SUMMARY AND CONCLUSIONS

Transcription

1 SUMMARY AND CONCLUSIONS 6.1 SUMMARY Dengue virus (DENV) is a mosquito-borne flavivirus and the most prevalent arbovirus affecting the tropical and subtropical regions of the world and causing serious morbidity and mortality. In the last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries and, in the present decade, from urban to rural settings. It causes a wide spectrum of illness from mild asymptomatic illness to severe fatal dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). Approximately 2.5 billion people live in dengue-risk regions with about 100 million new cases each year worldwide. The cumulative dengue diseases burden has attained an unprecedented proportion in recent times with sharp increase in the size of human population at risk. India is one of the seven identified countries in the South-East Asia region regularly reporting incidence of DF/DHF outbreaks and becoming major niche for dengue infection. The elucidation of the clinical profile in dengue fever is important for understanding the disease and patient management. Dengue is becoming endemic in the states of Andhra Pradesh and Telengana with seasonal variation. The clinical manifestations of Dengue are varied and confuse the physician with the other endemic diseases which were prevalent in this area, such as typhoid, hanta virus, leptospirosis, scrub typhus and enteroviral gastroenteritis. Early, sensitive and specific diagnosis is paramount for clinical decision making. Currently available diagnostic tests are limited in scope and utility. In view of the high mortality rate associated with the infection and to reduce the disease burden, it is imperative to diagnose the infection early in the course using a sensitive laboratory assay. In this study, we established and compared in-house developed NS1 serotype-specific Reverse Transcriptase PCR (RT-PCR) assay and NS1 antigen ELISA in the differentiation of dengue virus serotypes in 300 clinical samples. In-house developed NS1 serotype specific RT-PCR and CDC Real time PCR was performed on 300 samples based on days of onset of clinical illness. NS1 antigen ELISA showed sensitivity of 92% and 100% specificity. In-house developed NS 1 serotype specific RT-PCR assay, revealed a sensitivity 184

2 of 95% and 100% specificity. 100% concordance was observed between NS1 RT-PCR and CDC Real time PCR assay. Most predominant serotypes were Denv-2 and Denv-3. Highest detection rate was observed in patients who reported from 1-7 days post onset of illness. In this study, we established and compared in-house developed single step NS1 serotypespecific RT-PCR assay in the differentiation of dengue virus serotypes with samples collected from 300 patients with acute Dengue virus infections. Single-step denotes the inclusion of primers in one reaction tube that accomplishes both reverse transcription and cdna amplification. The single-step format was selected to improve workflow and decrease the opportunity for contamination. This study targets NS1 region of DENV by designing 4 sets of primers for differentiation of serotypes of dengue. NS1 Ag and RT-PCR assay, has the ability to improve the diagnostic algorithm contributing significantly to early diagnosis, clinical treatment and control of dengue. In the present study, the standardization and validation of a one step, single tube Reverse Transcription Loop-Mediated Isothermal Amplification Assay (RT-LAMP) targeting NS1 gene using the Genie II flourometer for rapid detection and serotyping of DENV. Performance of RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 clinically suspected cases of DENV. RT-LAMP was positive in 148/250 (59.2%) cases, while 92/250 (36.8%) were positive for anti-dengue IgM and IgG antibodies. The detection rate of acute DENV infection improved to 96% (240/250) when results of RT- LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 10 copies of viral RNA compared to 100 copies by conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential usefulness for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries like India. The detection sensitivity and specificity of the reported DENV NS1 serotype specific RT-PCR and RT-LAMP in freshly obtained blood samples from dengue infected patients, is compared with available test system for suitable algorithm. Dengue is the most rapidly spreading mosquito borne viral disease in the world, and as larger proportion of population is being affected, more unusual manifestations are being reported. Very few studies have documented the unusual manifestations of dengue from South India. This prospective study was undertaken from July, 2011 to June, 2013 to document the rare 185

