Stijn Deborggraeve

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. Conclusion/Significance This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Molecular Dipstick Test for Diagnosis of Sleeping Sickness

ABSTRACT Human African trypanosomiasis (HAT) or sleeping sickness is a neglected disease that affects poor rural populations across sub-Saharan Africa. Confirmation of diagnosis is based on detection of parasites in either blood or lymph by microscopy. Here we present the development and the first-phase evaluation of a simple and rapid test (HAT-PCR-OC [human African trypanosomiasis-PCR-oligochromatography]) for detection of amplified Trypanosoma brucei DNA. PCR products are visualized on a dipstick through hybridization with a gold-conjugated probe (oligochromatography). Visualization is straightforward and takes only 5 min. Controls both for the PCR and for DNA migration are incorporated into the assay. The lower detection limit of the test is 5 fg of pure T. brucei DNA. One parasite in 180 μl of blood is still detectable. Sensitivity and specificity for T. brucei were calculated at 100% when tested on blood samples from 26 confirmed sleeping sickness patients, 18 negative controls (nonendemic region), and 50 negative control blood samples from an endemic region. HAT-PCR-OC is a promising new tool for diagnosis of sleeping sickness in laboratory settings, and the diagnostic format described here may have wider application for other infectious diseases.

Molecular Tools for Diagnosis of Visceral Leishmaniasis: Systematic Review and Meta-Analysis of Diagnostic Test Accuracy

ABSTRACT Molecular methods have been proposed as highly sensitive tools for the detection of Leishmania parasites in visceral leishmaniasis (VL) patients. Here, we evaluate the diagnostic accuracy of these tools in a meta-analysis of the published literature. The selection criteria were original studies that evaluate the sensitivities and specificities of molecular tests for diagnosis of VL, adequate classification of study participants, and the absolute numbers of true positives and negatives derivable from the data presented. Forty studies met the selection criteria, including PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). The sensitivities of the individual studies ranged from 29 to 100%, and the specificities ranged from 25 to 100%. The pooled sensitivity of PCR in whole blood was 93.1% (95% confidence interval [CI], 90.0 to 95.2), and the specificity was 95.6% (95% CI, 87.0 to 98.6). The specificity was significantly lower in consecutive studies, at 63.3% (95% CI, 53.9 to 71.8), due either to true-positive patients not being identified by parasitological methods or to the number of asymptomatic carriers in areas of endemicity. PCR for patients with HIV-VL coinfection showed high diagnostic accuracy in buffy coat and bone marrow, ranging from 93.1 to 96.9%. Molecular tools are highly sensitive assays for Leishmania detection and may contribute as an additional test in the algorithm, together with a clear clinical case definition. We observed wide variety in reference standards and study designs and now recommend consecutively designed studies.

T. cruzi OligoC-TesT: a simplified and standardized polymerase chain reaction format for diagnosis of Chagas disease.

Background PCR has evolved into one of the most promising tools for T. cruzi detection in the diagnosis and control of Chagas disease. However, general use of the technique is hampered by its complexity and the lack of standardization. Methodology We here present the development and phase I evaluation of the T. cruzi OligoC-TesT, a simple and standardized dipstick format for detection of PCR amplified T. cruzi DNA. The specificity and sensitivity of the assay were evaluated on blood samples from 60 Chagas non-endemic and 48 endemic control persons and on biological samples from 33 patients, 7 reservoir animals, and 14 vectors collected in Chile. Principal Findings The lower detection limits of the T. cruzi OligoC-TesT were 1 pg and 1 to 10 fg of DNA from T. cruzi lineage I and II, respectively. The test showed a specificity of 100% (95% confidence interval [CI]: 96.6%–100%) on the control samples and a sensitivity of 93.9% (95% CI: 80.4%–98.3%), 100% (95% CI: 64.6%–100%), and 100% (95% CI: 78.5%–100%) on the human, rodent, and vector samples, respectively. Conclusions The T. cruzi OligoC-TesT showed high sensitivity and specificity on a diverse panel of biological samples. The new tool is an important step towards simplified and standardized molecular diagnosis of Chagas disease.

Trypanosoma lewisi or T. lewisi-like infection in a 37-day-old Indian infant.

Trypanosomes were observed in the peripheral blood smear of a 37-day-old Indian infant admitted off feeds, with fever and convulsions. Trypanosoma (Herpetosoma) lewisi was identified in the blood. The species identification was confirmed by morphometry, polymerase chain reaction, and sequencing. Human infection with this organism is rare. Only seven cases of this infection have been reported previously in humans. The cases reported are reviewed to develop a composite picture of this disease.

Epidemiology and clinical features of patients with visceral leishmaniasis treated by an MSF clinic in Bakool region, Somalia, 2004-2006.

