Investigation of Chikungunya virus genotype at tertiary care center in Western Maharashtra, India

Background & objectives: Chikungunya is a reemerging arbovirus infection. Laboratory diagnosis can be done by Classical test involving Rapid Immunochromatography, Enzyme-Linked Immunosorbent assay and Molecular methods. The present study was undertaken to know the genotype of the Chikungunya virus (CHICKV) among patients suspected of CHICKV and investigated by virus culture, partial sequencing, Rapid Immunochromatography, and Enzyme-linked Immunosorbent assay (ELISA). To understand different techniques used in Chikungunya diagnosis viz., virus culture, partial sequencing along with Immunochromatography and ELISA. Methods: This is a prospective, laboratory-based study at a tertiary care center. Lateral flow chromatography and ELISA was carried out on serum samples. All 50 samples were cultured and indirect Immunofluorescence was performed on positive samples at Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth Medical College Pune, Maharashtra, India. Virus isolates were subjected to partial sequencing for identification of genotype after confirmation by PCR. Statistical Package of Social Science (SPSS) version 22.0 software was used to calculate the Receiver operating curve (ROC) for different tests. Results: Out of 50 samples, 20 were positive by Immunochromatography, 23 by ELISA, and 3 by culture, PCR confirmed CHIKV isolates and sequencing identified genotypes as East Central South African type. Interpretation & conclusion: CHIKV culture isolates of East Central South African type lineage were predominantly found in the present study. These are also common genotypes present in Asia including India.

Rapid culture and identification of human parainfluenza viruses / 医学研究生学报

Objective Parainfluenza virus is an important pathogen of lower respiratory tract infections in infants and young children.This study was to search for a method for rapid culture and identification of human parainfluenza viruses from nasal swabs. Methods Nasal swab specimens were collected from 0-5 years old children with acute respiratory tract infection.The specimens were inoculated onto 96 plates with prefabricated LLC-MK2 cells and then centrifuged for 1 hour at 3000 r/min and also inoculated using the traditional culture method, followed by addition of virus mainte-nance medium containing 4 μg/mL TPCK trypsin.The cytopathic effect was observed daily, and hemagglutination and blood absorption tests were done at 2, 5, and 8 days after inoculation.In case of posi-tive result of either test, the specimen was subjected to immunofluo-rescence staining. Results Six strains of parainfluenza virus were isolated from the 83 nasal swab specimens, with a positive rate of 7.2%.There was a significant difference in the rate of separation be-tween the rapid and traditional culture methods after 2 days of culturing (7.2%vs 0%, P<0.05).The infected cells produced a cy-topathic effect that characterized by syncytium and crush formation.Hemagglutination and blood adsorption tests were positive at 4℃and negative at the room temperature.Immunofluorescence staining exhibited specific apple green fluorescence. Conclusion The method for rapid culture and identification of human parainfluenza viruses in nasal swab specimens was successfully established, which can be used to obtain and identify parainfluenza viruses with virulence and biological activity in 2 days.

Comparison of Culture, Direct Immunofluorescence Assay, and Multiplex Reverse Transcriptase PCR for Detection of Respiratory Viruses

BACKGROUND: Rapid detection of causative viruses is important for the management of acute respiratory illnesses. To increase the detection rate and decrease the turnaround time (TAT) and cost, we used 24-well plates instead of R-mix shell vials and changes the report time from once on day 3 to twice on days 1 and 5 of culture. The detection rate and TATs of each culture method, direct immunofluorescence assay (DFA), and multiplex reverse transcriptase polymerase chain reaction (mPCR) were compared. METHODS: Among 2,062 nasopharyngeal swabs (NPSs) received from January 2009 to January 2011, 707 NPSs were cultured in R-mix shell vials and 1,355 NPSs were cultured in 24-well plates. We analyzed 538 NPSs simultaneously using DFA, mPCR, and culture and compared the detection rate for 7 viruses (adenovirus, influenza A and B virus; parainfluenza virus 1, 2, and 3; and respiratory syncytial virus [RSV]). RESULTS: The detection rate when using shell vials was 28.4% (201/707) and was 29.4% (399/1,355) on day 1 and 33.3% (452/1,355) on day 5 when using 24-well plates. In addition, of the 53 viruses that were detected on day 5, 34 were adenovirus, 7 were parainfluenza virus, 4 were influenza A virus, 3 were influenza B virus, 4 were RSV, and 1 was a mix of influenza B and parainfluenza virus. The TAT when using shell vials and 24-well plates was 4.8 days and 2.5 days, respectively. The detection rate for the 7 respiratory viruses using culture, DFA, and mPCR was 24.3%, 20.8%, and 38.5%, and the TAT was 3.7 days, 1.0 day, and 1.4 days, respectively. CONCLUSIONS: Using 24-well plates for virus culture is an efficient method for the detection of respiratory viruses.

