|Year : 1999 | Volume
| Issue : 4 | Page : 241-246
Laboratory investigations on viral and chlamydia trachomatis infections of the eye : Sankara nethralaya experiences
Vision Research Foundation, Sankara Nethralaya, Chennai, India
H N Madhavan
Vision Research Foundation, Sankara Nethralaya, Chennai
Purpose: To review our experiences on the laboratory investigations of viral and chlamydial conjunctivitis, congenital cataract and acute retinal inflammations seen from 1990 to 1998 at Sankara Nethralaya, Chennai, India.
Methods: Conjunctival swabs/scrapings from 1061 patients with conjunctivitis were investigated. Nested polymerase chain reaction (nPCR) and restriction fragment length polymorphism (RFLP) techniques were applied on 74 conjunctival swabs during the 1996 outbreak of acute viral conjunctivitis. The occurrence of Rubella virus in 86 lens aspirates of congenital cataract was investigated. Tests were performed for the association of Herpes simplex virus (HSV), Varicella zoster virus (VZV) and Cytomegalovirus (CMV) with acute retinal inflammation in 32 patients.
Results: The causative agents of conjunctivitis were Adenovirus in 13.8%, HSV in 2.2% and C. trachomatis in 20.9% of the patients. Epidemics were due to Adenovirus type 4 in 1991, type 3 in 1992-93 and type 7a in 1996. PCR was 37.9% more sensitive in detecting Adenovirus than virological methods. RFLP identified the conjunctivitis epidemic strain of 1996 as Adenovirus 7a. Rubella virus was isolated from 8.1% of lens aspirates from congenital cataract. Nineteen of the 32 patients with acute retinitis had confirmed virus infections (VZV: 8; HSV: 5; and CMV: 6) and the rapid detection of the virus agent helped institute specific chemotherapy resulting in useful vision in some patients.
Conclusion: Laboratory investigations for diagnosis of viral and C. trachomatis ocular infections were useful in establishing the aetiology and determining the incidence of causative agents of specific ocular diseases
Keywords: Viral conjunctivitis, acute retinal necrosis, CMV retinitis, Rubella virus, Chlamydia trachomatis, congenital cataract
|How to cite this article:|
Madhavan H N. Laboratory investigations on viral and chlamydia trachomatis infections of the eye : Sankara nethralaya experiences. Indian J Ophthalmol 1999;47:241-6
|How to cite this URL:|
Madhavan H N. Laboratory investigations on viral and chlamydia trachomatis infections of the eye : Sankara nethralaya experiences. Indian J Ophthalmol [serial online] 1999 [cited 2014 Mar 9];47:241-6. Available from: http://www.ijo.in/text.asp?1999/47/4/241/14904
Although several viruses like Molluscum contagiosum, papilloma viruses, enteroviruses and paramyxoviruses are associated with ocular diseases, it is the Herpes virus group, adenoviruses, and the rubella virus that more commonly cause ocular infections. This review is based on our experience of virological investigations of ocular infections of patients reporting to Sankara Nethralaya, Chennai, India, during the 9-year period 1990-98. The ocular diseases considered in this review are viral conjunctivitis (of sufficient public health importance in India), congenital cataract due to rubella virus (a preventable viral disease), and acute viral retinal inflammation (a blinding ocular disease). We also review our investigations on C. trachomatis conjunctivitis because of the nature of the laboratory methods which are similar to those employed with virus. We have, however, excluded a review of our investigations of viral keratitis because of the large volume of the results and discussions required.
| Materials and Methods|| |
| Approaches to virological investigations|| |
The basic approaches to the virologicat investigations, collection and processing of specimens for detection and isolation of the infecting agent followed the procedures described by Schmidt and Emmons.
