Investigation of protective level of rabies antibodies in vaccinated dogs in Chennai, India.

BACKGROUND: Rabies is still endemic in India causing an estimated 20,000 human deaths a year. Free roaming dogs and unvaccinated owned dogs play a major role in the maintenance of the disease. Dog vaccination is the most crucial aspect of rabies prevention and control strategies; therefore vaccine immunogenicity and longevity are important determinants of the efficiency of rabies control efforts. METHODS: In this study at Madras Veterinary College, India, a total of 297 serum samples were collected from owned dogs that were vaccinated against rabies. Data regarding age, gender, breed, neuter status and last date of vaccination were collected at the time of blood collection. The level of rabies virus neutralising antibodies in the sera of these dogs was measured through rapid focus fluorescence inhibition test. The factors associated with protective level of rabies antibodies in vaccinated dogs were investigated through multivariable regression analysis. RESULTS: This cross-sectional investigation shows that only 40% (119/297) of the all the dogs in the study showed presence of protective level of anti-rabies antibodies, and 40% (72/180) of the dogs vaccinated within the last year showed presence of protective levels of antibodies causing concern about rabies vaccine quality and its impact on rabies control. The study also shows that older and neutered dogs are more likely to have protective titre among vaccinated dogs, while non-descript breed dogs are less likely to have a protective titre compared to pure breeds. CONCLUSION: In this study 60% (108/180) of young prima dogs and adult dogs did not show protective levels of antibodies within the year of last rabies vaccination, although they had previous vaccination history. This high percentage of apparent non-responders is a cause of concern of administration, distribution, storage, potency and quality management of vaccines in India.

Awareness of and willingness to use pre-exposure prophylaxis (PrEP) among people who inject drugs and men who have sex with men in India: Results from a multi-city cross-sectional survey.

INTRODUCTION: Pre-exposure prophylaxis (PrEP) is effective in reducing HIV transmission among key populations. In India, where PrEP is not currently part of the national HIV program, little is known about PrEP awareness, willingness to use PrEP, and barriers to uptake among people who inject drugs (PWID) and men who have sex with men (MSM). METHODS: We used respondent-driven sampling to accrue PWID and MSM in 22 sites from August 2016 to May 2017. Participants were asked about awareness of PrEP, willingness to use PrEP (following a brief description) and reasons why they might not be willing to use PrEP. Participants were also queried on preferences for PrEP delivery modality (oral vs. injectable). Multi-level logistic regression models were used to determine participant correlates of willingness to use PrEP. Estimates were weighted for the sampling method. RESULTS: A total of 10,538 PWID and 8,621 MSM who self-reported being HIV-negative were included in the analysis. Only 6.1% (95% confidence interval [CI]: 5.9, 6.3) of PWID and 8.0% of MSM (95% CI: 7.7, 8.4) were aware of PrEP. However, willingness to use PrEP was substantially higher in both groups: 52.4% of PWID and 67.6% of MSM. Participants commonly cited a perceived low risk for acquiring HIV infection, being perceived by others as being HIV-positive, and side effects as reasons why they would be unwilling to use PrEP. Among PWID, sharing needles and hazardous alcohol use were associated with increased willingness to use PrEP. Among MSM, having a main male partner and injection drug use were associated with increased willingness to use PrEP. Preference for daily oral or monthly injectable PrEP was similar among MSM (39.6%% vs. 41.7%,), while PWID were more likely to prefer oral to injectable administration routes (56.3% vs. 31.1%). CONCLUSIONS: As India plans to roll-out of PrEP in the public sector, our multi-city survey of PWID and MSM highlights the need for key population-focused education campaigns about PrEP and self-assessment of risk.

Local infiltration of rabies immunoglobulins without systemic intramuscular administration: An alternative cost effective approach for passive immunization against rabies.

Presently the dose of rabies immunoglobulin (RIG) which is an integral part of rabies post exposure prophylaxis (PEP) is calculated based on body weight though the recommendation is to infiltrate the wound(s). This practice demands large quantities of RIG which may be unaffordable to many patients. In this background, we conducted this study to know if the quantity and cost of RIG can be reduced by restricting passive immunization to local infiltration alone and avoiding systemic intramuscular administration based on the available scientific evidence. Two hundred and sixty nine category III patients bitten by suspect or confirmed rabid dogs/animals were infiltrated with equine rabies immunoglobulin (ERIGs) in and around the wound. The quantity of ERIG used was proportionate to the size and number of wounds irrespective of their body weight. They were followed with a regular course of rabies vaccination by intra-dermal route. As against 363 vials of RIGs required for all these cases as per current recommendation based on body weight, they required only 42 vials of 5ml RIG. Minimum dose of RIGs given was 0.25 ml and maximum dose given was 8 ml. On an average 1.26 ml of RIGs was required per patient that costs Rs. 150 ($3). All the patients were followed for 9 months and they were healthy and normal at the end of observation period. With local infiltration, that required small quantities of RIG, the RIGs could be made available to all patients in times of short supply in the market. A total of 30 (11%) serum samples of patients were tested for rabies virus neutralizing antibodies by the rapid fluorescent focus inhibition test (RFFIT) and all showed antibody titers >0.5 IU/mL by day 14. In no case the dose was higher than that required based on body weight and no immunosuppression resulted. To conclude, this pilot study shows that local infiltration of RIG need to be considered in times of non-availability in the market or unaffordability by poor patients. This preliminary study needs to be done on larger scale in other centers with long term follow up to substantiate the results of our study.