Comparison of R-mix Virus Culture and Multiplex Reverse Transcriptase-PCR for the Rapid Detection of Respiratory Viruses / 대한진단검사의학회지

BACKGROUND: Respiratory viral infections can become epidemic due to high contagiosity. Since there was no rapid diagnostic method for complete diagnosis in the past, diagnosis was solely made on the basis of clinical symptoms or the time of infection. With recent developments in rapid diagnostic methods like multiplex reverse transcriptase (RT)-PCR, R-mix virus culture, etc., early detection and effective treatment of respiratory viral infections is possible. Herein, we compared the efficiency of multiplex RT-PCR and the R-mix virus culture for the rapid detection of respiratory viruses. METHODS: We used 96 nasopharyngeal swab specimens for culturing respiratory viruses using R-mix (Diagnostics Hybrids Inc., USA). Afterwards, multiplex RT-PCR was performed using specimens stored at -70degrees C. RESULTS: R-mix virus culture yielded positive results in 34 cases (35.4%) and multiplex RT-PCR in 73 cases (76.0%). Both methods yielded identical results in 51 cases (29 positive cases and 22 negative cases). Among 45 cases that showed different results, 40 showed negative results in R-mix virus culture and positive results in multiplex RT-PCR, and 1 showed positive result in R-mix virus culture and negative result in multiplex RT-PCR. Different viruses were detected in the remaining 4 cases by both the methods. CONCLUSIONS: Multiplex RT-PCR provided faster results and had higher detection rates than R-mix virus culture. Further, unlike R-mix virus culture, multiplex RT-PCR can be used to identify new respiratory viruses. Therefore, multiplex RT-PCR is more useful than R-mix virus culture in the diagnosis of respiratory virus infection.

Identification of Adenovirus, Influenza Virus, Parainfluenza Virus, and Respiratory Syncytial Virus by Two Kinds of Multiplex Polymerase Chain Reaction (PCR) and a Shell Vial Culture in Pediatric Patients with Viral Pneumonia

PURPOSE: Early identification of causative agents in lower respiratory infection of pediatric patients can reduce morbidity and prevent an overuse of antimicrobials. Two kinds of multiplex polymerase chain reaction (PCR) and a commercial shell vial viral culture were performed to identify causative agents in pediatric patients. MATERIALS AND METHODS: Nasopharyngeal aspirates of 220 children diagnosed with viral pneumonia were obtained. Two kinds of multiplex PCR (Seeplextrade mark RV detection kit, and Labopasstrade mark RV detection kit), and a shell vial culture by R-Mix were performed. RESULTS: Positive samples from 220 total samples by two multiplex PCRs were 52.7% and 46.4%, respectively. We also cultured 103 samples that showed positive results of the adenovirus, influenza virus, parainfluenza virus, and respiratory syncytial virus (RSV) by two multiplex PCR. The RSV was most frequently detected in 53.0% (Seeplex) and 51.7% (Labopass) of patients. The detection rate of adenovirus (AdV) was 10.3% and 12.1%, influenza virus (IFV) A and B was 12.5% and 3.4%, and parainfluenza virus (PIFV) 1, 2, and 3 were 2.9% and 2.6%. Shell vial cultures showed concordant results with each multiplex PCR by 96.1% and 77.7%, respectively. Sequencing results were 90% consistent with multiplex PCR. CONCLUSION: Multiplex PCR showed more positivity than the shell vial culture and it can be an effective primary test. Other complementary efforts such as viral cultures and sequencing analysis could be considered, according to clinical and laboratory conditions.

Epidemiological Analysis of Influenza by Laboratory Surveillance in Incheon, 2003/2004~2004/2005 / 대한임상미생물학회지

BACKGROUND: Influenza is a highly contagious respiratory disease. Influenza virus, which causes epidemics every winter season, has the high possibility of appearing with new virus types every year due to antigen variation. Therefore, we intended to analyze the data on the epidemiology of influenza that had been acquired by laboratory surveillance in Incheon during the 2003/2004 and 2004/ 2005 seasons and to apply the knowledge to the control and prevention of influenza in Korea. METHODS: Specimens were inoculated into Madin-Darby canine kidney (MDCK) cells and, when cytopathic effect (CPE) was seen, culture supernatants were tested by mutiplex RT-PCR for typing and subtyping of influenza viruses. RESULTS: The first virus of the season was isolated at week 47 (3rd week on November) in 2003 during 2003/2004 and at week 43 (4th week on October) in 2004 during 2004/2005, which was about 4 weeks earlier than in the 2003/2004 season. From 532 specimens cultured for influenza virus during the 2003/2004 season. 330 (62.0%) viruses were isolated: 161 (48.8%) A/H3N2, 1 (0.3%) A/H1N1, and 168 (50.9%) B. During 2004/2005 season, 457 specimens were tested and 278 (60.8 %) were positive for influenza virus: 232 (83.5%) A/H3N2, 5 (1.8%) A/H1N1, and 38 (13.7%) B. The incidence of influenza was the highest in the school-age children and young adults of 7 to 19 years age group in both seasons. CONCLUSION: Influenza virus was isolated at a high rate (more than 60%) by the laboratory influenza surveillance system in Incheon during the 2003/2004 and 2004/2005 seasons: the predominant strain was influenza A/H3N2 subtype.

