The Current Status of Cytomegalovirus (CMV) Prevalence in the MENA Region: A Systematic Review

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus worldwide. According to the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), CMV infects people of all ages, and by the age of five, approximately one-third of children in the United States are infected. Although the infection is generally asymptomatic, it can cause severe disease in immunocompromised patients, transplant and transfusion recipients, as well as newborn neonates. The objective of this study is to systematically review published literature on CMV in the MENA region to estimate its incidence in the region and describe its epidemiological and clinical significance. The literature was searched through four scientific databases: PubMed, Scopus, Science Direct, and Web of Science. A total of 72 studies from 11 countries satisfied the inclusion criteria, covering a period from 1988–2019. The CMV IgG seroprevalence ranged from 8.7–99.2% (SD = 38.95%). CMV incidence in these countries ranged between 1.22% and 77% in transplant and transfusion recipients, with an increase in incidence with advanced age. However, the incidence rate was unclear for congenital CMV due to the variability of the reporting. This review highlights the need for more robust and well-designed studies to better estimate CMV incidence in the MENA region, standardize diagnostic criteria, and consider prophylactic and pre-emptive treatments to limit the morbidity and mortality of the disease.


Introduction
Cytomegalovirus (CMV) is a global herpesvirus that is highly prevalent worldwide. It is a ubiquitous virus with a prevalence of about 100% in both Africa and Asia, and 80% in Europe and North America [1]. It is a member of the family Herpesviridae and genus Cytomegalovirus [2]. CMV is classified as a β-herpesvirus (HHV-5) and considered to be the largest herpesvirus to infect humans, with a genome of nearly 240 kb [3]. According to the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO), CMV can infect people of all ages; over 50% of adults are infected with CMV by the age of 40, and approximately one in three children are infected with CMV by the age of five in the United States [4]. Although CMV infection is usually asymptomatic, it could lead to severe outcomes in immunosuppressed individuals, particularly transplant recipients and blood transfusion patients [5]. CMV disease can mimic a range of different manifestations and that satisfied all five inclusion criteria were included in this review. Studies were excluded from the review if: (i) the study consisted of a mixture of patients from within and without the MENA region, and it was not possible to differentiate the data, (ii) it is not clear to differentiate the origins of the patients, and (iii) the study investigated multiple viruses, including CMV, and the data could not be differentiated. A schematic of the search strategy and study selection process is shown in Figure 1. not be differentiated. A schematic of the search strategy and study selection process is shown in Figure 1.

Data Extraction and Analysis
The studies included in this systematic review were analyzed three times by the same individual to ensure accurate capture of the information. The analyzed data included the country of the study, period of study, number of subjects, type of the study, incidence/prevalence of CMV antibodies or infection, symptoms and complications, proposed risk factors and routes of transmission, as well as treatment and prevention.

Data Extraction and Analysis
The studies included in this systematic review were analyzed three times by the same individual to ensure accurate capture of the information. The analyzed data included the country of the study, period of study, number of subjects, type of the study, incidence/prevalence of CMV antibodies or infection, symptoms and complications, proposed risk factors and routes of transmission, as well as treatment and prevention.

Search Findings
The search yielded 4777 studies, of which 3745 citations remained after removing duplicates ( Figure 1). After screening the titles, abstracts, and keywords, 1724 citations were excluded. The removed citations included irrelevant studies, such as those on animal and plant viruses. The remaining 2021 citations were screened against the eligibility criteria. Of these, 218 studies were removed due to the lack of peer-reviewing, and 1096 were removed for not discussing the epidemiology of CMV in transplant and transfusion patients nor its congenital illness. Furthermore, 422 non-primary research studies were removed (i.e., review articles, editorials, communications, case-reports, etc.), two for being published in languages other than English with no English abstract, and 202 for not having CMV as a measure or outcome of the study. One study was excluded as it was retracted by the authors. Finally, five studies were excluded as they met the exclusion criteria. The remaining 72 studies covered 11 countries (Egypt, Iran, Iraq, Israel, Jordan, Kuwait, Oman, Palestine, Saudi Arabia, Sudan, and Tunisia) over 31 years, from 1988-2019. The highest number of studies was from Iran (n = 27), followed by Israel (n = 21). No studies meeting the eligibility criteria were found from Algeria, Bahrain, Djibouti, Libya, Morocco, Qatar, Syria, the United Arab Emirates, and Yemen.

