Prevalence of extended-spectrum-β-lactamase-producing Enterobacteriaceae: first systematic meta-analysis report from Pakistan

Background South-Asia is known as a hub for multidrug-resistant (MDR) bacteria. Unfortunately, proper surveillance and documentation of MDR pathogens is lacking in Pakistan. The alarming increase in the prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is a serious problem. From this perspective, we analysed published data regarding ESBL-producing Enterobacteriaceae in different regions of Pakistan. Methods A meta-analysis was performed to determine the prevalence of ESBL-producing Enterobacteriaceae in Pakistan. A Web-based search was conducted in electronic databases, including PubMed, Scopus and PakMedi Net (for non-indexed Pakistani journals). Articles published (in either indexed or non-indexed journals) between January 2002 and July 2016 were included in the study. Relevant data were extracted, and statistical analysis was performed using the Metaprop command of STATA version 14.1. Results A total of 68 studies were identified from the electronic data base search, and 55 of these studies met our inclusion criteria. Pakistan’s overall pooled proportion of ESBL-producers was 0.40 (95% CI: 0.34–0.47). The overall heterogeneity was significant (I2 = 99.75%, p < 0.001), and significant ES = 0 (Z = 18.41, p < 0.001) was found. OXA, SHV, TEM and CTX-M were the most commonly found gene variants for ESBLs in these studies. Conclusion The prevalence of ESBL-producing Enterobacteriaceae is high in Pakistan. Little is known about the annual frequency of ESBLs and their prevalence in different provinces of Pakistan. No data are available regarding ESBL frequency in Baluchistan. This underscores an urgent demand for regular surveillance to address this antimicrobial resistance problem. Surveillance to better understand the annual ESBL burden is crucial to improve national and regional guidelines.


Background
Antimicrobial resistance has been declared a global threat to public health, as a massive increase in this problem has been observed in different parts of the world [1]. Although the magnitude of the antimicrobial resistance problem differs by country and geographical region, South-Asia is considered to be a major region for multidrug-resistant (MDR) bacteria [2]. The reported frequency of MDRs is increasing, putting strain on the public health organizations that are attempting to control this issue in many countries [3].
MDRs are posing a treatment challenge, and are emerging as a major cause of morbidity and mortality worldwide. Unfortunately, proper surveillance and documentation of such pathogens is very limited, especially in developing countries. It has been estimated that more than 70% of antibiotic resistance occurs in the Asia-pacific region of the world, making antimicrobial resistance extremely problematic for Asian countries [1]. In Pakistan, ESBLs are especially problematic in terms of their contribution to the MDR bacteria problem. From this perspective, we analysed all of the available data regarding the prevalence of ESBL-producing isolates in different regions of Pakistan.
Antimicrobial resistance is on the rise. There are many factors associated with increasing antimicrobial resistance, one of which is ESBL production. The distribution of ESBLs differs in different communities, and every community must design their own protocol regarding the prevention and treatment of such infections [41]. Developed countries have annual surveillance systems to monitor the impact of antibiotic resistance as well as to determine the causative agents of antibiotic resistant infections. Such surveillance systems are often inadequate in developing countries. This meta-analysis will improve understanding of the distribution and epidemiology of ESBLs with different gene variants in Pakistan. This study also highlights the need to use molecular techniques to determine the different gene variants associated with ESBL-producing bacteria in Pakistan. To our knowledge, this is the first meta-analysis report from Pakistan, which would aid in updating the national treatment guidelines for ESBL infections. The purpose of this study was to determine the pooled prevalence of ESBL-producing Enterobacteriaceae with different gene variants in Pakistan.

Study design
This is a descriptive, meta-analysis study and is comprised of different studies reported from within Pakistan.

Literature search and strategy
A Web-based search using the key words: ESBLs, Pakistan, ESBL genes and Enterobacteriaceae, were performed using the electronic databases PubMed, Scopus, PakMedi Net and Web of Science in September 2016. Articles published in the English language were included in the study. A comprehensive search was carried out for publications on the subject of ESBLs from Pakistan. Furthermore, the references cited within the articles were also carefully screened to look for additional relevant publications.

