BackgroundSevere acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD).MethodsNasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples.ResultsCustom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 and 94.66 – 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more.ConclusionThe custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.

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In the treatment of influenza, Neuraminidase inhibitors (NAIs) (Oseltamivir and Zanamivir) play a major role. The emergence of variants of influenza A (H1N1) pdm09 virus resistant to Oseltamivir is a matter of great concern as it limits its usage. Therefore, vigilant monitoring for Oseltamivir-resistant viruses has been recommended by the World Health Organization (WHO). Our study aimed to screen the influenza A (H1N1) pdm09 virus for NAI drug resistance during the outbreak of 2015-16 in North-Western India. A total of 640 H1N1pdm09 virus-positive samples were screened for drug resistance to Oseltamivir by WHO allelic discrimination real-time RT-PCR protocol. The allelic discrimination PCR protocol can detect the presence of single nucleotide polymorphisms (SNPs), the H275Y mutation is detected by this method which causes resistance to Oseltamivir. Sanger sequencing of partial fragment of NA gene (fragment IV), of 90 samples were performed to confirm the presence of NA-H275Y mutation. Neuraminidase susceptibility of 20 randomly selected isolates to Oseltamivir was tested using NA inhibition chemiluminiscence based assay. Among 640 H1N1pdm09 positive samples tested, H275Y mutation was detected in one sample (0.15%) by PCR and confirmed by Sanger sequencing also. All the 20 isolates tested for NAI susceptibility by NA star assay were found to be sensitive to Oseltamivir. WHO allelic discrimination PCR is an easy, rapid and sensitive method for high-throughput detection of resistance to Oseltamivir. Systematic regular drug resistance surveillance of Influenza A is essential to monitor the emergence and spread of drug-resistant strains.

SETTING: Patients with presumed multidrug-resistant tuberculosis (MDR-TB) and undergoing MDR-TB treatment from Rajasthan, India.OBJECTIVE: To compare the GenoType® MTBDRsl v.1.0 (MTBDRsl) assay capacity to detect resistance to ofloxacin, amikacin, capreomycin, kanamycin and ethambutol in Mycobacterium tuberculosis with phenotypic drug susceptibility testing (DST) using MGIT™960™ in sputum samples and isolates.DESIGN: Fifty-three smear-positive sputum samples were tested directly by MTBDRsl and 205 MDR-TB isolates were processed using MTBDRsl and DST for five drugs on MGIT960. DNA sequencing was performed in isolates with discordance in the results between the two methods for the gyrA, gyrB and rrs genes.RESULT: Sensitivity and specificity of MTBDRsl was found to be respectively 93.1% and 100% for fluoroquinoline, respectively 75–78% and 100% for aminoglycosides/cyclopeptides, respectively 70% and 92% for ethambutol and respectively 92.3% and 100% for extensively drug-resistant (XDR) TB detection. On sequencing eight discordant isolates for quinolones, mutations were seen in 12.5% of the gyrB gene and among 20 discordant isolates for aminoglycosides/cyclopeptides in the rrs gene in 15% isolates. The turnaround time was 2 days for MTBDRsl vs. 10 days for MGIT960.CONCLUSIONS: MTBDRsl can be used as an initial rapid test for detecting XDR-TB, resistance to quinolones and aminogycosides/cyclopeptides in smear-positive sputum samples.

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