Case A

Background:  Sexually transmitted diseases are a major public health problem in the U.S.  Several organisms, including bacteria and viruses, can be transmitted through sexual contact. Neisseria gonorrheae (which causes gonorrhea) and Chlamydia trachomatis can cause urethritis inflammation of the urethra) in males and females, and pelvic inflammatory disease (PID) in women.  PID is infection of the uterus and fallopian tubes that can lead to chronic pelvic pain, infertility, ectopic pregnancy, abscess formation, and internal bleeding.  Organisms that cause genital ulcer disease (GUD) include the bacteria Haemophilus ducreyi (which causes chancroid), Treponema pallidum (syphilis), and herpes simplex virus type 2 (genital herpes).  GUD results in painful genital lesions in both males and females.  These organisms can be detected by culture methods, antibody-based tests such as ELISA, and nucleic acid amplification tests such as PCR.  Multiplex PCR can test for several organisms simultaneously.  Treatment is primarily antibiotics (or anti-viral drugs for Herpes simplex virus).

Case A. The Soldier’s Unexpected Gift. Robert Jr. is a 22 year old soldier who very recently returned to his hometown in the Midwestern region of the United States. Lucky for Robert, his assignment in Asia ended just in time to return to Old School University to complete a degree in Science and Mathematics Education. To celebrate his return, Robert’s girlfriend Jenna, a foreign exchange student, gathered lots of food, alcohol, and party favors for a night of celebration with friends. Several days later, the director of Old School University’s student health department issued an alert to the university president and the local department of health to report a dramatic increase in the number of genital ulcer disease (GUD) cases on campus. There were lots of rumors about an outbreak of HIV or gonorrhea on campus, however no official warnings were released by the university.  Three days after the party, Robert became very worried upon noticing an unusual, inflamed ‘bump’ on his penis during a shower. However, he resisted a trip to student health… he hoped it would simply go away. Two days later, the bump began bleeding. Immediately, Robert called Jenna and urged her to go with him to the student health facility to be tested for what he thought may be HIV.  But rather than test them for HIV, the student health nurse took an endocervical swab sample from Jenna and a urethral swab sample from Robert.  DNA was extracted from the swab samples and multiplex PCR was performed to test for five different sexually-transmitted diseases.

Procedure:

You will generate three separate images (two gels and a 96-well plate):

1. Control gel using standard PCR: Use the individual primers to amplify portions of the control DNA, and run each PCR product in a separate lane. This is the ‘standard PCR’ procedure. If the target DNA is present, the primers will amplify the following sizes of DNA fragments (in kilobases):

       Haemophilus ducreyi, 1.24
       Herpes simplex, 0.34
       Neisseria gonorrheae, 0.55
       Chlamydia trachomatis, 1.93
       Treponema pallidum, 0.40

Click on the bands on the gel to verify that the PCR products match the sizes above. Use the Gel menu to show sizes for bands in each lane of the gel (the default is to show distance moved for each band). When you click on a band, the size also shows up in the field at the bottom of the Data Screen.

2. Samples gel using standard PCR: Use a single combined primer set to perform ‘multiplex PCR’ on the DNA samples listed below. This will determine if one or more of the five disease-causing organisms are present in each sample. You can then use the control gel to determine which if any are present in a particular sample.

3. Samples plate using 96-well PCR: Use this procedure on the DNA samples listed below, testing each sample for the five sexually transmitted organisms.  Each sample should be tested separately for each the five sexually transmitted organisms (the folder includes five individual primer sets, in five separate files, the same ones that are used for the control gel above). Use five columns on the plate (for the five organisms), and six rows (for the four individuals plus the negative and positive controls).

DNA samples:

• Robert
• Jenna
• Another male student at the same college (Student 1)
• Another female student at the same college (Student 2)
• Negative Control
• Positive control, containing the following:
• Haemophilus ducreyi (chancroid)
• Herpes simplex virus type 2 (herpes)
• Neisseria gonorrheae (gonorrhea)
• Chlamydia trachomatis serovar D (Chlamydia)
• Treponema pallidum (syphilis)

1. What organism is most likely causing Robert’s genital ulcer?
2. Was his girlfriend Jenna also infected?
3. Were the other college students tested infected with the same organisms? 
4. Do these results give you any information about how the diseases are being spread?
5. What would you recommend as a treatment for Robert?
6. How would you discuss with Robert the importance of preventing the spread of these infections?
7. How likely is it that one person could be infected with multiple STIs? In other words, is this case strictly hypothetical, or is it something that could actually happen?