Study selection procedures and criteria
Study selection was carried out by three authors in three steps independently (SH, SA and SR). As a first step, all of the titles and abstracts that were related to the study question were reviewed, and these were included in a group of eligible articles with irrelevant articles being excluded. All articles in the initially selected group were further screened in a second step by reviewing the full details of the articles. As a third step, selected articles were evaluated by other authors specifically for metaanalysis (RAK, NA, H H, SR), which was conducted using software STATA version 14.1 (College Station, Texas, USA) as previously described [42].
All studies were included based on the following criteria 1) studies that reported the prevalence of ESBLs in any province of Pakistan; 2) studies on bacterial strains isolated from human specimen; 3) all relevant national and international full text original research articles; 4) studies with confirmed ESBLs using phenotypic detection methods; 5) studies that used molecular techniques for ESBL gene variants.

Data extraction
The data were extracted by SH, SA and SR from the eligible studies and included. The data extracted from eligible studies consisted of; year of publication, year in which study was conducted, name of authors, location where the study was conducted (different provinces of Pakistan), sample size, strains detected ESBL, phenotypic detection techniques including; the double disc synergy test (DDST), the combination disc test (CDST), the epsilometric test (E-Test) and molecular detection techniques for gene variants (PCR) (Tables 1 and 2). Data were extracted and analysed twice to remove any discordance.

Statistical analysis and reporting
Statistical analysis was performed using the Metaprop command in STATA version 14.1 (College Station, Texas, USA) to pool the published data regarding the predominance of ESBLs in different regions of Pakistan. Statistical heterogeneity was calculated using the I 2 statistic (measure of inconsistency) at the significance level of 5%. Heterogeneity was used to study the variation in studies using the I 2 statistic. The p-values (typically considered significant at 0.05) were used for converting meta-analysis results to defined/known tests of statistics. Random-effects model (REM) was used to estimate the pooled prevalence and 95% CI. A funnel plot and Begg tests were performed to evaluate the publication partiality using data graphically and statistically.

Distribution of articles describing ESBLs in Pakistan
Electronic database searches yielded a total of 68 studies. A total of 55 articles reviewed from four provinces of Pakistan included 14 (25.4%) from Punjab, 23 (41.8%) from the Islamabad/Rawalpindi, 9 (16.4%) from the KPK, while the remaining 9 (16.4%) were from the Sindh. No studies were found from Baluchistan province ( Fig. 3 and Table 3). The maximum number of articles on this subject was published in year 2013, followed by 2011, with the number of published articles in Pakistan decreasing afterwards (Fig. 4). In total, 42 (76.4%) of the articles reviewed included cases from in-patient and outpatient departments (OPD), 10 (15%) included patients attending in-patient departments, and 3 (6.3%) included patients attending OPD. A total of 21, 232 ESBLbacterial isolates were included in the analysis. A total of 53 (96.4%) of the reviewed studies were conducted on both adults and children, while only 2 (3.6%) studies were based solely on the paediatric population. No studies were found on male and females separately ( Table 2).

Laboratory methods used to estimate the proportion of ESBLs
For the variable phenotype methods, out of 55 studies, 48 (87.3%) had performed the double disc synergism test (DDST). However, only 13 (23.6%) had performed the combination disc test (CDST), and only 5 (9.09%) had performed the epsilometric test (E-Test). None of the studies had used the broth minimum inhibitory concentration (MIC) method. Out of 55 studies, 28 (50.9%) were published in local journals while the remaining 27 (49.1%) were in international journals ( Table 2).

Molecular methods used to estimate the proportion of ESBLs
For molecular detection methods, 6 (11%) out of 55 articles reported PCR-based gene detection methods. Among these, 50% of articles reported CTX-M group as the most prevalent group, and CTXM-1 as the most commonly found gene variant. Additionally, CTX-M and TEM combinations were found in 33% of selected articles. One study reported the association of qnrB genes with the appearance of the ESBLs phenotype ( Table 1).

Prevalence of ESBLs and their distribution in different geographical areas of Pakistan
Based on the available data (Table 1) Fig. 2 and Table 2).

