Burden of Rubella virus infection among females attending tertiary care hospitals of Odisha, India: a need for adult women vaccination

ABSTRACT

Rubella is a contagious disease caused by rubella virus leading to adverse outcomes among pregnant women including abortions, low birth weight, stillbirths and congenital rubella syndrome (CRS) in the baby. If not pregnant, the clinical manifestations are mild and self-limiting. In this hospital based cross-sectional study, 1985 blood samples were collected from females attending outpatient services of various hospitals to serologically detect Rubella infection. Rubella antibodies namely Immunoglobulin M (IgM)/Immunoglobulin G (IgG) were detected through enzyme linked immunosorbent assay (ELISA) or by identifying virus through polymerase chain reaction (PCR). From the total enrolled participants, 1951 samples were tested with age ranging from 16 to 38 years. Among the positive samples, about 60% patients had IgG antibodies as compared to less than 1% IgM and 0.40% by PCR. Out of 1951 samples, 7/849 (0.82%) and 651/1070 (60.8%) had IgM & IgG rubella antibodies respectively. The odds of having abortion was [OR-13.14 (4.94–34.97)] among anti-rubella positive and primi-gravida [OR-43.6 (5.9–322)] women. Therefore, vaccination of women against rubella before planning of pregnancy or at adolescence seems to be the need of hour to avoid the ill consequences during pregnancy as well as for the new born baby.

KEYWORDS:

• Epidemiology
• rubella virus
• pregnancy
• vaccination
• India
• sero-surveillance

Rubella is an acute, mild viral illness caused by rubella virus, belonging to the Togaviridae family.¹ It is a contagious disease which spreads through droplets. Clinically, it presents as rash which usually appears after two weeks of infection followed by mild fever and sore throat. Sometimes arthralgia, orchitis and inflammation of nerves may appear.² Although clinical manifestations are mild and self–limiting yet, it may manifest adverse effects if exposed at the time of pregnancy. Rubella exhibits teratogenic effect during pregnancy, especially in the first trimester,³ capable of causing serious congenital defects such as low birth weight,⁴ stillbirth⁵ and even abortion of the fetus. The virus can potentially cross placental barrier causing congenital rubella syndrome (CRS) in the baby.⁶ This may affect eyes,⁷ ears and even heart of the developing child. Hepatomegaly and jaundice may also be seen in these newborns.⁸ Acute encephalitis syndrome is another clinical manifestation of the virus affecting babies born to the infected mothers.⁹ It is estimated that, the rubella infection during 8–10th week of pregnancy may potentially have adverse effects on 90% of the newborn infants causing multiple defects. If the infection is acquired during 11–16th week of pregnancy, the risk of harm to the fetus reduces by 10–20%. The chances of adverse effects on fetus are rarely seen in infections after 16 weeks of gestation.¹⁰

This infection has a global distribution affecting a larger human population. Although, rubella vaccination has successfully reduced the infection yet, World Health Organization (WHO) estimates about 100,000 children are born with CRS every year worldwide.¹¹ Before the introduction of vaccination, outbreaks occurred in spring and summer.¹ Infection is uncommon in pre-school children but outbreaks involving school children and young adults have been reported.

Rubella is a potentially eradicable or vaccine preventable disease. With the vaccination programs in place, many countries have succeeded in controlling fatal CRS but India has yet not initiated rubella vaccination in a programmatic manner. The disease spreads faster as approximately fifty percent of the infected individuals are asymptomatic. The asymptomatic nature of this infection emphasizes to conduct a sero-surveillance of rubella through which the actual burden of the infection will be known. Hence, this study was planned to estimate the prevalence of rubella infection/exposure and its effects on pregnancy by analyzing the samples collected from women attending hospitals in Odisha, India.

A hospital based observational study was conducted among the female patients consulting in the Obstetrics & Gynecology (Obs. & Gyn.) department of various hospitals across Odisha, India. Based on the patient load in the region, five tertiary care hospitals were selected. Females of the reproductive age group, 16–45 years, consulting at Obs. & Gyn. outpatient department for regular checkup were included in this study which permitted us in managing ineligibility and non-response. We excluded debilitating patients who could not give consent. Participants not willing to respond/give samples in mid of the interview were also excluded.

