Human Brucellosis: Seroprevalence amongst Patients attending the General Out-patient Department (GOPD), Federal Teaching Hospital Gombe, Nigeria

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Kudi A.A., Ahmed R.A., Baba-Ali F.

Bio-global Diagnostic & Research Laboratories, Opp. State Specialist Hospital Main Gate, Jekadafari Road, Gombe
Uba A., Tahir F., Yusuf I.Z.

Department of Medical Microbiology & Immunology Federal Teaching Hospital Gombe, Gombe State
Bubalu J.L

Faculty of Science, Biological Science Department Abubakar Tafawa Balewa University, Bauchi

All correspondence to: Kudi A.A, kclifeglobal@yahoo.co.uk

ABSTRACT
Background
Brucellosis is a disease of domestic, livestock and wild animals with serious zoonotic implications in man. Its spread is worldwide and transmission to humans is by contact with fluids from infected animals or derived food products such as unpasteurized milk and cheese. The clinical picture of the disease in man is so strange and protean; easily bewildered with other infectious and noninfectious diseases, leading to diagnostic delays and late onset of therapy. The intention of this study was to determine and document the prevalence of Brucellosis in patients presented with acute febrile illnesses resembling malaria and/or typhoid infections using the serological technique.
Methods: A retrospective study of a two-year laboratory records of cases of brucellosis among patients with febrile illnesses resembling malaria and/or typhoid fever seen at the General out-patient department (GOPD), Federal Teaching Hospital, Gombe between 2012 and 2014. Blood samples routinely collected from 246 patients were allowed to clot and the sera extracted were tested for Brucella antibodies using Rose Bengal plate test (RBPT). Standard test protocol described by Alton et al., (1975) and Morgan et al., (1978) was followed. Results obtained were statistically analysed using tables and chi-square test at 95% confidence level.
Results: Out of the 246 samples analysed, 56.6% were from the males, while 43.4% from females between the ages 0-60yrs. Our record shows 32.5% positivity for brucellosis, while the remaining 67.5% were negative. Among the males tested, 14.5% were found positive for the disease, while among the females 18.0% was recorded, (p>0.05). Majority (13.3%) that tested positive for brucellosis were between the ages 31- 40 years. The least (3.6%) affected were seen in ages 21-30 and 51-60 years.
Conclusion: Brucellosis among the population tested was considered to be high. Symptoms clinically thought to be malaria and/or typhoid fever were seen to be cases of brucellosis. There was no significant association between sex and age in the rate of infection (p>0.05). Routine laboratory test request for Brucella antibody among patients with febrile illness in our Community is recommended. Co-ordinated public enlightment on zoonosis, with special emphasis to brucellosis in Gombe State was encouraged.

Key words: Brucellosis, Zoonosis, Febrile illnesses, Pyrexia of unknown origin, Gombe

INTRODUCTION

Brucellosis is an infectious disease caused by bacteria of the genus Brucella (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003; Kaltungo et al., 2014). The disease is caused by several species of Gram-negative facultative intracellular bacteria of the genus (Sofian et al., 2008). It is a neglected debilitating zoonosis and is recognized as an occupational hazard with a high prevalence in developing countries
(Mabel et al., 2013). Primarily, it is a disease of domestic, livestock and wild animals with serious zoonotic implications in man; causing huge economic losses to the livestock industry. Cattle, goats, pigs, sheep, horses and dogs play an important role in the transmission of this disease to man. It is defined as a contagious systemic bacterial disease of ruminants, characterized by inflammation of the genital organs and fetal membranes, abortion, sterility and formation of localized lesions in the lymphatic system and joints (WHO, 1971, CDC, 2005).

Brucellosis remains a main source of disease in humans and animal husbandry worldwide (Corbel, 1997).

