Map showing 18 Governorates of Iraq and bordering countries. Governorates are coloured by number of human rabies cases per 100,000 population 2001–2010. Population estimates were taken from a recent household survey [13]. Governorate and country boundaries are approximate. doi:10.1371/journal.pntd.0002075.g002 

Context in source publication

Context 1

... was initiated in the Baghdad region in April 2010. Here we report results of laboratory diagnosis and virus characterisation from these initial sampling efforts alongside official surveillance data for human rabies across Iraq. Human rabies is notifiable in Iraq through regional public health offices in each of the 18 Governorates (provinces). Private and public health centres and hospitals report rabies cases based on a clinical definition of encephalitis, combined with hydropho- bia and history of animal bite. There is no routine laboratory diagnosis undertaken. The regional public health offices report to the Zoonoses Section of the Centre for Disease Control (CDC) in Baghdad, who also collate post-exposure prophylaxis and reported animal bite numbers. These anonymized data on human rabies cases, animal bites and post-exposure prophylaxis were reviewed for the period 2001–2010. Analysis of the data was approved by the AHVLA Ethics Committee. Differences in rabies incidence between groups (age, sex and rural/urban habitation) were assessed using Chi-squared tests. Expected frequencies for age and area of habitation were taken from a separate recent household survey undertaken by others [13](Table 1). Brain samples. Between April 2010 and July 2011, forty clinically ill animals (38 dogs, 2 cattle) in the Greater Baghdad area were euthanased by private veterinary surgeons or Iraqi State Veterinary Company staff. Cases were selected where rabies could not be ruled out on the basis of clinical signs. Reported signs included one or more of: abnormal behaviour or vocalisation, aggression, hyper-salivation and neurological signs. A pool of brain tissues including brain stem were removed at post mortem and stored frozen. All samples were transported frozen on dry-ice to the OIE Reference Laboratory at the Animal Health and Veterinary Laboratories Agency, Weybridge UK. Brain samples were tested using a standard fluorescent antibody test (FAT) for lyssavirus antigen [14] and viral RNA was extracted from all brain samples in duplicate using enzymatic disruption (MELT, Ambion ) according to the manufacturers protocol. RNA was reverse transcribed and a hemi-nested Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) was undertaken on each sample as described previously [15]. PCR products were purified ( Qiagen ) and sequenced as described previously [16]. At least one forward and one reverse primer were used to determine consensus sequences which were aligned using ClustalX2 (version 1.2). Bayesian Markov Chain Monte Carlo (MCMC) phylogenetic analysis of the resulting consensus nucleoprotein sequences was implemented using the BEAST package (version 1.4.8) [17] with a panel of rabies viruses (RABVs) from neighbouring countries (Table S1). A relaxed molecular clock (uncorrelated lognormal) and GTR substitution model with a proportion of invariant sites were chosen over other evolutionary models based on comparison of multiple runs using Tracer (v1.4). A chain length of 10,000,000 was used and parameters were logged every 1,000 trees to ensure effective sample sizes were . 200 and posterior distribution was normal. The maximum clade credibility (MCC) tree was chosen using TreeAnnotator (v 1.4.8) after the first 10% of trees were discarded and the resulting tree was visualised using FigTree (v1.2). Data on reported rabies cases were supplied by all 18 regional public health offices. In the 10 years between 2001 and 2010, there was an average of 17 (SD 6.9) human rabies cases reported annually in Iraq (Figure 1a). There was a three-fold increase in reported cases between 2003 and 2005 and, although the number of cases has varied from year to year, there has not been less than 15 cases reported per year since 2005. Human rabies incidence for Iraq during 2009 is estimated from these data at 0.89 deaths per million population, using a population estimate of 30 million [13] (Table 1). Children are over represented among rabies cases in Iraq. An estimated 40% of the population is under 15 years of age [13], yet 63% of cases occur in this age group (X 2 = 48.4, p = 0.0001). Rabies is also more frequently reported in rural areas than urban areas, with 83% of cases reported in rural areas despite only 29% of the population living in rural areas [13] (X 2 = 283, p = 0.0001). However, there has also been an apparent three-fold increase in the number of cases reported in Baghdad over the past ten years (albeit not statistically significant), with an average of 2 cases per year reported in between 2001 and 2002, and 6 cases reported per year between 2009 and 2010 (Figure 1b). There is an extreme bias towards males, with eight cases in males reported for every one case in a female despite a population sex ratio of 1:1 [13] (X 2 = 122, p = 0.0001). There