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ORIGINAL ARTICLE
Year : 2002  |  Volume : 50  |  Issue : 4  |  Page : 295-299

Microbiological study of neonatal conjunctivitis with special reference to chlamydia trachomatis.


Department of Paediatrics, RP Centre for Ophthalmic Sciences, AIIMS, New Delhi, India

Correspondence Address:
M Mohile
Department of Paediatrics, RP Centre for Ophthalmic Sciences, AIIMS, New Delhi
India
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Source of Support: None, Conflict of Interest: None


PMID: 12532494

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  Abstract 

PURPOSE: To study the microbial agents, chiefly Chlamydia trachomatis and other bacteria, in neonatal conjunctivitis. METHODS: Conjunctival specimens from 70 newborns with conjunctivitis were subjected to bacterial culture and sensitivity testing, monoclonal antibody based C. trachomatis antigen detection test and species-specific Chlamydia antibody detection in the sera of babies and their mothers, by micro-immunofluorescence assay. RESULTS: Bacteria were isolated from 35 (50%) babies; the majority (20, 57.14%) were Staphylococcus epidermidis. C. trachomatis antigen was detected in conjunctival smears of 17 (24%) babies, and 6 (35.29%) of them were positive for other bacteria. Six babies and their mothers tested positive for C. trachomatis Ig G antibodies. At follow-up after 14 weeks, 6 (35.29%) of the Chlamydia antigen-positive babies were found to have developed recurrent conjunctivitis. CONCLUSION: C. trachomatis is responsible for almost a quarter of all cases of neonatal conjunctivitis, with recurrences in 35% of cases. Bacteria could be isolated from 50% of the patients though the exact role of Staphylococcus epidermidis, isolated from 28.65% of the neonatal conjunctivitis cases, remains unclear.

Keywords: Chlamydia trachomatis, newborns, conjunctivitis, antigen detection, immunofluorescence assay, antibody detection


How to cite this article:
Mohile M, Deorari AK, Satpathy G, Sharma A, Singh M. Microbiological study of neonatal conjunctivitis with special reference to chlamydia trachomatis. Indian J Ophthalmol 2002;50:295-9

How to cite this URL:
Mohile M, Deorari AK, Satpathy G, Sharma A, Singh M. Microbiological study of neonatal conjunctivitis with special reference to chlamydia trachomatis. Indian J Ophthalmol [serial online] 2002 [cited 2019 May 22];50:295-9. Available from: http://www.ijo.in/text.asp?2002/50/4/295/14763

CT A - C: C TRACHOMATIS SEROTYPES A - C ; CT D - K: C TRACHOMATIS SEROTYPES D - K; L1-L3: C TRACHOMATIS SEROTYPES L1 - L3 ; C PN.: C PNEUMONIAE; C PSITT: C PSITTACII; CT AG: C TRACHOMATIS ANTIGEN

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CT A - C: C TRACHOMATIS SEROTYPES A - C ; CT D - K: C TRACHOMATIS SEROTYPES D - K; L1-L3: C TRACHOMATIS SEROTYPES L1 - L3 ; C PN.: C PNEUMONIAE; C PSITT: C PSITTACII; CT AG: C TRACHOMATIS ANTIGEN

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Conjunction in newborns is a commonly encountered problem.[1] The majority of infectious neonatal conjunctivitis cases are of bacterial aetiology.[2],[3] Though most of these cases are benign, some of them may progress to systemic complications or visual loss. C. trachomatis and N.gonorrhoea, the two well described agents associated with ophthalmia neonatorum, are known to be associated with systemic complications and severe visual loss.[4][5][6] In some infants with Chlamydia-associated conjunctivitis, the infection persists for a longer period with pannus formation and scarring, and the babies later develop pharyngitis and pneumonia if left untreated.[4] With the increasing incidence of chlamydial genital infection throughout the world,[7],[8] there is an increased perceived risk of neonatal chlamydial conjunctivitis.[9],[10] The exact determination of the aetiology in neonatal conjunctivitis cases may help improve initial management and control, thereby preventing further complications.

This study was undertaken to determine the association of conjunctivitis with different bacterial agents, specifically C. trachomatis, in 70 newborns.


