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Post-pandemic influenza A (H1N1) virus detection by real-time PCR and virus isolation
Korean J. Microbiol 2019;55(1):25-32
Published online March 31, 2019
© 2019 The Microbiological Society of Korea.

Ali Mohamed Zaki1, Shereen El-Sayed Taha1,*, Nancy Mohamed Abu Shady2, Asmaa Saber Abdel-Rehim3, and Hedya Said Mohammed4

1Medical Microbiology and Immunology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
3Internal Medicine Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
4Chest Diseases Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Correspondence to: E-mail: dr_shereentaha@med.asu.edu.eg;
Tel.: +20-1119997002
Received December 17, 2018; Revised December 28, 2018; Accepted December 28, 2018.
Abstract

Influenza A (H1N1) virus caused a worldwide pandemic in 2009–2010 and still remains in seasonal circulation. Continuous surveillance activities are encouraged in the post pandemic phase to watch over the trend of occurrence every year, this is better to be done by a rapid and sensitive method for its detection. This study was conducted to detect proportions of occurrence of influenza A virus (H1N1) in patients with influenza-like illness. Samples from 500 patients with influenza or influenza-like clinical presentation were tested by real-time reverse transcription polymerase chain reaction (RT-PCR) and virus tissue culture. Among the total 500 participants, 193 (38.6%) were females and 307 (61.4%) males. Seventy-one patients (14.2%) were positive for H1N1 virus infection with real-time RT-PCR while 52 (10.4%) were positive by tissue culture. Non-statistically significant relation was found between age and gender with the positivity of H1N1. Sensitivity and specificity of real-time RT-PCR was 98.08% and 95.54%, respectively, in comparison to virus isolation with accuracy 95.8%. This study showed that H1N1 virus was responsible for a good proportion of influenza during the post-pandemic period. Real-time RT-PCR provides rapidity and sensitivity for the detection of influenza A virus (H1N1) compared with virus isolation and thus it is recommended as a diagnostic tool.

Keywords : influenza A virus, H1N1, post pandemic, real-time RT-PCR and tissue culture, seasonal influenza
Body

Influenza is a respiratory illness caused by viruses belonging to the family Orthomyxoviridae. Four influenza virus genera are included in this family (influenza virus A, influenza virus B, influenza virus C, and influenza virus D) and are classified according to differences in their internal glycoproteins nucleoprotein (NP) and matrix (M). Influenza viruses contain a single-stranded negative sense segmented RNA genome. Influenza A viruses are divided into various subtypes based on the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). There are 18 HA (H1–H18) and 11 NA (N1–N11) subtypes of influenza A viruses, that possibly form 198 theoretical HA/NA combinations (Vemula et al., 2016).

Influenza A viruses gained major public health concern as they circulate continually in human, swine, equine, and avian populations and cause global epidemics in humans and animals (Webby and Webster, 2003). Following the emergence of the novel strain of Influenza A virus H1N1 in March 2009 in Mexico, the H1N1 virus spreads rapidly throughout the world (WHO, 2009). Although WHO announced the end of pandemic (H1N1) 2009, influenza A (H1N1) 2009 virus still remains in the circulation as a seasonal virus for several years, and its behavior cannot be predicted (WHO, 2010). Surveillance of influenza-like illness cases from tertiary care hospitals gives a chance for analysis of the host factors and trend of infection (Kar et al., 2016).

Seasonal influenza is an acute, contagious respiratory infection caused by seasonal influenza viruses that circulate worldwide (WHO, 2016). Differentiating influenza illness from infections caused by other respiratory pathogens according to their clinical presentation is very difficult (Demicheli et al., 2000). So, diagnosis can only be confirmed by laboratory tests. Diagnostic methods currently used for detection of influenza viruses include rapid antigen tests, viral culture, enzyme immunoassay, and molecular tests such as real-time reverse transcription polymerase chain reaction (RT-PCR) and conventional RT-PCR (Ruest et al., 2003).

