Detecting Disease Carrying Ticks with a New Microbiological rapidSTRIPE Test System

By: Manuela Fabienke, Microbiology Focus Edition 1.1

By Manuela Fabienke, Scanbec …. m.fabienke@scanbec.de

Disease-carrying ticks are becoming more common as global climate change alters our weather; therefore, there is greater concern about the increased potential for parasitic diseases infecting humans after bites from these organisms. But today, detection systems are available for the rapid identification of disease-causing microorganisms found in ticks.

Scanbec GmbH is developing and producing rapid innovative molecular test systems for the detection of microorganisms. In 2003, they developed a test system called HybriScan ®, which is now distributed by Sigma- Aldrich. Applications for the HybriScan system are in the food and beverage industry, water analysis, and medical diagnostics. The adaptability of the HybriScan technology makes possible the development of beneficial universal methods and is a good base for developing new testing systems. Scanbec, an accredited laboratory, also provides micro- and molecular-biological services.

Due to increasing temperatures and changing climatic conditions, ticks have become more and more dangerous. These organisms are able to transmit parasitic diseases, the most prominently known of which is called Borreliosis or Lyme disease.

Removing and then analyzing the tick itself, rather than testing the human who has been bitten, for evidence of pathogens is of extraordinary value to both the attending doctor and the concerned person. Everyone receives fast and reliable results, allaying fears about infection faster than ever before. Scanbec uses a new rapid test system called rapidSTRIPE assay that is able to detect and identify borreliosis (Lyme disease) pathogens in ticks within two hours; the test is conducted in collaboration with the company Analytik Jena AG bio solutions.

There has been a proven increase in tick populations within the last few years; thus the risk of being bitten by a tick has drastically increased as well. Before the new system, people who were bitten by ticks had to wait four to six weeks after being bitten to be tested; the waiting time served to allow the person’s immune system time to produce antibodies against the pathogens, antibodies that would then be detected using common blood tests. The direct detection of pathogens in the tick enables diagnosis and treatment of infection in its earliest stages.

Using this new test method, the removed tick can be fixed on a strip of tape and sent to Scanbec’s laboratory by regular mail.

The detection of Borrelia that are transmitted via ticks occurs by a direct amplification of target DNA isolated from the tick sample. The analysis is performed in three steps: First, the tick is mechanically destroyed in order to isolate total DNA from the tick tissue (rapidSTRIPE Tick DNA Exytraction Kit; Analytik Jena). Then the Borrelia DNA is specifically amplified using rapid PCR (SpeedCycler; Analytik Jena). During PCR, Borrelia-specific primers are used to amplify the pathogen’s DNA, detecting and amplifying all different Borrelia species. The last step entails the detection of the amplified Borrelia DNA using a stripe assay (Lateral Flow Assay; Analytik Jena). The amplified Borrelia DNA is applied to the test strip and the results become visible after a few minutes. Two violet lines indicate the presence of Borrelia species bacteria in the tick. One violet line indicates that the tick was free of all Borrelia species (Figure 1).

Figure 1.Borrelia lateral flow assay (Analytik Jena)

The accuracy of the rapid tick test for the detection and identification of Borrelia species was validated by the company Analytik Jena AG. Four hundred ticks were tested, and all positive results were then sequenced to verify the obtained results and to classify the detected Borrelia species. The sample pool of 400 included ticks of the following types: Ixodes ricinus, Dermacentor reticulatus, Ixodes hexagonus, and Haemaphysalis concinna.

After the detection of Borrelia in a tick, the identification stage of the test system begins and the particular species of Borrelia is determined by sequencing. With these results, the attending physician can predict which symptoms are likely to manifest in the patient. For example, B. garinii mediates neuroborreliosis (a disorder of the central nervous system), and B. afzelii is known to mediate Erythema migrans (causes areas of reddened skin 5 to 6.8 cm in diameter). As treatment of borreliosis is complicated and difficult, knowledge of which Borrelia species has infected the patient can be of great help.

It is important to note that a positive result for the presence and identification of Borrelia species is only the first part of a larger process to deal with the infection. Scanbec recommends in all cases of positive Borrelia test results that the person in question follow up promptly with a doctor for examination and further testing, including early-stage blood or serum sample collection to monitor the patient’s serological processes (Borrelia antibody status), to see whether symptoms of borreliosis emerge and to ensure effective treatment with the appropriate antibiotics.

Scanbec also offers test systems (Analytik Jena AG) for the detection and identification of pathogenic Rickettsia, Babesia, and Anaplasma species in ticks. These tests work on the same principle as the Borrelia test system, differing only in the species-specific PCR primers used for rapidPCR (SpeedCycler; Analytik Jena) amplification of the DNA of the respective microorganisms.

Like Borrelia, all three of these pathogens can be transmitted by ticks to humans. A growing tick population, caused by global climate change, increases the likelihood of tick-borne infections like borreliosis/Lyme disease, tick-borne meningoencephalitis (TBEVirus), and rickettsiosis in humans.

Rickettsia species are found in the intestine and intestinal epithelia of arthropods like ticks. With this disease, detection during the early stages of infection means an easy treatment with antibiotics, thus the new rapid test time is key to a better outcome for the patient.

A recent survey on the proliferation of pathogens in ticks revealed that Rickettsia species are occurring with unexpected frequency in ticks. Rickettsia are able to survive only in endothelial cells; therefore, traditional testing for these bacteria involved proliferation by means of cell culture, which is highly time- and costintensive. Further, reliable confirmation of the presence of Rickettsia is only possible by nucleic acid based detection by PCR. This new system provides a faster and more cost-effective alternative to these traditional tests.

The new tick test for detection of Rickettsia was also validated by Analytik Jena AG. Again, a sample pool of 400 ticks was examined for the occurrence of Rickettsia to evaluate the specificity and accuracy of Rickettsia detection results.

Rickettsia positive samples were sequenced to verify the obtained results and to classify the detected Rickettsia species. The following tick species were examined: Ixodes ricinus, Dermacentor reticulatus, Ixodes hexagonus, and Haemaphysalis concinna.

Ticks are also able to transmit animal pathogens like Babesia bovis and Babesia bigemenia (tick fever or cattle fever). Both Babesia species are animal pathogens that usually affect mice, cattle, horses, and dogs; in exceptional cases, however, humans can be infected after being bitten by a tick carrying the protozoa. Babesia divergens and Babesia microti are also widespread through Europe and North America and can cause influenza-like symptoms such as fever, ague, arthralgia, thrombopenia, haemolytic anaemia, and haemoglobinuria in humans.

Ticks feeding on infected animals absorb the Babesia with erythrocytes. Within the intestine of the tick, the Babesia bacteria are released from the erythrocytes and start to proliferate. The Babesia then divide into thousands of sporozoites, the infectious stage of the parasite, in the saliva of the tick and are transmitted to a new host when the tick moves on.

Anaplasma phagocytophila is able to proliferate in leukocytes, especially in granulocytes. Transmission of anaplasmosis occurs, according to the pertinent literature, mainly via ticks. Anaplasma phagocytophila (formally known as Ehrlichia phagocytophila) causes human granulozytic anaplasmosis (HGA) and is prominent in southern and eastern Europe. Humans infected with anaplasmosis exhibit symptoms like fever, head and limb pain, gastrointestinal and pulmonary symptoms, and exanthema.

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