  Materials and Methods Top



  Study population Top


Seventy neonates with conjunctivitis and their mothers were included in the study. The infants were from rooms in the obstretics or paediatrics wards, and the paediatric out-patient department of the All India Institute of Medical Sciences, New Delhi, India. Details of mode of delivery, birth weight and day of onset of conjunctivitis were noted in each patient. Each patient underwent a detailed ophthalmic examination including recordings of the discharge, eye redness, chemosis and lid swelling. Detailed systemic clinical examination included a search for lymphadenopathy and pneumonia. The conjunctivitis was graded into 3 categories; (i) sticky eyes: (ii) mucopurulent conjunctivitis: and (iii) purulent conjunctivitis. The infants were followed for a duration of 14 weeks.


  Specimen collection Top


After informed consent from the mothers, conjunctival specimens were collected from each of the infants with sterile cotton-tipped swabs for (i) C. trachomatis antigen detection assay and (ii) bacterial culture and antibiotic sensitivity testing. A smear was made from the swab taken for Chlamydia antigen detection on a clean teflon-coated glass slide and transported to the laboratory. The slides were air dried, fixed in methanol for 10 minutes and preserved at -20°C until tested by direct immunofluorescence assay (DFA). After informed consent from parents blood was collected from the infants by pricking the heel and from their mothers by pricking the finger. Blood was collected in 5mm x 3mm cellulose sponges (J Weiss and Sons, UK). The saturated sponges were transported to the laboratory in tightly stoppered sterile glass vials and stored at -70°C till tested for Chlamydia antibodies.


  Bacterial culture and sensitivity Top


The swabs for bacterial culture and sensitivities were transported to the laboratory in a sterile stoppered vial and cultured immediately on blood agar, chocolate agar and thioglycolate broth. The inoculated plates and tubes were incubated at 37°C (the chocolate agar plate in a candle jar) for a minimum of 48 hours before a negative culture report was made. The isolated bacteria were identified using standard procedures.[11] The antimicrobial susceptibility of the isolated bacteria were tested against framycetin (100μg), chloramphenicol (10μg), gentamicin (10μg), tobramycin (10μg), cloxacillin (1ug) on Muller Hinkton agar medium by Kirby Bauer's disc-diffusion method.[12]


  Direct immunofluorescence test (DFA) for Chlamydia antigen detection Top


This was done using a C.trachomatis direct specimen test kit (Syva Microtrak, UK), according to the manufacturer's instructions. Briefly, the conjunctival smears were brought to room temperature. The smears were covered with 30 ml of the FITC conjugated C trachomatis murine monoclonal antibodies and incubated for 15 minutes at room temperature. The slides were washed in distilled water, air dried, mounted and observed under 100X objective of a fluorescent microscope. (Nikon, Japan). A positive and negative control smear (provided with the kit) was included with the test.


  Micro immunofluorescence assay (MIF) for detection of C. trachomatis Ig G and Ig M antibodies Top


To each of the vials containing blood-soaked cellulose sponges, 375ul of phosphate buffer saline (PBS) at pH 7.2 was added (final serum dilution 1:8).[13] MIF assay was performed on teflon-coated glass slides pre-coated with Chlamydia antigens: C. trachomatis serovars A-C, D-K, L1-L3, C. pneumoniae and C. psittacii as separate dots. (IO International, UK) in 4 two-fold serum dilutions using a standard procedure according to manufacturer 's instructions.[14] The antigen slides were stored at -20°C and the slide to be used was taken out 10 minutes prior to testing. Fluorescein isothiocyanate conjugated rabbit anti-human immunoglobulin (Ig G or Ig M) at working dilution (1:32) was used as the second antibody. The slides were washed in PBS pH 7.2 followed by distilled water, air dried, mounted and observed under the 100X objective of a fluorescent microscope. Positive and negative control specimens (provided with the kit) were included in the test.


  Management Top


Normally, the eyes of the neonates were cleaned with sterile normal saline after birth and no prophylactic antimicrobial drops were instilled. After detection of conjunctivitis, they were treated with gentamicin and with tobramycin eye drops, chloramphenicol eye drops or soframycin eye drops, depending on the antimicrobial sensitivity test result. The infants positive for C. trachomatis antigen also received oral erythromycin 40 - 50 mg/ kg of body weight /day for 14 days. They were followed up to 14 weeks for possible occurrence of pneumonia. The mothers of babies positive for C. trachomatis were informed and sent along with their partners to the Gynaecology Department for investigations and treatment for Chlamydial infections.