This study was conducted from February 2012 to March 2017 at the Regional Virology Research and Diagnostic Laboratory (VRDL) of Regional Medical Research Center, Bhubaneswar. Blood samples were collected from participants who gave written informed consent. Approximately, 2 ml of blood sample was collected from each participant for serological evaluation of rubella virus through enzyme linked immunosorbent assay (ELISA) kits manufactured by NovaTec Immundiagnostica GmbH, Germany. Then, these sera samples were classified as positive/negative based on anti-rubella virus Immunoglobulin M (IgM)/Immunoglobulin G (IgG) antibodies respectively. Polymerase chain reaction (PCR) was used to diagnose Rubella virus using suspected patients’ serum. PCR was performed taking viral RNA as template using Qiagen kit, Germany. The PCR product of 185 bp Deoxyribo Nucleic Acid (DNA) was identified in 2% agarose gel electrophoresis. Nested PCR was done with the final volume of 25 µl. Further, the final product was processed in 2% agarose gel electrophoresis to obtain the desired product size of 143bp. PCR was performed in the samples found negative for both IgG and IgM test.

The IgG/IgM tests for rubella antibodies was qualitatively done as quantitative ELISA kits were not available at the time of this study. The cutoff value for determining IgG was more than 0.2 NTU (NovaTec Units). We followed kit specific criteria for assay to be considered valid, with the value of substrate blank observed as absorbance value <0.100; negative control as absorbance value< cutoff; cutoff control as absorbance value 0.150–1.300 and positive control as absorbance value> cutoff. If these criteria were not met, test was considered invalid and hence, repeated. The cutoff is the mean absorbance value of the cutoff control. NTU is determined by sample (mean) absorbance value multiplied * 10/ cutoff.

Data were analyzed using STATA v.16 (STATA Corp, Texas) software. Descriptive analysis was done to summarize data in frequency and percentage. Further, Chi-square test was performed to calculate the association between recent and past infection with their correlates such as age, pregnancy status and history of abortion. This study was approved by the Institutional Ethics Committee of the ICMR-Regional Medical Research Center, Bhubaneswar, Odisha. Informed written consent was obtained from participants prior to the screening and enrollment.

A total of 1985 human blood samples were collected during the study period. All participants were females with age ranging from 16 to 38 years. Samples were received in cold chain for the investigation of rubella infection. We estimated IgM and IgG antibodies against rubella along with PCR test results of 1951 samples (Table 1(A)). Thirty-four samples could not be processed either due to insufficient quantity of the sample received or error in processing.

Table 1. Rubella infection status and its distribution across various demogrpahic characteristics

(A) Number of samples tested for rubella antibodies through various screening tests
Screening test conducted			Number of samples tested				Number positive (%)
IgG ELISA			1070				651 (60.8)
IgM ELISA			849				7 (0.82)
PCR			246				1 (0.40)
IgM +PCR			239				1 (0.41)
IgG+PCR			233				1 (0.42)
IgG+IgM			277				4 (1.44)
One sample is positive for all the tests.
(B) Age wise distribution of patients with their infection status.
Age group (in years)	Number of patients enrolled (n = 1951)			Anti-rubella virus IgM positive/total patients (%)[7/849 (0.82)]		Anti-rubella virus IgG positive patients (%)[651/1070(60.8)]			Rubella virus PCR positive (%)[1/246(0.40)]
16–20	373			3/158(1.9)		128/191(41.27)			0/62(0)
21–25	950			2/413(0.48)		307/513(59.84)			0/105(0)
26–30	541			2/232(0.86)		182/305(59.67)			1/59 (1.69)
31–35	84			0/43(0)		31/58(53.44)			0/17(0)
36–38	3			0/3(0)		3/3(100)			0/3(0)
(C) Anti-rubella IgG antibodies across age groups.
Age group (in years)		IgG positive			IgG negative			Statistical analysis
16–20		128			63			X^2 = 3.9, *p < .05
21–35		520			356
*Significant at p < .05
(D) Anti-rubella IgM antibodies across age groups.
Age group (in years)		IgM positive			IgM negative			Statistical analysis
16–20		3			155			X^2 = 2.7, *p < .10
21–35		4			684

*Significant at p < .10.

Further, participants were categorized into five age groups in the intervals of five years each. The last age group composed of all left out participants from 36 to 38 years age. We estimated rubella infection to be highest among 21–25 years age group. We observed IgM antibodies to be highest (1.9%) amongst 16–20 years of age as shown in (Table 1(B)). The prevalence of IgM antibodies is more commonly seen in adolescents reflecting recent or new infection whereas amongst young adults, IgG antibodies were more prevalent which indicates past infection.

To evaluate the status of recent and past infection, two groups were formed based on the age (Table 1C & D). Group 1 comprised of adolescents aged 16–20 years and group 2 consisted of young adults (majority of the participants) 21–35 years of age. We observed a significant association (p < .05) between IgG antibodies reflecting past infection across different age groups (Table 1C).

