Real time-PCR, ELISA, as an identification methods for Detection respiratory syncytial virus in children

The study have been done to detecting HRSV infections among hospitalized children under 10 years old in Salahaldin Governorate, in addition to compare different diagnostic methods for detection of this virus. Nasopharyngeal Swabs (NPS) and whole blood samples were taken from 400 hospitalized children between end of August 2013, to the end of May 2014, each sample was analyzed for HRSV by Real time –PCR, IgM and IgG specific to HRSV determining from blood specimens in serum using enzyme linked immune sorbent assay (ELISA) technique. Nasopharyngeal Swab was more sensitive than the whole blood specimens for the detection of HRSV ,which is found in 1% by RT-PCR, in 81.9% by IgM –anti HRSV antibodies and in79.5% by IgG-HRSV antibodies. Autumn – winter periodwas considered as the most common time for HRSV infections with peak in September and February.


Introduction
Respiratory Tract infection (RTIs) is one of the important causes of deaths among children especially in developing countries [1].It is caused by abroad spectrum of microbial agents viruses, on account for the largest number of respiratory tract infections [2].Among these respiratory viruses are human respiratory syncytial virus (HRSV) [3].HRSV belongs to the family paramyxoviridae, is important cause of Acute Respiratory Tract infection in children and represent a high percentage of the pediatric medical admission due to pneumonia and Bronchiolitis [4].Human respiratory syncytial virus is a liner single stranded (SS) negative sense RNA consisting of 15,191 base pairs (bp) [5].The laboratory diagnosis of HRSV infections is mostly done by Enzyme linked Immuno sorbent assay (ELISA) [6].The diagnosis can also be done by real timepolymerase chain reaction assay RT-PCR [7].HRSV has not been studied in Salahaldin Governorate of Iraq and no laboratory method have been used to detect this virus among patients in Salahaldin Governorate.The frequency of this virus as causative agent among hospitalized children with respiratory tract infections have not been clarified.The laboratory diagnostic methods that are routinely done in Iraqi medical laboratories and hospitals, do not include the diagnosis of HRSV in their schedule investigations, and generally no regular diagnostic methods are done for respiratory viruses in Iraqi hospitals.Where as (RTIs) is so common and as RTIs in Iraq is a real problem, this study was prepared and its aim was to detect HRSV in respiratory specimens of children with (RTIs)

Materials and Methods
This study was carried out on patients attended the general hospital in Baiji in the central of Baiji city and to the teaching hospital of Tikrit city.The patients were the children less than 10 years old and they were admitted to these hospitals with respiratory tract infections.A total of 400 children who required medical treatment were included both sexes were chosen.The period of the study was started from the end of August 2013 to the end of May 2014.

Collection of Specimens
Four milliliters (mls) of venous blood were collected from each child.The blood then was divided into two parts: The first part (3ml) was kept into a test tube without anticoagulant and centrifuged for 15 minutes at 3000 revolution per minute (RPM), the serum was then putted into one aliquot stored in one microcentrifuged (Eppendorf) tube labeled with patient's code number and frozen upon reaching the deep freeze at -70 till tested.Once thawed refreezing was avoided.The second part (1ml) was put into a test tube with anticoagulant, ethylene diamine tetramide (EDTA), and then mixed on a roller, labeled with patient's code number and frozen upon reaching the deep freeze at -70 till tested.Once thawed refreezing was avoided.One nasopharyngeal Swab (NPSs) was taken from each child by flocked swab applicators, thus one applicator was applied for one nostril.).This step includes the following: -Two l of the oligonucleotides primers (2 primers of HRSV) were added into a single 200 l microcentrifuge tube; and 9l of the eluted solution of the sample (template) were added to the same tube to get a final concentration of 11l.The tube was then subjected to short centrifugation for few seconds at 5000 rpm before putting it in thermal-cycler which was programmed for preheat at 105 C and heating 10 minutes at 65 C for sample denaturation and annealing, then chilling at 4C for 4 minutes.2. The second substep (called reverse transcription2, RT2).This step included the preparation of cDNA master mix by addition of 4l of the 5x rxn reaction buffer, 2l DTT, 2l of 10 l mMdNTPs, and 1l Cycle Script reverse transcriptase were collected in to a single microcentrifuge tube to get a final concentration of 9l.The final 9l concentration was added to the previous 11l to get reaction volume of 20l which was put in the thermal cycler.The cycler was programmed for the first strand cDNA synthesis by incubation at 50C for 60 minutes, then incubation at 95C for 5 minutes to step reaction.The product then was used for PCR amplification. 1.After reverse transcription, the reaction mix was prepared according to the table below for each cDNA sample.

