Activity 3.1.2: Rapid Pathogen Identification



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Activity 1.1.3: Using DNA to Identify Pathogens Extension

Purpose

Now that you have been introduced to using DNA to identify pathogens you will act as investigators to determine the cause(s) of an outbreak and to learn what information may be helpful and what information is identified but is not useful in your investigation.

As the investigators you will be supplied with pathology reports and DNA sequence data, which was received in increments over a series of days. These reports include symptoms and fragmentary sequence data. You will need to query genome databases with short (500-900 base pair) DNA sequences. From this information you will be able to make decisions as to which pathogens or pathogen families are present. Based on the correlation of symptoms and database matches you will rank and hypothesize which pathogens are responsible for the outbreak in order to better fight the disease. This process must support daily updates based on availability of new data.

Equipment

• Computers with Internet access

• Lab notebook

Procedure

As the primary investigators on this case you will need to document and report a variety of information including:

• Patient symptoms

• PCR data

• Entomology micrographs

• Shotgun sequencing

The following approach to the data analysis will aid you in your work:

a. Highlight or underline important components of the case history that appear integral to reaching a conclusion or diagnosis (i.e., lab data and risk factors such as recent vacations, careers, or lifestyle).

b. Generate a list of appropriate species that relate to situation being studied and produce symptoms or diseases similar to what has been presented in the case history.

c. Begin to reduce the list by eliminating candidates from it by using the information from the case history. Review each candidate with your fellow scientists until you reach an agreement.

d. Once the responsible organism has been identified, then determine a treatment strategy, for other at-risk individuals, and approaches to halting the progression of infection, making sure they are feasible.

Scenario:

It is a hot and humid July in Washington, D.C. The presidential race is heating up, outpacing the local mercury. The GOP campaign headquarters, located just off the Potomac River and surrounded by several large meditation ponds, is especially feeling the heat.

With millions of dollars fueling the GOP machine, the mood of the re-election team has been confident. However, panic is setting in at GOP campaign headquarters as an unknown illness appears to be affecting the staff.

With several campaign staffers affected and the impending presidential election in the not so distant future, the campaign-medical liaison, Dr. Shrub, decides to bring in a team of crack shot scientists.

Immediately, the team scramble and depart on their Biocom jet, which doubles as a mobile laboratory. Initial reports from Dr. Shrub are somewhat confusing.

Day 1

July 12, 2011

Republican HQ

Bill

A 21-year-old male intern who is on summer break from Stevenson University where he is a long jumper on the track team has reported a 24-hour history of painful urination. The young man reports urethral discharge. Urine appears clear, although many white blood cells are present. The young man gives a history of heterosexual activity with five partners over the past 6 months. He said that all his partners were clean.

Sophia

A 44-year-old Mexican-American media relations advisor with a keen interest in pre-Columbian artifacts reports a 3-day-old high fever. She also has severe joint pains, with a head and backache. She reports infrequent eye pain, nausea, and vomiting.

Max

A 33-old male volunteer in the mailroom, who works as textile worker in a fur store in the evenings was admitted into the Georgetown Hospital emergency room suffering from breathing difficulties and coughing spasmodically. Dr. Shrub was called and he ordered a chest x-ray which revealed widening of the mediastinum and pleural effusions. Lymph glands in the upper arm were swollen. He also observed vesicular lesions and black, necrotic skin tissue on his hands.

Dr. Shrub is overwhelmed, but it appears that he is doing decent background work on the patients. Yet, there are still a lot of unknowns in this campaign HQ.

From the signs and symptoms listed above make some preliminary diagnosis and record this information in your lab notebook.

Day 2

July 13

Bill

He still has burning urination. Dr. Shrub requested an STD screen on Bill. He will have PCR samples run.

Sophia

Her high fever continues and so do her severe joint pains, and headaches and backaches. She reports infrequent, eye pain, nausea and vomiting.

Max

The vesicular lesions are increasing as well as the black, necrotic skin tissue on his hands. Mail samples are screened for possible powder. Samples of mail are taken to test for bioterrorist agents.

New patients:

Justin

A 30-year old African-American male works as a pressroom assistant at GOP HQ. He enjoys doing tai chi by the ponds after work. He has been home sick for 4 days with a high fever and severe joint pains, with head and backache. Justin just called in to tell that his condition has worsened. He also reports frequent nausea and vomiting, and soreness around his eyes.

