Farhat Kazmi* , Hafiz Aamer**, Sadia Iqbal****, Saima Chaudhry***, Mateen Izhar*****, Ayaz Ali Khan******
Correspondence: “Dr. Saima Chaudhry ” <firstname.lastname@example.org>
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J Pak Dent Assoc 2010;19(3): 177 – 179
This study was done to assess the efficacy of saliva as a screening tool in Hepatitis C infection when collected in a sterile test tube by simple spitting process. Anti Hepatitis C Virus antibodies (Anti-HCV) were detected in salivary samples of patients and healthy controls without using any special salivary collection device.
Paired serum and oral fluid collections were obtained from 50 HCV positive cases and twenty five negative individuals. Saliva samples were collected in a sterile disposable plastic test tube by simple spitting of un-stimulated saliva by the study subjects. A modification of the serum HCV ELISA assay was developed to improve test accuracy for an oral fluid substrate. The presence or absence of anti-HCV antibodies in the blood serum was taken as gold standard against which anti-HCV levels in saliva were compared.
Overall sensitivity of saliva to detect Anti HCV antibodies was found to be 94.2% while specificity was 100%.
This simple method of oral fluid collection proved to be an effective alternative to special collection Saliva can be used as a cost effective screening tool for initial screening of Hepatitis C infection in high risk populations.
Hepatitis C infection, Anti HCV Antibodies, Saliva, Screening, Pakistan.
Hepatitis C virus (HCV) is a RNA virus that leads to chronic hepatitis and, ultimately, to liver cirrhosis and hepatocellular carcinoma 1.
Worldwide 170 million people are chronically infected with HCV, almost 3-4 million persons are newly infected each year and all are at risk of developing liver cirrhosis
and liver cancer 2. The prevalence of HCV infection in Pakistan is 4.9% with rates as high as 13.1% in certain areas 1.
The provisional diagnosis of hepatitis C is generally made based finding of anti HCV antibodies in serum of patients having clinical signs and symptoms of the disease 4. However, in recent years due to the invasive nature of blood sample collection and hazards of cross infection, the need for finding other body fluids as an alternative to blood has immensely increased 5. Other body fluid that is closest in composition to blood serum is saliva. Saliva is known as the mirror of blood and is being used successfully for diagnostic and monitoring purposes in various infectious diseases 6. The benefits of saliva are not only non-invasive detection of diseases but it also provides a cost effective approach for the screening of large population. Diagnosis of diseases via salivary analysis is also a more practical choice for children, elderly and medically compromised population 6.
Anti HCV antibodies have been successfully detected in saliva of infected patients with sensitivity values of 84.1% to 98.2% and specificity values of 99.1% to 100% by various researchers 7-13. This high detection rate in oral fluid makes it an ideal tool for screening of Hepatitis C infection in high risk populations. HCV is spreading like an epidemic in Pakistan. There is a strong need for screening the population of the country to know the exact prevalence of the disease. Analysis of anti HCV antibodies in saliva seems to be the ideal tool for screening but the problem using saliva is the expensive devices needed for collection of the fluid. Different collection devices like Orasure 13, 14 or Oracol 10, are available in the market and these have been used by various researchers. In low resource settings like Pakistan screening of infection through these devices can be costly. Therefore, the present study was designed to find out the sensitivity and specificity of saliva in detecting anti-HCV antibodies as compared to serum by collecting the patient’s sample in a sterile tube via simple spitting process
This cross-sectional pilot study was conducted in the department of gastroenterology and microbiology, Shaikh Zayed Medical Complex, Lahore, Pakistan. A total of 50 diagnosed hepatitis C positive patients were included in the study. Control group comprising of age matched 25 healthy subjects were recruited from the dental outdoor of the same hospital. The consent proforma for each subject was duly completed and ethical approval was obtained from hospital’s Ethical Committee.
