Development of a latex agglutination test with recombinant variant surface glycoprotein for serodiagnosis of surra.

Serodiagnosis of surra is commonly performed with the CATT/Trypanosoma evansi direct agglutination test. This antibody detection test is based on lyophilised bloodstream form trypanosomes propagated in rats and presenting the predominant variant surface glycoprotein (VSG) RoTat 1.2 on their surface. Recently, the N-terminal fragment of VSG RoTat 1.2 has been expressed as a recombinant protein in the yeast Pichia pastoris and showed diagnostic potential in ELISA. This recombinant antigen has now been incorporated in a latex agglutination test, the rLATEX/T. evansi. In this study, we compared the diagnostic accuracy of rLATEX/T. evansi and CATT/T. evansi with immune trypanolysis (TL) as reference test on a total of 1717 sera from camels, horses, bovines, water buffaloes, dogs and sheep. The rLATEX/T. evansi displayed a slightly better agreement with TL than CATT/T. evansi (kappa [κ] respectively 0.84 and 0.72). The sensitivities of rLATEX/T. evansi (84.2%, 95% CI 80.8-87.1) and CATT/T. evansi (84.0%, 95% CI 80.6-87.0) were similar, but rLATEX/T. evansi was significantly more specific (97.7%, 95% CI 96.7-98.4) than CATT/T. evansi (89.4%; 95% CI 87.6-91.1). We consider the rLATEX/T. evansi an alternative for the CATT/T. evansi, with the advantage that the use of a purified recombinant antigen leads to a more standardised diagnostic test with an improved specificity. Moreover, it eliminates the use of laboratory animals and can be easily scaled-up, e.g. in biofermentors.

Characterization of Trypanosoma brucei gambiense variant surface glycoprotein LiTat 1.5.

At present, all available diagnostic antibody detection tests for Trypanosoma brucei gambiense human African trypanosomiasis are based on predominant variant surface glycoproteins (VSGs), such as VSG LiTat 1.5. During investigations aiming at replacement of the native VSGs by recombinant proteins or synthetic peptides, the sequence of VSG LiTat 1.5 was derived from cDNA and direct N-terminal amino acid sequencing. Characterization of the VSG based on cysteine distribution in the amino acid sequence revealed an unusual cysteine pattern identical to that of VSG Kinu 1 of T. b. brucei. Even though both VSGs lack the third of four conserved cysteines typical for type A N-terminal domains, they can be classified as type A.

Human African trypanosomiasis in a Belgian traveller returning from the Masai Mara area, Kenya, February 2012.

A Belgian traveller was diagnosed with human African trypanosomiasis (HAT) due to Trypanosoma brucei rhodesiense nine days after visiting the Masai Mara area in Kenya. He presented with an inoculation chancre and was treated with suramin within four days of fever onset. Two weeks earlier, HAT was also reported in a German traveller who had visited the Masai Mara area. Because no cases have occurred in the area for over 12 years, this may indicate a focal cluster of HAT.

Low specificities of HIV diagnostic tests caused by Trypanosoma brucei gambiense sleeping sickness.

The accuracy of diagnostic tests for HIV in patients with tropical infections is poorly documented. Human African trypanosomiasis (HAT) is characterized by a polyclonal B-cell activation, constituting a risk for false-positive reactions to diagnostic tests, including HIV tests. A retrospective study of the accuracy of HIV diagnostic tests was performed with 360 human African HAT patients infected with Trypanosoma brucei gambiense before treatment and 163 T. b. gambiense-infected patients 2 years after successful treatment in Mbuji Mayi, East Kasai, Democratic Republic of the Congo. The sensitivities, specificities, and positive predictive values (PPVs) of individual tests and algorithms consisting of 3 rapid tests were determined. The sensitivity of all tests was 100% (11/11). The low specificity (96.3%, 335/348) and PPV (45.8%, 11/24) of a classical seroconfirmation strategy (Vironostika enzyme-linked immunosorbent assay [ELISA] followed by line immunoassay) complicated the determination of HIV status, which had to be determined by PCR. The specificities of the rapid diagnostic tests were 39.1% for Determine (136/348); 85.3 to 92.8% (297/348 to 323/348) for Vikia, ImmunoFlow, DoubleCheck, and Bioline; and 96.6 to 98.3% (336/348 to 342/348) for Uni-Gold, OraQuick, and Stat-Pak. The specificity of Vironostika was 67.5% (235/348). PPVs ranged between 4.9 and 64.7%. Combining 3 different rapid tests resulted in specificities of 98.3 to 100% (342/348 to 348/348) and PPVs of 64.7 to 100% (11/17 to 11/11). For cured HAT patients, specificities were significantly higher for Vironostika, Determine, Uni-Gold, and ImmunoFlow. T. b. gambiense infection decreases the specificities of antibody detection tests for HIV diagnosis. Unless tests have been validated for interference with HAT, HIV diagnosis using classical algorithms in untreated HAT patients should be avoided. Specific, validated combinations of 3 HIV rapid tests can increase specificity.

