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Investigation of suspected viral hepatitis outbreaks in North West India
Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Original article
Investigation of suspected viral hepatitis outbreaks in North West India☆,☆☆ Mini P. Singh a,1, Manasi Majumdar a,2, Kapil Goyal a, P.V.M. Lakshmi b, Deepak Bhatia c,3, R.K. Ratho a,⁎ a b c
Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh, India 160012 School of Public Health, Postgraduate Institute of Medical Education & Research, Chandigarh, India 160012 Integrated Disease Surveillance Project, Directorate of Health & Family Welfare, Punjab, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 18 June 2015 Received in revised form 8 December 2015 Accepted 9 December 2015 Available online xxxx Keywords: Hepatitis E virus HEV-antigen Outbreak Phylogenetic analysis Saliva
a b s t r a c t Hepatitis E (HEV) infection is diagnosed on the basis of serum anti-HEV IgM detection. In outbreaks, early diagnostic method is important for prompt control measures. This study compared 3 diagnostic methods in 60 serum samples collected in suspected HEV outbreaks. The suitability of saliva samples for antibody detection was also evaluated in 21 paired serum saliva samples. The anti-HEV IgM, HEV-Ag, and HEV-RNA were detected in serum samples of 52 (86.66%), 16 (26.66%), and 18 (30%) patients, respectively. The concordance between serum and saliva IgM was found to be 76.91%. The positivity of PCR and HEV-Ag detection was 100% within 1 week of illness which declined to 5–10% thereafter. The outbreak was attributed to HEV genotype 1, subtype 1a, and the clinical and environmental strains clustered together. HEV-antigen and RNA were an early diagnostic marker with 96.66% concordance. Saliva samples can be used as an alternative in outbreak setting. © 2015 Published by Elsevier Inc.
1. Introduction Viral hepatitis E, a waterborne hepatitis, has extensive epidemic potential, though focal outbreaks and sporadic cases are also frequently observed. Overall, the WHO (2014) has reported approximately 20 million hepatitis E infections, more than 3 million acute cases, and nearly 56,600 deaths due to hepatitis E. The causative agent was identified as a novel putative hepatitis E virus (HEV) during a massive waterborne epidemic of hepatitis which occurred in Delhi and caused 30,000 cases during 1955–1956 (Ramalingaswami and Purcell, 1988). This virus contributes to 30–70% of sporadic hepatitis cases in the Indian subcontinent (Aggarwal, 2011). Drinking water contaminated with sewage is the commonest mode of HEV transmission in developing nations like India. The virus is endemic in North India with documented hepatitis E outbreaks being reported from Mandi-Gobindgarh in the year 2005 (Bali et al., 2008; Kumar et al., 2006; Prinja et al., 2008) and Lalru in the year 2010 (Majumdar et al., 2013a). HEV is now regarded as the major cause of sporadic as well as epidemic hepatitis, which is no longer
restricted to Asia and the developing countries. Increasing number of cases is being documented from developed world such as United States and Europe (Clemente-Casares et al., 2003; Echevarría, 2014; Huang et al., 2002; Meng et al., 1997; Riveiro-Barciela et al., 2012). The present study reports a waterborne outbreak of viral hepatitis which occurred almost simultaneously in Nawanshahar (Punjab) and Palsora a suburb of Chandigarh in March–April, 2012. The detection of early warning signals, well-timed investigations, and application of specific control measures are important to control the outbreak. Hence, the present study compared the anti-HEV IgM, HEV-antigen, and HEV-RNA detection for the early diagnosis of a suspected HEV outbreak. The usefulness of collecting noninvasive clinical samples like saliva for diagnosis of HEV was also evaluated. Further, phylogenetic analysis of the viral strains from human and environmental samples was carried out for tracing the source of infection. 2. Materials and methods 2.1. Study design
☆ Financial grant received: Nil. ☆☆ Conflict of interest: None declared. ⁎ Corresponding author. Tel.: +91-172-2755170; fax: +91-172-2744401. E-mail addresses: [email protected] (M.P. Singh), [email protected] (M. Majumdar), [email protected] (K. Goyal), [email protected] (P.V.M. Lakshmi), [email protected] (D. Bhatia), [email protected] (R.K. Ratho). 1 2 3
Tel.: +91-172-2755199. Tel.: +91-9872186879. Tel.: +91-172-2621506; fax: +91-172-2620234.
In the month of March–April 2012, the Department of Virology, Postgraduate Institute of Medical Education and Research, Chandigarh, was informed about a suspected outbreak of enterically transmitted viral hepatitis from 2 different places, Nawanshahar in Punjab and Palsora, a village near Chandigarh, North India. An active surveillance of the affected region was carried out, and approximately 3 mL of blood samples was collected from 27 icteric patients from Nawanshahar and 33
http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002 0732-8893/© 2015 Published by Elsevier Inc.
Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
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M.P. Singh et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
patients from Palsora aseptically by trained health care personnel after obtaining a written informed consent. Additionally, 21 matched saliva samples were also collected from Nawanshahar patients. All the samples were transported in cold chain to the virology laboratory. Since the clinical samples were received during an outbreak for diagnostic purposes, prior ethical clearance could not be obtained. However, the study was retrospectively approved by the Institute Ethics Committee of Postgraduate Institute of Medical Education and Research, Chandigarh, as per national guidelines.
2.2. Descriptive epidemiology Nawanshahar district of Punjab and Palsora village of Chandigarh are situated at a distance of 93.8 km in the northwest part of India at a latitude of 31°N and longitude of 76°E with moderate rainfall and highest and lowest temperatures being 40 °C and 25 °C, respectively, in the months of March–April, when the outbreak occurred. Both the townships are in suburban areas with low socioeconomic living conditions, and at both places, sewage work was undertaken before the outbreak. An investigation team visited houses and collected information about cases, drinking water quality, source of water supply, and drainage system. The existing blueprint of the water supply pipelines and drains was also examined.
