Accepted Manuscript

Trichomonas vaginalis is most frequently detected in women at the age of peri-/pre-

menopause--an unusual pattern for a sexually transmitted pathogen

Shlomo M. Stemmer, MD, MS, MBA, Eli Mordechai, Ph.D., Martin E. Adelson, PhD,
Scott E. Gygax, PhD, David W. Hilbert, PhD

PII: S0002-9378(17)32481-X
DOI: 10.1016/j.ajog.2017.12.006
Reference: YMOB 11975

To appear in: American Journal of Obstetrics and Gynecology

Received Date: 4 August 2017

Revised Date: 7 November 2017
Accepted Date: 6 December 2017

Please cite this article as: Stemmer SM, Mordechai E, Adelson ME, Gygax SE, Hilbert DW, Trichomonas
vaginalis is most frequently detected in women at the age of peri-/pre-menopause–an unusual pattern
for a sexually transmitted pathogen, American Journal of Obstetrics and Gynecology (2018), doi:
10.1016/j.ajog.2017.12.006.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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1 Trichomonas vaginalis is most frequently detected in women at the age of peri-/pre-menopause--

2 an unusual pattern for a sexually transmitted pathogen

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4 Shlomo M. Stemmer, MD, MS, MBA,a, b; Eli Mordechai, Ph.D. b, Martin E. Adelson, PhD b;

5 Scott E. Gygax, PhD b; and David W. Hilbert, PhD b

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7 Virtua Hospital, Department of Obstetrics and Gynecology, Voorhees, NJ 08043

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8 Medical Diagnostic Laboratories, Hamilton, NJ 08690

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10 Running title: Detection rates of T. vaginalis infection by age and state
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12 Corresponding author: dhilbert@mdlab.com

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14 Keywords: trichomoniasis, chlamydia, metronidazole, qPCR

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16 Conflict of interest and financial disclosure: This study was funded by Medical Diagnostic

17 Laboratories. Shlomo M. Stemmer, MD, is a paid consultant for Medical Diagnostic

18 Laboratories (MDL).

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19 Abstract

20 Background: Trichomonas vaginalis is the most common non-viral sexually-transmitted

21 infection (STI). However, as it is not a reportable disease in the United States, there is limited

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22 information on the age of infected individuals and their geographic distribution.

23 Objective: To evaluate the detection rates of Trichomonas vaginalis infection compared to

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24 Chlamydia trachomatis by age and state in a commercial laboratory setting .

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25 Methods: Quantitative Real-time PCRs (qPCRs) were used to detect the presence of T. vaginalis

26 and C. trachomatis in cervico-vaginal samples obtained during gynecological exams. A total of

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27 1,554,966 and 1,999,077 samples from females age 10 to 79 were retrospectively analyzed for

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the presence of T. vaginalis and C. trachomatis, respectively.
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29 Results: The highest detection rate of an infection with T. vaginalis was between ages 47 to 53.

30 For C. trachomatis the highest detection rate was between ages 14 to 20. T .vaginalis detection
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31 rate distribution by age shows a bimodal pattern with first peak at ages 21-22 (4.0-4.1%) and a
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32 higher second peak at ages 48 to 51 (5.4 -5.8%). C. trachomatis prevalence distribution by age

shows a maximum peak of 8.6% at age 17 and a rapid decline thereafter. In general, the detection
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34 rates of both pathogens were higher in the southeast and in states along the Mississippi River
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35 Valley than in other parts of the country. A nucleotide polymorphism associated with T.
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36 vaginalis metronidazole resistance (ntr6TVK80STOP) was not associated with age and was found

37 most frequently in specimens from New Mexico and Vermont.

38 Conclusions: The detection rate of T. vaginalis does not appear to decrease with age as observed

39 for C. trachomatis and reaches maximum rates in women ages 48 to 51 years old. The

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40 geographic distribution of T. vaginalis appears to broadly similar to that of other STIs. The

41 ntr6TVK80STOP polymorphism did not have a specific association with age or geography.

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58 Introduction

59 Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is the world’s most common

60 non-viral sexually transmitted infection (STI), with an estimated 143 million new cases per year

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61 globally 1. In the United States (US) it is estimated that there are 3.7 million new infections per
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62 year and the overall prevalence among reproductive-aged women is 3.1% 3. Populations with

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63 an elevated prevalence of T. vaginalis infection include African American women (13%) 3,

women attending STI clinics (26%) 4 and incarcerated women (14 - 32%) 5, 6. In comparison, the

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65 prevalence among non-Hispanic white women is only 1.3% ³. Symptoms in women include a

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66 malodorous green-yellow vaginal discharge, vulvar irritation and colpitis macularis (i.e.

