An epidemiological synthesis of emerging and re-emerging zoonotic disease threats in Cameroon, 2000–2022: a systematic review

Highlights • From 2000 to 2022, 35 zoonoses (viral, bacterial, and parasitic) were reported in Cameroon.• Human and environmental factors influence the dynamics of emerging/re-emerging zoonoses.• Toxoplasmosis, dengue, brucellosis, and avian influenza were the most reported zoonoses.• Hospital-based cross-sectional studies accounted for the majority of studies on humans.• Community-level zoonoses risks and incidence/prevalence are underestimated.


Introduction
Over time, interactions between humans, animals, and their environment have changed significantly, leading to the heightened threat of infectious diseases, with some emerging and others re-emerging [1] . A majority (61%) of emerging infectious diseases have an animal origin, with animals either acting as reservoirs, vectors, or hosts of these pathogens that are transmissible to humans [2] . Zoonotic diseases involve a wide range of pathogens (bacteria, viruses, parasites, fungi, protozoa, and non-viral agents) [3] , and their emergence and re-emergence tant to devise effective and targeted interventions that assist prevention strategies through informing/educating public health leaders, veterinarians, environmental officers, communities, and other One Health actors [6] .
Approximately 70% of residents in Cameroon are involved in smallscale agriculture. Cameroon has a geostrategic position in the Central African sub-region (Economic Community of Central African States, EC-CAS) and is bordered by six countries including Nigeria and the Republic of the Congo. Coupled with its diverse landscape ranging from forests to plains, Cameroon is an ideal location for exploring risk from a broad range of zoonotic threats [7] . Also, a portion of the Congo Basin -a historical hotspot for zoonotic emergence -is located in the southern region of Cameroon. This risk is enhanced due to the typical hunting and butchering activities, consumption of bush animals, livestock husbandry, and frequent contact with wild animals [8 , 9] .
The Congo Basin region is home to one of the largest and most biologically diverse rainforests in the world. It has a huge forest population of non-human primates and other animal reservoirs of potential and actual human pathogens. The region is believed to be the origin of several important emerging human viruses, including HIV, multiple arthropodborne viruses (including chikungunya virus, Zika virus, Usutu virus, and Crimean-Congo haemorrhagic fever virus), monkeypox virus, and Ebola virus; a recent rhabdovirus called Bas-Congo virus also originated from this area [10] . Repeated sporadic outbreaks of Ebola and evidence that different HIV lineages have been independently transmitted to humans from their primary animal hosts multiple times in the past, indicate that there remains a persistent threat of not only the emergence in humans of novel viruses, but also the continuing re-emergence of globally relevant infectious pathogens.
Specific details of the burden of zoonotic diseases in Cameroon are not well understood. Epidemiological data on various zoonotic diseases across different health districts of Cameroon are abundant in the literature [11 , 12] , and there are some systematic reviews of specific zoonotic diseases like leptospirosis and monkeypox [13 , 14] , as well as groups of zoonotic diseases like those caused by bacterial and viral infections, from Africa as a whole [15] . The national zoonoses programme, unifying the approaches to the risk management of these threats, was established in 2014. In 2016, the programme produced a prioritization list for zoonotic diseases, five of which were set as top priorities using a semi-quantitative review approach: anthrax, bovine tuberculosis, Ebola and Marburg virus disease, highly pathogenic avian influenza, and rabies [7] . There is no comprehensive report of all zoonoses groups in Cameroon.
A synopsis of epidemiological data on zoonotic diseases would be a valuable step in understanding, through an evidence-based systematic search and synthesis of published findings, the threat pathogen exposures of Cameroonian communities related to zoonotic diseases. This would provide a basis to inform zoonotic disease prioritization and the identification of capacity and community awareness to prevent and prepare for zoonotic outbreaks. This systematic review and meta-analysis was conducted to determine the prevalence and distribution of priority and other zoonoses reported in humans and animals (with evidence of human transmission) in Cameroon between January 2000 and May 2022.

