Challenges in the diagnosis and management of sleeping sickness in Tanzania: a case report.

In Tanzania sleeping sickness presents a serious threat to human health with a country-wide average of 400 cases reported annually. Both wild and domestic animals have been found to play a significant role in the epidemiology of sleeping sickness. Serengeti National Park in northern Tanzania, has experienced a number of sleeping sickness epidemics since 1922. The epidemics were associated with abundant game animals in the areas and Glossina swynnertoni was incriminated as the main vector. However since 2001 there has been no case of sleeping sickness reported from the park. This case report highlights on the possibility of resurgence and challenges in the diagnosis and management of sleeping sickness in Serengeti. A 38 years old Tanzanian man working in the Serengeti National Park who had experienced various tsetse bites was presented with a febrile condition and history of unsuccessful case management at different health facilities. Blood and cerebrospinal fluid (CSF) samples were examined for the presence oftrypanosomes using wet film, Field's stain and concentration techniques. Typanosoma brucei rhodesiense were detected in both the blood and CSF samples. The patient was treated successfully with melarsoprol. The results of this case study highlight the possibility of resurgence of sleeping sickness in the park hence calls for the need to create more awareness among the community and clinicians. There is need for early reporting to health facility and strengthening the diagnostic capacity of healthcare facilities in and around national parks endemic for sleeping sickness.

Factors influencing individual and community participation in the control of tsetse flies and human African trypanosomiasis in Urambo District, Tanzania.

This study was carried out to assess the knowledge and level of individual and community participation in the control of Human African trypanosomiasis in Urambo District, western Tanzania. Semi structured questionnaires were used to collect information from individuals at house hold level. Retrospective data of HAT was sought from the medical officers in-charge of health facilities. The results indicate that, 191 (90.5%, n = 211) individuals knew tsetse flies and 187 (88.6%, n = 211) knew HAT. All nine key informants reported that, the communities were aware of HAT while seven key informants reported that, the communities were aware of health risks associated with tsetse bites in human. There was poor knowledge about the role played by animals in the transmission of HAT (26.7%, n = 187). Majority of those who knew HAT (n = 187) were willing to contribute labour (70.1%) and money (64.2%) to tsetse and HAT control whereas amongst those who knew tsetse flies, 66.5% and 60.7% were willing to contribute labour and money, respectively. Amongst those who knew any HAT control technique (n = 108), 78.7% and 82.4% were willing to contribute money and labour respectively. A total of 454 cases of HAT were reported in the area from 1999 to 2006. It is concluded that, the factors influencing individual and community participation include the knowledge of tsetse, HAT and control measures.

African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA.

Control of human African trypanosomiasis (HAT) is dependent on accurate diagnosis and treatment of infected patients. However, sensitivities of tests in routine use are unsatisfactory, due to the characteristically low parasitaemias in naturally infected individuals. We have identified a conserved sequence in the repetitive insertion mobile element (RIME) of the sub-genus Trypanozoon and used it to design primers for a highly specific loop-mediated isothermal amplification (LAMP) test. The test was used to analyse Trypanozoon isolates and clinical samples from HAT patients. The RIME LAMP assay was performed at 62 degrees C using real-time PCR and a water bath. DNA amplification was detectable within 25min. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. The amplicon was unequivocally confirmed through restriction enzyme NdeI digestion, analysis of melt curves and sequencing. The analytical sensitivity of the RIME LAMP assay was equivalent to 0.001 trypanosomes/ml while that of classical PCR tests ranged from 0.1 to 1000 trypanosomes/ml. LAMP detected all 75 Trypanozoon isolates while TBR1 and two primers (specific for sub-genus Trypanozoon) showed a sensitivity of 86.9%. The SRA gene PCR detected 21 out of 40 Trypanosoma brucei rhodesiense isolates while Trypanosoma gambiense-specific glycoprotein primers (TgsGP) detected 11 out of 13 T. b. gambiense isolates. Using clinical samples, the LAMP test detected parasite DNA in 18 out of 20 samples which included using supernatant prepared from boiled blood, CSF and direct native serum. The sensitivity and reproducibility of the LAMP assay coupled with the ability to detect the results visually without the need for sophisticated equipment indicate that the technique has strong potential for detection of HAT in clinical settings. Since the LAMP test shows a high tolerance to different biological substances, determination of the appropriate protocols for processing the template to make it a user-friendly technique, prior to large scale evaluation, is needed.