2.3. Description of the patient population The mean age of patients affected was 30.48 ± 14.58 years (95% confidence interval [CI]: 26.72–34.25), and the male:female ratio was 1.6:1. The predominant clinical manifestations were yellow urine (95%), fatigue (85%), yellow eye (81.66%), anorexia (75%), vomiting (65%), fever (61.66%), pain abdomen (58.33%), pruritis (30%), and arthalgia (21.66%), respectively. Liver enzymes were found to be raised, mean levels of aspartate aminotransferase (618.6 ± 115.8 IU/mL), alanine aminotransferase (694 ± 131.4 IU/mL), and mean bilirubin level was found to be 6.288 ± 3.975 mg/dL. Pregnant women with jaundice were not reported in these 2 outbreaks.
2.4. Antibody detection Sixty serum samples were tested for the following viral markers: anti-hepatitis A virus (HAV) IgM (ImmunoComb® II HAV Ab, Orgenics, Israel) and anti-HEV IgM (ImmunoVision, Springdale, AR) using commercially available enzyme-linked immunosorbent assay (ELISA) kits as per the manufacturer's instructions. Anti-HEV IgM detection system used a combination of recombinant HEV ORF-2 and ORF-3 peptide. The manufacturer of this kit has documented a sensitivity and specificity of N99% as per the kit insert. Neat saliva samples from 21 patients were diluted to 1:4 dilutions in sample diluent (i.e., 100 μL saliva + 400 μL sample diluent) and tested for the detection of anti-HEV IgM using commercially available ELISA kit (ImmunoVision).
2.5. HEV-antigen detection Sixty serum samples were tested using a commercially available HEV-antigen ELISA kit (Wantai, Beijing, China). This is a solid phase sandwich ELISA, in which the microwell strips were precoated with rabbit anti-HEV antibodies directed against ORF-2 protein. The absorbance was measured at 450 nm. According to the manufacturer's information provided, HEV-Ag cutoff = 0.12 + NC (the mean absorbance value for 3 negative controls) was calculated. Sample with absorbance higher than cutoff values were considered positive.
2.6. RNA extraction and reverse transcription–polymerase chain reaction (RT-PCR) RNA extraction was carried out from all the 60 serum samples using QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD). Briefly, 140 μL of serum was used for RNA extraction as per the manufacturer's guideline which was finally eluted in 50 μL of elution buffer. A 10-μL volume of the RNA was reverse transcribed to cDNA on the same day using M-MuLV RT and random hexameric primers (Fermentas, Hanover, MD). Nested PCR protocol was used for amplification using primers targeting the ORF 1 gene as previously described (Majumdar et al., 2013b). The amplicon of 343 bp was visualized by 2% agarose gel electrophoresis following ethidium bromide staining. With each run, 1 positive and negative control was included. Positive control was a recombinant plasmid (pIRES-neoORF1), a kind gift from Dr Shahid Jameel, ICGEB, New Delhi. Representative 6 clinical strains from Nawanshahar and 5 from Palsora, sewage samples from both the places, and tap water sample from Palsora were sequenced for confirmation of the etiological agent.
2.7. Sewage and tap water sampling Samples of untreated sewage were collected from sewage pumping stations of Nawanshahar and Palsora. A sterile glass bottle was lowered into the flowing water to collect approximately 1 L of sewage and transported to the laboratory in cold chain. Further, an early morning, free-flowing 1-L tap water sample was collected in a sterile glass bottle from a final distribution tap in Palsora. The tap water was muddy, and visible particulate materials were appreciated. Adequate personal safety precautions were taken while collecting and processing the samples. Materials used for sample collection and processing were decontaminated by autoclaving.
2.8. Sewage and tap water sample processing Briefly, 40-mL sewage sample was ultracentrifuged (110,000 × g for 1 h at 4 °C) to pellet down both the viral particles and suspended material. The sediment was eluted using 4 mL 0.25 N glycine buffer, pH 9.5. Suspended solids were then separated by centrifugation at 12,000 × g for 15 min. Viruses were finally pelleted down using a second ultracentrifugation step (110,000 × g for 1 h at 4 °C) and resuspended in 0.1-mL phosphate-buffered saline (Clemente-Casares et al., 2003). From the virus concentrate, RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen), cDNA was synthesized using M-MuLV reverse transcriptase enzyme (Fermentas), and HEV ORF1 gene was amplified as described above followed by sequencing and phylogenetic analysis.
2.9. Sequence analysis and phylogenetic tree construction HEV-specific RNA-dependent RNA polymerase (RdRp) gene was amplified from 11 clinical (6 from Nawanshahar and 5 from Palsora) and 3 environmental samples, i.e., sewage samples collected from Palsora, Nawanshahar, and the tap water of Palsora were sequenced. ABI chromatogram files were viewed using Finch TV 1.4.0, following sequence drafting with bioedit 7.0.9 software. For the confirmation of these sequences, database search was implemented using BLAST program available at NCBI Web site. For sequence comparison, standard representative strain sequences were retrieved from GenBank as previously described (Majumdar et al., 2015). Clustal X 2.0.11 program was used for multiple sequence alignment followed by Molecular Evolutionary Genetics Analysis (Tamura et al., 2007) for phylogenetic tree construction.
Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
M.P. Singh et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
3. Results
3
Table 2 Table showing the sensitivity and specificity of detecting HEV-antigen in serum samples, taking HEV-RNA positivity in serum samples as gold standard (n = 60).