Protocol registration
The protocol for this systematic review was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines [16] ( Supplementary Material File S1 ); this review has been registered in the International Prospective Register of Systematic Reviews of the National Institute for Health Research (NIHR, UK) (PROSPERO ID: CRD42022333059).

Evidence gathering
The peer-reviewed literature was searched across six databases: Embase, PubMed, CINAHL, Scopus, African Index Medicus, and Cochrane. The search was conducted on May 30, 2022, using the following keywords: (emerging OR new OR re-emerging OR neglect * ) AND ( "zoonotic disease * " OR "animal disease * " OR vector-borne OR "cross-species transmission " OR "interspecies transmission " OR rabies OR anthrax OR "avian influenza " OR Ebola OR Marburg OR "Bovine tuberculosis " OR "Lassa Fever " "Yellow Fever " OR "Crimean-Congo Hemorrhagic Fever " OR Plague OR "Yersinia pestis " OR Leptospirosis OR "Nipah virus ") AND (Cameroon) ( Supplementary Material File S2 ). Additional evidence was obtained from the grey literature, including reports from the World Health Organization (WHO) Disease Outbreak News, health district and country reports (District Health Information Software 2 and National Zoonoses Program database), and expert network consultations.

Eligibility criteria
Studies were included if they (1) reported zoonoses data and were published between January 1, 2000, and May 30, 2022; (2) involved human subjects of any age and animals related to human infections; (3) the study population was from any of the 10 regions of Cameroon (Adamawa, Centre, East, Far North, Littoral, North, North West, South, South West, and West regions); (4) the study was published in English; and (5) the article included an abstract and full text; this latter requirement was waived for the grey literature, such as district and government reports. All types of study designs except case reports were considered.
Unpublished studies, conference abstracts, protocols, reviews, letters, interventions (trials), studies conducted in other countries involving travellers from Cameroon, and animal diseases that are not known to be transmissible to humans were excluded.

Outcome measures
Outcomes abstracted from the retrieved articles included the following: for each represented zoonotic disease, when available, the total cases per year by district; case incidence by year, by district; disease prevalence by year (cross-sectional seroprevalence studies), by district; mortality rate by year among incident cases, by district; demographics of those directly impacted (cases and communities/region with case load); source of transmission and reservoir connected to the crossover event or vector; and for human-to-human or human-vector-human outbreaks defined as five or more cases within 30 days, the days between the first case and last case, days between the first case and having attained half of the cases, and estimated incubation period.
Additionally, narratives around spillover events were captured, where applicable (how the spillover happened and how it was controlled). From this, themes were identified and tagged.

Data abstraction and risk of bias
Selected articles were imported into Rayyan software, a semiautomated web and mobile-based tool for systematic reviews [17] . The software identified duplicates, and one of the authors verified and removed the duplicates. Two authors (N.B.T. and F.S.W.) independently screened the titles and abstracts, based on the eligibility criteria stated above to validate their selection, and screened the full text for selected articles. A third author (D.N.) resolved all conflicts after the title/abstract screening and again after full-text screening. The team developed a standardized data abstraction form coherent with the study objectives and outcome measures. This data abstraction template was pilot-tested on a subset of articles and then tested for face and construct validity.
The risk of bias across studies was assessed using the tool developed by Hoy and colleagues [18] , a method that relies on the GRADE work-ing group (Grades of Recommendation, Assessment, Development and Evaluation) and Cochrane approaches. The tool assesses external validity (items 1-4), for example "Was some form of random selection used to select the sample, OR, was a census undertaken? " and internal validity (items 5-10), for example "Was the study instrument that measured the parameter of interest shown to have reliability and validity (if necessary)? " for each study, where a score of 1 was given if the item was reported, 0 if it was not reported, and 0.5 for 'no information' ( [18] p.4). N.B.T. and D.N. independently scored the studies, and the risk of bias was classified as low (8.5-10), moderate (5)(6)(7)(8), or high (0-5.5).

