Use of Polyclonal Antibody for the Diagnosis of Human African Trypanosomiasis

Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3rd immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4th immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.

Specificity of serological screening tests and reference laboratory tests to diagnose gambiense human African trypanosomiasis: a prospective clinical performance study

Background Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis(gHAT).Presently,they preselect individuals for microscopic confrmation,but in future"screen and treat"strategies they willidentify individuals for treatment.Variability in reported specificities,the development of new rapid diagnos-tic tests(RDT)and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests.Methods During active screening,venous blood samples from 1095 individuals from Cote d'ivoire and Guinea were tested consecutively with commercial(CATT,HAT Sero-K-SeT,Abbott Bioline HAT 2.0)and prototype(DCN HAT RDT,HAT Sero-K-SeT 2.0)gHAT screening tests and with a malaria RDT.Individuals with≥1 positive gHAT screening test underwent microscopy and further immunological(trypanolysis with T.b.gambiense LiTat 1.3,1.5 and 1.6;indirect ELISA/Tb.gambiense;T.b.gambiense inhibition ELISA with T.b.gambiense LiTat 1.3 and 1.5 VSG)and molecular reference laboratory tests(PCR TBRN3,18S and TgsGP;SHERLOCK 18S Tids,7SL Zoon,and TgsGP;Trypanozoon S2-RT-qPCR 18S2,177T,GPl-PLC and TgsGP in multiplex;RT-qPCR DT8,DT9 and TgsGP in multiplex).Microscopic trypanosome detection confrmed gHAT,while other individuals were considered gHAT free.Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar.Results One gHAT case was diagnosed.Overall test specificities(n=1094)were:CATT 98.9%(95%CI:98.1-99.4%);HAT Sero-K-SeT 86.7%(95%CI:84.5-88.5%);Bioline HAT 2.082.1%(95%CI:79.7-84.2%);DCN HAT RDT 78.2%(95%CI:75.7-80.6%);and HAT Sero-K-SeT 2.078.4%(95%CI:75.9-80.8%).In malaria positives,gHAT screening tests appeared less specific,but the difference Was significant only in Guinea for Abbott Bioline HAT 2.0(P=0.03)and HAT Sero-K-Set 2.0(P=0.0006).The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100%(n=399)and 93.0-100%(n=302),respectively.Among 44 reference laboratory test positives,only the confrmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity.Conclusions Although a minor effect of malaria cannot be excluded,gHAT RDT specificities are far below the 95%minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment.Unless specificity is improved,an RDT-based"screen and treat"strategy would result in massive overtreatment.In view of their inconsistent results,additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying,among screening test positives,those at increased suspicion for gHAT.

Performance of clinical signs and symptoms,rapid and reference laboratory diagnostic tests for diagnosis of human African trypanosomiasis by passive screening in Guinea:a prospective diagnostic accuracy study