| Conjunctivitis|| |
Investigations were carried out for the detection of viral agents and Chlamydia trachomatis on conjunctival swabs or scrapings from 1061 patients with conjunctivitis (acute, chronic and recurrent) during the 9-year period (1990-98). The specimens were collected in Hank's balanced salt solution (HBSS) or 2SP (0.1M sucrose in 0.2M phosphate-buffered saline at pH 7.2) medium and processed for detection of adenovirus, Herpes simplex virus (HSV) and Chlamydia trachomatis as described earlier.,, Briefly, the methods were as follows: smears made from these specimens on clean microscopic slides were fixed in cold acetone and stained by fluorescent antibody tests (FAT) for the detection of the adenovirus group-specific antigen, and HSV and C. trachomatis antigens. Adenovirus antigen was tested by indirect FAT using rabbit anti-adenovirus antiserum (National Institute of Health, USA) and Fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit immunogiobulin (Dako, Denmark). Adenoviruses were isolated by inoculating 100ml of the specimen in HBSS or 2SP medium into tubes of monolayer of HEp-2 cells obtained from the National Facility for Animal and Tissue Cell Culture (NFATCC), Pune, India. When cytopathic effects involved about 80% of cells, they were scraped and stained by indirect FAT as described above. The viral isolates were identified by the micro-neutralisation test (NT), using the available anti-adenoviral antiserum types 1, 2, 3, 4, 5, 6, 7a, and 14 (NIAID antisera, ATCC, Rockville, MD, USA). For the detection of HSV, the smears were stained by indirect FAT using polyclonal rabbit antibody (DAKO, Denmark) against the virus and FITC-conjugated swine anti-rabbit immunoglobulin serum (DAKO, Denmark), or direct FAT using FITC-conjugated polyclonal rabbit anti-HSV antibody. HSV was isolated by inoculating the specimen into a Vero cell line (NFATCC); the isolated viruses were identified by FAT and neutralisation test using type-specific (HSV 1 and 2) rabbit anti-HSV serum (DAKO, Denmark).
From 1995 onwards, investigations were carried out for the presence of Enterovirus 70 (EV 70) and Coxsackie virus A 24 variant antigens. Indirect FAT was done for this purpose using EV 70 and Coxsackie A 24 monoclonal antibodies (Chemicon, USA) and FITC-conjugated anti-mouse antiserum (DAKO, Denmark).
C. trachomatis was detected on direct smears by the FAT. A specimen was considered positive by FAT for C. trachomatis if three or more typical elementary and / or inclusion bodies were identified [Figure - 1] using FITC-conjugated monoclonal antiserum directed against the major outer membrane protein (MOMP) (MicroTrak, Syva, USA). Cultures for C. trachomatis were carried out on duplicate cover slip cultures of cyclohexamide-treated stationary McCoy cell cultures (NFATCC) in shell vials. The growth of the organism was detected by FAT using FITC-conjugated monoclonal antibody against MOMP of C. trachomatis (MicroTrak, Syva, USA). The presence of more than one punctate or round fluorescent green body [Figure - 2] (not yellow) indicated chlamydial elementary or inclusion bodies.
| Polymerase chain reaction for the detection of adenovirus in conjunctival specimens|| |
Nested polymerase chain reaction (nPCR) for the detection of adenoviruses was standardised and described by us earlier. This nPCR was further applied on a total of 74 conjunctival swabs from 58 patients (both eyes in 16 and one eye in 42 patients) during an outbreak of acute conjunctivitis between August and November 1996. The sensitivity and specificity of nPCR were determined using DNA from ATCC VR-846 adenovirus serotype 2 (Ad 2) and DNA of viruses and bacteria that commonly cause conjunctivitis. We also applied the restriction fragment length polymorphism (RFLP) technique on the amplified products of the conjunctival specimens using restriction enzymes Hae III and Hinf I (Bangalore Genei, India). Standardised RFLP technique was applied on the amplified products of conjunctival specimens obtained during the outbreak of acute conjunctivitis in late 1996.
| Congenital cataract due to Rubella virus|| |
Eighty six lens aspirates of clinically diagnosed cases of congenital cataract were investigated for the presence of rubella virus. Each specimen was collected in 1ml of HBSS. The specimen was mixed well using a Cyclomix (Remi, India) and 100 ml lots were inoculated into BHK 21 and Vero cell lines (NFATCC). No cytopathogenic effects were seen. After one week of incubation, smears were made by scraping the cells from the tubes. The smears were stained by indirect FAT using suitably diluted hyperimmune anti-rubella human serum and FTTC-conjugated polyclonal anti-human immunoglobulin (DAKO, Denmark). The hyperimmune anti-rubella serum was prepared from sterile pooled convalescent sera of patients with proven rubella virus infection and standardised to the dilution required to detect only rubella antigen and not other virus antigens commonly associated with ocular clinical specimens. If the virus was not isolated in the first passage, three more blind passages were made and scraped cells were stained for the growth of the virus before the specimen was declared negative for rubella virus.