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"strawberry cervix"). However, up to 85% of infected women are asymptomatic
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68 asymptomatic women at STI clinics were infected with T. vaginalis , indicating that
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69 asymptomatic colonization is common. Complications associated with T. vaginalis infection
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70 include adverse pregnancy outcomes (e.g. pre-term labor and low birth weight infants) and
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71 increased susceptibility to HIV infection 9. While traditionally diagnosed by microscopy and/or

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72 culture, molecular methods, such as quantitative real-time polymerase chain reaction (qPCR)
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73 have proven to be much more sensitive . Most infections can be successfully treated with
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74 metronidazole 7, although resistance has been reported in 4.3% - 9.6% of isolates 12, 13.
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75 Most STIs have the highest prevalence among younger individuals and decrease in frequency
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76 with increasing age. For example, the prevalence of chlamydia is 3.4% among women aged 20-

77 24 years and decreases to 1.2% at ages 25-30 and to < 1% thereafter 14. Similarly, the prevalence

78 of gonorrhea is the highest between the ages 15-24 (≥ 0.5%) and decreases to ≤ 0.2% thereafter
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79 . However, three studies evaluating the prevalence or detection rate of T. vaginalis have found

80 a very different pattern. Sutton et al. found that women 30-40 years of age had a prevalence of ≥

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81 3.6% compared to ≤ 2.3% for women 14-29 years of age 3. Ginocchio et al. found the highest
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82 rate (12%) in women ≥ 40 years of age compared to ≤ 8.5% in women < 40 years of age .

83 Finally, Stemmer et al. found the highest detection rate (6.3%) was in women 46-55 years old 16.

84 These studies evaluated a relatively modest number of specimens—3,754; 7,593 and 78,428,

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85 respectively 3, 15, 16. Lastly, a recent study of 9,186 women in the United Kingdom found that age

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86 was an independent risk factor for T. vaginalis infection 17.

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87 According to the Centers for Disease Control and Prevention (CDC), the highest prevalence of

88 C. trachomatis is found in Alaska and the Southeastern US, with rates exceeding 600 cases per

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89 100,000 individuals in Mississippi, Louisiana and Alabama 14. Lower rates were reported for the

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mid-west, western and northeastern states 14. Detection rates using nucleic acid amplification in a

91 commercial laboratory setting were also highest in specimens from the Southeastern US (7.9%)
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92 in comparison to other regions (≤ 6.6 %) 15. Similar results were obtained for T. vaginalis, with a

93 14% detection rate in specimens from the Southeastern United States and ≤ 9.5% detection rates
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94 in specimens from other parts of the country 15.

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95 The goals of this study were to evaluate the relationship between age and geography with T.
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96 vaginalis detection using a very large collection of specimens. As a comparison, we also

97 evaluated C. trachomatis detection rates over a similar time frame. Lastly, we assessed the
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98 detection rate of a nucleotide polymorphism associated with T. vaginalis metronidazole

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99 resistance 18.

100 Methods

101 Cervico-Vaginal Specimens

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103 Cervico-vaginal sample specimens sent to Medical Diagnostic Laboratories, L.L.C. (MDL), from

104 January 1, 2014, to December 31, 2014, by practicing obstetricians and gynecologists for

105 diagnostic testing for T. vaginalis (n = 1,554,966) and C. trachomatis (n = 1,999,077), and were

106 analyzed using quantitative real-time polymerase chain reactions. Specimens were processed

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107 using a computer-generated number to protect patient anonymity, and clinical information was

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108 not available for this retrospective study. Only one specimen was analyzed per patient. Age, state

109 of residence, and positivity status for T. vaginalis and C. trachomatis were the only information

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110 used for this analysis. The specimens were obtained from a mixture of urban, suburban and rural

111 practices. With regards to payment for T. vaginalis testing, 45.9% of patients had commercial

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112 insurance and 31.1% had government-provided insurance. Patients’ dates of birth were obtained

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from their health care provider and confirmed by MDL before submission of payment claim to

third party payers or receipt of cash payment. Cervico-vaginal specimens were collected by the
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115 evaluating physician using a OneSwab® (Medical Diagnostic Laboratories) and placed in 2 ml of
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116 UTM™-RT transport medium (Copan Diagnostics) and were transported to the laboratory within
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117 24 hours for analysis.

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119 Molecular Analysis

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121 DNA was extracted from the specimens using the X-tractor Gene automated nucleic acid
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122 extraction system (Corbett Robotics) according to the manufacturer's instructions. C. trachomatis

123 and T. vaginalis were detected using qPCRs performed using a CFX384 real-time thermocycler

124 (Bio-Rad). These assays have been validated as having a sensitivity of ≤ 100 copies of template

125 and specificity against a broad panel of bacteria, fungi and viruses found in the lower female

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126 genital tract. All 384 well reaction plates included four separate no-template control wells and

127 six positive control wells with 102, 104 or 106 copies of the qPCR target template. Each assay

128 evaluated 400 nl of extracted DNA. The assay used to detect the ntr6TV(K80STOP)

129 polymorphism associated with T. vaginalis metronidazole resistance has been described

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130 previously 18.

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132 Statistical Analysis

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134 Positivity rates, normality tests and correlation analysis were performed using GraphPad Prism

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135 5.01. The D'Agostino and Pearson omnibus normality test was used to evaluate the distribution

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of positivity rates by age. Ninety-five percent confidence internals were determined using the

asymptotic (Wald) method based on normal approximation. State-by-state and age-by-age

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138 pairwise comparisons were performed by Fisher's exact tests with False Discovery Rate to
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139 control for multiple comparisons using R 3.4.2 and the package "RVAideMemoire". An alpha of
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140 0.05 was used to determine significance. Heat maps of the United States were generated using

141 Plotly 2.0.

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143 Results
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144 We first analyzed the positivity rates for T. vaginalis as a function of age (Fig. 1). We observed

145 that the positivity rate steadily increased from 10 years (0.5%) through 22 years (4.1%) and then

146 slowly decreased through 31 years (3.6%). After this point, the positivity rate begins to slowly

147 and steadily increase again until it reaches a peak at age 48 (5.8%) and then begins to drop,