Atypical rabies encephalitis in a six-year-old boy: clinical, radiological, and laboratory findings.

A 6-year-old boy from India developed an atypical form of rabies following a stray dog bite and as a consequence of not receiving the standard World Health Organization recommended post-exposure prophylaxis for category III wounds. Serial rising rabies virus neutralizing antibody titres in serum and cerebrospinal fluid by rapid fluorescent focus inhibition test helped confirm the diagnosis of rabies. The child has survived for 4 months since the onset of illness, albeit with neurological sequelae.

Cellular immune response following pre-exposure and postexposure rabies vaccination by intradermal and intramuscular routes.

PURPOSE: Immunization against rabies in humans induces protective neutralizing antibodies; however, the induction of type 1 or type 2 cytokine mediated cellular immune responses following rabies vaccination is not understood. Hence, the present study investigated cellular cytokine responses in vaccinated individuals. MATERIALS AND METHODS: The study groups included healthy rabies antigen naive controls (n=10), individuals who received intradermal primary (n=10) or booster pre-exposure vaccination (n=20) and subjects who received postexposure rabies vaccination either by intradermal (n=18) or intramuscular (n=20) routes. The antigen specific cellular responses were analyzed by stimulating peripheral blood mononuclear cells with a rabies vaccine antigen in the interferon-γ (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunospot (ELISpot) assay. These responses were compared to the rabies virus neutralizing antibody (RVNA) titers that were measured by rapid fluorescent focus inhibition test. RESULTS: We observed that cellular and humoral immune responses to primary intradermal rabies vaccination could be greatly enhanced by a booster vaccine; and both type 1 and type 2 cytokine responses were significantly elevated. The magnitude of type 1 and type 2 cytokine responses did not differ significantly among the intramuscular and intradermal routes of postexposure vaccination. The number of cells producing IFN-γ and IL-4 correlated significantly with the levels of RVNA. CONCLUSION: Both type 1 and type 2 cellular cytokine responses are strongly induced after rabies vaccination and directly correlate with levels of RVNA titers. The neutralizing antibody as well as the type 1 and type 2 cytokine responses may be important for vaccine induced protective responses against rabies.

Comparative study on the immunogenicity and safety of a purified chick embryo cell rabies vaccine (PCECV) administered according to two different simulated post exposure intramuscular regimens (Zagreb versus Essen).

Despite availability of effective rabies vaccines, India has the highest global mortality rate for rabies. Low socio-economic communities are most affected due to lack of awareness of the disease and poor compliance to post-exposure prophylactic regimens. Currently, the only approved intramuscular regimen for post-exposure prophylaxis (PEP) against rabies in India is the Essen regimen, which consists of 5 injections administered over 5 separate days in a period of one month. The high number of doses and clinical visits, however, are major reasons for non-compliance, and thus a shorter regimen would be beneficial. In a simulated PEP trial in healthy, adult subjects, this study evaluated whether purified chick embryo cell vaccine (PCECV), administered according to the WHO-recommended 4-dose/3 visit Zagreb vaccination regimen is of equal immunogenicity and safety as the standard Essen regimen in Indian subjects. Two hundred and 50 healthy adults were enrolled and randomized into a Zagreb or Essen group, each receiving PCECV according to their respective regimen. Blood samples were collected on Days 0, 7, 14 and 42 and analyzed using the rapid fluorescent focus inhibition test (RFFIT). By Day 14, all subjects across both groups attained rabies virus neutralizing antibody (RVNA) concentrations of ≥ 0.5IU/ml. The Zagreb regimen was then demonstrated to be immunologically non-inferior to the Essen regimen by Day 14, which was the primary endpoint of the study. No safety issues were noted and the occurrence of adverse events was similar in both groups (17% and 15%, respectively). NCT01365494. CTRI No.: CTRI/2011/07/001857.

