Detection of trypanosomes in suspected sleeping sickness patients in Uganda using the polymerase chain reaction.

Diagnosis of sleeping sickness (trypanosomiasis) is difficult because of the fluctuating levels of parasitaemia encountered in patients. In the present study we found that the polymerase chain reaction (PCR) demonstrated trypanosome infection in 20 out of 35 (57.1%) blood samples and in 21 out of 34 (61.7%) cerebrospinal fluid (CSF) samples collected from an area endemic for sleeping sickness in north-west Uganda. A total of 14 blood samples and 13 CSF samples that were positive for trypanosomes by double centrifugation were also positive by PCR, demonstrating good concordance between the two methods. However, 6 (28.6%) of the 21 blood samples that were parasitologically negative were positive by PCR, while 8 (38.0%) out of 21 CSF samples that were negative by double centrifugation were positive by PCR. These 14 negative samples could therefore be from sleeping sickness cases even though a positive PCR test is not evidence for the presence of trypanosomes. Furthermore, of these 8 CSF samples, 4 had been designated as early cases, based on the absence of trypanosomes and on a count of < or = 5 white blood cells (WBC) per microliter. This suggests that some late-stage cases could potentially be missed according to the present criteria, and it is therefore important to perform clinical trials to determine whether these cases could be treated successfully with the first-stage drug alone. The remaining four CSF samples had been classified as late-stage cases, based on a count of > 6 WBC per microliter, even though trypanosomes could not be detected in these samples by either double centrifugation or PCR. A cut-off point of 5 WBC per microliter, which is used as a rule of thumb to stage sleeping sickness patients, seems to leave some late-stage cases undetected since trypanosomes were detected in four CSF samples from suspected cases with < 5 WBC per microliter.

PIP: This study evaluates the effectiveness of polymerase chain reaction (PCR) in detecting trypanosomes in the blood and cerebrospinal fluid (CSF) of suspected sleeping sickness patients in Uganda. A total of 35 blood samples and 34 CSF samples were analyzed. Trypanosomes were detected in 20 of 35 (57.1%) blood samples, and in 21 of 34 (61.7%) CSF samples by PCR. However, 6 (28.6%) of the 20 blood samples that were parasitologically negative were positive by PCR, while 8 (38.0%) out of 21 CSF samples that were negative by double centrifugation were positive by PCR. These 14 negative samples could therefore be from sleeping sickness cases even though a positive PCR test is not an evidence for the presence of trypanosomes. Furthermore, of these 8 CSF samples, 4 had been designated as early cases, based on the absence of trypanosomes and on a count of 5 or fewer white blood cells (WBCs) per microliter. The remaining 4 CSF samples had been classified as late-stage cases, based on a count of 6 WBCs per microliter, even though trypanosomes could not be detected in these samples by either double centrifugation or PCR. Usually, 5 WBCs per microliter is considered to be the cut-off point in the staging and treatment of sleeping sickness patients. In conclusion, it is imperative to carry out a detailed clinical study on the use of PCR for trypanosomiasis diagnosis and staging of patients in order to demonstrate the relation between PCR and the outcome of treatment.

Parasitological detection of Trypanosoma brucei gambiense in serologically negative sleeping-sickness suspects from north-western Uganda.

Forty-five parasitologically confirmed cases of sleeping sickness were diagnosed in north-western Uganda using a combination of two or three techniques. Forty of the cases were positive by the card agglutination test for trypanosomiasis (CATT), four were negative and one was not screened by the CATT. Trypanosomes isolated from the four CATT-negative but parasitologically positive cases were propagated for detailed biochemical genetic analysis. The aim was to demonstrate whether these four stocks lacked the LiTat 1.3 gene which encodes the antigen on which the CATT is based. All the DNA extracts isolated from these CATT-negative stocks and from six CATT-positive stocks of Trypanosoma brucei gambiense were targeted for amplification by the three variable-surface-glycoprotein genes thought to be ubiquitous in T. b. gambiense. The LiTat 1.3 gene was shown to be present in all 10 stocks. Trypanosome carriers may be CATT-negative because the CATT is not sensitive enough, because their parasites lack the LiTat 1.3 gene, or because their parasites have this gene but do not express it. The four sleeping-sickness cases who gave negative CATT results in the present study have very important implications in the diagnosis of T. b. gambiense infections using the CATT. Following treatment of the CATT-positive cases, the CATT-negative carriers of the trypanosomes remain as human reservoir hosts for continuous infection of the population. Because CATT-negative individuals are rarely examined further, the general prevalence of parasitologically positive but CATT-negative cases is unclear. This study demonstrates the value of co-ordinated use of serological and parasitological techniques in the diagnosis of Gambian sleeping sickness.