Comparison of a chemiluminescence immunoassay and an enzyme immunoassay for detection of IgM antibodies against measles, mumps, rubella, cytomegalovirus (CMV), Epstein Barr virus (EBV), and human herpes virus (HHV) -1 and -2 infections.

PURPOSE: Outbreaks of vaccine-preventable viral diseases have been increasingly reported globally over the past few years. The burden of congenital viral infections, their impact on physical and mental development and the resulting economic loss to the family and the community are also well known. IgM antibody detection has been convenient in the diagnosis of acute viral infections, particularly in settings with limited resources where molecular tests are not feasible. METHODS: This is a comparative study between a chemiluminescence immunoassay (Liaison, DiaSorin, Saluggia, Italy) and an enzyme linked immunosorbent assay (ELISA) (Euroimmun, Lubeck, Germany) for the detection of IgM antibody against measles, mumps, rubella, CMV, EBV and HHV-1 and -2 viruses using a total of 345 samples. Results are expressed as agreement using kappa statistics. RESULTS: In this study, CLIA is perfectly comparable to ELISA for the detection of IgM antibodies against measles (0.86) and mumps (0.92) with a moderate agreement for rubella (0.52), CMV (0.57), EBV (0.50), and HHV-1 and -2 (0.47) assays. However, a PABAK (prevalence-adjusted bias-adjusted kappa) showed improved agreement for rubella (0.64), CMV (0.65), EBV (0.60), and HHV-1 and -2 (0.88) assays. CONCLUSIONS: IgM antibody assays (CLIA and ELISA) against measles and mumps virus can be comparably used depending on the laboratory setup, throughput and expertise.

Changing epidemiology of human enteroviruses (HEV) in a hand, foot and mouth disease outbreak in Vellore, south India.

PURPOSE: Hand Foot and mouth disease (HFMD) is a major childhood exanthematous disease causing outbreaks that have become a major public health threat in recent years. In Vellore district of Tamil Nadu, south India, occasional outbreaks are common among the paediatric age group, most commonly in those under 5years of age (U5s). CoxsackieA6, A4, A5, A9, A10, B2 and B5 are the common serotypes causing outbreaks. This study aimed to identify the molecular serotype of the causative agent, co-circulating in this region. METHODS: Adapting the WHO case definition, cases during an HFMD outbreak between October and December 2017, were identified by a clinical criterion of fever, mouth ulcers and rash in the extremities. Vesicle fluid from these lesions were collected in viral transport medium and transported cold to the Clinical Virology laboratory of a tertiary care hospital in Vellore. Identification of the causative agent was undertaken by two real time PCRs (EV1 and EV2) followed by sequencing the VP1-2C region and constructing a phylogenetic tree. RESULTS: Among the 31 HFMD patients included in this study, 23 (74.2%) were U5s, 3 (9.7%) were between 6 and 15 years and the remaining 5 (16.1%) were adolescents (>15 â€‹yrs). The outbreak ran a mild clinical course, with 22(71%) patients having fever as a prodromal symptom. Papulovesicular lesions characteristic of HFMD were present on all 31 (100%) patients' palms and soles, buttocks of 19 (61.3%), oral mucosa of 12 (38.7%), and all over the body in 4 (12.9%) patients. Coxsackie A6(75%) and Coxsackie A16(25%) were the pathogens associated with this outbreak. CONCLUSIONS: Changing epidemiology of HFMD was seen in this outbreak since; other serotypes apart from the classical Coxsackievirus serotypes causing HFMD outbreak were also found co-circulating. EV1 PCR was a better screening assay than EV2 PCR in this region. Continued surveillance and molecular serotyping are necessary for HFMD outbreaks in any region.

Clinical Features of Four West Nile Virus Cases and Its Molecular Characterization from a South Indian Tertiary Care Hospital.