Data synthesis
The management of abstracted data was accomplished in Microsoft Excel 2021. Meta-analysis was considered only when the level of bias and data harmonization were appropriate. An a priori power analysis for the test of significance of the variance component for each abstracted outcome was conducted for zoonotic diseases with at least five ( k ) reported studies, with the following parameters: degrees of freedom, df, k -1 = 4; significance level, = 0.5; the standard difference for the difference between disease proportions and mean, standard deviation (SD) = 6; and variance component, 2 = 0.24 [19] . Thus, a power of 53.0% was obtained for a test of variation using five studies. Ngaya and colleagues [20] developed a Stata command (metaprop) for prevalence studies, which was employed in the present study to compute the 95% confidence intervals (CI) using the Wilson score interval, because some case counts were expected to be close to zero. Also, Freeman-Tukey double arcsine transformation was used to account for variance between studies, and the I 2 statistic was used to assess the heterogeneity of the studies by zoonotic disease [20] . The power analysis was done using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA) and the meta-analysis was conducted using Stata/BE 17.0 (StataCorp LLC, College Station, TX, USA).
ArcGIS Pro version 3.0.0 (Esri Inc.) was used to produce map visualizations of the data, and shapefiles were obtained through DIVA-GIS spatial data repository, with GADM version 1.0 (Database of Global Administrative Areas) as the primary source.

Results
In total, 4142 articles were identified from the different databases (Embase = 1841; PubMed = 1231; CINAHL = 74; Scopus = 967; African Index Medicus = 0; and Cochrane = 29), of which 816 were duplicates. After screening, 64 articles were included for data abstraction and 12 were included from the literature cited in the articles retained for fulltext screening, such as systematic reviews; overall, 76 studies were included for data synthesis ( Figure 1 ).

Viral zoonotic diseases reported
Several of the studies that reported viral zoonotic diseases investigated these in animals and only a few involved human participants (8/27). Studies of zoonotic infections found a broad range of diseases, with a sample size ranging from 35 (in a family-based investigation of human T-lymphotropic virus (HTLV) subtype 3 and other simian retroviruses in a Bakola Pygmy 60-year-old man who had a positive test: 14.3% tested positive for HTLV-1 and 25.7% HTLV-2, and 14.3% for simian foamy virus (SFV)) to 4478 (domestic birds sampled between 2016 and 2018 in Adamawa, Centre, South, and West Regions for highly pathogenic avian influenza A (H5N1) with a positivity rate of 7.3%). Between 2009 and 2012, some studies reported low proportions of swine flu (H1N1) among commercial pigs and birds (0.6-5.6%) and humans (5.2%) in the Centre, Littoral, North, and West Regions. In a study by Steffen et al., 1.3% of the 240 healthy individuals tested in the South Region, tested positive for antibodies against Ebola virus (formerly Ebola Zaire). A majority of dogs (66%) presenting at veterinary clinics in the Centre, East, Littoral, North West, and West Regions (2010-2016) tested positive for rabies virus. A study conducted in October 2017 in the Centre and South Regions among 125 healthy individuals working at a primate sanctuary and living in villages around the sanctuary, reported evidence of monkeypox infection; 34.4% of individuals who had never received the smallpox vaccine compared to 6.3% of those who had previously received the smallpox vaccine, tested positive for antibodies (IgG) against monkeypox.

Bacterial zoonotic diseases reported
Overall, seven bacterial zoonoses were reported in the included studies ( n = 13). The prevalence of brucellosis among cattle and other livestock has predominantly been investigated, and a majority in the Adamawa region (5/6), with a period prevalence of 1.3% in Vina Division, Adamawa (January-November 2013) to 12.6% in Bamenda, North West (February 2017-January 2018) among pastoral cattle. In two studies conducted by Ndip

Parasitic zoonotic diseases reported
For studies that reported toxoplasmosis prevalence, positivity rates for prior infections (IgG) were greater than 20%. Most of these studies focused on pregnant women visiting different clinics in their first, second, or third trimester. In one such study performed in April-June 2014, of 643 pregnant women (age 27.1 ± 2.51 years, 39.5% second trimester and 50.7% third trimester) presenting at Penka-Michel and Menoua clinics, West Region, 35.8% were seroreactive. Similarly, a January-April 2015 study at Douala Regional Hospital and two private clinics, revealed that 78.6% were seroreactive. In June 2019-May 2020, Ayeah and colleagues investigated for the presence of antibodies against Toxoplasma gondii in neonates in two hospitals in the Centre Region via cord blood         Simian foamy virus (SFV) 9.7% seropositive among exposed (24.1% seropositive exposed to apes and 3.6% exposed to monkeys); of these, 90% PCR integrase-positive 24.1% positive cases exposed to apes and 3.6% to monkeys

