Concept of Coinfections in Lyme Disease
Nick S. Harris, Ph.D., ABMLI
There have been reports in the literature for the past several years on the detection of coinfections of Babesiosis or Ehrlichiosis in patients with Lyme disease. The prevalence or frequency of these diseases as coinfections in humans is not known. It is known, however, that 10 to 20% of the same ticks that carry B. burgdorferi also carry the Babesia and/or Ehrlichia organisms.
Both Babesia and Ehrlichia, in their commonly recognized acute clinical presentation, result in patients who are quite sick with fever, malaise, myalgia, nausea and other symptoms. Because of these symptoms, patients usually receive prompt treatment. Laboratory markers for these acute diseases are high antibody titers: greater than 1:640 for IgM and IgG. We have seen some samples with titers greater that 1:4000. In acute Babesia, anemia and the presence of piroplasm in red cells are also detected.
Now that the medical community is aware of potential coinfections, physicians often request tests for Babesia and Ehrlichia when they order tests for Lyme disease. We and others have observed in some patients the presence of low level titers (less than 1:320) of Babesia and/or Ehrlichia antibodies in patients with Lyme disease. We have investigated these Babesia and/or Ehrlichia antibodies extensively and they do not appear to be cross reactions with host B. burgdorferi antibodies or due to B. burgdorferi antigens.
In the normal healthy population, in individuals with a normal immune system, Babesia and/or Ehrlichia may be a self-limiting subclinical disease that requires no medical intervention. We and other investigators think the possibility exists that some patients with Lyme disease may have a subclinical infection of Babesia and/or Ehrlichia and that these subclinical infections impact the resolution of the primary disease, Lyme.
Any idea needs to be tested. For this reason we have developed and are developing additional tests to examine Babesia and Ehrlichia coinfections. The new test developed and available is a PCR for B. microti DNA and soon a Fluorescence In-situ Hybridization Assay (FISH) for B. microti RNA will be available. Additional information is provided in this newsletter.
References for Babesia and Ehrlichia:
- Benach JL, Coleman JL, Habicht GS. Serologic evidence for simultaneous occurrences of Lyme disease and babesiosis. J Infect Dis. 1981;144:473-477.
- Grunwaldt E, Barbour AG, Benach JL. Simultaneous occurrence of babesiosis and Lyme disease. N Engl J Med 1983;308:1166.
- Anderson JF, Mintz ED, Gadbaw JJ, Magnarelli LA. Babesia microti, human babesiosis, and Borrelia burgdorferi in Connecticut. J Clin Microbiol 1991;29:2779-83.
- Magnarelli LA, Dumler SJ, Anderson JF, Johnson RC, Fikrig E. Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera. J Clin Microbiol 1995;33:3054-57.
- Krause PJ, Telford SR, Spielman A, et al. Concurrent Lyme disease and babesiosis: evidence for increased severity and duration of illness. JAMA 1996;275:1657-60.
- Mitchell PD, Reed KD, Hofkes JM. Immunoserologic evidence of coinfection with Borrelia burgdorferi, Babesia microti, and human granulocytic Ehrlichia species in residents of Wisconsin and Minnesota. J Clin Microbiol 1996;34:724-727.
- Fishbein DB, Dawson JE, Robinson LE. Human ehrlichiosis in the United States, 1985 to 1990. Ann Intern Med 1994;120:736-43.
- Dumler JS, Bakken JS. Ehrlichial diseases of humans: emerging tick-borne infections. Clin Infect Dis 1995;20:1102-10.
- Anderson BE, Dawson JE, Jones DC, Wilson KH. Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J Clin Microbiol 1991;29:2838-42.
- Walker DH, Barbour AG, Oliver JH, et.al. Emerging bacterial zoonotic and vector-borne diseases: ecological and epidemiological factors. JAMA. 1996;275:463-469.
- Goodman JL, Nelson C, Vitale B, et al. Direct cultivation of the causative agent of human granulocytic ehrlichiosis. N Engl J Med 1996;334:209-15.
- Persing DH, Mathiesen D, Marshall WF, et al. Detection of Babesia microti by polymerase chain reaction. J Clin Microbiol 1992;30:2097-103.
