although test results were slightly more accurate for women with normal cytologic findings at baseline (area under the ROC curve, 0.70; 95% CI, 0.61–0.78) than those for women with ASCUS (area under the ROC curve, 0.62; 95% CI, 0.55–0.69) or LSIL (area under the ROC curve, 0.60; 95% CI, 0.55–0.65), the difference was not statistically significant (P=.25 and P=.10, respectively). For women with HSIL at enrollment, the area under the curve was only 0.46 (95% CI, 0.38–0.54), which is no better than a random classification. The cutoff values of HPV-16 DNA loads for a given sensitivity varied with baseline cervical cytologic findings, e.g., corresponding to a sensitivity of 95%, the values of the log10-transformed virus loads were 0.65, 1.81, and 2.05 for women with normal cytology, ASCUS, and LSIL, respectively, which resulted in specificities of 34.70%, 22.73%, and 24.10%, accordingly.

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Discussion

In this study of ALTS participants with HPV-16 infection at enrollment, we found that the amount of baseline HPV-16 DNA increased as a function of the 2-year cumulative risk of CIN-3. The virus load-related risk was not explained by factors previously shown to be related to the risk for cervical neoplasia, including age, use of hormonal contraceptives, and lifetime number of sex partners or by different managements of the study arms. Because the HPV-16 DNA load was measured without knowledge of any clinical or epidemiologic information and the diagnosis of cervical lesion was extensively reviewed by a panel of expert pathologists, potential bias in ascertainment of exposure and outcome was minimized. It is also unlikely that the risk association resulted from differential access to health care, because ALTS participants came from a screened population of women who were referred to the trial because of a mildly abnormal results of a Pap smear; in this well-controlled trial setting, the majority of participants were examined and followed and their condition diagnosed according to criteria of the standardized protocol.

The risk association observed in this large-scale longitudinal setting—perhaps the largest so far of studies with similar objectives—agrees with previous reports that higher HPV DNA loads were related to the prevalence of [1, 7, 9–11] and progression to [2–4, 12, 13] high-grade CIN. This study further extends these reports by showing that HPV-16 DNA load-associated risk of CIN-3 varied with cervical cytologic findings at the time of virus load measurement, from a substantial risk increase for women with normal cytologic findings to a lack of association for women with HSIL.

One explanation for the effect of cervical cytologic status on the association between the virus load and the risk of CIN-3 could be that the number of abnormal epithelial cells in cervical exfoliative samples increases as the severity of cervical cytologic status increases and that the increased number of cells that reflect active HPV infections makes the virus load contributed by CIN-3 lesions less apparent. As shown in a previous study of women with CIN-3, the HPV DNA load was highly correlated with the number of ASCUS and LSIL cells present in cervical samples [32]. Another possibility is that the effect of cervical cytologic status on the association between the virus load and the risk of CIN-3 is mediated by the size of the cervical lesion evaluated. The cervical lesion size is an important determinant of virus load detected in cervical samples [44]. As the size of cervical lesions increase, so does the severity of cervical cytologic status; the increase is usually more substantial for lesions that surround the site of CIN-3 [45]. These nearby lesions made important contributions to the virus load in cervical samples [32], because specimens obtained by scraping favor collection of cells shed from these extensive lesions, even in the presence of CIN-3. Consequently, variations in the risk association with respect to cervical cytologic status could be explained by differences in the size and severity of the surrounding lesions. Furthermore, because of aggressive follow-up, the majority of CIN-3 lesions in ALTS participants were small. Thus, a fraction of the HPV-16 load contributed by cells shed from these lesions is more likely to be masked by an increasing severity of cytologic status.

In view of the risk association in women with normal cytologic findings at baseline, one would expect that determination of the HPV-16 DNA load would be useful for predicting the risk of underlying CIN-3. As indicated by the ROC analysis, however, the added value was too small to be clinically useful. Even among women with normal cytologic findings, the status of only ∼70% of randomly drawn pairs of women with and women without CIN-3 would be accurately classified by the test. A rough guide for classifying the accuracy of a good diagnostic test requires an area of at least 0.85 in the traditional academic point system. Possible explanations for a lack of clinical usefulness of the test include wide variations in virus load within disease grades and a substantial overlap of virus loads between women with and women without CIN-3 [4, 7, 32].

Nonetheless, the clinical implication of the present study is that, for a given amount of HPV-16 DNA, the risk of developing a histopathologic abnormality varies according to the cervical cytologic status. Because a Pap smear supplemented by an HPV test is likely to continue to be the major approach for cervical cancer screening, physicians could one day apply this implied finding to their clinical management of women with HPV-16 infection. It should be cautioned, however, that the impact associated with the HPV-16 DNA load may not be generalizable to other HPV types because the associations between the risk of CIN-3 and the virus load differ according to HPV type [28, 46].

Several limitations of the study should be addressed. Because women in the ALTS had ASCUS or LSIL detected during referral cytologic analysis, normal cytologic findings, ASCUS/LSIL, and HSIL at enrollment, when the amount of HPV-16 DNA was measured, can also be interpreted as cytologic regression, persistent mild abnormality, and cytologic progression, respectively. Thus, the risk associated with a given quantity of HPV-16 DNA in women with a particular cytologic status may not be generalizable to the risk associated with the same HPV-16 DNA load in general populations. In addition, an extent of misclassification of disease status that may bias the effects of cervical cytologic status down towards was less substantial in this, as compared to general, population, because in ALTS, cervical cytology and histology were initially diagnosed by the clinical center pathologists and then meticulously reviewed by a panel of export pathologists. Second, although a rigorous tracking system was implemented in the ALTS, ∼23% of HPV-16-positive women exited the trial before their final scheduled visit. Biases in assessment of the 2-year cumulative risk could have been introduced if a loss to follow-up had been differentially related to the virus load. In opposition to this, the amount of HPV-16 DNA at baseline was highly comparable between women who attended the last visit and those who did not, and the additional adjustment for the number of follow-up visits did not change the risk estimates substantially. Finally, we recognize that women included in this study tested positive for HPV-16 at enrollment and that no distinction was made between new and previously existing infections. However, although the virus load may fluctuate during the course of natural infection, the comparison of the virus load at the same time point between the groups is still valid [47].

In summary, a higher HPV-16 DNA load was associated with an increased risk of CIN-3, but the association varied with the cervical cytologic findings at the time of virus load measurement, which underscores the importance of cervical cytologic status in the interpretation of the HPV-16 DNA load-related risk of having an underlying histopathologic condition. However, the clinical usefulness of determining the HPV-16 DNA load at a single point in time for detection of CIN-3 was minimal, even in women with normal cytologic findings.

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Acknowledgments

We thank the ALTS Group, for providing the biological specimens and HPV typing results, and Dr. Kathrin Jansen, for providing the HPV-16 DNA standard.

This study was part of a project ancillary to the ALTS clinical trial, but the findings reported here are our own and not those of the ALTS Group.

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Footnotes

  • Potential conflicts of interest: L.A.K. receives research funds from Merck Research Laboratories. All other authors: none reported.

  • Presented in part: 24th International Papillomavirus Conference and Clinical Workshop, Beijing, China, 3–10 November 2007 (abstract PS 26–1).

  • Financial support: Public Health Service (grant CA 84396 to L.F.X.).

  • Received December 8, 2007.
  • Accepted February 12, 2008.
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  1. J Infect Dis. (2008) 198 (3): 324-331. doi: 10.1086/589715
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