Sunita Gulati1, Bo Zheng1, Sanjay Ram1, George W. Reed2, Guillermo E. Madico3, Xianbao He3, Robin R. Ingalls3, Christine Chaisson4, Wenjing Le5,

Xiaohong Su5 and Peter A. Rice1

 

Neisseria gonorrhoeae and Chlamydia trachomatis co- Infection: More than just a Coincidence 淋病奈瑟菌与沙眼衣原体共感染：不只是巧合

1Division of Infect. Diseases and Immunol. and 2Preventive and Behavioral Medicine, Department of Medicine, University of Massachusetts School of Medicine, Worcester MA 016053Section of Infectious Diseases, Department of Medicine, Boston University Medical Center, Boston, MA 021184Data Coordinating Center, Boston University School of Public Health, Boston, MA 02118 5Nanjing STD Clinic, Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing 210042, PRP

Study Design Two epidemiologic studies of Neisseria gonorrhoeae (Ng) and Chlamydia

trachomatis (Ct) transmission in heterosexual partners:淋病奈瑟菌和沙眼衣原体异性性伴间传播的流行病学研究

1) Lin, J-s et al JID, 1998: Conducted in Boston 2) Ongoing: Conducted in Nanjing

Study design enhances likelihood that transmission occurs from men to women.研究设计旨在保证由男向女的病原体传播方向

1) Men are eligible if they have >2 female sex-partners in the 30 days prior to the onset of their symptoms: men are eligible and enrolled if they have a Gram’s stain positive for Gram-negative intracellular diplococci (GNIDs): Enrolled men may also have Ct infection: 59/191 (31% co-infection); Boston 11/46 (24% co-infection); Nanjing 2) Women are eligible/enrolled if they have been sex-partners of men 2 weeks before to 2 weeks after male symptoms developed and have had no other sex-partners; in Nanjing, >95% women are married

Comparison of Gonococcal Genotypes Between Sex-Partners性伴间感染淋球菌的基因型比较 -确定传播关系

tbpB porB porB tbpBtbpB-1 P.IB~1 P.IB~1 tbpB-1tbpB-2 P.IB~2 P.IB~2 tbpB-2

tbpB-21 P.IB~2 P.IB~2 tbpB-21tbpB-2 P.IB~2tbpB-3 P.IB~22

tbpB-15 P.IB~28 P.IB~28 tbpB-15tbpB-4 P.IB~3 P.IB~3 tbpB-4tbpB-2 P.IB~3 P.IB~3 tbpB-2tbpB-5 P.IB~8 P.IB~8 tbpB5

tbpB-17 P.IB~8 P.IB~8 tbpB-17tbpB-2 P.IB~25 P.IB~25 tbpB-2tbpB-6 P.IB~5 P.IB~5 tbpB-6

tbpB-16 P.IB~29 P.IB~29 tbpB-16tbpB-7 P.IB~9 P.IB~9 tbpB-7tbpB-3 P.IB~24 P.IB~24 tbpB-3tbpB-3 P.IB~23 P.IB~23 tbpB-3tbpB-2 P.IB~23 P.IB~23 tbpB-2tbpB-3 P.IB~4 P.IB~4 tbpB-3

tbpB-10 P.IB~20tbpB-18 P.IB~15 P.IB~15 tbpB-18

P.IB~15 tbpB-18tbpB-13 P.IB~27 P.IB~27 tbpB-13tbpB-22 P.IB~31 P.IB~31 tbpB-22tbpB-8 P.IB~30 P.IB~30 tbpB-8tbpB-4 P.IB~26 P.IB~26 tbpB-4tbpB-8 P.IB~21tbpB-8 P.IB~10tbpB-2 P.IB~10tbpB-2 P.IB~10tbpB-8 P.IB~10tbpB-8 P.IB~10tbpB-8 P.IB~10tbpB-8 P.IB~10 P.IB~10 tbpB-8tbpb-19 P.IB~19 P.IB~19 tbpb-19tbpB-8 P.IB~6tbpB-2 P.IB~7tbpB-2 P.IB~7 P.IB~7 tbpB-2tbpB-8 P.IB~7 P.IB~7 tbpB-8tbpB-8 P.IB~7 P.IB~7 tbpB-8

tbpB-20 P.IB~7 P.IB~7 tbpB-20tbpB-9 P.IA~1 P.IA~1 tbpB-9tbpB-2 P.IA~2 P.IA~2 tbpB-2

tbpB-12 P.IA~2 P.IA~2 tbpB-12tbpB-11 P.IA~3 P.IA~3 tbp-11tppB-14 P.IA~4 P.IA~4 tppB-14