Factors Influencing Individual and Community Participation in the Control of Tetse Flies and Human African Trypanosomiasis in Urambo District; Tanzania

This study was carried out to assess the knowledge and level of individual and community participation in the control of Human African trypanosomiasis in Urambo District; western Tanzania. Semi structured questionnaires were used to collect information from individuals at house hold level. Retrospective data of HAT was sought from the medicalofficers in-charge of health facilities. The results indicate that; 191 (90.5 ; n = 211) individuals knew tsetse flies and 187(88.6; n=211) knew HAT. All nine key informants reported that; the communities were aware of HAT while seven key informants reported that; the communities were aware of health risks associated with tsetse bites in human. There was poor knowledge about the role played by animals in the transmission of HAT (26.7; n=187). Majority of those who knew HAT (n = 187) were willing to contribute labour (70.1) and money (64.2) to tsetse and HAT control whereas amongst those who knew tsetse flies; 66.5 and 60.7 were willing to contribute labour and money; respectively. Amongst those who knew any HAT control technique (n = 108); 78.7 and 82.4were willing to contribute money and labour respectively. A total of 454 cases of HAT were reported in the area from 1999 to 2006. It is concluded that; the factors influencing individual and community participation include the knowledge of tsetse; HAT and control measures

Characterisation of the Trypanosoma brucei rhodesiense isolates from Tanzania using serum resistance associated gene as molecular marker.

Serum resistance associated (SRA) gene has been found to confer resistance to the innate trypanolytic factor (TLF) found in normal human serum; thus allowing Trypanosoma brucei brucei to survive exposure to normal human serum. This study was carried out to examine the presence of SRA gene and identify the origin of T. b. rhodesiense isolates from three districts in Tanzania, namely Kibondo, Kasulu and Urambo. Twenty-six T. b. rhodesiense isolates and two references T. b. rhodesiense isolates from Kenya were examined for SRA gene using simple Polymerase Chain Reaction technique. The gene was found to be present in all 26 T. b. rhodesiense isolates including the two references isolates from Kenya. The SRA gene was confirmed to be specific to T. b. rhodesiense since it could not be amplified from all other Trypanozoon including T. b. gambiense; and gave an amplified fragment of the expected size (3.9kb), confirming that all these isolates were T. b. rhodesiense of the northern variant. Although the geographic distributions of T. b. gambiense and T. b. rhodesiense are clearly localized to west/central Africa and eastern Africa, respectively, natural movement of people and recent influx of large number of refugees into Tanzania from the Democratic Republic of Congo, could have brought T. b. gambiense in western Tanzania. The overlap in distribution of both of these pathogenic sub-species could result in erroneous diagnoses since both trypanosome sub-species are morphologically identical, and currently serologic methods have low specificity. Both the susceptible and resistant T. b. rhodesiense isolates possessed the SRA gene suggesting that there is no correlation between drug resistance and presence of SRA gene. The use of SRA gene helps to confirm the identity and diversity of some of the isolates resistant to various drugs.

Glossina dynamics in and around the sleeping sickness endemic Serengeti ecosystem of northwestern Tanzania.