Epidemiological Findings
The reviewed studies covering CMV incidence in transplant recipients included renal transplants (n = 15), hematopoietic stem cell transplant (HSCT; n = 4), bone marrow transplants (BMT; n = 3), liver transplants (n = 3), and coronary artery bypass graft (n = 1). Country-wise, the majority of the studies were from Iran (n = 19). The remaining studies were published from Saudi Arabia (n = 4), Egypt (n = 2), Israel (n = 2), Kuwait (n = 2), Iraq (n = 1), Jordan (n = 1), Oman (n = 1), Sudan (n = 1), and Tunisia (n = 1). Overall, CMV infection among transplant recipients has been reported with an incidence ranging from 1.22% to 72%, regardless of prophylactic treatment. On the other hand, the anti-CMV IgG seroprevalence rate ranged between 8.7% and 99.2% (SD = 38.95), being mostly reported in Iran (Table 1). Fewer studies reported the IgM seroprevalence (n = 3), which ranged from 1.6% to 9.6%. The design and criteria to describe CMV disease in these studies were inconsistent (Table 1). Furthermore, multiple studies reported an association between increased incidences of CMV and advanced age. Other risk factors that were found include anti-rejection therapy, previous exposure to CMV, serological mismatch between the donor and recipient, as well as, several immunological determinants. However, there was a lack of CMV epidemiological studies from several countries in the region (Algeria, Bahrain, Djibouti, Lebanon, Libya, Morocco, Palestine, Qatar, Syria, the UAE, and Yemen), rendering the actual incidence of CMV across the region undetermined. As for congenital CMV, the studies were also inconsistent with their diagnosis and reporting of the results. Therefore, accurate incidence/prevalence data could not be deduced. Furthermore, the majority of the studies report infected cases with no incidence/prevalence data. The 38 studies with epidemiological information included in this review came from Israel (n = 19), Iran (n = 9), Iraq (n = 2), Egypt (n = 2), Kuwait (n = 1), and Oman (n = 1).

Discussion
To the best of our knowledge, this is the first systematic review study that investigated the epidemiology and status of CMV in immunocompromised patients in the MENA region. A total of 72 out of 3745 screened studies, covering 11 countries, were examined transplantation and blood transfusion recipients as well as the congenital CMV.

