Project 5.1.1B Evidence Report #2
Culture Results
Patients present with elevated white blood cell count (left shift) and persistent fever, suggesting infection. Both blood and urine culture samples were collected from each patient and were sent to the lab for analysis.
Blood culture is used to detect the presence of bacteria or yeast in the blood and to identify any microorganisms that may be present. A blood sample is cultured on nutrient media and monitored for growth of microorganisms.
Urine culture tests for the presence of microorganisms in the urine. A small sample of the urine is placed on an agar plate and incubated at body temperature. A microorganism that is present should grow in isolated colonies after 24-48 hours. A colony is then smeared on a clean slide and subjected to Gram-staining and further identification tests.
Results
The blood culture results for Patients 1, 3, 4, and 5 come back negative. The sample from Patient 2 was inconclusive and will be redrawn. Doctors begin to rule out PCP pneumonia, Cryptococcus or other infection by yeast, and aspergillus contaminated food products.
Urine culture samples from Patients 1, 3, 4, and 5 showed no growth. The urine culture results for Patient 2 reveal greater than 100,000 organisms/ml. Pink, Gram-negative rods are detected under a microscope.
Environmental Testing Report
All five patients now present with respiratory symptoms. The differential diagnosis is consistent with poisoning. Suspected agents include phosphine, a fast-acting poison used to kill outdoor pests. Therefore, an environmental testing team was sent to search the residence of each patient. Various pest traps were found at more than one home, but there was no evidence of phosphine poison at any patient residence. Two of the more rural residences were located next to farms. When interviewed, both farm owners mentioned spraying with both herbicides and pesticides; however, no evidence of either chemical was found on the property of a patient.
The environmental team determined that two of the homes are supplied by public water and two rely on wells. Water samples were taken from each well, as well as from the lake where Patient 3 had been camping.
PCR-based testing was run to determine the presence or absence of coliform bacteria in each source. Samples were run on a gel and analyzed.
Lane 1: 200bp ladder
Lane 2: Positive control
Lane 3: Negative control
Lane 4: Well sample – Patient 1 and 2
Lane 5: Well sample – Patient 5
Lane 6: Lake sample
Chemical assays from each source were all within normal limits for lead, nitrates, chloride, copper, pH, and sulphate.
Zoonotic disease cannot be ruled out at this time.
Interview Log
Epidemiologists are searching for patterns among patients and for additional clues as to how the disease in question is transferred. Additional information was gathered regarding specific responses on the illness complaint worksheet. All patients were interviewed on their first day of admission.
Patient 1
Q: It says here that you are a biology teacher. Do you use any harmful chemicals at work?
A: I do set up some labs that require chemicals, but as this is the time of the year when we study anatomy, I have been doing a lot of dissections with the students. Last week students dissected a pig heart. Companies do not use formaldehyde to package these specimens anymore, so I believe they are relatively safe.
Q: You said you just returned from a conference in Beijing. Was it a big event?
A: Oh yes, there were thousands of teachers from around the world there to exchange teaching strategies and ideas. The sessions were interesting, but I spent way too many hours cramped in small rooms with tons of people. The only parts of the city I managed to see were the subway and a few restaurants. By the end of the conference, I was tired and ready to come home.
Patient 2
Q: It says on your intake sheet that you are restoring a barn at your house. What does that entail?
A: We live on old farmland and are converting this space to an extra bedroom/guest house. We have just gotten things to the point where we can put up new walls. It took a long time to sweep out all of the hay and clean the area. Luckily, my fiancé did a good job clearing out the old farm equipment before he left for China.
Q: You noted that your nephew was sick. Is he feeling better?
A: Actually, it has been a week and he still has a fever. I talked to him the other night, and he could not stop coughing.
Patient 3
Q: Could you tell me a bit more about your hiking trip?
A: The past few months have been so rainy and unpleasant. My friends and I were looking forward to getting out in the nice weather and taking a break from school. It was a relaxing trip. We found a great campsite near a lake and slept out under the stars. I did have a hard time sleeping. The sound of animals moving around was hard to ignore. I also kept scratching my mosquito bites, some of which became pretty raw.
Q: What did you eat and drink out there?
A: We brought some bottled water, but filtered what else we needed from the lake. I am pretty picky when it comes to food, so I brought all of my own snacks in my pack. We did bring some meats to cook over the fire and kept them in a cooler at our camp.
Patient 4
Q: What can you tell me about your job?
