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.

Isoenzyme comparison of Trypanozoon isolates from two sleeping sickness areas of south-eastern Uganda.

The study characterized 151 Trypanozoon isolates from south-east Uganda by isoenzyme electrophoresis. Stocks were from a range of hosts, including man, cattle, pigs, dogs and Glossina fuscipes fuscipes: 104 isolates were from the Busoga area, 47 were from the Tororo district. Stocks were characterized on thin layer starch gel using eight enzyme systems: ALAT, ASAT, ICD, MDH, ME, NHD, NHI, PGM. Enzyme profiles were generally typical of East Africa; new patterns for ICD and ME were detected. Trypanosomes were classified on the basis of their profile by similarity coefficient analysis and the unweighted pair-group method using arithmetic averages (UPGMA). The majority of trypanosomes were classified in one or other of two genetically distinct groups which corresponded to the strain groups busoga and zambezi, both of which are associated with Rhodesian sleeping sickness in East Africa. Contingency table analyses indicated associations between certain isoenzymes of ICD and PGM, according to host and geographical origin. Significant relationships between trypanosome strain group and geographic origin were also demonstrated for some host groups.

The isolation and genetic heterogeneity of Trypanosoma brucei gambiense from north-west Uganda.

Fifty-two samples of blood were taken from sleeping sickness patients in north-west Uganda. All samples failed to infect immunosuppressed mice. Ten cryopreserved blood samples were fed to laboratory bred Glossina morsitans morsitans; eight flies developed midgut infections from which procyclic cultures were established in vitro. Isoenzyme electrophoretic analysis of 9 enzymes revealed that 7 of the 8 trypanosome isolates had a combination of enzyme patterns already described for Trypanosoma brucei gambiense. The eighth isolate had a different aspartate aminotransferase polymorphism which placed it in a new zymodeme. Analysis of polymorphisms in genes for 3 variant surface glycoproteins (VSGs) confirmed that the 8 Ugandan trypanosome isolates were T.b.gambiense and revealed further heterogeneity. The VSG 117 gene was present in all the isolates in a pattern of fragments (equivalent to AnTat 1.8) characteristic for T.b.gambiense. For two other VSG genes characteristic of T.b.gambiense, the LiTat 1.3 gene was present in all the isolates, while the AnTat 11.17 gene was present in only 2 of the 8 isolates.

Characterization by isoenzyme electrophoresis of Trypanozoon stocks from sleeping sickness endemic areas of south-east Uganda.

An epidemic of sleeping sickness, which started in 1976 in a focus within the county of Luuka in Central Busoga, has spread to cover the three districts of Busoga and large parts of the neighbouring districts of Tororo and Mukono. Forty-three isolates of the subgenus Trypanozoon from Busoga and Tororo (27 from man, 9 from cows, 2 from pigs and 5 from tsetse flies) were compared by thin-layer starch-gel electrophoresis for seven enzymes. Thirty zymodemes were identified; 17 of them were found circulating in the human population. The zymodemes seen previously in Busoga were still circulating together with several new ones. Of the 16 isolates from cattle, pigs and tsetse flies, only two had the same profile, indicating a high degree of diversity. Two zymodemes from cows and a pig were identical to those found in man, implicating domestic stock in the transmission of human disease in south-east Uganda. A computer analysis of the results produced six main zymodeme groups. One comprised only isolates from man; two were composed of isolates from man, domestic animals and tsetse; and three consisted of stocks from domestic animals only. These groups quite probably indicate the different cycles of transmission involving man, tsetse fly and domestic stock.