| Acute retinal inflammation|| |
Thirty two patients with acute retinal inflammation during this 9-year period (1991-98) were investigated for the detection of HSV, Varicella zoster virus (VZV), and Cytomegalovirus (CMV). The clinical diagnoses made in these cases were: acute retinal necrosis (27 patients), multifocal choroiditis (1), granulomatous uveitis (1), and CMV retinitis (3). The methods used were as described by us earlier. Aqueous humor (AH) and vitreous fluid (VF) collected as diagnostic tap or during therapeutic vitrectomy were tested for the presence of these viruses by FAT on direct smears and by virus isolation in diploid cell lines (MRC-5 and WI38 obtained from NFATCC) and Vero cell line. Detection of HSV in the direct smear and by isolation from the intraocular specimens was made as described above. For detection of CMV in the direct smears and identification of the isolate, indirect FAT was done using the monoclonal anti-CMV serum and rabbit anti-mouse serum obtained from DAKO, Denmark. Optimally diluted VZV hyperimmune human serum was used for detection and identification of the virus in the specimen by indirect immunofluorescence using FITC conjugated anti-human globulin antibody (DAKO, Denmark). The hyperimmune anti-varicella zoster virus serum was prepared from sterile pooled convalescent sera of patients of proven varicella zoster virus infection and standardised to the dilution to detect only VZV antigen and not other virus antigens commonly associated with ocular clinical specimens. Serum samples were assayed for IgG and IgM antibodies to HSV, VZV and CMV enzyme-linked immunosorbent assay (ELISA). ELISA tests were done on the first serum samples as soon as they were received for tentative initial diagnosis and they were later tested again along with subsequent samples (if available) at one sitting for assay of rise in antibody titres. For the assay of anti-HSV antibodies, microwell modules (Nunc-immuno module, Denmark) coated with HSV antigen prepared from HSV type 1 strain # 753166 supplied by the National Institute of Virology (NIV), Pune, India were used. This antigen was prepared as described by Fortier et al with virus grown on Vero cell line. ELISA tests for assay of antibodies to CMV (Diamedix, USA) and for VZV (Novum Diagnostica, Germany & Melotest, Spain) were done. Results of the ELISA tests were recorded using an automatic microplate reader (Model EL 311, Bio-Tek, USA or Dynatech, USA).
Sera of all patients were tested for antibodies to human immunodeficiency virus types 1 and 2 (HIV 1 and 2) by Immuno Comb (Orgenics Ltd., Israel) and Tridot (J. Mitra, India). If the serum was positive for HIV antibody, it was reconfirmed at the YRG-Centre, Chennai by the western blot method.
| Results|| |
| Conjunctivitis|| |
Among the 1061 patients with conjunctivitis (acute, chronic and recurrent) investigated during 1990 to 1998, the causative agents were adenovirus in 146 (13.8%), HSV in 23 (2.2%) and C. trachomatis in 222 (20.9%) patients. Adenoviruses were isolated mostly during epidemics of acute conjunctivitis. The serotypes of adenovirus isolated during this period are given [Table - 1]. Serotypes isolated during these epidemics were Adenovirus type 4 in 1991, type 3 in 1992-93 and type 7a in 1996. However, 20 isolates could not be identified due to the non-availability of several type specific antisera. EV70 in one and Coxsackievirus A 24 in four patients were detected only by FAT in direct smears in the sporadic cases during 1996-97.
