Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 1, 2011, pp. 37-41

Indian Journal of Medical Microbiology, Vol. 29, No. 1, January-March, 2011, pp. 37-41

Original Article

Pandemic Influenza A (H1N1) 2009 in India: Duration of virus shedding in patients under antiviral treatment

I Gandhoke, DS Rawat, A Rai, S Khare, RL Ichhpujani

Head of Division of Microbiology, National Centre for Disease Control, (NCDC), Government of India, DGHS, Ministry of Health and Family Welfare, 22-Shamnath Marg, Delhi - 110 054, India

Correspondence Address: S Khare, Head of Division of Microbiology, National Centre for Disease Control, (NCDC), Government of India, DGHS, Ministry of Health and Family Welfare, 22-Shamnath Marg, Delhi - 110 054, India shashikhare@hotmail.com

Date of Submission: 21-May-2009
Date of Acceptance: 30-Nov-2010

Code Number: mb11009

PMID: 21304193

DOI: 10.4103/0255-0857.76522

Abstract

Keywords: Influenza pandemic, Oseltamivir, real-time polymerase chain reaction, virus shedding

Introduction

Influenza A viruses are important viral pathogens causing significant mortality and morbidity throughout the world. Annual influenza epidemics are estimated to affect 5-15% of the global population. Although most cases are mild, this still causes severe illness in 3-5 million people and around 250,000 to 500,000 deaths worldwide. In industrialised countries, severe illness and death occurs mainly in high-risk populations of infants, the elderly and chronically ill patients. In addition to these annual epidemics, Influenza A virus strains caused three major pandemics during the 20th century: the Spanish flu in 1918, Asian flu in 1957 and Hong Kong flu in 1968-1969. These pandemics were caused by strains of Influenza A virus that had undergone major genetic changes and for which the population did not possess significant immunity. ^[1],[2]

The new strain of Influenza A subtype H1N1 which is thought to be a reassortment of four known strains endemic in human, birds and pigs, declared by World Health Organization (WHO) as 2009 pandemic influenza virus, has established itself as a dominant influenza strain and is causing outbreaks in most parts of the world. Transmission of the new Influenza A strain is from human to human, and the virus spreads typically via coughing, sneezing or touching contaminated surfaces and then touching the nose or mouth. Symptoms which could last up to a week are similar to those of seasonal flu, and may include fever, sneezes, sore throat, cough, running nose, difficulty in breathing, headache and muscle or joint pain. The virus continues to move through susceptible population, indicating that the pandemic is here to stay for many more months to come. The clinical picture of this pandemic influenza has been reported to be consistent across all countries. The majority of patients continue to experience mild to moderate illness with self-recovery or resolving infection with antiviral therapy. The virus can cause very severe and fatal illness in young and healthy people, though the numbers of such cases remain small. Most ICU hospitalisation and deaths have been of persons, having underlying conditions like asthma, diabetes, heart disease or weakened immune system. ^[3],[4],[5]

The screening of the pandemic Influenza A (H1N1) virus in overseas travellers with influenza like illness started in India since the last week of April 2009, following the guidelines set by Centre for Disease Control and Prevention. ^[6] The first confirmed case of Influenza A (H1N1) virus was detected on 14 May 2009, and within a month, the spread of virus was like wildfire. Till 31 August, 8531 patients had been tested and 1186 confirmed cases had been detected. In accordance with the estimated period of infectivity with Influenza A virus, the current Centre for Disease Control and Prevention infection control guidelines for the prevention and control of influenza advocate that patients hospitalised with suspected or confirmed influenza be placed under standard isolation precautions and droplet isolation for 5 days on exhibiting symptoms suggesting the onset of illness. ^[7]

This study was undertaken at the institute to see the period of infectivity in the patients who contracted the current pandemic Influenza A (H1N1) infection in India. Around 150 laboratory confirmed cases of different age groups were included in the study. Additional samples, daily, were requested from these patients after antiviral therapy was started, till they showed no virus on repeat testing of positive patients with real-time reverse transcription-polymerase chain reaction (real-time RT-PCR).

Materials and Methods

Throat swabs and nasal/nasopharyngeal swabs collected by clinicians in virus transport medium (VTM) from patients exhibiting influenza like symptoms were received at the designated reference laboratory in triple packaging cold conditions from all over the country. Samples were accompanied by duly filled performa with demographic characteristics, date of symptom onset, co-morbidities, travel/contact history, antiviral treatment, etc. Initially, all samples were collected before administration of antiviral therapy. All the respiratory specimens were processed in BSL-3 lab within 2-3 hours of receipt of the samples.

RNA isolation was done in these samples using QIAmp^® Viral RNA Mini Kit or RNeasy^® Mini Kit from Qiagen, USA. Reverse transcription and amplification of the target genes was carried out by real-time RT-PCR using CDC real-time RT-PCR protocol for detection and characterisation of Swine Influenza (version 2009). ^[8] Primers and dual labelled probes were procured from CDC and SuperScript^TM III Platinum^® one-step quantitative RT-PCR kit from Invitrogen, California, USA, was used. One-step quantitative RT-PCR probe hydrolysis (TaqMan-ABI) package kit with Agpath-ID^TM and one-step RT-PCR kit supplied by Applied Biosystems-Ambion, USA, were also used later. In each sample, four target genes were amplified: Influenza A, Swine Influenza A, Swine H1 and RNaseP. A sample was declared positive when it showed amplification in all four target genes. One hundred fifty positive patients belonging to different age groups were selected for the study. Repeat samples for these patients under antiviral treatment, as per the guidelines laid down by the Government of India, were collected and tested everyday till no amplification for three specific target genes of the virus was seen on two consecutive days.