Overview - Diagnostic Tests for Babesiosis
Jyotsna Shah, Ph.D. - Director, Research & Development
Babesiosis is caused by an intraerythrocytic parasite, Babesia microti, which is similar in effect to Plasmodium falciparum, the causative agent of malaria. Recently another species of Babesia, Wa-1, has been described as a human pathogen. The intermediate hosts for B. microti and Wa-1 are the same ticks that transmit the Lyme disease bacteria, Borrelia burgdorferi. Symptoms of Babesiosis are somewhat similar to some of the symptoms of Lyme disease: fatigue, malaise, myalgia, arthalgia, chills and fever. Usually the fever is high. The disease is particularly life threatening in splenectomized patients. Diagnostic tests for Babesiosis currently available and those under development are discussed.
Babesiosis is usually diagnosed by the microscopic examination of Giemsa-stained thin blood smears. This method is neither very specific nor sensitive.
Detection of B. microti IgM and IgG antibodies in serum by indirect immunofluorescence assay (IFA) is the routine method. This assay is a serological test in an IFA format. Diagnosis is based upon indirect detection of B. microti-specific IgG or IgM antibodies in serum. Diagnosis based on antibody response requires the seroconversion of infected individuals towards production of anti-B. microti antibodies. 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 exhibit antibody;
- Antibodies often persist long after the symptoms have disappeared;
- Polyclonal antibody based tests lack specificity. Thus, a positive IFA test result only suggests exposure, not active infection. A negative test also does not rule out the disease.
We have developed a nucleic acid based diagnostic assay for direct detection of B. microti from blood. This type of test has enhanced performance compared to the microbiological and immunological methods currently available for detection of parasites. Some of the advantages are:
- Increased sensitivity: i.e., the ability to detect a target parasite in a given sample more frequently;
- Accurate identification of bio-chemically unusual strains of the target Babesia, even if it has a different outer membrane protein;
- Direct assay for presence of the parasite and subsequent potential to quantify the etiological agent;
- Assay independent of the host's immune response schedule - much earlier detection possible;
- Direct testing permitting monitoring the efficacy of an antibiotic regime;
- Potential to detect said etiological agent in samples of tissue normally low in antibody titer such as skin.
PCR test for B. microti from whole blood
IGeneX has developed a three-step PCR-based diagnostic assay for B. microti. It consists of:
- Hybridization/Selection: The first step of the assay "hybridization/selection" specifically removes the "common PCR inhibitors" from blood and selects and purifies the DNA of interest.
- PCR Amplification: The purified B. microti fragment is PCR amplified with B. microti specific primers. The B. microti specific primer, as used herein, refers to a synthetically produced nucleic acid sequence that, by design, contains specific nucleotide sequences. This sequence "hybridizes' or binds specifically to B. microti DNA and not to DNA from other Babesia species (B. equi, B. rohaini, B. caballi, B. bigemia, B. bovis, B. gibsoni, B. species) under predetermined PCR conditions. Therefore, only B. microti and specific DNA is amplified.
- Detection: The PCR amplified B. microti DNA fragment is detected by agarose gel electrophoresis. Thus any of the "right size" amplified product detected by agarose gel electrophoresis would be B. microti specific. We have not tested the Wa-1 strain of Babesia. It is possible that the Wa-1 strain of Babesia may be detected by this method.
Assays Under Development
Fluorescence In-situ Hybridization Assay (FISH) for Direct Detection of B. microti RNA
Babesiosis is usually diagnosed by the examination of Giemsa-stained thin blood smears. This method is neither very specific nor sensitive. We are currently developing a fluorescence in-situ hybridization (FISH) assay that can detect viable B. microti parasites directly on blood smears, i.e., active infection. It is based upon two fundamental principles: Hybridization directly on a thin blood smear with a fluorescent-labeled B. microti specific probe and identification of B. microti parasites within the red blood cells by a dark field fluorescent microscope. The B. microti specific probe refers to a synthetically produced DNA sequence that, by design, contains nucleotide sequences which allow it to hybridize on a slide specifically to B. microti nucleic acid sequences.
How Many Days of Urine Collection are Necessary?
We have observed that Lyme patients with active disease may be positive in the LUAT (Lyme Urine Antigen Test). Some patients are positive most of the time they are tested, but the majority of patients are only sporadically positive. Several physicians have developed and use antibiotic challenge protocols which enhance the killing of the B. burgdorferi, thus enhancing the antigenuria. Even with these protocols, the patients may not be positive in the LUAT every time that they are tested.
Studies have been performed and analyzed by statistical experts to help find the balance between the number of samples to be tested, the time of the day to be tested and the detection of a positive LUAT. The results of these studies indicate that three morning samples give the best probability for a positive result. (One antibiotic protocol uses alternate days and one uses consecutive days). While not every sample may be positive, three morning samples is the most cost-effective compromise for the best chance of obtaining a single positive result.
