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Presumptive diagnosis of brucella epididymoorchitis by modified cold ZN staining of testicular pus sample.

Sir,

Brucellosis is a well known zoonotic disease. Consumption of unpasteurised milk and milk products is a common mode of transmission of brucella to humans. Rare modes of transmission include transplacental transfer, breast feeding, respiratory and even sexual intercourse (1).

Brucellosis known for its varied manifestations is common in north Karnataka (2,3). Therefore, a very high index of suspicion is necessary for early diagnosis. Deep-seated chronic infections leading to various complications and a lot of morbidity are strongly associated with untreated brucellosis (4,5). Epididymoorchitis is a frequent genitourinary complication and up to 20 per cent of men with brucellosis suffer from this complication (6,7).

Protean manifestations of brucellosis, lack of awareness about the disease and slow growth of the causative agent make the laboratory diagnosis difficult. Culture is the gold standard for laboratory diagnosis of brucellosis. But, culture being less sensitive and often delayed, serology is the mainstay of diagnosis (4,8-10). Therefore, importance of rapid diagnostic techniques cannot be overemphasized. Use of modified cold ZN stain on blood culture broths for presumptive and early identification of brucella has been documented11. We present here a case of Brucella epididymoorchitis, where modified cold ZN staining of pus smears was used for rapid presumptive diagnosis of brucella infection.

A 53 year old diabetic male, came to Surgery OPD at SDM Medical College Hospital, Dharwad (Karnataka, India) in the last week of September 2007 with complaints of fever for one week and pain and swelling in both the testes since 5 days. On examination, the patient had mild fever (99[Degrees]F), moderate hepatosplenomegaly and bilaterally inflamed testes. A presumptive diagnosis of bilateral epididymoorchitis was made at this stage, the patient was admitted and was put on amoxicillin-clavulanic acid (1.2 g b.d., i.v.,) metronidazole (500 mg t.i.d., i.v.) and diclofenac sodium (25 mg i.m.)

The laboratory investigations revealed following findings; fasting blood sugar--252 mg/dl and postprandial blood sugar--445 mg/dl. HIV by Tridot (BioMed Industries, Himachal Pradesh) and HBsAg by Hepacard (BioMed Industries, Himachal Pradesh) were negative. Ultrasonography (USG) abdomen showed hepatomegaly with grade II fatty infiltration and non specific moderate splenomegaly. USG scrotum showed bilateral epididymoorchitis with right testicular abscess and left sided hydrocele.

Widal test for enteric fever (Span Diagnostics Ltd., Surat, India) and quantitative buffy coat (QBC) for malarial parasite (QBC Diagnostics Inc., Philipsburg, PA, USA) were negative. In our laboratory every sample received for Widal test is also tested with Rose Bengal Plate (agglutination) Test for Brucellosis (RBPT) [Indian Veterinary Research Institute [IVRI], Izatnagar, India]. The RBPT for this sample was strongly positive. The serum was therefore, subjected to standard tube agglutination test (SAT--suspension of pure smooth culture of Brucella abortus strain 99 in phenol saline, IVRI, Izatnagar) to know the titre of brucella agglutinins. The patient showed alarmingly high titres of 10,240 IU/ml with SAT.

The patient was posted for right sided orchidectomy. The right testicle showed pus pockets, which yielded at least 15 ml of frank pus. The pus was collected in sterile plain bulb and was transported to the microbiology laboratory immediately. Blood for culture was also collected and processed.

The pus was plated on chocolate agar and MacConkey's agar. Two tubes each of thioglycollate broth and brain heart infusion broth were inoculated simultaneously. Pus smears were stained by Gram stain and modified cold ZN stain (12). Gram stain performed on several smears showed only pus cells and no organisms. However, the modified cold ZN stained smears showed acid-fast coccobacilli arranged in groups of 5 to 10 along with pus cells (Fig.).

Because of the initial suspicion of brucella infection all the procedures were carried out in the bio-safety cabinet (Kartos International, Class II, Type A). The culture plates revealed growth after 48 h incubation. The growth on chocolate agar showed tiny grey colonies and MacConkey's agar showed nonlactose fermenting tiny colonies. Growth from both the media was provisionally and separately identified as brucella species with the help of following reactions. Gram stain--Gram negative coccobacilli, modified cold ZN stain--acid fast coccobacilli, oxidase positive, catalase--positive, motility--non-motile, urease test--positive.

Slide agglutination of growth from chocolate agar showed strong agglutination with high titre brucella antiserum stored in our laboratory. The blood culture, after 5 days of incubation, also grew a similar organism with the entire set of characteristics identical with the growth from pus. The isolates were confirmed as Brucella species by PCR at The Maratha Mandal Dental College Molecular Biology Laboratory, Belgaum, Karnataka. The isolates were also sent to Indian Veterinary Research Institute (IVRI), Izatnagar for identification, and were identified as Brucella melitensis biotype 1. The patient was treated with rifampicin (450 mg b.d.) and doxycycline (100 mg b.d.) for a period of 6 wk after orchidectomy. His fever subsided, left sided hydrocele resolved completely, and his blood culture was negative for brucella after completion of treatment.