3.1. Determination of serological markers in serum samples
HEV-RNA
The 60 serum samples collected from icteric patients during the 2 outbreaks were tested for anti-HEV IgM, HEV-antigen, and anti-HAV IgM. Out of 60 blood samples tested, 52 (86.66%) were positive for anti-HEV IgM; and 16 (26.66%), for HEV-antigen. Anti-HAV IgM alone could be detected in 4 (6.66%) samples, while both anti-HAV IgM and anti-HEV IgM could be detected in 2 (3.33%) patients suggesting dual infection with HAV and HEV. 3.2. Comparison of anti-HEV IgM detection in paired serum and saliva samples Paired serum and saliva samples were obtained from 21 icteric patients from Nawanshahar outbreak. Both the serum and saliva samples were simultaneously tested for anti-HEV IgM antibodies. A total of 15 serum samples showed the presence of anti HEV IgM antibodies, out of which 10 (10/15; 66.6%) were also positive for anti-HEV IgM antibodies in saliva (χ 2 = 7.636; P b 0.0057), thus showing a concordance of 76.91%. Considering anti-HEV IgM positivity in serum as the gold standard, the sensitivity and specificity of detecting anti-HEV IgM in saliva samples were found to be 66.67% and 100%, respectively (Table 1). 3.3. Relationship between HEV-RNA and HEV-antigen HEV-RNA was detected in 18 of 60 blood samples tested (30%). HEVantigen and RNA detection picked 1 extra case which was missed by anti-HEV IgM detection in the serum. This sample was collected during the initial part of the illness (1–3 days). A high concordance of (96.66%) was observed between HEV-RNA and HEV-Ag detection. All the 16 samples positive for HEV-Ag were found to be positive for HEV-RNA (100%), and 4.5% of HEV-Ag negative samples (2/44) were found to be positive for HEV-RNA (χ 2 = 50.91; P b 0.0001) (Table 2). 3.4. Comparison between molecular and serological methods with respect to sample age The sample age was defined as the day of sample collection after the onset of symptoms. HEV-RNA and antigen were detected in 100% of patient samples collected within 1–7 days of illness, and their positivity declined to 10.2% and 5.12%, respectively, in samples collected after 7 days of illness. In contrast, anti-HEV IgM positivity in serum was 100% in samples collected 4th day onwards (Fig. 1).
HEV-antigen
Positive
Negative
Positive Negative Total Validity parameters Sensitivity Specificity Positive predictive value Negative predictive value
16 2 18 Values in % 88.89 100 100 95.45
0 16 42 44 42 60 95% CI (lower–upper) 65.29–98.62 91.59–100.00 79.41–100 84.53–99.44
Total
Concordance between HEV-RNA and HEV-Ag detection was found to be 16 + 42/ 60 = 96.66%, 100% samples positive for HEV-Ag were positive for HEV-RNA (16/16), and 4.5% of HEV-Ag–negative samples (2/44) were found to be positive for HEV-RNA (χ2 = 50.91; P b 0.0001).
submitted to GenBank (Accession numbers: JX857991.1 to JX858004.1). Phylogenetic analysis was performed using neighbor joining method, and a bootstrap value of 1000 replicates was used for statistical verification. The clustering topology of the phylogram revealed that the strain responsible for this outbreak belonged to HEV genotype 1, subtype 1a. The sequences from the 2 outbreaks formed separate clusters, and the HEV sequences retrieved from the clinical strains clustered with the respective environmental strains. The sequences retrieved from the sewage clustered with the sequences obtained from patients of the respective outbreaks giving a direct epidemiological link between the sewage contaminations of drinking water leading to the outbreak (Fig. 2).
4. Discussion This study documents 2 focal single peak outbreaks of hepatitis E which occurred at Nawanshahar, Punjab, and Palsora, Chandigarh North India, during March–April 2012. The phylogenetic analysis of HEV strains from sewage, tap water, and hepatitis patients attributed this outbreak to the sewage contamination of drinking water. The main reasons for the outbreak were found to be a) sewerage work going on in the city for past 2 months prior to the outbreak; b) use of tullu-pumps which are documented to create negative pressure in water pipes, leading to contamination with sewage water when drinking water supply is periodically off through leaky joints; c) water supply to the affected area was through a tube well, whose chlorination machine was out of order. Detection of early warning signals, well-timed
3.5. Phylogenetic analysis of HEV strains and epidemiological link HEV-RNA could be detected in both the sewage samples collected from Nawanshahar and Palsora and the tap water sample of Palsora. The sequences from 11 clinical and 3 environmental samples were Table 1 Table showing the sensitivity and specificity of detecting anti-HEV IgM in saliva, taking anti-HEV IgM positivity in serum samples as gold standard (n = 21). HEV serum IgM HEV saliva IgM
Positive
Negative
Total
Positive Negative Total Validity parameters Sensitivity Specificity
10 5 15 Values in % 66.67 100
0 10 6 11 6 21 95% CI (lower–upper) 38.41–88.05 54.05–100.00
Concordance between anti-HEV serum and saliva IgM detection was found to be 10 + 6/ 21 = 76.19%; 66.6% (10/15) samples positive for anti-HEV IgM in serum were also found positive for anti-HEV IgM in saliva (χ2 = 7.636; P b 0.0057).
Fig. 1. Line diagram showing percentage positivity of detection of anti-HEV IgM, HEVantigen, and HEV-RNA in serum samples with respect to sample age (n = 53).
Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
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Fig. 2. Phylogenetic analysis of 6 clinical strains from Nawanshahar, 5 from Palsora, sewage samples from both the cities, and tap water sample from Palsora. Phylogenetic analysis of strains causing HEV outbreaks at Nawanshahar and Palsora. Neighbor-joining tree constructed for the conserved RdRp region (ORF1) of HEV. The avaian strain of HEV (EF206691.1) is included as an outgroup. Bootstrap values of 1000 replicates were used for statistical verification. The HEV-PUL are the Palsora strains which clustered along HEV genotype 1, subtype 1a, strains reported from India along with the HEV-PUL sewage and HEV-TAP water strain. Similarly, HEV-NS are Nawanshahar strains which formed a separate cluster within HEV genotype 1, subtype 1a, and clustered along with HEV-NS sewage strain. Strain representation: genotype 1a, ●; genotype 1b, □; genotype 1c, ○; genotype 1d, ◊; genotype 1e, ʋ; genotype 2, ♦; genotype 3, ■; genotype 4, ▲.
investigation, and application of specific control measures like distribution of chlorine tablets in the community can avert the spread of the outbreak and impede on the predictable morbidity and mortality. Therefore, investigation of these outbreaks is important to identify the exact etiological agent to formulate recommendations, which can help to manage the future outbreaks systematically. Due to the vast development in the diagnostic modalities for HEV in the last decade, it has become crucial to understand their applicability with respect to the clinical course of the disease. The conventional anti-HEV IgM ELISA suffers a wide range of variations in sensitivity and specificity (Dawson et al., 1992; Favorov et al., 1992, 1994, 1996;
Khudyakov and Kamili, 2011). Recombinant protein-based assays have been reported to be more sensitive than synthetic peptide-based tests. In the present study, an ELISA kit based on a cocktail of HEV ORF-2 and 3 recombinant proteins was used. Overall, the HEV IgM positivity in the present study was found to be 86.66% which is in agreement with Favorov et al. (1992) showing IgM positivity in 40–100% epidemic samples. In the era of molecular techniques, detection of HEV-RNA by RT-PCR is considered to be the gold standard (Lin et al., 2000; Myint et al., 2006; Takahashi et al., 2005; Zhang et al., 2009). During this outbreak, 30% of the patients were positive for HEV-RNA. HEV-RNA could detect 1
Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
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additional case which was missed by anti-HEV IgM. This patient had an illness of 2 days duration, thus explaining the absence of antibody response. This patient was also positive for HEV-antigen. The HEV-RNA and HEV-antigen could be detected in (100%) of samples with a sample age of up to 7 days, and it declined rapidly to 10% and 5%, respectively, when tested in samples with a sample age exceeding 7 days. Therefore, the timing of serum collection and selection of the assay are both important for establishing a successful diagnosis. Further, a concordance of 96.66% was observed between the detection of HEV-RNA and HEVantigen. There were only 2 samples (4.5%) which were positive for HEV-RNA and negative for HEV-antigen detection. Recently, Zhao et al. (2015) had reported a concordance of 77.8% and 80.8% between HEV-RNA and HEV-antigen detection in the serum and stool samples, respectively, from acute HEV patients in Hebei province, China. The high concordance between these 2 tests has immense diagnostic implications since the detection of HEV-RNA is confined to specialized laboratories and it requires sophisticated instruments and expertise in addition to the maintenance of cold chain, making the shipment of samples to the referral laboratories difficult. In an outbreak setting, the detection of HEV-antigen by ELISA appears to be a feasible alternate system for peripheral laboratories with a prospect of wider usage in endemic resource poor countries. Conventionally, the laboratory diagnosis and epidemiological surveys for HEV require the collection of approximately 3–5 mL of blood through venipuncture requiring a trained phlebotomist. In many instances, phlebotomy in needle-phobic febrile individuals, especially children, becomes difficult which leads to significant drop in the number of total individuals screened during outbreak situation. A previous study has shown the utility of testing dried blood samples for antiHEV IgM during an outbreak with a sensitivity and specificity of 91% and 100%, respectively, compared to the blood samples using area under receiver operating characteristic curve analysis for calculating cutoffs (Singh et al., 2014). The present study has demonstrated the potential use of saliva as an alternate less invasive sample for diagnosing HEV in an outbreak set up with a sensitivity and specificity of 66.67% and 100% respectively. Saliva samples have been used for the detection of antibody, antigen, and nucleic acid for a large number of viruses including cytomegalovirus (Ross et al., 2014), rubella (Ben Salah et al., 2003; Vijaylakshmi et al., 2006), varicella zoster (Talukder et al., 2005), mumps (Reid et al., 2008), and hepatitis C virus (Scalioni Lde et al., 2014). The collection of saliva samples is a less invasive, safe, and less expensive method which makes it suitable for screening and epidemiological purposes. Though the sample size in the present study is a limiting factor, it sheds definitive light on the applicability of collecting saliva samples as an alternate for the surveillance purposes. The phylogenetic analysis based on RdRp region detected HEV genotype-1, subtype-1a, in all the clinical and environmental samples. The HEV-RNA could be detected in the un-concentrated sewage samples from both Nawanshahar and Palsora as reported previously by Arankalle et al. (2006) during investigation of hepatitis outbreak at Kerala. Traditionally, the concentration of viruses in the water samples is carried out using ultra-filtration technology (Verma and Arankalle, 2010). Due to frank contamination and visible particulate deposition, the Palsora tap water samples also demonstrated a positive result for HEV-RT-PCR. In both the outbreak localities, the sewage line was found closely placed to drinking water pipelines with rusted walls. Leakage in the sewage line led to the mixing of sewage water which was further potentiated by the use of tullu-pumps by the local residents which created negative pressure leading to the sucking of waste water into the intermittently supplied drinking water. These outbreaks occurred in 2 geographically close areas at a parallel time frame but formed 2 different clusters on phylogenetic analysis. In conclusion, the present study documents detection of HEVantigen and HEV-RNA as early diagnostic markers of HEV infection. Further, saliva can be considered as a less invasive alternative sample for detection of anti-HEV IgM in outbreak settings.
