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March 13, 2014

Challenging Infectious Diseases in Zambia With a 100-Yen Diagnostic Kit
New diagnostic kit opens a door to early diagnosis and treatment for tuberculosis and trypanosomiasis

A key to preventing infectious diseases from spreading is early detection and treatment of the patients. In developing countries, however, it doesn’t get to the stage of finding the patients because of the high inspection cost in many cases.

One in three people is infected by tuberculosis

photoThe diagnostic kit developed by the project team.

The spread of tuberculosis is a danger. Though tuberculosis is regarded as a disease of the past in Japan, it is reported that one third of the global population is still infected now and 1.4 million people lose their lives annually. The infection is especially prevalent in Africa, and one in every three people is infected in Zambia. In addition, co-infection among HIV-infected people who have low immunity and an epidemic caused by an increase in multidrug-resistance tuberculosis with antibiotics tolerance are the cause of great concern.

On the other hand, infection with African trypanosomiasis (sleeping sickness, 1) in sub-Saharan Africa is widely spread, with an estimated 50,000 people killed every year. In Zambia many cases are reported where the symptoms become severe because of misdiagnosis as malaria, and result in death. Swift action is also required in these cases.

photoProfessor Yasuhiko Suzuki, middle, teaches procedures for gene amplification to Zambian researchers.

Following a request from the government of Zambia, the Japan International Cooperation Agency (JICA), along with the Japan Science and Technology Agency (JST), implemented a project, “Establishment of Rapid Diagnostic Tools for Tuberculosis and Trypanosomiasis and Screening of Candidate Compounds for Trypanosomiasis” (2), headed by Chief Advisor, Professor Yasuhiko Suzuki of the Research Center for Zoonosis Control at Hokkaido University. With joint efforts by Research Center for Zoonosis Control and others, they succeeded in developing an accurate, rapid and cheap diagnostic kit for tuberculosis and African trypanosomiasis (3).

The traditional tuberculosis exam involves a combination of a sputum smear exam, the checking of the germ by a microscope, and an incubation exam, which involves incubating the sputum to increase the number of germs. The result of a sputum exam is available quickly; however, detection is not possible if the quantity of germs is small. That is why the incubation exam is implemented to make a final diagnosis, even though the result of a sputum smear exam is negative.

In Zambia, it takes two to five days to obtain the results of a sputum smear exam. Moreover, if the result of the sputum smear exam is negative, the incubation exam as the next step is omitted in most cases. Even when an incubation exam is implemented, the diagnostic accuracy can be low because the skill of individual technicians differs and such exams are prone to human errors. Also, since it requires two to four weeks to see the result of an incubation exam, it could take up to a month before the patient starts receiving treatment, or in some cases the hospital could no longer contact the patient by the time the exam result is available. There is also a risk of worsening symptoms while waiting for the result and thereby spreading the infections around.

Trypanosomiasis, on the other hand, is diagnosed by checking parasites in the blood with a microscope. The accuracy is low too, so early detection is difficult.

Achieving diagnosis on the day of the check and reduced cost

photoExpert Mari Miller carries out a diagnostic test

To address these challenges, the development of the new diagnostic kit applied a technique to amplify genes of tuberculosis germs or parasites, extracted from the sputum or blood of the patients. With this, the time required from the exam to its result was reduced to approximately an hour, and the accuracy is now close to 100 percent. Thanks to the new technology, 60 to 70 percent of overlooked patients with the traditional exam can now be found on the day of the exam. “The new diagnostic kit will make a point-of-care diagnosis (4) common in rural areas, away from the town,” expert Mari Miller expects.

In addition, the use of an inexpensive reagent developed by Hokkaido University made it possible to reduce the cost of the new exam to approximately 100 yen, while other quick diagnostic methods cost 1,000 yen to 2,500 yen. “The cheaper cost will enable more people to be inspected and the risk of infection spread will be significantly reduced. Moreover, it is expected to lead to early detection and treatment,” Miller explains.

“It is a breakthrough in Zamia to be able to see the results on the same day of the inspection, and to move forward to a treatment of the patient straight away. This is a great achievement,” said Masaru Iizuka, an expert who has been in charge of operational coordination since the start of the project in 2009.

Aiming for more accurate exam result

photoExpert Kiichi Kajino from Hokkaido University diagnoses trypanosomiasis using the new diagnostic kit at a rural health center.

It was a challenge to put the diagnostic kit into practical use in Zambia. At a demonstration experiment, the team encountered an unexpected problem: variability in the exam results depending on the procedure of each Zambian lab staff member. For the exam, they blend several types of defrosted liquid reagent. The quantity of each reagent comes in by microliter and the test requires procedural accuracy on the part of the staff. However, the accuracy of the exam wasn’t kept accurate because some engaged in idle talk while in the process, and others failed to follow the procedure and cut corners.

To tackle the problem, the team developed a dried reagent as an alternative to the liquid reagent. It is easier to manage temperature with the dried reagent compared with the liquid reagent that requires low temperature control. Also the procedure of mixing the reagent can be simplified so individual variability is minimized, and thus exam accuracy is improved. On top of that, for a fluorescence detector to detect genes, the team developed a small-sized detector powered by size AA batteries so that it works in the area where the power situation is unstable. With all these efforts, it became possible to keep exam results accurate even in rural areas.

As the diagnostic kit was developed and introduced toward the end of the project, there was a timeline concern over whether data collection by demonstration experiment could be completed before project completion in November 2013. But the team made it, and the research data was praised by the Ministry of Health in Zambia. Iizuka and Miller both recalled that it was a rush until the last moment, but the achievement was the fruit of the teamwork of each project stakeholder.

The transferred procedure has already been put into a practical use in Zambia, and is also attracting interest from other countries in Africa and Asia. Moreover, the procedure has the possibility of being applicable to other infectious diseases. There are large hopes for the 100-yen diagnostic kit.

Notes:

1. A zoonotic infectious disease caused by trypanosomiasis, with the parasites carried by tsetse flies. Transmission occurs through blood sucking by tsetse flies, as well as contact with infected livestock, and causes sleeping disorders. Also known as African sleeping sickness.
2. A JST and JICA program for research projects targeting global issues and involving partnerships between researchers in Japan and developing countries. Science and Technology Research Partnership for Sustainable Development (SATREPS).
3. The primer (strand of nucleic acid used to copy a targeted nucleic acid) used for the diagnostic kit is different for tuberculosis and trypanosomiasis. For tuberculosis, sputum is used as the specimen, and for trypanosomiasis, blood is used. The reaction temperature after adding the specimen is different for each. The small-sized fluorescence detector can be used for both kits.
4. Procedures or equipment that doesn’t involve transporting the patient’s specimen elsewhere to have it checked at a dedicated inspection-specialized facility, but allows the exam result to be checked at the place of the exam.

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