We investigated the dynamics of Glossina spp. and their role in the transmission of trypanosomiasis in the sleeping sickness endemic Serengeti ecosystem, northwestern Tanzania. The study investigated Glossina species composition, trap density, trypanosome infection rates, and the diversity of trypanosomes infecting the species. Tsetse were trapped using monopyramidal traps in the mornings between 06:00 to 11:00 and transported to the veterinary laboratory in Serengeti National Park where they were sorted into species and sex, and dissected microscopically to determine trypanosome infection rates. Age estimation of dissected flies was also conducted concurrently. Tsetse samples positive for trypanosomes were subjected to PCR to determine the identity of the detected trypanosomes. Out of 2,519 tsetse trapped, 1,522 (60.42%) were G. swynnertoni, 993 (39.42%) were G. pallidipes, three (0.12%) were G. m. morsitans, and one (0.04%) was G. brevipalpis. The trap density for G. swynnertoni was between 1.40 and 14.17 while that of G. pallidipes was between 0.23 and 9.70. Out of 677 dissected G. swynnertoni, 63 flies (9.3%) were infected, of which 62 (98.4%) were females. A total of 199 G. pallidipes was also dissected but none was infected. There was no significant difference between the apparent densities of G. swynnertoni compared to that of G. pallidipes (t = 1.42, p = 0.18). Molecular characterization of the 63 infected G. swynnertoni midguts showed that 19 (30.2%) were trypanosomes associated with suid animals while nine (14.3%) were trypanosomes associated with bovid animals and five samples (7.9%) had T. brucei s.l genomic DNA. Thirty (47.6%) tsetse samples could not be identified. Subsequent PCR to differentiate between T. b. brucei and T. b. rhodesiense showed that all five samples that contained the T. brucei s.l genomic DNA were positive for the SRA molecular marker indicating that they were T. b. rhodesiense. These results indicate that G. swynnertoni plays a major role in the transmission of trypaniosomiasis in the area and that deliberate and sustainable control measures should be initiated and scaled up.

Characterisation of the Trypanosoma brucei rhodesiense isolates from Tanzania using serum resistance associated gene as molecular marker

Serum resistance associated (SRA) gene has been found to confer resistance to the innate trypanolytic factor (TLF) found in normal human serum; thus allowing Trypanosoma brucei brucei to survive exposure to normal human serum. This study was carried out to examine the presence of SRA gene and identify the origin of T. b. rhodesiense isolates from three districts in Tanzania, namely Kibondo, Kasulu and Urambo. Twenty-six T. b. rhodesiense isolates and two references T. b. rhodesiense isolates from Kenya were examined for SRA gene using simple Polymerase Chain Reaction technique. The gene was found to be present in all 26 T. b. rhodesiense isolates including the two references isolates from Kenya. The SRA gene was confirmed to be specific to T. b. rhodesiense since it could not be amplified from all other Trypanozoon including T. b. gambiense; and gave an amplified fragment of the expected size (3.9kb), confirming that all these isolates were T. b. rhodesiense of the northern variant. Although the geographic distributions of T. b. gambiense and T. b. rhodesiense are clearly localized to west/central Africa and eastern Africa, respectively, natural movement of people and recent influx of large number of refugees into Tanzania from the Democratic Republic of Congo, could have brought T. b. gambiense in western Tanzania. The overlap in distribution of both of these pathogenic sub-species could result in erroneous diagnoses since both trypanosome sub-species are morphologically identical, and currently serologic methods have low specificity. Both the susceptible and resistant T.b. rhodesiense isolates possessed the SRA gene suggesting that there is no correlation between drug resistance and presence of SRA gene. The use of SRA gene helps to confirm the identity and diversity of some of the isolates resistant to various drugs.

Drug-resistance of Trypanosoma b. rhodesiense isolates from Tanzania.