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Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
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Original article
Investigation of suspected viral hepatitis outbreaks in North West India☆,☆☆ Mini P. Singh a,1, Manasi Majumdar a,2, Kapil Goyal a, P.V.M. Lakshmi b, Deepak Bhatia c,3, R.K. Ratho a,⁎ a b c
Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh, India 160012 School of Public Health, Postgraduate Institute of Medical Education & Research, Chandigarh, India 160012 Integrated Disease Surveillance Project, Directorate of Health & Family Welfare, Punjab, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 18 June 2015 Received in revised form 8 December 2015 Accepted 9 December 2015 Available online xxxx Keywords: Hepatitis E virus HEV-antigen Outbreak Phylogenetic analysis Saliva
a b s t r a c t Hepatitis E (HEV) infection is diagnosed on the basis of serum anti-HEV IgM detection. In outbreaks, early diagnostic method is important for prompt control measures. This study compared 3 diagnostic methods in 60 serum samples collected in suspected HEV outbreaks. The suitability of saliva samples for antibody detection was also evaluated in 21 paired serum saliva samples. The anti-HEV IgM, HEV-Ag, and HEV-RNA were detected in serum samples of 52 (86.66%), 16 (26.66%), and 18 (30%) patients, respectively. The concordance between serum and saliva IgM was found to be 76.91%. The positivity of PCR and HEV-Ag detection was 100% within 1 week of illness which declined to 5–10% thereafter. The outbreak was attributed to HEV genotype 1, subtype 1a, and the clinical and environmental strains clustered together. HEV-antigen and RNA were an early diagnostic marker with 96.66% concordance. Saliva samples can be used as an alternative in outbreak setting. © 2015 Published by Elsevier Inc.
1. Introduction Viral hepatitis E, a waterborne hepatitis, has extensive epidemic potential, though focal outbreaks and sporadic cases are also frequently observed. Overall, the WHO (2014) has reported approximately 20 million hepatitis E infections, more than 3 million acute cases, and nearly 56,600 deaths due to hepatitis E. The causative agent was identified as a novel putative hepatitis E virus (HEV) during a massive waterborne epidemic of hepatitis which occurred in Delhi and caused 30,000 cases during 1955–1956 (Ramalingaswami and Purcell, 1988). This virus contributes to 30–70% of sporadic hepatitis cases in the Indian subcontinent (Aggarwal, 2011). Drinking water contaminated with sewage is the commonest mode of HEV transmission in developing nations like India. The virus is endemic in North India with documented hepatitis E outbreaks being reported from Mandi-Gobindgarh in the year 2005 (Bali et al., 2008; Kumar et al., 2006; Prinja et al., 2008) and Lalru in the year 2010 (Majumdar et al., 2013a). HEV is now regarded as the major cause of sporadic as well as epidemic hepatitis, which is no longer
restricted to Asia and the developing countries. Increasing number of cases is being documented from developed world such as United States and Europe (Clemente-Casares et al., 2003; Echevarría, 2014; Huang et al., 2002; Meng et al., 1997; Riveiro-Barciela et al., 2012). The present study reports a waterborne outbreak of viral hepatitis which occurred almost simultaneously in Nawanshahar (Punjab) and Palsora a suburb of Chandigarh in March–April, 2012. The detection of early warning signals, well-timed investigations, and application of specific control measures are important to control the outbreak. Hence, the present study compared the anti-HEV IgM, HEV-antigen, and HEV-RNA detection for the early diagnosis of a suspected HEV outbreak. The usefulness of collecting noninvasive clinical samples like saliva for diagnosis of HEV was also evaluated. Further, phylogenetic analysis of the viral strains from human and environmental samples was carried out for tracing the source of infection. 2. Materials and methods 2.1. Study design
☆ Financial grant received: Nil. ☆☆ Conflict of interest: None declared. ⁎ Corresponding author. Tel.: +91-172-2755170; fax: +91-172-2744401. E-mail addresses: [email protected] (M.P. Singh), [email protected] (M. Majumdar), [email protected] (K. Goyal), [email protected] (P.V.M. Lakshmi), [email protected] (D. Bhatia), [email protected] (R.K. Ratho). 1 2 3
Tel.: +91-172-2755199. Tel.: +91-9872186879. Tel.: +91-172-2621506; fax: +91-172-2620234.
In the month of March–April 2012, the Department of Virology, Postgraduate Institute of Medical Education and Research, Chandigarh, was informed about a suspected outbreak of enterically transmitted viral hepatitis from 2 different places, Nawanshahar in Punjab and Palsora, a village near Chandigarh, North India. An active surveillance of the affected region was carried out, and approximately 3 mL of blood samples was collected from 27 icteric patients from Nawanshahar and 33
http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002 0732-8893/© 2015 Published by Elsevier Inc.
Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
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patients from Palsora aseptically by trained health care personnel after obtaining a written informed consent. Additionally, 21 matched saliva samples were also collected from Nawanshahar patients. All the samples were transported in cold chain to the virology laboratory. Since the clinical samples were received during an outbreak for diagnostic purposes, prior ethical clearance could not be obtained. However, the study was retrospectively approved by the Institute Ethics Committee of Postgraduate Institute of Medical Education and Research, Chandigarh, as per national guidelines.
2.2. Descriptive epidemiology Nawanshahar district of Punjab and Palsora village of Chandigarh are situated at a distance of 93.8 km in the northwest part of India at a latitude of 31°N and longitude of 76°E with moderate rainfall and highest and lowest temperatures being 40 °C and 25 °C, respectively, in the months of March–April, when the outbreak occurred. Both the townships are in suburban areas with low socioeconomic living conditions, and at both places, sewage work was undertaken before the outbreak. An investigation team visited houses and collected information about cases, drinking water quality, source of water supply, and drainage system. The existing blueprint of the water supply pipelines and drains was also examined.