148 although it remains > 5% through 53 years and > 4% through 60 years. We compared the

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149 detection rate for specimens from patients at each to every other age (Fig. 2). In general, the

150 detection rates for specimens from patients aged 40 - 55 were greater than those from patients

151 aged < 40 (upper left quadrant) and those from patients aged 60 - 80 were less than those of

152 patients aged < 60 (lower right quadrant). We next evaluated specimens for the positivity rate of

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153 C. trachomatis as a function of patient age (Fig. 3). From 10 years (the lowest age evaluated in

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154 the study) through approximately 27 years we observe a normal distribution (D'Agonisto and

155 Pearson omnibus normality test, P > 0.05), beginning with very low positivity rates (≤ 0.9% at

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156 10-11 years), peaking at 8.6% at 17 years and then dropping down to 2.0% by 27 years. After

157 this point we observe a steady slow decline from 27-46 years, with positivity rates decreasing

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158 from 2.0% to 0.4%. From 47-79 years the positivity rate was ≤ 0.3%. We also compared the

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detection rate for specimens from patients at each age to each other (Fig. 4). Overall, the

detection rates were greater for specimens from patients aged 16 - 30 than every other age group
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161 evaluated.
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162 The specimens analyzed in this study were obtained from all 50 states as well as from
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163 Washington D.C (Tables 1 and 2). The states from which the greatest number of specimens were

164 collected and analyzed for testing (% of all C. trachomatis tests, % of all T. vaginalis tests) were
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165 Texas (19.0, 19.5), Florida (13.9, 14.3), Pennsylvania (8.8, 8.6), New Jersey (7.4, 8.1), Georgia
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166 (6.0, 6.4) and Arizona (5.6, 6.1). Kentucky, Louisiana, Ohio, Illinois, Maryland, Michigan,
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167 Alabama, Virginia, Tennessee, Missouri, California, Delaware and Oklahoma all accounted for

168 1-5% of total tests for both pathogens, and all other states each individually accounted for < 1%

169 of total tests performed. Positivity rates by state are listed in Tables 1 and 2. With regards to T.

170 vaginalis positivity (Fig. 5), the highest rates were found among specimens from Mississippi

171 (9.0%), Wisconsin (7.8%), South Carolina (7.4%), Illinois (7.0%), Arizona (6.7%), Alabama

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172 (5.7%), Louisiana (5.7%), Kansas (5.5%), Georgia (5.3%), Washington D.C. (5.2%) and

173 Tennessee (5.2%). The lowest T. vaginalis positivity rates were among specimens from

174 Arkansas, Hawaii and North Dakota at 0.0%, followed by New Hampshire (0.4%), Minnesota

175 (1.1%), Massachusetts (1.7%), Utah (1.8%), New York (1.8%), Maine (1.8%) and California

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176 (1.8%). With regards to state-by-state comparisons (Fig. 6), we found that Alabama, Florida,

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177 Mississippi, Wisconsin and South Carolina had significantly greater detection rates than nearly

178 every other state that could be evaluated. It is important to note that there were no significant

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179 comparisons for Alaska, Hawaii, Montana, North Dakota or South Dakota due to the low number

180 of specimens evaluated (n ≤ 77).

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The states with the highest positivity rates for C. trachomatis (Fig. 7) were Maine (6.4%), South

182 Dakota (4.6%), Louisiana (4.3%), Alabama (4.0%) and Wisconsin (4.0%). The states with the
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183 lowest positivity rates for C. trachomatis were Arkansas, Hawaii and North Dakota at 0.0%,

184 followed by Delaware (1.1%), New York (1.2%), Minnesota (1.2%), New Hampshire (1.3%)
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185 and Massachusetts (1.5%). With regards to state-by-state comparisons (Fig. 8), we found that
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186 detection rates in Alabama, Georgia, Louisiana, Mississippi South Carolina and Wisconsin were

187 significantly greater than most other states. Although the detection rate was greatest for Maine, it
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188 was only significantly greater than one other state, due to the small number of specimens
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189 evaluated from this state (n = 73). We also evaluated the positivity rates of the pathogens for
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190 correlations at the state level (Fig. 9) and found a weak positive correlation (R-squared = 0.29, P

191 < 0.0001) between the positivity rates.

192 Last, we analyzed our detection rate for a specific polymorphism (K80STOP) in the ntr6TV gene
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193 of T. vaginalis which is associated with metronidazole resistance . Overall, 876 out of 58,891

194 T. vaginalis-positive specimens were positive for this polymorphism, resulting in a positivity rate

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195 of 1.5%. We evaluated the positivity rate as a function of age (Fig. 10) but there was no clear

196 pattern, with rates seemingly fluctuating at random. There were no significant differences in

197 detection rates between specimens from patients of different ages. When evaluated for detection

198 rate by geography, we found the highest positivity rates for this polymorphism were found

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199 among specimens from Vermont (14.3%), New Mexico (11.1%), West Virginia (5.6%) and

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200 California (5.3%) (Table 3, Fig. 11). We failed to detect the ntr6TV (K80STOP) polymorphism in

201 specimens from the following states: Arkansas, Colorado, Hawaii, Massachusetts, Maine,

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202 Montana, New Hampshire, North Dakota, South Dakota, Washington Wisconsin or Wyoming.