Development and evaluation of a new immunohistochemistry-based test for the detection of rabies virus neutralizing antibodies.

Rabies claims about 55,000 human lives and many hundreds of thousands of livestock every year, worldwide. Despite a heavy disease burden, laboratory facilities to diagnose the infection remain scarce in most countries of the developing world where the disease is endemic. Rapid Fluorescent Focus Inhibition Test (RFFIT) and Fluorescent Antibody Virus Neutralization Test (FAVN) are the common tests done in the rabies diagnostic laboratories to detect and quantitate Rabies Virus Neutralizing Antibodies (RVNA). RFFIT is most often employed in confirming seroconversion following prophylactic vaccination, and to aid ante-mortem diagnosis in suspected cases of rabies. Though this remains one of the most sought-after diagnostic services in rabies laboratories, the requirements for expensive anti-rabies fluorochrome antibody conjugate and a fluorescent microscope restrict its performance to only a few reference laboratories. Cost-effective laboratory diagnostic methods employing affordable technology are a need of the hour in the rabies-endemic countries. In this study we have developed a new immunohistochemistry-based neutralization test and extensively evaluated it along with RFFIT. One hundred and 20 human serum samples collected after post-exposure vaccination were subjected to both the tests for determining RVNA titers. The results obtained with the new test correlated significantly with those of RFFIT. Further validation of the inter- and intra- assay precision, lower limit of quantification (LLOQ) and specificity was also performed. The best correlation between the 2 methods, however, was observed only when the RVNA concentrations in the samples were>20 IU/mL. Overall, the immunohistrochemistry-based neutralization test yielded satisfactory results. We suggest that it might serve as a cost-effective alternative to RFFIT in low-resource settings in the developing countries.

Assessing safety and immunogenicity of post-exposure prophylaxis following interchangeability of rabies vaccines in humans.

Rabies post exposure prophylaxis with cell culture vaccines by either intramuscular route or intradermal route spans over a period of one month. World Health Organization recommends completing post exposure prophylaxis against rabies with the same cell culture or embryonated egg rabies vaccine and with same route of administration and any deviation from this shall be an exception. In the present study, the safety and immunogenicity of rabies post-exposure prophylaxis was studied prospectively in 90 animal bite cases that had interchangeability of rabies vaccines either by route of administration or brand/type and such changes had occurred due to logistical/financial problems. Among them, 47 had change in route of administration from intramuscular to intradermal or vice versa and 43 had change in the brand/type of cell culture rabies vaccine. All of them had category III rabies exposure and received equine rabies immunoglobulin along with the rabies vaccine. None of the study subjects had any adverse reactions. The rabies virus neutralizing antibody titers was assessed by rapid fluorescent focus inhibition test and all the vaccinees had titers ≥0.5 IU per mL on day 14 which is considered as adequate for protection against rabies. Thus, the present study showed that, rabies post-exposure prophylaxis was safe and immunogenic despite changes in the route of administration and brand/type of rabies vaccine.

Utility of real-time Taqman PCR for antemortem and postmortem diagnosis of human rabies.

Rabies, a fatal zoonotic viral encephalitis remains a neglected disease in India despite a high disease burden. Laboratory confirmation is essential, especially in patients with paralytic rabies who pose a diagnostic dilemma. However, conventional tests for diagnosis of rabies have several limitations. In the present study the utility of a real-time TaqMan PCR assay was evaluated for antemortem/postmortem diagnosis of rabies. Human clinical samples received for antemortem rabies diagnosis (CSF, saliva, nuchal skin biopsy, serum), and samples obtained postmortem from laboratory confirmed rabies in humans (brain tissue, CSF, serum) and animals (brain tissue) were included in the study. All CSF and sera were tested for rabies viral neutralizing antibodies (RVNA) by rapid fluorescent focus inhibition test (RFFIT) and all samples (except sera) were processed for detection of rabies viral RNA by real-time TaqMan PCR. All the 29 (100%) brain tissues from confirmed cases of human and animal rabies, and 11/14 (78.5%) CSF samples obtained postmortem from confirmed human rabies cases were positive by real-time TaqMan PCR. Rabies viral RNA was detected in 5/11 (45.4%) CSF samples, 6/10 (60%) nuchal skin biopsies, and 6/7 (85.7%) saliva samples received for antemortem diagnosis. Real-time TaqMan PCR alone could achieve antemortem rabies diagnosis in 11/13 (84.6%) cases; combined with RVNA detection in CSF antemortem rabies diagnosis could be achieved in all 13 (100%) cases. Real-time TaqMan PCR should be made available widely as an adjunctive test for diagnosis of human rabies in high disease burden countries like India.