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Sexually Transmitted Diseases:
doi: 10.1097/01.olq.0000187206.69006.6e
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Evaluation of Goat Blood as Substitute for Sheep Blood in Modified Thayer-Martin Agar Medium for Culture and Isolation of Neisseria gonorrhoeae

Chawdhury, Faisal Arif Hasan MSc; Sultana, Jinath MSc; Rahman, Motiur PhD

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From the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh

This study was conducted at the ICDDR, B: Centre for Health and Population Research with the support of cooperative agreement: HRN-A-00 to 96-90005 to 00 from USAID. ICDDR, B acknowledges with gratitude the commitment of USAID to the Centre’s research efforts.

Correspondence: Motiur Rahman, Laboratory Sciences Division, ICDDR, B, GPO Box-128, Dhaka-1000, Bangladesh. E-mail: motiur@icddrb.org.

Received for publication May 2, 2005, and accepted August 9, 2005.

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Abstract

Modified Thayer-Martin (MTM) medium containing goat blood (MTMGB) and sheep blood (MTMSB) was compared for culture of N. gonorrhoeae. Evaluation of 252 endocervical specimens and quantitative culture of 10 strains revealed no significant difference in isolation and growth. MTMGB can be a suitable and cost-effective alternative for MTMSB.

GONORRHEA IS A MAJOR sexually transmitted disease prevalent in both developed and developing countries. Culture and characterization of Neisseria gonorrhoeae can provide valuable information on gonococcal strain populations in the community and the emergence and spread of antibiotic resistant strains. Culture is essential for antimicrobial susceptibility monitoring of microorganisms. Microbiologic media play the most important role in culture and identification of the bacteria. The fastidious organism requires growth supplements, including blood and inhibitors including antimicrobial agents for proper growth of the organism of interest. Culture of N gonorrhoeae, the causative organism of the sexually transmitted infection (STI) gonorrhea, is often difficult due to its fastidious nature. Thayer and Martin reported successful development of a selective medium for the cultivation of gonococci in 1964.1 The Thayer-Martin (T-M) medium has been widely accepted for primary isolation of gonococcus from sites where these organisms are far outnumbered by the natural bacterial flora. The high degree of specificity and selective sensitivity of the T-M selective medium supplemented with sheep blood returned the culture method to favor for diagnosis of N gonorrhoeae.2 Subsequently, modified TM (MTM) medium containing saponin lysed sheep blood has been described and shown to have selective advantage when plates were reexamined after 24 to 36 hours.3 However, in many developing countries sheep blood is not readily available due to local animal husbandry practices and climatic conditions, making sheep blood relatively expensive in the setting of meager resources. Goat blood had been successfully used as a substitute for sheep blood for fastidious organisms, including Haemophilus influenzae and Streptococcus pneumoniae.4 Inspired by the previous finding, we have evaluated the sensitivity and specificity of modified Thayer-Martin medium containing sheep blood (MTMSB) and modified Thayer-Martin medium containing goat blood (MTMGB) media for culture and identification of N gonorrhoeae from endocervical swab and compared the growth of N gonorrhoeae by quantitative culture.

Sheep blood and goat blood were collected aseptically by jugular vein puncture from antibiotic-free sheep and goat and lysed with 10% saponin solution and were used for preparation of MTMSB and MTMGB medium, respectively. MTMSB and MTMGB media containing 10% sheep blood or 10% goat blood, isovitalex supplement (Oxoid Ltd, Basingstoke, Hampshire, United Kingdom) and VCNT (Oxoid Ltd) inhibitor were prepared as recommended elsewhere.5,6

The study was conducted at an STI clinic for female sex workers in Dhaka, Bangladesh, during June 2003 to December 2003. All women attending the clinic for STI service irrespective of symptoms of STI were eligible for enrollment. All eligible women were requested to participate in the study after receiving a brief description of the purpose and procedures of the study. Written informed consent was obtained from all participants. Trained physicians performed a speculum examination, and 2 endocervical swabs were collected from each subject and were streaked evenly over the surface of the MTMGB and MTMSB. The order of streaking was randomized between MTMGB and MTMSB. The plates were immediately placed in candle extinction jars and incubated at 36°C in presence of 5% to 10% CO2 with in 2 hours of collection. Evaluation of cultures of N gonorrhoeae was done after 24 and 48 hours by technicians masked of the composition of the media. Presumptive identification of N gonorrhoeae was done by colony morphology, oxidase and catalase test.7 Growths in primary plates were counted and described as heavy growth (>100 cfu/plate), moderate growth (<100 to >50 cfu/plate), and poor growth (<50 cfu/plate).

