Versions’ Comparison

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Table 4.11: Multivariate Analysis of Selected Risk Factors for Campylobacter Isolation in Stool.

95% Confidence Interval

Risk Factor Adjusted Odds Ratio Lower Upper p-value

Well water^a 13.57 0.07 0.44 <0.001 Consumption of Chicken^b 6.36 0.10 0.75 0.012 Raw milk Consumption^b 1.46 0.06 1.93 0.227 Animal Contact^c 6.32 0.11 0.76 0.012 Chicken Kept^d 0.01 0.40 2.75 0 .919 Sheep kept^d 1.30 0.26 1.43 0.255 Canned milk^e 5.26 0.10 0.83 0.022

Key

a = Well water as major source of drinking water.

b = Recent consumption of Chicken/Raw milk within the family.

c = Recent history of contact with chicken, duck, sheep, cow, cat or dog.

e = Consumption of canned milk as supplementary feed.

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CHAPTER FIVE 5.0 DISCUSSION

This study provides evidence that Campylobacter species are important agents of diarrhoeal disease among children in Zaria, North Western Nigeria. It agrees with the first reported isolation of Campylobacter in humans in the country, from the same centre in 1981.²² Therefore, the failure of Yakubu and Bello, the second researchers, to demonstrate Campylobacter in the centre, soon after the first report, may be due to other reasons, not the actual absence of the bacteria in the stool samples examined.⁵¹ The reasons could be: (1) Their subjects were mostly adults – isolations in the country were mostly from children less than 3 year of age, (2) Butzler’s medium was used – which give a poor yield in this environment, and (3) Routine stools samples were used – usually not suitable for Campylobacter isolation; as the samples were not usually submitted in time, This assertion is further strengthened by the isolation of Campylobacter in 1988, in 2.6% faecal samples of both nourished and undernourished children in the area.¹⁵⁴

The isolation of 40 Campylobacter species from 261 stools samples, giving a prevalence rate of 15.3%, showed great increase in isolation rate in this centre after three decades. The increase in the isolation rate from 1.69% in 1980, to 15.3% tallies with trends in South Western part of the country.^28,60 Isolation rates from that region rose from 5.2% and 12.4% to 16.5% and 19.1% in Lagos and Ile-Ife respectively.21,23-26,28

The value in this study is slightly lower, probably because the subjects were from both the urban and rural dwellers. Lower rates were reported for rural dwellers than for the urban dwellers.¹⁵⁵ However, the rate is higher than 8%

and 8.2% reported from Nsukka, South Eastern Nigeria and Ilorin, North Central Nigeria respectively.^156,157 The rate falls within the range of 8.0% and 17.4% observed in other

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developing countries like Iran, Tanzania, Ethiopia, China, India and Bangledash.^158-162 The isolation of Campylobacter in North Western Nigeria, from Chicken, sheep, Camel and raw milk, which are the major reservoirs for the human disease, further strengthens the role of Campylobacter as an agent of enteritis in the region.^52,163-167

As in other studies in the country and many reports from other developing countries,21,28,29,168,169

this study also shows more isolation from males than females, though it was not statistically significant (p-value > 0.5). This is in contrast to the report from Enugu and Onitsha in South eastern Nigeria, and reports from Burkina Faso, South Africa and Tehran were the rates were higher in females than the males.^170-173

As reported by earlier studies in Nigeria most of the isolates (90%) came from children under 2 years of age.1,21,24,28,156

This is similar to reports from Ghana and Pakistan¹⁷⁴ in contrast to findings from Tanzania, Egypt and Ethiopia.168,175-178

In the control group none of the faecal samples yielded Campylobacter. This is in agreement with previous studies in the country, especially reports from Ilorin and Lagos.^21,156,179 However, this is in contrast to the earlier study in this centre and reports from other parts of the country, in which isolation in the control groups were recorded.16,28,49,154,155

Asymptomatic excretion of Campylobacter is common in developing countries.^65,180-186 Isolation from healthy children in such countries may be due to early acquisition of the pathogen because of poor sanitation and contact with animals.^1,187 However, in tandem to present study, Campylobacter was isolated from only 0.6% of the healthy controls as against 13.5% of cases from a rural Northern India 2005.¹⁶⁰

The risk factor for Campylobacter enteritis among the infants and young children defer to those among adults, though there are few over lapse among the two groups.¹⁸⁸ The risk factors

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defer with the nature of the illness – sporadic or an outbreak, and also on socioeconomic status – developing or developed countries.6,15,59,88,189-194

In this study, by multivariate regression model, the identified independent risk factors for campylobacteriosis were; recent history of consumption of chicken with the family household, contact with animals that may harbour the pathogen, well water as the major source of drinking water for the family and consumption of canned milk as supplementary feed.