2.3. Description of the patient population The mean age of patients affected was 30.48 ± 14.58 years (95% confidence interval [CI]: 26.72–34.25), and the male:female ratio was 1.6:1. The predominant clinical manifestations were yellow urine (95%), fatigue (85%), yellow eye (81.66%), anorexia (75%), vomiting (65%), fever (61.66%), pain abdomen (58.33%), pruritis (30%), and arthalgia (21.66%), respectively. Liver enzymes were found to be raised, mean levels of aspartate aminotransferase (618.6 ± 115.8 IU/mL), alanine aminotransferase (694 ± 131.4 IU/mL), and mean bilirubin level was found to be 6.288 ± 3.975 mg/dL. Pregnant women with jaundice were not reported in these 2 outbreaks.
2.4. Antibody detection Sixty serum samples were tested for the following viral markers: anti-hepatitis A virus (HAV) IgM (ImmunoComb® II HAV Ab, Orgenics, Israel) and anti-HEV IgM (ImmunoVision, Springdale, AR) using commercially available enzyme-linked immunosorbent assay (ELISA) kits as per the manufacturer's instructions. Anti-HEV IgM detection system used a combination of recombinant HEV ORF-2 and ORF-3 peptide. The manufacturer of this kit has documented a sensitivity and specificity of N99% as per the kit insert. Neat saliva samples from 21 patients were diluted to 1:4 dilutions in sample diluent (i.e., 100 μL saliva + 400 μL sample diluent) and tested for the detection of anti-HEV IgM using commercially available ELISA kit (ImmunoVision).
2.5. HEV-antigen detection Sixty serum samples were tested using a commercially available HEV-antigen ELISA kit (Wantai, Beijing, China). This is a solid phase sandwich ELISA, in which the microwell strips were precoated with rabbit anti-HEV antibodies directed against ORF-2 protein. The absorbance was measured at 450 nm. According to the manufacturer's information provided, HEV-Ag cutoff = 0.12 + NC (the mean absorbance value for 3 negative controls) was calculated. Sample with absorbance higher than cutoff values were considered positive.
2.6. RNA extraction and reverse transcription–polymerase chain reaction (RT-PCR) RNA extraction was carried out from all the 60 serum samples using QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD). Briefly, 140 μL of serum was used for RNA extraction as per the manufacturer's guideline which was finally eluted in 50 μL of elution buffer. A 10-μL volume of the RNA was reverse transcribed to cDNA on the same day using M-MuLV RT and random hexameric primers (Fermentas, Hanover, MD). Nested PCR protocol was used for amplification using primers targeting the ORF 1 gene as previously described (Majumdar et al., 2013b). The amplicon of 343 bp was visualized by 2% agarose gel electrophoresis following ethidium bromide staining. With each run, 1 positive and negative control was included. Positive control was a recombinant plasmid (pIRES-neoORF1), a kind gift from Dr Shahid Jameel, ICGEB, New Delhi. Representative 6 clinical strains from Nawanshahar and 5 from Palsora, sewage samples from both the places, and tap water sample from Palsora were sequenced for confirmation of the etiological agent.
2.7. Sewage and tap water sampling Samples of untreated sewage were collected from sewage pumping stations of Nawanshahar and Palsora. A sterile glass bottle was lowered into the flowing water to collect approximately 1 L of sewage and transported to the laboratory in cold chain. Further, an early morning, free-flowing 1-L tap water sample was collected in a sterile glass bottle from a final distribution tap in Palsora. The tap water was muddy, and visible particulate materials were appreciated. Adequate personal safety precautions were taken while collecting and processing the samples. Materials used for sample collection and processing were decontaminated by autoclaving.
2.8. Sewage and tap water sample processing Briefly, 40-mL sewage sample was ultracentrifuged (110,000 × g for 1 h at 4 °C) to pellet down both the viral particles and suspended material. The sediment was eluted using 4 mL 0.25 N glycine buffer, pH 9.5. Suspended solids were then separated by centrifugation at 12,000 × g for 15 min. Viruses were finally pelleted down using a second ultracentrifugation step (110,000 × g for 1 h at 4 °C) and resuspended in 0.1-mL phosphate-buffered saline (Clemente-Casares et al., 2003). From the virus concentrate, RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen), cDNA was synthesized using M-MuLV reverse transcriptase enzyme (Fermentas), and HEV ORF1 gene was amplified as described above followed by sequencing and phylogenetic analysis.
2.9. Sequence analysis and phylogenetic tree construction HEV-specific RNA-dependent RNA polymerase (RdRp) gene was amplified from 11 clinical (6 from Nawanshahar and 5 from Palsora) and 3 environmental samples, i.e., sewage samples collected from Palsora, Nawanshahar, and the tap water of Palsora were sequenced. ABI chromatogram files were viewed using Finch TV 1.4.0, following sequence drafting with bioedit 7.0.9 software. For the confirmation of these sequences, database search was implemented using BLAST program available at NCBI Web site. For sequence comparison, standard representative strain sequences were retrieved from GenBank as previously described (Majumdar et al., 2015). Clustal X 2.0.11 program was used for multiple sequence alignment followed by Molecular Evolutionary Genetics Analysis (Tamura et al., 2007) for phylogenetic tree construction.
Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
M.P. Singh et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
3. Results
3
Table 2 Table showing the sensitivity and specificity of detecting HEV-antigen in serum samples, taking HEV-RNA positivity in serum samples as gold standard (n = 60).