A new format of the CATT test for the detection of human African Trypanosomiasis, designed for use in peripheral health facilities.

OBJECTIVES: To test the reproducibility and thermostability of a new format of the Card-Agglutination Test for Trypanosomiasis (CATT) test for Human African Trypanosomiasis (HAT), designed for use at primary health care facility level in endemic countries. METHODS: A population of 4217 from highly endemic villages was screened using the existing format of the CATT test (CATT-R250) on whole blood. All those testing positive (220) and a random sample of negatives (555) were retested in the field with the new format (CATT-D10). Inter-format reproducibility was assessed by calculating kappa. All samples testing positive on whole blood with either method were further evaluated in Belgium by CATT titration of serum by two observers, using both old and new format. CATT-D10 test kits were incubated under four temperature regimens (4, 37, 45 degrees C and fluctuating) with regular assessments of reactivity over 18 months. RESULTS: Inter-format reproducibility of CATT-D10 vs. CATT-R250 on whole blood performed by laboratory technicians in the field was excellent with kappa values of 0.83-0.89. Both inter- and intra-format reproducibility assessed by CATT titration were excellent, with 96.5-100% of all differences observed falling within the limits of +/-1 titration step. After 18 months, reactivity of test kits incubated under all four temperature regimens was still well above the minimum threshold considered acceptable. CONCLUSION: The CATT-D10 is thermostable and can be used interchangeably with the old format of the CATT test. It is highly suitable for use in peripheral health facilities in HAT-endemic countries.

Comparison of operational criteria for treatment outcome in gambiense human African trypanosomiasis.

OBJECTIVE: To develop a simple and standard operational decision tool for the diagnosis of relapse after treatment for human African trypanosomiasis (HAT), by evaluating the performance of several criteria currently used by HAT control programs and research projects. METHODS: We identified 10 different criteria for relapse, based on trypanosome presence and/or white blood cell count in cerebrospinal fluid, and compared their specificity, sensitivity and time to diagnosis on a data set containing 63 relapsed and 247 cured T.b. gambiense patients. RESULTS: At any time point, the criterion 'Trypanosomes present and/or a cerebrospinal white blood cell count > or =50/microl' allowed accurate and timely detection of HAT relapse, irrespective of disease stage. This criterion was 13-25% more sensitive (P < or = 0.013) than trypanosome detection alone and was >97% specific. Lumbar punctures at the end of treatment and at 3-month post-treatment provided limited clinical information. CONCLUSIONS: Adequate detection of relapse was possible with a simple criterion but these findings should be validated in a prospective study before adoption in clinical practice.

Aparasitemic serological suspects in Trypanosoma brucei gambiense human African trypanosomiasis: a potential human reservoir of parasites?

