|Year : 2017 | Volume
| Issue : 3 | Page : 417-420
Investigation of an outbreak of varicella in Chandigarh, North India, using a real-time polymerase chain reaction approach
Mini P Singh1, Ramanpreet Kaur1, Archit Kumar1, Madhu Gupta2, Shubha Garg1, RK Ratho1
1 Department of Virology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 School of Public Health, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Web Publication||12-Oct-2017|
Mini P Singh
Department of Virology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
Outbreaks of varicella are reported when susceptible population accumulates. This study reports a chickenpox outbreak in Burail in August 2014, wherein 20 laboratory-confirmed cases were identified by the detection of varicella zoster virus (VZV) DNA and VZV IgM antibodies. The viral load between vesicular swabs and serum samples from 8 patients with active lesions was found to have good correlation and further also related with disease severity. Real-time polymerase chain reaction can be useful for early diagnosis of an outbreak and vesicular swab can be used as a less invasive sample for assessing the disease severity.
Keywords: Outbreak, real-time polymerase chain reaction, varicella, vesicular swabs
|How to cite this article:|
Singh MP, Kaur R, Kumar A, Gupta M, Garg S, Ratho R K. Investigation of an outbreak of varicella in Chandigarh, North India, using a real-time polymerase chain reaction approach. Indian J Med Microbiol 2017;35:417-20
|How to cite this URL:|
Singh MP, Kaur R, Kumar A, Gupta M, Garg S, Ratho R K. Investigation of an outbreak of varicella in Chandigarh, North India, using a real-time polymerase chain reaction approach. Indian J Med Microbiol [serial online] 2017 [cited 2018 Apr 26];35:417-20. Available from: http://www.ijmm.org/text.asp?2017/35/3/417/216620
| ~ Introduction|| |
Varicella is a highly contagious disease caused by varicella zoster virus (VZV) belonging to the family Herpesviridae. The transmission of the disease is direct through skin lesion or airborne spread. The primary infection is self-limiting and results in acute varicella which usually presents as fever, malaise and a widely disseminated vesicular and pruritic rash predominantly involving the trunk and face. The disease has an incubation period of 10–21 days, with a high secondary attack rate of >85% among susceptible contacts.
Before the varicella vaccine was licensed, approximately 4 million cases, 10,500 hospitalisations and 105 deaths due to varicella were reported each year. The introduction of a live attenuated VZV vaccine led to substantial decline in morbidity and mortality due to varicella in the United States. Despite the high vaccination coverage, outbreaks still continued to occur in schoolgoing children between 2001 and 2005, resulting in the introduction of a two-dose vaccination schedule in 2006, which lead to a drastic decline in the number of cases. However, VZV vaccine is still not included in the Universal Immunisation Programme of India, and hence, sporadic outbreaks are still reported., The present study reports an outbreak in Burail area near Chandigarh, India.
| ~ Materials and Methods|| |
After receiving information regarding cases of maculopapulovesicular rash with or without fever in August 2014, active surveillance of the area was initiated. Samples were collected after written informed consent in case of adults/parents in case of children. This study was approved by the Institute Ethics Committee. In all, 28 clinical cases were identified and blood samples obtained from 26 cases and 27 age-matched and sex-matched controls. The serum samples obtained were then tested qualitatively for the presence of anti-VZV IgM and IgG antibodies using commercial kit (Demeditec Diagnostics, GmbH, Germany). Vesicular swabs were obtained from 8 cases showing active lesions. The disease severity was assessed as described earlier. The score index for mild was ≤7, moderately severe was 8–15 and for severe disease was ≥16.
Both the vesicular swabs and serum samples of active cases were subjected to DNA isolation using commercial kit (Qiagen, Germany) followed by real-time polymerase chain reaction (PCR) using 1X TaqMan Universal master mix II (Applied Biosystems, USA), 500 nM of each primer (Forward = 5'CCATCCGATTATGC CACCTTTACAG3'; Reverse = 5'TCGAACGTTA GAGCGCACAA3) and 250 nM of NED labelled probe (5'TTGGAGGAAAACGTCTTTTTT3') targeting ORF 28 (DNA polymerase) and run on ABI 7500 real-time instrument.(Applied Biosystems, USA).
The Spearman's correlation coefficient (rs) and Mann–Whitney U-tests were performed. The P < 0.05 was considered statistically significant The SPSS version 22.0 (IBM, USA) was used for the statistical calculation.
| ~ Results|| |
In the present outbreak, 28 suspected cases of chickenpox were identified with median age of 8 years (1.5–38 years) and male-to-female ratio of 0.55:1. The cases occurred from the 3rd week of May and lasted till the 2nd week of August 2014. The children with age <15 years were mainly affected (64.3%). Two pregnant women were also infected, with a gestation period of 32 weeks and 28 weeks, respectively. The cases mainly presented with maculopapulovesicular lesions (96.43%) and fever (82.14%). None of the patients had received acyclovir treatment.
