Babesiosis

Babesiosis is caused by an intraerythrocytic parasite, Babesia microti, which is similar in effect to Plasmodium falciparum, the causative agent of malaria.³⁶,⁴³ Recently, WA1, another species of Babesia, has been described as a possible human pathogen. The intermediate host for B. microti and WA1 is the same tick that transmits B. burgdorferi, the causative agent of Lyme disease. Symptoms of Babesiosis are similar to those of Lyme disease: fatigue, malaise, myalgia, arthralgia, chills, and fever. The disease is particularly life threatening in splenectomized patients. There are several diagnostic tests for Babesiosis.

Diagnostic Tests for Babesiosis

Overview

Detection of Babesia IgM and IgG Antibodies in Serum by the Indirect Immunofluorescent Antibody Assay (IFA)

This immunofluorescent assay (IFA) indirectly detects Babesia-specific IgG or IgM antibodies in patient serum. Red blood cells from Syrian hamsters, infected with Babesia parasites, are fixed on a glass slide. Patient serum is added, and the patient's B. microti-specific IgG or IgM antibodies bind to the parasites in the infected red blood cells. In the third step, a labeled anti-human antibody is added. Fluorescence occurs if Babesia-specific antibodies are present. The slides must be read with a fluorescent microscope.

Interpretation

Assays for Babesia are usually performed using IFA against cells containing the organism. For the IFA, patient serum is titered using doubling dilutions. These dilutions start at 1:8 or 1:10. Thus, an assay starting at 1:8 would have values at 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, etc., and an assay starting at 10 would have values at 1:20, 1:40, 1:80, 1:160, 1:320, 1:640; 1:1280, etc.

Cut-off ranges between a laboratory negative and a laboratory positive sample are comparable for most clinical laboratories. The laboratory positive must be statistically different (mean +/- 2SD) from the negative sample. This does not imply that a titer of 1:40 or 1:80 is clinically significant. In fact, a positive antibody test by itself implies nothing. However, a positive antibody test, with appropriate clinical symptoms (determined by a physician), can lead to a diagnosis.

Positive Babesiosis titers are generally 1:160 or higher. Early in disease the titers may rise 4-fold to 1:1280. Later in disease the titer falls. For this reason, the testing of paired samples 4 to 6 weeks apart improves the diagnostic efficiency.

Diagnosis based on antibody response requires the seroconversion of infected individuals toward production of anti-Babesia antibodies. Unfortunately, this approach does not always work because:

• At the height of Babesiosis (within weeks of the initial bite) a patient with fever may fail to have evidence of antibody.
• Antibodies often persist long after the symptoms have disappeared.
• Polyclonal antibody-based tests lack specificity.

Nucleic Acid Based Diagnostic Tests (PCR and FISH) For Babesiosis

Nucleic acid based diagnostic tests impart enhanced performance, when compared to currently available microbiological and immunological methods for the detection of parasites in test samples. Some of the advantages are:

• Increased sensitivity: The nucleic acid based tests are able to detect a specific parasite in a given sample more frequently.
• Increased specificity: Accurate identification of biochemically unusual strains of Babesia, and those with dramatically different outer membrane proteins, is possible.
• These are direct assays for the presence of the parasite and have the consequent potential to identify the etiological agent.
• The assay is independent of the host's immune response schedule. Therefore, much earlier detection of the parasite is possible.
• Direct testing allows the monitoring of the efficacy of an antibiotic regime.
• There is the potential to detect the etiological agent in samples of tissue normally low in antibody titers (such as skin).

IGeneX has developed two nucleic acid based assays for the direct detection of Babesia in clinical specimens: the PCR and the Fluorescent In-Situ Hybridization (FISH) assays. The PCR assay detects DNA and can be performed on fresh or archived clinical specimens. The FISH assay is performed on thin blood smears and detects the ribosomal RNA of Babesia (thereby indicating active infection).

The PCR Test for Babesiosis using Whole Blood

The PCR-based diagnostic assay for Babesiosis is highly specific and sensitive. It is a three-step assay, performed directly on whole blood. The three steps are:

• Hybridization/Selection
• Amplification of Babesia-specific DNA
• Detection of Babesia-specific amplified DNA fragments

Hybridization/Selection

The hybridization/selection step specifically removes the common PCR inhibitors from the clinical sample and, at the same time, selects, purifies, and concentrates the DNA fragment of interest, thereby improving sensitivity.

PCR Amplification

During the second step, the purified Babesia fragment is PCR amplified with Babesia-specific primers. The primer is a synthetically produced nucleic acid sequence that, by design and selection, contains Babesia-specific nucleotide sequences. Under predetermined PCR conditions, this sequence "hybridizes" or binds specifically to Babesia species and not to other bacteria or parasites or human DNA. Therefore, only Babesia-specific DNA is amplified.

Detection of Babesia-specific Amplified Products

In the third step, the PCR-amplified Babesia DNA fragment is detected by agarose gel electrophoresis. Any of the "right size" amplified product detected by agarose gel electrophoresis is Babesia-specific. It is possible that the WA1 strain of Babesia may be detected by a similar method.

The combination of these three steps imparts a very high specificity and sensitivity to the test.

In order to minimize DNA contamination, separate work stations are utilized for the set- up, reagent preparation, cycling, and gel electrophoresis.

It should be kept in mind that a negative result with the PCR assay implies only that Babesia DNA is not detected in the test sample. The PCR can be performed on ticks and EDTA whole blood, since the organism infects red cells. Samples have excellent stability if mixed with equal parts of 95% ethanol, special IGeneX PCR buffer, or are kept at -70^°C.

IGeneX, Inc.
795 San Antonio Rd., Palo Alto, CA 94303 USA
Tel. 650.424 1191 / 800.832.3200 Fax. 650.424.1196