Background There are few reports describing the epidemiology of visceral leishmaniasis (VL) in Somalia. Over the years 2002 to 2005, a yearly average of 140 patients were reported from the Huddur centre in Bakool region, whereas in 2006, this number rose to 1002 patients. Given the limited amount of information on VL and the opportunity to compare features with the studies done in 2000 in this part of Somalia, we describe the epidemiologic and clinical features of patients who presented to the Huddur treatment centre of Bakool region, Somalia, using data routinely collected over a five-year observation period (2002–2006). Methodology Methods used included the analysis of routine data on VL cases treated in the Huddur treatment centre, a retrospective study of records of patients admitted between 2004 and 2006, community leaders interviews, and analysis of blood specimens taken for parasite species identification in Antwerp Institute of Tropical Medicine. Principal Findings A total of 1671 VL patients were admitted to the Huddur centre from January 2002 until December 2006. Nearly all patients presented spontaneously to the health centre. Since 2002, the average patient load was stable, with an average of 140 admissions per year. By the end of 2005, the number of admissions dramatically increased to reach a 7-fold increase in 2006. The genotype of L. donovani identified in 2006 was similar to the one reported in 2002. 82% of total patients treated for VL originated from two districts of Bakool region, Huddur and Tijelow districts. Clinical recovery rate was 93.2% and case fatality rate 3.9%. Conclusions After four years of low but constant VL case findings, a major increase in VL was observed over a 16-month period in the Huddur VL centre. The profile of the patients was pediatric and mortality relatively low. Decentralized treatment centers, targeted active screening, and community sensitization will help decrease morbidity and mortality from VL in this endemic area. The true magnitude of VL in Somalia remains unknown. Further documentation to better understand transmission dynamics and thus define appropriate control measures will depend on the stability of the context and safe access to the Somali population.

Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

ABSTRACT There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P = 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P = 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P = 0.005), a trend also observed for the other qualitative PCR methods tested (P ≤ 0.05). Test positivity increased with increasing parasite burdens, as measured by real-time qPCR: OligoC-TesT and kDNA PCR detected 100% and 99% of positive samples when parasite burdens exceeded 74 and 49 parasites/ml, respectively. OligoC-TesT has high sensitivity for detection of canine Leishmania infections; its ease of operation and ease of interpretation are further advantages for veterinary diagnostic laboratories and for large-scale survey work in developing countries.

Diagnostic Accuracy of Loopamp Trypanosoma brucei Detection Kit for Diagnosis of Human African Trypanosomiasis in Clinical Samples

Background Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT), but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME) DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP) in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC). Methodology/Principal findings 142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9–91.8%) in the first run and 93.0% (95% CI 87.5–96.1%) in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4–96.3%) in the first run and 96.4% (95% CI 91.1–98.6%) in the second run. Reproducibility was excellent with a kappa value of 0.81. Conclusions/Significance In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions.

Leishmania OligoC-TesT as a Simple, Rapid, and Standardized Tool for Molecular Diagnosis of Cutaneous Leishmaniasis in Peru

ABSTRACT Molecular methods such as PCR have become attractive tools for diagnosis of cutaneous leishmaniasis (CL), both for their high sensitivity and for their specificity. However, their practical use in routine diagnosis is limited due to the infrastructural requirements and the lack of any standardization. Recently, a simplified and standardized PCR format for molecular detection of Leishmania was developed. The Leishmania OligoC-TesT is based on simple and rapid detection using a dipstick with PCR-amplified Leishmania DNA. In this study, we estimated the diagnostic accuracy of the Leishmania OligoC-TesT for 61 specimens from 44 CL-suspected patients presenting at the leishmaniasis clinic of the Instituto de Medicina Tropical Alexander von Humboldt, Peru. On the basis of parasitological detection and the leishmanin skin test (LST), patients were classified as (i) confirmed CL cases, (ii) LST-positive cases, and (iii) LST-negative cases. The sensitivities of the Leishmania OligoC-TesT was 74% (95% confidence interval (CI), 60.5% to 84.1%) for lesion aspirates and 92% (95% CI, 81.2% to 96.9%) for scrapings. A significantly higher sensitivity was observed with a conventional PCR targeting the kinetoplast DNA on the aspirates (94%) (P = 0.001), while there was no significant difference in sensitivity for the lesion scrapings (88%) (P = 0.317). In addition, the Leishmania OligoC-TesT was evaluated for 13 CL-suspected patients in two different peripheral health centers in the central jungle of Peru. Our findings clearly indicate the high accuracy of the Leishmania OligoC-TesT for lesion scrapings for simple and rapid molecular diagnosis of CL in Peru.

Recent progress in molecular diagnosis of sleeping sickness

This article will review the most recent progress in the molecular diagnosis of sleeping sickness and its potential role in patient management and disease control. While PCR remains restricted to research and reference laboratories, promising alternative molecular platforms have emerged over the last few years. Several loop-mediated isothermal amplification assays have been developed for detection and identification of the parasite with reported high analytical sensitivity and specificity. Simplified loop-mediated isothermal amplification formats have been designed and are undergoing evaluation studies in the field. Accurate diagnosis based on specific detection of the parasite’s ribosomal RNA has been made possible by the isothermal nucleic acid sequence-based amplification and by direct hybridization with fluorescent detection probes. In addition to the technological progress, the authors also discuss the diagnostic performance of molecular tests in the most recent clinical evaluation studies and briefly present some viewpoints for the near future.