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22 April 2018
PLATFORM2013: Sleeping sickness diagnosis improved by University of Zambia Print
Thursday, 12 December 2013 10:23

University of Zambia’s article in PLATFORM2013 discusses the re-emergence of sleeping sickness, a tsetse transmitted debilitating disease of humans and animals in Sub-Saharan Africa. Diagnosis of sleeping sickness in endemic regions remains unsatisfactory, but cost-effective Loop-mediated Isothermal Amplification [LAMP] has the potential to accurately address this problem.

Researcher: Prof. Boniface Namanagala | Head of Department | Paraclinical Studies | University of Zambia | Lusaka.

(Portrait: Vice-Chancellor Prof. Stephen Simukanga.)

Research in context
“The University of Zambia (UNZA) strives to meet society’s needs and aspirations through its motto of ‘service and excellence in teaching, research and public service,’” said Vice-Chancellor Prof. Stephen Simukanga, when discussing Research Uptake at the University. “The 2013 UNZA Research Uptake and Dissemination activity calendar is in line with research focus areas number five and seven of the 2013–2017 UNZA strategic plan. The highlighted study for the 2013 DRUSSA publication, currently being taken up by our Ministry of Health, describes a simple molecular procedure developed for detecting sleeping sickness, and will potentially benefit both local and international communities. Communications about this research will be disseminated through our planned Research Uptake initiatives on the University website, on radio, at media briefings and on institutional visits.”

 

Background
In Zambia, sleeping sickness, caused by trypanosoma brucei rhodesiense, is endemic in Luangwa river valley where increasing unpublished cases are being reported. Control of sleeping sickness heavily relies on accurate diagnosis and effective case management. Current available para- sitological methods used for sleeping sickness diagnosis are either not sensitive enough to, or unable to accurately identify species.

Polymerase chain reaction (PCR) is a laboratory test for the diagnosis of a wide variety of central nervous system (CNS) diseases and has higher sensitivity and specificity for trypanosoma detection than many other tests. However, the cost and skill implications of using this method are obstacles to its wide application in clinical settings in Sub-Saharan Africa where sleeping sickness is endemic. A low-cost alternative is loop-mediated isothermal amplification (LAMP), a novel strategy that amplifies DNA with high sensitivity and rapidity under isothermal conditions, producing large quantities of DNA within 60 minutes. This allows visual detection of amplicons, which are pieces of artificially amplified DNA or RNA that can be used for purposes that include detection and quantification of infectious agents by the naked eye. Advances in the development of trypanosome- specific LAMP have been made through identification of conserved sequences, including the human-serum resistance-associated (SRA) gene that defines trypanosoma brucei rhodesiense, andtherefore provides unequivocal identification of the parasite. In the present study at University of Zambia, the performance of SRA-LAMP was evaluated using blood and cerebral-spinal fluid (CSF) from 110 people from Zambia’s Luangwa and Zambezi river valleys.

Methodology
This study received ethical clearance from Zambia’s Ministry of Health and from the University of Zambia’s Biomedical Research Ethics Committee for the collection of human blood and CSF. The majority of the consenting participants were sampled in 2010 during a sleeping sickness screening exercise in Chama, a small town in the Eastern province of Zambia. The remainder of participants was made up of 10 people who were reported as suspected sleeping sickness patients between 2010 and 2013. Each of the 10 sleeping sickness patients exhibited malaria-like symptoms and mainly sought medical attention at the University Teaching Hospital, Lusaka, where they were admitted. After clinical examination, patient blood and CSF samples were collected. The presence of trypanosomes was detected by microscopic examination of the buffy-coat following centri- fugation of blood and leukocyte count (WBC) in the case of CSF. Thereafter, around 200μl blood or CSF was placed on a labelled FTA-Elute-card for DNA extraction according to manufacturer’s suggested protocol. A LAMP reaction of 25μl was performed using a Loopamp DNA Amplification Kit (Eiken Chemical, Japan) and the DNA extracted as described by Namangala et al., using primers recently described by Njiru et al. for SRA-LAMP. The reaction mixture was incubated at 64°C for 30 minutes in a heat block and then at 95°C for two minutes to terminate the reaction. The DNA produced by the LAMP was made visible using a transilluminator, which passes light through the DNA.