Out of 26 blood samples collected, 18 (69.23%) were found to have VZV IgM antibodies. Similarly, 18 (69.23%) patients were positive for VZV IgG antibodies. Both VZV IgM and IgG antibodies were detected in 14/26 (53.87%) patients. One out of 27 healthy controls was positive for VZV IgM antibodies, whereas 13/27 (48.15%) had IgG antibodies to varicella.
The VZV DNA could be demonstrated by real-time PCR in all the 8 vesicular swabs and 8 serum samples with mean viral load of 61,500 copies/μl (93.3–4.9 × 105 copies/μl) and 40,000 copies/μl (17.9–1.7 × 106 copies/μl), respectively [Table 1]. A statistically significant positive correlation was found between viral load of vesicular swabs and serum samples rs= 0.88; P = 0.004. Out of 8 patients with active disease, 2 had mild infection and 6 had moderate/severe illness. A significant difference was found in viral load (serum and vesicular swabs) of patients with moderate/severe illness (median = 90500 copies/μl and 1, 95,500 copies/μl) compared to patients with mild presentation (median = 30 copies/μl and 354 copies/μl) (P = 0.046) [Figure 1].
|Figure 1: Correlation of viral load with clinical disease severity in patients with active lesions. (a) Vesicular swabs and (b) serum|
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| ~ Discussion|| |
This study reports the investigation of an outbreak of varicella in Burail which is inhabited by population from neighbouring states of Chandigarh who have low socioeconomic status and is overcrowded. As observed earlier,, children <15 years of age were predominantly affected. Out of 28 cases, 20 were laboratory confirmed (18 confirmed by IgM testing and 2 additional samples positive by real-time PCR). Among 26 blood samples tested, 8 were found to be negative for anti-VZV IgM antibodies. Out of these 8 patients, 4 gave a history of previous varicella exposure and were positive for VZV IgG antibodies. Four patients did not have both anti-VZV IgM and IgG antibodies and gave a history of rash duration <5 days by which time the IgM antibody response may not have developed.
The detection and quantification of viremia during varicella infection may be potentially useful for diagnostic, prognostic and therapeutic monitoring purposes. In our study, real-time PCR could detect viremia in patients presenting as early as 3 days of illness. An early diagnosis in an outbreak situation is desirable for prompt initiation of control measures, to prevent the further spread of outbreak, especially to pregnant women and immunocompromised individuals.
Varicella diagnosis and management in pregnancy demands little more attention based on the mortality rates. Two pregnant women gave history of exposure to varicella cases, out of which one was detected to have fetal anencephaly during her level-II ultrasound at 18–20 weeks of gestation; however, she did not terminate her pregnancy and later acquired VZV infection. This patient had a high viral load in both serum and vesicular swab samples. She did not take any acyclovir treatment, and on follow-up, she had intrauterine fetal death. In contrast, the other pregnant lady who presented in convalescent phase received acyclovir treatment and delivered a healthy term baby.
The quantitative viral load detection has also been found useful in monitoring the treatment response of patients receiving antiviral therapy against VZV.
A limitation of our study was that the sample size of active cases was small to definitely comment on the correlation of viral load with disease severity. A positive correlation between viral load of vesicular swabs and serum samples suggests that vesicular swab samples can be used as an alternative to serum samples for viral load determination which is a less invasive procedure.
Majority (69.2%) of the cases identified in our study were primary cases of varicella as shown by the presence of VZM IgM antibodies. However, 4 patients had only anti-VZM IgG antibodies and were negative for anti-VZM IgM antibodies possibly suggesting reinfection. A limitation of our study was that the patients were not followed up as the basic aim of this study was to assess the role of real-time PCR in early diagnosis of varicella.
The introduction of varicella vaccine in the United States (1995) has drastically reduced the number of varicella cases, and thereby decreasing hospital admissions (88%) and childhood mortality (74%–92%) (2002). VZV vaccine can also be useful in outbreak settings if given within 3–5 days of exposure. This vaccine is found to have a good safety profile and high effectiveness, especially in immunocompetent individuals. The available evidence also support the use of two-dose vaccine over single dose as the second dose of varicella vaccine appears to be highly effective (93%) in preventing disease of any severity. Furthermore, the individuals receiving two doses of vaccine develop breakthrough disease less often, and when breakthrough disease does occur it is generally milder when compared to that unvaccinated individuals.,
This vaccine is presently recommended by the Indian Academy of Paediatrics to be given as two doses. However, it is still not a part of Universal Immunisation Programme of our country. This contributes to one of the major reasons for the repeated occurrence of outbreaks. Various concerns which have decreased the worldwide acceptance of this vaccine are fear of increase of Herpes zoster incidence, the fear of age shift of the disease towards older age groups, and the economic burden of universal vaccination implementation. The vaccine is currently available in India as Okavax (Aventis Pasteur) and Varilrix (GlaxoSmithKline) with approximate price of 1200–1500 INR.
The authors recommend that this vaccine should be included in the national immunisation schedule to control these outbreaks and a sustained use can also pave the way to a world without varicella.
Financial support and sponsorship
This research did not receive any specific grant from funding agencies in public, commercial or not-for-profit sectors. The work was carried out as a part of routine investigation of an outbreak of VZV.
Conflicts of interest
There are no conflicts of interest.
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