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

PURPOSE: Influenza epidemics and periodic pandemics occur worldwide resulting in significant mortality, morbidity and economic loss. There is need for a sensitive, rapid and cost-effective assay to detect, type and sub-type influenza viruses, as cell culture has a long turnaround time. MATERIALS AND METHODS: Nasopharyngeal swabs were collected from patients presenting with influenza-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 influenza A using primers for matrix gene and for influenza B using non-structural gene (NS) primers. All influenza 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 influenza by RT-PCR. Further typing analysis revealed 86 (63.2%) were typed as influenza A and 47 (34.5%) as influenza B viruses and 3 (2%) samples showed dual infection with influenza A and B. Of the 86 influenza A positive samples 48 (55.8%) were identified as seasonal influenza A/H1N1, 22 (25.6%) as A (H1N1) pdm09 and 16 (18.6%) as A/H3N2. Comparison of influenza positivity using virus culture revealed that only 97/136 (71.3%) were influenza positive. Sensitivity of viral detection was lowest for seasonal A/H1 (26/48; 54%), followed by H3N2 (11/16; 68.7%) and influenza B (38/47; 80.8%); all influenza A/H1N1pdm09 viruses were detected by both methods. CONCLUSION: RT-PCR is a sensitive, low cost and rapid screening test for diagnosing influenza infection during epidemics and pandemics. mRT-PCR increased the detection rates for influenza by 28.6% as compared with virus isolation and thus is a useful assay in both diagnostic and epidemiological settings in resource poor countries.

Induction of reversible antifertility with a crude ethanol extract of Citrullus colocynthis Schrad fruit in male rats.

A crude 50% ethanol extract of Citrullus Colocynthis Schrad was administered orally to male albino rats for evaluation of antifertility effects. The animals were divided into five groups: group A was a vehicle-treated control group; treatment groups B, C, and D received 100 mg/kg/day C. Colocynthis extract for periods of 20, 40, and 60 days, respectively, and group E animals received the extract at 100 mg/kg/day for 60 days followed by 60 days of recovery. For androgenicity evaluation of the extract, the animals were divided into four groups: group F animals were castrated 30 days before the experiment to serve as controls, and group G, H, and I were subjected to castration 30 days before the experiments, followed by administration of fruit extract (100 mg/kg/day p.o.), testosterone propionate (0.01 mg/rat/alternate day s.c.), and fruit extract along with testosterone propionate, respectively, for 30 days. Significantly reduced cauda epididymis sperm motility and density, number of pups, fertility, and circulatory levels of testosterone were observed in all treatment groups. The weights of testes, epididymis, seminal vesicle, and prostate were significantly decreased in groups B, C, and D. The weights of all organs in the different groups of the androgenicity study were markedly decreased in group F when compared with group A, in group G when compared with group F, and in group I when compared with group H, and increased in group H when compared with group F. The serum testosterone levels also showed a similar pattern. The concentration of testicular cholesterol was significantly elevated, while protein, sialic acid, acid and alkaline phosphatase concentrations were decreased. The histoarchitecture of the testes showed degenerative changes in the seminiferous epithelium, arrest of spermatogenesis at the secondary spermatocyte stage, cytolysis, and the lumen filled with eosinophilic material. Histometric parameters except Sertoli cell nuclear area and number of round spermatids showed marked alterations. All altered parameters restored to normal in group E. No changes were observed in body weight, litter size, hematology, and serum biochemistry. In conclusion, a 50% ethanol extract of C. Colocynthis showed an antiandrogenic nature, thereby reduced reversible infertility in male albino rats.

Antifertility effect of chronically administered Martynia annua root extract on male rats.

The effects of 50% ethanol extract of Martynia annua L. root on reproduction was studied on male rats. The study was divided into four groups of five animals each. The first group (I) received vehicle alone to serve as control. The second, third and fourth groups (II, III and IV) of animals were administered the root extract daily at 50 mg/kg body weight, po, 100 mg/kg body weight, po, and 200 mg/kg body weight, po, respectively, for a period of 60 days. Significant decreases in the weights of testes, epididymides, seminal vesicle and ventral prostate were observed. A dose related reduction in the testicular sperm count, epididymal sperm count and motility, number of fertile males, ratio between delivered and inseminated females and number of pups were observed. The testis showed a clear correlation between the dose and severity of lesions of seminiferous epithelium. In general, the seminiferous tubules appear reduced in size with a frequently filled eosinophilic material. Spermatogenesis arrested at the secondary spermatocyte stage. Pachytene spermatocytes were undergoing degeneration. Disorganisation and sloughing of immature germ cells were visible. Leydig cells were atrophied. No morphological changes were observed in Sertoli cells. Significant reduction in serum concentration of luteinising hormone and testosterone were observed. No distinct change in serum FSII concentration was recorded. The final body weights of all groups were elevated markedly. No alterations were recorded in any hematological parameters. It is concluded that the 50% ethanol extract of M. annua root produced dose related effects on male reproduction without altering general body metabolism.