There is another reason for multiple sampling: a UTI (Urinary Tract Infection) may cause a non-specific false positive. This false positive is due to the physical interference of a large number of bacteria or yeast in the washing-step in the assay. One of the general characteristics of a UTI is the presence of red and/or white blood cells in the urine. This is seen in normal circumstances as well, such as during menses or as part of the killing of B. burgdorferi in the bladder wall. If the laboratory notices red and/or white blood cells, it is noted on the report.
If red and/or white blood cells are present in all three samples and all three results have similar high values in the LUAT, it is possible that the positives are due to the UTI and not the shedding of the antigen. If only one sample has blood cells and is a high positive and one or both of the other two samples lack the red and/or white blood cells, this suggests that the high positive sample is a valid positive test and not due to a UTI cross reaction.
Explanation of IgM and IgG Western Blots for CPT Coding
Although the two tests described below share the same CPT code, they are distinctly different and are used either together or separately for different situations.
Lyme Western Blot IgM - This test is a sensitive indication of early Lyme infection. It is also useful for recurrent or persistent infection, when patients are seronegative by other techniques.
Lyme Western Blot IgG - This antibody test is a specific and sensitive indicator of exposure to B. burgdorferi. Sixty to seventy percent of patients with Lyme disease are positive to this assay between 2 and 4 months after infection. Some seronegative patients may seroconvert to a positive blot after treatment.
(Since many patients do not know when infection began, the use of these two tests, along with clinical symptoms, can aid the physician in a course of appropriate therapy.)
The body's response to an infection by bacteria, parasites, or virus is to make antibodies against the infectious agent. The body usually makes an IgM antibody followed by an IgG antibody. However, sometimes even in late Lyme disease, the only antibody detected is IgM. This may be an infection of an acute or recurrent disease process. The most accurate method to detect these antibodies is by a technique called the Western Blot. Since the IgM antibodies and the IgG antibodies are unique and different, two separate Western Blots (an IgM and an IgG) need to be performed on the patient's blood sample.
The CPT code for an IgM Western blot and for an IgG Western Blot are identical. Thus, insurance companies need to be billed for the CPT code 86617 times (x) 2. With other laboratory tests, Hepatitis B core antibody for example, there is a separate CPT code for IgG #86704 and for IgM # 86705.
Presented at the 11th Annual International Scientific Conference
on Lyme Disease and Other Tick-borne Disorders
April 25-26, 1998 - New York City, NYDirect Detection of Babesia microti by Polymerase Chain Reaction
Jyotsna Shah* and Nick S. Harris, IGeneX, Inc., 795 San Antonio Rd, Palo Alto, CA 94303
Babesiosis is caused by an intraerythrocytic parasite, Babesia microti. Babesiosis is usually diagnosed by the examination of Giemsa-stained thin blood smears. In addition, there are indirect immunofluorescence assays (IFA) using polyclonal antibodies directed against B. microti. However, all these tests lack specificity and sensitivity. Isolation and culture of these parasites as a diagnostic method would provide adequate specificity and sensitivity, however, it is not considered technically or economically feasible. We have developed a PCR test for direct detection of B. microti DNA from whole blood. The test was performed on 145 EDTA treated whole blood samples. 137 samples were from patients with Babesiosis-like symptoms and eight were from normal individuals. Sixty-two (43%) of the 145 blood samples were positive by PCR. None of the samples from the control group were positive.
At the same time immunofluorescence assays (IFA) for detection of B. microti specific IgG or IgM were performed on serum from 97 of the 145 samples. Sixty-four samples, including three from the control group, were positive by IFA. Thirty-four of these patients were also positive by PCR. None of the IFA positive samples from the control group were positive by PCR.
Four blood samples were obtained from one patient over a three month period. The first and second samples (2 weeks apart) were positive by PCR and also by IFA. The third and forth samples were negative by PCR, but positive by IFA. Two blood samples were obtained from the second patient, two months apart. Both samples were positive by PCR and IFA. Based on the data presented here, and the fact that the PCR assay is independent of the host's immune response schedule, much earlier detection is possible. Thus, the test, when used in conjunction with clinical symptoms for Babesiosis could improve patient management.
IGeneX, Inc.
795 San Antonio Rd., Palo Alto, CA 94303 USA
Tel. 650.424.1191 / 800.832.3200 Fax. 650.424.1196