A: I clean houses for my brother’s cleaning service. His company is often called in to clean up houses that have been abandoned or foreclosed upon. We get the houses ready to be placed on the market for resale.
Q: How often do you have chest pain? Do you have it monitored regularly?
A: I don’t have chest pain nearly as often as I used to. I am trying to cut down the level of stress in my life. My doctor has me taking medication for my cholesterol. I try to watch what I eat, but it’s hard when I am working so much. I am overdue for my annual checkup. The doctor said he would like to perform a stress test at the next visit.
Patient 5
Q: Do you have any brothers or sisters?
A: I have two brothers, but they are older and don’t want to play with me as much these days. They stay late at school and have lots of homework when they do get home.
Q: What type of things do you do when you play?
A: I like to play sports and hang out with my friends. I like watching movies, but my mom doesn’t let me watch TV if it is nice outside. I spend a lot of time in my backyard. Now that my brothers are big, they let me use their tree house.
Molecular Testing
A colleague from out of state sends you the following molecular testing results.
Lung tissue samples were collected (via biopsy) from a patient suffering symptoms similar to those in your cluster. RT-PCR was used to amplify the suspected disease agent, and the genetic information was sent to the lab for sequencing. The lab returned the following data.
agacgggcag ctgtgtctgc attggagacc aaactcggag aacttaagcg ggaactggc gatcttattg cagctcagaa attggcttca aaacctgttg atccaacagg gattgaacct gatgaccatc taaaggaaaa gtcatcattg agatatggaa atgttcttga tgtaaattcc attgacttag aagagccaag tgggcaaaca gctgattgga aatccatcgg actctacatt ctaagtttcg cattaccgat tattcttaaa gccttgtaca tgttatctac taggggccgt caaacaatca aagaaaacaa gggaacaaga attcgattca aggatgattc atcttatgaa gaagtcaatg ggatacgtaa gccaagacat ctgtatgttt ctatgccaac tgcccagtct
1. Visit the National Center for Biotechnology Information (NCBI) BLAST site at http://blast.ncbi.nlm.nih.gov/Blast.cgi to analyze the DNA sequence isolated from Patient 1’s sample.
2. From the menu under Basic BLAST, choose nucleotide blast.
3. Wait for the query page to open. Cut and paste the sequence data from the sequence above into the Enter Query Sequence box.
4. Locate the heading Choose Search Set. Click on the down arrow to reveal more choices.
5. Under Other Databases, choose Nucleotide Collection (nr/nt). Note that you can search information by amino acid sequences as well as nucleotide sequences. As you are looking strictly at DNA in this example, you will focus on nucleotides.
6. Locate the heading Program Selection. Choose Somewhat similar sequences –blastn.
7. Once all of the parameters are set, click the large blue BLAST button at the bottom of the page to search the genetic database for the entered sequence. This search may take a few moments.
8. Scroll down the results page to view the matches that are found. The descriptions of organisms or genes found at the top of the list have the most in common with the sequence that was entered.
Serology Results
Laboratory microscopy studies locate a virus in the tissue of Patient 2. The electron micrograph is shown below.
Image courtesy of Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Disease, CDC.
**The full citation can be found in the lesson document and the associated teacher resources.
Given this result and the results of the molecular testing, you further investigate potential viral invaders.
Immunohistochemistry
Immunohistochemistry locates antigens or proteins in tissue sections by the use of labeled antibodies as specific reagents. Through antigen-antibody interactions, antigens of interest are located by a marker such as a fluorescent dye, enzyme, or colloidal gold.
Because of Patient 3’s declining renal function, you take a small biopsy of the kidney. Tissue samples from this patient are placed on slides and incubated with antibody or serum samples available from the Centers for Disease Control (CDC) collection. IHC testing of patient tissues with specific monoclonal and polyclonal antibodies can be used to detect viral antigens and has proven to be a sensitive method for laboratory confirmation of some infections.
In your test the presence of the antigen is signaled by a red/pink stain, a fluorescent dye conjugated to the antibody. High power magnification showing immunostaining of renal interstitial capillaries using Peromyscus maniculatus serum is displayed below.
ELISA
Serum samples are collected from each patient to confirm the results of the immunostaining. ELISA was run using patient serum and antibodies against three viral invaders spread via animal vector – Lymphocytic Choriomeningitis, West Nile Virus, and Sin Nombre Virus (SNV).
All patient samples tested negative for Lymphocytic Choriomeningitis and West Nile. Results for SNV are shown below.
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Biomedical Innovation Project 5.1.1B Evidence Report #2 – Page 3