MALE FEMALE

*

*No gonococcal infection transmitted to the female

Chlamydial Genotypes between Ng exposed /infected Sex-Partners AND Pre-Existing Chlamydial Genotypes in Women 暴露并感染淋球菌的性伴和以前已感染沙眼衣原体女性的沙眼衣原体的基因分型

*Uninfected with Chlamydia trachomatis (Ct)

*

Transmission of N. gonorrhoeae (Ng) Infection to Women with and without Pre-Existing C. trachomatis (Ct) infection女性淋球菌接触者的沙眼衣原体感染可促进淋球菌的男向女传播

Women exposed to men with Ng*

Boston studyNg acquired Ng not acquired % Ng acquired P value **

Pre-existing Ct infection

23 3 87%0.03

No Ct infection 70 36 66%

Nanjing study

Pre-existing Ct infection

10 1 91%0.39

No Ct infection 17 7 71%

*In both studies, men were infected with Ng alone; Ng/Ct co-infected men were excluded.排除淋球菌 /沙眼衣原体共感染男性 **Fisher Exact test

Performance Characteristics of Ng qPCR淋球菌感染定量分析 -qPCR结果与 CFU线性相关

R2 = 0.9988

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

5,000 500 50 5

N. gonorrhoeae CFU / reaction

Co

lorim

etr

ic A

ss

ay

R = 0.6186

0

1

2

3

4

5

6

0 2 4 6 8

N. gonorrhoeae Culture Log10 CFU

N. g

on

orr

ho

ea

eq

PC

R L

og

10 C

FU

Primers:-papTM-F 5' CAGCATTCAATTTGTTCCGAGTC 3'papTM-R 5' Biotin-GAACTGGTTTCATCTGATTACTTTCCA 3'

papTM-probe 5' CGCCTATACGCCTGCTACTTTCACGC 3’

Ng strain FA1090

Transmission of Ng to Women Related to Numbers of Ng in the Male Sex-Partner AND Influence of Pre-Existing Ct

Infection in Women on Susceptibility to Ng Infection

淋球菌男向女转播效率与感染男性的淋球菌量和女性性伴是否原有沙眼衣原体感染相关

P<0.006

N=-12

N=-25

5/7* women ex-posed to ≤120 cfu/ml Ng had Pre-Existing Ct infection

2/12* women Had Pre-Existing Ct infection

*P=0.04 (Fisher Exact Test); correlating transmission of low level exposure to Ng (≤120 cfu/ml) infection with Underlying Ct infection in women

_________

Summary

• Ng transmission was enhanced from men to women with pre-existing Ct infection

女性已有沙眼衣原体感染可促进淋球菌由男向女的传播• Men with low level Ng infection (≤120 cfu/ml equivalents in

urine) failed to transmit Ng infection unless

Their Female sex-partner had pre-existing Ct infection:

5/7 (71%) of women with pre-existing Ct infection

exposed to low level Ng infection were infected

男性感染的淋球菌量低导致淋球菌的传播失败，除非女性性伴原已感染了沙眼衣原体，原已感染沙眼衣原体女性暴露于感染少量淋球菌的男性，有 71%获得感染

Animal Studies

InoculateC.muridarum 3.5 x 105 IFU Estrogen

Antibiotics (Vancomycin + Streptomycin)

Ng (FA1090) inoculation 2 x 105 CFU

Serum sample obtained todetermine Ng and Cm specifc antibodies

-4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

Vaginal swabs daily for: (1) Cm quantitative PCR, daily(2) Ng quantitative cultures, daily (3) Number of complement component 3 (C3) and Ig molecules bound per Ng organism Serum measurements of Cm- and Ng-specific antibody (whole cell ELISA), DAY 11

