SBIR-STTR Award

Plasmodium -Dual Probe Fluorescent In-Situ Hybridization (PFV- FISH) is a method of detecting and differentiating Plasmodium falciparum (PF) ribosomal RNA (rRNA) and P. vivax (PV) rRNA on a SINGLE air- dried thin blood smear. The assay uses PF and PV specific probes labeled with different fluorescent dyes. Thus, PF and PV shall fluoresce with different colors under specific dual pass filters. The assay is simple and inexpensive (< $5.00/test and a one time expense of ~$600 for filters or <$1700 for the microscope and filters). It consists of five steps -fixation, hybridization, washing, counterstaining and viewing the processed smear under a fluorescent microscope. The total assay time is less than 1 hour. The treatment for P. falciparum and P. vivax is different. Therefore, PFV-FISH assay would be very useful in areas where both P. falciparum and P. vivax are endemic. Specific Aims: Develop a simple and inexpensive Dual Probe FISH assay (PFV-FISH) kit, for direct detection of PF and PV on an air-dried whole blood smear. The kit is expected to provide the specificity equivalent to amplified DMA probe assays, and sensitivity equivalent to giemsa stained smear. The kit shall contain all the reagents and control smears. Phase I: (1) The feasibility study of the PFV - FISH Test to determine the sensitivity of the assay to detect and differentiate P. falciparum and P. vivax. (2) Determination of the Hybridization Conditions for a Dual Color PFV-FISH Assay. (3) Limited Specificity Study and Limit of Detection Study. (4) Assay Performance on 100 Clinical Samples. Generally clinical samples are not tested in Phase I. However, these are the only samples that can be used to study the assay. Culture samples are available for P. faciparum, but are not readily available. Phase II: (1) Assay Development - Optimization of the PFV-FISH. based on the results of the study performed on 100 clinical samples. (2) Set up Quality Control Procedures and Controls (3) Set up manufacturing facilities. (4) Perform an in-house study on clinical samples. (Number of samples to be tested shall be determined statistically by the Biostatistician, Dr. Stubbs.) (5) Identification and validation of Potential Clinical Sites India and Kenya for Phase III. Phase III: (1) Perform clinical trials in the US; and concurrently in Kenya and India. (2) Get FDA approval. (3) Marketing within US and Outside US. (4) Set up manufacturing plant in an underdeveloped country where malaria is endemic, e.g. Kenya or India.(4) Extend the assay to include P. ovate and P. malaria

Malaria can be a life-threatening disease, especially in children, if left untreated. According to WHO, 525,000 to 2.0 million African children die from Malaria every year. The current gold standard for diagnosis is examination of Giemsa stained smear by microscopy. However, when parasite levels are very low, or in mixed infections, the information obtained by examination of Giemsa stained smear by microscopy is limited. Under SBIR Phase II we have developed 3 kits, P-Genus, PF and PV FISH assay kits. These kits detect malaria parasites on blood smears prepared with a special reagent by Fluorescent in Situ Hybridization (FISH) technique, using specific fluorescent labeled DNA probes targeted to ribosomal RNA of viable parasites. P- Genus kit detects all species of Plasmodium. PF-FISH kit and PV-FISH kit detect and differentiate P. falciparum and P. vivax from other species of Plasmodium respectively, on any P-Genus screen positive blood sample. Since ribosomal RNA is the target, morphological information too is obtained. The assays are simple, semi-quantitative and detect all stages of the malaria parasites. The only requirement is a fluorescent microscope. We have validated a LED light unit with 2 filters that can be attached to a regular light microscope for reading the FISH assay results. The assays consist of six steps: smear preparation using proprietary reagent, fixation, hybridization, washing, counterstaining and viewing the processed smear under a fluorescent microscope. The total assay time is approximately 90 minutes. The limit of detection is between 2-3 parasites per 300 fields with 100X objective. Based on a study on 500 patients from Kenya, India and Peru with malaria- like symptoms, the sensitivity of the FISH assays was >95% whereas the sensitivity of Giemsa stained smear by microscopy was between 84 - 91%. In Phase IIB we will complete all the additional validation studies requested by FDA to file for 510(K). The specific aims are: (1) Finalization of study protocols; (2) Manufacture of 3 lots of kits and completion of stability stud of kits and reagents; (3) Analytical Sensitivity Study using patient samples positive for P. falciparum and P. vivax; (4) Reproducibility Study near limit of detection using patient samples; (5) Completion of Specificity Study (including Analytical Specificity and Interference Substances; (6) Clinical studies at 3 sites; (7) PCR on all clinical study samples and sequencing on all PCR positive samples (8) Analysis of data; and (9) Write 510(K) submission report. (10) Start marketing in countries that do not require FDA clearance. In Phase III (1) File for 510(K) (2) Set up infrastructure for marketing; (3) Increase the market share (4) Develop P. ovale, P. malariae and P. knowlesi specific FISH assays.