Detection of HRSV infection: Detection of HRSV from nasopharyngeal swab and whole blood samples using RT-PCR technique:
The nucleic acid RNA of HRSV was detected by real time -PCR technique from NPS and whole blood samples from 23(5.75%) patients, the results showed that 4 (17.39%)out of 23 (5.75%) were positive for HRSV, while 6 (26.1%) out of 23 patients were negative for HRSV from whole blood samples.On the other hand, the results showed that 13 (56.5%)out of 23 patients were negative for HRSV from NPS.All positive whole blood cases were negative in NPS, (Figure 1).Flocked swab was used for the collection of NPS, also whole blood samples were the only specimens used for antigen and nucleic acid detection.Other kinds of specimens like NPA, NPW, nasal wash, tracheal aspirates were not performed in this study, thought they are the mainly specimens which were used in most of other studies for the detection of HRSV antigen or nucleic acid [8,9,6].

Detection of HRSV using real time-PCR:
RNA of HRSV was detected for each children from NPS and whole blood specimens by using the molecular technique real time -PCR.The results showed that 4 (1%) children were positive while the remaining 396 (99%) children were negative (Figure 2).The prevalence of HRSV infections in the nearby countries revealed variations in frequency using RT-PCR, in a study done in Jordan it was 64% [14].and in Saudi Arabia study 54.4% of children were infected by HRSV [15].In Iran, astudy found that 45.6% of children under 5 years were infected with HRSV [16].In USA a study found that 24% of hospitalized children were infected with HRSV [17].And in Philippines another study found that only 8.4% of children were infected with HRSV [18].The variation in results from different countries and even from the same country reflected the sample size as some studies were taking small sample size, or due to the different techniques were used in different studies or due to the period of study as some studies were performed during only winter season, or due to the type of sample as some studies were chose only children with bronchiolitis and pneumonia but not all respiratory infections as a whole, more over geographical variations, environmental differences and control measures used might also contribute to the differences in the percentage of HRSV infections in different countries.

Seropositivity of anti -HRSV IgM antibodies among patients with RTI.
Measurement of IgM anti-HRSV antibodies seropositive showed that 68(81.9%)out of 83 children infected with HRSV had positive antibody titers, while only 3(0.9%) out of 317 patients (False positive), who were negative for HRSV, were having positive antibody titer.The total IgM anti-HRSV antibodies was 314 (78.3%) in all hospitalized children.The results were statistically not significant (p=0.3452).Seropositivity of IgM anti-HRSV antibodies among the 83 HRSV patients showed that positivity was found in 8 (80%) out of 10 patients aged <1 year, 10 (83.3%) out of 12 patients aged 1 to < 2 years, 9(69.2%)out of 13 patients aged 2 to <3 years, 7 (87.5%)out of 8 patients aged 3 to <4 years, 8(88.8%)out of 9 patients aged 4 to <5 years, 6(85.7%)out of 7 patients aged 5 to <6 years, 6(85.7%)out of 7 patients aged 6 to <7 years, 5(83.3%)out of 6 patients aged 7 to <8 years, 4(80%) out of 5 patients aged 8 to <9 years, and 5(83.3%)out of 6 patients aged 9 to <10 years.The highest seropositivity was found in the age group 4 to <5 years, while the lowest rate in the age group 2 to <3 years, the results among age groups were statistically not significant (p= 0.0218) (Figure 7).However, the results showed that IgM anti-HRSV antibodies as seroprevalence in children with positive HRSV antigen were significant and more frequent than in HRSV antigen negative children were important diagnostic tools [19].

