Diagnosis of rabies by polymerase chain reaction with nested primers.

A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for detection of rabies virus is described. The process consists of sample preparation, reverse transcription, two-step DNA amplification, and detection of the amplified product. RNA was extracted from animal and human brain by phenol-chloroform using guanidinium thiocyanate. Viral RNA was then amplified in a two-step PCR that used two sets of nested primers designed to amplify rabies nucleocapsid (N) sequence. Rabies nucleocapsid sequence was amplified from all brain samples from 95 dogs and 3 humans with rabies confirmed by fluorescent antibody (FAT) and mouse inoculation tests (MIT). Rabies-negative brain samples (110 dogs, 2 humans) were PCR-negative. The process requires < 24 h. Detection of viral RNA was still possible in brain material that was left at room temperature for 72 h. As little as 8 pg of rabies virus RNA could be detected. This technique could have practical applications as a confirmatory test to FAT at busy rabies diagnostic centers.

Regional distribution of rabies viral antigen in central nervous system of human encephalitic and paralytic rabies.

We studied the distribution of rabies viral antigen in the brain and spinal cord of 7 patients with rabies by immunohistochemical techniques. Four patients presented with encephalitis, the remaining 3 had paralysis. Neither the rabies viral antigen distribution nor inflammation paralleled clinical presentations. Patients who had survival times of 7 days or less (4/7) had a greater amount of antigen-positive neurons in brainstem and spinal cord regardless of the clinical type. Neuroglial cells were also found to contain rabies antigen. Our findings suggest that virus localization may not account for the difference in clinical manifestations.

Autoantibodies in falciparum malaria: a sequential study in 183 Thai patients.

Various autoantibodies were sequentially studied in 183 consecutive Thai adults infected with Plasmodium falciparum. On the first day of admission, 31.1% of the patients had positive fluorescent anti-nuclear antibodies (FANA) and 16.9% had positive smooth muscle antibodies (SMA). The incidences of positive FANA and SMA rose progressively with times when the patients returned for the 2 and 4 week follow-ups after discharge, although most of their malaria was cured. The majority of the positive FANA and SMA titres lay between 1:20 and 1:160. The positivity of the FANA and SMA did not correlate with the complications of malaria, nor the initial serum IgG or IgA levels. However, they significantly correlated with the initial hyper-IgM. More interestingly, almost all of the positive FANA were of the speckled type of nuclear staining. The antigen specificity of the speckled FANA were found not to be the double stranded DNA or the extractable nuclear antigens. The polyclonal B cell activation in active malarial infection was postulated.