High Resolution Melting Curve Analysis for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Clinical Isolates

Background: Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, however, its susceptibility is not routinely tested. High-resolution melting (HRM) curve analysis has been widely used for many applications. In this study, HRM assay was developed and evaluated for the detection of PZA resistance in Mycobacterium tuberculosis clinical isolates. Methods: Ninety five M. tuberculosis clinical isolates with different susceptibility patterns to anti-TB drugs were used to evaluate this assay. Isolates were phenotypically (Bactec MGIT 960) and genotypically (HRM and pncA gene sequencing) analysed for PZA resistance. Results: Bactec MGIT 960 analysis revealed that 29 of the 95 M. tuberculosis isolates were PZA resistant. In comparison to the Bactec MGIT 960, HRM showed a sensitivity of 47.7% and specificity of 74.6%, and the overall agreement between the two methods was 68.4%. Based on DNA sequencing, a correlation of 0.67 (significant at p-value pncA mutations was observed. PZA resistance was strongly associated with multi-drug resistant (MDR)-TB as it was shown in 79.3% of the MDR isolates included in the study. Conclusion: HRM is simple and useful for screening clinical M. tuberculosis isolates for PZA resistance, however, further modifications to improve its performance are required.

Zoonotic Pathogens Detected in Ticks in Kenyan Game Reserves

Little is known on tick-borne pathogens and their role in disease in game reserves in Kenya. Ticks were collected by sterile forceps from restrained cattle hide and placed into labeled falcon tubes. Ticks were screened for pathogens by High Resolution Melting (HRM) analysis and sequencing of specific RT-PCR products of Anaplasma, Ehrlichia, and Rickettsia species. A total of 317 ticks (281 adult ticks and 36 nymphs) comprising seven species were collected around the Tsavo National Reserve (TNR) in Taita Taveta County with Amblyomma gemma being the most commonly collected species (n = 135, 42.6%). From near Shimba Hill game reserve (SHNR), a total of 240 adult’s ticks were sampled, representing eight species, with again Amblyomma gemma being the most sampled species (n = 156, 65%). From Tsavo, a total of three pools of Rhipicephalus appendiculatus were positive for Theileria parva, two pools of Rhipicephaline evertsi for Anaplasma platys and one pool of Amblyomma variegatum nymphs for Rickettsia africae. Rickettsia africae, which causes African tick-bite fever, was detected in two pools of Am. variegatum and one pool of Amblyomma gemma collected near Shimba Hill game reserve. Rickettsia sp. and Anaplasma sp. were detected in Am. gemma and Rh. evertsi respectively. Rickettsia aeschlimannii was detected in a pool of Am. gemma. These findings highlight the risk of transmission of zoonotic pathogens to humans in regions with high human-wildlife interfaces. Of specific importance, we provide evidence of R. aeschlimannii in A. gemma for the first time, representing a potential new R. aeschlimannii vectors.