CMV in Transplantation Recipients in the MENA Region
CMV infects up to 60-100% of people in adulthood, and it is one of the main agents involved in infectious complications after transplantation [17]. It threatens the survival of transplant recipients and the function of the transplanted organ. Graft rejection and graft-versus-host disease (GVHD) are multisystem disorders that are common complications of transplantation. GVHD occurs when immune cells transplanted from a non-identical donor (the graft) recognize the transplant recipient (the host) as foreign, thereby initiating an immune reaction that causes disease in the transplant recipient [18]. A less well-established risk factor for GVHD is the CMV status of the donor and host [19][20][21][22][23]. Fortunately, it rarely results in mortality, with only one study reporting severe effects [20]. Generally, infection with CMV results in systemic viral syndrome, as shown in Table 1. The main manifestations are characterized by fever, malaise, vomiting, leukopenia, thrombocytopenia, and elevated liver enzymes.
Upper digestive tract symptoms and pain are also common [23][24][25][26]. Furthermore, respiratory illnesses, including pneumonia, have been reported in a few studies [24,[27][28][29]. Few studies reported other illnesses including hepatitis [25], urinary tract infection [24], rhinitis [28,29], skin conditions [23,24], and rarely, aortic plaques [30]. The disease caused by post-transplant CMV occurs due to the transplantation of an infected organ, reactivation of latent infection, or after primary infection in seronegative transplant patients [31,32]. The occurrence of disease caused by CMV in transplanted patients varies according to the organ transplanted, the serological match between recipient and donor, the immunosuppressive drugs used, and, most importantly, the interference of any additional diseases [17]. The incidence of CMV infection is 50% to 75% in patients undergoing heart-lung or lung transplantation and 50% in patients undergoing pancreas or kidney-pancreas transplantation. The incidence of CMV infection is 9% to 23% after heart transplantation, and 22% to 29% after liver transplantation [33]. In addition, 30% of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) and approximately 5% of patients undergoing autologous HSCT develop CMV disease [34]. Moreover, a study on CMV infections in the context of HSCT showed that 68% of pediatric patients who received umbilical cord blood transplantation were CMV seropositive [34].
Multiple studies investigated the association between the risk of CMV infection in transplant patients' and their demographics, including age, gender, and source of transplantation; however, results were inconsistent. A recently published study from Iran reported that CMV was diagnosed in 178 out of 725 (24.6%) kidney recipients, and showed that the incidence of CMV disease in kidney transplant patients within the age group 41-60 was four-fold more compared to those under 20 years old [27]. Similarly, a study conducted on HSCT in Iran revealed a positive correlation between the age of the recipients and CMV antigenemia [23]. On the other hand, other studies, such as in Saudi Arabia, failed to show this correlation [35]. These observations, along with those from other countries around the globe [36], indicate that age was a risk factor for CMV infection, especially in transplant patients [37,38]. For instance, a study reported that the risk of death was significantly increased in patients >38 years old, who underwent transplantation with peripheral blood, with an unrelated or mismatched donor, and who developed a CMV infection [39]. In addition, a study conducted on 200 allografted patients showed that recipients aged over 16 years were found to be at significant risk of CMV reactivation compared with younger patients (p = 0.007) [40].