PCR for detection of adenovirus was specific and sensitive enough to detect 0.0032 fg of the viral DNA. The results of PCR on 74 conjunctival specimens are shown in [Table - 2]. PCR positivity in 14 culture-negative specimens showed a significant increase in sensitivity from 14.9% to 53.8% (by 38.9%; McNemar test: p = 0.001) for the detection of adenovirus. The study suggests that PCR on conjunctival swabs is a rapid and reliable tool for the detection of in conjunctivitis. RFLP done on the amplified products of DNA of conjunctival specimens during the 1996 epidemic clearly showed a pattern similar to that of the standard strain of adenovirus 7a (ATCC VR-1084) as shown in [Figure - 3].
| Lens aspirates from congenital cataract|| |
Among the 86 lens aspirates obtained from congenital cataract, rubella virus was isolated from 7 (8.1%) specimens.
| Acute retinal inflammation|| |
Fifteen among the 32 patients had acute retinal inflammation due to a virus infection confirmed either by detection of the viral antigen in the direct smears [Figure - 4] or by isolation of the virus [Figure - 5] or by both the methods in AH or VF of 15 patients (VZV: 7, HSV: 2 and CMV: 6). The pattern of clinical diagnosis is shown in [Table - 3]. VZV was detected in AH of a case of granulomatous uveitis and the serum of this patient was positive for antibodies to HIV 1 and 2. CMV retinitis occurred in two patients who were on immunosuppressive therapy following renal transplantation. Significant serological response in the form of more than 4-fold rise in antibody titre was present in six patients in whom presence of the respective virus in the intraocular fluids was demonstrable (VZV:2, HSV:1 and CMV:3) [Table - 3]. Apart from these 15 patients, 4 others had serological evidence of virus infection (HSV: 3 and VZV: 1) in the form of a 4-fold rise in antibody titre for the respective virus. Thus, 19 patients in this series had acute retinal inflammation with proven viral infection. Fourteen other patients did not show any evidence of infection by these three viruses.
| Discussion|| |
Virological investigations are often not done in eye infections, especially in India, either because of lack of facilities or trained personnel. Often no importance is given to the required investigations, since most of these diseases are self-limiting in nature. At Sankara Nethralaya, Chennai, planned studies were initiated on the role of C. trachomatis and viruses in ocular infections, by applying virological techniques on the ocular specimens, especially for HSV, VZV, and CMV, to enable ophthalmologists to institute specific therapy and determine the incidence of types of viruses causing these diseases in our patients.
Viral conjunctivitis characterised by a rapid onset of redness, tearing, foreign body sensation, photophobia, lid swelling and visual disturbances, first in one eye followed by development of similar clinical features in the other eye, is commonly caused by adenoviruses and to some extent EV70 and Coxsackievirus A24 variant. Associated subconjunctival haemorrhages may be present. Adenovirus conjunctivitis has more often caused epidemics [12, 13] either as epidemic kerato-conjunctivitis (EKC) or pharyngo-conjunctival fever (PCF). EKC, predominantly caused by serotypes 8, 19 and 37 and less commonly by serotypes 2, 3, 4, 5, 7, 10, 11, 21, and 29 is the most serious manifestation of adenovirus kerato-conjunctivitis. PCF, a disease commonly affecting children, is most commonly caused by serotypes 3, 4, 5 and 7. Serotypes 1 to 11 and 19 are agents of nonspecific follicular conjunctivitis. Complete recovery without any sequelae is the common result. Though our experience indicates that adenoviruses were the most common causes of sporadic instances and epidemics of acute conjunctivitis in Chennai, there have been reports mainly of epidemics due to EV 70 and Coxsackie virus A 24 variant in India. We believe such studies were undertaken only when pandemics due to these viruses occurred. One of the important features of EV 70 conjunctivitis epidemic were its neurological manifestations. Coxsackievirus A24 was first isolated in 1970 during the Singapore epidemic and subsequent epidemics due to this virus have been identified in India and Sri Lanka.[l5],, Since detection of the virus by FAT on conjunctival smears was the method of choice, we performed direct smear diagnosis. However, our attempts to isolate these viruses in Vero and HEp-2 cell lines were unsuccessful. Since virus isolation was found to be difficult, diagnosis may mainly be made by serological means using the prototype strains J670/71-V1250. We wish to emphasise that only sporadic cases of infections due to these viruses were recorded in our laboratory during 1995-98. The occurrence of HSV conjunctivitis was uncommon. Our most important finding was the occurrence of a large (20.9%) number of laboratory-proven C trachomatis conjunctivitis. In our earlier study on consecutive patients of conjunctivitis during the period September 1991 to September 1992, 36.4% of patients had C. trachomatis conjunctivitis.