The clinical picture of brucellosis in man is so strange and protean that it can be easily bewildered with other infectious and noninfectious diseases, leading to diagnostic delays and late onset of therapy (Al Dahouk and Nockler, 2011). Malta, Rock, Gilbraltar, Crimean, Cyprus or Mediterranean fever, Bang’s disease, intermittent typhoid or Typho-malarial fever, undulant fever, etc, are just various synonyms for brucellosis (Al- Dahouk et al, 2003). Those suffering from the disease show unspecific symptoms, e.g. fever, chills, malaise, arthralgia, headache, tiredness and weakness. Therapeutic failure and relapses, chronic courses and severe complications like bone and joint involvement, neurobrucellosis and endocarditis are characteristic for the disease (Al- Dahouk et al, 2003).
About half a million human brucellosis cases are reported annually. However, according to the WHO (1997) estimates, the true frequency of the disease is 10-25-times higher than the reported number. The highest annual incidence rates are reported from the Middle Eastern countries, such as Syria, Iraq, Iran, and Saudi Arabia (Pappas et al., 2006). In Iran, where Brucellosis is endemic, the incidence of the disease is up to 34 per 100,000 per year in certain areas (Najafi et al., 2011). In Nigeria, it was reported that human brucellosis is hardly diagnosed in hospitals despite suggestions that the magnitude of the infection may be greater than appreciated (Njoku 1995; Rajis et. al, 2003).
Detailed studies confirming the problem of brucellosis in Nigeria’s livestock have been documented by several authors (Esuruoso, 1974; Falade, 1974; Falade, et al., 1974; Falade et al., 1975; Okon, 1980, Chukwu, 1987; Brisibe et al., 1993; Ajogi, 1997; Ajogi et al., 1998; Ogundipe et al., 1994; Ishola et al., 2001); with evidence of the spread of the disease in all parts of the country which is usually accompanied by severe economic losses. Serological prevalence rate of between 0.20% and 79.70% have been reported in various parts of the country to date. The infection has been reported in various animal species in Nigeria (Esuruoso and Hill, 1971; Esuruoso, 1974; Falade, 1974; Falade and Shonekan 1981; Falade et al., 1975; Okoh et al., 1978; Adamu and Ajogi, 1995). These demonstrate how brucellosis has been identified as an endemic and problematic disease in Nigeria. However, the infection is not static; it is evident from previous studies that prevalence varies at different times and locations. This is especially apparent where there is no control policy, like Nigeria. There is a pattern of low and high prevalence in specific areas of the country and prevalence variability also arises between herds in the same area (Nuru and Dennis, 1975). Although prevalence in brucellosis has been shown to be low in most dairy and private farms, it is actually on the increase among nomadic and semi-nomadic herds which contribute about 95% of all annual food population in Nigeria (Rikin, 1988).

Making a diagnosis of brucellosis may be difficult because of the unspecific symptoms and signs shared with other febrile illnesses, slow growth rate of the causative agent in blood culture, and the complexity of sero-diagnosis (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003). Presumptive diagnosis of brucellosis can be
made by the use of several serological tests to brucella antibodies, but the “gold standard” remains isolation and identification of the bacterium. Evidence of the infection either through serological or cultural examinations has been demonstrated in domestic livestock and humans in Nigeria (Ocholi et al., 1993). Most of the disease reports originated from Government herds where screening tests were easily carried out, while some originated from settled Fulani herds and private farms (Ocholi et al., 1993). The general situation is that the disease is more prevalent in Government-owned farms than in the nomadic herds (Esuruoso, 1974). Epizootiological investigations also revealed that results obtained varied depending on the region, area or animal group sampled (Ocholi et al., 1993). The distribution of the disease among humans is not well known but serological evidence has shown that the disease exists worldwide. Evidence of the presence of the disease in humans in Nigeria has also been published (Collard, 1962, Alausa, 1977, Alausa and Osoba, 1977, Falade, 1974 and Falade, 2002). It is a zoonosis and the disease in man is highly debilitating, though not considered to be fatal (Falade, 1974). Collard (1962) documented the first case of human brucellosis in Nigeria where Brucella antibodies were demonstrated in the sera of healthy persons in various parts of the country.
The intention of this study was to determine and document the prevalence of Brucellosis in patients presented with signs and symptoms of being ill (acute febrile illness) resembling malaria and/or typhoid infections in our hospital using the serological technique retrospectively. We suggest that similarities in signs and symptoms of diseases have often led to under diagnosis or misdiagnosis of brucellosis in humans. Most often, as the case may be, patient illness is dismissed as malaria, typhoid or pyrexia of an unknown origin (PUO); in that case, the disease go undiagnosed and untreated, leading to considerable suffering for those affected (John et al, 2002). We view brucellosis as one of the neglected zoonosis and most under reported or misdiagnosed disease in most hospitals in our locality. We found no known documented report of brucellosis among humans in Gombe and its environs; the few found reports were among domestic animals but not humans. Access to current documented cases of brucellosis in humans in our environment may be of paramount assistance in patients’ management thereby, contributing to quality healthcare delivery; the gap we made attempt to close.