Approximately 4 ml of the blood was drawn from each study subject by anti-cubital venepuncture in a 10 ml disposable syringe. Serum was separated and samples were stored in polypropylene tube at 200 C until assayed. Serum samples were analyzed for the serum anti HCV, using LG HCD 3.0 plus hepatitis C virus antibody diagnostic kit, according to instructions of supplier. Cut-off value for the test was 0.411. Samples with value less than the cut-off value were considered non-reactive and no further testing was done. Samples with values greater than or equal to the cut-off value are considered initially reactive and retested in duplicate, before final interpretation, sample was considered positive if either or both of the values of duplicate were greater than or equal to the cut-off value. Saliva samples were also collected from all subjects. Subjects were asked to refrain from eating, drinking, and smoking or oral hygiene procedures for at least one hour prior to saliva collection. Un-stimulated saliva was collected in a sterilized container by simple spitting process. Saliva was centrifuged and diluted by normal saline by 1: 1 ratio. Salivary anti-HCV antibody was detected using the same kit, however, the specimen volume was increased to 1 volume of saliva (100µl) to 1 volume of diluents (100µl) and the incubation period was increased to overnight (~ 20 h) at room temperature. None of the oral fluid sample was excluded. All HCV sero-positive and sero-negative samples were analyzed for the presence of HCV antibodies in serum and in saliva
Hepatitis C positive patients comprised of 15 males (30 %) and 35 females (70 %). Their age ranged from 16 yrs to 70 yrs with a mean of 45.60 ±10.61 years in females and 51.60± 13.60 in males. In the control group 10 (40%) were males and 15 (60%) were females. Considering serum HCV antibody test as gold standard, the sensitivity and specificity of saliva HCV antibody test was determined. Out of 50 HCV positive cases, 48 were correctly detected as positive suggesting an overall sensitivity of 96.0%. Out of 25 negative subjects the saliva HCV antibodies were found negative in all the 25 samples.
The specificity of using saliva as diagnostic fluid was 100%. The positive predictive value (PPV) was 100% while negative predictive value (NPV) was found to be 96% (Table 1).
Hepatitis C (HCV) infection is common in developing countries, where blood sampling and expensive sophisticated methods for detection are less available. The biggest concern with the use of blood in diagnosing HCV infection is reuse of disposable needles. The syringe which is used for sample collection from a patient who is HCV positive when reused without sterilization can lead to dramatic spread of infection 15. According to WHO 39.6% – 70% of injections are given with needles and syringes without sterilizing them between use 16. The other hazard is the unsafe practices by the health-care workers that include poor disposal of infected syringes that can result in needle-stick injuries and further spread of HCV infection among health care workers and the community 16. By not using injections for sample collection in a suspected patient we can reduce the chances of spread of infection in countries where reuse of infected syringes and improper hospital waste management is a problem. We have found saliva to be an effective tool for screening tool for identification of Hepatitis C infection. Different studies have analyzed saliva for detection of anti-HCV antibodies 13, 16.
All these studies are different from the present investigation in terms of the collection devices used. Most of the researchers have used Orasure to collect salivary samples 13, 14. Saliva collectors involves a sponge swab which is rubbed between the teeth and the gums for approximately 2 min. Oral fluid is later recovered by centrifugation at 2500 rpm for 15 minutes at 10ºC, and then stored at 20ºC until analysis. The samples were analyzed through Ortho HCV 3.0 assay revealing a specificity of 100% but the sensitivity was in a range of 75% to 84% which is lower as compared to our findings. De Cock et al. used Oracol device collection with Ortho HCV 3.0 reached the sensitivity of 84% 10. These differences in results could be attributed to the collection devices used, the modifications applied for testing saliva and the populations sampled. In the present investigation saliva collection was done by normal spitting process. Considering serum HCV test as gold standard, the sensitivity and specificity of saliva was comparable to serum in the present investigation. Saliva can be effectively used for screening, provisional diagnosis and monitoring of HCV infected individuals.
It can be invaluable for patients who are afraid of needles and also small children. The simplicity of this technique makes it a valuable tool for epidemiological studies and public health surveys in areas where HCV infection is spreading like an epidemic. Future studies on larger populations and employing molecular methods for detection of HCV infection in saliva are suggested in Pakistani population and other high risk groups to validate the present finding
Saliva is a suitable alternative to blood for screening of Hepatitis C infection in high risk populations. When collected in test tubes with simple spitting process the approach also becomes user friendly and cost effective.
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