Further, we analyzed association between rubella infection and the status of pregnancy i.e primi-gravida or multi-gravida. It was found that about 94.76% (543/573) pregnant women had anti-rubella virus antibodies. Significantly, anti-rubella virus antibodies were seen more among prima-gravida women 99.69% (326/327) as compared to 86.01% (29/246) multi-gravida women (p < .0001) (Table 2). The abortion rate was more amongst women 98.73% (389/394) having antibodies to rubella virus than non-infected women 85.54% (148/173) (p < .0001) (Table 2). The odds of having abortion was [OR-13.14 (4.94–34.97)] among anti-rubella positive and primi-gravida [OR-43.6 (5.9–322)] women.

Table 2. Relationship between Rubella virus antibodies with pregnancy status and abortion

Parameters		Anti-rubella virus positive	Anti-rubella virus negative	Statistical analysis	Odds ratio
Pregnancy status	Primi-gravida	326	1	X^2 = 37.4, *p < .00001	43.6 (5.9–322)
	Multi-gravida	217	29
Abortion	Yes	389	5	X^2 = 41.7, * p < .00001	13.14 (4.94–34.97)
	No	148	25

*Significant at p < .05

Serological data are more essential to estimate the epidemiology of any infectious disease, followed by which health policy makers can design the intervention program more effectively. Although, there are studies on rubella infection amongst pregnant women, but the data from the eastern region (Odisha) of India is scarce. In this study, more samples were collected from adult females as we wanted to focus on the effects of rubella serology on pregnancy in order to help policy makers design a vaccination or intervention strategy. Adolescent females of reproductive age group were tested for rubella antibodies to get a reference suggestive of intervention requirement. Also, these young adolescent girls are at the stage of becoming mothers in near future, thus, an emphasis on their health status is much required.

In India, about 10% to 28% pregnant women have no immunity to rubella virus^12–14 so, the risk of acquiring the infection during pregnancy is high. Various studies from Delhi, North India;¹⁵ Puducherry;¹⁶ and South India have reported rubella IgG sero-positivity in approximately 87% of pregnant women which is consistent with the findings of this hospital-based study where 94.76% pregnant women had IgG antibody. The presence of IgG antibody in adults indicates a past rubella infection which might have occurred during childhood. Positive IgG can be caused not only by infection but also may be due to vaccination.

Furthermore, presence of IgM were found more frequently among adolescents reflecting the initiation of the infection at an early age where it persists in acute stage. Most of the acute infection starts at an early age and continues to reflect in the form of IgM antibodies even in the adulthood. The childhood vaccination program against rubella initiated in the campaign mode is a good step towards eliminating rubella. Since, the campaign is relatively new, the chances of rubella infection amongst young women of reproductive age does not decrease as they would have not been vaccinated through this campaign in their childhood. This is supported by this study which shows a large number of adult women have IgG antibodies reflective of past infection. The chances of re-infection have been demonstrated by the recent reports which further poses a greater challenge.¹⁷ Most of the re-infection occurs in the subjects previously vaccinated. Also, the present study reports the rate of abortion is higher among primi-gravida in comparison with multi-gravida which could be due to the developed immune tolerance in the later pregnancies.

In India, rubella vaccination has yet not been included in routine immunization program. Hence, a small fraction of population who are capable of affording vaccination gets vaccinated against rubella. This study suggests that prevalence of antibodies is high among adult females who require intervention at an early age.

The strength of this study is that a large number of samples for rubella virus were collected and screened. But samples from 34 participants could not be processed. Another limitation of the study is that PCR could be performed in a limited number of samples only.

Rubella vaccine should be included in the childhood vaccination program in order to decrease the burden of disease, but yet, it is not enough as the immunity following vaccination decreases over time. Current vaccination campaign along with add on immunization is required for women of childbearing age.¹⁸ This should potentially be given to all females in their early adolescent years preferably 16–20 years of age. It will help in bringing down the ill effects of rubella virus amongst young adults and hence on pregnancy and its adverse outcomes on their child.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Authors contributions

PS conceptualised the idea. JS, SS and BD carried out diagnostic tests. AS, PS and SP did the statistical analysis, manuscript writing and editing. PS and SP monitored investigations in the laboratory. SP supervised the study and provided overall guidance.

Acknowledgments

We would like to thank Ms Shubhadra Priyadarshini for supporting with statistical analysis of the study. The authors are also thankful to anonymous volunteers who participated in this study. We are also grateful to Indian Council of Medical Research, New Delhi for providing resources and operational support through VRDL laboratory of RMRC, Bhubaneswar.