203 The rates in West Virginia and California were significantly greater than that of many other

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204 states, whereas the rate in Wisconsin was significantly lower than many other states (Fig. 12). It

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is important to note that although Vermont had the greatest detection rates, it was significantly

greater than only one other state (Wisconsin) due to the low number of specimens evaluated (n =
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207 7). In contrast, New Mexico, with the second greatest detection rate, had a significantly greater
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208 detection rate than nine other states due, in part, to the larger number of specimens evaluated (n
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209 = 63).

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211 Discussion
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212 Previous studies have found the prevalence of T. vaginalis, unlike most STIs, to be higher in
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213 women older than 30 years of age 3, 15, 16. We have replicated these findings using a much larger

214 specimen collection evaluating 20-fold greater specimens than any prior study, thus allowing us

215 to determine the peak age of T. vaginalis detection rate more precisely as between 46 and 55

216 years of age. The reason for this later in life peak is unknown. One possible explanation is an

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217 increase in sex partners and unprotected sexual activity 19. However, one would expect to see a
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218 similar pattern with other STIs, such as chlamydia and gonorrhea, which is not the case .

219 Therefore, we hypothesize that the age-related increase in T. vaginalis detection rate is due to

220 either increased susceptibility to T. vaginalis infection in these women or re-activation of a pre-

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221 existing latent T. vaginalis infection in asymptomatic women.

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222 T. vaginalis infection is associated with abnormal vaginal flora that is deficient in

Lactobacillus20. As Lactobacillus is lost during menopause due to decreased estrogen production

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224 and thus reduced glycogen concentration in vaginal secretions , one may speculate that this

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225 mechanism is responsible for the age-related increase we observed. However, a similar increase

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in susceptibility to gonorrhea and chlamydia has also been noted in women with abnormal

227 vaginal flora . Another possibility is altered mucosal immunology in the lower reproductive
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228 tract. For example, menopause is associated with a reduction in B cells and CD8+ T cells in the

229 ectocervix23. Menopause also leads to a decrease in the cervico-vaginal concentration of

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230 secretory leukocyte protease inhibitor (SLPI) , which protects mucosal tissues from serine
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231 proteases. As cysteine proteases produced by T. vaginalis degrade SLPI 25, we speculate that this

232 protein is protective against infection and its deficiency after menopause could contribute to our
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233 observed increase in detection rates. Although pregnant women also have altered mucosal
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234 immunity in the lower genital tract , no association was found between pregnancy and T.
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235 vaginalis infection 3. An alternate explanation is that T. vaginalis can persist after an initial

236 episode in a dormant, undetected fashion and then is re-activated during or after menopause.

237 Although T. vaginalis does not form cysts, an environmentally-resistant pseudocyst form has
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239 differentiate into the infectious trophozoite form and proliferate in response to hormonal,

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240 epithelial and/or immunological changes associated with menopause. Lastly, the endocervical

241 atrophy that accompanies menopause 28 may provide protection against pathogens that infect this

242 site, such as C. trachomatis29 or N. gonorrhea 30

, but does not provide protection against T.

243 vaginalis, which infects both the vagina and the cervix 31.

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244 With regards to geographic distribution, we observed that detection rates for both pathogens

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245 were greater in the Southeastern US (Mississippi, Louisiana, and Alabama) and along the

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246 Mississippi River Valley (Missouri, Indiana and Wisconsin) than in other parts of the country.

247 Our results for C. trachomatis are largely consistent with those of the CDC, which found that

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248 Mississippi, Louisiana and Alabama are the states with the 2nd-4th greatest prevalence of this

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disease 14. Other states where our results were concordant with those of the CDC include South

250 Carolina, Oklahoma, Texas, South Dakota and Illinois. States where the CDC reports a high
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251 prevalence where we found a low detection rate include New Mexico, Arkansas and New York.

252 Conversely, we found high detection rates for Wisconsin and Kansas whereas the CDC-reported
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253 prevalence is low. One simple explanation for these discrepancies is that the CDC is reporting all
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254 cases state-wide whereas our detection rate determination is limited to specimens sent to our

255 laboratory. In states where our detection rate is higher than expected we may be receiving
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256 specimens from patients where the T. vaginalis prevalence is greater, such as among black
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257 women or those with lower socioeconomic status 32. One possible explanation for this pattern
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258 is that the Southern US is the region with the highest frequency of douching , which is

259 associated with T. vaginalis infection 3. Conversely, in states where our detection rate is lower

260 than anticipated we may be receiving specimens from patients who are disproportionately white

261 and/or of higher socioeconomic status.

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262 Strengths of our study include a very large sampling population from all states and ages 10 to 80

263 years old. Additionally, we used a sensitive and specific qPCR test and measured the detection of

264 ntr6TV(K80STOP) polymorphism associated with T. vaginalis metronidazole resistance.

265 However, it is important to note that this polymorphism is associated with in vitro resistance to

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266 metronidazole and its association with metronidazole treatment failure has not yet been

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267 demonstrated. Our analysis was limited by sampling that was not generally random. Women may

268 have been tested during annual gynecological examination, for newly diagnosed pregnancy or

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269 due to concerns of a STI. However, because most women infected with T. vaginalis are

270 asymptomatic 7, the use of clinical considerations for T. vaginalis detection is largely inaccurate.

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271 As there is no routine screening for T. vaginalis, our testing population may be skewed towards

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symptomatic patients which would bias our detection rates upward. Another weakness is the lack

of demographic data on the women tested, specifically number of sexual partners and racial
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274 distribution. In addition, we do not know if the infection was recurrent, asymptomatic or if there
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275 was a co-infection with other pathogens.

