Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases.

Purpose: Infl uenza epidemics and periodic pandemics occur worldwide resulting in signifi cant mortality, morbidity and economic loss. There is need for a sensitive, rapid and cost-effective assay to detect, type and sub-type infl uenza viruses, as cell culture has a long turnaround time. Materials and Methods: Nasopharyngeal swabs were collected from patients presenting with infl uenza-like illness (ILI) at AIIMS OPD and Primary Health Centre Ballabhgarh (Haryana). From June 2007 to January 2009 and then from September to November 2009, of 1567 specimens collected, 544 were randomly selected and were tested by virus culture using Madin-Darby Canine Kidney (MDCK) cells and by reverse transcription polymerase chain reaction (RT-PCR) for infl uenza A using primers for matrix gene and for infl uenza B using non-structural gene (NS) primers. All infl uenza A positives were sub-typed using primers for HA and NA genes of A/H1, A/H3. A separate multiplex RT-PCR having primers from matrix and HA genes of pandemic A (H1N1) pdm09 viruses was carried out on samples collected after September 2009. Results: Of the 544 samples, 136 (25%) were positive for infl uenza by RT-PCR. Further typing analysis revealed 86 (63.2%) were typed as infl uenza A and 47 (34.5%) as infl uenza B viruses and 3 (2%) samples showed dual infection with infl uenza A and B. Of the 86 infl uenza A positive samples 48 (55.8%) were identifi ed as seasonal infl uenza A/H1N1, 22 (25.6%) as A (H1N1) pdm09 and 16 (18.6%) as A/H3N2. Comparison of infl uenza positivity using virus culture revealed that only 97/136 (71.3%) were infl uenza positive. Sensitivity of viral detection was lowest for seasonal A/H1 (26/48; 54%), followed by H3N2 (11/16; 68.7%) and infl uenza B (38/47; 80.8%); all infl uenza A/H1N1pdm09 viruses were detected by both methods. Conclusion: RT-PCR is a sensitive, low cost and rapid screening test for diagnosing infl uenza infection during epidemics and pandemics. mRT-PCR increased the detection rates for infl uenza by 28.6% as compared with virus isolation and thus is a useful assay in both diagnostic and epidemiological settings in resource poor countries.

Effect of cholesterol on multiplication, lipid metabolism and lysosomal enzymes of Hartmanella culbertsoni.

Addition of sonicated dispersions of cholesterol to peptone-salt-vitamin medium resulted in the metabolism of the sterol by Hartmanella culbertsoni. Trophozoite multiplication was stimulated at 1–5 mg/litre, but retarded at 10-20 mg/litre. When cholesterol was added to the medium, incorporation of [1,2—14C] –acetate into neutral lipid, phospholipid, non-saponifiable and cholesterol fractions of the amoebae was significantly reduced. Cholesterol ester was detected in the medium but phospholipids were not released. Addition of cholesterol stimulated the activity of lysosomal acid phosphatase, acid deoxyribonuclease and cathepsin Β but did not affect 5'-nucleotidase, adenosine triphosphatase, alkaline phosphatase, glucose-6-phosphatase, succinate dehydrogenase and cytochrome C oxidase.