Out of the three variables tested in relation to recent history of consumption of risky food in the household, only consumption of Chicken was found to be independently associated with increased campylobacteriosis. Many studies have shown chicken to be a major source of campylobacteriosis.59,157,159,178,195-199

The risk of developing Campylobacter due to consumption of chicken in the household might be attributed to cross contamination of food, feeding utensils and hands that handle raw chicken.^59,200-202 The contaminated; foods, feeding utensils and the hand (of mother/care giver) served as vehicle for the transmission of the agent, during food preparation, assisted feeding or breast feeding.^203-205 The finding in the present study similar to findings in Cameroon, Denmark, Netherlands and Norway.201,205-207

History of consumption raw milk within the household was shown to be potential risk factor for Campylobacter infection. However, on multivariate analysis it was shown to be non-significant. Though raw milk on itself is potential source of the pathogen, it may not be a major source of cross-contamination within the household as compared to chicken.199,208,209

History of consumption of vegetables in the household was not statistically significant.

This is in contrast to the work of Fullerton et al, where eating fruits or vegetables prepared in the home was identified as potential risk of Laboratory-confirmed Campylobacter infection in

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infants.¹⁸⁸ Tenkate and Stafford as in the present study did find history of consumption of vegetables as a significant risk factor among infants and young children.²⁰⁹

Close contact with animals as reported in other studies has an increased risk of Campylobacteriosis.198,209,197

This has been shown to be the typical transmission route of Campylobacter for infants and young children particularly in developing countries.15,207,209-211

The children got in contact with the bacteria by direct contact with the animals that harbour the organisms on their body or indirectly through the contaminated environment.²¹² The environment became contaminated by the animals shedding the organism from their body or by excreting the agents in their faeces.180,189,211,213,214

Evidence from this study suggested keeping of Chicken and Sheep in the household as associated risk factors for campylobacteriosis as reported in some studies.163,189,209,212,213,215

However, both were shown not to be independent risk factors by multivariate analysis. Presence of animal(s) in the household may therefore, be a ‘confounder’, the real risk could be the direct or indirect contact with the animal(s), their products or the environment they contaminate.189,205,212,213,215

Almost all the children (97.5%) whose stool yielded Campylobacter isolates were on one form of supplementary foods or the other. Molbak et al reported a significant correlation between Campylobacter excretion and use of supplementary feed among the Liberian children.¹⁸¹ The use of canned (formula) milk was independently associated with Campylobacteriosis in the present study. The feed got contaminated as result of poor handling, preparation and storage, or through contaminated feeding utensils.216,217,218,219

These utensils are often not properly cleaned after used.211,219,220

Additionally, children on formula feed are more likely to be partially breast fed or not to be on breast milk at all.^221-225 Therefore, more prone to

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infection by Campylobacter and other enteric pathogens due to deficiency or lack in breast milk induced/associated immunity.225,226-230

Consumption of raw milk have been traditionally associated epidemic Campylobacter enteritis.167,231-236

Similar to other reports of sporadic campylobacteriosis, consumption of raw milk was not a significant risk factor in this study.^237-241 This is however in contrast report from USA, were consumption of raw milk was identified as a significant independent risk factor endemic sporadic Campylobacter enteritis.^189,208

It should be noted, however, that only two children with positive Campylobacter isolates in their stools were on raw milk as supplementary feed.

Well as a source of drinking water is a risk factor, similar to a reports from Ethiopia, Sweden and Netherland where presence of a well and consumption of the untreated water was identified as a risk factor for Campylobacter infections in children under the age of 24 months.189,242-244

Other studies too have identified the use of untreated water as a source for both sporadic and outbreak of Campylobacteriosis. 88,188,207,245-247

The use of the untreated, contaminated water has been associated with cross-contamination in home-made food; especially in the preparation of infant feeds.217,248-250

The well waters, in the place of this study are most likely contaminated at source.²⁵¹ Many of them are shallow wells that were fed by contaminated water bodies or get contaminated by the nearby gutters or drainages.²⁵² Poultry (chicken and ducks) and domestic animals, that can be source (s) of Campylobacter species, are usually seen around such wells.^253,254

Additionally, irrespective of the source; well, bore hole or pipe borne water, the majority of the households in this study got supply of water through water hawker, vendors or distributor who Know little about hygiene. Osumanu reported higher incidence of diarrhoea in children in children whose household depend on water from vendors.²⁵⁵ Those that do not patronized the

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water sellers, the water is fetched by adults or by children; who often contaminate the water while playing.

