Brucellosis in Riyadh, Saudi Arabia. A microbiological and clinical study

820 TRANSACTIONS OPTHE ROYALSOCIETY OF TROPICALMEDICINEAND HYGIENE,VOL. 77, No. 6, 820.824(1983)

Brucellosis

in Riyadh,

Saudi

Arabia. study

A microbiological

and clinical

A. M. KAMBAL’*, E. S. MAHGOUB**, G. A. JAMJOOM** AND M. N. H. CHOWDHURY** ‘*College of Applied Medical Sciences and **Dept. of Pathology, College of Medicine, King Saud University, P.O. Box 2925, Riyadh, Saudi Arabia

Summary

During a period of two years, 30 cases of brucellosis were positively diagnosed from a total of 209 patients who reported with prolonged fever for investigation. Diagnosis was made both by blood culture and serological tests. The latter included slide and tube agglutination in all cases and an enzyme linked immunosorbent assay (ELISA) in 16. 11 cases (36.7%) gave negative results by the slide-agglutination screening test used at the recommended single serum dilution of 1 : 80. This was due to the prozone phenomenon as they gave positive results upon further dilution in the tube agglutination test. 13 of the 16 tested by ELISA were positive for both IgM and IgG and three were positive for IgG only. Of the six cases that were positive by culture, five grew BruceZlu melitensis and one B. abortus.

Introduction Brucellosis is primarily a disease of animals which can be transmitted to man. It is of major economic importance in livestock. Man becomes infected through the consumption of raw milk or milk products, contact wiih products of conception or wssiblv meat from infected animals. Transmission irom ian to man may occur rarely (CHRISTIE, 1974). The disease runs a chronic course and is a common cause of prolonged fever in some countries. More than 20,000 cases are reported every year from all over the world with some countries, e.g., Argentina, Greece and Italy each reporting more than 1000 cases a year (KAUFMANN & MARTONE, 1980). The disease has also been reported from some Middle Eastern and African countries, e.g., Iran, Turkey, Chad and Kenva (ABDUSSALAM & FEIN, 1976). HASSAN & FARED (i974). stated that brucellbsis is ihe third most common infective cause of fever of unknown origin in Cairo. As far as we know, there is no information on brucellosis from Saudi Arabia. This paper highlights our findings in cases of brucellosis encountered at King Abd-Al Aziz Teaching Hospital Laboratory from Riyadh and the surrounding areas in Saudi Arabia. It also clearly shows how cases could easily be missed if laboratory diagnosis was limited to screening by the slideagglutination test only. The prozone phenomenon, shown by a significant proportion of sera, is described. Materials and Methods This study was undertaken on 209 patients who reported with prolonged fever (more than two weeks duration) between September 1, 1980, and October 18, 1982 in the King Abd-Al Aziz Teaching Hospital and the University’s Paediatrics wards at the Riyadh Maternity and Children’s Hospital. Clinical details of the patients were obtained either from in-patients’ case notes or by verbal questioning of out-patients by both the treating medical officer and the clinical microbiologist. Diagnosis was by blood culture and serological tests. Blood was cultured using Difco Blood Culture medium No. TSBW/SPS-1712-37-4 and THIOL

0355-37-g. At least two blood culture sets, 10 ml each, were taken from each in-patient and one set (two bottles) from each out-patient. Blood culture bottles were incubated at 37”C, subcultured after two and seven days and then once weekly for eight weeks as recommended by STOKES& RIDGWAY(1980). Subculture was done on duplicate blood agar plates incubated with and without CO* Pure growths of isolated organisms were identified by slide agglutination using the Wellcome monospecific B. aborrusand B. melitensis antisera, Code No. ZMOl and ZM02. In vitro antibiotic sensitivity tests were done by the disc diffusion method using the Oxford strain of Staphylococcusaureusas a control organism (STOKES& FUDGWAY,1980). For serological tests, one serum sample was collected from each patient. For slide and tube agglutination tests, the Wellcome stained Brucella Antigens No. ss14and 15 were used. The slide screening test was done at a dilution of 1 : 80, as recommended by the manufacturer. The tube agglutination was done on doubling dilutions from 1 : 20 to 1 : 20,480. The methods used were those described in the instruction sheets supplied. ELISA was performed using reagents supplied by Dynatech Company (Switzerland) and the recommended procedure. Antihuman immunoglobulin conjugates were used for detecting IgG and IgM antibodies separately. Automatic washing and absorption readings were done using the Flow Laboratories’ Multiwash and Multiscan instruments, respectively. Results The map (Fig. 1) shows the localities from which patients came. 80% of patients were from the central region of Saudi Arabia and the remainder from the south western region. Most (73.3%) were resident or partially resident in rural agricultural areas, such as El-Kharj Oasis, and 26.7% were from urban areas. Most (83.3%) had some contact with domestic dairy animals and 76.6% gave a history of drinking raw milk mainly from goats and cows and, in one patient, camel. Nearly half the patients were between five and 20 years old but the youngest was aged 18 months. The male to female ratio was 7 : 3. Three patients (10%) were veterinary staff, 14 (46.7%) were farmers and their families, ten (33.3%) were students and in three patients (10%) the occupation was unspecified.

