| Case A | Case B |
Plasmodium sequences include chromosome 5 plus 18S rRNA. Primers that distinguish Plasmodium species amplify 18S rRNA sequences. The nested PCR protocol is not necessary with the Case It software, but it replicates the protocol used in a clinical or research lab, where the Plasmodium DNA needs to be amplified before species-specific primers are used.
Reference: Johnston et al., 2006. PCR as a Confirmatory Technique for Laboratory Diagnosis of Malaria, J. Clin. Microb. doi:10.1128/JCM.44.3.1087–1089
Case A. Sergio Román was glad to be surrounded by his family during the holidays. This would be the first Christmas without his older sister, Milagros, who died after contracting malaria during a missionary visit to Columbia. She was diagnosed when she returned to Puerto Rico with a high fever that came and went every few days. Milagros took the drugs they prescribed and seemed to recover, but a month later the fevers returned, and this time she did not recover. Sergio was frustrated because he did not understand why the drugs did not work for Milagros. Other members of the mission group, including Sergio’s younger sister, Rosario, had also become infected but recovered fully after the drug treatments. Both were treated with the same dosage of chloroquine. It did not seem fair that a disease that was supposedly eradicated from Puerto Rico many years ago should take such a toll on Sergio’s family. Milagros and her family had agreed to allow her blood samples to be used for research into drug resistant malaria.
Procedure: To determine which species of Plasmodium infected the two sisters, perform a nested PCR by first amplifying DNA isolated from their blood with a general Plasmodium primer set. Then use a second round of PCR, amplifying the first PCR product using species-specific primers.
The products expected with the specific primers are 205 bp for P. falciparum, 120 bp for P. vivax, 800 bp for P. ovale, and 144 bp for P. malariae. Band size is 120 bp, so Both Milagros and Rosario were infected with P. vivax.
Bioinformatics: SIngle nucleotide polymorphisms (SNPs) in the multidrug resistance gene pfmdr-1 have been associated with resistance to the drug treatments used for Milagros. DNA samples from Milagros and Rosario were tested by using PCR with primers that amplify regions containing SNPs. Both yield 3 bands (182, 135, and 91 bp). Sequences of the PCR products were compared by exporting the sequences to the Export field and aligning them with MEGA. The sequences of the 135 and 91 bp bands are identical. However, alignment of the 182 bp band sequence reveals a SNP at position 139:
Case B. Jennie could not wait to tell her best friend the news. “You’re kidding me! Your dad actually bought you that ‘round-the-world’ ticket you’ve been asking for every birthday since you turned 18?” Synove looked at her best friend incredulously and continued, “I just can’t believe it!”
“Yup.” Jennie answered, looking a little smug. But then her face brightened as she smiled, telling Synove, “But, he said I couldn’t go alone. So he bought TWO tickets and told me to pick a friend to go with me! That’s you!”
“No way!” Synove shouted. “Where are we going to go?”
“Well, I thought we’d start off heading to Europe first just to get acclimated to traveling on our own. Maybe start in Paris and bike to southern Spain. Then cross the Straits of Gibraltar to Morocco and head down west Africa. Then we hop a flight Victoria Falls in Zimbabwe; safari in Botswana and Namibia; Cape Town; and up the east coast for a break in Dar es Salaam. From there we can fly to India and bum around a bit before heading to Thailand and Cambodia. Then we visit my extended family in Taiwan before catching a flight to Japan and from there back to Seattle. What do you think?” Jennie asked.
“Wow. Sounds great! Do we have to get any shots before we go?”
“I’m pretty sure, but I’ll ask my mom tonight.” Jennie said.
“Of course! A doctor would know…”
“Well girls, you’re going to need lots of shots before you go on this world tour of yours.” said Jennie’s mother. You’ll need Yellow Fever, Hepatitis A and B, Typhoid, Rabies, and Meningococcal. Jennie I know you’ve had it, but Synove, do you know if you were vaccinated for Polio as a child? If not, you’ll need that. For India, Thailand, and Cambodia you’ll probably need to get Japanese encephalitis too if you’re going to be in rural areas. You should probably get Dengue Fever immunizations too, just to be on the safe side. Good thing you’ve got about eight months before you leave. All these shots are going to take a while.”
“Mom, what about malaria? Do we need to worry about that?”
About a week after returning from their trip, Jennie began to feel ill. She was feverish, felt weak, had a headache, and just ached all over. Both she and Synove thought it was just the flu. But one afternoon, Synove couldn’t wake Jennie from a nap she was taking between classes and called an ambulance. Based on Jennie’s recent travels and her symptoms, the physician at the hospital orders a blood analysis, which revealed evidence of Plasmodium.
Procedure: A nested PCR was performed by first amplifying DNA isolated from her blood with a general Plasmodium primer set. A second round of PCR was then performed, amplifying the first PCR product using species-specific primers. Jennie has a positive result for the PCR, and her band is the same size as the P. faciparum band.