The serological and parasitological tests used for Trypanosoma brucei gambiense human African trypanosomiasis (HAT) diagnosis have low specificity and sensitivity, respectively, and in the field, control program teams are faced with subjects with positive serology but negative parasitology who remain untreated. The aim of this work was to explore, using PCR tool, the significance of these aparasitemic serological suspects. Since discordant PCR results have been observed earlier with different extraction methods, two DNA extraction methods were compared (the Chelex 100 resin and the DNeasy Tissue kit). The study was conducted on 604 blood samples: 574 from parasitologically confirmed patients, aparasitemic serological suspects and endemic controls collected in Côte d'Ivoire and 30 from healthy volunteers collected in France. No significant differences were observed between the PCR results obtained with the two extraction methods. Concerning PCR, problems of reproducibility and discordances with both serological and parasitological test results were observed, mainly for the aparasitemic serological suspects. In addition to previous results that pointed to the existence of non-virulent or non-pathogenic trypanosome strains and of individual susceptibility leading to long term seropositivity without detectable parasitaemia but positive PCR, the results of this study support the notion of a long lasting human reservoir that may contribute to the maintenance or periodic resurgences of HAT in endemic foci.

Detection of trypanosome-specific antibodies in saliva, towards non-invasive serological diagnosis of sleeping sickness.

OBJECTIVE: The detection of trypanosome-specific antibodies in saliva is technically feasible, and, if clinically validated, could become an attractive option for non-invasive diagnosis of sleeping sickness. We wanted to optimize the test format of an enzyme-linked immunosorbent assay (ELISA)-based antibody detection system. METHODS: Different ELISA formats for antibody detection in serum and saliva were developed and standardized. Saliva and serum samples were collected from 78 patient and 128 endemic control samples, and sensitivity and specificity of saliva ELISAs, serum ELISAs and the card agglutination test for trypanosomiasis (CATT), were evaluated. RESULTS: All ELISA formats showed sensitivity and specificity above 90%. Saliva ELISAs showed a similar test performance as serum ELISAs and the CATT on whole blood or serum. CONCLUSIONS: This study confirms the potential of trypanosome-specific antibody detection in saliva.

Review Article: cerebrospinal fluid in human African trypanosomiasis: a key to diagnosis, therapeutic decision and post-treatment follow-up.

Human African trypanosomiasis is a lethal parasitic infection with neurological involvement. Examination of the cerebrospinal fluid (CSF) plays an essential role in diagnosis, selection of treatment and post-treatment follow-up. This paper reviews clinical presentation, diagnosis and treatment of the disease, with emphasis on CSF characteristics and interpretation of the CSF results for therapeutic decision and post-treatment follow-up.

Recombinant RoTat 1.2 variable surface glycoprotein as antigen for diagnosis of Trypanosoma evansi in dromedary camels.

The transcript encoding a predominant Trypanosoma evansi variable surface glycoprotein RoTat 1.2 was cloned and expressed as a recombinant protein in Spodoptera frugiperda and Trichoplusia ni (insect) cells. Its potential as an antigen for specific detection of antibody in serum of dromedary camels affected by surra, was evaluated. In ELISA, the reactivity of the recombinant RoTat 1.2 VSG was similar to that of native RoTat 1.2 VSG. An indirect agglutination reagent was therefore prepared by coupling the recombinant RoTat 1.2 VSG onto latex particles. The performance of the latex agglutination test was evaluated on camel sera, and compared with the performance of CATT/T. evansi and LATEX/T. evansi tests, using the immune trypanolysis assay with T. evansi RoTat 1.2 as a reference test. The relative sensitivity and specificity of the latex coated with recombinant RoTat 1.2 VSG, using a 1:4 serum dilution, were respectively, 89.3 and 99.1%. No differences were observed between the performance of latex coated with recombinant RoTat 1.2 VSG and LATEX/T. evansi or CATT/T. evansi. Here, we describe the successful use of the recombinant RoTat 1.2 VSG for detection of specific antibodies induced by T. evansi infections.

Human African trypanosomiasis: quantitative and qualitative assessment of intrathecal immune response.