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276 In contrast to chlamydia, trichomoniasis is not a reportable disease and therefore we do not have

277 extensive data regarding its epidemiology in the US. In general, we found a similar pattern for T.
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278 vaginalis detection as for C. trachomatis, with states in the Southeastern US and along the
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279 Mississippi River Valley having greater detection rates. These results are generally consistent
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280 with those of a prior study examining the US distribution of T. vaginalis detection, which found
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281 the highest rates in the Southeastern US . We also evaluated the detection rate of the

282 ntr6TV(K80STOP) nucleotide polymorphism that is associated with metronidazole resistance in

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283 T. vaginalis . The two states with the highest detection rates were Vermont (14%) and New

284 Mexico (11%). We also found high detection rates in several regions of the country, including

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285 the west coast (California and Oregon), the Midwest (north Dakota, Idaho and Oklahoma), and

286 the Northeast (new York and Rhode Island) along with Iowa and West Virginia. The higher

287 detection rates in these areas may indicate a greater degree of metronidazole resistance and

288 potential treatment failure with this agent. However, as routine testing for metronidazole

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289 susceptibility is not available to most physicians, there is no way at this time to test this

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290 hypothesis.

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291 In summary, we have evaluated a very large number of specimens (n = ~1.5 million for T.

292 vaginalis, n = ~1.9 million for C. trachomatis), and confirmed and extended prior analyses of the

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293 relationship between T. vaginalis maximum detection rates in women aged 46 to 55 years old.

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Explanations for this pattern, biological and/or behavioral, should be investigated. We also

295 provide data regarding the geographical distribution of the detection rates, which have only been
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296 reported in one prior study examining a relatively small number of specimens (n= 7,593) 15. We

297 found that the geographic distribution of detection rates for T. vaginalis in the US is broadly
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298 similar to those of other STIs. Lastly, we provide the first report of the detection rate for a T.
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299 vaginalis nucleotide polymorphism associated with metronidazole resistance 18 and found higher

300 rates in several geographically discontinuous clusters. Overall, these data broaden our
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301 understanding of the epidemiology of the most common non-viral STI and can serve as a
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302 foundation for further studies.

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303 Acknowledgements

304 This study was funded by Medical Diagnostic Laboratories. Shlomo M. Stemmer, MD, is a paid

305 consultant for Medical Diagnostic Laboratories (MDL). We would like to thank Janet Cohen for

306 assistance in data analysis.

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347 14. CDC. Sexually Transmitted Disease Surveillance, 2014.

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348 15. GINOCCHIO CC, CHAPIN K, SMITH JS, et al. Prevalence of Trichomonas vaginalis and coinfection

349 with Chlamydia trachomatis and Neisseria gonorrhoeae in the United States as determined by the
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350 Aptima Trichomonas vaginalis nucleic acid amplification assay. Journal of clinical microbiology
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351 2012;50:2601-8.

352 16. STEMMER SM, ADELSON ME, TRAMA JP, DORAK MT, MORDECHAI E. Detection rates of

353 trichomonas vaginalis, in different age groups, using real-time polymerase chain reaction. Journal

354 of lower genital tract disease 2012;16:352-7.

355 17. NICHOLLS JE, TURNER KME, NORTH P, et al. Cross-sectional study to evaluate Trichomonas

356 vaginalis positivity in women tested for Neisseria gonorrhoeae and Chlamydia trachomatis,

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357 attending genitourinary medicine and primary care clinics in Bristol, South West England.

358 Sexually transmitted infections 2017.

359 18. PAULISH-MILLER TE, AUGOSTINI P, SCHUYLER JA, et al. Trichomonas vaginalis metronidazole

360 resistance is associated with single nucleotide polymorphisms in the nitroreductase genes ntr4Tv

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361 and ntr6Tv. Antimicrobial agents and chemotherapy 2014;58:2938-43.

362 19. STEMMER S. Reply to letter: increased prevalence of Trichomonas vaginalis in mid-aged women

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363 is linked to sexual activity and not to hormonal changes. Journal of lower genital tract disease

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364 2013;17:e33-4.

365 20. BALKUS JE, RICHARDSON BA, RABE LK, et al. Bacterial vaginosis and the risk of trichomonas

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366 vaginalis acquisition among HIV-1-negative women. Sexually transmitted diseases 2014;41:123-

367 8.

368 21.
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PABICH WL, FIHN SD, STAMM WE, SCHOLES D, BOYKO EJ, GUPTA K. Prevalence and

369 determinants of vaginal flora alterations in postmenopausal women. The Journal of infectious
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370 diseases 2003;188:1054-8.
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371 22. WIESENFELD HC, HILLIER SL, KROHN MA, LANDERS DV, SWEET RL. Bacterial vaginosis is a
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372 strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infection. Clinical

373 infectious diseases : an official publication of the Infectious Diseases Society of America
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374 2003;36:663-8.

375 23. TRIFONOVA RT, LIEBERMAN J, VAN BAARLE D. Distribution of immune cells in the human
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376 cervix and implications for HIV transmission. American journal of reproductive immunology
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377 2014;71:252-64.

378 24. SHIMOYA K, ZHANG Q, TEMMA K, et al. Secretory leukocyte protease inhibitor levels in

379 cervicovaginal secretion of elderly women. Maturitas 2006;54:141-8.