Although viral culture has been the “gold standard” for laboratory diagnosis, it requires specimen storage and transport in viral media maintained at ultralow temperatures to optimally preserve infectious viral particles (Krafft et al., 2005). This process requires time that may hinder quick clinical management and unfortunately negative viral culture does not exclude influenza infection (Chauhan et al., 2013). Accurate and rapid diagnosis is critical and of paramount importance for minimizing further spread, lower costs due to the illness, and prevent the inappropriate use of antibiotics through timely implementation of appropriate vaccines and antiviral treatment and prophylaxis where available (Centers for Disease Control and Prevention, 2002; Whiley et al., 2009). Molecular techniques such as conventional and real-time RT-PCR provide the rapidity and enhanced sensitivity for detection and typing and subtyping of influenza viruses (Dhakad et al., 2015). This study aimed to detect trend proportions of occurrence of influenza A virus (H1N1) in patients with influenza-like illness using real-time reverse transcription polymerase chain reaction (RT-PCR) and virus tissue culture.

Materials and Methods

This study was conducted, during three years period from November 2014 to December 2017, on 500 patients with uncomplicated influenza or influenza-like clinical presentation from outpatient’s clinic of pediatric, chest and internal medicine clinics of Ain Shams University Hospitals.

Uncomplicated influenza: ILI (Influenza-like illness) is characterized by sudden onset of constitutional and respiratory symptoms such as fever, cough, sore throat, rhinorrhea, bony aches and headache which usually resolves after 3–7 days although cough can persist for > 2 weeks.

The study was approved by the ethics committee and informed consent was obtained from those who agreed to participate after explaining the study and its goals to them.

Thorough history and examination were performed with emphasis on the age and sex of the patients and the duration of the symptomatic period. combined throat and nasal swabs were collected in viral transport medium and transported to the laboratory on ice. At the laboratory, samples were vortexed thoroughly and split into three aliquots.

Virus isolation

It was done on madin-darby canine kidney (MDCK) continuous cell line and eagle’s minimum essential medium (MEM-E) [Biological Products Company, Vaccines & Drugs (VACSERAEVYVAC)]. The MDCK cells were cultured on T25 flasks, were supplemented with streptomycin (100 µg/ml), penicillin (100 IU/ml), L-glutamine (2 mM), 1% nonessential amino acids and 10% fetal bovine serum and were incubated at humid atmosphere with 5% CO2 at 37ºC till confluent within three to four days. A volume of 5 × 103 MDCK cells was transferred to small tubes. After one day and just before inoculation of the samples, MDCK cells were washed with sterile PBS and sample was added and incubated for 1 h at 37°C then maintenance media, similar to the growth media used previously, but no fetal bovine serum was added and instead 1% lactalbumin hydrolysate (Sigma) and 0.5 μg/ml of trypsin (Sigma) was added. Cells were incubated at 37°C for 7 days and were observed daily under an inverted microscope for cytopathic effect (CPE). By the end of the week, cells were tested for the presence of the virus using the same PCR used for direct specimen detection of the virus.

RT-PCR for direct detection of influenza A virus (H1N1)

Aliquots for molecular testing were stored at -70°C until tested. Viral genomic RNA was extracted from the supernatants of the patient samples by using a QIAamp RNA extraction kit (QIAGEN), according to the protocol suggested by the manufacturer. Clinical samples were homogenized by vortexing for 30 sec, and 140 μl was used for the extraction of viral genomic RNA. The RNA was eluted from the columns with 50 μl of elution buffer.

Real-time RT-PCR was done using Quantitect Probe RT- PCR master mix (QIAGEN). The following primers and probe prepared by (Mol Biol) were used: forward primer (H1SWS) 5’-CAT TTG AAA GGT TTG AGA TAT TCC C-3’. Backward primer (H1SWAs1) 5’-GGA CAT GCT GCC GTT ACACC-3’. TaqMan probe (H1SWP) FAM-5’-ACA AGT TCA TGG CCC AAT CAT GAC TCG-3’-BBQ (Schweiger and Biere, 2009).