Results

One thousand one hundred eighty-six confirmed cases of Influenza A (H1N1) were detected out of 8531 tested cases till August 2009. The main clinical features manifested by the positive patients were fever, sore throat and cough. Some of the patients (5%) had pneumonia and breathlessness. Age and gender wise analysis of positive cases showed that infection was more common in patients of age group 1-30 years. Out of total 150 positive patients selected for the study, 74% (111/150) were in the age group of 1-30 years, 17.33% (26/150) were in 31-45 years age group and 8.67% (13/150) were in >45 years age group. Male patients (55.33%) numbered slightly more than female patients (44.67%) [Table - 1]. Most of the patients [(149/150) 99.33%] were without any major underlying health condition and only one patient (0.67%) was diabetic and asthmatic. All these patients had been quarantined and were being treated with antiviral Oseltamivir (Roche, USA). Ten patients (6.67%) turned negative after 3 days of antiviral treatment, 20 patients (13.33%) turned negative on the 4th day, 65 (43.33%) turned negative on the 5th day, 8 (5.33%) on the 6th day, 26 (17.33%) on the 7th day, 7 (4.67%) on the 8th day, 8 (5.33%) on 9th day, and 5 (3.33%) cases turned negative on 10th day. One patient who was diabetic and had asthma showed prolonged viral shedding that lasted till 18 days and was being treated in the ICU of a government hospital.

The infection resolved completely within 3-10 days in all the patients except one. In 63.33% (95/150) of the cases, the infectivity period was up to 5 days, and in 36% it was from 6 to 10 days. Mean duration for viral shedding was found to be 6.5 days and median period for viral shedding was 7 days. Maximum number of patients 72.55% (37/51) from the age group of 18-30 years showed infectivity period up to 5 days, while the least number of patients [38.46% (5/13)] recovered in 5 days in the age group >45 years [Table - 1]. [Figure - 1],[Figure - 2],[Figure - 3] show the amplification in all the three specific targets of Influenza A (H1N1) virus on 7 consecutive days.

Discussion

Outbreaks and spread of pandemic Influenza A (H1N1) virus are inevitable. Till 31 August, 1186 positive cases were detected at the designated reference lab. All the patients enrolled under the study were symptomatic and confirmed positive for Influenza A (H1N1) by real-time RT-PCR and were undergoing antiviral treatment. In our study, we have found that nearly 80% of the patients showed relief in symptoms like decreasing body temperature, relief in body aches, uneasiness, etc., within 2 days of treatment, though actual virus shedding was observed from 3 to 10 days, as demonstrated by positive results of RT-PCR. Also, 63.33% (95/150) Influenza A (H1N1) positive cases turned negative up to the 5th day, 80% Influenza A (H1N1) positive cases turned negative up to the 7th day [Figure - 1],[Figure - 2],[Figure - 3] but significant number of patients [14% (21/150)] remained infective up to the 10th day, indicating that isolation for 5 days was not sufficient, as currently recommended by the Centre for Disease Control and Prevention to control influenza in acute settings. Our findings are in accordance with some other reports. ^{[9],[10],[11]} Fast recovery was seen in the age group 18-30 years and recovery was least in the age group >45 years. Although prolonged virus shedding up to 18 days [Figure - 4],[Figure - 5],[Figure - 6][Figure - 7] was observed in a 66-year-old woman with the underlying health condition, finally she recovered fully with the Oseltamivir treatment, showing no drug resistance. Prolonged virus shedding in patients under antiviral treatment has been found to be associated with drug resistant virus; that is, during the course of treatment the patients stop responding to antiviral to which they were responding earlier and the symptoms of illness start reappearing. ^[12],[13] It is also recommended that contact between patients taking antiviral and people not taking the drug should be minimized.

Real-time RT-PCR is a very sensitive technique for detecting the Influenza A (H1N1) infection but it can detect inactive virus also sometimes. In our study, the patient was declared negative when on two consecutive days his/her sample showed no amplification. However, in the present study, we could not include patients who were not administered antiviral therapy as some studies have shown a significant reduction in the duration of virus shedding with neuraminidase inhibitor treatment. ^[14] Everyone who had contracted Influenza A (H1N1) infection was treated with Oseltamivir in India to limit the spread of infection.

Around 80% Influenza A (H1N1) positive cases turned negative up to the 7th day and almost all (~99%) turned negative by the 10th day, except occasional cases.

Acknowledgement

Support from the Centre for Disease Control and Prevention is duly acknowledged for supplying reagents for testing. We thank for the support of hospitals from all states of India for sending samples and technical support from staff of Microbiology and Biochemistry & Biotechnology is acknowledged.

References

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