Seropositivity of anti -HRSV IgG antibodies among patients with RTI.
The seropositivity of anti-HRSV IgG antibodies specific to HRSV in the study group showed that 66(79.5%)out of 83 HRSV infected patients were positive, while 251(79.2%)out of 317 patients HRSV-negative patients were IgG seropositive (Table 4-7).The difference in IgG Seropositivity between the HRSV infected patients and those who were not infected with HRSV was not statistically significant (p=0.366).Positive IgG anti-HRSV antibodies among the 83 HRSV patients infected children were found in 8(66.7%)out of 12 in age group <1 year, 9(75%) out of 12 in age group 1 to <2 years, 8 (80%) out of 10 were aged 2 to <3 years, 7(77.8%)out of 9 were aged 3 to <4 years, 7(87.5%)out of 8 were aged 4 to <5 years, 6(75%) out of 8 were aged 5 to <6 years, 7(87.5%)out of 8 were aged 6 to <7 years, 5 (83.3%) out of 6 were aged 7 to <8 years, 4(100%) out of 4 were aged 8 to <9 years, and 5 (83.3%) out of 6 were aged 9 to <10 years.The highest Seropositivity is found in the age group 8 to <9 years, while the lowest is in the age group <1 year, the results among age groups were not statistically significant (p= 0.3209) (Figure 8).Positive IgG anti HRSV antibodies percentage among HRSV-negative children was (66.7%), (75%), (80%), (77.8%), (87.5%), (75%), (87.5%), (83.33%), (100%), (83.33%) in age groups <1 year, 1 to <2 years, 2 to <3 years, 3 to <4 years, 4 to < 5 years, 5 to <6years, 6to <7 years, 7 to <8 years, 8 to <9 years, 9to <10 years respectively, the results showed the presence of wavy increase in a percentage of children with positive IgG anti-HRSV antibodies in HRSV infected and non-HRSV infected group (Figure 9).The results revealed that 79.5% of HRSV positive patients were having positive titers, while 79.2% of HRSV-negative patients were IgG seropositive.The differences in the two groups were statistically not significant, and the high rate of previous infections with HRSV among children under 10 years, or in infants under 1 year of age, may be because the transport of IgG anti -HRSV antibodies from mothers to infants during intrauterine life.A previous study by [20].Revealed that 83% of the pregnant women as well as all the babies of these mothers were anti-HRSV IgG positive .IgG anti-HRSV antibodies were investigated among all patients in this study and the total prevalence rate was 79.2%.This result was similar to a previous study done in Baghdad/ Iraq, in which 79% of infants with ARTIs were having positive titers of antibodies [11].Beside that the patients in the previous study were limited to one year old infants, while in this study, 66.7% of the age group< 1 year old was IgG seropositive.
The results showed the presence of wavy increase in the percentages of children with positive IgG anti -HRSV antibodies in HRSV infected group and HRSV non -infected group.This result points to the cumulative increase in HRSV infections with the increase in the age of children.A study by [21].In Thailand found that the prevalence of IgG anti -HRSV antibodies was increasing with age: from 6 to 11 months, 11.76%; from 12 to 17 months, 41.67%; from 18 to 23 months, 60.87%; from 24 to 29 months, 88.24%; from 30 to 35 months, 78.57%; from 36 to 41 months, 94.44% and from 42 to 60 months 100%.This figure showed increase in the frequency of infections which started in January, presented in February peaked and declined in March.In this figure also another peak, started from August, peaked in September and declined in October and November, Thus Autumn-Winter period was the most common for HRSV infections, while Summer is the least for HRSV infections.Salahaldin Governorate is located in the middle of Iraq with some mountains and hills like Hemreen and Makhool in some parts of this Governorate.The main annual temperature is high as like as Baghdad temperature and the climate is dry and windy in Winter.The HRSV infections were detected all the year in this study.Autumn-Winter period was the most common for HRSV infections, with peak in September and another peak in February.The differences in viral distribution and seasonality might be attributed to the climate and geographical factors.Most of other studies from different regions all over the world like Pakistan, Japan and Jordan showed Winter season predominance of HRSV infections, [22,23,24].Though in Thailand and in similar climate countries, the annual peak of HRSV hospitalization among children is occurring between July and October which are the rainfall months in this country [25].In Winter, inhalation of cold air will slow the mucociliary escalator and reduce phagocytic activity of leukocytes, thereby increasing susceptibility to infection [26].May be because that people's stay indoors in Winter will increase the risk of HRSV transmission and especially among children who catch their infections from older people.