Other vector-borne viral zoonotic diseases reported
The majority of studies that reported vector-borne zoonoses focused on dengue virus fever (14/21) Figure 2 ).

Meta-analysis by zoonotic disease to assess heterogeneity
Four of the reported diseases -brucellosis, dengue, influenza, and toxoplasmosis -had the number of studies sufficient for a power of ≥ 53%. It was expected that the reported prevalence across studies would vary for reasons other than sampling error (diagnostic tool, study population, and study period), which explains the SD = 6 and use of a  Figures S1-S4) show the pooled prevalence, I 2 statistics, and Pvalues associated with these. The pooled prevalence estimates should not be interpreted as representative of the burden of the specified zoonotic diseases across the national territory because of the expected variation in meta-analyses of prevalence studies. For the meta-analysis, all four pooled prevalence estimates among those surveyed in the different studies (febrile and non-ill community members) were associated with an I 2 statistic greater than 75% (high inter-study heterogeneity) and P < 0.01: brucellosis (random-effects pooled estimate proportion, ES 0.05%, 95% CI 0.03-0.07; I 2 = 90.91%; n = 6), dengue (ES 0.13%, 95% CI 0.06-0.22; I 2 = 98.90%; n = 12), avian and swine influenza virus (ES 0.10%, 95% CI 0.04-0.20; I 2 = 98.29%; n = 8), and toxoplasmosis (ES 0.49%, 95% CI 0.35-0.63; I 2 = 98.39%; n = 11). Figure 3 summarizes the results of the risk of bias analysis for the included studies. Of the 76 studies, eight had a high risk of bias, 64 a moderate risk, and four a low risk. For studies with a high risk of bias, they failed almost entirely in terms of external generalizability, and all of the included studies where information was provided were not representative of the national population in terms of demographics like age, sex, or occupation. All of the studies used the proper numerator and denominator in estimating prevalence, and all except two studies used appropriate diagnostic tools that have been shown to have acceptable validity and reliability. The studies checked 'Yes' for a majority of the questions regarding internal validity; 373 out of 456 (81.8%).