West Nile virus (WNV) is currently a significant reemerging virus of the 21st century. It belongs to the family Flaviviridae and genus Flavivirus. Although it is primarily transmitted by the Culex spp of mosquitoes, other routes of transmission are also well defined. Of eight lineages described, Lineage 1a has been reported from many parts of South India and is known to cause neuroinvasive illness. Many tests and serological techniques have been described to diagnose WNV infection such as complement fixation, neutralization, heamagglutination inhibition, ELISA, and PCR for molecular confirmation. The latter far outweighs the limitations inherent in the other tests. WNV infection is being reported from Vellore for the first time after 1968. This paper aims to describe four cases of WNV infection causing central nervous system manifestations with its molecular characterization. West Nile virus infection was diagnosed with the available molecular techniques such as PCR and sequencing, which emphasizes the need for considering West Nile virus in the differential diagnosis of acute meningoencephalitis and the wider availability of molecular diagnostic tests.

Phylogenetic analysis of partial pre-membrane and envelope sequences of dengue viruses circulating in India.

OBJECTIVE: Phylogenetic characteristics of circulating Indian dengue viruses (DENV) were analysed using partial pre-membrane (PrM) and envelope (E) sequences. An immunodominant region was analysed for mutations, and alignment with common DENV PCR primers and probes was determined. METHODS: Published Indian PrM and E DENV sequences were analysed with hitherto unpublished PrM sequences from this study site. Alignments of DENV were checked for mutations in an immunodominant region and against the commonly used PCR primers and probes. RESULTS: All four serotypes of DENV circulate in India. Genotype (G) GIII and GI of DENV-1 co-circulated in the south with significant PrM mutations before and after 2012. DENV-2 American genotype was first reported after which the Cosmopolitan genotype co-circulated with it in the southwest. The Cosmopolitan strain has been the only DENV-2 genotype circulating, although an Asian American genotype was recently reported. Significant mutations were found in the E region of DENV-2 strains. DENV-3 strains were GIII across the country. DENV-4 GI from the south and west has now spread across India. No significant mutations were found for DENV-3 or DENV-4. Indian strains showed mutations in an immunodominant region of the E gene and in the regions targeted by commonly used PCR primers and probes. CONCLUSIONS: The genetic variability of Indian DENV with co-circulation of multiple genotypes suggests that genotype surveillance is crucial to determining the composition of dengue vaccines and understanding their contribution to epidemiology, virus fitness and pathogenesis. Some mutations seen in an immunodominant region of the E gene may allow these viruses to evade host immune cells. The mutations in the regions targeted by commonly used primers and probes necessitate higher degeneracy.

A Low Incidence of Cytomegalo Virus Infection Following Allogeneic Hematopoietic Stem Cell Transplantation Despite a High Seroprevalence.

Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality following allogeneic stem cell transplantation (SCT). We wanted to study if the high sero-prevalence seen in our population translated into a high incidence of CMV infection following SCT. This is a retrospective analysis of patients who underwent allogeneic SCT between January 2008 and December 2012 at our centre. 475 patients underwent allogeneic SCT for malignant (46.5%) and non-malignant (53.5%) haematological disorders. 463 (97.4%) SCT recipients and 403 (84.8%) SCT donors were IgG seropositive for CMV. CMV reactivation within 100 days post SCT was seen in 174 (36.6%) at a median of 41 days (range 10-100) post SCT. Ganciclovir was used in 166 patients (95.4%) for a mean duration of 16 days (range 5-32). 157 patients (90%) responded to therapy. Sixty-six patients (42.3%) had secondary reactivation of the virus. Use of a male donor (p = 0.000), donor and recipient age > 15 (p = 0.005 and 0.000), unrelated donor (p = 0.000), degree of HLA mismatch (p = 0.000), occurrence of acute GVHD (p = 0.000) and steroid refractory acute GVHD (p = 0.026) were identified as risk factors for CMV reactivation while early neutrophil recovery (< 15 days) was found to be protective (p = 0.004). On multivariate analysis, male donor (p = 0.042), degree of HLA mismatch (p = 0.006), the occurrence of acute GVHD (p = 0.000) and steroid refractory acute GVHD (p = 0.031) continued to remain significant. 5-year overall survival was significantly better in patients without CMV reactivation compared to those who developed reactivation of CMV (68.9 ± 3.7 vs 58.2 ± 4.9% p = 0.004). The incidence of CMV infection does not seem to be higher despite a high sero-prevalence of CMV. However, patients who developed CMV infection post SCT had inferior outcomes.

