Veerle Lejon

Options for Field Diagnosis of Human African Trypanosomiasis

SUMMARY Human African trypanosomiasis (HAT) due to Trypanosoma brucei gambiense or T. b. rhodesiense remains highly prevalent in several rural areas of sub-Saharan Africa and is lethal if left untreated. Therefore, accurate tools are absolutely required for field diagnosis. For T. b. gambiense HAT, highly sensitive tests are available for serological screening but the sensitivity of parasitological confirmatory tests remains insufficient and needs to be improved. Screening for T. b. rhodesiense infection still relies on clinical features in the absence of serological tests available for field use. Ongoing research is opening perspectives for a new generation of field diagnostics. Also essential for both forms of HAT is accurate determination of the disease stage because of the high toxicity of melarsoprol, the drug most widely used during the neurological stage of the illness. Recent studies have confirmed the high accuracy of raised immunoglobulin M levels in the cerebrospinal fluid for the staging of T. b. gambiense HAT, and a promising simple assay (LATEX/IgM) is being tested in the field. Apart from the urgent need for better tools for the field diagnosis of this neglected disease, improved access to diagnosis and treatment for the population at risk remains the greatest challenge for the coming years.

Intrathecal immune response pattern for improved diagnosis of central nervous system involvement in trypanosomiasis

Diagnosis of central nervous system (CNS) involvement in human African trypanosomiasis is crucial in determination of therapy. Cerebrospinal fluid (CSF) and serum immunoglobulin concentrations, blood-CSF barrier dysfunction, pattern of intrathecal immunoglobulin synthesis, trypanosome-specific antibody synthesis, and CSF lactate concentrations were analyzed in 272 patients with Trypanosoma brucei gambiense infection. As part of the 2- or 3-class immune response, the predominant intrathecal IgM synthesis was the most sensitive (95%) marker for inflammation of the brain. We propose to replace the World Health Organization (WHO) criteria (white blood cell count >5 cells/microL and presence of trypanosomes in CSF) with a new approach for stage determination in trypanosomiasis: CNS involvement is diagnosed only in patients with >20 cells/microL or with intrathecal IgM synthesis, independent of the presence of trypanosomes in CSF. Compared with the use of these new criteria, the WHO criteria incorrectly classified 49 of 234 patients in the meningoencephalitic stage and 7 of 38 patients in the hemolymphatic disease stage. We also show that trypanosomiasis-related immunoglobulin patterns are of value in differential diagnosis.

Equivalence Trial of Melarsoprol and Nifurtimox Monotherapy and Combination Therapy for the Treatment of Second-Stage Trypanosoma brucei gambiense Sleeping Sickness

BACKGROUND Treatment of second-stage sleeping sickness relies mainly on melarsoprol. Nifurtimox has been successfully used to cure melarsoprol-refractory sleeping sickness caused by Trypanosoma brucei gambiense infection. METHODS An open, randomized trial was conducted to test for equivalence between the standard melarsoprol regimen and 3 other regimens, as follows: standard melarsoprol therapy (3 series of 3.6 mg/kg/day intravenously [iv] for 3 days, with 7-day breaks between the series); 10-day incremental-dose melarsoprol therapy (0.6 mg/kg iv on day 1, 1.2 mg/kg iv on day 2, and 1.8 mg/kg iv on days 3-10); nifurtimox monotherapy for 14 days (5 mg/kg orally 3 times per day); and consecutive 10-day melarsoprol-nifurtimox combination therapy (0.6 mg/kg iv melarsoprol on day 1, 1.2 mg/kg iv melarsoprol on day 2, and 1.2 mg/kg/day iv melarsoprol combined with oral 7.5 mg/kg nifurtimox twice a day on days 3-10). Primary outcomes were relapse, severe adverse events, and death attributed to treatment. RESULTS A total of 278 patients were randomized. The frequency of adverse events was similar between the standard melarsoprol regimen and the other regimens. Encephalopathic syndromes occurred in all groups and caused all deaths that were likely due to treatment. Relapses (n=48) were observed only with the 3 monotherapy regimens. CONCLUSION A consecutive 10-day low-dose melarsoprol-nifurtimox combination is more effective than the standard melarsoprol regimen.

