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## **Meet the editors**

#### Patricia Price, Editor

The focus of my work is HIV disease - specifically the consequences of beginning ART with low CD4 T-cell counts. This has included studies of Immune Restoration Diseases associated with opportunistic infections that are common in the developing world. We showed that poor control of HCV replication marks Indonesian patients experiencing relapses of HCV disease on ART. A larger study in Jakarta will correlate "the footprint" of chronic CMV infection with clinical endpoints in patients beginning ART. We are also studying CMV immunology at Royal Perth Hospital/University of Western Australia. We are addressing the role of CMV in immunological aging – comparing the footprint of CMV in HIV patients and renal transplant recipients with that seen in healthy individuals of a similar age.

#### Nandini Makwana, Editor

I am pursuing a PhD at the University of Western Australia, and my interest is in the effects of Cytomegalovirus on Natural Killer cells and adaptive immune response in renal transplant patients. I have expertise in tissue culture work, and have worked on Japanese Encephalitits, West Nile and Bagaza virus in National Institute of Virology, India, which is also a WHO collaborating centre for Arboviruses and Haemorrhagic fever reference and research. I have also done in-vitro screening of anti-HIV compounds and microbicides at the National AIDS Research Institute, India, and have gained expertise on setting up human cervical explant cultures. My area of interest is virology and immunology but I also hold a postgraduate certificate degree in clinical research and clinical data management.

#### Samantha Brunt, Editor

Following on from my B.Sc in biomedical science which was completed during 2012, my PhD work focuses on Cytomegalovirus antibody production in HIV patients. This is an interest of a larger project investigating CMV immunity and how it relates to accelerated aging and increased prevalence of age associated diseases, such as cardiovascular disease. I am continuing investigations into the 'footprint' CMV infection leaves on a person's immune system, focusing on B-cell activation and also prevalence of auto-antibodies relative to CMV antibody. My honours work demonstrated a correlation of high CMV antibody titres and increased risk of cardiovascular disease in HIV patients stable on therapy.

Contents

**Preface VII**

**Infection 1**

Patricia Price

Chapter 1 **Hearing Loss in Children with Congenital Cytomegalovirus**

Matthew Bates, Kunda Musonda and Alimuddin Zumla

Ana Maria Sampaio, Ana Carolina Guardia, Arlete Milan, Elaine Cristina Ataíde, Rachel Silveira Bello Stucchi, Sandra Botelho Cecilia

Satoshi Iwasaki and Shin-ich Usami

Chapter 2 **Human Cytomegalovirus (HCMV) Infection in**

Chapter 3 **Management of CMV-Associated Diseases in Immunocompromised Patients 41** A.L. Corona-Nakamura and M.J. Arias-Merino

Chapter 4 **Cytomegalovirus Infection in Liver Transplantation 63**

Costa and Ilka de Fatima Santana Ferreira Boin

Chapter 5 **The Footprint of CMV Infection May Last a Lifetime 77**

Chapter 7 **Cytomegalovirus Tegument Proteins and the Development of**

Chapter 6 **The Oncogenicity of Human Cytomegalovirus 95** Prakash Vishnu and David M. Aboulafia

**Novel Antiviral Therapeutics 111**

John Paul III Tomtishen

**Sub-Saharan Africa 17**

### Contents

#### **Preface XI**

Preface

viduals at risk.

diabetes.

cardiovascular changes.

CMV is a prevalent viral pathogen that rarely causes recognisable symptoms in immu‐ nocompetent hosts. CMV is the strangest virus. It has 200 genes but needs only 30 to replicate. It infects everyone but few people have heard of it. We carry it all our lives but it can rarely be isolated from blood. Up to 23% of CD8 T-cells can be specific for CMV in older healthy adults, despite undetectable viral load. CMV-specific T-cells have a "worn out" phenotype, likely gained from lifelong surveillance of persistent CMV

CMV causes distinct patterns of disease in different classes of immunodeficient pa‐ tients. This includes retinitis in HIV patients, pneumonitis after transplantation and deafness in babies. This book includes chapters focussing on different groups of indi‐

CMV reactivation (in previously seropositive recipients) or primary infection (in any recipient) is the most common infectious complication in renal transplantation. In the 1970's one in three recipients experienced pathologies associated with CMV. In most countries, prophylaxis (valganciclovir) is routinely administered for 12-26 weeks after transplantation. However CMV remains a significant cause of graft loss and the opti‐ mal duration of prophylaxis is unclear. In the longer term, CMV reactivations are im‐ plicated in deterioration in renal function, cardiovascular disease and perhaps type II

CMV has also been anecdotally linked with cardiovascular disease and in vitro studies show increased expression of adhesion molecules in endothelial tissues infected with CMV. One study linked T-cell responses to CMV with cardiovascular symptoms (athe‐ rosclerosis) in HIV patients. CMV-DNA has been found in tissues removed during sur‐ gery for abdominal aortic aneurysm and CMV seropositivity is more frequent in coronary artery disease requiring surgery. In HIV patients, stronger T-cell responses to CMV (considered an indication of exposure to CMV rather protection) associate with

In this book we also consider CMV in the developing world where it has been compre‐ hensively ignored. However this must change as ART is rolled out for HIV patients worldwide and transplantation becomes feasible in South America, Africa and Asia.

Finally we need to understand how infection of so few cells with CMV has such dra‐ matic effects. Perhaps CMV has effects even broader than we realise. CMV is a highly

We need to understand the dynamic role of CMV in these populations.

which re-awakens in response to stresses or inflammation.

### Preface

CMV is a prevalent viral pathogen that rarely causes recognisable symptoms in immu‐ nocompetent hosts. CMV is the strangest virus. It has 200 genes but needs only 30 to replicate. It infects everyone but few people have heard of it. We carry it all our lives but it can rarely be isolated from blood. Up to 23% of CD8 T-cells can be specific for CMV in older healthy adults, despite undetectable viral load. CMV-specific T-cells have a "worn out" phenotype, likely gained from lifelong surveillance of persistent CMV which re-awakens in response to stresses or inflammation.

CMV causes distinct patterns of disease in different classes of immunodeficient pa‐ tients. This includes retinitis in HIV patients, pneumonitis after transplantation and deafness in babies. This book includes chapters focussing on different groups of indi‐ viduals at risk.

CMV reactivation (in previously seropositive recipients) or primary infection (in any recipient) is the most common infectious complication in renal transplantation. In the 1970's one in three recipients experienced pathologies associated with CMV. In most countries, prophylaxis (valganciclovir) is routinely administered for 12-26 weeks after transplantation. However CMV remains a significant cause of graft loss and the opti‐ mal duration of prophylaxis is unclear. In the longer term, CMV reactivations are im‐ plicated in deterioration in renal function, cardiovascular disease and perhaps type II diabetes.

CMV has also been anecdotally linked with cardiovascular disease and in vitro studies show increased expression of adhesion molecules in endothelial tissues infected with CMV. One study linked T-cell responses to CMV with cardiovascular symptoms (athe‐ rosclerosis) in HIV patients. CMV-DNA has been found in tissues removed during sur‐ gery for abdominal aortic aneurysm and CMV seropositivity is more frequent in coronary artery disease requiring surgery. In HIV patients, stronger T-cell responses to CMV (considered an indication of exposure to CMV rather protection) associate with cardiovascular changes.

In this book we also consider CMV in the developing world where it has been compre‐ hensively ignored. However this must change as ART is rolled out for HIV patients worldwide and transplantation becomes feasible in South America, Africa and Asia. We need to understand the dynamic role of CMV in these populations.

Finally we need to understand how infection of so few cells with CMV has such dra‐ matic effects. Perhaps CMV has effects even broader than we realise. CMV is a highly

#### XII Preface

evolved virus. The last chapter discusses the need for novel therapeutics to reduce the impact of CMV infection in at risk groups.