Other sources of water examined, though not statistically significant may be potential risk factors.^256-260 The use of sachet water popularly known as ‘Pure water’ is gaining popularity in this region as in other parts of the country. The water usually got contaminated during packing, storage, distribution and hawking.²⁶¹ The major dams in Zaria, as reported by Agbogu et al, were already contaminated by domestic sewage, the waste stabilization pond, recreational activities in the water bodies, herd watering and drain from abattoir.²⁵¹ Campylobacter species were demonstrated from a major river that supply many dams in the region.²⁵² Bottled water in contrast to a previous report was not a risk factor in the present study.⁸⁵

Though, maternal educational level attained is a significant socioeconomic risk determinant of campylobacteriosis.^193,262 In the present study similar to reports from Egypt and Burkina Faso, there were no significant association between Campylobacter isolation and level of maternal education.^63,173 This is in contrast to reports from Peru and Ethiopia, where low level of maternal education was associated with increased incidence of Campylobacter enteritis in under five children.^180,242

In contrast to developed worlds, where lower level of parental education is associated with increased risk of Campylobacteriosis, due to greater exposure of children to raw products.²⁶³ In the present study, though not statistically significant, there was an apparent lower isolation rate in tandem with developing countries where lower level of maternal education is associated with decreased incidence of Campylobacter enteritis.^193,264 Breast feeding habit is strongly related to mother’s education, those with none or lower level (primary or secondary) of education are more likely to breast fed than those mothers with higher level.^265,266 Hence, their children are more likely to be protected by breast milk immunity against Campylobacter enteritis

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and other diarrhoeal diseases.^225-230 Furthermore, children of highly educated mothers (usually more economically viable) are more likely to be on formula fed and to be with caretakers/nannies most of the time; who often have little knowledge about safe food handling and hygiene.^267,268 This is because such mothers, work longer hours outside the home.²⁶⁶ However, the effect of increasing education appears to be more protective for children in wealthy families than for children in poor families.²⁶⁹

Gentamicin, the drug commonly used for Campylobacter bacteriamia, shows 100%

sensitivity similar to other reports from Nigeria.^39,270-272 In this study the resistance rate of less than 8% is consistent with reports from Ethiopia, Burkina Faso and Germany.169,172,173,273,274

The 4% gentamicin resistance reported from Ilorin, Nigeria is generally low compared to resistance rate (10% - 17%) from other countries, like India, China and South Africa.160,162,173

The low level of gentamicin resistance seen in Nigeria so far, may thus be attributable to relatively low and exclusive use of the drug in human medicine.

The pattern of resistance noted for nalidixic acid and ciprofloxacin in Campylobacter generally is either slightly higher levels of nalidixic acid over ciprofloxacin or equal levels in the same set of isolates.¹⁵⁰ However, in this study the level of resistance for ciprofloxacin is slightly higher to that of nalidixic acid. Mutations that confer resistance to ciprofloxacin also confer resistance to nalidixic acids.²⁷⁴ while some mutations confer only ciprofloxacin resistance.^275,276 The different pattern in this study may be due to high selection pressure on ciprofloxacin. The drug is prescribed rationally or irrationally virtually for any clinical condition; unlike nalidixic acid that is restricted for some cases of urinary tract infections only.²⁷⁶

Evidence from this study and other studies, show sensitivity to ciprofloxacin is still high in Nigeria.^39,170,178 Resistance to the flouroquinolone, is however bound to occur, following the use of the drug for agriculture, as it happened in the developed countries when the drug was

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licensed for use in agriculture.^279,278 Veterinary drugs containing ciprofloxacin, enrofloxacin and ofloxacin, are being used in poultry for various purposes, including prophylaxis.²⁷⁸

In Nigeria all the isolates in Ile-Ife, Ilorin and Osogbo were reported to be sensitive to ciprofloxacin.^39,194,195 This indicates that ciprofloxacin can still be use empirically as an alternative to erythromycin (the first drug of choice) in treatment of Campylobacter enteritis when indicated.

Low level tetracycline resistance (5%) is reported in this study, though the drug is widely used both in human and in animal husbandry.²⁷⁹ This is in contrast to report from Taiwan with tetracycline resistance of 93.4%.²⁸⁰ The level is lower than reports from Ilorin (12.0%) and Ile-Ife (12.5%).^39,156 Although all the isolates from Osogbo and Ile-Ife were sensitive to tetracycline,^39,271 a study 2 decades earlier showed up 82% tetracycline resistance in the country.^281, Within 2 years, tetracycline resistance increased from 40% to 60% in Bahrain and Saudi Arabia.^282,283 However, resistance to tetracycline in both developed and developing countries, generally stands between 20% and 40%.160,162,284

The high level resistance in tetracycline in those countries, may not be unconnected with widespread use in both humans and animals.²⁷⁹ Unfortunately, similar trend has already began in the Southern part of the country, where resistant to tetracycline in humans and chickens has reached 20% and 35% respectively in 2012.²⁸⁴ Here in the North west, tetracycline resistance of 71.4% has already been reported among thermophilic Campylobacter species isolated from meat.²⁸⁵

