The main Babesiosis diagnostic tests available are microbiological and serological. Unfortunately these tests have low specificity and sensitivity. Two molecular tests, the Polymerase chain reaction (PCR) and Fluorescent In-Situ Hybridization (FISH) tests, developed by IGeneX have great potential. Using 100 mice models the sensitivity and the specificity of the two tests will be determined and compared with the serological test.
Babesiosis is a disease caused by an intracellular parasite, Babesia, which infects erythrocytes. Four main species of this parasite are known to infect humans; Babesia microti, B-MOI, B. divergens and B.duncani. Babesiosis is transmitted by ticks and has symptoms such as myalgia, malaise, fatigue, chills, fever and arthralgia.
The first and most important step in the treatment and/or management of any disease is the diagnosis of the disease. It is therefore imperative to come up with highly specific, sensitive and accurate diagnostic tests. Various diagnostic tests for Babesiosis exist, the main ones being microbiological and serological tests. Unfortunately these tests have low specificity and sensitivity. Two molecular tests, the Polymerase chain reaction (PCR) and Fluorescent In-Situ Hybridization (FISH) tests, developed by IGeneX have great potential.
The objective of this study is to determine the specificity and sensitivity of the Babisiosis PCR and FISH diagnostic tests. The study shall compare the two tests with the serological test.
The study is a comparative study comparing three diagnostic techniques: the serological test, the PCR and the FISH test. A hundred mice will be used for the study; 50 will be infected with B.microti and 50 will be free of infection and be used as control. Blood samples from the two sets will be collected and analysed for the parasite using the three diagnostic techniques.
2.1 Procedures and principles of serological test
The serological test, Immunofluorescent Antibody Assay (IFA) is based on indirect detection of Babesia IgM or IgG immunoglobulins in the serum prepared from the mice blood samples. The first step of the assay involves the fixing of erythrocytes from B.microti infected Syrian hamsters on a glass slide. Serum from the mice is then added and IgM/IgG immunoglobins from infected mice bind to the protozoa in the infected erythrocytes. Finally labelled anti-mice antibody is added. Fluorescence, observed via fluorescent microscopy, is indicative of positive Babisiosis diagnosis. This test requires the formation of antibodies by the infected subject thus may give misleading results at certain stages of the disease. Within the first few weeks of infection there may be no evidence of antibodies and long after the disease symptoms have disappeared antibodies may persist. Furthermore there are polyclonal antibody-based tests that have low specificity.
2.1 Principles and procedures of the PCR Babisiosis test
Polymerase chain reaction (PCR) refers to invitro amplification of DNA fragments. The PCR-babiosis test involves three steps. The selection step removes PCR inhibitors from the samples and selects, purifies as well as concentrates the B.microti DNA fragments from the blood sample thus improves sensitivity. The next step is the mixing of the purified DNA with synthetic Babesia-specific primers. The mixture is run in a PCR under predetermined conditions. The primers are designed to specifically bind to the Babesia DNA thus only the Babesia DNA is amplified. The next step involves the detection of the PCR products by dot-blot. In this step the PCR-products are transferred to a nitrocellulose membrane with probes that are specific to the Babesia DNA. Blue dots appear on the membrane in infected blood sample.
2.2 Babesia FISH Assay
Fluorescence in situ hybridization (FISH) is a technique that enables researchers to visualize and map genetic material. Unlike other molecular techniques FISH is very versatile because it can be performed on cells that are not actively dividing. FISH has found wide application in diagnosis of cancers. Babesia FISH diagnostic test specifically detects ribosomal RNA (rRNA) of the parasite in infected blood. The whole blood is fixed on a glass slide and hybridized to a fluorescent labelled probe specifically targeting B. microti rRNA. After washing the excess probe the smear is counterstained for viewing under photomicroscopy at 1000X. The rRNA is found in the cytoplasm thus a fluorescent signal is observed while maintaining the parasite morphology.
Static software (SPSS) will be used to calculate if the differences between the test methods are statistically significant. In addition the percentage sensitivity and specificity of each method will be calculated. The results of this analysis will be presented in tables.
The PCR and FISH tests are expected to have better performance than the serological tests due to high sensitivity and specificity (because the DNA and rRNA detected are specific to specific Babesia species). Furthermore since the tests do not depend on the host’s immunological response early diagnosis is possible. The tests may also allow detection of the parasites in tissues normally low in antibody levels such as the skin. Finally since they are both direct tests the efficiency of the therapeutic intervention can be monitored.
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