Momar Ndao

Multiplexed Real-Time PCR Assay for Discrimination of Plasmodium Species with Improved Sensitivity for Mixed Infections

ABSTRACT The implementation of real-time PCR for the diagnosis of malaria has been hampered by poor sensitivity for the detection of mixed infections. We have optimized a method that enhances the sensitivity of detection of minor species in mixed infections within a single multiplex reaction. Our assay uses species-specific forward primers in combination with a conserved reverse primer and largely overcomes primer competition for the minor species DNA. With a blind panel of clinical samples, we successfully identified the species in 13/16 mixed infections. This assay was further validated with 91 blood samples and demonstrated a specificity and sensitivity for single infections of 100% compared with nested PCR as the “gold standard.” This test has been implemented for routine confirmation of malaria species in Alberta, Canada. In comparison with species identification by microscopy, the real-time PCR test demonstrated greater sensitivity for the identification of species causing low-level and mixed infections and for the discrimination of Plasmodium species other than Plasmodium falciparum. Our experience supports a role for real-time PCR in the identification of malarial species in conjunction with microscopy.

Comparison of Blood Smear, Antigen Detection, and Nested-PCR Methods for Screening Refugees from Regions Where Malaria Is Endemic after a Malaria Outbreak in Quebec, Canada

ABSTRACT The importation of malaria into a region where it is not endemic raises many concerns, including the timely delivery of appropriate care, safety of the blood supply, and the risk of autochthonous transmission. There is presently no consensus on the best way to screen mobile populations for malaria. Between August 2000 and March 2001, 535 refugees arrived in Quebec, Canada, from Tanzanian camps. Within 4 weeks of resettlement of the first group of 224, the McGill University Centre for Tropical Diseases noted an outbreak of malaria across the province (15 cases over a 3-week period). This group (group 1) was traced and screened for malaria between 3 and 4 months after arrival in Canada. Subsequent groups of 106 and 205 refugees were screened immediately upon arrival in Canada (group 2) and immediately prior to their departure from refugee camps (group 3), respectively. A single EDTA-blood sample was obtained from 521 refugees for testing by thick and thin blood smears (groups 1 and 2), antigen detection (ICT Malaria Pf and OptiMAL; group 1 only), and nested PCR (all groups). Overall, 98 of 521 refugees were found to be infected (18.8%). The vast majority of infections (81 of 98) were caused by Plasmodium falciparum alone. Using PCR as the “gold standard,” both microscopy (sensitivity, 50%; specificity, 100%) and antigen detection (ICT sensitivity, 37.5%; ICT specificity, 100%; OptiMAL sensitivity, 29.1%; OptiMAL specificity, 95.6%) performed poorly. None of the PCR-positive subjects were symptomatic at the time of testing, and only two had recently had symptoms compatible with malaria (with or without diagnosis and treatment). Active surveillance of migrants from regions of intense malaria transmission can reduce the risk of morbidity in the migrant population and mitigate against transmission to the host population. Our data demonstrate that PCR is, by far, the most powerful tool for such surveillance.

Diagnosis of Parasitic Diseases: Old and New Approaches

Methods for the diagnosis of infectious diseases have stagnated in the last 20–30 years. Few major advances in clinical diagnostic testing have been made since the introduction of PCR, although new technologies are being investigated. Many tests that form the backbone of the “modern” microbiology laboratory are based on very old and labour-intensive technologies such as microscopy for malaria. Pressing needs include more rapid tests without sacrificing sensitivity, value-added tests, and point-of-care tests for both high- and low-resource settings. In recent years, research has been focused on alternative methods to improve the diagnosis of parasitic diseases. These include immunoassays, molecular-based approaches, and proteomics using mass spectrometry platforms technology. This review summarizes the progress in new approaches in parasite diagnosis and discusses some of the merits and disadvantages of these tests.

Emergence of Sylvatic Echinococcus granulosus as a Parasitic Zoonosis of Public Health Concern in an Indigenous Community in Canada

Within a remote Canadian Indigenous community, at least 11* of people had antibodies against Echinococcus granulosus and E. granulosus eggs were detected in 6* of environmentally collected canine fecal samples. Dog ownership, hunting, and trapping were not risk factors for seropositivity, suggesting that people are most likely exposed to E. granulosus through indirect contact with dog feces in the environment. In this situation, human exposure could be most effectively curtailed by preventing consumption of cervid viscera by free-roaming dogs.

Reversible Cysteine Protease Inhibitors Show Promise for a Chagas Disease Cure

ABSTRACT The cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of Chagas disease. Thus, inhibitors of cruzipain are considered promising anti-T. cruzi chemotherapeutic agents. Reversible cruzipain inhibitors containing a nitrile “warhead” were prepared and demonstrated 50% inhibitory concentrations (IC50s) as potent as 1 nM in baculovirus-generated cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated antiparasitic behavior in the 5 to 10 μM IC50 range; however, trypomastigote production from the amastigote form was ∼90 to 95% inhibited at 2 μM. Two key compounds, Cz007 and Cz008, with IC50s of 1.1 and 1.8 nM, respectively, against the recombinant enzyme were tested in a murine model of acute T. cruzi infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for benznidazole, the control compound, at 50 mg/kg.