Background Passive diagnosis of human African trypanosomiasis(HAT)at the health facility level is a major component of HAT control in Guinea.We examined which clinical signs and symptoms are associated with HAT,and assessed the performance of selected clinical presentations,of rapid diagnostic tests(RDT),and of reference laboratory tests on dried blood spots(DBS)for diagnosing HAT in Guinea.Method The study took place in 14 health facilities in Guinea,where 2345 clinical suspects were tested with RDTs(HAT Sero-K-Set,rHAT Sero-Strip,and SD Bioline HAT).Seropositives underwent parasitological examination(reference test)to confirm HAT and their DBS were tested in indirect enzyme-linked immunoassay(ELISA)/Trypanosoma brucei gambiense,trypanolysis,Loopamp Trypanosoma brucei Detection kit(LAMP)and m18S quantitative PCR(qPCR).Multivariable regression analysis assessed association of clinical presentation with HAT.Sensitivity,specificity,positive and negative predictive values of key clinical presentations,of the RDTs and of the DBS tests for HAT diagnosis were determined.Results The HAT prevalence,as confirmed parasitologically,was 2.0%(48/2345,95%CI:1.5–2.7%).Odds ratios(OR)for HAT were increased for participants with swollen lymph nodes(OR=96.7,95%CI:20.7–452.0),important weight loss(OR=20.4,95%CI:7.05–58.9),severe itching(OR=45.9,95%CI:7.3–288.7)or motor disorders(OR=4.5,95%CI:0.89–22.5).Presence of at least one of these clinical presentations was 75.6%(95%CI:73.8–77.4%)specific and 97.9%(95%CI:88.9–99.9%)sensitive for HAT.HAT Sero-K-Set,rHAT Sero-Strip,and SD Bioline HAT were respectively 97.5%(95%CI:96.8–98.1%),99.4%(95%CI:99.0–99.7%)and 97.9%(95%CI:97.2–98.4%)specific,and 100%(95%CI:92.5–100.0%),59.6%(95%CI:44.3–73.3%)and 93.8%(95%CI:82.8–98.7%)sensitive for HAT.The RDT’s positive and negative predictive values ranged from 45.2–66.7%and 99.2–100%respectively.All DBS tests had specificities≥92.9%.While LAMP and m18S qPCR sensitivities were below 50%,trypanolysis and ELISA/T.b.gambiense had sensitivities of 85.3%(95%CI:68.9–95.0%)and 67.6%(95%CI:49.5–82.6%).Conclusions Presence of swollen lymph nodes,important weight loss,severe itching or motor disorders are simple but accurate clinical criteria for HAT referral in HAT endemic areas in Guinea.Diagnostic performances of HAT Sero-K-Set and SD Bioline HAT are sufficient for referring positives to microscopy.Trypanolysis on DBS may discriminate HAT patients from false RDT positives.

Integrating innovations:a qualitative analysis of referral non-completion among rapid diagnostic test-positive patients in Uganda’s human African trypanosomiasis elimination programme

Background:The recent development of rapid diagnostic tests(RDTs)for human African trypanosomiasis(HAT)enables elimination programmes to decentralise serological screening services to frontline health facilities.However,patients must still undertake multiple onwards referral steps to either be confirmed or discounted as cases.Accurate surveillance thus relies not only on the performance of diagnostic technologies but also on referral support structures and patient decisions.This study explored why some RDT-positive suspects failed to complete the diagnostic referral process in West Nile,Uganda.Methods:Between August 2013 and June 2015,85%(295/346)people who screened RDT-positive were examined by microscopy at least once;10 cases were detected.We interviewed 20 RDT-positive suspects who had not completed referral(16 who had not presented for their first microscopy examination,and 4 who had not returned for a second to dismiss them as cases after receiving discordant[RDT-positive,but microscopy-negative results]).Interviews were analysed thematically to examine experiences of each step of the referral process.Results:Poor provider communication about HAT RDT results helped explain non-completion of referrals in our sample.Most patients were unaware they were tested for HAT until receiving results,and some did not know they had screened positive.While HAT testing and treatment is free,anticipated costs for transportation and ancillary health services fees deterred many.Most expected a positive RDT result would lead to HAT treatment.RDT results that failed to provide a definitive diagnosis without further testing led some to question the expertise of health workers.For the four individuals who missed their second examination,complying with repeat referral requests was less attractive when no alternative diagnostic advice or treatment was given.Conclusions:An RDT-based surveillance strategy that relies on referral through all levels of the health system is inevitably subject to its limitations.In Uganda,a key structural weakness was poor provider communication about the possibility of discordant HAT test results,which is the most common outcome for serological RDT suspects in a HAT elimination programme.Patient misunderstanding of referral rationale risks harming trust in the whole system and should be addressed in elimination programmes.