Follow‐up of Card Agglutination Trypanosomiasis Test (CATT) positive but apparently aparasitaemic individuals in Côte d'Ivoire: evidence for a complex and heterogeneous population

Summary The aetiological diagnosis of human African trypanosomiasis (HAT) is based on the detection of the parasite, but currently available parasitological tests have low sensitivity and are hampered by fluctuating parasitaemia. The identification of seropositive individuals on whom to focus parasitological examination is based on antibody detection by means of the Card Agglutination Trypanosomiasis Test (CATT/T.b.gambiense). A complicating phenomenon is the occurrence of serologically positive but parasitologically unconfirmed results (isolated CATT positivity). This work presents a two‐year longitudinal serological, parasitological and molecular follow‐up of CATT‐positive individuals including repeated examinations of each individual, to study the evolution over time of seropositivity at both the population and the individual levels. At the population level, the rate of seropositivity decreased during the first months of the survey, and afterwards showed remarkable stability. At the individual level, the results reveal the extreme heterogeneity of this population, with subjects showing fluctuating results, others with a short transient CATT positivity, and subjects that maintain their seropositivity over time. The stability of seropositivity and the pattern of results obtained with both immunological and parasitological examinations support the view that individual factors, such as immune response to infection, might be involved in the isolated CATT positivity phenomenon.

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.

A combined CXCL10, CXCL8 and H-FABP panel for the staging of human African trypanosomiasis patients

Background Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic tropical disease. It progresses from the first, haemolymphatic stage to a neurological second stage due to invasion of parasites into the central nervous system (CNS). As treatment depends on the stage of disease, there is a critical need for tools that efficiently discriminate the two stages of HAT. We hypothesized that markers of brain damage discovered by proteomic strategies and inflammation-related proteins could individually or in combination indicate the CNS invasion by the parasite. Methods Cerebrospinal fluid (CSF) originated from parasitologically confirmed Trypanosoma brucei gambiense patients. Patients were staged on the basis of CSF white blood cell (WBC) count and presence of parasites in CSF. One hundred samples were analysed: 21 from stage 1 (no trypanosomes in CSF and ≤5 WBC/µL) and 79 from stage 2 (trypanosomes in CSF and/or >5 WBC/µL) patients. The concentration of H-FABP, GSTP-1 and S100β in CSF was measured by ELISA. The levels of thirteen inflammation-related proteins (IL-1ra, IL-1β, IL-6, IL-9, IL-10, G-CSF, VEGF, IFN-γ, TNF-α, CCL2, CCL4, CXCL8 and CXCL10) were determined by bead suspension arrays. Results CXCL10 most accurately distinguished stage 1 and stage 2 patients, with a sensitivity of 84% and specificity of 100%. Rule Induction Like (RIL) analysis defined a panel characterized by CXCL10, CXCL8 and H-FABP that improved the detection of stage 2 patients to 97% sensitivity and 100% specificity. Conclusion This study highlights the value of CXCL10 as a single biomarker for staging T. b. gambiense-infected HAT patients. Further combination of CXCL10 with H-FABP and CXCL8 results in a panel that efficiently rules in stage 2 HAT patients. As these molecules could potentially be markers of other CNS infections and disorders, these results should be validated in a larger multi-centric cohort including other inflammatory diseases such as cerebral malaria and active tuberculosis.

Improved latex agglutination test for detection of antibodies in serum and cerebrospinal fluid of Trypanosoma brucei gambiense infected patients

A rapid latex agglutination test (LATEX/T. b. gambiense) for detection of antibodies in patients infected with Trypanosoma brucei gambiense is presented. The reagent is coated with a mixture of three variable surface antigens of bloodstream form trypanosomes. Two hundred and forty sera and 79 CSF samples from patients with parasitologically confirmed trypanosome infection along with 173 sera and 38 CSF samples from non-trypanosomiasis patients have been tested. At 1:16 serum dilution, test specificity was 99%, while sensitivity ranged from 83.8 to 100% depending on the geographical origin of the samples. Undiluted CSF samples from non-trypanosomiasis and from first stage patients scored negative while 42 out of 66 CSF samples from second stage patients were positive. Stability and reproducibility of the lyophilized reagent were excellent.

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 °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 ≥ 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 ≥ 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.

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.

Rapid diagnostic test for sleeping sickness.

Sleeping sickness, or human African trypanosomiasis, is a potentially life-threatening vectorborne disease of sub-Saharan Africa. Data on a prototype rapid diagnostic test for this condition are presented.