> **Patricia Price** University of Western Australia, Australia

> > **Nandini Makwana** Pune University, India

**Chapter 1**

**Hearing Loss in Children with Congenital**

Sensorineural hearing loss (SNHL) is a common birth defect. The genetic origins of SNHL can be identified in half of the prelingual cases; in the others, SNHL is caused by environmental or unidentified genetic factors. The most common environmental cause of SNHL is congenital cytomegalovirus (CMV) infection. CMV is also the most common cause of intrauterine and congenital viral infection, affecting 0.5% to 2.5% of all live neonates [1]. While 90% of CMVinfected children are asymptomatic at birth, 10% of those exhibit clinically apparent sequelae at birth, including SNHL, mental retardation, motor disability, and microcephaly [1-4]. Recent studies have revealed that children with asymptomatic congenital CMV infection are at risk of late-onset SNHL and/or deterioration of SNHL during early childhood. These developments may not appear until months or even years following birth. The frequency of SNHL associated with asymptomatic congenital CMV infection reportedly ranges from 13% to 24% [5-9]. Although asymptomatic CMV infection is associated with a lower incidence of SNHL than symptomatic CMV infection, SNHL caused by congenital CMV often remains undiagnosed because maternal screening for CMV infection is not routinely conducted and the detection of

Hearing loss is detected in approximately 50% of children with symptomatic congenital CMV infection. In 66% of these patients, hearing loss will deteriorate [3, 11]. Children with symptomatic congenital CMV infection are easily identified at birth. In children with symptomatic infection, intrauterine growth retardation and petechiae have been associat‐ ed with the development of hearing loss [12]. SNHL is diagnosed in 7%–25% of children with asymptomatic congenital CMV infection. Rates of delayed-onset SNHL, progressive SNHL, and improvement of SNHL are reported to be 11%–18%, 23%–62%, and 23% –

and reproduction in any medium, provided the original work is properly cited.

© 2013 Iwasaki and Usami; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

SNHL during newborn hearing screening (NHS) tests is difficult [7, 10].

**Cytomegalovirus Infection**

Satoshi Iwasaki and Shin-ich Usami

http://dx.doi.org/10.5772/56160

**1. Introduction**

47%, respectively [5-9].

Additional information is available at the end of the chapter

**Samantha Brunt** University of Western Australia, Australia

### **Hearing Loss in Children with Congenital Cytomegalovirus Infection**

Satoshi Iwasaki and Shin-ich Usami

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56160

#### **1. Introduction**

evolved virus. The last chapter discusses the need for novel therapeutics to reduce the

**Patricia Price**

**Nandini Makwana** Pune University, India

**Samantha Brunt**

University of Western Australia, Australia

University of Western Australia, Australia

impact of CMV infection in at risk groups.

VIII Preface

Sensorineural hearing loss (SNHL) is a common birth defect. The genetic origins of SNHL can be identified in half of the prelingual cases; in the others, SNHL is caused by environmental or unidentified genetic factors. The most common environmental cause of SNHL is congenital cytomegalovirus (CMV) infection. CMV is also the most common cause of intrauterine and congenital viral infection, affecting 0.5% to 2.5% of all live neonates [1]. While 90% of CMVinfected children are asymptomatic at birth, 10% of those exhibit clinically apparent sequelae at birth, including SNHL, mental retardation, motor disability, and microcephaly [1-4]. Recent studies have revealed that children with asymptomatic congenital CMV infection are at risk of late-onset SNHL and/or deterioration of SNHL during early childhood. These developments may not appear until months or even years following birth. The frequency of SNHL associated with asymptomatic congenital CMV infection reportedly ranges from 13% to 24% [5-9]. Although asymptomatic CMV infection is associated with a lower incidence of SNHL than symptomatic CMV infection, SNHL caused by congenital CMV often remains undiagnosed because maternal screening for CMV infection is not routinely conducted and the detection of SNHL during newborn hearing screening (NHS) tests is difficult [7, 10].

Hearing loss is detected in approximately 50% of children with symptomatic congenital CMV infection. In 66% of these patients, hearing loss will deteriorate [3, 11]. Children with symptomatic congenital CMV infection are easily identified at birth. In children with symptomatic infection, intrauterine growth retardation and petechiae have been associat‐ ed with the development of hearing loss [12]. SNHL is diagnosed in 7%–25% of children with asymptomatic congenital CMV infection. Rates of delayed-onset SNHL, progressive SNHL, and improvement of SNHL are reported to be 11%–18%, 23%–62%, and 23% – 47%, respectively [5-9].

© 2013 Iwasaki and Usami; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Thus, the incidence of asymptomatic CMV infection and resulting SNHL may be higher, making it the leading cause of SNHL in children. Treatment of children with congenital CMV infection can prevent late-onset SNHL and/or deterioration of SNHL during early childhood. Cochlear implantation is also effective for the development of speech perception and auditory skills for deaf children with congenital CMV infection. Therefore, early identification of congenital CMV infection is very important.

improvement of hearing loss from the initial abnormal ABR (profound unilateral loss and

ABR: unilateral moderate hearing loss

Hearing Loss in Children with Congenital Cytomegalovirus Infection

http://dx.doi.org/10.5772/56160

3

ABR: unilateral profound hearing loss

ABR: normal

**Table 1.** Initial symptoms and audiologic results during the first 6 months of life in 18 children with congenital CMV

**Age Hearing loss Characteristic**

Delayed-onset Progressive

Fluctuating Improvement

Improvement

Delayed-onset Progressive

Cochlear implantation

Normal speech development

Normal speech development

Cochlear implantation

(39 mo)

Fluctuating Normal speech development

(29 mo)

**Initial hearing loss Results of follow-up audiologic examination Outcome**

profound

moderate

profound

**Table 2.** Results of longitudinal audiologic examinations in 5 children with SNHL caused by asymptomatic CMV

mild

26 mo Bilateral

Case 4 Bilateral severe 17 mo Normal Fluctuating

SNHL: sensorineural hearing loss. This table is cited from reference [11].

36 mo Bilateral

**Symptoms Audiologic examinations**

Case 1 Not found Automatic ABR: unilateral REFER

Case 6-16 Not found ABR: normal

ABR: auditory brainstem response. This table is cited from reference [11].

Ventricular dilatation

Ventricular dilatation Heart anomaly

Case 2 Unilateral moderate 53 mo Unilateral

Case 3 Unilateral profound 53 mo Unilateral

Case 2 Not found ABR: unilateral moderate hearing loss Case 3 Not found ABR: unilateral profound hearing loss Case 4 Not found ABR: bilateral severe hearing loss Case 5 Not found Automatic ABR: bilateral PASS

profound bilateral loss, respectively).

Case 17 Microcephaly

Case 18 Microcephaly

Case 1 Unilateral moderate (Unilateral REFER)

Case 5 Normal

infection.

(Bilateral PASS)

infection.

### **2. Epidemiology of hearing-impaired children with congenital CMV infection**

Of the 12,599 pregnant women included in a prospective study [13] conducted where from June 1996 to December 2003, maternal ages were as follows: <20 years, 1.6%; 20–24 years, 14.7%; 25–29 years, 41.4%; 30–34 years, 28.6%; 35–39 years, 7.9%; and >40 years, 0.8%. The annual seropositivity rate decreased over the 8-year study period, particularly during the last 4 years. The seropositivity rate of CMV immunoglobulin G (IgG) antibody was 75.3% in the sample as a whole. The seronegativity rate was 23.6%, and the percentage of cases borderline positive for IgG antibody was 1%. The seronegativity rate of CMV IgM antibody was 94.8% in the sample as a whole. The seropositivity rate was 2.2%, and 3% of cases were borderline positive for CMV IgM antibody. During the study period, in the cases positive for IgM antibody (n = 146), borderline positive for IgM antibody (n = 73), and borderline positive for IgG antibody (n = 14) and in cases with seroconversion of IgG antibody (n = 3), neonatal urine was analyzed for CMV DNA. Seroconversion of CMV IgG antibody occurred in 0.32% of the 929 cases negative for IgG antibody. Congenital CMV infection was identified in 18 infants by polymer‐ ase chain reaction (PCR) analysis of urine. Follow-up was conducted in these cases.