Real-time RT-PCR was done, using step one real-time machine (Applied Biosystems), in a 25-μl final volume. Briefly, 5 μl of extracted RNA was added to a master mixture composed of an enzyme mixture (heterodimeric recombinant RTs omniscript and sensiscript and hotstart Taq DNA polymerase), 400 μM each deoxynucleoside triphosphate, 20 U of RNase inhibitor (RNaseOUT; Invitrogen), and H1N1-specific primers, each at a final concentration of 20 pmol with the following parameters for amplification: The optimized profile in the thermal cycler (step one real-time PCR from Applied Biosystems) was 50°C for 30 min and 95°C for 15 min, followed by 40 amplification cycles (with each cycle consisting of denaturation at 94°C for 30 sec, annealing and extension at 60°C for 1 min. Results were obtained in real-time by the software of the machine and appear as amplification curve with a specific threshold cycle (CT) cycle that is inversely proportional to the initial amount of sample RNA (Fig. 1).

Fig. 1.

Amplification plot of H1N1 positive samples.



Statistical analysis

Data were analyzed using a personal computer statistical software package version 5 (Stat Soft Inc.). Quantitative data were statistically represented in terms of minimum, maximum, mean, and standard deviation (SD). Comparison between two groups was done using independent student t-test. Qualitative data were statistically represented in terms of numbers and percentages. Comparison between different groups was done using chi-square test. The validity of real-time RT-PCR in relation to virus isolation was estimated by sensitivity, specificity, positive predictive value and negative predictive value. A P-value < 0.05 was considered statistically significant and P > 0.05 was considered non-significant.

Results

The age of the study population ranged from 1–94 years, with a Mean ± SD age 26.892 ± 18.53 years. Of the total participants, 193 (38.6%) were females and 307 (61.4%) males. Table 1 shows that 71 patients out of 500 (14.2%) were diagnosed with H1N1 virus infection with real-time RT-PCR; whereas, 52 (10.4%) were detected by tissue culture. It also shows the distribution of cases according to sex; 46 male patients (64.8%) and 25 female patients (35.2%) were detected by real-time RT-PCR while 33 males (63.5%) and 19 females (36.5%) were detected by tissue culture. No statistically significant relation was found between gender and H1N1 infection.

Relation between of influenza A (H1N1) infection and gender

Test Sex Total Chi square test P value

Male Female



N % N % N %
PCR Positive 46 14.55 25 12.95 71 14.2 0.4009 0.5266
Negative 261 85.45 168 87.05 429 85.8

Culture Positive 33 10.75 19 9.84 52 10.4 0.1041 0.747
Negative 274 89.25 174 90.16 448 89.6


Table 2 illustrates the distribution of cases according to age. We can observe that influenza A (H1N1) primarily affected the younger population, with patients in the 0~45 years age group accounting for about 86%. Non-statistically significant relation was found between age and H1N1 infection as shown in Table 3.

Distribution of influenza A (H1N1) infection by age group

Age groups (Years) PCR positive Culture positive
N = 71 N = 52


N % N %
0∼15 20 28.2 18 34.6
> 15∼30 23 32.4 14 26.9
> 30∼45 18 25.4 13 25
> 45∼60 7 9.8 4 7.7
> 60 3 4.2 3 5.8

Relation between of influenza A (H1N1) infection and age

Test Age (years) t* P value

Mean SD
PCR Positive 27.51 17.64 0.3033 0.7618
Negative 26.79 18.67

Culture Positive 26.52 19.06 0.1510 0.8800
Negative 26.93 18.47


Clinical manifestations of Influenza among the infected population are summarized in Table 4; with cough being the most common symptom 65 (91.5%), followed by fever 57 (80.2%), dyspnea 50 (70.4%), rhinorrhea 21 (29.6%), diarrhea 10 (14.1%), and nasal congestion being the least common symptom 8 (11.3%).

Clinical manifestations of influenza in the infected population

Signs and symptoms PCR positive N = 71
Cough 65 (91.5%)
Fever 57 (80.2%)
Dyspnea 50 (70.4%)
Rhinorrhea 21 (29.6%)
Diarrhea 10 (14.1%)
Nasal congestion 8 (11.3%)


Among the positive patients, 29 (40.84%) had comorbid conditions. Hypertension was found in (31.03%), diabetes in (20.69%), chronic lung disease (13.79%) cardiovascular diseases in (13.79%), current smokers (10.34%), and pregnancy in (10.34%) as shown in Table 5.