380 25. DRAPER D, DONOHOE W, MORTIMER L, HEINE RP. Cysteine proteases of Trichomonas vaginalis

381 degrade secretory leukocyte protease inhibitor. The Journal of infectious diseases 1998;178:815-

382 9.

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383 26. WIRA CR, FAHEY JV, RODRIGUEZ-GARCIA M, SHEN Z, PATEL MV. Regulation of mucosal

384 immunity in the female reproductive tract: the role of sex hormones in immune protection against

385 sexually transmitted pathogens. American journal of reproductive immunology 2014;72:236-58.

386 27. PEREIRA-NEVES A, RIBEIRO KC, BENCHIMOL M. Pseudocysts in trichomonads--new insights.

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387 Protist 2003;154:313-29.

388 28. TAY SK, SINGER A. The effects of oral contracdeptive steroids, menopause and hormonal

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389 replacement therapy on the cervical epithelium. In: Jordan J, ed. The Cervix. USA: Wiley-

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390 Blackwell, 2006.

391 29. BRUNHAM RC, PAAVONEN J, STEVENS CE, et al. Mucopurulent cervicitis--the ignored

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392 counterpart in women of urethritis in men. The New England journal of medicine 1984;311:1-6.

393 30. BHATTACHARYYA MN, JEPHCOTT AE, MORTON RS. Diagnosis of gonorrhoea in women:

394
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comparison of sampling sites. British medical journal 1973;2:748-50.

395 31. BOEKE AJ, DEKKER JH, PEERBOOMS PG. A comparison of yield from cervix versus vagina for
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396 culturing Candida albicans and Trichomonas vaginalis. Genitourinary medicine 1993;69:41-3.
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397 32. CRICHTON J, HICKMAN M, CAMPBELL R, BATISTA-FERRER H, MACLEOD J. Socioeconomic

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398 factors and other sources of variation in the prevalence of genital chlamydia infections: A

399 systematic review and meta-analysis. BMC public health 2015;15:729.

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400 33. ARAL SO, MOSHER WD, CATES W, JR. Vaginal douching among women of reproductive age in

401 the United States: 1988. American journal of public health 1992;82:210-4.
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402
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405

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406 Figure Legends

407

408 Figure 1. T. vaginalis positivity as a function of age

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409 Figure 2. Year-by-year comparison of T. vaginalis positivity rates. Colored spaces indicate

410 comparisons where a significant different (P < 0.05) between ages was observed. The color of

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411 the space corresponds to the state with the greater positivity rate (blue for the state listed on the

412 X-axis and red for the state listed on the Y-axis).

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413 Figure 3. C. trachomatis positivity as a function of age

U
414 Figure 4. Year-by-year comparison of C. trachomatis positivity rates. Colored spaces indicate

415
AN
comparisons where a significant different (P < 0.05) between ages was observed. The color of

416 the space corresponds to the state with the greater positivity rate (blue for the state listed on the
M
417 X-axis and red for the state listed on the Y-axis).
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418 Figure 5. Map of T. vaginalis positivity rates (%)

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419 Figure 6. State-by-state comparison of T. vaginalis positivity rates. Colored spaces indicate
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420 comparisons where a significant different (P < 0.05) between states was observed. The color of

421 the space corresponds to the state with the greater positivity rate (blue for the state listed on the
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422 X-axis and red for the state listed on the Y-axis).
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423 Figure 7. Map of C. trachomatis positivity rates (%)

424 Figure 8. State-by-state comparison of C. trachomatis positivity rates. Colored spaces indicate

425 comparisons where a significant different (P < 0.05) between states was observed. The color of

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426 the space corresponds to the state with the greater positivity rate (blue for the state listed on the

427 X-axis and red for the state listed on the Y-axis).

428 Figure 9. Correlation of positivity rates for C. trachomatis and T. vaginalis testing by state. The

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429 red line represents a linear regression of the two positivity rates.

430 Figure 10. Positivity for ntr6TV(K80STOP) as a function of age. Ages for which there were no

RI
431 positive specimens are displayed as having a value of 0 for presentation purposes on a

SC
432 logarithmic scale.

433 Figure 11. Map of ntr6TV(K80STOP) positivity rates (%) among T. vaginalis-positive

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434 specimens.
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435 Figure 12. State-by-state comparison of ntr6TV(K80STOP) positivity rates. Colored spaces
M
436 indicate comparisons where a significant different (P < 0.05) between states was observed. The

437 color of the space corresponds to the state with the greater positivity rate (blue for the state listed
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438 on the X-axis and red for the state listed on the Y-axis).
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439
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440
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Table 1. Results of T. vaginalis testing. States are ordered by positivity rates.