The symptoms at birth and sequelae observed during the first 6 months of life in the 18 children with congenital CMV infection are shown in Table 1. Among these infants, 2 children (11.1%) were symptomatic and the remaining 16 (88.9%) were asymptomatic. In this study, newborn infants were considered symptomatic if central nervous system involvement such as micro‐ cephaly or ventricular dilatation was detected. SNHL was detected in 1 child (50%) with symptomatic infection and in 4 children (25%) with asymptomatic infection. Profound unilateral SNHL had developed in the child with symptomatic infection. In the 4 children with asymptomatic infection, the severity of SNHL varied from mild unilateral loss to profound bilateral loss. Of the 4 children, unilateral SNHL was identified in 3 (75%). Mild unilateral SNHL occurred in 2 children (66.7%), and profound unilateral loss occurred in 1 child (33.3%). Profound bilateral SNHL occurred in 1 child with asymptomatic infection. The unilateral hearing loss in case 1 was detected by a neonatal automatic auditory brainstem response (ABR) screener. SNHL in the other 3 children was detected by conventional ABR. Table 2 shows a summary of the findings from longitudinal audiological evaluations in the 5 children with asymptomatic congenital CMV infection. On subsequent audiological testing, delayed-onset SNHL was detected in 2 children who had passed the newborn hearing screening (NHS) test (1 bilateral and 1 unilateral). Two cases (40%) had progressive hearing loss and 2 (40%) had improvement of hearing loss from the initial abnormal ABR (profound unilateral loss and profound bilateral loss, respectively).

ABR: auditory brainstem response. This table is cited from reference [11].

Thus, the incidence of asymptomatic CMV infection and resulting SNHL may be higher, making it the leading cause of SNHL in children. Treatment of children with congenital CMV infection can prevent late-onset SNHL and/or deterioration of SNHL during early childhood. Cochlear implantation is also effective for the development of speech perception and auditory skills for deaf children with congenital CMV infection. Therefore, early identification of

**2. Epidemiology of hearing-impaired children with congenital CMV**

ase chain reaction (PCR) analysis of urine. Follow-up was conducted in these cases.

The symptoms at birth and sequelae observed during the first 6 months of life in the 18 children with congenital CMV infection are shown in Table 1. Among these infants, 2 children (11.1%) were symptomatic and the remaining 16 (88.9%) were asymptomatic. In this study, newborn infants were considered symptomatic if central nervous system involvement such as micro‐ cephaly or ventricular dilatation was detected. SNHL was detected in 1 child (50%) with symptomatic infection and in 4 children (25%) with asymptomatic infection. Profound unilateral SNHL had developed in the child with symptomatic infection. In the 4 children with asymptomatic infection, the severity of SNHL varied from mild unilateral loss to profound bilateral loss. Of the 4 children, unilateral SNHL was identified in 3 (75%). Mild unilateral SNHL occurred in 2 children (66.7%), and profound unilateral loss occurred in 1 child (33.3%). Profound bilateral SNHL occurred in 1 child with asymptomatic infection. The unilateral hearing loss in case 1 was detected by a neonatal automatic auditory brainstem response (ABR) screener. SNHL in the other 3 children was detected by conventional ABR. Table 2 shows a summary of the findings from longitudinal audiological evaluations in the 5 children with asymptomatic congenital CMV infection. On subsequent audiological testing, delayed-onset SNHL was detected in 2 children who had passed the newborn hearing screening (NHS) test (1 bilateral and 1 unilateral). Two cases (40%) had progressive hearing loss and 2 (40%) had

Of the 12,599 pregnant women included in a prospective study [13] conducted where from June 1996 to December 2003, maternal ages were as follows: <20 years, 1.6%; 20–24 years, 14.7%; 25–29 years, 41.4%; 30–34 years, 28.6%; 35–39 years, 7.9%; and >40 years, 0.8%. The annual seropositivity rate decreased over the 8-year study period, particularly during the last 4 years. The seropositivity rate of CMV immunoglobulin G (IgG) antibody was 75.3% in the sample as a whole. The seronegativity rate was 23.6%, and the percentage of cases borderline positive for IgG antibody was 1%. The seronegativity rate of CMV IgM antibody was 94.8% in the sample as a whole. The seropositivity rate was 2.2%, and 3% of cases were borderline positive for CMV IgM antibody. During the study period, in the cases positive for IgM antibody (n = 146), borderline positive for IgM antibody (n = 73), and borderline positive for IgG antibody (n = 14) and in cases with seroconversion of IgG antibody (n = 3), neonatal urine was analyzed for CMV DNA. Seroconversion of CMV IgG antibody occurred in 0.32% of the 929 cases negative for IgG antibody. Congenital CMV infection was identified in 18 infants by polymer‐

congenital CMV infection is very important.

2 Manifestations of Cytomegalovirus Infection

**infection**

**Table 1.** Initial symptoms and audiologic results during the first 6 months of life in 18 children with congenital CMV infection.

SNHL: sensorineural hearing loss. This table is cited from reference [11].

**Table 2.** Results of longitudinal audiologic examinations in 5 children with SNHL caused by asymptomatic CMV infection.

In this prospective study, the rates of delayed-onset SNHL, progressive SNHL, and improve‐ ment of SNHL were 12%, 40%, and 40%, respectively. Although a low rate of fetal CMV infection was observed, the results of the present study regarding the rate of SNHL are in accordance with the findings of those previous studies. The prevalence of congenital CMV infection is affected by the socioeconomic and geographic differences, but it seems to be no differences on characteristics of hearing loss induced by congenital CMV infection.

was defined as a decrease in hearing of ≥10 dB at 1 or more frequencies. Fluctuating hearing loss was defined as a decrease in hearing of >10 dB followed by an improvement of >10 dB at 1 or more frequencies. To analyze congenital CMV infection, CMV DNA quantitative PCR (qPCR) analysis was performed. Prior to qPCR analysis, total DNA, including genomic DNA and CMV DNA, was extracted from preserved dried umbilical cords. The results of this study revealed that in 9.0% (12/134) of children, SNHL could be attributed to congenital CMV infection. CMV DNA from preserved umbilical cords was detected in 8.7% (4/46) of children with bilateral SNHL and 9.1% (8/88) of those with unilateral SNHL. Congenital CMV infection caused bilateral severe-to-profound SNHL, bilateral mild-to-moderate SNHL, unilateral severe-to-profound SNHL, and unilateral mild-to-moderate SNHL in 14.3% (4/28), 0% (0/18), 9.6% (7/73), and 6.7% (1/15) of hearing-impaired children, respectively. This study also revealed that both congenital and late-onset SNHL could be caused by congenital CMV

**Hearing loss Gender Hearing level Severe-profound HL Mild-moderate HL**

(75.4%)

28 (20.9%)

72 (54.5%)

Table 4 shows the clinical characteristics of 12 children in whom CMV DNA was identified. Of these 12 children, bilateral SNHL was detected in 4 and unilateral SNHL in 8. All 4 children with bilateral SNHL had late-onset profound SNHL. Hearing fluctuation and PASS at the NHS test were confirmed in 3 children (75%). Of the 8 children with unilateral SNHL, detectable defects were confirmed in 2 children. Hearing fluctuation was detected in only 1 child (12.5%).

Retrospective diagnosis of congenital CMV infection is important to improve our understand‐ ing of the etiology of pediatric SNHL. In previous reports (Table 5), the frequency of congenital CMV infection in children with bilateral SNHL has varied from 3% to 36% because of variations in parameters (number of subjects, severity of SNHL) and methods [CMV IgM testing, DNA urinalysis, DNA from dried blood spots (DBS) in Guthrie cards] [19-24]. In 2 Japanese studies based on the retrospective diagnostic method of analysis of preserved dried umbilical cords, congenital CMV infection was detected in 10%–12% of children with bilateral SNHL [25, 26];

M: male, F: female. R: right, L: left. B: better ear, W: worse ear. This table is cited from reference [16].

No inner ear anomaly was found in any of the 8 children with unilateral SNHL.

**Table 3.** Summary of characteristics of children with bilateral or unilateral hearing loss.

M: 70, F: 64 101

71.7 dB [L]

13.6 dB (B)

M: 31, F: 15 71.8 dB [R]

M: 39, F: 49 89.5 dB (W)

HL: hearing loss. Diagnostic age: age diagnosed as hearing loss.

**(n) (dB) n Diagnostic age n Diagnostic age**

34.4±34.7 mo 33

16.6±19.9 mo 18

41.2±36.6 mo 15

(24.6%)

Hearing Loss in Children with Congenital Cytomegalovirus Infection

http://dx.doi.org/10.5772/56160

5

(13.4%)

(11.2%)

48.8±38.7 mo

11.1±39.1 mo

40.3±36.8 mo

infection.