Naomi

A 28-year old Chinese-American female coordinates public opinion surveys for the campaign and will meet with hundreds of field officers a week who are roaming the streets of D.C. while conducting public opinion surveys. She has complained of headaches and neck pain. She enjoys meditating by the ponds before and after work.

Ponds

Dr. Shrub suggests that it may be worth while to take water samples from the Potomac River and the meditation ponds to test for infectious agents. What should you look for?

DAY 3

July 14

The following information is found in database searches.

Use the nucleotide BLAST page, Use Blastn or search for short, nearly exact matches. Refer to the following URL:

National Center for Biotechnology Information. (n.d.) BLAST. Retrieved October 18, 2006 from

You can make your search faster by restricting the database searched to only viral or bacterial sequences.

Select Viruses [ORGN] in the Option section of the page.

If you do not receive hits, try increasing the Expect value; to, 10000.You might also take a look at the Program selection guide, linked on the Blast page.

Follow-up on Patients:

Bill

Now only sequence information is required to confirm the results.

1 CCGTCAAATT CCAATTCCCG CCCGAAGTCG TGCGCGTCCT TGCAGGCATG GGTAACAACC

61 TGAACCAAAT AGCCAAGGCC CTGAACACCG CCGCAAAGGT CGGCACGTTG GGCAATGTGG

121 AAGCACTCAA GGCGACGACC GAGCTGGCAG CGTTGGAACG TTCCTTAAAT TCCCTACGGG

180 ATTTTTTAGC CAAAGAAAAG AACGGATGGC AGTCCCAATG ATTGTGCAGT TTTTCAATAG

It would be wise to start treating Bill. What should he be given?

Sophia

Her high fever continues and so do her severe joint pains, and headaches and backaches. She reports frequent eye pain, nausea, and vomiting.

This 362-nucleotide sequence was produced by sequencing a sample of Lupita’s lymph using universal primers (4 nucleotides).

1 gaagtccggc cttccgagag ctagctgtcc gccgcggccc ccgcacgccg ggcagccgtc

61 cctcgccgcc tcgggcgcgc caccatgggg ccccggctca gcgtctggct gctgctgctg

121 cccgccgccc ttctgctcca cgaggagcac agccgggccg ctgcgaaggg tggctgtgct

181 ggctctggct gtggcaaatg tgactgccat ggagtgaagg gacaaaaggg tgaaagaggc

241 ctcccggggt tacaaggtgt cattgggttt cctggaatgc aaggacctga ggggccacag

301 ggaccaccag gacaaaaggg tgatactgga gaaccaggac tacctggaac aaaagggaca

361 ag

Max

Samples of his lesions were taken and universal primers were used to sequence.

The following 119-nucleotide sequence was generated:

1 TTCCCTTCAA CTTGAAGCAC TTTCTTTATC AATTGAATCC CAAACCAAAA TCCCTCTCCT

61 ACTCAAGCGT AACAATTTAT GAGTATAAGA ATATTTTCCA CCCTTCAATG TAATGTAAA

The powder found in the mail was negative for amplification (PCR) and sequencing. It was found to be of non-biological origin.

Justin

His fever has resided somewhat after receiving Tylenol from Dr. Shrub. However, his frontal headache continues and he now has rash on his torso that is rapidly expanding.

Naomi

Her joint pain and aches continue. She is allergic to Tylenol but has taken some ibuprofen which has reduced her fever. A rash just appeared on her arm.

Ponds

Water samples were taken from the pond closest to GOP HQ. Sequencing with universal primers revealed two sequences. The first was 348 nucleotides:

1 atggaaggga aacgtgccct cctgtggctg ctactgattg ctgcagcggc ctttcaattg

61 tccgcccagc actggtcgca cggactcagt ccaggtggca agagggaagc tcacactctg

121 tcagaagtga tggaaggtct accaaagagg agtgcatcgc tttgtgggag tgaatacagg

181 gacggttccc catataaaag gccagataga cttgaacaac tgcttaatct gatggaggga

241 gaaaatgtag cttatgactg aacaacaaaa aatgaacaga ttcaataaaa cactgcagtg

301 tcttctaaat aaaaaaaaaa aaaaaaaaaa aaaaaaaa

The second shotgun sequence was 180 nucleotides:

1 aaaactgaat tagctaaatt attagctaaa caattatttg gttcagaaaa agaattaata

61 agatttgata tgagtgaata tatggaaaaa cattcaattt caagattaat tggttcacct

121 ccaggttata taggttattc agaaggagga caattaacag aacaagtttt taaaaaacct

DAY 4

July 15

Bill

What is Bill’s final diagnosis and how will you treat him?