3.1. Determination of serological markers in serum samples
HEV-RNA
The 60 serum samples collected from icteric patients during the 2 outbreaks were tested for anti-HEV IgM, HEV-antigen, and anti-HAV IgM. Out of 60 blood samples tested, 52 (86.66%) were positive for anti-HEV IgM; and 16 (26.66%), for HEV-antigen. Anti-HAV IgM alone could be detected in 4 (6.66%) samples, while both anti-HAV IgM and anti-HEV IgM could be detected in 2 (3.33%) patients suggesting dual infection with HAV and HEV. 3.2. Comparison of anti-HEV IgM detection in paired serum and saliva samples Paired serum and saliva samples were obtained from 21 icteric patients from Nawanshahar outbreak. Both the serum and saliva samples were simultaneously tested for anti-HEV IgM antibodies. A total of 15 serum samples showed the presence of anti HEV IgM antibodies, out of which 10 (10/15; 66.6%) were also positive for anti-HEV IgM antibodies in saliva (χ 2 = 7.636; P b 0.0057), thus showing a concordance of 76.91%. Considering anti-HEV IgM positivity in serum as the gold standard, the sensitivity and specificity of detecting anti-HEV IgM in saliva samples were found to be 66.67% and 100%, respectively (Table 1). 3.3. Relationship between HEV-RNA and HEV-antigen HEV-RNA was detected in 18 of 60 blood samples tested (30%). HEVantigen and RNA detection picked 1 extra case which was missed by anti-HEV IgM detection in the serum. This sample was collected during the initial part of the illness (1–3 days). A high concordance of (96.66%) was observed between HEV-RNA and HEV-Ag detection. All the 16 samples positive for HEV-Ag were found to be positive for HEV-RNA (100%), and 4.5% of HEV-Ag negative samples (2/44) were found to be positive for HEV-RNA (χ 2 = 50.91; P b 0.0001) (Table 2). 3.4. Comparison between molecular and serological methods with respect to sample age The sample age was defined as the day of sample collection after the onset of symptoms. HEV-RNA and antigen were detected in 100% of patient samples collected within 1–7 days of illness, and their positivity declined to 10.2% and 5.12%, respectively, in samples collected after 7 days of illness. In contrast, anti-HEV IgM positivity in serum was 100% in samples collected 4th day onwards (Fig. 1).
HEV-antigen
Positive
Negative
Positive Negative Total Validity parameters Sensitivity Specificity Positive predictive value Negative predictive value
16 2 18 Values in % 88.89 100 100 95.45
0 16 42 44 42 60 95% CI (lower–upper) 65.29–98.62 91.59–100.00 79.41–100 84.53–99.44
Total
Concordance between HEV-RNA and HEV-Ag detection was found to be 16 + 42/ 60 = 96.66%, 100% samples positive for HEV-Ag were positive for HEV-RNA (16/16), and 4.5% of HEV-Ag–negative samples (2/44) were found to be positive for HEV-RNA (χ2 = 50.91; P b 0.0001).
submitted to GenBank (Accession numbers: JX857991.1 to JX858004.1). Phylogenetic analysis was performed using neighbor joining method, and a bootstrap value of 1000 replicates was used for statistical verification. The clustering topology of the phylogram revealed that the strain responsible for this outbreak belonged to HEV genotype 1, subtype 1a. The sequences from the 2 outbreaks formed separate clusters, and the HEV sequences retrieved from the clinical strains clustered with the respective environmental strains. The sequences retrieved from the sewage clustered with the sequences obtained from patients of the respective outbreaks giving a direct epidemiological link between the sewage contaminations of drinking water leading to the outbreak (Fig. 2).
4. Discussion This study documents 2 focal single peak outbreaks of hepatitis E which occurred at Nawanshahar, Punjab, and Palsora, Chandigarh North India, during March–April 2012. The phylogenetic analysis of HEV strains from sewage, tap water, and hepatitis patients attributed this outbreak to the sewage contamination of drinking water. The main reasons for the outbreak were found to be a) sewerage work going on in the city for past 2 months prior to the outbreak; b) use of tullu-pumps which are documented to create negative pressure in water pipes, leading to contamination with sewage water when drinking water supply is periodically off through leaky joints; c) water supply to the affected area was through a tube well, whose chlorination machine was out of order. Detection of early warning signals, well-timed
3.5. Phylogenetic analysis of HEV strains and epidemiological link HEV-RNA could be detected in both the sewage samples collected from Nawanshahar and Palsora and the tap water sample of Palsora. The sequences from 11 clinical and 3 environmental samples were Table 1 Table showing the sensitivity and specificity of detecting anti-HEV IgM in saliva, taking anti-HEV IgM positivity in serum samples as gold standard (n = 21). HEV serum IgM HEV saliva IgM
Positive
Negative
Total
Positive Negative Total Validity parameters Sensitivity Specificity
10 5 15 Values in % 66.67 100
0 10 6 11 6 21 95% CI (lower–upper) 38.41–88.05 54.05–100.00
Concordance between anti-HEV serum and saliva IgM detection was found to be 10 + 6/ 21 = 76.19%; 66.6% (10/15) samples positive for anti-HEV IgM in serum were also found positive for anti-HEV IgM in saliva (χ2 = 7.636; P b 0.0057).
Fig. 1. Line diagram showing percentage positivity of detection of anti-HEV IgM, HEVantigen, and HEV-RNA in serum samples with respect to sample age (n = 53).
Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
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Fig. 2. Phylogenetic analysis of 6 clinical strains from Nawanshahar, 5 from Palsora, sewage samples from both the cities, and tap water sample from Palsora. Phylogenetic analysis of strains causing HEV outbreaks at Nawanshahar and Palsora. Neighbor-joining tree constructed for the conserved RdRp region (ORF1) of HEV. The avaian strain of HEV (EF206691.1) is included as an outgroup. Bootstrap values of 1000 replicates were used for statistical verification. The HEV-PUL are the Palsora strains which clustered along HEV genotype 1, subtype 1a, strains reported from India along with the HEV-PUL sewage and HEV-TAP water strain. Similarly, HEV-NS are Nawanshahar strains which formed a separate cluster within HEV genotype 1, subtype 1a, and clustered along with HEV-NS sewage strain. Strain representation: genotype 1a, ●; genotype 1b, □; genotype 1c, ○; genotype 1d, ◊; genotype 1e, ʋ; genotype 2, ♦; genotype 3, ■; genotype 4, ▲.
investigation, and application of specific control measures like distribution of chlorine tablets in the community can avert the spread of the outbreak and impede on the predictable morbidity and mortality. Therefore, investigation of these outbreaks is important to identify the exact etiological agent to formulate recommendations, which can help to manage the future outbreaks systematically. Due to the vast development in the diagnostic modalities for HEV in the last decade, it has become crucial to understand their applicability with respect to the clinical course of the disease. The conventional anti-HEV IgM ELISA suffers a wide range of variations in sensitivity and specificity (Dawson et al., 1992; Favorov et al., 1992, 1994, 1996;
Khudyakov and Kamili, 2011). Recombinant protein-based assays have been reported to be more sensitive than synthetic peptide-based tests. In the present study, an ELISA kit based on a cocktail of HEV ORF-2 and 3 recombinant proteins was used. Overall, the HEV IgM positivity in the present study was found to be 86.66% which is in agreement with Favorov et al. (1992) showing IgM positivity in 40–100% epidemic samples. In the era of molecular techniques, detection of HEV-RNA by RT-PCR is considered to be the gold standard (Lin et al., 2000; Myint et al., 2006; Takahashi et al., 2005; Zhang et al., 2009). During this outbreak, 30% of the patients were positive for HEV-RNA. HEV-RNA could detect 1
Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002
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additional case which was missed by anti-HEV IgM. This patient had an illness of 2 days duration, thus explaining the absence of antibody response. This patient was also positive for HEV-antigen. The HEV-RNA and HEV-antigen could be detected in (100%) of samples with a sample age of up to 7 days, and it declined rapidly to 10% and 5%, respectively, when tested in samples with a sample age exceeding 7 days. Therefore, the timing of serum collection and selection of the assay are both important for establishing a successful diagnosis. Further, a concordance of 96.66% was observed between the detection of HEV-RNA and HEVantigen. There were only 2 samples (4.5%) which were positive for HEV-RNA and negative for HEV-antigen detection. Recently, Zhao et al. (2015) had reported a concordance of 77.8% and 80.8% between HEV-RNA and HEV-antigen detection in the serum and stool samples, respectively, from acute HEV patients in Hebei province, China. The high concordance between these 2 tests has immense diagnostic implications since the detection of HEV-RNA is confined to specialized laboratories and it requires sophisticated instruments and expertise in addition to the maintenance of cold chain, making the shipment of samples to the referral laboratories difficult. In an outbreak setting, the detection of HEV-antigen by ELISA appears to be a feasible alternate system for peripheral laboratories with a prospect of wider usage in endemic resource poor countries. Conventionally, the laboratory diagnosis and epidemiological surveys for HEV require the collection of approximately 3–5 mL of blood through venipuncture requiring a trained phlebotomist. In many instances, phlebotomy in needle-phobic febrile individuals, especially children, becomes difficult which leads to significant drop in the number of total individuals screened during outbreak situation. A previous study has shown the utility of testing dried blood samples for antiHEV IgM during an outbreak with a sensitivity and specificity of 91% and 100%, respectively, compared to the blood samples using area under receiver operating characteristic curve analysis for calculating cutoffs (Singh et al., 2014). The present study has demonstrated the potential use of saliva as an alternate less invasive sample for diagnosing HEV in an outbreak set up with a sensitivity and specificity of 66.67% and 100% respectively. Saliva samples have been used for the detection of antibody, antigen, and nucleic acid for a large number of viruses including cytomegalovirus (Ross et al., 2014), rubella (Ben Salah et al., 2003; Vijaylakshmi et al., 2006), varicella zoster (Talukder et al., 2005), mumps (Reid et al., 2008), and hepatitis C virus (Scalioni Lde et al., 2014). The collection of saliva samples is a less invasive, safe, and less expensive method which makes it suitable for screening and epidemiological purposes. Though the sample size in the present study is a limiting factor, it sheds definitive light on the applicability of collecting saliva samples as an alternate for the surveillance purposes. The phylogenetic analysis based on RdRp region detected HEV genotype-1, subtype-1a, in all the clinical and environmental samples. The HEV-RNA could be detected in the un-concentrated sewage samples from both Nawanshahar and Palsora as reported previously by Arankalle et al. (2006) during investigation of hepatitis outbreak at Kerala. Traditionally, the concentration of viruses in the water samples is carried out using ultra-filtration technology (Verma and Arankalle, 2010). Due to frank contamination and visible particulate deposition, the Palsora tap water samples also demonstrated a positive result for HEV-RT-PCR. In both the outbreak localities, the sewage line was found closely placed to drinking water pipelines with rusted walls. Leakage in the sewage line led to the mixing of sewage water which was further potentiated by the use of tullu-pumps by the local residents which created negative pressure leading to the sucking of waste water into the intermittently supplied drinking water. These outbreaks occurred in 2 geographically close areas at a parallel time frame but formed 2 different clusters on phylogenetic analysis. In conclusion, the present study documents detection of HEVantigen and HEV-RNA as early diagnostic markers of HEV infection. Further, saliva can be considered as a less invasive alternative sample for detection of anti-HEV IgM in outbreak settings.
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Please cite this article as: Singh MP, et al, Investigation of suspected viral hepatitis outbreaks in North West India, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.12.002