Total (N=134)

Bilateral HL (N=46)

Unilateral HL (N=88)

Because they develop later, both delayed-onset and progressive hearing loss frequently remain undiagnosed during universal newborn hearing screening (NHS) test [7, 10]. The 1994 Joint Committee on Infant Hearing [14] pointed out that additional hearing evaluations after universal NHS are required to detect delayed-onset hearing loss. Combined neonatal screening for CMV infection and repeated auditory evaluation should be considered, particularly for children with asymptomatic congenital CMV infection. Counseling of pregnant women on prevention of CMV infection is also important.

#### **2.1. Retrospective study of congenital CMV infection**

Hearing loss in children with congenital CMV infection often presents at birth; however, in many instances, it may develop after months or even years. One report stated that children with normal hearing at 6 months of age develop hearing loss at a rate of approximately 1% per year; the cumulative risk of late-onset hearing loss is substantial (6.9%) in a population of in‐ fants with asymptomatic congenital CMV infection [15]. Speech is often delayed in children with bilateral hearing loss. For cases of severe bilateral SNHL, Ogawa et al. [16] reported that congenital CMV infection could be diagnosed through the detection of CMV DNA in the dried umbilical cord. In addition, genetic defects (particularly those related to *GJB2*) were identified in 15% and 30% of the children, respectively. However, the etiology of pediatric SNHL, in‐ cluding mild to moderate and unilateral SNHL, remains uncertain. In a study of congenital CMV infection retrospectively diagnosed by the detection of CMV DNA extracted from dried umbilical cord specimens, the prevalence of CMV in children with unilateral or bilateral SNHL was investigated. In many of these cases, SNHL developed several months or even years after birth.

In total, 134 patients (70 males and 64 females) with bilateral (n = 46; 34.3%) or unilateral (n = 88; 65.7%) SNHL were evaluated. These cases were referred to the Department of Otolaryng‐ ology, Shinshu University School of Medicine from May 2008 to September 2009 (Table 3) [17]. The age of these children ranged from 1 month to 138 months (mean age: 37.7 ± 36.2 months). In children with bilateral SNHL, both genetic testing for deafness and CMV DNA analysis were performed. For children with unilateral SNHL, CMV DNA analysis and genetic testing for gene mutations of *GJB2, Mitochondrial1555* were performed. Objective audiometric evaluation was performed for each patient using ABR and auditory steady-state evoked response systems (MASTER 580-NAVPRO; NIHON KOHDEN Co., Ltd, Tokyo, Japan). Behavioral audiological tests and/or pure-tone audiometry were also performed. Hearing levels were classified into 2 categories on the basis of the severity of hearing loss in the worse ear as severe (>70 dB) to profound (>90 dB) and mild (20–40 dB) to moderate (41–70 dB). Followup hearing assessments were performed at intervals of 6–12 months. Progressive hearing loss was defined as a decrease in hearing of ≥10 dB at 1 or more frequencies. Fluctuating hearing loss was defined as a decrease in hearing of >10 dB followed by an improvement of >10 dB at 1 or more frequencies. To analyze congenital CMV infection, CMV DNA quantitative PCR (qPCR) analysis was performed. Prior to qPCR analysis, total DNA, including genomic DNA and CMV DNA, was extracted from preserved dried umbilical cords. The results of this study revealed that in 9.0% (12/134) of children, SNHL could be attributed to congenital CMV infection. CMV DNA from preserved umbilical cords was detected in 8.7% (4/46) of children with bilateral SNHL and 9.1% (8/88) of those with unilateral SNHL. Congenital CMV infection caused bilateral severe-to-profound SNHL, bilateral mild-to-moderate SNHL, unilateral severe-to-profound SNHL, and unilateral mild-to-moderate SNHL in 14.3% (4/28), 0% (0/18), 9.6% (7/73), and 6.7% (1/15) of hearing-impaired children, respectively. This study also revealed that both congenital and late-onset SNHL could be caused by congenital CMV infection.

HL: hearing loss. Diagnostic age: age diagnosed as hearing loss.

In this prospective study, the rates of delayed-onset SNHL, progressive SNHL, and improve‐ ment of SNHL were 12%, 40%, and 40%, respectively. Although a low rate of fetal CMV infection was observed, the results of the present study regarding the rate of SNHL are in accordance with the findings of those previous studies. The prevalence of congenital CMV infection is affected by the socioeconomic and geographic differences, but it seems to be no

Because they develop later, both delayed-onset and progressive hearing loss frequently remain undiagnosed during universal newborn hearing screening (NHS) test [7, 10]. The 1994 Joint Committee on Infant Hearing [14] pointed out that additional hearing evaluations after universal NHS are required to detect delayed-onset hearing loss. Combined neonatal screening for CMV infection and repeated auditory evaluation should be considered, particularly for children with asymptomatic congenital CMV infection. Counseling of pregnant women on

Hearing loss in children with congenital CMV infection often presents at birth; however, in many instances, it may develop after months or even years. One report stated that children with normal hearing at 6 months of age develop hearing loss at a rate of approximately 1% per year; the cumulative risk of late-onset hearing loss is substantial (6.9%) in a population of in‐ fants with asymptomatic congenital CMV infection [15]. Speech is often delayed in children with bilateral hearing loss. For cases of severe bilateral SNHL, Ogawa et al. [16] reported that congenital CMV infection could be diagnosed through the detection of CMV DNA in the dried umbilical cord. In addition, genetic defects (particularly those related to *GJB2*) were identified in 15% and 30% of the children, respectively. However, the etiology of pediatric SNHL, in‐ cluding mild to moderate and unilateral SNHL, remains uncertain. In a study of congenital CMV infection retrospectively diagnosed by the detection of CMV DNA extracted from dried umbilical cord specimens, the prevalence of CMV in children with unilateral or bilateral SNHL was investigated. In many of these cases, SNHL developed several months or even

In total, 134 patients (70 males and 64 females) with bilateral (n = 46; 34.3%) or unilateral (n = 88; 65.7%) SNHL were evaluated. These cases were referred to the Department of Otolaryng‐ ology, Shinshu University School of Medicine from May 2008 to September 2009 (Table 3) [17]. The age of these children ranged from 1 month to 138 months (mean age: 37.7 ± 36.2 months). In children with bilateral SNHL, both genetic testing for deafness and CMV DNA analysis were performed. For children with unilateral SNHL, CMV DNA analysis and genetic testing for gene mutations of *GJB2, Mitochondrial1555* were performed. Objective audiometric evaluation was performed for each patient using ABR and auditory steady-state evoked response systems (MASTER 580-NAVPRO; NIHON KOHDEN Co., Ltd, Tokyo, Japan). Behavioral audiological tests and/or pure-tone audiometry were also performed. Hearing levels were classified into 2 categories on the basis of the severity of hearing loss in the worse ear as severe (>70 dB) to profound (>90 dB) and mild (20–40 dB) to moderate (41–70 dB). Followup hearing assessments were performed at intervals of 6–12 months. Progressive hearing loss

differences on characteristics of hearing loss induced by congenital CMV infection.

prevention of CMV infection is also important.

4 Manifestations of Cytomegalovirus Infection

years after birth.

**2.1. Retrospective study of congenital CMV infection**

M: male, F: female. R: right, L: left. B: better ear, W: worse ear. This table is cited from reference [16].

**Table 3.** Summary of characteristics of children with bilateral or unilateral hearing loss.

Table 4 shows the clinical characteristics of 12 children in whom CMV DNA was identified. Of these 12 children, bilateral SNHL was detected in 4 and unilateral SNHL in 8. All 4 children with bilateral SNHL had late-onset profound SNHL. Hearing fluctuation and PASS at the NHS test were confirmed in 3 children (75%). Of the 8 children with unilateral SNHL, detectable defects were confirmed in 2 children. Hearing fluctuation was detected in only 1 child (12.5%). No inner ear anomaly was found in any of the 8 children with unilateral SNHL.

