Ante-mortem Diagnosis of Human Rabies Cases Using SYBR Green Real-Time PCR

Rabies, as the most important zoonotic disease, is transmitted through a bite or scratch by an infected domestic or wild carnivores and bats or contact of open wound with infected saliva. The fluorescent antibody test (FAT) is the "gold standard" diagnostic method for suspected brain samples. For close monitoring of unknown encephalitis, rabies surveillance, and also the limitations for post-mortem diagnosis of rabies in human and performing fast prophylactic measures for other individuals in contact with rabid patients, ante-mortem diagnosis based on molecular methods such as real-time polymerase chain reaction (PCR) seems to be more reliable. In this study, we detected 2 positive rabid cases using SYBR Green real-time PCR for the first time in Iran.

In this study, 3 saliva samples at intervals up to 6 hours were collected from any of the nine suspected patients with nonspecific symptoms between March 2016 and March 2017. Total RNA extraction, cDNA synthesis and real-time PCR were performed along with confirmed negative and positive controls. Then, we tracked the patients for follow-up and understanding of their status. On brain samples of patients who died, FAT and MIT (mouse inoculation test) were performed to obtain definitive results.

In this study, the patients were 4 females and 5 males, between 8 and 80 years old from different geographical areas of Iran. The ante-mortem saliva samples of 2 out of nine patients who died were positive by SYBR Green real-time PCR. Positive results of FAT test on these samples confirmed the presence of rabies virus infection in their brains and also the ante-mortem diagnosis results.

The results of this study suggest that SYBR Green real-time PCR technique on saliva sample can be used as an applicable method for ante-mortem diagnosis of rabies to avoid infection of other people such as the treating medical staff or family members of the patient.