Discussion
Although epidemiological data on various zoonotic diseases from the different health districts are found scattered across the literature, to date there are no clearly available specific details of the burden of zoonotic diseases in Cameroon. This systematic review provides a one-stop resource for understanding, through an evidence-based systematized approach, the threat of pathogen exposures of Cameroonian communities related to zoonotic diseases. Overall, 35 unique zoonotic diseases reported in at least one region of Cameroon between January 2000 and May 2022 were identified. This concerted effort is consistent with the National Program for the Prevention and Fight Against Emerging and Re-emerging Zoonoses (NPPFERZD) and One Health Cameroon (national zoonoses programme) goal to have a unified and informed approach to risk management (community awareness, capacity strengthening, and prioritization) of zoonotic threats [7] .
From the studies that were reviewed, there is clearly a pattern in the number of research groups that have investigated particular groups of zoonotic diseases. Emphasis has been placed on an array of viral zoonoses: over the second decade of the study period of interest (2011-2022), more studies focused on vector-borne zoonoses; in the first decade, more studies focused on other viral zoonoses. Be-  Continued sides these, studies have been limited to brucellosis and toxoplasmosis, and other zoonotic threats have been investigated quite sparingly. The south-eastern portion of Cameroon and scattered parts of the Centre and North West Regions covered by the Congo Basin rainforest (16 674 023 hectares) teem with a unique biodiversity, including about 335 mammal species, 874 bird species, and 218 amphibian species [21,22] . Additionally, Cameroon is characterized by live market networks where livestock and other food products are exchanged. This clustering and movement within and between regions is a catalyst that drives the spread of diseases through interactions of infected and susceptible people at the community level, including the spatial overlap of non-human primate density with human activities, which transfers to trucking routes where bushmeat is sold [23] . A majority of the studies included in this review were hospital-based cross-sectional studies, and among those investigating animals, most studied cattle and swine in select farms or markets in the North, Adamawa, and North West regions. Also, epidemiological data that are available through the District Health Information System of the Ministry of Public Health are mostly case counts as reported by the respective health districts [24] . These are passive surveillance results; the actual burden is likely underestimated, as suggested by the current study findings. Increased vigilance for zoonotic disease events is indicated in order to better inform risk management efforts.
This systematic review also identified a need for more robust population research and case finding, consistent with other systematic reviews conducted by different research groups for specific zoonotic diseases like leptospirosis and monkeypox, or groups of zoonotic dis-  [13-15 , 25] . Differentiating zoonotic diseases as emerging or re-emerging is time and location specific. Emerging zoonoses would include those that have either never occurred before or have affected a small proportion of individuals and whose incidence is increasing, whereas re-emerging zoonoses include those that are well known but whose incidence has significantly increased or the hostenvironment-vector interaction has changed. It would be misleading to interpret the epidemiological data from this systematic review as indicative of the trend of the different zoonotic diseases that have been indexed.
For the pathogens with at least five studies, heterogeneity was greater than 75%. This is expected with a meta-analysis of prevalence studies because of the difference in the sampling frame, sampling technique, sampling size, diagnostic tool, and differing biomarkers reported as a result [26] .

Limitations
Although an evidence-based systematic approach was used to synthesize the literature and abstract the epidemiological data related to zoonoses in Cameroon, the findings should be interpreted with caution. First, the search strategy applied included the names of Cameroon and WHO-designated priority zoonotic diseases in addition to general terms. As such, it is possible that some literature databases did not return articles where the words zoonotic disease or animal disease or vectorborne or cross-species transmission or interspecies transmission were not used. Nonetheless, care was taken to use indexed/controlled terms for each database, such as MeSH terms in PubMed. Second, based on the risk of bias analysis, 73 out of the 76 studies included utilized a sampling frame that was not representative of the target population and none were representative of the national demographics. Third, some zoonotic diseases of known aetiology such as hepatitis E virus infection, fascioliasis, and microsporidiosis were excluded; human African trypanosomiasis was excluded because of indistinctness with the subspecies (zoonotic Trypanosoma brucei rhodesiense versus non-zoonotic Trypanosoma brucei gambiense ) in some studies [27][28][29] . Severe acute respiratory syndrome coronavirus 2 (SARS -CoV -2) was intentionally not indexed, as while animal-human-animal cycles have occurred, the pandemic is dominated by human-to-human transmission, confounding observation of this dynamic [30] . Fourth, although additional evidence was obtained from the grey literature, including country reports (District Health Information Software 2 and the National Zoonoses Program), the data were not included in this review because of ethical concerns and unbalanced data by reporting year. Fifth, social disturbance, including conflict, drought, population migration, and other stresses on communities, are incompletely accounted for in the literature and are factors important to the health security risk. Last, all of the studies included were cross-sectional studies and, as such, these are only point and period prevalence data. There were no data on the severity or duration of the infections, and no inference can be made about the demographic risk factors for the respective diseases.

Conclusions
This systematic review bridges some existing gaps in the understanding of the landscape of zoonoses and exposes critical gaps in the surveillance and reporting of zoonotic diseases in Cameroon. There is evidence of viral, bacterial, and parasitic zoonoses across the territory, many of which have epidemic potential. The SARS-CoV-2 pandemic and monkeypox epidemic underscore the critical role of pandemic preparedness. Therefore, there is a need to study definitive reservoirvector-acquisition associations and to strengthen passive surveillance systems and reporting of active or sentinel surveillance findings. In Cameroon, an improved understanding of specific groups and communities at higher risk than others for emerging and re-emerging zoonotic spillover events will allow careful prioritization of limited resources for better One Health risk management.