Retrospective diagnosis of congenital CMV infection is important to improve our understand‐ ing of the etiology of pediatric SNHL. In previous reports (Table 5), the frequency of congenital CMV infection in children with bilateral SNHL has varied from 3% to 36% because of variations in parameters (number of subjects, severity of SNHL) and methods [CMV IgM testing, DNA urinalysis, DNA from dried blood spots (DBS) in Guthrie cards] [19-24]. In 2 Japanese studies based on the retrospective diagnostic method of analysis of preserved dried umbilical cords, congenital CMV infection was detected in 10%–12% of children with bilateral SNHL [25, 26]; however, these studies included few subjects (10–26 cases). In children with unilateral SNHL, CMV DNA from preserved umbilical cords was detected in 9.1% (8/88). The frequency of congenital CMV infection was similar in children with unilateral and bilateral SNHL. It has been speculated that approximately 10% of SNHL in children is caused by congenital CMV infection. Few reports have examined the frequency of congenital CMV infection using retrospective diagnostic methods in children with unilateral SNHL. However, using the CMV DNA detection method, 25% (1/4) [16] and 19% (8/42) [19] of children with unilateral SNHL were diagnosed with congenital CMV infection.

and CMV DNA positivity were detected in 32.1% (9/28) and 14.3% (4/28) of patients, respec‐ tively [17]. The diagnostic rate has been concluded to be 46.4% (13/28). If analysis of CMV DNA from preserved dried umbilical cords could be combined with genetic testing for deafness, approximately 50% of cases of bilateral severe-to-profound hearing loss in children could be

NR: not reported. NHS: newborn hearing screening. DBS: dried blood spot. UC: umbilical cord. qPCR: quantitative PCR.

HL: hearing level. SNHL: sensorineural hearing loss. Re.: republic. This table is cited from reference [16].

**Table 5.** List of previous reports on children with congenital CMV nfection.

**CMV positive rate Diagnostic**

Hearing Loss in Children with Congenital Cytomegalovirus Infection

**Total Bilateral Unilateral**

Barbi et al. [19] 2003 > 40 dBHL 9/79 (11.4%) 1/37 (2.7%) 8/42 (19%) DBS, qPCR Italy Ogawa et al. [16] 2007 > 20dB, nonsyndromic SNHL 10/67 (10.5%) 9/63 (14.3%) 1/4 (25%) US, PCR Japan Samileh et al. [21] 2008 > 40 dBHL 33/95 (34.7%) NR/75 NR/20 Cerologic test Iran Stehel et al. [22] 2008 NHS refer 16/256 (6%) 16/256 (6%) NR DNA from urine USA Walter et al. [43] 2008 unexplained SNHL 8/35 (22.9%) NR NR DSS, qPCR UK Mizuno et al. [44] 2008 only bilateral 3/45 (6.7%) 3/45 (6.7%) 0 UC, qPCR Japan

Jakubikova et al. [20] 2009 > 60 dBHL, NHS refer 4/71 (5.6%) 4/55 (7.3%) 0/16 (0%) Cerologic test Slovak Re. Boudewyns et al. [45] 2009 NHS refer, > 20 dB 4/55 (7.3%) NR NR DBS, qPCR Belgium

Choi et al. [18] 2009 NHS refer 13/479 (2.7%) 13/479 (2.7%) NR DBS, qPCR USA Tagawa et al. [26] 2009 > 70 dB, deaf school children 3/26 (11.5%) 3/26 (11.5%) 0 (0%) UC, qPCR Japan Kimani et al. [46] 2010 NHS refer 11/109 (10.1%) 8/92 (8.8%) 3/17 (17.6%) DBS, qPCR USA Adachi et al. [47] 2010 NHS refer, >35dB, bilateral 13/77 (17%) 13/77 (17%) 0 US, qPCR Japan

**methods**

http://dx.doi.org/10.5772/56160

**Country**

7

Congenital CMV infection is also often diagnosed by detecting CMV DNA in urine within the first 2 weeks of life and serological testing for CMV-specific IgM antibody from mother and child [28]. In recent years, the detection of CMV DNA by retrospective methods has been more valuable not only in diagnosing congenital CMV infection during later stages of life but also in identifying children at highest risk of late-onset and progressive SNHL. Some reports have stated that DBS stored on Guthrie cards has been used for the retrospective diagnosis of congenital CMV infections [18, 29]. Similarly, preserved umbilical cords have been recently used in Japan [25, 26, 30]. The sensitivity varies widely depending on the DNA extraction method in the DBS case. Some investigators have reported sensitivities of 71%–100% and specificities of 99%–100% [19, 29]. In this study, the qPCR method and preserved umbilical

cords were used because they were useful for more accurate detection of CMV DNA.

detected.

**Reference Year Subjects**

F: female, M: male. Mo: month. HL: hearing loss. R: right, L: left. NHS: newborn hearing screening. Diagnostic age: age diagnosed as hearing loss. This table is cited from reference [16].

**Table 4.** Clinical data of CMV DNA-positive children

#### **2.2. Genetic hearing loss and congenital CMV infection**

Genetic testing for deafness has become valuable for precise diagnosis of hearing loss. The most frequently implicated gene in nonsyndromic hearing loss is *GJB2*, the most prevalent gene responsible for congenital hearing loss worldwide. *GJB2, SLC26A4, CDH23*, and mito‐ chondrial 12s ribosomal RNA (rRNA) are the other major genes that cause hearing loss in Japan. One study stated that genetic mutations were responsible for deafness in 40%–45% of children with congenital hearing loss [27]. In our study [17], 10 gene mutations associated with deafness (*GJB2*, n = 7; *SLC26A4*, n = 3) were identified in 21.7% (10/46) of children with bilateral SNHL. In children with bilateral severe-to-profound SNHL, gene mutations causing deafness

HL: hearing level. SNHL: sensorineural hearing loss. Re.: republic. This table is cited from reference [16].

**Table 5.** List of previous reports on children with congenital CMV nfection.

however, these studies included few subjects (10–26 cases). In children with unilateral SNHL, CMV DNA from preserved umbilical cords was detected in 9.1% (8/88). The frequency of congenital CMV infection was similar in children with unilateral and bilateral SNHL. It has been speculated that approximately 10% of SNHL in children is caused by congenital CMV infection. Few reports have examined the frequency of congenital CMV infection using retrospective diagnostic methods in children with unilateral SNHL. However, using the CMV DNA detection method, 25% (1/4) [16] and 19% (8/42) [19] of children with unilateral SNHL

**Unilateral Severity Average HL**

**(R/L: dB)**

**Onset NHS**

were diagnosed with congenital CMV infection.

**Bilateral**/

 F 60 mo Bilateral Profound 87.5/108.8 Late Pass F 52 mo Bilateral Profound 87.5/110.0 Late Pass M 50 mo Bilateral Profound 100.0/100.0 Late Pass M 62 mo Bilateral Profound 110.0/46.3 Likely late −

5 M 6 mo Unilateral Profound 32.5/103.8 Congenital Refer (L)

 M 65 mo Unilateral Profound 107.5/17.5 Unknown − M 50 mo Unilateral Profound 6.3/100.0 Unknown − F 98 mo Unilateral Profound 110.0/15.0 Unknown − F 55 mo Unilateral Profound 15.0/92.5 Late Pass F 2 mo Unilateral Profound 90.0/18.3 Congenital Refer (R)

11 M 80 mo Unilateral Severe 13.3/70.0 Unknown − 12 F 44 mo Unilateral Moderate 15.0/58.3 Late Pass

Genetic testing for deafness has become valuable for precise diagnosis of hearing loss. The most frequently implicated gene in nonsyndromic hearing loss is *GJB2*, the most prevalent gene responsible for congenital hearing loss worldwide. *GJB2, SLC26A4, CDH23*, and mito‐ chondrial 12s ribosomal RNA (rRNA) are the other major genes that cause hearing loss in Japan. One study stated that genetic mutations were responsible for deafness in 40%–45% of children with congenital hearing loss [27]. In our study [17], 10 gene mutations associated with deafness (*GJB2*, n = 7; *SLC26A4*, n = 3) were identified in 21.7% (10/46) of children with bilateral SNHL. In children with bilateral severe-to-profound SNHL, gene mutations causing deafness

F: female, M: male. Mo: month. HL: hearing loss. R: right, L: left. NHS: newborn hearing screening.

Diagnostic age: age diagnosed as hearing loss. This table is cited from reference [16].

**2.2. Genetic hearing loss and congenital CMV infection**

**Table 4.** Clinical data of CMV DNA-positive children

**Case no. Sex Diagnostic age**

6 Manifestations of Cytomegalovirus Infection

and CMV DNA positivity were detected in 32.1% (9/28) and 14.3% (4/28) of patients, respec‐ tively [17]. The diagnostic rate has been concluded to be 46.4% (13/28). If analysis of CMV DNA from preserved dried umbilical cords could be combined with genetic testing for deafness, approximately 50% of cases of bilateral severe-to-profound hearing loss in children could be detected.

