Monitoring for multidrug-resistant Plasmodium falciparum isolates and analysis of pyrimethamine resistance evolution in Uige province, Angola.

OBJECTIVES: To assess the extent of drug resistance in Uige through molecular genetic analysis and to test whether the dhfr triple mutant alleles present in Angola are of southeast Asian origin. METHODS: Seventy-one samples of blood from children admitted to the Pediatric Emergency Unit of Uige Provincial Hospital in 2004 were screened for resistance mutations at pfcrt, pfmdr1, pfdhfr, pfdhps and pfATPase6. RESULTS: Mutations in pfcrt (codon76), pfmdr1 (codon86), pfdhfr (codons 51, 59, 108) and pfdhps (codons 436, 437) were common. Among the 66 isolates for which we were able to determine complete genetic information 13.7% carried all seven of these mutations. Flanking microsatellite analysis revealed the triple mutant pfdhfr was derived from the southeast Asian lineage, while the N51I+S108N double mutant pfdhfr alleles are a local origin. pfATPase6 mutations were rare and S769N was not found. CONCLUSION: The parasite population of Uige Angola has high frequency mutations in pfcrt, dhfr and dhps associated with resistance to chloroquine and sulphadoxine pyrimethamine, reflecting past reliance on these two drugs which were the mainstay of treatment until recently. Our findings show that drug resistance in Uige has occurred through a combination of local drug pressure and the regional and international dispersal of resistance mutant alleles.

Diagnosing human African trypanosomiasis in Angola using a card agglutination test: observational study of active and passive case finding strategies.

OBJECTIVE: To assess the operational feasibility of detecting human African trypanosomiasis by active and passive case finding using the card agglutination test with serial dilution of serum to guide treatment. SETTING: Trypanosomiasis control programme in the Negage focus, northern Angola, during a period of civil war. DESIGN: Observational study. PARTICIPANTS: 359 patients presenting themselves to health centres with symptoms (passive case finding) and 14,446 people actively screened in villages. MAIN OUTCOME MEASURES: Whole blood and serological tests at different dilutions using the card agglutination test, and detection of parasites by microscopy. RESULTS: Active case finding identified 251 people with a positive card agglutination test result, 10 of whom had confirmed parasites. In those presenting for investigation 34 of 51 with a positive card agglutination test result at the dilution of 1:8 or more used to guide treatment had parasites in blood, lymph node fluid, or cerebrospinal fluid, compared with 10 of 76 in those detected by active case finding: positive predictive values of 67% for passive case detection and 13% for active case detection. Only at a cut-off dilution more than 1:32 was the positive predictive value in active case detection reasonable (46%) and at this dilution 40% of microscopically proved cases were missed. CONCLUSIONS: The card agglutination test is useful for initial screening in active detection of cases with human African trypanosomiasis but, given the toxicity of the drugs, serology using the card agglutination test should be not used alone to guide treatment after active case finding. A second confirmatory test is needed.

Real-time PCR for detection of Trypanosoma brucei in human blood samples.

We have developed a real-time PCR assay for detection of Trypanosoma brucei DNA in human blood samples. The PCR was conducted with newly designed primers targeting the 177-bp repeat satellite DNA in T. brucei and with Sybr Green to monitor the amplicon accumulation. DNA purification using Chelex 100 resin was performed on blood samples collected on Whatman FTA cards and was shown to be a simple and quantitative method as revealed by real-time PCR. The detection limit of the assay was 100 trypanosomes per mL blood, corresponding to an analytical sensitivity of 0.1 genome equivalents. Trypanosome DNA was detected in all blood samples from sleeping sickness patients and, furthermore, the identity of the amplicon was confirmed in all assays by dissociation analysis. Although template DNA from blood samples was amplified with significantly lower efficiency than genomic DNA, similar efficiency between all assays ensured quantitative results. No amplicon product was obtained with samples from uninfected individuals. The results indicate that the real-time PCR assay described is a rapid and sensitive method suitable for the detection of T. brucei in human blood samples in routine clinical laboratory practice.

A novel and accurate diagnostic test for human African trypanosomiasis.

INTRODUCTION: Human African trypanosomiasis (sleeping sickness) affects up to half a million people every year in sub-Saharan Africa. Because current diagnostic tests for the disease have low accuracy, we sought to develop a novel test that can diagnose human African trypanosomiasis with high sensitivity and specificity. METHODS: We applied serum samples from 85 patients with African trypanosomiasis and 146 control patients who had other parasitic and non-parasitic infections to a weak cation exchange chip, and analysed with surface-enhanced laser desorption-ionisation time-of-flight mass spectrometry. Mass spectra were then assessed with three powerful data-mining tools: a tree classifier, a neural network, and a genetic algorithm. FINDINGS: Spectra (2-100 kDa) were grouped into training (n=122) and testing (n=109) sets. The training set enabled data-mining software to identify distinct serum proteomic signatures characteristic of human African trypanosomiasis among 206 protein clusters. Sensitivity and specificity, determined with the testing set, were 100% and 98.6%, respectively, when the majority opinion of the three algorithms was considered. This novel approach is much more accurate than any other diagnostic test. INTERPRETATION: Our report of the accurate diagnosis of an infection by use of proteomic signature analysis could form the basis for diagnostic tests for the disease, monitoring of response to treatment, and for improving the accuracy of patient recruitment in large-scale epidemiological studies.

Retaking sleeping sickness control in Angola.

Africa is severely affected by a resurgence of human African trypanosomiasis (HAT) at epidemic proportions. We report the results of the first 5 years of a HAT control programme in northern Angola run by the non-governmental organization (NGO) ANGOTRIP. In the period between 1996 and 2001, 13 426 patients were screened for HAT. The mortality rate of patients in stage II who were treated with melarsoprol fell from 7.5% to 2.9%, possibly as a result of training and the standardization of treatment protocols. A total of 191,578 people in three provinces of Angola were screened for HAT. Vector control activities were initiated using Lancien traps. Our experiences reflect the connection between war and the increasing incidence of disease, but also demonstrate that HAT control is possible by dedicated NGOs in close cooperation with national institutions even under extremely difficult circumstances.