A.

M.

KAMBAL

et

821

al.

Table

I-Result

Antigen B. B B. B.

Fig. 1. Map of Saudi Arabia showingthe numbersand localities of the 30 patientswith brweilosis.

30. 28--

23-

15-

2

o-

I: L4

ll-

it

3

% iit 2 P

9

53-

abortus melitensis abortis &a melitensis

of slide screening

test

No. of positive sera

No. of negative sera

Total

18 18

::

ii

19

11

30

Table I shows the results of slide screening tests. 11 serawere negative, even when both B. abortus and B. melitensis antigens were used simultaneously. Table II shows the results of the tube agglutination test. These results showed significantly that titres of most of the serawere between 1 : 1280 and 1 : 5120. Fig. 3 shows the titres and prozone levels in the 15 serawhich gave negative results at a dilution of 1 : 80 by the tube agglutination method. Of these, five sera gave a prozone of 1 : 80, seven 1 : 160 and two 1 : 320 with at least one of the antigens used. The remaining serum gave a prozone of 1 : 640 with both antigens. 16 sera were treated by ELISA, 13 of these were positive for both IgG and IgM and three for IgG only. Five blood cultures grew B. mditensis, one grew B. abortus and 24 were negative. The growth of these strains was noted at different times: the strain of B. abortus grew after a week; two strains of B. melitensis grew after one week, two after two weeks and one after a month’s incubation. All the strains were sensitive to tetracycline, streptomycin, sulphamethoxazole and ampicillin, and moderately sensitive to trimethoprim. All strains were resistant to penicillin. Table III shows the various drug regimens tried in the treatment of patients. The doses, route of administration and duration of treatment are also shown. Two of the three patients who relapsed on tetracycline therapy were treated with cotrimoxazole for six weeks. The remainder and the one who relapsed on cotrimoxazole therapy, were treated with tetracycline and streptomycin. Discussion

lClinml

Feature

Fig. 2. The commonclinical featuresencounteredin 30 patients with bmcellosis.

As shown in Fig. 2 the outstanding clinical features in these patients were fever, followed by musculoskeletal pains. Of the six patients whose blood culture proved positive, four had obvious signs of septicaemia, high fever (>39.6”C) with rigors, and were more toxic than the other patients. In 90% of patients the duration of the illness was less than three months. One female patient aborted during the febrile episode of the illness. 30% of the patients had a haemoglobin of < 11 g/dl. Leucopenia (<4 x 109/L) was found in 43.3% of patients. The mean E.S.R. was raised (46 mm/hour).

These findings show the importance of brucellosis as a cause of prolonged fever in Saudi Arabia. The incidence of brucellosis was 14.3% compared to 3.3% for enteric fever in the same patient population. In spite of the present large foreign community in Saudi Arabia, all brucellosis patients but one were Saudi nationals, whereas all the enteric fever patients but one were expatriates living in the country. Though no reports of brucellosis in Saudi Arabia are available, this high incidence is not surprising because of the widespread practice of drinking raw milk from goats, cows and camels. Brucellosis in camels has been reported by different authors (OKOH, 1979; WAGHELA et al., 1978; KULSHRESTHA et al., 1975; MATHUR & BHARGAVA, 1979). However, in our series only one patient gave history of drinking raw camel’s milk. Expatriates visiting endemic areas should beware of drinking raw milk. Our study showed that brucellosis occurred mostly among farmers, shepherds and their families; similar findings have been reported from other countries

822

BRUCELLOSIS

Table

II-Results

of the tube agglutination

IN

SAUDI

ARABIA

test

Antigen

Titre 1:80 1 1

B. abmtus B. melitensis

1:160 :

1:320 1:640 : ii

1:1280 1:2560 : 10 11

1:5120 1:10240 7 1 5 1

20480. 10240.

2560. 1280. e .E

640.

B

320.

