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Challenging TaqMan probe-based real-time PCR and loop-mediated isothermal amplification (LAMP): the two sensitive molecular techniques for the detection of toxoplasmosis, a potentially dangerous opportunistic infection in immunocompromised patients

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Abstract

Due to defects and drawbacks of most conventional diagnostic methods including serology for the diagnosis of toxoplasmosis as a dangerous opportunistic infection in immunocompromised individuals, the accurate, rapid, and sensitive detection of infection in such patients is essential. In this study, the TaqMan probe-based real-time PCR and, a relatively new nucleic acid amplification method, the loop-mediated isothermal amplification (LAMP) technique was compared based on the repetitive elements (RE) sequence to detect Toxoplasma gondii (T. gondii) DNA in blood samples of immunocompromised individuals. During this study, 119 blood samples from immunocompromised cancer patients with renal failure, undergoing dialysis were studied. After DNA extraction from blood samples using the salt extraction method, the molecular techniques of TaqMan probe-based real-time PCR and LAMP were used to investigate the contamination of the samples with T. gondii, based on the 529 bp (RE) sequence of T. gondii. The analytical sensitivity of LAMP and real-time PCR was evaluated by duplicating the five-step serial dilutions of T. gondii tachyzoites from 0.25 to 5×105 spiked tachyzoites per milliliter of the Toxoplasma seronegative blood sample. The extracted DNA from other parasites and human chromosomal DNA were used to determine the specificity of the molecular methods. The obtained results were analyzed using Kappa statistical test and SPSS22 software. Out of 119 studied samples, 7 (5.8%) and 5 (4.2%) samples were positive for Toxoplasma by TaqMan probe-based real-time PCR and LAMP, respectively. The limits of detection of TaqMan probe-based real-time PCR and RE-LAMP in negative serum samples were one and five tachyzoites (CT 38), respectively. Both real-time PCR and LAMP methods were 100% specific for Toxoplasma detection. Positive results were obtained only with T. gondii DNA, while other DNA samples were negative. The TaqMan probe-based real-time PCR based on the RE sequence showed higher sensitivity to T. gondii DNA detection in blood samples of cancer patients and serial dilutions of parasitic tachyzoites. The results show that TaqMan probe-based real-time PRC is a sensitive and specific method for the detection of toxoplasmosis in immunocompromised individuals, as well as the LAMP assay, which can be used as a suitable alternative diagnostic method for the detection of toxoplasmosis in such patients, without need the for any expensive equipment.

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Funding

The funds for this study has been provided by the Vice-Chancellor for Research and Technology of Guilan University of Medical Sciences, Rasht, Iran.

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Authors and Affiliations

  1. Department of Pharmacology, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran

    Bahram Soltani Tehrani & Mansureh Safari Kavishahi

  2. Department of Biochemistry and Biophysics, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran

    Ebrahim Mirzajani

  3. Hepatitis Research Center, Lorestan University of Medical Science, Khorramabad, Iran

    Shirzad Fallahi

  4. Department of Parasitology and Mycology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran

    Shirzad Fallahi

  5. Department of Medical Parasitology and Mycology, Faculty of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran

    Kourosh Manouchehri Naeini

  6. Research Center for Fascioliasis and Parasitic Diseases, Guilan University of Medical Sciences, Rasht, Iran

    Mohammad Reza Mahmoudi

  7. Cellular and Molecular Research Center, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran

    Bahram Soltani Tehrani, Ebrahim Mirzajani, Vajiheh Eskandari & Nozhat Zebardast

Authors
  1. Bahram Soltani Tehrani
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  2. Ebrahim Mirzajani
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  3. Shirzad Fallahi
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  4. Kourosh Manouchehri Naeini
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  5. Mohammad Reza Mahmoudi
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  6. Mansureh Safari Kavishahi
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  7. Vajiheh Eskandari
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  8. Nozhat Zebardast
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Correspondence to Nozhat Zebardast.

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Communicated by Erko Stackebrandt.

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Soltani Tehrani, B., Mirzajani, E., Fallahi, S. et al. Challenging TaqMan probe-based real-time PCR and loop-mediated isothermal amplification (LAMP): the two sensitive molecular techniques for the detection of toxoplasmosis, a potentially dangerous opportunistic infection in immunocompromised patients. Arch Microbiol 202, 1881–1888 (2020). https://doi.org/10.1007/s00203-020-01903-1

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  • DOI: https://doi.org/10.1007/s00203-020-01903-1

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