In addition to age, several other factors have been identified in association with CMV incidence in SOT and HSCT recipients. One of the most significant risk factors is the extensive use of immunosuppressive drugs for patients undergoing organ transplantation. For instance, Taherimahmoudi et al. suggested that immunosuppression therapy using lymphocyte-depleting antibodies, namely anti-thymocyte globulin (ATG) therapy, is considered as one of the leading risk factors for CMV disease [41]. Charfeddine et al. reported that 12 out of the 16 patients who received kidney transplants suffered from CMV infections. Additionally, 6 out of those 12 patients developed acute rejection episodes due to CMV infection after administrating additional immunosuppressive treatment [21]. Such high frequency of infection was attributed to intensive antirejection therapy (including azathioprine, prednisone, cyclosporine, and ATG), among other factors such as previous exposure to CMV prior to transplantation [42], serological mismatch between both donor and recipient (e.g., CMV-negative recipients (R−)) received grafts from CMV-positive donors (D+)), and immune system factors.
Several immune correlates were identified as predictors for post-transplant CMV infection. One of them is mannose-binding-lectin (MBL), an innate humoral immunity protein that is important for pathogen opsonization and activation of complement pathways. One study documented a positive correlation between the infection and low levels of MBL. MBL deficiency compromises the innate immune response, resulting in a higher risk of developing post-transplant CMV disease and increasing the necessity of prophylactic treatment [43]. Other factors include CD 56 + T-cell levels, HLA mismatch, and GVHD. CD56 + T-cells (also known as NK-like T cells or cytokine-induced killer cells) have a potent cytotoxic effect on CMV, and a significant increase in their levels in renal transplant patients is suggestive of current CMV infection [44]. As for HLA, CMV disease was found to be influenced by more than one HLA allele mismatch [45]. Moreover, some studies have shown that specific alleles can promote or protect from post-transplant CMV disease. For instance, Khalifa et al. reported that 33.3% of CMV pp65 antigenemia positive patients in SOT have the HLA-A * 02 genotype, while patients with HLA-A * 01 (57.1%) had a protective effect against CMV infection [46]. Another study showed that kidney recipients with the HLA-B44 allele are more susceptible to CMV infection after transplantation, while carriers of the HLA-B8 allele are naturally protected from CMV infection [47]. Furthermore, a study showed that specific single-nucleotide polymorphisms (SNPs) in the loci of co-stimulatory molecules CTLA4 and CD28, which function in the regulation of T-cell activation, influence active CMV infection in kidney transplant patients [48]. Overall, these studies suggest the use of HLA typing as a predictor for CMV infection within the context of personalized medicine for prophylactic post-transplantation treatment.
The high risk of CMV infection and transmission through organ transplantation is universal. The prevalence across the globe ranges from 45% in developed countries to near 100% in developing countries [1]. The health burden of CMV infection and its manifestations seen in the MENA region is similar to that seen in other populations [49,50]. Even though the risk is high for transplant patients in the MENA region, only a few studies suggested screening for CMV in donors. For instance, in Saudi Arabia, screening for CMV and other pathogens before transplantation, especially in HSCT, was found to improve patient safety and mitigate the risk of accidental CMV infection [51]. One of the suggested approaches to protect from post-transplant CMV infection is the treatment with antibodies targeting CMV antigens (pp. 28, 150), which was shown to be effective in kidney transplantation [52]. On the other hand, Shibolet et al. demonstrated the development of late CMV disease and the occurrence of rejection episodes in liver transplantation recipients, regardless of the early use of antiviral prophylaxis [29]. In conclusion, due to the potential abnormalities associated with CMV infection and various morbidities, the establishment of preventive measures, especially vulnerable populations, including transplant recipients, is required.

