Mohammad Ridhuan Mohd Ali1, Amira Wahida Mohd Safee2, Nor Hayati Ismail3, Roslinda Abu Sapian4, Hani Mat Hussin3, Nabilah Ismail2, Chan Yean Yean5. 1. Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia; Secretariat National Institutes of Health (NIH), Ministry of Health Malaysia, c/o Institut Pengurusan Kesihatan, Jalan Rumah Sakit Bangsar, 59000 Kuala Lumpur, Malaysia. 2. Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. 3. Kota Bharu Public Health Laboratory, Jalan Kuala Krai, 16010 Kota Bharu, Kelantan, Malaysia. 4. Secretariat National Institutes of Health (NIH), Ministry of Health Malaysia, c/o Institut Pengurusan Kesihatan, Jalan Rumah Sakit Bangsar, 59000 Kuala Lumpur, Malaysia. 5. Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia; Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Electronic address: yeancyn@yahoo.com.
Abstract
BACKGROUND: Early diagnosis of leptospirosis is important for ensuring better clinical management and achieving better outcomes. Currently, serological assays suffer from inconsistent performance and are less useful for early diagnosis of leptospirosis. As an alternative, qPCR is more sensitive, specific and able to detect the presence of leptospiral DNA during the acute phase of the infection. Meanwhile, most molecular assays do not detect the non-pathogenic group of Leptospira, even though these groups may also infect humans, although less frequently and less severely. METHODS: A set of primers and probe targeting rrs genes of 22 Leptospira spp. were designed and evaluated on 31 Leptospira isolates, 41 other organisms and 65 clinical samples from suspected patients. RESULTS: The developed assay was able to detect as low as 20 fg Leptospira DNA per reaction (equivalent to approximately 4 copies) and showed high specificity against the tested leptospiral strains. No cross amplification was observed with the other organisms. During the evaluation of the confirmed clinical specimens, the developed assay was able to correctly identify all positive samples (n = 10/10). One amplification was observed in a negative sample (n = 1/55). The sequencing of the PCR product of the discordant sample revealed that the sequences were similar to those of L. interrogans and L. kirschneri. CONCLUSION: The findings suggest that the developed Taqman qPCR assay is sensitive, specific and has potential to be applied in a larger subsequent study.
BACKGROUND: Early diagnosis of leptospirosis is important for ensuring better clinical management and achieving better outcomes. Currently, serological assays suffer from inconsistent performance and are less useful for early diagnosis of leptospirosis. As an alternative, qPCR is more sensitive, specific and able to detect the presence of leptospiral DNA during the acute phase of the infection. Meanwhile, most molecular assays do not detect the non-pathogenic group of Leptospira, even though these groups may also infect humans, although less frequently and less severely. METHODS: A set of primers and probe targeting rrs genes of 22 Leptospira spp. were designed and evaluated on 31 Leptospira isolates, 41 other organisms and 65 clinical samples from suspected patients. RESULTS: The developed assay was able to detect as low as 20 fg Leptospira DNA per reaction (equivalent to approximately 4 copies) and showed high specificity against the tested leptospiral strains. No cross amplification was observed with the other organisms. During the evaluation of the confirmed clinical specimens, the developed assay was able to correctly identify all positive samples (n = 10/10). One amplification was observed in a negative sample (n = 1/55). The sequencing of the PCR product of the discordant sample revealed that the sequences were similar to those of L. interrogans and L. kirschneri. CONCLUSION: The findings suggest that the developed Taqman qPCR assay is sensitive, specific and has potential to be applied in a larger subsequent study.