Quantitative determination of viable N gonorrhoeae in MTMGB and MTMSB plates was done on 8 randomly selected isolates from enrolled subjects and 3 World Health Organization (WHO) control strains by serial dilution agar plating procedure using 8 different N gonorrhoeae isolates.8 Briefly, bacterial suspension in tryptic soy broth was prepared from overnight culture of N gonorrhoeae and was adjusted to 3 × 108 cfu/ml.6 A 10-fold serial dilution of bacterial suspension was spread on MTMGB and MTMSB plates (Becton Dickinson) and incubated at 36°C in 5% to 10% CO2 for 24 to 48 hours, and colonies were counted by colony counter. WHO control strains A, D, and E were used as control in the quantitative culture. In order to investigate the possible role of serotype and serovar on the growth of N gonorrhoeae on MTMSB and MTMGB media, serotyping of the isolates was done. Serotype of the isolates was determined by a coagglutination test using Phadebact Monoclonal GC kit according to the manufacturer’s instructions (Boule Diagnostics AB, Huddinge, Sweden). Serovar of the isolates was also determined by a coagglutination method using a panel of 14 monoclonal antibodies to protein I (Boule Diagnostics AB), as described elsewhere.

A total of 252 female sex workers were enrolled in the study, and an endocervical swab was collected from all of them. Among the enrolled subjects, 121 were symptomatic and 131 were asymptomatic. Of these 252 endocervical specimens, a total of 65 (25.8%) endocervical swabs were positive for N gonorrhoeae, including 36 (29.8%) from symptomatic and 29 (22.1%) from asymptomatic subjects. Sixty-four were positive on MTMSB and 62 were positive in MTMGB plates (Table 1). The sensitivity, specificity, positive predictive value and negative predictive value of MTMGB plates for isolation of N gonorrhoeae were 95%, 100%, 98%, and 100%, respectively. There was no significant difference in colony morphology, size, and number of colonies of isolates from symptomatic and asymptomatic subjects on both types of plates (Table 1). Among the 65 positive isolates, 14%, 46%, and 4% isolates had heavy, intermediate, and poor growth on MTMSB plates compared to 11%, 49%, and 2% on MTMGB plates, respectively. Among the isolates, 41 (63%) belonged to serotype A, including 26 (63%) Arost serovar, and 24 (37%) to serotype B, including 6 (25%) Byvupt serovar. There was no significant difference in serotype and serovar of the isolates grown in MTMSB and MTMGB (data not shown). Two isolates that did not grow on MTMGB belonged to serovar Arost and Byoptr, and 1 isolate that did not grow on MTMSB belonged to serovar Arst.

Table 1
Table 1
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Quantitative determination of the number of viable bacteria in MTM plate containing goat blood or sheep showed no significant difference in number of colonies in any of the test and control isolates at 10−5 and 10−6 dilution (Table 2). Furthermore, no significant difference in growth was observed between serotype A and serotype B isolates.

Table 2
Table 2
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Culture of N gonorrhoeae is expensive as it requires growth supplements, inhibitors, and sheep blood. Although growth supplements and inhibitors are commercially available, the commercial availability of sheep blood remains a problem in many countries in southeast Asia due to local animal husbandry practice or cost. The current study was motivated by the unavailability of sheep blood for establishment of gonococcal culture and antimicrobial susceptibility monitoring in a facility in eastern Nepal. Our data suggest that there is no significant difference in isolation of N gonorrhoeae using MTM plates containing sheep blood or goat blood, and blood from neither animal significantly favored any serotype specific recovery of the organism. We anticipate that MTM plates containing goat blood could be used as a suitable substitute for sheep blood in areas where sheep blood is not easily available or maintenance of sheep is not favored by climatic conditions or local culture.

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References

1. Thayer JD, Martin JE Jr. A selective medium for the cultivation of N. gonorrhoeae and N. meningitidis. Public Health Rep 1964; 79:49–57.

2. Thayer JD, Martin JE Jr. Improved medium selective for cultivation of N. gonorrhoeae and N. meningitidis. Public Health Rep 1966; 81:559–562.

3. Riddell RH, Buck AC. Trimethoprim as an additional selective agent in media for the isolation of N gonorrhoeae. J Clin Pathol 1970; 23:481–483.

4. Gratten M, Battistutta D, Torzillo P, Dixon J, Manning K. Comparison of goat and horse blood as culture medium supplements for isolation and identification of Haemophilus influenzae and Streptococcus pneumoniae from upper respiratory tract secretions. J Clin Microbiol 1994; 32:2871–2872.

5. Laboratory diagnosis of gonococcus. Available at: http://w3.whosea.org/bct/book33/1h.htm.

6. Van Dyck E, Meheus AZ, Piot P. Laboratory Diagnosis of Sexually Transmitted Diseases. Geneva: World Health Organization; 1999:109–112.

7. Ho BS, Feng WG, Wong BK, Egglestone SI. Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples. J Clin Pathol 1992; 45:439–442.

8. Cappucino J, Sherman N. Microbiology: A Laboratory Manual. 2nd ed. 1993:75.

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