OBJECTIVE: To determine the drug resistance of Trypanosoma brucei rhodesiense strains isolated from sleeping sickness patients in Tanzania. METHOD: We first screened 35 T. b. rhodesiense strains in the mouse model, for sensitivity to melarsoprol (1.8, 3.6 and 7.2 mg/kg), diminazene aceturate (3.5, 7 and 14 mg/kg), suramin (5, 10 and 20 mg/kg) and isometamidium (0.1, 1.0 and 2 mg/kg). A 13 isolates suspected to be resistant were selected for further testing in vitro and in vivo. From the in vitro testing, IC(50) values were determined by short-term viability assay, and MIC values were calculated by long-term viability assay. For in vivo testing, doses higher than those in the initial screening test were used. RESULTS: Two T. b rhodesiense stocks expressed resistance in vivo to melarsoprol at 5 mg/kg and at 10 mg/kg. These strains had high IC(50) and MIC values consistent with those of the melarsoprol-resistant reference strain. Another isolate relapsed after treatment with 5 mg/kg of melarsoprol although it did not appear resistant in vitro. One isolate was resistant to diminazene at 14 mg/kg and another was resistant at both 14 and 28 mg/kg of diminazene. These two isolates had high IC(50) values consistent with the diminazene-resistant reference strain. Two isolates relapsed at a dose of 5 mg/kg of suramin, although no isolate appeared resistant in the in vitro tests. Two isolates were resistant to isometamidium at 1.0 mg/kg and had higher IC(50) values. Two isolates were cross-resistant to melarsoprol and diminazene and one isolate was cross-resistant to suramin and isometamidium. CONCLUSION: The reduced susceptibility of T. b. rhodesiense isolates to these drugs strongly indicates that drug resistance may be emerging in north-western Tanzania.

Knowledge, attitudes and practices on tsetse and sleeping sickness among communities living in and around Serengeti National Park, Tanzania.

A study was undertaken to investigate knowledge, attitudes and practices about sleeping sickness (human African trypanosomiasis) among communities living in and around Serengeti National Park (SENAPA). Structured questionnaires were administered to a total of 1490 consenting participants. Of the respondents, 924 (62%) knew sleeping sickness, and 807 (87.3%) knew the right place to seek healthcare. Of 924 who knew sleeping sickness, 386 (42%) said the disease was present in the areas they live. Most respondents (85.4%) knew that sleeping sickness infections were acquired in the bush and forest. The most common (69.3%) sources of information about sleeping sickness were relatives and friends. Symptoms of sleeping sickness mentioned included abnormal sleep (45.2%), fever (35.3%), body malaise (14.5%), headache (7.6%) and lymph node enlargement (6.1%). Of 1490 people interviewed 90.4% knew tsetse flies and 89.8% had been bitten by tsetse flies. The majority (86.6%) of the respondents knew that sleeping sickness is transmitted through a tsetse bite. Activities that exposed people to tsetse bites included working in tsetse infested bushes/forests, grazing livestock in tsetse infested areas and hunting game animals. In conclusion, communities living in and around SENAPA were knowledgeable about tsetse and sleeping sickness. The communities can thus understand and support community based tsetse and sleeping sickness control programmes to ensure success.

Community knowledge on HIV/AIDS and its relationship with sexual practices in Tabora and Igunga Districts, Western Tanzania.

HIV/AIDS represents one of the critical challenges to human development in sub Saharan Africa. This study was carried out to assess the knowledge of HIV/AIDS and its relationship with sexual practices among communities in Tabora and Igunga Districts in western Tanzania. The study employed both qualitative and quantitative methods, which included interviews and group discussions. A total of 568 participants (female = 49%; males = 51%) were involved in the study. Two hundred and eighty-four of the respondents were adults (> 25 years) and 284 were youths of 12 - 25 years. The results showed although the knowledge of the disease and its prevention was high (90%) among the community, some gaps regarding the knowledge on modes of transmission were observed. About 17.2% of the respondents reported to have multiple sexual partners and only about half of the respondents reported the use of condoms. The level of education correlated significantly with the individual knowledge on HIV/AIDS (P = 0.003). There was no significant difference between urban and rural communities on their knowledge on HIV/AIDS (P > 0.05). Health education on HIV/AIDS prevention needs to be strengthened and improved to include cognitive behavioural interventions that emphasize attitude changes, negotiation skills and decision-making skills that could be effective in changing and maintaining safe sexual behaviour.