Quantitative and qualitative techniques for assessment of the intrathecal humoral immune response in human African trypanosomiasis were compared, and their diagnostic potential for detection of the meningo-encephalitic stage of the disease was evaluated. Total and trypanosome specific immunoglobulin G (IgG) and IgM intrathecal synthesis were studied in paired cerebrospinal fluid (CSF) and blood samples of 38 trypanosomiasis patients and in three controls using Reiber's formulae. The presence of CSF-specific oligoclonal IgG and of trypanosome-specific antibodies was determined using iso-electric focusing followed by immunoblotting and antigen-driven immunoblots. The intrathecal IgG fraction (16% positive) and oligoclonal IgG detection (24% positive) were insensitive for detection of an intrathecal humoral immune response. Trypanosome-specific IgG synthesis, reflected by the IgG antibody index (AI) (26% positive), was confirmed by the presence of oligoclonal specific IgG (47% positive), but the latter was more sensitive. Although the detection technique failed for oligoclonal IgM, the intrathecal IgM fraction (42% positive) and the IgM AI (32% positive) indicated that the meningo-encephalitic stage of the disease is characterized by a dominant intrathecal IgM response, which was higher than the IgG response. The highest combination of diagnostic sensitivity and specificity for the meningo-encephalitic stage of trypanosomiasis was observed for quantitative IgM determinations.

The challenge of Trypanosoma brucei gambiense sleeping sickness diagnosis outside Africa.

Sleeping sickness is a lethal African disease caused by parasites of the Trypanosoma brucei subspecies, which is transmitted by tsetse flies. Occasionally, patients are reported outside Africa. Diagnosis of such imported cases can be problematic when the infection is due to Trypanosoma brucei gambiense, the chronic form of sleeping sickness found in west and central Africa. The low number of trypanosomes in the blood and the non-specific, variable symptoms make the diagnosis difficult, particularly when the index of suspicion is low. When the trypanosomes have penetrated into the central nervous system, neuropathological signs become apparent but even at this stage, misdiagnosis is frequent. Rapid and correct diagnosis of sleeping sickness can avoid inappropriate or delayed treatment and even death of the patient. In this article, an overview on diagnosis of imported cases of T b gambiense sleeping sickness is given, and possible pitfalls in the diagnostic process are highlighted. Bioclinical parameters that should raise the suspicion of sleeping sickness in a patient who has been in west or central Africa are discussed. Techniques for diagnosis are reviewed. A clinician suspecting sleeping sickness should contact a national reference centre for tropical medicine in his or her country, or the WHO, Geneva, Switzerland, or the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, for clinical consultation and provision of specific diagnostic tests. Appropriate drugs for sleeping sickness treatment are also provided by WHO and the CDC.

Towards saliva-based screening for sleeping sickness?

A pilot study was carried out on the detection of trypanosome-specific antibodies in saliva for diagnosis of sleeping sickness. All twenty-three saliva samples of parasitologically confirmed Trypanosoma brucei gambiense patients tested positive in an indirect enzyme-linked immunosorbent assay, whereas all 14 saliva samples of a negative control group remained negative. Trypanosome-specific antibody levels in patient saliva correlated with antibody levels in serum, but were about 250-fold lower. Eight of 23 undiluted saliva samples of sleeping sickness patients tested positive in CATT/T. b. gambiense and two of 23 in LATEX/T. b. gambiense. All fourteen saliva samples of the negative control group were also positive in CATT/T. b. gambiense, as were four of 14 in LATEX/T. b. gambiense. CATT and LATEX were thus inappropriate for antibody detection in saliva. These results indicate that trypanosome-specific antibody detection in saliva is possible. This could lead to the development of a simple, non-invasive, reliable saliva field test for diagnosis of sleeping sickness.

Stage determination and therapeutic decision in human African trypanosomiasis: value of polymerase chain reaction and immunoglobulin M quantification on the cerebrospinal fluid of sleeping sickness patients in Côte d'Ivoire.