  Results Top


Twenty eight (40%) babies were males and 42 (60%) babies were females. Thirty-eight babies were delivered vaginally and 32 babies were delivered by caesarean section. The onset of conjunctivitis was before 4 days of birth in 59 (70%) babies and before 7 days of birth in 62 (88.57%) babies. In 41 (59%) babies the conjunctivitis was unilateral and in 29 (41%) it was bilateral. The conjunctivitis was graded mild (sticky eyes) in 25 (35.71%) babies, mucopurulent in 30 (42.85%) and purulent in 15 (21.42%) babies. On systemic evaluation none of the infants had any abnormal findings including lymphadenopathy.

The results of bacterial isolation are given in [Table - 1]. Bacteria were isolated from the conjunctival specimens in 35 (50%) infants. The distribution of bacterial isolation in 3 grades of conjunctivitis was as follows: 12 (48%) infants with sticky eyes, 16 (53.33%) infants with mucopurulent conjunctivitis; and 7 (46.66%) infants with purulent conjunctivitis. Significant difference was not observed between the types of bacteria from the 3 categories of conjunctivitis. Staphylococcus epidermidis was the most common bacterium in all 3 categories. The in-vitro antimicrobial sensitivity patterns are shown in [Table - 2]. No correlation could be observed between type of organism, infant gender and laterality with the severity of the disease.

C. trachomatis antigen could be detected in conjunctival smears in 17 (24%) infants by DIF assay. Twelve of 17 (70.58%) infants had developed conjunctivitis within 4 days of birth and in 10 (58.82%) of them the conjunctivitis was unilateral. Nine of 38 (23.68%) of the vaginally delivered infants developed Chlamydial conjunctivitis compared to 8 of 32 (25%) of those delivered by caesarean section (p= 0.89). It was noted that 20 of 30 caesarean sections were due to early rupture of membrane. None of the infants had received any prophylactic eye drops. Ig G antibody against Chlamydiae could be detected in the sera of 6 infants and their mothers by MIF assay. These 6 infants also tested positive for the Chlamydia antigen. Three of 6 infants had C. pneumoniae species specific antibodies and 3 of them had Chlamydia group specific antibodies [Table - 3]. The antibody pattern observed in the infants and their respective mothers were identical. In the remaining 11 cases of C. trachomatis conjunctivitis the babies and the mothers were negative for Chlamydia antibodies. C. trachomatis Ig M antibody was not detected in any of the infants and their mothers. Bacteria could be isolated from conjunctival smears of 6 of 17 Chlamydia antigen positive infants - S. epidermidis from 4, S. aureus from one and Diphtheroids from one.

In the follow-up period up to 14 weeks, 6 infants initially positive for Chlamydia antigen developed recurrent conjunctivitis; only one of these 6 babies was positive for Chlamydia antigen at 14 weeks. None of the babies developed pneumonia.


  Discussion Top


Conjunctivitis in newborns is an important entity. It is usually assumed that the affected infants acquire the infection from mothers' birth canals, though some may acquire the infections from their immediate surroundings. Therefore it is an indirect indicator of reproductive tract microbial carriage in their mothers. The results of this study indicate that 24% of the infants had Chlamydial conjunctivitis, as demonstrated by Syva Microtrak antigen detection assay using monoclonal antibodies. This antigen detection test has reported sensitivity and specificity of 96.23% and 99.5%[15] against isolation of Chlamydia in tissue culture; this is still considered the "gold standard" for diagnosis of Chlamydial infections.[16] In this particular study group the incidence of Chlamydial conjunctivitis appears high, but due to lack of information it could not be compared with past incidences. In a report from the United States, C. trachomatis was found to be the commonest microbial cause of neonatal conjunctivitis and could be demonstrated in 51% of the infants by antigen detection and/or on tissue culture.[6] At present C. trachomatis is the commonest cause of sexually transmitted infections in the world[7],[8] and infants born to mothers with Chlamydia infection of cervix are at 60-70% risk of acquiring Chlamydial infection.[17],[18]