Comorbid conditions in the infected population

Comorbid conditions N = 29
Hypertension 9 (31.03%)
Diabetes mellitus 6 (20.69%)
Chronic lung diseases 4 (13.79%)
Cardiovascular diseases 4 (13.79%)
Current smoking 3 (10.34%)
Pregnancy 3 (10.34%)


Seasonal distribution of influenza A (H1N1) virus infection is illustrated in Table 6. Most of the cases presented in winter followed by autumn and may extend to late springtime.

Seasonal distribution of influenza A (H1N1) virus infection

Season (n = 71) No %
Winter (December, January, February) 42 59.15
Spring (March, April, May) 10 14.08
Summer (June, July, August) 0 0
Autumn (September, October, November) 19 26.76


From Table 7 we can notice that there is discrepancy between tissue culture and real-time RT-PCR results as there are 20 patients that were negative by tissue culture while positive with real-time RT-PCR and one patient was positive with tissue culture while negative by real-time RT-PCR. Sensitivity and specificity of real-time RT-PCR were 98.08% and 95.54%, respectively, in comparison to virus isolation with accuracy 95.8%.

The performance of real time RT-PCR in relation to conventional viral culture

Test Culture Total S SP PPV NPV Accuracy

Positive Negative
PCR Positive 51 20 71 98.08% 95.54% 72.83% 99.77% 95.8%
Negative 1 428 429

Total 52 448 500

S, sensitivity; SP, specificity; PPV, positive predictive value; NPV, negative predictive value


Discussion

The 2009 pandemic influenza A(H1N1) virus [hereafter referred to as influenza A (H1N1) pdm09] which was detected in 2009 in the United States causing a global influenza pandemic, is now a seasonal influenza virus that co-circulates with other seasonal viruses (National Institute for Communicable Diseases, 2017).

A rapid, specific and sensitive method to detect influenza virus is very important for effective patient management and surveillance of newly emerging influenza strains, vaccine development and to detect the spread of virus in early stages, thus preventing epidemics or pandemics of influenza. Among diagnostic tools available for detecting influenza viruses, molecular tests are the most sensitive, specific and rapid. Real-time RT-PCR is one of the important and effective molecular tests (Lopez et al., 2011).

Seventy-one patients out of 500 (14.2%) and 52 (10.4%) were diagnosed with H1N1 virus infection by real-time RT-PCR and tissue culture, respectively. This finding is similar to the findings of a study conducted by Samra et al. (2011) (17.35%), Mehta et al. (2013) (23%) and Amaravathi et al. (2015) (17.12%). However, higher detection rates were found by Chudasama et al. (2013) who reported that 29.6% of cases having influenza-like symptoms were positive for A (H1N1) influenza. Studies performed by Tulloch et al. (2009) and Torres et al. (2010) also reported figures of 40.9% and 45.9% as positive, respectively. Also, Cheung et al. (2017) reported that H1N1 predominated in 2013∼2014 and 2015∼2016 seasons in Hong Kong. Differences in attack rate may be due to geographic, demographic and socioeconomic factors, living conditions, cultural factors, health status, and genetic predispositions.

As regards sex predominance, the current study demonstrated that male patients were predominately affected by H1N1 infection although there was no statistically significant relation. This agrees to a study conducted by Jordi et al. (2009) on 32 critically ill patients in Spain and reported that 73.3% of them were male and 26.7% of them were female.

On the contrary, Domínguez-Cherit et al. (2009), who performed their study on 58 critically ill patients in Mexico, demonstrated that 53.4% of them were female and 46.6% were male. Furthermore, Kumar et al. (2009), studied 168 critically ill patients in Canada and found that 67.3% of them were female and 32.7% of them were male. However, Mehta et al. (2013) and Amaravathi et al. (2015) found that cases were equally distributed in both genders. This may be due to differences in underlying diseases and conditions which predispose to flu infection. Muscatello et al. (2011) stated that sex was not associated with influenza disease.

Influenza A (H1N1) virus primarily affected the younger population (86% of cases). Still, no statistically significant relation was found between age and positivity of H1N1.

Other studies investigated the age factor in H1N1. Dudley et al. (2009) clarified that the age group of 20∼39 years was the predominantly affected group. Khattab et al. (2014) found that the mean age of the patients who survived was 30.75 years, and the mean age of the patients who died was 28.9 years (range 11∼60 years), therefore accounting for 91.2% of cases under the age of 40 years. Amaravathi et al. (2015) reported that the majority of cases were in the younger age group (11∼40 years), accounting for 61.36% with a mean age of 31.15 years.