In human African trypanosomiasis (HAT), two disease stages are defined: the first, or haemo-lymphatic stage, and the second, or meningo-encephalitic stage. Stage determination forms the basis of therapeutic decision and is of prime importance, as the drug used to cure second-stage patients has considerable side-effects. However, the tests currently used for stage determination have low sensitivity or specificity. Two new tests for stage determination in the cerebrospinal fluid (CSF) were evaluated on 73 patients diagnosed with HAT in Côte d'Ivoire. The polymerase chain reaction (PCR) detecting trypanosome DNA (PCR/CSF) is an indirect test for trypanosome detection whereas the latex agglutination test detecting immunoglobulin M (LATEX/IgM) is an indicator for neuro-inflammation. Both tests were compared with classically used tests, double centrifugation and white blood cell count of the CSF. PCR/CSF appeared to be the most sensitive test (96%), and may be of use to improve stage determination. However, its value for therapeutic decision appears limited, as patients whose CSF was positive with PCR were successfully treated with pentamidine. This result confirms those of previous works that showed that some patients with trypanosomes in the CSF could be treated successfully with pentamidine. LATEX/IgM, which depending on the cut-off, showed lower sensitivity of 76% and 88%, but higher specificity of 83% and 71% for LATEX/IgM 16 and LATEX/IgM 8 respectively, appears more appropriate for therapeutic decision making.

Susceptibility of trypanotolerant West African Dwarf goats and F1 crosses with the susceptible Sahelian breed to experimental Trypanosoma congolense infection and interactions with helminth infections and different levels of diet.

Forty pure West African Dwarf (WAD) goats and 35 of its F1 crosses with the Sahelian breed were used in a multifactorial experimental design to evaluate the effects of an experimental Trypanosoma congolense infection and interactions with natural helminth infections and different levels of diet on health and productivity of these two breeds. Trypanosome infection caused a severe drop in packed cell volume (PCV), but this was not significantly affected by breed. Neither deworming nor diet had any effect on the course of anaemia after trypanosome infection. The mean score of parasitaemia tended to be higher in crossbreeds than in WAD goats although this was not significant (P>0.05). Similarly, the antibody response to trypanosome infection was not significantly different between breeds. Parasitaemia level was significantly influenced by the level of diet with the group under high supplementation having a higher mean parasitaemia score than the group under low supplementation. Weight loss due to trypanosome infection tended to be greater in crossbreeds than in WAD goats (P>0.05). During this study, there was no difference in mean helminth egg output between crossbred and WAD goats. However, between weeks 4 and 10 after trypanosome infection (corresponding to a period of heavy rainfall and highly infective pastures), the mean egg output was higher in the crossbreeds. The immunosuppressive effect of trypanosome infections was revealed by a lower antibody response to Haemonchus contortus in infected animals compared to the uninfected controls. Trypanosome infection tended to increase strongyle egg output. This study did no reveal any superior trypanotolerance of WAD goats compared to crossbreeds.

Interleukin (IL)-6, IL-8 and IL-10 in serum and CSF of Trypanosoma brucei gambiense sleeping sickness patients before and after treatment.

Serum and cerebrospinal fluid (CSF) concentrations of interleukin (IL)-6, IL-8, IL-10, tumour necrosis factor-alpha and interferon-gamma were determined in 46 Trypanosoma brucei gambiense sleeping sickness patients in DR Congo, before and after treatment. According to their CSF cell number before treatment, patients were classified as early-stage (0-5 cells/microL), intermediate-stage (6-20 cells/microL) or late-stage patients (> 20 cells/microL). In serum, slightly higher IL-8 concentrations were found in early-stage patients compared to intermediate- or late-stage patients. These high IL-8 levels dropped after treatment. Higher IL-10 concentrations were detected in serum of patients in intermediate or late stage compared to early-stage patients. In both intermediate- and late-stage groups, serum IL-10 decreased after treatment. In CSF, elevated concentrations of IL-6, IL-8 and especially of IL-10 were observed in late-stage T. b. gambiense patients. After treatment, these concentrations dropped to levels similar to those of the other patients. Tumour necrosis factor-alpha was detected only in a few serum and CSF samples, which were scattered over the different patient groups. Interferon-gamma was detected in serum of 5 patients and remained undetectable in CSF.

IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test.