MATERIALS AND METHODS
Study Area
Gombe, the capital city of Gombe State is located in the Guinea Savannah of North-Eastern part of Nigeria in West Africa. The State is bordered by Borno State to the East, Yobe State to the North-East, Taraba State to the South, Adamawa State to the South-East and Bauchi State to the West. It occupies a total land area of 20,265 square kilometres and has a population of about 2,353,879. The State is made up of 11 Local Government Areas (Jibrin, 2003, GomSACA, 2008). The principal ethnic groups of State are the Tera, Tangale, Fulani, Waja and Bolewa. The people are predominantly farmers and as is the culture, they keep and rear domestic animals and poultry for economic purposes.

Ethical Consideration and Confidentiality
Approval for this work was obtained from the Federal Teaching Hospital (FTH), Gombe Research and Ethics Committee. Patients’ consent was obtained at the time of sample collection to run the test. Throughout the test analysis, confidentiality of health information of the patient was maintained.

Sample Collection
Whole blood samples were collected from 246 patients with febrile illnesses resembling malaria and/or typhoid seen at the General out-patient department (GOPD), Federal Teaching Hospital, Gombe from 2012 to 2014. The samples were routinely collected in the Medical Microbiology & Immunology Laboratory of the same hospital by the attending Phlebotomist. From each patient, 4mls of whole blood was collected in a sterile screw capped non-anticoagulant 5ml-capacity disposable (plastic) bottles using sterile disposable syringe with a 21G needle. The blood was allowed to clot and then centrifuged at 1500 rpm to separate the serum from the red blood cells. Using a sterile disposable Pasteur pipette, the serum extracted was transferred into a sterile screw capped vial, labeled with a laboratory number that correspond with the subject identification. The labeled serum sample was ready for analysis (Alton et al., 1975). Where there was delay in the analysis, the sample was stored at -20oC until required for testing. Demographic characteristics of each patient were noted at the time of sample collection.

Serological Testing
Each sample was screened for Brucella antibodies using Rose Bengal Plate Test (RBPT). The test was carried out using the standard protocol described by Alton et al., (1975) and Morgan et al., (1978). Procedure: Serum samples and antigen were brought to room temperature (22 ± 4°C). Approximately 25µl of each serum was placed on a white tile and an equal volume of antigen was placed near each serum spot. Both were mixed thoroughly using a clean wooden rod and read for agglutination immediately after a 4-minute period. The agglutination reactions were recorded as positive (+) or negative (-) depending on whether there was agglutinations or not. The reagents in the kit were reconstituted and the test procedure was carried out according to manufacturers’ instructions. Individuals were considered as positive based on a positive RBPT result. The necessary quality control (QC) was carried out on the reagent used using known positive and negative controls. All safety precautions were followed according to the manufacturer’s instructions on the kit insert.

Statistical Analysis
Data collected were analysed using tables and chi-square tests. Results were considered as significant if the chi-square p-value was < 0.05 otherwise, non-significant if the p-value was > 0.05.

DISCUSSION
Similarities in signs and symptoms of diseases have often led to under diagnosis or misdiagnosis of brucellosis in humans. It was postulated that making a diagnosis of brucellosis is difficult because of the unspecific symptoms and signs shared with other febrile illnesses (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003). The clinical picture of brucellosis is so strange and protean that it can be easily bewildered with other infectious and noninfectious diseases (Al Dahouk and Nockler, 2011). As the case may be, patient illness is dismissed as malaria, typhoid or pyrexia of unknown origin (PUO). This leads to diagnostic delays and late onset of therapy; therefore, laboratory investigation is always needed to confirm etiologic agent (Al Dahouk and Nockler, 2011).