[FIGURE OMITTED]

Early diagnosis of brucellosis will be vital not only for early initiation of specific antibiotic therapy to the patient but also for safety of the laboratory personnel handling the samples. Direct microscopy of blood and bone marrow is found to be less useful in diagnosis of brucellosis (13). Though molecular diagnostic techniques like real time PCR is very useful for rapid diagnosis of this condition, it is not yet available for routine use in countries like India14. Culture, therefore, is the gold standard for unequivocal diagnosis of brucellosis (8).

In the present case, a presumptive identification of brucella epididymoorchitis was done in about 15 min after receiving the pus sample. To conclude, rapid presumptive diagnosis of brucellosis by modified cold ZN stain of exudates is possible especially when there is a strong serological evidence of brucella infection. The staining technique does not need great expertise or sophisticated laboratory equipment and hence can be easily used at peripheral laboratories. However, further confirmation of the test result by culture is necessary. As brucellosis is one of the most commonly encountered laboratory acquired infections, presumptive diagnosis of brucellosis would be of great help in the safety of laboratory workers as well (15,16).

Acknowledgment

The authors thank Dr Kishor Bhat, Maratha Mandal Dental College Molecular Biology Laboratory, Belgaum, Karnataka, for PCR confirmation of isolates as Brucella species and Dr B.S. Patil, Professor and Head, Department of Medicine, SDM College of Medical Sciences & Hospital, Dharwad, for support and critical suggestions.

References

(1.) Madkour M, Brucellosis: Overview. In: Madkour's brucellosis manual, 2nd ed. Berlin: Springer; 2001. p. 2-3.

(2.) Hemashettar BM, Patil CS. Brucellosis among practicing veterinarians. Indian J Med Microbiol 1991; 9 : 45-7.

(3.) Mantur BG, Biradar MS, Bidri RC, Mulimani MS, Veerappa, Kariholu P. Protean clinical manifestations and diagnostic challenges of human brucellosis in adults: 16 years experience in an endemic area. J Med Microbiol 2006; 55 : 897-03.

(4.) Centers for Disease Control and Prevention (CDC). Laboratory- acquired brucellosis-Indiana and Minnesota 2006. CDCMMWRMorb Mortal Wkly Rep 2008; 57 : 39-42.

(5.) Smits HL, Kadri SM. Brucellosis in India: A deceptive infectious disease. Indian J Med Res 2005; 122 : 375-84.

(6.) Martinez AN, Solera J, Corredoira J, Beato JL, Alfario EM, Atienzar M, et al. Epididymoorchitis due to Brucella melitensis: A retrospective study of 59 patients. Clin Infect Dis 2001; 33 : 2017-22.

(7.) Akinci E, Bodur H, Cevik MA, Erbay A, Eren SS, Ziramanl N, et al. A complication of brucellosis: Epididymoorchitis. Int J Infect Dis 2006; 10 : 171-7.

(8.) Jordi S, Miquel V. Laboratory diagnosis of brucellosis in a rural endemic area in northern Spain. Int Microbiol 2004; 7 : 53-8.

(9.) Mantur BG, Amarnath SK, Shinde RS. Review of clinical and laboratory features of human brucellosis. Indian J Med Microbiol 2007; 25 : 188-202.

(10.) Mantur BG, Mulimany MS, Mangalagi SS, Patil AV. Brucella epididymoorchitis--report of 5 cases. Indian J Med Microbiol 2001; 19 : 208-11.

(11.) Joshi PA, Kulkarni RD, Powar RM. Modified cold ZN staining for presumptive identification of Brucella. Indian J Med Res 2005; 121 : 108-10.

(12.) Alton CG, Jones LM, Peitz DE. Laboratory techniques in brucellosis, 2nd ed. Geneva: World Health Organization; 1975.

(13.) Forbs BA, Sahm D, Weissfeld SA. Bailey and Scott's diagnostic microbiology. 11th ed. USA: Mosby Inc; 2006. p. 487-9.

(14.) MIno A, Colmenero JD, Baeza NG, Clavijo E, Morata P. Rapid diagnosis of Brucella epididymoorchitis by real time polymerase chain reaction. Urology 2006; 176 : 2290-3.

(15.) Noviello S, Gallow R, Kelly N, Limberger RJ, DeAngelis K, Louis K, et al Laboratory acquired brucellosis. Emerg Infect Dis 2004; 10 : 1848-50.

(16.) Yagupsky P, Barron EJ. Laboratory exposures to Brucellae and yimplications for bioterrorism. Emerg InfectDis 2005; 11 : 1180-5.

R.D. Kulkarni *, Sneha K. Chunchanur Ajantha G.S., Shubhada C. & Pavitra Jain

Department of Microbiology

SDM College of Medical Sciences & Hospital

Dharwad 580 004, Karnataka, India

* For correspondence: atul410@yahoo.com
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Title Annotation:Correspondence
Author:Kulkarni, R.D.; Chunchanur, Sneha K.; G.S., Ajantha; C., Shubhada; Jain, Pavitra
Publication:Indian Journal of Medical Research
Article Type:Case study
Geographic Code:9INDI
Date:Oct 1, 2009
Words:1458
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