State Positive Specimens (n) Negative Specimens (n) Total Specimens (n) Positivity Rate (%) 95% Confidence Interval (%)
MS 577 5,861 6,438 9.0 8.3 - 9.7
WI 77 907 984 7.8 6.1 - 9.5
SC 147 1,852 1,999 7.4 6.3 - 8.5
IL 3,678 48,839 52,517 7.0 6.8 - 7.2
AR 301 4,197 4,498 6.7 6 - 7.4
AL 2,123 35,343 37,466 5.7 5.5 - 5.9

PT
LA 3,191 52,880 56,071 5.7 5.5 - 5.9
KS 725 12,484 13,209 5.5 5.1 - 5.9
GA 5,246 93,978 99,224 5.3 5.2 - 5.4
DC 321 5,861 6,182 5.2 4.6 - 5.8

RI
TN 1,558 28,336 29,894 5.2 4.9 - 5.5
MO 1,306 25,499 26,805 4.9 4.6 - 5.2
DE 731 14,751 15,482 4.7 4.4 - 5.0
NC 510 10,818 11,328 4.5 4.1 - 4.9

SC
OH 1,642 35,887 37,529 4.4 4.2 - 4.6
IN 420 9,781 10,201 4.1 3.7 - 4.5
KY 2,094 48,767 50,861 4.1 3.9 - 4.3
OK 879 20,520 21,399 4.1 3.8 - 4.4
MI 1,310 31,630 32,940 4.0 3.8 - 4.2

U
AK 2 50 52 3.8 0.0 - 9.0
NE 55 1,376 1,431 3.8 2.8 - 4.8
SD 3 77 80 3.8 0.0 - 8
WY
MD
TX
9
1,889
10,596
233
50,838
292,725
AN 242
52,727
303,321
3.7
3.6
3.5
1.3 - 6.1
3.4 - 3.8
3.4 - 3.6
PA 4,619 129,516 134,135 3.4 3.3 - 3.5
FL 6,853 216,096 222,949 3.1 3 - 3.2
M
VA 1,036 32,234 33,270 3.1 2.9 - 3.3
VT 7 222 229 3.1 0.9 - 5.3
NJ 3,628 122,747 126,375 2.9 2.8 - 3.0
WA 98 3,402 3,500 2.8 2.3 - 3.3
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CT 84 3,053 3,137 2.7 2.1 - 3.3

NM 63 2,270 2,333 2.7 2 - 3.4
IA 48 1,802 1,850 2.6 1.9 - 3.3
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CO 173 6,783 6,956 2.5 2.1 - 2.9

NV 303 12,329 12,632 2.4 2.1 - 2.7
OR 24 962 986 2.4 1.4 - 3.4
WV 54 2,330 2,384 2.3 1.7 - 2.9
ID 24 1,130 1,154 2.1 1.3 - 2.9
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AZ 1,865 92,766 94,631 2.0 1.9 - 2.1

RI 28 1,403 1,431 2.0 1.3 - 2.7
CA 378 21,032 21,410 1.8 1.6 - 2.0
ME 1 55 56 1.8 0.0 - 5.3
NY
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116 6,308 6,424 1.8 1.5 - 2.1

UT 28 1,528 1,556 1.8 1.1 - 2.5
MA 64 3,798 3,862 1.7 1.3 - 2.1
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MN 6 545 551 1.1 0.2 - 2.0

NH 1 231 232 0.4 0.0 - 1.2
HI 0 16 16 0.0 0-0
MT 0 19 19 0.0 0-0
ND 0 8 8 0.0 0-0
Total 58,891 1,496,075 1,554,966 3.8 3.8 - 3.8
 ACCEPTED MANUSCRIPT

Table 2. Results of C. trachomatis testing. States are ordered by positivity rates.

State Positive Specimens (n) Negative Specimens (n) Total Specimens (n) Positivity Rate (%) 95% Confidence Interval (%)
ME 5 73 78 6.4 1 - 11.8
SD 5 104 109 4.6 0.7 - 8.5
LA 3,212 70,666 73,878 4.3 4.2 - 4.4
MS 599 13,382 13,981 4.3 4 - 4.6
AL 1,702 40,820 42,522 4 3.8 - 4.2
WI 96 2,299 2,395 4 3.2 - 4.8

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KS 725 17,744 18,469 3.9 3.6 - 4.2
IL 2,286 59,414 61,700 3.7 3.6 - 3.8
OK 913 24,426 25,339 3.6 3.4 - 3.8
SC 84 2,278 2,362 3.6 2.9 - 4.3
MO 1,332 37,270 38,602 3.5 3.3 - 3.7

RI
TX 13,334 365,520 378,854 3.5 3.4 - 3.6
GA 4,096 115,805 119,901 3.4 3.3 - 3.5
NE 60 1,753 1,813 3.3 2.5 - 4.1
IN 539 16,598 17,137 3.1 2.8 - 3.4

SC
TN 1,218 38,437 39,655 3.1 2.9 - 3.3
IA 129 4,198 4,327 3 2.5 - 3.5
AR 174 5,767 5,941 2.9 2.5 - 3.3
NM 89 3,072 3,161 2.8 2.2 - 3.4
NV 488 17,094 17,582 2.8 2.6 - 3

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AZ 3,040 107,972 111,012 2.7 2.6 - 2.8
KY 2,150 77,113 79,263 2.7 2.6 - 2.8
OH 1,738 61,908 63,646 2.7 2.6 - 2.8
OR
ID
CO
NC
62
85
335
483
2,238
3,182
12,845
18,483
AN 2,300
3,267
13,180
18,966
2.7
2.6
2.5
2.5
2.0 - 3.4
2.1 - 3.1
2.2 - 2.8
2.3 - 2.7
UT 52 2,036 2,088 2.5 1.8 - 3.2
M
VA 1,006 39,919 40,925 2.5 2.3 - 2.7
MI 1,054 42,149 43,203 2.4 2.3 - 2.5
MT 4 165 169 2.4 0.1 - 4.7
WA 98 4,358 4,456 2.2 1.8 - 2.6
WY 6 272 278 2.2 0.5 - 3.9
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DC 136 6,267 6,403 2.1 1.7 - 2.5

