Sensitivity and specificity of HAT Sero-K-SeT, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by Trypanosoma brucei gambiense: a case-control study.

BACKGROUND: Human African trypanosomiasis (HAT) is a life-threatening infection affecting rural populations in sub-Saharan Africa. Large-scale population screening by antibody detection with the Card Agglutination Test for Trypanosomiasis (CATT)/Trypanosoma brucei (T b) gambiense helped reduce the number of reported cases of gambiense HAT to fewer than 10 000 in 2011. Because low case numbers lead to decreased cost-effectiveness of such active screening, we aimed to assess diagnostic accuracy of a rapid serodiagnostic test (HAT Sero-K-SeT) applicable in primary health-care centres. METHODS: In our case-control study, we assessed participants older than 11 years who presented for HAT Sero-K-SeT and CATT/T b gambiense at primary care centres or to mobile teams (and existing patients with confirmed disease status at these centres) in Bandundu Province, DR Congo. We defined cases as patients with trypanosomes that had been identified in lymph node aspirate, blood, or cerebrospinal fluid. During screening, we recruited controls without previous history of HAT or detectable trypanosomes in blood or lymph who resided in the same area as the cases. We assessed diagnostic accuracy of three antibody detection tests for gambiense HAT: HAT Sero-K-SeT and CATT/T b gambiense (done with venous blood at the primary care centres) and immune trypanolysis (done with plasma at the Institute of Tropical Medicine, Antwerp, Belgium). FINDINGS: Between June 6, 2012, and Feb 25, 2013, we included 134 cases and 356 controls. HAT Sero-K-SeT had a sensitivity of 0·985 (132 true positives, 95% CI 0·947-0·996) and a specificity of 0·986 (351 true negatives, 0·968-0·994), which did not differ significantly from CATT/T b gambiense (sensitivity 95% CI 0·955, 95% CI 0·906-0·979 [128 true positives] and specificity 0·972, 0·949-0·985 [346 true negatives]) or immune trypanolysis (sensitivity 0·985, 0·947-0·996 [132 true positives] and specificity 0·980, 0·960-0·990 [349 true negatives]). INTERPRETATION: The diagnostic accuracy of HAT Sero-K-SeT is adequate for T b gambiense antibody detection in local health centres and could be used for active screening whenever a cold chain and electricity supply are unavailable and CATT/T b gambiense cannot be done.

Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection.

We used the cysteine proteinase B (cpb) gene family of the trypanosomatid genus Leishmania as a target to develop rapid, specific, and easy-to-use polymerase chain reaction (PCR) tests to discriminate Leishmania infantum, Leishmania donovani, Leishmania tropica, Leishmania aethiopica, and Leishmania major. Identification of all 5 Old World species and validation of intraspecies variability are features lacking in other species-specific PCRs. Amplicon analysis was done on agarose gels and was further simplified by using an oligochromatography dipstick to detect L. infantum and L. donovani products. Because the analytical sensitivity is lower than that of certain other species- and genus-specific PCRs, our assays are especially valuable for use on cultured isolates or directly on cryostabilates. As such, they can be implemented by research and health centers having access to culturing, DNA isolation, and PCR.

A simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis.

BACKGROUND: Definite diagnosis of Leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. However, microscopy generally shows low sensitivity and requires invasive sampling. METHODS: We describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified Leishmania DNA. A phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (CL), 12 with mucocutaneous leishmaniasis (MCL), and 43 with visceral leishmaniasis (VL) from Peru, Kenya, and Sudan. RESULTS: The lower detection limits of the assay are 10 fg of Leishmania DNA and 1 parasite in 180 microL of blood. The specificity was 98.3% (95% confidence interval [CI], 91.1%-99.7%) and 95.6% (95% CI, 85.2%-98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% CI, 81.8%-97.7%), 91.7% (95% CI, 64.6%-98.5%), and 86% (95% CI, 72.7%-93.4%) for lesions from patients with CL or MCL and blood from patients with VL, respectively. CONCLUSIONS: The Leishmania OligoC-TesT showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. The assay is a promising new tool for simplified and standardized molecular detection of Leishmania parasites.

Survival, immune responses and tissue cyst production in outbred (Swiss white) and inbred (CBA/Ca) strains of mice experimentally infected with Neospora caninum tachyzoites.

The present work compared inbred (CBA/Ca) and outbred (Swiss white) strains of mice for their capacity to cope with a Neospora caninum infection and to consistently produce tissue cysts. In each experiment Swiss white and CBA/Ca mice were given three different doses of NC-1 tachyzoites. Lymphoproliferative and humoral responses as well as cytokine production were evaluated eight weeks after infection (PI) whereas tissue cyst production and histopathology were assessed 4, 6 and 10 weeks PI in immunosuppressed mice. Tissue cysts were observed 10 weeks after infection only in CBA/Ca mice receiving the two highest inoculum doses. Furthermore this strain showed the highest specific lymphoproliferative response. A mixed cytokine response with elevated IFN-gamma and fairly low IL-4 and IL-10 secretion was recorded. In both strains, no lesions were observed in the tissues of infected mice. This study indicates that CBA/Ca female mice infected with 5 x 10(6) NC-1 tachyzoites represent a useful model for the study of specific maternal immune responses in pregnant animals.

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells.

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.