The prominent clinical manifestations found in this study were cough and fever and this finding was similar to studies reported in Vietnam by Hien et al. (2004), in New Zealand by Dee and Jayathissa (2010), in China by Mu et al. (2010), in India by Chudasama et al. (2013) and Amaravathi et al. (2015), in Egypt by Khattab et al. (2014).

However, Waleed (2010) reported that fever was the main complaint occurring in 93.3%, followed by muscle/joint pain 53.3%, sore throat and dry cough 40%, shortness of breath 33.3%, productive cough and vomiting 26.7%, diarrhea and headache 20%, and abdominal pain and sneezing were the least occurring symptoms, with 6.7% each. The differences in the clinical presentations could be due to difference in the number of cases included in these studies.

Of all the positive patients, 29 (40.84%) had comorbid conditions. Hypertension was the most common comorbidity followed by diabetes, chronic lung diseases, cardiovascular diseases, current smoking, and pregnancy. This result agrees to that found in other studies performed by Puvanalingam et al. (2011), Jagannatha et al. (2011), and by Chudasama et al. (2011), Mehta et al. (2013), Singh and Sharma (2013), and Amaravathi et al. (2015).

Most influenza virus (H1N1) infected cases presented in winter followed by autumn and late spring. This result was concordant with studies done by Laguna-Torres et al. (2009), Puzelli et al. (2009), and Sadeq et al. (2014). This may be due to climatic parameters, such as absolute humidity, temperature, and rainfalls. This was in line with the Northern Hemisphere seasonality of influenza which affects the northern hemisphere from November to April (WHO, 2016).

Twenty real-time RT-PCR positive samples were found to be culture negative. So, the diagnostic yield for positive samples increased by 26.76%. This discrepancy was explained by Zambon et al. (2001) who stated that culture may miss up to 46% of influenza positive samples, especially in patients with an advanced clinical course of disease. Another explanation returned back to the use of antiviral medication in those patients or the presence of non-viable viruses in specimens. The quality of the collected specimen, virus titer in the original clinical sample and following propagation in cell culture both can act as parameters in determining the sensitivity and specificity of diagnostic method and especially virus isolation since an intact virus is required (Peaper and Landry, 2014).

One culture positive sample was found to be PCR negative, which is likely a true influenza positive sample. A possible explanation for this discrepancy is very small viral load in this sample. Another possible explanation may be the presence of inhibitors in the PCR reaction as RNases are present in respiratory tract specimens that may gradually digest naked viral RNA.

Similar discrepancy and an increase in diagnostic yield were found by Angione et al. (2013). Furthermore, Seifi and Ghannad (2014) showed that 20.3% of patients were positive using PCR, in comparison with 8.9% positive results detected by cell culture. Vontas et al. (2015) reported that 34% of cases were positive by real-time RT-PCR while by virus isolation were 26%.

Sensitivity and specificity of real-time RT-PCR were 98.08% and 95.54%, respectively, in comparison to virus isolation with accuracy 95.8%. Similar findings were found by López et al. (2011) reported sensitivity, specificity, positive predictive value, and negative predictive value: 95.6, 82.3, 78.3, and 96.5%, respectively. Gohar (2012) found them 96.6%, 95.8%, 96.6%, and 95.8%, respectively. Also, Angione et al. (2013) who reported 100% specificity, 100% positive predictive value (PPV) and 94% negative predictive value (NPV) with an accuracy of 97.5%. In addition, other studies performed by Espy et al. (2006), Zitterkopf et al. (2006), Jain et al. (2014), and Hiergeista et al. (2016) support these findings. The efficiency and performance of RT-PCR systems are strongly dependent on the quality of the primer sets and probes applied for the amplification of nucleic acid target regions (Behzadi et al., 2016).

Conclusion

H1N1 virus was responsible for a good proportion of influenza during the post-pandemic period. Real-time RT-PCR provides rapidity and sensitivity for the detection of influenza A virus (H1N1) compared with virus isolation and thus it is recommended as a diagnostic tool.

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