Discussion
To address the issue of MDR bacteria, it is necessary to raise awareness about the magnitude of the problem by collecting data about antibiotic-resistance in various countries and regions [1,29,43]. The scarcity of studies available from Pakistan warrants attention for future research. Limited data regarding the overall predominance of ESBLs from Pakistan are available, but with no studies specifically from Baluchistan. This is the first meta-analysis about the extent of the ESBL problem in the Pakistani population. This is the first metaanalysis regarding the extent of the ESBL problem in the Pakistani population. However, this meta-analysis finds a high percentage of ESBL-producing Enterobacteriaceae across different geographical regions of Pakistan [5,7,12,20,[44][45][46][47][48][49][50]. This meta-analysis is comprised of different studies reported from within Pakistan. The overall pooled proportion for ESBLs in this meta-analysis for Pakistan was 40% (Table 2). In China, a nationwide survey comprised of 30 different hospitals reported a 46% ESBL proportion, which is quite close to the data reported in our study [51]. A survey conducted in the hospitals of East Africa reported an overall pooled ESBL proportion of 42% (95% CI: 0.34-0.50) [52]. Previous research showed a considerably higher frequency of ESBL in Asian and African countries compared to developed countries [53]. For instance, the German population showed the estimated ESBL proportion in the range of 10 to 15% [54]. Similarly, a report was published from the US in 2012, which was based on the surveillance of ESBLs in nine census regions of the US, and they reported 4 to 12% resistance due to ESBLs [55]. However, among the Asian continent, an increase in ESBL mediated resistance was observed among the Japanese community, where the pooled ESBL proportion increased from 6.3% to 20% in 9 years [56].
These results indicate an extensive and statistically significant degree of disparity in ESBL proportion estimates (p < 0.05). The variation in ESBL occurrence reported in this systematic report may depend on several factors, including the socio-economic status of a society and the availability of antibiotics [57]. Moreover, differences in the sensitivity and specificity of the different methods applied in determining the proportions are also contributing factors. The majority of studies used purely phenotypic approaches, while some studies used molecular methods along with phenotypic testing [7,9,16,17,44,47,49,58,59]. Differences in ESBL proportions have been documented from all over the world established by hospital or community-based surveys [60][61][62]. In a study conducted in Ha'Emek Medical Center Israel> 50% ESBL prevalence was reported for communityacquired infections [63]. Whereas studies in Egypt, Cameroon, Bamako, Spain, China, Saudi Arabia, United Kingdom, United States, Latin America    [63][64][65][66][67][68]. Due to limited resources and a lack of infrastructure, only a few articles (11%) have investigated the molecular characterization and presence of ESBL encoding genes [4,7,16,24,29,44,45,50,51,58]. OXA, SHV, TEM and CTX-M were the most commonly found gene variants in these studies for ESBLs [7,16,17,50,69]. The CTX-M group was found to be prevalent in 50% of studies reporting utilization of PCR-based molecular detection methods. The CTXM-15 gene variant of CTX-M group 1 has already been reported in many studies in the Asian continent [70][71][72][73]. In particular, CTXM and TEM is a common gene variant combination [7,50]. One study reported the association of qnrB gene variants with the appearance of an ESBL phenotype, as this gene is involved in fluoroquinolone resistance [17]. However, few studies have reported the incidence of NDM genes, which are responsible for Carbapenemresistance [52]. There are many reasons for variations in ESBL prevalence in the four studied regions of Pakistan. There may be substrate preferences, higher use of any specific class of antibiotics, co-resistances to other classes of antibiotics, poor health and diagnostic facilities [74,75]. With this limited available information to hand, it is challenging to plan intensive and effective interventions for combating the problem of resistance.

Conclusion and recommendations
This meta-analysis indicated that there is a high ESBL burden in Pakistan. Few papers are available that address the annual frequency of ESBLs and their distribution in different provinces of Pakistan. No paper is available regarding the frequency of ESBLs in Baluchistan. Only 6 papers that reported gene detection were found. Detection of gene variants in β-lactamaseproducing bacteria is essential information for the appropriate and effective treatment of patients. This underscores an urgent demand for regular surveillance to address this antimicrobial resistance issue. National and regional guidelines would be based upon such surveillance in order to understand the annual ESBLs burden. Effective measures such as the establishment of active surveillance and infection control programmes, emphasizing hand hygiene together with coherent antibiotic policies in hospitals and clinics should be implemented to stop and manage the spread of ESBLs in hospitals and communities.