New Inflammation-Related Biomarkers during Malaria Infection

Malaria is one of the most prevalent infectious diseases worldwide with more than 250 million cases and one million deaths each year. One of the well-characterized malarial-related molecules is hemozoin (HZ), which is a dark-brown crystal formed by the parasite and released into the host during the burst of infected red blood cells. HZ has a stimulatory effect on the host immune system such as its ability to induce pro-inflammatory mediators responsible for some of the malaria related clinical symptoms such as fever. However, the host serum proteins interacting with malarial HZ as well as how this interaction modifies its recognition by phagocytes remained elusive. In the actual study, using proteomic liquid chromatographic mass spectrometry (LC-MS/MS) and immunochemical approaches, we compared the serum protein profiles of malaria patients and healthy individuals. Particularly, we utilized the malarial HZ itself to capture serum proteins capable to bind to HZ, enabling us to identify several proteins such as apolipoprotein E (ApoE), serum amyloid A (SAA), gelsolin, complement factor H and fibrinogen that were found to differ among healthy and malaria individual. Of particular interest is LPS binding protein (LBP), which is reported herein for the first time in the context of malaria. LBP is usually produced during innate inflammatory response to gram-negative bacterial infections. The exact role of these biomarkers and acute phase responses in malaria in general and HZ in particular remains to be investigated. The identification of these inflammation-related biomarkers in malaria paves the way to potentially utilize them as diagnostic and therapeutic targets.

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease.

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease is described. The identified inhibitors bearing an amino nitrile warhead in P1 exhibit low nanomolar in vitro potency against cruzipain. Further SAR in P2 portion led to the identification of compounds, such as 26, that have a unique selectivity profile against other cysteine proteases and offering new opportunities for safer treatment of Chagas disease.

Discovery and validation of serum biomarkers expressed over the first twelve weeks of Fasciola hepatica infection in sheep.

Abstract Serum biomarkers associated with Fasciola hepatica infection of Corriedale sheep were analysed during the first 12 weeks of infection using surface-enhanced laser desorption ionisation time of flight mass spectrometry (SELDI-TOF MS). In the discovery phase of analysis, pooled sera collected at week 0 and at each week p.i. to week 12 were fractionated by anion-exchange chromatography and the protein mass fingerprints obtained in individual fractions were in the M/z range 1.5–150kDa. A total of 2302 protein clusters (peaks) were identified that varied between time-points following infection with peaks increasing or decreasing in intensity, or showing transient variation in intensity, during the 12 weeks of parasite challenge. In the validation phase, candidate biomarkers in sera of individual sheep at weeks 3 and 9 p.i. were analysed, identifying 100 protein peaks, many of which are small peptides <10kDa in size: 54% of these peaks were up-regulated in intensity at week 3 or 9 p.i. Twenty-six biomarkers were chosen for further study, ranging in size from 1832 to 89,823Da: six biomarkers were up-regulated at weeks 3 and 9 p.i., 16 biomarkers were up-regulated only at week 9 p.i. and four biomarkers were down-regulated at week 9 p.i. Two biomarkers up-regulated at week 9 were identified as transferrin (77.2kDa) and Apolipoprotein A-IV (44.3kDa), respectively. The results show that the interaction between the host and F. hepatica is complex, with changes in biomarker patterns beginning within 3 weeks of infection and either persisting to weeks 9–12 or showing transient changes during infection. Identification of biomarkers expressed during ovine fasciolosis may provide insights into mechanisms of pathogenesis and immunity to Fasciola and may assist in the rational development and delivery of vaccines.

The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

Background Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Methodology/Principal findings Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. Conclusions/Significance A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.

Reprofiled drug targets ancient protozoans: drug discovery for parasitic diarrheal diseases.

Recently, we developed a novel automated, high throughput screening (HTS) methodology for the anaerobic intestinal parasite Entamoeba histolytica. We validated this HTS platform by screening a chemical library containing US Food and Drug Administration (FDA)-approved drugs and bioactive compounds. We identified an FDA-approved drug, auranofin, as most active against E. histolytica both in vitro and in vivo. Our cell culture and animal studies indicated that thioredoxin reductase, an enzyme involved in reactive oxygen species detoxification, was the target for auranofin in E. histolytica. Here, we discuss the rationale for drug development for three parasites which are major causes of diarrhea worldwide, E. histolytica, Giardia lamblia and Cryptosporidium parvum and extend our current finding of antiparasitic activity of auranofin to Entamoeba cysts, G. lamblia and C. parvum. These studies support the use of HTS assays and reprofiling FDA-approved drugs for new therapy for neglected tropical diseases.