FL 5,964 271,651 277,615 2.1 2 - 2.2
MD 1,167 55,848 57,015 2 1.9 - 2.1
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PA 3,507 173,345 176,852 2 1.9 - 2.1

CA 596 30,138 30,734 1.9 1.7 - 2.1
CT 87 4,499 4,586 1.9 1.5 - 2.3
WV 77 3,910 3,987 1.9 1.5 - 2.3
NJ 2,658 145,710 148,368 1.8 1.7 - 1.9
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RI 26 1,518 1,544 1.7 1.1 - 2.3

VT 6 372 378 1.6 0.3 - 2.9
MA 91 5,887 5,978 1.5 1.2 - 1.8
NH 4 311 315 1.3 0.1 - 2.5
MN 11 875 886 1.2 0.5 - 1.9
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NY 86 7,113 7,199 1.2 0.9 - 1.5

DE 291 26,273 26,564 1.1 1 - 1.2
HI 0 21 21 0 0-0
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AK 0 64 64 0 0-0
ND 0 9 9 0 0-0
Total 58,891 1,496,075 1,554,966 2.8 2.8 - 2.8
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Table 3. Detection of ntr6TV(K80STOP) in T. vaginalis-positive specimens. States are ordered by

positivity rates. NA indicates Not Applicable (no positive specimens from that state to analyze).

ntr6TV(K80STOP)- ntr6TV(K80STOP)- Total T. vaginalis- 95% Confidence Interval

Positivity Rate
State positive negative positive (%)
(%)
specimens (n) specimens (n) Specimens (n)
VT 1 6 7 14.3 0.0 - 40.2
NM 7 56 63 11.1 3.3 - 18.9

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WV 3 51 54 5.6 0.0 - 11.7
CA 20 358 378 5.3 3.0 - 7.6
NY 5 111 116 4.3 0.6 - 8.0
IA 2 46 48 4.2 0.0 - 9.9
ID 1 23 24 4.2 0.0 - 12.2

RI
OK 37 842 879 4.2 2.9 - 5.5
OR 1 23 24 4.2 0.0 - 12.2
AR 12 289 301 4.0 1.8 - 6.2

SC
RI 1 27 28 3.6 0.0 - 10.5
UT 1 27 28 3.6 0.0 - 10.5
KY 56 2038 2,094 2.7 2.0 - 3.4
AZ 41 1824 1,865 2.2 1.5 - 2.9
AL 42 2081 2,123 2.0 1.4 - 2.6

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KS 13 712 725 1.8 0.8 - 2.8
LA 56 3135 3,191 1.8 1.3 - 2.3
NE 1 54 55 1.8 0.0 - 5.3
IL
TX
FL
62
177
100
3616
10419
6753
AN 3,678
10,596
6,853
1.7
1.7
1.5
1.3 - 2.1
1.5 - 1.9
1.2 - 1.8
TN 24 1534 1,558 1.5 0.9 - 2.1
M
IN 6 414 420 1.4 0.3 - 2.5
MS 8 569 577 1.4 0.4 - 2.4
SC 2 145 147 1.4 0.0 - 3.3
CT 1 83 84 1.2 0.0 - 3.5
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MI 16 1294 1,310 1.2 0.6 - 1.8

MO 15 1291 1,306 1.1 0.5 - 1.7
PA 53 4566 4,619 1.1 0.8 - 1.4
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VA 11 1025 1,036 1.1 0.5 - 1.7

NV 3 300 303 1.0 0.0 - 2.1
GA 45 5201 5,246 0.9 0.7 - 1.1
OH 14 1628 1,642 0.9 0.5 - 1.3
NC 4 506 510 0.8 0 - 1.6
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NJ 20 3608 3,628 0.6 0.4 - 0.8

DE 4 727 731 0.5 0 - 1.0
MD 10 1879 1,889 0.5 0.2 - 0.8
DC 1 320 321 0.3 0.0 - 0.9
C

AK 0 2 2 0.0 0-0
CO 0 173 173 0.0 0-0
MA 0 64 64 0.0 0-0
AC

ME 0 1 1 0.0 0-0
MN 0 6 6 0.0 0-0
NH 0 1 1 0.0 0-0
SD 0 3 3 0.0 0-0
WA 0 98 98 0.0 0-0
WI 0 77 77 0.0 0-0
WY 0 9 9 0.0 0-0
HI 0 0 0 NA NA
MT 0 0 0 NA NA
ND 0 0 0 NA NA
Total 879 58012 58,891 1.5 1.4 - 1.6
 ACCEPTED MANUSCRIPT

Figure 1.

Total Tested
Total Positive
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Positivity Rate
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# Samples (Log)

% Positive

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Figure 3.

Total Tested
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5 Positivity Rate 10

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# Samples (Log)

% Positive
3 6

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10 20 30 40 50 60 70 80

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Figure 5.

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Figure 8.

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Figure 9.

10 r2 = 0.29
T. vaginalis (% Positivity)

9 P < 0.0001

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Figure 10.

ntr6TV (K80STOP)

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Positivity Rate
Total Tested
4 8
Total Positive

RI
# Samples (Log)

3 6

% Positive

SC
2 4

1 2

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0
10 20 30 40 50 60
AN
70 80
0

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Figure 11.

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Figure 12

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