Polymerase chain reaction proved to be a reliable and valuable technique for the detection of adenoviruses in conjunctival specimens, and in the diagnosis of adenovirus conjunctivitis. RFLP could specifically identify the epidemic strain of 7a. Application of PCR and RFLP techniques may be of great value in the further study of adenoviral conjunctivitis.
One of the important clinical features associated with congenitally acquired Rubella infection is cataract. Rubella virus can be recovered from most infants from nasopharyngeal secretions and other organ systems with severe congenitally acquired Rubella. Since Rubella virus persists for 1-3 years in the lens, its isolation is diagnostic of congenital cataract due to this virus. In our study, nearly 8% of lenses with congenital cataract yielded Rubella virus, which we believe formed a significant percentage of this disease. This could have been prevented if immunisation against this virus infection been mandatory for girls in our country.
Viral infections of the retina, if left untreated, often lead to blindness. Acute retinal necrosis (ARN), a distinct ocular inflammatory syndrome, is characterised by a severe, occlusive vasculitis primarily involving the arteries of the retina as well as the choroid, resulting in full-thickness, diffuse necrotising retinitis. There is direct and indirect evidence indicating the involvement of the herpes family of viruses as the aetiologic agents. CMV retinitis, a clinically specific condition, is increasingly seen due to the rise in HIV infections and the increasing use of immunosuppressive drugs. CMV retinitis is often the initial manifestation of HIV infection. The key to potential sight preservation is the prompt and accurate aetiological diagnosis, as specific chemotherapy is available. In this series, predominant among the virus as causing ARN was VZV, followed by HSV and CMV. Almost all our patients with proven intraocular viral infections were apparently healthy except for one HIV-positive patient with VZV infection and two CMV retinitis patients who were on immunosuppressive agents because of renal transplants. Rapid virological diagnosis did help in prompt institution of specific chemotherapy and supportive measures resulted in useful vision in some of the patients as reported by us earlier. In this report, we described the clinical and virological features of 5 cases of viral retinitis. One patient with CMV retinitis showed excellent response to intravenous ganciclovir and another with HSV retinitis responded to acyclovir treatment. Both patients at admission had visual acuity of counting fingers in both eyes and on specific therapy had a final visual acuity of 6/6, N6 in one eye and improved vision in the other eye.
Diagnostic virology is done for a variety of reasons, from aetiologic diagnosis to epidemiological study. Local needs and the available resources determine the methods chosen. The microscopic techniques described here can be followed as an annexe to a bacteriology laboratory. Substantial resources in terms of virologists, trained technicians and laboratory facilities are needed for virus isolation. Serological tests using commercially available reagents may be performed for the commonly encountered viruses but the results require careful evaluation and interpretation. Finally, molecular biological techniques should be employed by larger diagnostic virology laboratories because of their specialised needs and the cost factor.
| References|| |
|1.||Hooks JJ. Ocular virology. In: Tabbara KF, Hydink RA, editors. Infections of the Eye. 2nd ed. New York, USA: Little Brown & Co; 1996. p 99-113. |
|2.||Schmidt NJ, Emmons RW. General principles of laboratory diagnostic methods for viral, rickettisial and chlamydial infections. In : Schmidt NJ, Emmons RW, editors. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. 6th ed. Washington DC: American Public Health Association; 1989. p 1-36. |
|3.||Madhavan HN, Rao SK, Natarajan K, Seethalakshmi G, Jayanthi I, Roy S. Evaluation of laboratory tests for diagnosis of chalmydial infections in conjunctival specimens. Indian J Med Res 1994;100:5-9. |
