Molecular characterisation of rickettsial diseases in adults presenting to Teluk Intan Hospital / Sa’adiah Abdul Malek

Rickettsioses are emerging infectious diseases caused by Rickettsia species, a genus of obligate intracellular gram-negative bacteria found in various arthropods vectors such as ticks, lice, fleas, and chiggers. The three main diseases caused by Rickettsia species are scrub typhus (e.g. Orientia tsutsugamushi, Orientia chuto), murine typhus (e.g. Rickettsia typhi), and spotted fever (e.g. Rickettsia conorii, Rickettsia helvetica). Rickettsioses is one of the most common agriculture and animal farming-associated zoonotic diseases, and it is potentially fatal if left unrecognised and untreated. Infections in humans are manifested as acute undifferentiated febrile illnesses, headaches and rashes following the bites from various arthropods vectors. The disease has often been overlooked due to its similar symptoms to other vector-borne diseases such as dengue fever, leptospirosis, and malaria. Misdiagnosis and improper antibiotic therapy may result in severe complications and even death if not addressed promptly. The exact burden of rickettsial diseases in Malaysia is limited due to the disease being underdiagnosed. Serology methods such as indirect immunoperoxidase and indirect hemagglutination tests are commonly used in the diagnosis of the infection. However, these techniques are laborious and require reference serum and antigens. Moreover, it can only detect a limited number of rickettsia species and strains. Hence, the molecular technique is the method of choice for studying rickettsia's genetic diversity as it can detect the causative agents at the genomic level. Therefore, this study aimed to identify and characterise the rickettsia species among adults presented to Teluk Intan Hospital by polymerase chain reaction and DNA sequencing. Briefly synthetic DNA of the 56-kDa type-specific antigen (TSA), the 17-kDa, and the OmpB, were utilised as templates and positive controls in the validation of PCR for scrub typhus, murine typhus, and spotted fever, respectively, using published primers. The PCR product was visualised on a 2% agarose gel. A total of 141 blood samples that fulfilled the inclusion and exclusion criteria were collected from adults presented to Teluk Intan Hospital, Perak, Malaysia, from November 2019 to November 2020. Only one sample was detected positive for scrub typhus and sequence-verified to be Orientia tsutsugamushi strain UT302, which was initially isolated from Thailand. The experimental findings have demonstrated that the optimised PCR methods are rapid, straightforward, precise, and reproducible. The number of positive cases was lower than anticipated; this was most likely due to the hospital being gazetted as a COVID-19 hospital during the global COVID-19 pandemic in 2010. This has greatly affected patient recruitment as the number of patients visiting the hospital has been reduced. In conclusion, from this study, we have detected and identified an Orientia tsutsugamushi strain TA 763 from a hospitalised patient at Teluk Intan Hospital using the molecular technique. This finding is important as the identified strain has not been previously discovered in Malaysia, hence it is not part of the antigen control used in the serology test performed in Malaysian government hospitals. This could be one of the reasons that cause the underdiagnosis of rickettsia in the country. The findings of this study will be useful for future rickettsia epidemiological studies and improvement of the diagnostic methods.