CMV in Blood Transfusion Recipients in the MENA Region
CMV infection not only compromises transplantation but can also compromise the effectiveness of blood-transfusions, leading to transfusion-transmitted CMV infection, especially in immunocompromised patients. Transfusion-transmitted CMV infection occurs mainly due to the re-activation of the latent virus in WBCs. Many studies were performed to assess the seropositivity rates in different MENA region countries. Only a few studies, conducted mainly in Iran and Egypt, reported CMV infection after blood transfusion in the MENA region. The type of transfusion mainly discussed by the articles included in this review is whole blood transfusion; this is because most of the patients that need regular and frequent whole blood donations are anemic and thalassemic patients such as transfusion of other blood products like plasma transfusion, which is safer since most of the time it is autologous. That is why all research done on CMV seroprevalence was about patients with thalassemia who received multiple types of blood from different donors that could have been positive for the virus. A recent study in Mashhad, Iran, showed that out of 1008 blood samples that were tested for CMV antibodies, 99.2% were found to be positive [53]. In another study from Iran, Sepehrvand et al.
reported that the high incidence of CMV antibodies that were present in transfusion patients' blood is a result of receiving blood from CMV infected donors [54].
Furthermore, Mahmoud et al. showed that frequent blood transfusions among thalassemic children in Upper Egypt exposed them to a higher risk of transfusion-transmitted infections, including CMV. In this study, high rates of CMV infections were reported in children receiving a blood transfusion, and the infection was positively correlated with increasing age and the duration of the thalassemia [55]. These high rates of CMV infections are attributed to the high CMV seroprevalence among blood donors in Egypt, as reported by Gawad et al. In their study, 96.6% of blood donors (out of 88 tested blood samples) were CMV seropositive [56].
Overall, these findings demonstrate a high rate of prevalence of CMV infections in the MENA region, and as such, posing serious implications for the blood transfusion practices if proper screening measures are not implemented. High CMV positivity in transfusion patients, highlighted clearly in some of MENA countries ( Table 1), assures that there is a crucial need for mitigation plans to reduce CMV transmission in transfusion patients.
In terms of disease morbidity, one of the common complications of cCMV observed in the MENA region is abnormal brain sonography. Hadar et al. reported a 67% occurrence of abnormal brain sonography in infants born to mothers with primary maternal infection, and 8.3% in infants born to mothers with non-primary infection [89]. Another common complication of cCMV is hearing loss. A retrospective study in Israel correlated early cCMV infection with hearing loss [79]. In addition, cCMV could lead to hepatic damage, including hepatitis and cholestatic disease [88]. The frequency of which (6.6%) was found to be less than was previously expected and is far out shadowed by CNS involvement (84.6%) and hearing loss (53.8%) [88]. In all cases, however, rapid antiviral treatment showed improvement in symptoms, albeit over prolonged periods [79,88]. As for mortality, higher rates were found in association with primary cCMV infections compared to recurrent infections, and abortions were reported at all pregnancy stages [73,90]. Jahromi et al. found that there is an association between seropositivity and abortion rate [91].
Nevertheless, there is substantial variability in the definitions of symptomatic and asymptomatic cCMV infection, study designs, and the methods of determining CMV infection. This variability, along with the variability in study design and detection methods, created difficulty in assessing the overall status of cCMV in the region ( Table 2). Similar variations are also seen in the US and Germany, suggesting complicated disease manifestations, depending on the genetic, health, and nourishment status of the infants and their mothers [92][93][94][95]. Still, the incidence rate of cCMV in the MENA region seems to be higher than in other populations. Schlesinger et al. reported an incidence of 945/135,000 (0.7%) births in 2005 in Israel [96,97]. This incidence rate is similar to that found in the United States (0.6-0.7%) [98]. The rate is, however, higher than in Europe, to which Israel's healthcare system is more similar. For instance, Sweden and the UK have incidence rates of 4.6/1000 births (0.46%) and 3.2/1000 births (0.32%), respectively, and a decades-long prospective study in Denmark found the incidence rate to be 0.4% [99,100].