Vaginal swabs daily for: (1) Cm quantitative PCR, daily(2) Ng quantitative cultures, daily (3) Number of complement component 3 (C3) and Ig molecules bound per Ng organism Serum measurements of Cm- and Ng-specific antibody (whole cell ELISA), DAY 11

Tim

ing

of

pro

ced

ure

sT

imin

g o

f p

roce

du

res

DAYDAY

Experimental Design: Mouse Model of co-Infection (Cm and Ng)

实验小鼠共感染模型

C

Cm – Ng

Ng

Cm

Mice Groups A

B

F

E

0

8

6

4

2Lo

g1

0 C

FU

p=0.029

61 2 3 4 5 7 8 9 10

DAYS

100

0

755025

% I

nfe

cted p=0.0022

DAYS

61 2 3 5 7 8 9 104

Cm - Ng Cm

GROUPS

p=0.45

0

40302010

AU

C l

og

10 I

FU

eq

uiv

.

p=0.96

91 2 3 4 5 6 7 80- 1- 2

DAYS

Lo

g1

0 I

FU

eq

uiv

.0

54

21

3

91 2 3 4 5 6 7 80- 1- 2

DAYS

p=0.96100

0

75

50

25

% I

nfe

cted

DNg - CFU Cm - IFU

Co-Infection of Female Mice with N. gonorrhoeae (Ng) and C. muridarum (Ct) 共感染与单独感染小鼠的病原体体内生存 /清除时间和存活菌量分析

Cm - Ng Ng

GROUPS

15

35302520

AU

C l

og

10 C

FU

p=0.0002

Number (No.) C3 and Ig Molecules per Ng Organism

阴道局部 C3 和 Ig比较方法 - C3 或 Ig 分子浓度 / 淋球菌量

0

8

6

4

2

Lo

g1

0 C

FU

p=0.029

61 2 3 4 5 7 8 9 10

DAYS

Cm – Ng

Ng

Mice Groups

- Vaginal Samples from Day 1-3 (and Day 4-6) from each mouse were pooled- Ng captured to an ELISA plate coated with rabbit anti-Ng - Number of Ng organisms captured determined using an Ng mAb (calibrated) - Amount of C3, IgG, IgM and IgA bound to captured Ng determined- Density of C3, IgG, IgM and IgA (number of molecules per org.) determined

The Journal of Infectious Diseases 1999;179:124–35

No. of molecules Ng organism =

Number of C3 and Ig molecules per org

Sample - Pool of vaginal washes from Day 1-3 of each mouse 采集自感染1~3天的阴道冲洗液（ C3 或 Ig / 菌量）

IgG IgM IgA

Anti-Ng

Anti-Cm

Ng and Cm Specific Antibodies (Whole Cell ELISA) in

Mouse Serum, 10 days after Ng inoculation病原体共感染与单独感染血清特异性抗体的比较

P= <0.0001P= 0.005P= 0.0026

Mouse groups (N=10 per group)

ns nsns

CONCLUSIONS

Pre-existing Ct infection in women is associated with an increased risk of acquiring Ng from their male sex-partner女性原有沙眼衣原体感染与淋球菌男向女传播风险增加相关

Low-level Ng infection in men is more readily transmitted to female sex-partners with pre-existing Ct infection低菌量的淋球菌感染男性更易向原有沙眼衣原体感染的女性传播

Pre-existing Cm infection in BALB/c mice results in a longer period of carriage and

an increased burden of Ng in a co-infection model—corroborating co-infection in humans共感染小鼠模型中淋球菌的生存时间和存活菌量增加 -与人体研究结果一致

Pre-existing Cm infection in BALB/c mice results in fewer immmunoglobulin (Ig)--antibody and complement (C3) molecules binding per Ng organism in the face of increased numbers of Ng present in co-infection原有沙眼衣原体感染致使阴道局部淋球菌特异性 C3 和 Ig分子浓度下降，淋球菌菌量增加

Speculation: Concomitant Chlamydia infection results in changes in the surface of Ng that results in inhibition of Ig and C3 binding推测：合并感染的衣原体改变淋球菌表面结构抑制 Ig 和 C3

结合 Pre-existing Cm infection in BALB/c mice suppresses immune (Ig)--antibody

responses to Ng infection 衣原体感染小鼠抑制淋球菌特异性抗体反应