|4.||Madhavan HN. Evaluation of rapid culture method for detection of adenovirus from conjunctival specimens. Invest Ophthalmol Vis Sci (Abstract) 1992;33:S853. |
|5.||Madhavan HN, Ong KH, Sekar B, Radhika G. Rapid diagnosis of herpes simplex virus infection. Indian J Virol 1991;7:22-25. |
|6.||Schmidt NJ. Cell culture procedures for diagnostic virology. In : Schmidt NJ, Emmons RW, editors. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. 6th ed. Washington DC: American Public Health Association 1989: p 37-100. |
|7.||Hierholzer JC. Adenoviruses. In : Schmidt NJ, Emmons RW, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. 6th ed. Washington DC: American Public Health Association; 1989. p 219-64. |
|8.||Suresh D, Lilly TK, Roy S, Madhavan HN. Development and use of nested polymerase chain reaction for the detection of adenovirus from conjunctivitis specimens. J Clin. Virology 1998;11:77-84. |
|9.||Saitoh-Inagawa W, Oshima A, Aoki K, Itoh N, Isobe K, Uchio E, et al. Rapid diagnosis of adenoviral conjunctivitis by PCR and restriction fragment length polymorphism analysis. J Clin Microbwl 1996;34:2113-16. [PUBMED] [FULLTEXT]|
|10.||Madhavan HN, Biswas J, Gopal L, Sharma T. Investigations in the virological aetiology in acute retinal inflammation. Indian J Med Res 1995;101:233-37. [PUBMED] |
|11.||Fortier B, Chabrolle AD, Huranx JM. Comparison of an ELISA technique with quantal micro-neutralisation test for seroryping of HSV-1 and HSV-2 infected patients. J Virol Methods 1982;11:11-20. |
|12.||Guyer B, O' Day DM, Hierholzer JC, Schatfner W. Epidemic keratoconjunctivitis. A community outbreak of mixed adenovirus type 8 and type 19 infection. J Infect Dis 1975;132:142-50. |
|13.||Kaiwar R, Pal SR, Gangwar DN, Saigal AK, Biswas J, Sporadic cases of acute haemorrhagic conjunctivitis due to Enterovirus 70. Indian J Med Res 1983;78:447-49. |
|14.||Christopher S, John TJ, Charles V, Ray S. Coxsackie virus A24 variant EH 24/70 and enterovirus type 70 in an epidemic of acute haemorrhagic conjunctivitis-a preliminary report. Indian J Med Res 1975;63:27-30. |
|15.||Gogate SS, Padbidri VS. Studies on an outbreak of conjunctivitis in Bombay during 1979. Indian J Pathol Microbiol 1981;24:257-59. [PUBMED] |
|16.||Broor S, Kishore J, Dogra V, Satpathy G, Seth P, An epidemic of acute haemorrhagic conjunctivitis caused by Coxsackie A24 variant. Indian J Med Res 1992;95A:253-55. |
|17.||Wadia NH, Irani PF, Katrak SM. Lumboscaral radiculomyelitis associated with pandemic acute haemorrhagic conjunctivitis. Lancet 1973; i:350-52. |
|18.||Yin MM, Lim KH, Hoa YA. A Coxsackie virus type A24 epidemic of acute haemorrhagic conjunctivitis. Southeast Asian J Trop Med Pub Health 1976;7:1-5. |
|19.||Higgins PG, Chapman TED. Coxsackie A24 and acute haemorrhagic conjunctivitis in Sri Lanka. Lancet 1977; i:361. |
|20.||Rao SK, Madhavan HN, Prema P, Sitalakshmi G, Natarajan K, Garg D. Ocular chlamydial infections- clinicomicrobiological correlation. Cornea 1996;15:62-65. |
|21.||Duker JS, Blumenkranz MS. Diagnosis and management of acute retinal necrosis (ARN) syndrome. Surv Ophthalmol 1991;35:327-43. [PUBMED] |
|22.||Ludwig IH, Zegarra H, Zakov ZN. The acute retinal necrosis syndrome - possible Herpes simplex retinitis. Ophthalmology 1984;91:1659-64. [PUBMED] |
|23.||Yoser SL, Forester DJ, Rao NA. Systemic viral infections and their retinal and choroidal manifestations. Surv Ophthalmol 1993;37:313-52. |
|24.||Hennis HL, Scott AA, Apple DJ. Cytomegalovirus retinitis. Surv Ophthalmol 1989;34:193-203. [PUBMED] |
|25.||Gross JG, Bozzette SA, Mathews WC, Spector SA, Abramson NS, McCutchan JA, et al. Longitudinal study of cytomegalovirus retinitis in acquired immune deficiency syndrome. Ophthalmology 1990;97:681-86. |
|26.||Biswas J, Madhavan HN, Gopal L, Badrinath SS. Clinical features and virologic studies in viral retinitis. Indian J Ophthalmol 1995;43:63-68. [PUBMED] |
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5]
[Table - 1], [Table - 2], [Table - 3]