Our findings in this study show that more males (56.6%) presented with febrile illness that resembles malaria and/or typhoid fever at our Hospital’s out-patient department (GOPD) than females (43.4%). However, there was no significant difference between the sexes in the clinical presentation (p>0.05). As high as 32.5% prevalence rate of brucellosis in this study was recorded among the patients tested. This finding seems to agree with other workers who reported – though in domestic animals, serological prevalence rate of brucellosis of between 0.20% and 79.70% in various parts of Nigeria (Esuruoso and Hill, 1971; Esuruoso, 1974; Falade, 1974; Falade and Shonekan 1981; Falade et al., 1975; Okoh et al., 1978; Adamu and Ajogi, 1995). Other researchers recorded in humans close to the range of 28 to 57% among high risk group such as abattoir/butchers workers (Falde 1974; Ocholi 1993; Edu, 2005). In contrast however to our findings, Maryceline et al., (2001) recorded as low as 5.2% sero-positivity among
patients with PUO in Maiduguri, northeastern Nigeria. Perhaps geographical location and other demographic factors play some roles to this variation. The distribution of the disease among humans is not well known but serological evidence has shown that the disease exists worldwide and it is a zoonosis (Falade, 1974). Our findings therefore, has further agrees with other published reports that the presence of the disease in humans in Nigeria exist (Collard, 1962; Alausa, 1977; Alausa and Osoba, 1977; Falade, 1974; Falade, 2002). The first case in Nigeria was documented by Collard (1962) among apparently healthy persons in various parts of the country.
Other workers further reported cases among abattoir workers, nomads and those handling meat and other animal products. Mabel et al. (2013) recorded 32.7% seroprevalence among butchers in Abuja Nigeria; this is almost comparable to our findings in this study. Asanda and Agbede (2001) suggested that infection seems more associated with humans engaged in livestock and livestock product activities than those engaged in other productive ventures. Occupational status of the subject in this study was not determined; we therefore acknowledged this as one of the limitations, however, we could assume that contact with infected animals or animal products, such as meat, milk and other animal products among the population was inevitable, hence the high prevalence recorded. It was reported that the infection is not static; the prevalence varies at different times and locations (Rikin, 1988).
We noticed in this study that the females have the highest prevalence of brucellosis with 18.0% as against the males (14.5%). However, there was no significant difference in the rate of infection among the sexes (p>0.05). Our findings here tally with the report of Maryceline et al., (2001) where sex has no significance role in the distribution of brucellosis. This is in contrast with the findings recorded by Marbel (2013) among the abattoir workers and butchers in Abuja Nigeria, where males were significantly more infected with Brucella. This should be expected since males (in terms of occupation) are more involved in the handling of animals and animal products such as raw meat, unpasteurized milk and cheese. Such activities have been documented as significantly associated with Brucella infectivity (Hannah et. al., 2011); butchering in particular was noted to be a male-dominated activity thereby, the males stand at high risk of being infected with Brucella.
Our findings indicated that the highest seropositivity (13.3%) was in the age group 31-40 years, while the least was found in the ages between 21-30 and 51-60 years. However, in contrast to our findings, other workers reported individuals who tested positive to the brucellosis test were within the ages 18-25 years, while the least affected were those above 41 years of age (Marbel, 2013). The differences in the rate of infection among the ages in this study was found to be statistically significant (p<0.05). Reasons for the difference in the rate of infection was not immediately known to us, but it could be speculated that the ages 31-40 years is the most productive and active years thereby, frequency in contact with infected animals/products was high, as such, chances of getting infected therefore, most likely was significantly increased. Maryceline et al., (2001) however reported that age does not significantly affect the distribution of brucellosis.he limitation to the current study as observed and stated earlier was lack of determining the level of association of the subject with animals/animal product and brucellosis to confirmed zoonosis. However, infection with Brucella has always been reported to be the primary risk factor associated with keeping sheep, goats and other small animals in the household (Hannah et. al., 2011). It was generally observed that keeping and rearing goats, sheep, pigs and Cattles in our community is the practice for economic reasons; we may therefore, speculate that the high seropositivity (32.5%) recorded in our study was somewhat expected. The study was also limited in determining the level of knowledge of brucellosis in the community. Howbeit, with the high prevalence rate of infection recorded in this study, it is most likely that the level of awareness among the subjects, especially the zoonotic role in transmission, control and preventive measures was very low. In such situation therefore, and with the fact that infection is often subclinical, with high morbidity for both humans and animals (Colmenero et al. 1996), we again speculate that the disease is wide spread and diagnosis is either missed, under diagnosed or unreported in most of our healthcare settings.
Conclusion
The study reveals that among the population tested for brucellosis, there was no significant association between sex and age in the rate of infection. To the contrary however, other workers have reported significant difference in the rate of infection among the male abattoir workers/butchers (Falde 1974; Ocholi 1993; Edu, 2005). Among all that presented with febrile illnesses in our Centre, 32.5% would have been thought, dismissed or treated as having malaria, typhoid fever or PUO, but for the serological test which indicated brucellosis. This shows the significance of Brucella antibody testing on all patients presented with acute febrile illnesses in our healthcare facilities. We therefore advocate regular or routine laboratory test requests for diagnosis of brucellosis as is the case with other endemic infectious diseases, such as malaria and typhoid fever in our Community. The practice may enhance adequate and prompt diagnosis of acute febrile illnesses. Co-ordinated public enlightment and education programme generally on zoonosis, with special emphasis to brucellosis’ prevention and control should be instituted by the authorities concern in Gombe State and North eastern Nigeria as a whole.

 

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