Laboratory Diagnosis of CMV in the MENA Region
CMV diagnosis was traditionally performed by serologic testing and viral cultures from multiple samples; however, molecular diagnosis is currently the standard [116]. A similar trend can be seen in the MENA region with the diagnosis of CMV. In the 1980s, culture and serological methods were used to detect CMV. The trend moved towards molecular testing, with polymerase chain reaction (PCR) becoming more prominent over time. In the reviewed literature, multiple diagnostic laboratory tools were used, including serology, PCR, antigenemia assays, immunohistochemistry, and culture.
Serological methods indirectly provide evidence of current or prior infection by detection of antibodies in serum. The presence of anti-CMV IgM antibodies can be used to diagnose a recent or acute infection, or at least a fourfold increase in IgG antibody titer in subsequent specimens obtained two to four weeks apart [116]. However, IgM antibodies can persist for several months, leading to false-positive results for acute infection [117]. Thus, a definite diagnosis of CMV infection cannot be obtained from serological tests. Nevertheless, serological antibody detection remains a cost-effective screening tool. Serological methods alone, or in conjunction with other methods, are employed in the MENA region to screen for CMV before and after transplantation [23,47,52,57,63,68,70,[118][119][120][121][122][123][124]. Moreover, serology is used in Israel to screen pregnant women for CMV infection. Although no formal regulations exist, still, most hospitals perform routine screening for anti-CMV antibodies using the criteria mentioned earlier to establish a diagnosis [74,125,126]. Once the presence of CMV is established in the mother, congenital CMV is diagnosed either by pre-natal amniocentesis followed by culture or PCR, or by PCR or antigenemia assays in the urine of the infant [73,75,76,78,80,104,108,127].
In recent years, PCR has become prominent in CMV testing in the MENA region. PCR is highly sensitive and specific. However, its main drawback is that it cannot differentiate between latent and active virus [116]. Additionally, there is variability in the viral loads obtained by different laboratories for the same specimen. In response, the WHO developed an international quantitative standard to standardize measurements and facilitate studies to correlate viral load with the development of the disease [128,129]. Nevertheless, the variability among assays continues to exist due to differences in specimen types, DNA extraction methods, as well as the PCR protocol [130][131][132]. Currently, PCR is commonly used in the MENA region for the diagnosis of CMV. It is used to test for congenital CMV in amniotic fluid or urine [73,75,76,78,80,104,108,127], to monitor CMV infection after transplant [41,62,[133][134][135][136], as well as end-organ testing when a specific organ is affected, such as HIV associated CMV retinitis [137].
Finally, CMV antigenemia, immunohistochemistry, and culture methods are used in the MENA region as a definite diagnosis of CMV infection. Antigenemia is used in the diagnosis of congenital CMV and the monitoring of viremia in transplant patients. The assay is based on the detection of the CMV pp65 protein in peripheral blood leukocytes. Their advantage over measuring anti-CMV antibodies and PCR is that it can detect the active virus, correlating the result of the assay with viremia [116]. Immunohistochemistry assays use labeled antibodies directed towards CMV antigens (early antigen, immediate early antigens, pp65, and late antigen) and are visualized under the microscope. A positive result in immunohistochemistry indicates the presence of CMV [138]. In the MENA region, this method was only used in studies of tissue biopsies of invasive CMV infections in the case of cancer [139][140][141][142]. This usage is in line with the international consensus for the diagnosis of CMV in invasive tissue disease [138]. As for culture, conventional methods have been replaced by the more rapid shell-vial cultures [116]. Shell-vial culture involves the centrifugation of a patient specimen onto a cell monolayer in a vial, which speeds up the time to the result. The virus is then detected in the monolayer using fluorescent antibodies. The MENA region also sees the same trend of replacing traditional cell culture with shell-vial cultures. In Israel, shell-vial culture is the method in use for the diagnosis of congenital CMV from urine specimens from the infants [72,89,102,118,143,144].
While we summarized in this review the techniques used in CMV diagnosis, they do not necessarily reflect the status of CMV diagnosis in the region. The reviewed studies did not cover all the countries in the MENA region; thus, a more in-depth analysis was not possible. Nevertheless, the information does reflect the availability of the diagnostic tools in the region during the study period. However, even though the diagnostic tools are available and widely used in the region, there is no information on country-wide screening guidelines and policies. Instead, hospitals implement their own practices and guidelines on screening.

Conclusions
The literature search yielded a total of 72 primary research articles covering 11 out of the 21 MENA countries over 31 years. The studies on transplant and transfusion recipients reported the incidence of CMV with uncertainty about the disease. Although infection with CMV virus is common among graft recipients in this region (up to 77%), the actual rate of clinically confirmed CMV disease was unclear as most of the studies depend solely on seroprevalence reports. Multiple other studies exist outside of those included in this review; however, they do not measure the incidence of CMV. Although some studies assessed the frequency of transplantation, the CMV incidence as a critical outcome was not reported in many of the studies. Similarly, with blood transfusion, studies report CMV seroprevalence with no clear indication of clinical burden. The same can be observed with cCMV, where substantial variability in cCMV reporting criteria leads to a less-than-accurate picture about the status of cCMV in the MENA region. The low number of published articles, the low number of reporting countries, the inconsistency in measurement and detection protocols, as well as the variability in endpoint evaluation of the clinical illness, are all factors that limited the ability to generalize the information across the region. Thus, there is an incessant need to conduct well-designed studies under the umbrella of the WHO to estimate the burden of CMV disease on transplant recipients in the MENA region and to standardized both the prophylactic and preemptive treatment and predict the subsequent morbidity and mortality.