Efficacy of Indirect Immunofluorescent Antibody Test in Herpes Simplex Keratitis

Herpes simplex virus keratits(HSK) is one of the most common external eye diseases that cause corneal blindness, Therefore early diagnosis and proper treatment of HSK are essential. However it is frequently misdiagnosed because it shows non-specific corneal lesion than other infectious corneal disease. And also diagnosis of HSK mostly rely on clinical examination and patient history. We evaluated suspicious HSK patients by indirect immunofluofluorescent(IF) antibody test and analyzed its efficacy in the early diagnosis of HSK. Among 47 patients(47 eyes), 37 patients were suspicious heretic keratitis and others not. Dendritic keratitis patients existed in 17 out of 37 patients and they were evaluated with virus culture and indirect IF test. The result of indirect IF test was confirmed under the immunofluorescent microscope and for virus culture the specimens were inoculated on Vero cells(monkey kidney cells). The positive results of indirect IF test was 28 out of 37 suspicious HSK patients and 1 out of 10 non-suspicious HSK patients. Dendritic HSK patients showed IF positive in 15 out of 17 patients(82.3%). Sensitivity of indirect IF test in suspicious HSK patients was 75.7%(2837) and 88.2%(15/17) in dendritic HSK patients. Indirect IF test was all positive(14/14) in dendritic HSK patients that showed culture positive. From these results, indirect IF test has a high sensitivity in early diagnosis of HSK and might be ussful as a rapid diagnostic tool in HSK patients.

Efficacy of Indirect Immunofluorescent Antibody Test in Herpes Simplex Keratitis

Herpes simplex virus keratits(HSK) is one of the most common external eye diseases that cause corneal blindness, Therefore early diagnosis and proper treatment of HSK are essential. However it is frequently misdiagnosed because it shows non-specific corneal lesion than other infectious corneal disease. And also diagnosis of HSK mostly rely on clinical examination and patient history. We evaluated suspicious HSK patients by indirect immunofluofluorescent(IF) antibody test and analyzed its efficacy in the early diagnosis of HSK. Among 47 patients(47 eyes), 37 patients were suspicious heretic keratitis and others not. Dendritic keratitis patients existed in 17 out of 37 patients and they were evaluated with virus culture and indirect IF test. The result of indirect IF test was confirmed under the immunofluorescent microscope and for virus culture the specimens were inoculated on Vero cells(monkey kidney cells). The positive results of indirect IF test was 28 out of 37 suspicious HSK patients and 1 out of 10 non-suspicious HSK patients. Dendritic HSK patients showed IF positive in 15 out of 17 patients(82.3%). Sensitivity of indirect IF test in suspicious HSK patients was 75.7%(2837) and 88.2%(15/17) in dendritic HSK patients. Indirect IF test was all positive(14/14) in dendritic HSK patients that showed culture positive. From these results, indirect IF test has a high sensitivity in early diagnosis of HSK and might be ussful as a rapid diagnostic tool in HSK patients.

Epidemiological and Clinical Analysis of Influenza and Viruses Isolation during Winter of 1996-1997 / 감염

BACKGROUND: Although influenza has been a leading cause of global morbidity and mortality, we have few data regarding the epidemiological and clinical characteristics of influenza activity in Korea. Since an outbreak of influenza was recognized during winter of 1996-1997, we analyzed the epidemiological and clinical features of influenza activity in the hospital setting. METHODS: All clinical specimens requested for isolation of influenza virus at Samsung Medical Center from October 1996 to April 1997 were included. Mardin- Darby canine kidney (MDCK) cell line was used for virus culture. Isolated viruses were confirmed with immunostain followed by subtyping. The demographic and clinical characteristics of the patients were reviewed retrospectively. RESULTS: Ninety-eight influenza viruses were isolated from 461 patients (21.3%). Influenza A and B virus was isolated from 58 (54 children and 4 adults) and 40 pediatric patients, respectively. One of 31 influenza A viruses were confirmed as A/Wuhan/359/95-like strain and 5 of 12 influenza B viruses were confirmed as B/Guangdong/8/97-like strains. Two distinctive peaks of influenza activity were recognized and the most common age of patients was less than 1 year for influenza A, and 3 to 5 years for influenza B. Common lower respiratory infections were pneumonia followed by croup, bronchiolitis and laryngitis. CONCLUSION: We analyzed the epidemiological and clinical features of influenza activity during winter of 1996-1997. Although this study was performed not in the community but in the hospital setting, the morbidity caused by influenza may not be low in Korea. Therefore, nationwide surveillance for influenza activity is warranted.