Estimating the economic and social consequences for patients diagnosed with human African trypanosomiasis in Muchinga,Lusaka and Eastern Provinces of Zambia(2004-2014)

Background:Acute human African trypanosomiasis(rHAT)caused by Trypanosoma brucei rhodesiense is associated with high mortality and is fatal if left untreated.Only a few studies have examined the psychological,social and economic impacts of rHAT.In this study,mixed qualitative and quantitative research methods were used to evaluate the socio-economic impacts of rHAT in Mambwe,Rufunsa,Mpika and Chama Districts of Zambia.Methods:Individuals diagnosed with rHAT from 2004 to 2014 were traced using hospital records and discussions with communities.Either they,or their families,were interviewed using a structured questionnaire and focus group discussions were conducted with affected communities.The burden of the disease was investigated using disability adjusted life years(DALYs),with and without discounting and age-weighting.The impact of long-term disabilities on the rHAT burden was also investigated.Results:Sixty four cases were identified in the study.The majority were identified in second stage,and the mortality rate was high(12.5%).The total number of DALYs was 285 without discounting or age-weighting.When long-term disabilities were included this estimate increased by 50%to 462.The proportion of years lived with disability(YLD)increased from 6.4%to 37%of the undiscounted and un-age-weighted DALY total.When a more active surveillance method was applied in 2013-2014 the cases identified increased dramatically,suggesting a high level of under-reporting.Similarly,the proportion of females increased substantially,indicating that passive surveillance may be especially failing this group.An average of 4.9 months of productive time was lost per patient as a consequence of infection.The health consequences included pain,amnesia and physical disability.The social consequences included stigma,dropping out of education,loss of friends and self-esteem.Results obtained from focus group discussions revealed misconceptions among community members which could be attributed to lack of knowledge about rHAT.Conclusions:The social and economic impact of rHAT on rural households and communities is substantial.Improved surveillance and strengthening of local medical services are needed for early and accurate diagnosis.Disease prevention should be prioritised in communities at risk of rHAT,and interventions put in place to prevent zoonotic disease spill over from domestic animals and wildlife.Supportive measures to mitigate the long-term effects of disability due to rHAT are needed.

MRI Signal Abnormalities of the Optic Tracts, a Marker of Meningoencephalitis Caused by Trypanosoma Gambiense (TG)—A Delayed Patho-Radlological Correlation

Parasitic meningoencephalitis presents several etiologies which sometimes depend on their geographical location. They require thorough blood and cerebrospinal fluid check-up for directing an efficient treatment. Clinicians and radiologists are constantly looking for specific signs that could point to a particular etiology. The meningoencephalitis caused by Human African Trypanosomiasis (HAT) due to Trypanosoma brucei gambiense (TG) is a rare disease characterized by a slow progression, over years sometimes. Its non-specific presentation either clinically or in imaging can lead to misdiagnosis and thus, delay the treatment. However, involvement of the optic tracts seems to be characteristic of this condition, on old data from animal experimentation and recent high-field MRI data. MRI is the best current technique to explore the brain, cranial nerves, and visual pathways. In this article, we are going to present two observations of meningoencephalitis caused by HAT and then discuss some specific aspects of this neglected and re-emerging disease.

Evaluation of the TbgI_(2) and TbgI_(17) Tandem Repeat Antigens as Potential Antigens for the Diagnosis of Trypanosoma brucei rhodesiense

Human African trypanosomiasis (HAT) affects up to half a million people every year in sub-Saharan Africa. Interruption of transmission of the disease by early diagnosis and treatment is core to the control and eventual elimination of HAT. The routine diagnostic method for HAT is light microscopy of blood samples. The present study sought to evaluate the potential of TbgI2 and TbgI17 tandem repeat antigens as candidates for the diagnosis of Trypanosoma brucei rhodesiense. The expressed proteins were purified and the antigenic reactivity evaluation was done using multiplex assay using sera obtained from HAT patients. Receiver operating characteristic analysis showed that recombinant antigen, TbgI2 had high sensitivity for sera from patients infected with T. b. rhodesiense with the area under the curve being 0.577 and a sensitivity of 0.641 and specificity 0.650. The results suggest that TbgI2 is a potential biomarker for T. b. rhodesiense HAT serodiagnostic tests.