3 manifestations of dengue fever in 175 hospitalized patients and compare them with the WHO dengue classification system. The clinical diagnosis was confirmed by either the detection of NS1 Ag by ELISA, Dengue IgM or IgG and/or a RT-PCR and CDC Real time PCR for DENV RNA. The daily profiles of the hematological and biochemical investigations were followed and recorded. Unusual and rare manifestations of dengue were documented among 115 patients (66%). Hepatitis was observed in 70% of cases. Pleural effusion was seen in 11%, acute renal failure in 10%, neurological complications like encephalitis in 7.4%, myocarditis in 9% and bleeding gastric ulcers in 3.4% of the cases in our study. Dengue serotype 2 was more prevalent in patients with unusual manifestations of dengue in our study. WHO classification system does not include unusual and rare manifestations, hence it is essential to be aware of these manifestations and closely monitor them for better clinical management and outcome of patients. To the best of our knowledge this is the first report of rapid detection and differentiation of Dengue using NS1 serotype specific RT-PCR assay and NS1 RT-LAMP assay with real time fluorometer (Genie II from Optigene, U.K.) from South India which can be potentially used for seroepidmiological studies of dengue in the early stages of infection, disease prevention, and control activities in dengue endemic regions like India. 6.2 Characteristics Assessed in Evaluation of Diagnostic Accuracy Performance characteristics, sensitivity and specificity when compared with the CDC 1-4 Real time RT-PCR assay as standard test and Operational characteristics including the time taken to perform the test, its technical simplicity or ease of use, user acceptability and the stability of the test under user conditions were evaluated for NS1 serotype specific RT-PCR assay and NS1 RT-LAMP assay for rapid detection and typing of Dengue virus were found to meet the STARD guidelines. Both the assays have been appropriately evaluated and shown to meet defined performance targets in patients with DENV infection. 186

4 6.3 Conclusion 1. In conclusion, this study substantiates that in comparison to NS1 Ag rapid and early NS1 MAC-ELISA, NS1 serotype specific RT-PCR assay is an effective tool for diagnosis of Dengue virus infection, especially within the first few days of illness. In endemic countries like India where Dengue and Chikungunya coexist, early detection and serotype differentiation by NS1 assay can help in early confirmation and management of this vulnerable group. NS1 Ag and RT-PCR assay, on a single serum sample of a suspected case, has the ability to improve the diagnostic algorithm contributing significantly to the serotype identification, clinical treatment and control of dengue viral infections. 2. This study demonstrated that NS1 antigen detection provides a sensitive and specific approach to dengue diagnosis especially within first 5 days of illness, when the levels of viremia was higher, using single serum sample compared to antibody detection. Thus NS1 antigen detection can be a useful tool for early detection of dengue in patients with high viremia levels who have higher risks of developing complications during their illness. 3. Our experience with RT-LAMP emphasized its utility as a rapid, simple, easy, cost effective, isothermal, highly sensitive and specific field applicable technique for detection and differentiation of dengue virus serotypes which overcomes the deficiencies present in existing techniques. Its applicability in tertiary care institutes is emphasized by its ability in viral quantification and evaluation of viraemia in patients. It can suitably be introduced in zonal/peripheral hospitals as an adjunct to existing immunological tests acting as parallel controls. RT-LAMP assay in OptiGene Genie II instrument may be employed as the new gold standard for timely and accurate diagnosis and serotyping of dengue in clinical care, disease surveillance, disease prevention, and control activities in endemic countries like India. 4. WHO classification system although includes unusual and rare manifestations of dengue, lack of awareness among primary physicians, may jeopardize the clinical diagnosis. Since these manifestations are a result of micro vascular leaks, they are considered to have an impact on the outcome of the patients. Hence, it is essential to be aware and closely monitor these unusual and rare manifestations for better clinical management and outcome of patients. 187

5 6.4 Future Prospects of the study To use the NS1 serotype specific RT-PCR assay and NS1 RT-LAMP assay for rapid detection and typing of Dengue virus in large scale population based epidemiological studies to document early and acute DENV infection and prevention of epidemics in future. 188

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