Congenital CMV infection is also often diagnosed by detecting CMV DNA in urine within the first 2 weeks of life and serological testing for CMV-specific IgM antibody from mother and child [28]. In recent years, the detection of CMV DNA by retrospective methods has been more valuable not only in diagnosing congenital CMV infection during later stages of life but also in identifying children at highest risk of late-onset and progressive SNHL. Some reports have stated that DBS stored on Guthrie cards has been used for the retrospective diagnosis of congenital CMV infections [18, 29]. Similarly, preserved umbilical cords have been recently used in Japan [25, 26, 30]. The sensitivity varies widely depending on the DNA extraction method in the DBS case. Some investigators have reported sensitivities of 71%–100% and specificities of 99%–100% [19, 29]. In this study, the qPCR method and preserved umbilical cords were used because they were useful for more accurate detection of CMV DNA.

#### **3. Diagnosis of congenital CMV infection**

#### **3.1. Detection methods**

The gold standard for diagnosis of congenital CMV infection is isolation of the virus from urine or saliva in the first 2 weeks of life. However, asymptomatic congenital CMV infection in children who develop SNHL after the first 2 weeks following birth cannot be diagnosed on the basis of viral isolation from urine or saliva. Detection of CMV DNA in infant blood or the umbilical cord using PCR assays is a more feasible method for identifying children with lateonset SNHL. The method involves analysis of blood stored as DBS on Guthrie cards. In Japanese culture, the dried umbilical cord is generally stored at home as a memento of the birth. These specimens are suitable for retrospective diagnosis of congenital CMV infection. The sensitivity varied widely depending on the DNA extraction method from DBS on Guthrie cards. Some investigators reported sensitivities of 71-100% and specificities of 99-100% [19, 29]. The qPCR method and dried umbilical cord could be useful for more precise detection of CMV DNA.

formed using the following primers and TaqMan® probe: GJB2-2F: 5′-ACGTCCACGT‐ TAGGATGAGG-3′, GJB2-2: 5′-GTATGTGGCGCTTCTCTCGT-3′, and GJB2-2 TaqMan probe: 5′-FAM- AACCTGTGCACCACAGCGCC -TAMRA-3′. The initial preheating steps were performed for 2 min at 50°C and 10 min at 95°C. Following this, qPCR was per‐ formed for 43 cycles of 15 s at 95°C and 60 s at 60°C. After qPCR analysis, relative CMV concentrations in each sample were evaluated as ΔCt (delta cycle threshold), which was calculated by determining the threshold cycle of CMV qPCR minus that of *GJB2* qPCR.

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The invader assay described by Abe [32] was used for genetic testing for deafness.

**4.1. Cochlear implantation in children deafened by symptomatic CMV infection**

**4.2. Cochlear implantation in children deafened by asymptomatic CMV infection**

The effectiveness of cochlear implantation in children deafened as a result of symptomatic congenital CMV infection has been evaluated by various groups, but there are only limited outcome data for deaf children with asymptomatic CMV infection. Children with asympto‐ matic congenital CMV infection have a better prognosis than symptomatic children, but it is difficult to evaluate the SNHL because children with asymptomatic congenital CMV infection

Cochlear implantation for the correction of congenital deafness is an effective way to ensure the development of speech recognition. Cochlear implantation in children deafened by symptomatic CMV infection has been reported [33, 34]. The prognosis of children with symptomatic CMV infection is worse than that of those with asymptomatic CMV infection with regard to cognitive and neurological development. It has been suggested that cochlear implantation should be contraindicated for infants with symptomatic CMV infection and deafness because they are less likely to develop spoken language [35]. In contrast, other reports [33, 34] have suggested that cochlear implantation may improve quality of life, even if progress is slower or lesser than that expected in congenitally deaf children not infected with CMV. Pyman et al. [35] suggested that the prognosis in terms of linguistic outcome after cochlear implantation is poorer for CMV-infected deaf children than for other congenitally deaf children because of coexisting central disorders. Wide variation in speech perception and intelligibility after cochlear implantation has also been reported in children deafened by symptomatic CMV infection [33]. In that report, poor development in these areas was observed in 50% of children with symptomatic CMV infection, whereas development similar to that in congenitally deaf children not infected with CMV was evident in 31% of children and devel‐ opment better than that in noninfected congenitally deaf children was evident in 19% of children. In addition, a recent study has shown that deafness caused by symptomatic congen‐ ital CMV infection associated with motor and cognitive delays is not a contraindication for cochlear implantation. Early diagnosis of hearing loss and subsequent cochlear implantation

**4. Treatment for hearing loss induced by**

is important for successful speech perception [34].

**congenital CMV infection**

#### **3.2. Serological method**

Diagnosis of symptomatic CMV infection is easier in children who display cognitive or neuromuscular abnormalities than in asymptomatic children with CMV infection. Without neonatal viral screening, the prevalence of SNHL caused by asymptomatic CMV infection remains undetermined. To diagnose primary CMV infection, a serological method has been used [31]. Pregnant women who test positive for CMV IgG seroconversion or CMV IgM antibody may transmit the virus to the fetus. Production of IgM antibody persists for 6–9 months [28]; therefore, a CMV IgM-positive result alone does not accurately predict the risk of fetal infection.

#### **3.3. Detection of CMV DNA from umbilical cord**

For the detection of congenital CMV infection, CMV DNA qPCR analysis was performed. Prior to qPCR analysis, total DNA, including genomic DNA and CMV DNA, was extract‐ ed from preserved dried umbilical cords. The procedure is as follows. Each 5-mm tissue section was incubated in a lysis buffer containing proteinase K and incubated overnight at 56°C. Total DNA was extracted using the DNeasy® Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. The total amount of DNA was measured using the Qubit® Fluorometer with Quant-iT™ dsDNA BR Assay Kit (Life technologies-Invitrogen, Carlsbad, CA, USA). Total DNA (10 pg) was analyzed using the Step One Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and TaqMan® Universal Master Mix II (Applied Biosystems). The qPCR primers and TaqMan® probe used for CMV DNA qPCR analysis were as follows: US14-1F: 5′- ACGTCCACGTTAGGATGAGG-3′, US14-1R: 5′-GTATGTGGCGCTTCTCTCGT-3′, and US14-1 TaqMan probe: 5′-FAM- AACCTGTGCACCACAGCGCC -TAMRA-3′. To quantify the input DNA amount in each sample, qPCR of each genomic region was also per‐ formed using the following primers and TaqMan® probe: GJB2-2F: 5′-ACGTCCACGT‐ TAGGATGAGG-3′, GJB2-2: 5′-GTATGTGGCGCTTCTCTCGT-3′, and GJB2-2 TaqMan probe: 5′-FAM- AACCTGTGCACCACAGCGCC -TAMRA-3′. The initial preheating steps were performed for 2 min at 50°C and 10 min at 95°C. Following this, qPCR was per‐ formed for 43 cycles of 15 s at 95°C and 60 s at 60°C. After qPCR analysis, relative CMV concentrations in each sample were evaluated as ΔCt (delta cycle threshold), which was calculated by determining the threshold cycle of CMV qPCR minus that of *GJB2* qPCR. The invader assay described by Abe [32] was used for genetic testing for deafness.