5 8 .& % Q

160. 80. (80. Case Number 10 0 0 1 = Brucella

nbortus

m=

Bruce&a

melitensk

[

= Prozone

Fig. 3. The titre and prozone levels in the 15 cam of bmcellosis which were negative at the dilution of 1 : 80.

(ALAUSA & AWOSEYI, 1976; MAKARAM et al., 1982). Unlike other studies (GILBERT et al., 1980; HENDERSON& HILL, 1972) there were no abattoir workers in our group of patients. Ten of our patients had visited endemic areas briefly and contracted brucellosis soon afterwards. Similar occurrences have been reported by other workers (DE NIE & ANSINK SCHIPPER, 1981; SCHWARZ& CONEN, 1981). A high percentage (26.6%) of our patients were children less than 10 years old, in contrast to MAKARAM et al. (1982) whose patients were predominantly adults. This might point to consumption of raw milk as the main source of infection in our group of patients. From the laboratory aspect our results emphasize the importance of the prozone phenomenon in influencing the serological diagnosis of brucellosis. Of 30 sera, 15 gave negative results at a dilution of 1 : 80 by the tube dilution method. 11 cases(36.6%) would have been completely missed if the diagnosis had been

based on the slide screening test alone. We believe that this is a major cause of missing the diagnosis of brucellosis in our laboratory previously and possibly also in other laboratories in the country. As the methods for diagnosing brucellosis in most casesare mainly serological, it is very important to guard against the prozone phenomenon. Several explanations have been proposed for the prozone phenomenon which has been particularly noticed in this study. These include the formation of soluble antigen-antibody complexes at high antibody concentrations, and the presenceof high IgA level and of blocking antibodies from IgA and IgG classes (DRUTZ & GRAYBILL, 1980; NORMAN & MCCULLOUGH, 1980; WICHER, 1981). According to McNAUGHT et al. (1977), the specified antibody is IgG’ which blocks IgG’ and IgM antibodies. CRUICKSHANK et al. (1974) attributed the prozone phe-

nomenon to a high protein concentration which increases the molecular net charge that leads to repulsion which, in turn, opposes antibody binding

A.

Table III-Drug

M.

KAMBAL

823

et al.

Therapy Dose and route of administration

Drugs

Duration of treatment

No. of patients

No. of patients relapsed (%)

5

cl (0)

Streptomycin (St) and Tetracycline (TC)

1.0 g/day/l/ML 500 mg 6-hourly/orally

St + TC for 2 weeks then TC for 4 weeks

Tetracycline

500 mg 6-hourly/orally

6 weeks

Streptomycin (St) and Cotrimoxazole (Cot)

40 mg/kg/day/I/M

St + Cot for 2 weeks then Cot for 4 weeks

6**

0 (0)

Cotrimoxazole

2 tablets (adult) twice daily orally

6 weeks

5

1 (20)

Ampicillin

(TC)

(Cot)

10 ml of suspension twice daily orally OR 2 tablets (paediatric) twice daily orally

125 mg 6-hourly

13

3 (23.1)

one ** no follow up

orally

** Children <8 years old Ampicillin was given before the patient was diagnosed as Brucellosis. effect. We therefore recommend that whenever brucellosis is suspected, serological diagnosis by the tube agglutination method should be routinely done up to a minimum dilution of 1 : 640 to avoid the prozone phenomenon, as one of our sera gave a positive agglutination at a dilution of 1 : 640. Though we did not carry out serological tests such as Coombs, radioimmunoassay and indirect fluorescent antibody tests, yet they are still recommended and valid as diagnostic tests for brucellosis, as shown by CERNYSEVA et al. (1977), PARRATT et al. (1977), WHITE (1978), EDWARDS et al. (1970). From our experience in the treatment of brucellosis, as judged by the absence of relapse, we are inclined to favour a combination of tetracycline and streptomycin in adults and cotrimoxazole and streptomycin in children. Brucellosis seems to be a common disease in Saudi Arabia. It should always be kept in mind in the differential diagnosis of patients having prolonged fever. Perhaps a reference laboratory for brucellosis, using all or some of the above mentioned serological tests and better culture techniques, will help in the proper investigation, diagnosis and control of brucellosis in Saudi Arabia. Acknowledgements We are indebted to our colleagues in the Departments of Medicine and Paediatrics, King Saud University for supplying relevant clinical information. We would also like to thank Mrs. Maybeth Magown, Mr. Ahmed Khirat, Mr. Mohammed Zein and Mr. Mohammed Mahgoub for their technical help. Our thanks are extended to Mr. Gordon Lees for drawing the figures.

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824

BRUCELLOSIS

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31st March,

1983.