Molecular Characterization and Prevalence of Trypanosoma Species in Cattle from a Northern Livestock Area in Cote d’Ivoire

Background: African animal trypanosomiasis (AAT) is caused mainly by Trypanosoma congolense, T. vivax, and T. brucei brucei and is the major constraint for livestock productivity in Sub-Saharan African countries. Information about animal trypanosomiasis status in Ivory Coast is missing, especially regarding molecular epidemiology. Therefore, this study intended to apply molecular tools to identify and characterize trypanosomes in Ivory Coast for sustainable control. Methods: 363 cattle blood samples were collected from Ferkessedougou Region in northern Ivory Coast in 2012. Buffy coat technique (BCT) and species-specific PCR assays were used to detect trypanosome species. Results: Out of 363 cattle examined with BCT, 33 were found positive with all trypanosomes species accounting for an average of 9.09% prevalence whereas polymerase chain reaction (PCR) using species-specific primers showed that 81 out of 363 cattle were infected with trypanosomes with an overall prevalence of 22.31%. Trypanosoma congolense savanah type, T. Vivax and T. brucei sl. accounted for 28.39%, 49.38% and 23.45% of the infection rate respectively. No infection with T. congo forest?type was detected. T. vivax infection was the most prevalence in the area investigated compared to the two other trypanosome species. Mixed infections with different trypanosomes species were detected accounting for 7.32% of prevalence. Regarding sexrelated prevalence, male cattles were slightly more infected than female but the difference was not significant. Conclusion: Our results showed that there was a high prevalence of AAT in livestock in Ferkessedougou Area. There is therefore a need to strengthen control policies and institute measures that help prevent the spread of the parasites for sustainable control of animal trypanosome in this area.

Evaluating the impact of targeting livestock for the prevention of human and animal trypanosomiasis,at village level,in districts newly affected with T.b.rhodesiense in Uganda

Background:Uganda has suffered from a series of epidemics of Human African Trypanosomiasis(HAT),a tsetse transmitted disease,also known as sleeping sickness.The area affected by acute Trypanosoma brucei rhodesiense HAT(rHAT)has been expanding,driven by importation of infected cattle into regions previously free of the disease.These regions are also affected by African Animal Trypanosomiasis(AAT)demanding a strategy for integrated disease control.Methods:In 2008,the Public Private Partnership,Stamp Out Sleeping Sickness(SOS)administered a single dose of trypanocide to 31486 head of cattle in 29 parishes in Dokolo and Kaberamaido districts.This study examines the impact of this intervention on the prevalence of rHAT and AAT trypanosomes in cattle from villages that had(HAT^(+ve))or had not(HAT^(-ve))experienced a recent case of rHAT.Cattle herds from 20 villages were sampled and screened by PCR,pre-intervention and 6-months post-intervention,for the presence or absence of:Trypanosoma brucei s.l.;human infective T.b.rhodesiense;Trypanosoma vivax;and Trypanosoma congolense savannah.Results:Post-intervention,there was a significant decrease in the prevalence of T.brucei s.l.and the human infective sub-species T.b.rhodesiense in village cattle across all 20 villages.The prevalence of T.b.rhodesiense was reduced from 2.4%to 0.74%(P<0.0001),with the intervention showing greater impact in HAT^(-ve) villages.The number of villages containing cattle harbouring human infective parasites decreased from 15/20 to 8/20,with T.b.rhodesiense infection mainly persisting within cattle in HAT^(+ve) villages(six/eight).The proportion of T.brucei s.l.infections identified as human infective T.b.rhodesiense decreased after the intervention from 8.3%(95%CI=11.1-5.9%)to 4.1%(95%CI=6.8-2.3%).Villages that had experienced a recent human case(HAT^(+ve) villages)showed a significantly higher prevalence for AAT both pre-and post-intervention.For AAT the prevalence of T.vivax was significantly reduced from 5.9%to 0.05%post-intervention while the prevalence of T.congolense increased from 8.0%to 12.2%.Conclusions:The intervention resulted in a significant decrease in the prevalence of T.brucei s.l.,human infective T.b.rhodesiense and T.vivax infection in village cattle herds.The proportion of T.brucei s.l.that were human infective,decreased from 1:12 T.brucei s.l.infections before the intervention to 1:33 post-intervention.It is clearly more difficult to eliminate T.b.rhodesiense from cattle in villages that have experienced a human case.Evidence of elevated levels of AAT in livestock within village herds is a useful indicator of risk for rHAT in Uganda.Integrated veterinary and medical surveillance is key to successful control of zoonotic rHAT.