Results and discussion
SRA-LAMP confirmed sleeping sickness in 9.1% of the participants. LAMP was not only sensitive, but also very specific and confirmed the microscopic observation of the trypanosomes in patients. The presence of trypanosoma brucei rhodesiense only in patient blood but not in CSF, coupled with ≤5 WBCs/mm3 in CSF, signified early-stage Human African trypanosomiasis (HAT), which was treated with suramin1,6. However, the presence of the parasites in both blood and CSF, coupled with >5 WBCs/mm3 in CSF, indicated late-stage HAT, which was treated with melarsoprol1,6. All 10 treated sleeping sickness patients eventually recovered. The 100 other suspected sleeping sickness cases from Chama that tested negative by microscopy also tested negative by LAMP, demonstrating their good concordance.

The 10 sleeping sickness cases reported herein were all detected through passive surveillance. It is possible that these cases could represent several others that may be unreported considering that sleeping sickness affects the underprivileged marginalised remote rural communities. According to Odiit et al., around 39% of rhodesiense sleeping sickness cases and 92% of the deaths it causes are unreported. Indeed, there are several unpublished sporadic sleeping sickness cases reported at local health centres mainly linked to National Parks. Of note, in 2008 alone, 12 cases were reported among Zambia Wildlife Authority staff assigned to the then newly openedMbambanda-Zaro sanctuary in Chama.

 

According to these preliminary results, availed to policy-makers, i.e. the Zambian Ministry of Health, LAMP testing confirmed the standard staging criteria (microscopy and WBC in CSF), which led to successful treatment of all the patients. Accurate staging of sleeping sickness, i.e. ascertaining what stage the illness is at, is critical for appropriate therapeutic decisions. Despite its simplicity, LAMP is highly sensitive, specific, rapid and user-friendly. Such a cost-effective technique has the potential to be the future principal molecular tool in sleeping sickness diagnosis and staging in resource- limited sleeping sickness-endemic regions like Zambia.

Join the Research Uptake Conversation

If this piece of evidence-based development research from SSA is of use to you, please continue the Research Uptake Conversation by contacting the University of Zambia.

Website: www.unza.hm

DRUSSA Research Uptake Leader at University of Zambia
Dr. John Simwinga
Deputy Director
Research Capacity Building And Dissemination/Senior Lecturer 
j.simwinga@unza.zm

DRUSSA Research Uptake Past Leader at University of Zambia
Prof. Imasiku A. Nyambe
Director
Directorate of Research and Graduate Studies
inyambe@unza.zm

DRUSSA Research Uptake Champions at University of Zambia

Dr W. N.M. Mwenya
Deputy Vice-Chancellor
dvc@unza.zm

Dr Vitalicy Chifwepa
University Librarian
vchifwepa@unza.zm

Researcher and article writer
Prof. Boniface Namangala
Head of Department
Department of Paraclinical Studies
boniface_1020@yahoo.com
b.namangala@unza.zm

PLATFORM2013 in digital
Read this article on pages 32 and 33 of PLATFORM2013 – a print and digital publication from the DRUSSA Universities in Sub-Saharan Africa [SSA] – aimed at accessibly communicating evidence-based development research with the goal of deepening its reach and impact in the region. 

Follow the PLATFORM2013 blog campaign daily until 16 January 2014. You’ll find the blog schedule on PLATFORM2013 articles from the DRUSSA Universities here

Twitter @DRUSSAfrica #PLATFORM2013 #ResearchUptake (Tweet about this on your twitter account)

 

 

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