Human African Trypanosomiasis and challenges to its control in Urambo; Kasulu and Kibondo Districts; western Tanzania

A study was carried out to determine the prevalence and management of Human African Trypanosomiasis (HAT) in Urambo; Kasulu and Kibondo districts of western Tanzania. Parasitological surveys for trypanosome and other blood parasites were conducted in selected villages. Interviews with health workers were conducted to explore facility capacity to diagnose and manage HAT. Community knowledge on tsetse and availability of trypanocidal drugs was explored. Results showed that; although health facility records showed HAT is an important public health problem in the three districts; typanosomes were found in 0.6of the examined individuals in Urambo district only. Malaria parasites with a prevalence of 12.1; 19.7and 9.7; in Urambo; Kibondo and Kasulu; respectively were detected in blood samples from the same individuals examined for trypanosomes. There was poor capacity for most of the health facilities in the diagnosis; treatment and control of HAT. In both districts; communities were knowledgeable of the tsetse identity (82.4) and had experienced tsetse bites (94). The majority (91.4) of the community members knew that they were at risk of acquiring HAT. However; only 29of the respondents knew that anti-trypanocidal drugs were readily available free of charge from health care facilities. Late treatment seeking behaviour was common in Kasulu and Urambo districts. In conclusion; health facilities in western Tanzania are faced with problems of poor capacity to diagnose and manage HAT and that treatment seeking behaviour among the communities at risk is poor. Efforts should be made to strengthen the capacity of the health facility to handle HAT cases and health education to the population at risk

A multi-centre evaluation of the card indirect agglutination test for trypanosomiasis (TrypTect CIATT).

A version of the card indirect agglutination test for trypanosomiasis, the TrypTect CIATT, was evaluated for the diagnosis of Trypanosoma brucei gambiense and T. b. rhodesiense sleeping sickness. The results of this antigen-detection test indicated high relative sensitivity (99.3%) and specificity (99.4%), and also much higher prevalences of infection in the general population of endemic foci (27.9% for T. b. gambiense and 21.8% for T. b. rhodesiense) than detected by parasitological diagnosis (1.6% and 1.1%, respectively). TrypTect CIATT detected (and could therefore be used for the diagnosis of) non-patent infections. Among the suspected cases (i.e. those initially found to be parasite-negative but to be antigen-positive), trypanosomes were detected in 29 (4.2%) of those checked at a 3-month follow-up, and 17 more such suspects when they were followed up at 6-18 months. Moreover, a high proportion of blood samples from a random sample of the rest of the suspects tested positive for trypanosome-specific DNA by PCR (79.9% for T. b. gambiense and 13.9% for T. b. rhodesiense). ELISA also demonstrated the presence of anti-trypanosome antibodies in many of the suspects tested (63%, 38%, 24% and 66.9% of those in Cameroon, Côte d'Ivoire, Tanzania, and Malawi, respectively). A follow-up of 164 patients treated with melarsoprol revealed that, by 9 months post-treatment, 113 (69.0%) had no detectable trypanosome antigens in their peripheral blood. The test could therefore be used for evaluating chemotherapeutic cure, as well as for diagnosis.

Genetic diversity among Trypanosoma brucei rhodesiense isolates from Tanzania.

We compared 19 stocks of Trypanosoma brucei rhodesiense collected in 1991 and 1994 from Tanzania with representative stocks from other foci of Rhodesian sleeping sickness in Zambia, Kenya and Uganda. Stocks were characterized by isoenzyme electrophoresis, restriction fragment length polymorphisms in variant surface glycoprotein genes and random amplification of polymorphic DNA; the banding patterns obtained were coded for numerical analysis. In addition, the Tanzanian stocks were compared by pulsed field gel electrophoresis. Overall the Tanzanian stocks formed a homogeneous group and the predominant genotype isolated in 1991 was still present in the 1994 sample, although at a reduced level. The Tanzanian stocks were distinct from representative stocks from other East African foci. This observation does not support the proposal that there are northern and southern strains of T. b. rhodesiense, but is consistent with the view that T. b. rhodesiense stocks form a mosaic of different genotypes varying from focus to focus in East Africa.