Public Health Relevance Statement:


Public Health Relevance:
Malaria is a serious, often fatal, parasitic disease caused by infection of the red blood cells with protozoan parasites of the Plasmodium species, P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. According to the latest WHO estimates, there were about 219 million cases of malaria in 2010 and an estimated 660 000 deaths. 90% of deaths occur in Africa, the most affected continent. In Africa a child dies every minute from malaria. The six highest burden countries in the WHO African region (in order of estimated number of cases) are: Nigeria, Democratic Republic of the Congo, United Republic of Tanzania, Uganda, Mozambique and Cote d'Ivoire. These six countries account for an estimated 103 million (or 47%) of malaria cases. In South East Asia, the second most affected region in the world, India has the highest malaria burden (with an estimated 24 million cases per year), followed by Indonesia and Myanmar.1 Even though malaria is a frequently encountered disease in many developing countries, it is difficult to make the right diagnosis relying on clinicl signs only. Drug selection for the treatment of malaria depends on species of malaria present. Delayed or missed diagnosis of falciparum malaria increases the risk of complicated or severe disease, which may be fatal vulnerable populations. P. falciparum from much of the world is largely chloroquine resistant and thus the standard treatment for P. vivax cannot be used. To prevent unnecessary anti-malarial treatment and future drug-resistance strains of malaria parasites, it is important to confirm clinical suspicious with a good laboratory test. In light of he changing drug policies of many African countries, including Tanzania and Kenya, where the expensive artemisinin combination therapy (ACT) drugs are prescribed as first-line treatment, a good laboratory confirmation will also have an impact on the economics. The Giemsa stain is helpful. However, when parasite levels are very low, or in mixed infections, the information obtained by examination of Giemsa stained smear by microscopy is limited, and in some cases, biased, by the inability to devote the necessary amount of time to the examination of blood smears. PCR would help. Unfortunately it is time-consuming and expensive. Thus, FISH Tests for detection of Plasmodium and for differentiation of P. falciparum and P. vivax in air-dried blood smears has potential in the rapid diagnosis of this disease. Advantages: P-Genus and PFV FISH Assays 1. P- Genus FISH Assay detects all five species of Plasmodium, P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. Therefore the Genus FISH assay can be used for screening. Any sample that is positive can be tested further if necessary with the PFV- FISH Assay to determine whether the infection is due to P. falciparum, P. vivax, both or neither. 2. Specificity of the assays is equivalent to the amplified assays. 3. Sensitivity of te assays when performed by a "typical microscopist" should be the same as or better than Giemsa stained smear performed by an "expert microscopist' 4. Ability to perform the tests on an air-dried whole blood smear. 5. Results can be obtained in two hours or less would be valuable. 6. Provides parasite morphology as the Giemsa stained smear. 7. Easy to perform. 8. Low Cost.

Project Terms:
Accounting; Affect; Africa; African; Air; Antimalarials; artemisinine; Artemisinins; base; Biological Assay; Blood; Blood specimen; Cessation of life; Child; Chloroquine resistance; Clinical; Clinical Research; Combined Modality Therapy; cost; Country; Data Analyses; Democratic Republic of the Congo; Detection; Developing Countries; Diagnosis; diagnosis standard; Disease; DNA Probes; Drug Prescriptions; Drug resistance; Economics; Erythrocytes; Falciparum Malaria; Far East; fluorescence microscope; Fluorescent in Situ Hybridization; Future; Giemsa stain; Gold; Grant; Hour; India; Indonesia; Infection; Ivory Coast; Kenya; Label; Laboratories; Left; Life; Light; Light Microscope; Malaria; Marketing; Microscopy; Morphology; Mozambique; Nigeria; Parasites; Parasitic Diseases; Patients; Peru; Pharmaceutical Preparations; Phase; Plasmodium; Plasmodium falciparum; Policies; Preparation; prevent; Principal Investigator; Process; programs; Protocols documentation; public health relevance; rapid diagnosis; Reading; Reagent; Reporting; Reproducibility; Research Infrastructure; resistant strain; Ribosomal RNA; Risk; sample fixation; Sampling; screening; Selection for Treatments; Site; Small Business Innovation Research Grant; Specificity; Staging; standard care; Symptoms; Tanzania; Techniques; Testing; Time; Uganda; validation studies; Vulnerable Populations; Whole Blood; Writing