Sophia

What was Sophia’s sequence sample and was it helpful in the diagnosis?

Her high fever continues and so do her severe joint pains, with headaches and backaches. She reports a large rash on her chest and arms.

After hearing about the focus on the water in the meditation ponds, she lets on about a recent addition she added to the water. She had recently purchased some Inca artifacts from Peru from eBay-Peru in late January, 2011. The Peruvian dealer also sent some Inca holy water from Lambayeque to bless the arrival of the artifacts to their new home. Knowing that this was “sacred water,” Sophia poured it into the nearest meditation pond in order to cleanse the republican campaign.

Max

What was he infected with and how will you treat him?

Justin

His fever, headache, and rash continue. He also has some bleeding of the gums and nose. It is starting to look critical.

This 362-nucleotide sequence was produced by sequencing a sample Justin’s rash-pustule using universal primers (4 nucleotides).

1 gaattctggt tgatcctacc agtaatatac gcttgtctca aaggttaagc catgcatgtc

61 taagtacaaa cagatttaat gtgaaaccgc ataaggctca gtataacagc tataatttac

121 aagatcattt aactagttac ttggataact gtggaaaatc tagagctaat acatgcaaaa

181 tgcaggaacc tcgcggaacc tgtgcaatta ttagtcaaac caatcgtcct ccgtgacgct

241 ggagttgaaa tctggataat tttgttgatc gtatggtctc gcaccgacga cagatctttc

301 aaatatctgc cctatcaact attgatggta gtatagagga ctaccatggt tgcaacgggt

361 a

Naomi

Her joint pain and aches continue. Her rash just spread to her legs.

This 120-nucleotide sequence was produced by sequencing a sample Naomi’s lymph using universal primers (4 nucleotides).

1 ataggagtcgtcatcacatggataggaatgaattcacgcagcacctcactgtctgtgtcactagtattag

61 gggagtcgtgacattgtatttgggagttatggtgcaggccgatagtggttgcgttgtgagttggaaaaa

Ponds

More water samples were taken from the pond closest to GOP HQ. Sequencing with universal primers revealed two sequences. The first was 129 nucleotides:

1 tttctgttta gtttcattac agatctggac atcctgtccc ttctagcttc atcagcttag

61 tagtctaagt agctcaagaa atcccgataa atcctttcct tccctgttac aaatgtatta

121 actaacctt

The second shotgun sequence was 249 nucleotides:

1 cacgttgaac gcatattgca catcgtacta ccagtacgat gtacacattt ttgagtgcct

61 atatttatcc attcaactat acgcgccgcc cgcgcgcgta tgcgtagtga tgttttcccg

121 ccttcagtgc gcggtaaaac attgaagata gtcagacgtg gtggtgacac accgcggttg

181 atgaatacat cccactatgg cgcgctcgct cgccttgtgt tgtattccat cattcactaa

241 ctaactccct

DAY 5

FINAL REPORT

July 20th

Instructions:

Trace path of infection for each patient. Draw out a flow chart (Outbreak style) and label unknown or speculative pathways with question marks. Make sure this is done for each patient. This information will hopefully reduce the risk of future infection.

For each patient, write out the primary evidence that brought you to your conclusion. It is important to rule out false positives in this line of investigation.

Generally, you want multiple criteria confirmed before making your diagnosis.

Be sure to answer the following questions:

• Is everyone treated?

• Are they going to be ok?

• Can this be prevented in the future?

Final Report

• Identify pathogen and path of infection for each patient.

• Evidence for pathogen identification.

• Recommendation for prevention of similar scenarios.

Conclusion

1. What is more definitive in pathogen identification: clinical information or sequence data? Defend your answer.

2. What variables must be considered when engineering a computer program to sleuth an outbreak scenario?

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