### **4. Treatment for hearing loss induced by congenital CMV infection**

**3. Diagnosis of congenital CMV infection**

The gold standard for diagnosis of congenital CMV infection is isolation of the virus from urine or saliva in the first 2 weeks of life. However, asymptomatic congenital CMV infection in children who develop SNHL after the first 2 weeks following birth cannot be diagnosed on the basis of viral isolation from urine or saliva. Detection of CMV DNA in infant blood or the umbilical cord using PCR assays is a more feasible method for identifying children with lateonset SNHL. The method involves analysis of blood stored as DBS on Guthrie cards. In Japanese culture, the dried umbilical cord is generally stored at home as a memento of the birth. These specimens are suitable for retrospective diagnosis of congenital CMV infection. The sensitivity varied widely depending on the DNA extraction method from DBS on Guthrie cards. Some investigators reported sensitivities of 71-100% and specificities of 99-100% [19, 29]. The qPCR method and dried umbilical cord could be useful for more precise detection of

Diagnosis of symptomatic CMV infection is easier in children who display cognitive or neuromuscular abnormalities than in asymptomatic children with CMV infection. Without neonatal viral screening, the prevalence of SNHL caused by asymptomatic CMV infection remains undetermined. To diagnose primary CMV infection, a serological method has been used [31]. Pregnant women who test positive for CMV IgG seroconversion or CMV IgM antibody may transmit the virus to the fetus. Production of IgM antibody persists for 6–9 months [28]; therefore, a CMV IgM-positive result alone does not accurately predict the risk

For the detection of congenital CMV infection, CMV DNA qPCR analysis was performed. Prior to qPCR analysis, total DNA, including genomic DNA and CMV DNA, was extract‐ ed from preserved dried umbilical cords. The procedure is as follows. Each 5-mm tissue section was incubated in a lysis buffer containing proteinase K and incubated overnight at 56°C. Total DNA was extracted using the DNeasy® Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. The total amount of DNA was measured using the Qubit® Fluorometer with Quant-iT™ dsDNA BR Assay Kit (Life technologies-Invitrogen, Carlsbad, CA, USA). Total DNA (10 pg) was analyzed using the Step One Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and TaqMan® Universal Master Mix II (Applied Biosystems). The qPCR primers and TaqMan® probe used for CMV DNA qPCR analysis were as follows: US14-1F: 5′- ACGTCCACGTTAGGATGAGG-3′, US14-1R: 5′-GTATGTGGCGCTTCTCTCGT-3′, and US14-1 TaqMan probe: 5′-FAM- AACCTGTGCACCACAGCGCC -TAMRA-3′. To quantify the input DNA amount in each sample, qPCR of each genomic region was also per‐

**3.1. Detection methods**

8 Manifestations of Cytomegalovirus Infection

CMV DNA.

**3.2. Serological method**

of fetal infection.

**3.3. Detection of CMV DNA from umbilical cord**

#### **4.1. Cochlear implantation in children deafened by symptomatic CMV infection**

Cochlear implantation for the correction of congenital deafness is an effective way to ensure the development of speech recognition. Cochlear implantation in children deafened by symptomatic CMV infection has been reported [33, 34]. The prognosis of children with symptomatic CMV infection is worse than that of those with asymptomatic CMV infection with regard to cognitive and neurological development. It has been suggested that cochlear implantation should be contraindicated for infants with symptomatic CMV infection and deafness because they are less likely to develop spoken language [35]. In contrast, other reports [33, 34] have suggested that cochlear implantation may improve quality of life, even if progress is slower or lesser than that expected in congenitally deaf children not infected with CMV. Pyman et al. [35] suggested that the prognosis in terms of linguistic outcome after cochlear implantation is poorer for CMV-infected deaf children than for other congenitally deaf children because of coexisting central disorders. Wide variation in speech perception and intelligibility after cochlear implantation has also been reported in children deafened by symptomatic CMV infection [33]. In that report, poor development in these areas was observed in 50% of children with symptomatic CMV infection, whereas development similar to that in congenitally deaf children not infected with CMV was evident in 31% of children and devel‐ opment better than that in noninfected congenitally deaf children was evident in 19% of children. In addition, a recent study has shown that deafness caused by symptomatic congen‐ ital CMV infection associated with motor and cognitive delays is not a contraindication for cochlear implantation. Early diagnosis of hearing loss and subsequent cochlear implantation is important for successful speech perception [34].

#### **4.2. Cochlear implantation in children deafened by asymptomatic CMV infection**

The effectiveness of cochlear implantation in children deafened as a result of symptomatic congenital CMV infection has been evaluated by various groups, but there are only limited outcome data for deaf children with asymptomatic CMV infection. Children with asympto‐ matic congenital CMV infection have a better prognosis than symptomatic children, but it is difficult to evaluate the SNHL because children with asymptomatic congenital CMV infection are at risk of development of delayed onset SNHL and progressive SNHL. As a result, they are also at risk of late-onset learning difficulties and/or progressive learning difficulties.

**5. Conclusion**

Congenital CMV infection is a major cause of bilateral and unilateral SNHL in children. In total, 9.0% of SNHL cases of unknown causes (bilateral SNHL: 8.7%, unilateral SNHL: 9.1%) are attributed to congenital CMV infection. Screening tests such as the detection of CMV DNA from preserved dried umbilical cords and genetic testing are important for the detection of SNHL in children. Using this combined methodology, detection of the cause of SNHL is

Hearing Loss in Children with Congenital Cytomegalovirus Infection

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11

Cochlear implantation is effective to ensure the development of speech perception and auditory skills in deaf children with asymptomatic congenital CMV infection. No significant difference in growth of meaningful auditory integration was observed between the overall pediatric cochlear implant population not infected with CMV and that with asymptomatic CMV infection. Implementation of CMV screening models is important to prevent late-onset

These works were supported by grants for Research on Sensory and Communicative Disorders from Ministry of Health, Labour and Welfare and grants for Scientific Research (C) from

1 Department of Hearing Implant Sciences, Shinshu University School of Medicine, Matsu‐

2 Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto

[1] Hagay, Z. J, Biran, G, Ornoy, A, & Reece, E. A. Congenital cytomegalovirus infection: a long-standing problem still seeking a solution. Am J Obstet Gynecol (1996). , 174,

Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan.

possible in approximately 50% of children with hearing loss.

SNHL and deterioration of hearing loss.

and Shin-ich Usami2\*

\*Address all correspondence to<EMAIL_ADDRESS>
**Acknowledgements**

**Author details**

Satoshi Iwasaki1

moto City, Japan

City, Japan

**References**

241-5.

 This study examined 2 deaf infants before and after cochlear implantation using the Infant/Toddler Meaningful Auditory Integration Scale (IT-MAIS) [36]. Vocalization behavior in case 1 was observed 6 months after implementation and showed slow improvement but finally overtook after 36 months. After 3 months of cochlear implant use, the 2 children responded to speech and environmental sounds in everyday situations and interpreted sounds in a meaningful way. They continued to improve at 36 months postoperatively. IT-MAIS scores in these 2 children were similar to the mean scores in the 5 congenitally deaf children without CMV infection. No difference was observed in the effect of early cochlear implantation for deafness induced by CMV infection between the groups of children. Another group reported that significant improvement in auditory and language skills could be achieved in cochlear implanted children with asymp‐ tomatic CMV infection, but they did not achieve the same levels of outcome as congenitally deaf children without CMV infection [37]. They found a wide variation in the outcome of cochlear implantation in these children and speculated that the variation is related to the degree of cognitive impairment. There are only a few studies available on outcomes of cochlear implanted children with asymptomatic CMV infection. Therefore, more studies will be needed to evaluate the effectiveness of cochlear implantation in these children.

#### **4.3. Treatment for hearing-impaired children with congenital CMV infection**

To prevent late-onset and/or deterioration of SNHL, treatment with intravenous ganciclovir (GCV) and/or oral valganciclovir (VGCV) has been recommended in children with sympto‐ matic congenital CMV disease involving the central nervous system [38-41]. In previous reports, treatment with intravenous GCV was initiated within the first 10–14 days of life for 2–6 weeks, and GCV doses ranged from 5 to 12 mg/kg twice daily. One report revealed that in 5 of 9 children with congenital CMV infection and SNHL, treatment with intravenous GCV induced improvement of SNHL in 2 children and prevented deterioration of SNHL in 5 children [38]. Another report revealed that in 4 of 6 children with congenital CMV infection and SNHL, treatment with intravenous GCV induced improvement of SNHL in 2 children and no deterioration of SNHL in 4 children during the 21-month observation period [39]. Im‐ provement of SNHL or maintenance of normal hearing was reported in 84% of children treated with intravenous GCV and 59% of untreated children. Deterioration of SNHL was reported in 21% of treated children and 68% of untreated children [40]. According to these reports, good results have been observed in the group of children treated with GCV. Treatment with intravenous GCV and oral VGCV can prevent the development of SNHL during an 18-month administration period [41]. Treatment with intravenous GCV has been investigated in hearingimpaired children with asymptomatic congenital CMV infection. No SNHL was found for 4 – 11 years in 12 children with asymptomatic congenital CMV infection treated with intravenous GCV, but SNHL developed in 2 of 11 untreated children [42]. Unfortunately there is no evidence for the efficacy of longer treatment with oral VGCV.