Chloramphenicol showed high level sensitivity rate of 90%, which is similar to rates from Pakistan and Ethiopia with sensitivity rate of 83% and 100% respectively.^45,169 Higher resistance rates of between 21% and 50% were reported from South Africa and Egypt.^173,177 Previous studies in Nigeria did not include chloramphenicol in their antimicrobial susceptibility panel.39,270-272,281

Therefore, resistance to chloramphenicol could not be ascertained wither it is on

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the increase or otherwise. Though, a more recent study in the country from Osogbo suggests increased resistance, as the reported sensitivity rate is only 55%.²⁸⁴

The sensitivity rate (82%) to erythromycin, the drug of choice for Campylobacter enteritis, in this study is similar to earlier reports from Lagos.^270,281 More recent works from Ilorin and Ile-Ife, however showed 100% sensitivity to erythromycin,^39,272 similar to reports from Germany, Greece and Egypt.^286-288 The Resistant rate of more than 18% in this study is high compared to reports from Serbia (12.5%), Ethiopia (12.5%), Saudi Arabia (7.3%), China (6.3%) and India (6.1%) and United Arab Emirates (1.2%).160,162,169,281-283

Although, in a report from Lagos in 1994,²¹ only 20.8% of the isolates were sensitive, majority of reports in the country demonstrated a sustained high level antimicrobial activity by the macrolide.39,156,270-272,281

Erythromycin, therefore, can still be used as drug of choice. Recent trend may show Erythromycin Resistant Campylobacter in the country emerging but not widespread.

Co-trimoxazole, a drug commonly used empirically to treat diarrhoea in children, showed very high level resistance in the study. This resistance to co-trimoxazole is as expected since C. jejuni is characteristically resistant to the synergistic combination of trimethoprim and sulphomethoxazole.²⁸⁹ The resistant rate of 50% was proportionately lower to previous studies in Nigeria, Pakistan and Spain, where levels as high as 79 - 96.7% have been reported.39,45,155,156,290,291

The resistance level of 36% from South Africa though lower than in this study, is still high.¹⁶⁹ The use of sulphonamides for both human and veterinary medicine for several decades partly explain the high resistance rate.^291,292 Furthermore, once sulphonamide resistance becomes established it persist even in the absence of selection pressure.²⁹³ Empirical co-trimoxazole, therefore, should be used with caution to treat diarrhoeal illness in paediatrics lest the cause is Campylobacter specie.

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Amoxicillin resistance rate of 55% is the worst among the antibiotics tested for in the study. Generally previous studies in Nigeria do not check for amoxicillin susceptibility. Rather they tested for other beta-lactam penicillins like ampicillin and penicillin. The reported resistance rates for the beta-lactams in those studies stood at 6.3% - 100%.28,155,156,281,294

The least resistance rate was from Ile-Ife in 2002,³⁹ but the rate in the country is generally high, usually above 80%.^39,272,281 Similarly, other countries like India and South Africa also show high level of resistance to ampicillin, 81.6% and 100% respectively.^160,172 Therefore, the use of amoxicillin and other beta-lactam penicillins empirically to treat suspected cases of Campylobacter enteritis and non-enteric campylobacteriosis alike, should be with caution.

Earlier studies in the country reported 9.6 – 12.5% beta lactamase production in their isolates.^21,270 In the present study none of the isolates demonstrated beta lactamase production.

This is similar to later reports by Aboderin et al, Samuel et al, and Adekunle et al in 2004, 2006 and 2009 respectively, from other parts of Nigeria.^39,271,272 However, it is in contrast to many reports from developing and developed countries, where between 22% - 92% beta lactamese production were reported.160,172,294,295

Although resistance to amoxicillin as in other beta lactams is usually associated with beta-lactamase production, other mechanisms could be involved.²⁹⁵ This may explain the zero demonstration of beta lactamase production in this study despite high rate of amoxicillin resistance. Similar findings were reported by Samie et al from South Africa, where 94% – 100%

of their isolates were resistant to beta-lactams (ampicillin, amoxicillin and penicillin G), but only 22.4% were beta-lactamase positive.¹⁷².

As with other reports in the country Campylobacter jejuni was the major cause of gastro enteritis than C. coli. The most important biotype was C. jejuni type I.^49,282 However, unlike the

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reports from the other parts of the country where only types I and II of the C. jejuni were isolated,28,49,156,282 C. jejuni type III and type IV were also demonstrated. The role of these biotypes in human campylobacteriosis in the region may be related to the use of sheep meat as important food source in the area as it was the case in Saudi Arabia where C. jejuni type IV was the most important isolate.^21,283 There are reported correlation between the biotypes isolated in humans and the biotypes in the possible animal source(s).^296-299