From the present study no significant association could be found between vaginal delivery and Chlamydial conjunctivitis. The incidence of deliveries by caesarean section was high (45.71%) in this study group. However the incidence of Chlamydial conjunctivitis was not significantly different between the infants delivered by caesarean section or by vaginal mode. A significant association could have been expected if the Chlamydial conjunctivitis was to be solely acquired by passage through infected mother's birth canal. The high incidence of caesarean sections as a mode of delivery along with high incidence of early onset conjunctivitis in the infants suggest a possibility of intrauterine Chlamydial infection in these infants due to early rupture of membrane. Indeed early rupture of membrane is the commonest indication for caesarean section in our Institute. Majority (70%) of the Chlamydia conjunctivitis occurred within 4 days of birth, corroborating the reports that neonatal Chlamydial conjunctivitis usually occurs within a few days of birth.[4],[5]

In this study group bacteria could not be isolated in 50% infants. Similar observations have been made by others.[1],[3],[6],[19] S. epidermidis was the most common isolated bacterial species (28.6%) followed by S. aureus (10%). Gram-negative bacteria could be isolated from 4.3% of infants. This spectrum of bacterial isolation was similar to that observed by other investigators from different parts of the world.[2],[3],[19] In an earlier study, S. epidermidis was the predominant bacterial species isolated from mild neonatal conjunctivitis and S. aureus was the predominant bacterial species isolated from purulent neonatal conjunctivitis.[20] Even though S. epidermidis was the most commonly isolated bacterial species, the exact role of this organism in the causation of conjunctivitis in newborn babies remains unclear. These organisms might have been the part of the normal flora that the infants. The 4 isolates of Diphtheroids also were most likely part of the normal conjunctival flora.

Specific serum Ig M antibody against Chlamydia is raised only when there is systemic Chlamydial infection.[21] Therefore diagnosis based on specific Chlamydial Ig M detection has a poor sensitivity and specificity for diagnosis of Chlamydial conjunctivitis. In the present study Chlamydia Ig M could not be detected in any of the infants. Chlamydia Ig G was detected in 6(8.5%) infants and their mothers. In these infants the Ig G possibly represented placental transfer of the maternal antibodies.[22] In presence of C. trachomatis antigen, absence of C. trachomatis antibodies in 11 of 17 neonates is puzzling, though it might indicate early stages of infection before development of systemic antibodies. An earlier study has reported passively transferred Chlamydial Ig G could be demonstrated in 4.4% of the apparently healthy newborns infants.[23] It may also be possible that all the genitally infected mothers did not possess serum antibodies. Since cervical samples from mothers could not be investigated for C. trachomatis in the present study, the exact situation remains unclear. The significance of the Ig G antibodies in infants is not clear, as the Chlamydia antibody has been demonstrated in infants who later developed Chlamydial infections as well as in those who did not develop infection.[23]

Since none of the babies received any routine prophylaxis apart from normal saline cleaning, the conjunctivitis cases without any microbial isolation, could not be due to reaction to any chemicals. Moreover, in 6 infants with C. trachomatis conjunctivitis, conjunctival discharge was persistent even at 14 weeks' follow-up in spite of the oral erythromycin therapy. At this stage the Chlamydia antigen could only be detected in one of them. The exact reason for the recurrence could not be ascertained. Newer therapies like azithromycin may be helpful in these cases.

From the present study, it appears that Chlamydial infections contribute substantially (almost a quarter) to neonatal conjunctivitis in this study group and serological tests alone are not helpful in diagnosis.

 
  References Top

1.
Di Bartolomeo S, Mirta DH, Janer M, Rodriguez FMR, Sauka D, Magarinos F, et al. Incidence of Chlamydia trachomatis and other potential pathogens in neonatal conjunctivitis. Int J Infect Dis. 2001;5:139-43.  Back to cited text no. 1
    
2.
Prentice MJ, Hutchinson GR, Taylor Robinson D. A microbiological study of neonatal conjunctivae and conjunctivitis. Br J Ophthalmol 1977;61:601-7.  Back to cited text no. 2
    
3.
Molgaard TL, Nielsen PB, Kacrn J. A study of the incidence of neonatal conjunctivitis and of its bacterial causes including Chlamydia trachomati. Acta Ophthalmol. 1984;62:461-71.  Back to cited text no. 3
    
4.
Hobson D. Chlamydial infection in neonates. New Eng J Med 1977:296:398.  Back to cited text no. 4
    
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Chandler JW, Alexander ER, Pfiffer TA, Wang SP, Holmesk K, English M. Ophthalmia neonatorum associated with maternal Chlamydial infection. Trans. Am Acad Ophthalmol Otolaryngo. 1977;83:302-8.  Back to cited text no. 5
    