An increased IgM concentration in cerebrospinal fluid (CSF), occurring as a consequence of massive intrathecal IgM synthesis, is a marker of interest for diagnosis of the meningo-encephalitic stage in human African trypanosomiasis. However, in current practice, IgM in CSF is not determined because of the lack of a simple and robust test that is applicable in African rural regions where the disease prevails. We describe the development of a sensitive semiquantitative card agglutination test, LATEX/IgM, for IgM quantification in CSF. The test is simple and fast and the lyophilized reagent remains stable even at 45 degrees C. CSF end-titres obtained with LATEX/IgM parallel the IgM concentrations determined by nephelometry and enzyme-linked immunosorbent assay. Detection of intrathecal IgM synthesis is the most sensitive marker for CNS involvement in sleeping sickness. At a cut-off value of >or= 8, the sensitivity and specificity of LATEX/IgM for intrathecal IgM synthesis are 89.4 and 92.7%. As a consequence, patients with LATEX/IgM end-titres >or= 8 are likely to have intrathecal IgM synthesis, thus central nervous system involvement and therefore should be treated accordingly. Further studies should concentrate on the relationship between the LATEX/IgM end-titres, presence of intrathecal IgM synthesis and occurrence of treatment failures in patients treated with pentamidine.

Comparison of different DNA preparation protocols for PCR diagnosis of Human African Trypanosomosis in Côte d'Ivoire.

During a medical survey the sleeping sickness focus in Bonon, Ivory Coast, PCR with Trypanosoma brucei specific primers (TBR 1-2 from Parasitology 99 (1989) 57) was tested on DNA derived from blood samples. DNA purification using a chelating resin was performed either on whole blood or on the buffy coat prepared in two different ways. The preparation based on whole blood performed better than those using the buffy-coat. Using this first method, the sensitivity was 100% on parasitologically confirmed patients, and the specificity was 92%. However, problems of reproducibility of the technique were pointed out, particularly on samples from serologically positive but apparently aparasitemic individuals. It is suggested that the PCR could help in the diagnosis of Human African Trypanosomosis, but the use of other primers should be investigated.

Blood-cerebrospinal fluid barrier and intrathecal immunoglobulins compared to field diagnosis of central nervous system involvement in sleeping sickness.

Diagnosis of central nervous system (CNS) involvement in sleeping sickness is crucial in order to give an appropriate treatment regimen. Neurological symptoms occur late, therefore field diagnosis is based on white blood cell count, total protein concentration and presence of trypanosomes in cerebrospinal fluid (CSF). More sensitive and specific parameters are now available. Blood-CSF barrier (B-CSFB) dysfunction, intrathecal total and specific immunoglobulin synthesis were evaluated in 95 patients with and without obvious meningoencephalitis, and compared to field criteria.B-CSFB dysfunction is a rather late event in the course of CNS involvement and correlates with the presence of trypanosomes, neurological signs and intrathecal polyspecific and specific immune response. IgM intrathecal response and particularly IgM antibody index are early markers of CNS invasion. We showed that 29% of patients with CSF abnormalities but without trypanosome detection in the field had no neuro-immunological response. In contrast, patients with normal CSF according to field diagnosis showed an intrathecal immune response in 31% of the cases.Field diagnosis can therefore fail to determine neurological involvement but can also provide false positive results. Improved criteria including B-CSFB dysfunction and IgM detection are needed in order to provide an adapted treatment regimen.

[Diagnosis of sleeping sickness stage: towards a new approach]. / Le diagnostic du stade dans la maladie du sommeil: vers une nouvelle approche.

Diagnosis of the neurological disease stage in Trypanosoma brucei (T.b.) gambiense infection is essential to select an optimal chemotherapy. The actual parameters for stage determination, the cerebrospinal fluid (CSF) cell count, total protein concentration and trypanosome detection, are insufficiently specific and sensitive. In order to identify new parameters for stage determination, we studied the neuro-inflammatory immune response in the central nervous system, notably the intrathecal humoral immune response in sleeping sickness patients. The presence of intrathecal IgM synthesis was identified as an excellent marker of central nervous system involvement. However, intrathecal IgM detection cannot be performed under field conditions. As a consequence of the strong intrathecal IgM synthesis, extremely high concentrations of IgM are found in the CSF of sleeping sickness. We therefore developed a latex agglutination field test (LATEX/IgM) indicative for intrathecal IgM synthesis and CNS involvement in sleeping sickness. Based on our observations on the intrathecal immune response and with LATEX/IgM, we propose a new approach for stage determination in sleeping sickness.