Trypanosoma brucei rhodesiense infection in a Chinese traveler returning from the Serengeti National Park in Tanzania

Background:Human African trypanosomiasis(HAT)is one of the most complex parasitic diseases known to humankind.It usually occurs in endemic areas in Africa,but is occasionally detected in returning travelers and migrants in non-endemic countries.Case presentation:In August 2017,a case of HAT was diagnosed in China in a traveler returning from the Masai Mara area in Kenya and the Serengeti area in Tanzania.The traveler visited Africa from 23 July to 5 August,2017.Upon return to China,she developed a fever(on 8 August),and Trypanosoma brucei rhodesiense infection was confirmed by laboratory tests(on 14 August)including observation of parasites in blood films and by polymerase chain reaction.She was treated with pentamidine followed by suramin,and recovered 1 month later.Conclusions:This is the first imported rhodesiense HAT case reported in China.This case alerts clinical and public health workers to be aware of HAT in travelers,and expatriates and migrants who have visited at-risk areas in Africa.

Control of malaria and other vector-borne protozoan diseases in the tropics: enduring challenges despite considerable progress and achievements

Vector-borne protozoan diseases represent a serious public health challenge,especially in the tropics where poverty together with vector-favorable climates are the aggravating factors.Each of the various strategies currently employed to face these scourges is seriously inadequate.Despite enormous efforts,vaccines-which represent the ideal weapon against these parasitic diseases—are yet to be sufficiently developed and implemented.Chemotherapy and vector control are therefore the sole effective attempts to minimize the disease burden.Nowadays,both strategies are also highly challenged by the phenomenon of drug and insecticide resistance,which affects virtually all interventions currently used.The recently growing support from international organizations and governments of some endemic countries is warmly welcome,and should be optimally exploited in the various approaches to drug and insecticide research and development to overcome the burden of these prevalent diseases,especially malaria,leishmaniasis,Human African Trypanosomiasis(HAT),and Chagas disease.

Support for research towards understanding the population health vulnerabilities to vector-borne diseases:increasing resilience under climate change conditions in Africa

Background:Diseases transmitted to humans by vectors account for 17%of all infectious diseases and remain significant public health problems.Through the years,great strides have been taken towards combatting vectorborne diseases(VBDs),most notably through large scale and coordinated control programmes,which have contributed to the decline of the global mortality attributed to VBDs.However,with environmental changes,including climate change,the impact on VBDs is anticipated to be significant,in terms of VBD-related hazards,vulnerabilities and exposure.While there is growing awareness on the vulnerability of the African continent to VBDs in the context of climate change,there is still a paucity of research being undertaken in this area,and impeding the formulation of evidence-based health policy change.Main body:One way in which the gap in knowledge and evidence can be filled is for donor institutions to support research in this area.The collaboration between the WHO Special Programme for Research and Training in Tropical Diseases(TDR)and the International Centre for Research and Development(IDRC)builds on more than 10 years of partnership in research capacity-building in the field of tropical diseases.From this partnership was born yet another research initiative on VBDs and the impact of climate change in the Sahel and sub-Saharan Africa.This paper lists the projects supported under this research initiative and provides a brief on some of the policy and good practice recommendations emerging from the ongoing implementation of the research projects.Conclusion:Data generated from the research initiative are expected to be uptaken by stakeholders(including communities,policy makers,public health practitioners and other relevant partners)to contribute to a better understanding of the impacts of social,environmental and climate change on VBDs(i.e.the nature of the hazard,vulnerabilities,exposure),and improve the ability of African countries to adapt to and reduce the effects of these changes in ways that benefit their most vulnerable populations.