6.
Barry WC, Teare EL, Uttley AHC, Wilson SA, McManus TJ, Limks Gamsu H, et al. Chlamydia trachomatis as a cause of neonatal conjunctivitis. Arch Dis Child 1986;61:797-99.  Back to cited text no. 6
    
7.
Piot P. Islam MQ. Sexually transmitted diseases in the 1990s; Global epidemiology and challenges for control. Sex. Trans. Dis. 1994;21:S7-S13.  Back to cited text no. 7
    
8.
Chlamydia trachomatis genital infection- United States, 1995. MMWR Morb. Morta. Wkly Rep.1997;46:193-98.  Back to cited text no. 8
    
9.
Valencia C, Prado V, Rios M, Cruz MA, Pilorget JJ. Prevalence of Chlamydia trachomatis in neonatal conjunctivitis: Determination by indirect fluorescence & gene amplification. Rev. Med Chil 2000;128:758-65.  Back to cited text no. 9
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10.
Salpietro CD, Bisignano G, Fulia F, Marino A, Barberi I.Chlamydia trachomatis conjunctivitis in the new born. Arch Pediatr 1999;6: 317-20.  Back to cited text no. 10
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11.
Colle JG, Miles RS. Tests for identification of bacteria. In: Collee JG, Dugid JP, Frases AG, Marmion BP, editors. Mackie and Mc Cartney's Practical Medical Microbiology, 13th ed. Churchill Livingstone. 1989.Vol 2, pp141-60.  Back to cited text no. 11
    
12.
Brauer AW, Kirby WMM, Sherris JC, Truck M. Antibiotic sensitivity testing by a standard disc method. Am J Clin Pathol. 1966;45:493-97  Back to cited text no. 12
    
13.
Darougar S, Treharne JD, Minassian D, El Sheikh H, Dines RJ, Jones BR. Rapid serological test for diagnosis of Chlamydial ocular infections. Br J Ophthalmol. 1978;62:503-8.  Back to cited text no. 13
    
14.
Treharne JD, Darougar S, Jones BR. Modification of the microimmunofluorescence test to provide a routine serodiagnostic test for Chlamydial infections. J Clin Pathol. 1977;30:510-17.  Back to cited text no. 14
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15.
Uyeda CT, Welborn P, Ellison BN, Shunk K, Tsaoux B. Rapid diagnosis of Chlamydial infections with Microtrack Direct Test. J Clin Microbiol 1984; 20:948-50.  Back to cited text no. 15
    
16.
Ridgway GL, Taylor Robinson D. Current problems in microbiology. Chlamydial infections: Which laboratory test? J Clin Pathol 1991;41:1- 5.  Back to cited text no. 16
    
17.
Schacter J. Grossman M., Sweet RC, Holt J, Jordon C, Bishop E. Prospective study of perinatal transmission of C. trachomatis. JAMA: 1986;255:3374-77.  Back to cited text no. 17
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Frommel GT, Rothenberg R, Wang SP, McIntosh K. Chlamydial infection of mothers and their infants. J Paediatr 1979;95:28-32.  Back to cited text no. 18
    
19.
Sandstrom KI. Etiology and diagnosis of neonatal conjunctivitis. Acta Paediatr Scand. 1987;76:221-27.  Back to cited text no. 19
    
20.
Titiyal JS. Clinico-bacteriological study of conjunctival flora of new-born in health and disease. Thesis Submitted to Faculty of All India Institute of Medical Sciences. 1987 p 35-40.  Back to cited text no. 20
    
21.
Schacter J, Grossman M, Azimi PH. Serology of C. trachomatis in infants. J Infect Dis.1982;146:530-35.  Back to cited text no. 21
    
22.
Hemmerschalg MR, Anderka M, Semine DZ, Mc Comb D, Mc Cormak WM. Prospective study of maternal and infantile infection with Chlamydia trachomatis. Paediatr. 1979;64:142-48.  Back to cited text no. 22
    
23.
Singh M. Khedkar G, Satpathy G. Sero-prevalence of Chlamydia infection in under-five children. Ind. Paediatr. 1996;33:841-44.  Back to cited text no. 23
    



 
 
    Tables

  [Table - 1], [Table - 2], [Table - 3]


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