Performance of an in-house real-time PCR assay for detecting Cytomegalovirus infection among transplant patients from a tertiary care centre.

Background: Quantitative Cytomegalovirus (CMV) polymerase chain reactions are increasingly being used for monitoring CMV DNAemia in haematopoietic stem cell transplants and solid organ transplants. Objective: In this study, a commercial CMV viral load assay was compared with an in-house viral load assay. Materials and Methods: A total of 176 whole-blood samples were tested for CMV DNAemia using both assays. Results: Our evaluation showed a difference of 1 log10copies/ml between the two assay systems in determining CMV viral loads in the clinical samples. Conclusion: The in-house viral load assay had a better correlation with clinical findings compared to the commercial assay. Quality assessment of these assays was done by the United Kingdom National External Quality Assessment Scheme (UKNEQAS), an external proficiency testing programme, and by the National Institute for Biological Standard and Control (NIBSC) standard. For UKNEQAS and NIBSC standards, the bias between the assays was 0.73 log10and 0.85 log10, respectively. This difference is well within the acceptable range already reported in the literature.

Targets for intervention to improve virological outcomes for patients receiving free antiretroviral therapy in Tamil Nadu, India.

Operational research to identify factors predicting poor clinical outcomes is critical to maximize patient care and prolong first-line regimens for those receiving free antiretroviral therapy (ART) in India. We sought to identify social or clinical factors amenable to intervention that predict virological outcomes after 12 months of ART. We examined a retrospective cohort of consecutive adults initiating free nonnucleoside reverse transcriptase inhibitor-based regimens. Individuals remaining in care 12 months post-ART initiation were tested for HIV viral load and surveyed to identify barriers and facilitators to adherence, and to determine clinic travel times and associated costs. Uni- and multivariate logistic regression identified factors predicting HIV viral load >200 copies/mL after 12 months of ART. Of 230 adults initiating ART, 10% of patients died, 8% transferred out, 5% were lost to follow-up, and 174/230 (76%) completed 12 months of ART, the questionnaire, and viral load testing. HIV viral load was <200 copies/mL in 140/174 (80%) patients. In multivariate models, being busy with work or caring for others (OR 2.9, p < 0.01), having clinic transport times ≥ 3 hours (OR 3.0, p = 0.02), and alcohol use (OR 4.8, p = 0.03) predicted viral load >200 copies/mL after 12 months of ART. Clinical outcomes following ART are related to programmatic factors such as prolonged travel time and individual factors such as being busy with family or using alcohol. Simple interventions that alter these factors should be evaluated to improve clinical outcomes for populations receiving free ART in similar settings.

Pharmacy and self-report adherence measures to predict virological outcomes for patients on free antiretroviral therapy in Tamil Nadu, India.

Over 480,000 individuals receive free antiretroviral therapy (ART) in India yet data associating ART adherence with HIV viral load for populations exclusively receiving free ART are not available. Additionally estimates of adherence using pharmacy data on ART pick-up are not available for any population in India. After 12-months ART we found self-reported estimates of adherence were not associated with HIV viral load. Individuals with <100% adherence using pharmacy data predicted HIV viral load, and estimates combining pharmacy data and self-report were also predictive. Pharmacy adherence measures proved a feasible method to estimate adherence in India and appear more predictive of virological outcomes than self-report. Predictive adherence measures identified in this study warrant further investigation in populations receiving free ART in India to allow for identification of individuals at risk of virological failure and in need of adherence support.