Conclusions
Pneumonia was the main clinical disease in HRSV in hospitalized children and the infections were higher among age group <4-5 years.

Figure 1 :
Figure 1: Detection of HRSV by the real time -PCR technique in nasopharyngeal swab (NPS) and whole blood specimens for infection children.

Figure 2 :
Figure 2: Frequency of HRSV infections among patients in the study using real time -PCR technique.

Figure 9 :
Figure 9: Percentage of anti-HRSV IgG antibodies among patients infected with RTI.

FrequencyFigure 10 :
Figure 10: Seasonal distributions of HRSV infections among hospitalized children in the study

of respiratory syncytial virus (all species) Nucleocapsid (N) gene by RT-PCR kit:
This kit is designed and manufactured by primer design TM genesig company/ United Kingdom.

kit for IgM anti -HRSV antibodies detection (Cusabio Company/ China.Cat.NO.CSB- E13790h
This is the second step in real time PCR, and also divided into two sub-steps.1.The first substep is (called reverse transcription 1, RT1 ).This ELISA test kit is used for the qualitative determination of human anti RSV antibody (IgM) concentrations in serum.The kit compounds are the following: 1-Microplate wells: containing 96 test well coated with RSV antigen.2-Negative control: one vial containing 0.5 ml.3-Positive control: one vial containing 0.5 ml.4-Sample diluent: one bottle containing 12 ml.5-HRP-conjugate: one bottle containing 12 ml.

ELISA test kit for IgM anti-HRSV antibodies qualitative determination concentrations in serum. -Test procedure, according to supplied company kit
. One hundred l of negative control, positive control were added, controls need test in duplicate.5.One hundred l of sample diluent were added to rest wells, then 10 l of sample per well were added then covered with the adhesive strip provided and incubated for 30 minutes at 37 C.6.A respiratory and washing each well was done for 5 times.Washing by filling each well with wash buffer (200l) by autowasher, and standing for 20 seconds and completed removal of liquid at each step for good performance after the last washing, any remaining wash buffer was removed by a separating or, decanting with inverted and bloted the plate against clean paper towels.7. Then 100 l of HRP-conjugate was added to each well (not to blank well), and covered the microtiter plate with a new adhesive strip and incubated for 20 minutes at 37 C.8.The aspiration/ wash process for 5 times as in step 6 was repeated.9. Fifty l of substrate A and 50 l substrate B were added to each well and incubated for 10 minutes at 37 C with protected from light.10.Then 50 l of stop solution was added to each well with gently tap the plate to ensure through mixing.11.Blank well was taken as zero and the optical density (OD) of each well was determined within 10 minutes by using a microplate reader set to 450 nm.
-Test procedure, according to supplied company kit: 1.All reagents and samples were prepared and brought to room temperature (18-25C).2.The assay layout sheet was used to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and stored unused wells at 4 C.3. A blank well was set without any solution.4while OD.Sample/ OD.  2.1: positive.While OD.Sample/ OD. < 2.1: negative.