is regional variation in the number of reported cases, with Governorates in the centre of the country reporting the highest incidence per 100,000 population during 2001–2010 (Figure 2 and Table S2). Rabies prophylaxis is available in Iraq, although is not always initiated, and is rarely completed. The five-dose (Essen) regime is most frequently followed, and although a large proportion of dog bite victims receive the first vaccination (75%) a much lower number complete the full course (7%). From 2002 to 2004 there were less than 1000 dog bites reported annually in Baghdad, corresponding to an incidence of 20 (95% CI 18.76–21.24) bites per 100,000 people, based on a population estimate of 5 million [18]. In the years between 2007 and 2010 the average had increased to 3300 bites reported per year (Figure 1c), corresponding to an annual incidence of 46 (95% CI 44.27–47.40) bites per 100,000 people, using a population estimate of 7.2 million [19] (paired incident rate test, p , 0.0001) (Figure 1c). Three out of 40 brain samples were positive for rabies virus by both FAT and RT-PCR. The three positive samples yielded unique partial nucleoprotein gene (N-gene) sequences (Genbank accession numbers JX524176-8). Phylogenetic analysis using a 400 base pair region of the N-gene showed that the viruses are closely related, forming a well supported clade separated from other published sequences (Figure 3). A relaxed molecular clock model applied to these data (assuming constant virus population size) suggests they share a common ancestor approximately 22 years ago (95% HPD 14–32 years) with viruses in the cosmopolitan lineage of RABV, from neighbouring countries including Turkey, Iran, and Syria. Rabies is a preventable disease, and yet the data presented here demonstrate that it remains a significant public and animal health challenge in Iraq. All except two of the 18 Governorates reported human rabies cases during the period of study, indicating that rabies is endemic and widespread across the country. The reported incidence of human rabies far exceeds that reported by some neighbouring countries. Incidence during 2009 is estimated from these data at 0.89 deaths per million population, compared with 0.025 for Turkey and 0.02 for Iran [11]. There was an increase in reported cases for the whole country after 2003 and a three-fold increase in reported cases in Baghdad in the ten years between 2001 and 2010. This increase coincides with a period of intense conflict in Iraq, with the potential to have widespread direct and indirect consequences on disease control. These include well documented affects of the migration of health professionals and restrictions on travel, resources and sanitary conditions previously reported to cause increases in infectious diseases such as typhoid, measles and mumps in Iraq [6,20]. As rabies is a zoonotic disease, with domestic dogs as primary reservoir host in many regions, changes to the free-roaming dog population will also have a large effect on human rabies incidence [3]. The effects of conflict on municipal services and disruption of human habitation are likely to have an impact on the urban dog population. The doubling of reported dog bites in Baghdad reported here coincide with the increase in human rabies cases and anecdotal reports of a mass expansion of the free-roaming dog population in Baghdad. It is likely therefore, that the increase in human rabies is due to an increase in the free roaming dog population and associated increase in dog bites. In addition to the increase in risk of zoonotic disease, the dog population increase has animal welfare implications. Historical approaches to manage dog populations in Baghdad have included pro-active culling in areas where large accumulations of free roaming dogs are reported. Although this temporarily results in fewer free roaming dogs, it is increasingly recognised that indiscriminate culling is not a long-term solution to dog population control, or to reducing rabies prevalence, has welfare implications and can make the situation worse [3,12]. Animal birth control (ABC) programs, where sterilisation is combined with rabies vaccination, have proven effective in reducing or stabilising free-roaming dog populations, and also reducing rabies incidence [21,22] but they may not be necessary in many socioeconomic settings [3,23,24,25]. Therefore, where dog population management is used, it should be according to international guidelines and only after assessment of the local dog population [26]. Promotion of responsible dog ownership and appropriate legislative measures are also recommended as longer term solutions for controlling rabies in Iraq [12]. As with all studies using human rabies surveillance data, the data presented here have limitations. The low reported numbers of human rabies cases in Baghdad preclude robust statistical comparison, meaning that the apparent increase in cases in Baghdad could be the result of annual variation rather than a genuine increase in cases. In addition, human rabies cases are currently only diagnosed based on clinical data without laboratory confirmation. The incidence ...