Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Diagnosis plays a central role in the control of human African trypanosomiasis (HAT) whose mainstay in disease control is chemotherapy. However, accurate diagnosis is hampered by the absence of sensitive techniques for parasite detection. Without concentrating the blood, detection thresholds can be as high as 10,000 trypanosomes per milliliter of blood. The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) are promising molecular diagnostics that generally yield high sensitivity and could improve case detection. Recently, these two tests were coupled to oligochromatography (OC) for simplified and standardized detection of amplified products, eliminating the need for electrophoresis. In this study, we evaluated the diagnostic accuracy of these two novel tests on blood specimens from HAT patients and healthy endemic controls from D.R. Congo and Uganda. Both tests exhibited good sensitivity and specificity compared to the current diagnostic tests and may be valuable tools for sensitive and specific parasite detection in clinical specimens. These standardized molecular test formats open avenues for improved case detection, particularly in epidemiological studies and in disease diagnosis at reference centres

Diagnostic accuracy of PCR in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study.

BACKGROUND:The polymerase chain reaction (PCR) has been proposed for diagnosis, staging and post-treatment follow-up of sleeping sickness but no large-scale clinical evaluations of its diagnostic accuracy have taken place yet. METHODOLOGY/PRINCIPAL FINDINGS:An 18S ribosomal RNA gene targeting PCR was performed on blood and cerebrospinal fluid (CSF) of 360 T. brucei gambiense sleeping sickness patients and on blood of 129 endemic controls from the Democratic Republic of Congo. Sensitivity and specificity (with 95% confidence intervals) of PCR for diagnosis, disease staging and treatment failure over 2 years follow-up post-treatment were determined. Reference standard tests were trypanosome detection for diagnosis and trypanosome detection and/or increased white blood cell concentration in CSF for staging and detection of treatment failure. PCR on blood showed a sensitivity of 88.4% (84.4-92.5%) and a specificity of 99.2% (97.7-100%) for diagnosis, while for disease staging the sensitivity and specificity of PCR on cerebrospinal fluid were 88.4% (84.8-91.9%) and 82.9% (71.2-94.6%), respectively. During follow-up after treatment, PCR on blood had low sensitivity to detect treatment failure. In cerebrospinal fluid, PCR positivity vanished slowly and was observed until the end of the 2 year follow-up in around 20% of successfully treated patients. CONCLUSIONS/SIGNIFICANCE:For T.b. gambiense sleeping sickness diagnosis and staging, PCR performed better than, or similar to, the current parasite detection techniques but it cannot be used for post-treatment follow-up. Continued PCR positivity in one out of five cured patients points to persistence of living or dead parasites or their DNA after successful treatment and may necessitate the revision of some paradigms about the pathophysiology of sleeping sickness

Sensitivity and specificity of PCR on blood for diagnosis of HAT.

<p>Note. CI = confidence interval.</p

Classification of study participants according to reference standards for diagnosis, staging and follow-up (FU).

<p>At each follow-up time point, the number of patients attending is given. Patient groups reaching a final outcome of treatment failure during follow-up and excluded from analysis at subsequent time points, are indicated with an arrow.</p

Specificity and sensitivity of PCR on CSF during post-treatment follow-up.

<p>Note. CI = confidence interval.</p

A five month follow up of an efficacy study for a <i>T. evansi</i> experimentally infected goat model, using the haematocrit centrifugation technique (HCT), after treatment with Diminazene, DB 75, DB 867 or DB 1192 at various compound doses.

<p>The results are depicted as negative (−) or positive (+) for motile trypanosomes observed in goat blood samples, along with goats, which died (<i>D</i>) or were removed from the project (<i>Rem</i>) during the study period.</p

A five month follow up of an efficacy study for a <i>T. evansi</i> experimentally infected goat model, using the polymerase chain reaction (PCR), after treatment with Diminazene, DB 75, DB 867 or DB 1192 at various compound doses.

<p>The results are depicted as negative (−) or positive (+) for the presence of parasite DNA in goat blood samples, along with goats, which died (<i>D</i>) or were removed from the project (<i>Rem</i>) during the study period.</p

A five month follow up of an efficacy study for a <i>T. evansi</i> experimentally infected goat model, using a card agglutination test (CATT/<i>T. evansi</i>), after treatment with Diminazene, DB 75, DB 867 or DB 1192 at various compound doses.

<p>Results are depicted as strongly positive (+++), intermediate (++), weak (+) or negative (−) for agglutination, when goat plasma was mixed with an antigenic reagent, along with goats, which died (<i>D</i>) or were removed from the project (<i>Rem</i>) during the study period.</p

Pharmacokinetic parameters determined for a treatment schedule of four applications of diminazene (at 5 mg/kg), DB 75, DB 867 and DB 1192 (at 2.5 mg/kg and 1.25 mg/kg), based on average plasma concentration levels taken from <i>T. evansi</i> experimentally infected goats.

<p>Pharmacokinetic parameters determined for a treatment schedule of four applications of diminazene (at 5 mg/kg), DB 75, DB 867 and DB 1192 (at 2.5 mg/kg and 1.25 mg/kg), based on average plasma concentration levels taken from <i>T. evansi</i> experimentally infected goats.</p