#### **5. Conclusion**

are at risk of development of delayed onset SNHL and progressive SNHL. As a result, they are also at risk of late-onset learning difficulties and/or progressive learning difficulties.

10 Manifestations of Cytomegalovirus Infection

A prospective study was conducted on deaf children with asymptomatic CMV infection to assess the development of speech perception and auditory skills. This study examined 2 deaf infants before and after cochlear implantation using the Infant/Toddler Meaningful Auditory Integration Scale (IT-MAIS) [36]. Vocalization behavior in case 1 was observed 6 months after implementation and showed slow improvement but finally overtook after 36 months. After 3 months of cochlear implant use, the 2 children responded to speech and environmental sounds in everyday situations and interpreted sounds in a meaningful way. They continued to improve at 36 months postoperatively. IT-MAIS scores in these 2 children were similar to the mean scores in the 5 congenitally deaf children without CMV infection. No difference was observed in the effect of early cochlear implantation for deafness induced by CMV infection between the groups of children. Another group reported that significant improvement in auditory and language skills could be achieved in cochlear implanted children with asymp‐ tomatic CMV infection, but they did not achieve the same levels of outcome as congenitally deaf children without CMV infection [37]. They found a wide variation in the outcome of cochlear implantation in these children and speculated that the variation is related to the degree of cognitive impairment. There are only a few studies available on outcomes of cochlear implanted children with asymptomatic CMV infection. Therefore, more studies will be needed

to evaluate the effectiveness of cochlear implantation in these children.

evidence for the efficacy of longer treatment with oral VGCV.

**4.3. Treatment for hearing-impaired children with congenital CMV infection**

To prevent late-onset and/or deterioration of SNHL, treatment with intravenous ganciclovir (GCV) and/or oral valganciclovir (VGCV) has been recommended in children with sympto‐ matic congenital CMV disease involving the central nervous system [38-41]. In previous reports, treatment with intravenous GCV was initiated within the first 10–14 days of life for 2–6 weeks, and GCV doses ranged from 5 to 12 mg/kg twice daily. One report revealed that in 5 of 9 children with congenital CMV infection and SNHL, treatment with intravenous GCV induced improvement of SNHL in 2 children and prevented deterioration of SNHL in 5 children [38]. Another report revealed that in 4 of 6 children with congenital CMV infection and SNHL, treatment with intravenous GCV induced improvement of SNHL in 2 children and no deterioration of SNHL in 4 children during the 21-month observation period [39]. Im‐ provement of SNHL or maintenance of normal hearing was reported in 84% of children treated with intravenous GCV and 59% of untreated children. Deterioration of SNHL was reported in 21% of treated children and 68% of untreated children [40]. According to these reports, good results have been observed in the group of children treated with GCV. Treatment with intravenous GCV and oral VGCV can prevent the development of SNHL during an 18-month administration period [41]. Treatment with intravenous GCV has been investigated in hearingimpaired children with asymptomatic congenital CMV infection. No SNHL was found for 4 – 11 years in 12 children with asymptomatic congenital CMV infection treated with intravenous GCV, but SNHL developed in 2 of 11 untreated children [42]. Unfortunately there is no

Congenital CMV infection is a major cause of bilateral and unilateral SNHL in children. In total, 9.0% of SNHL cases of unknown causes (bilateral SNHL: 8.7%, unilateral SNHL: 9.1%) are attributed to congenital CMV infection. Screening tests such as the detection of CMV DNA from preserved dried umbilical cords and genetic testing are important for the detection of SNHL in children. Using this combined methodology, detection of the cause of SNHL is possible in approximately 50% of children with hearing loss.

Cochlear implantation is effective to ensure the development of speech perception and auditory skills in deaf children with asymptomatic congenital CMV infection. No significant difference in growth of meaningful auditory integration was observed between the overall pediatric cochlear implant population not infected with CMV and that with asymptomatic CMV infection. Implementation of CMV screening models is important to prevent late-onset SNHL and deterioration of hearing loss.

#### **Acknowledgements**

These works were supported by grants for Research on Sensory and Communicative Disorders from Ministry of Health, Labour and Welfare and grants for Scientific Research (C) from Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan.

#### **Author details**

Satoshi Iwasaki1 and Shin-ich Usami2\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Hearing Implant Sciences, Shinshu University School of Medicine, Matsu‐ moto City, Japan

2 Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto City, Japan

#### **References**

[1] Hagay, Z. J, Biran, G, Ornoy, A, & Reece, E. A. Congenital cytomegalovirus infection: a long-standing problem still seeking a solution. Am J Obstet Gynecol (1996). , 174, 241-5.

[2] Stagno, S, Pass, R. F, Cloud, G, Britt, W. J, Henderson, R. E, Walton, P. D, et al. Pri‐ mary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and clinical outcome. JAMA (1986). , 256, 1904-8.

[15] Rosenthal, L. S, Fowler, K. B, Boppana, S. B, Britt, W. J, Pass, R. F, Schmid, D. S, et al. Cytomegalovirus shedding and delayed sensorineural hearing loss: results from lon‐ gitudinal follow-up of children with congenital infection. Pediatr Infect Dis J (2009). ,

Hearing Loss in Children with Congenital Cytomegalovirus Infection

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13

[16] Ogawa, H, Suzutani, T, Baba, Y, Koyano, S, Nozawa, N, Ishibashi, K, et al. Etiology of severe sensorineural hearing loss in children: independent impact of congenital

cytomegalovirus infection and GJB2 mutations. J Infect Dis (2007). , 195, 782-8.

nosis using preserved umbilical cord. Acta Ololaryngol (2011). , 131, 976-82.

pass newborn hearing screening. Pediatr Infect Dis (2009). , 28, 1095-8.

[17] Furutate, S, Iwasaki, S, Nishio, S, Moteki, H, & Usami, S. Clinical profile of hearing loss in children with congenital cytpmegalovirus (CMV) infection: CMV DNA diag‐

[18] Choi, K. Y, Schimmenti, L. A, Jurek, A. M, Sharon, B, Daly, K, Khan, C, et al. Detec‐ tion of cytomegalovirus DNA in dried blood pots of Minnesota infants who do not

[19] Barbi M Binda SCaroppo S, Ambrosetti U, Corbetta C, Sergi P. A wider role for con‐ genital cytomegalovirus infection in sensorineural hearing loss. Pediatr Infect Dis J

[20] Jakubikova, J, Kabatova, Z, Pavlovcinova, G, & Profant, M. Newborn hearing screen‐ ing and strategy for early detection of hearing loss in infants. Int J Pediatr Otorhino‐

[21] Samileh, N, Ahmad, S, Mohammad, F, Framarz, M, Azardokht, T, & Jomeht, E. Role of cytomegalovirus in sensorineural hearing loss of children: a case-control study

[22] Stehel, EK, Shoup, AG, & Owen, . . Newborn hearing screening and detection of con‐

[23] Grosse, S. D, Ross, D. S, & Dollard, S. C. Congenital cytomegalovirus (CMV) infec‐ tion as a cause of permanent bilateral hearing loss: a quantitative assessment. J Clin

[24] Foulon, I, Naessens, A, Foulon, W, Casteels, A, & Gordts, F. Hearing loss in children with congenital cytomegalovirus infection in relation to the maternal trimester in

[25] Ogawa, H, Baba, Y, Suzutani, T, Inoue, N, Fukushima, E, & Omori, K. Congenital cy‐ tomegalovirus infection diagnosed by polymerase chain reaction with the use of ore‐ served umbilical cord in sensorineural hearing loss children. Laryngoscope (2006). ,

[26] Tagawa, M, Tanaka, H, Moriuchi, M, & Moriuchi, H. Retrospective diagnosis of con‐ genital cytomegalovirus infection at a school for the deaf by using preserved dried

which the maternal primary infection occurred. Pediatrics (2008). e, 1123-7.

Tehran, Iran. Int J Pediatr Otorhinolaryngol (2008). , 72, 203-8.

genital cytomegalovirus infection. Pediatrics 2008;121:970-5.

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[15] Rosenthal, L. S, Fowler, K. B, Boppana, S. B, Britt, W. J, Pass, R. F, Schmid, D. S, et al. Cytomegalovirus shedding and delayed sensorineural hearing loss: results from lon‐ gitudinal follow-up of children with congenital infection. Pediatr Infect Dis J (2009). , 28, 515-20.

[2] Stagno, S, Pass, R. F, Cloud, G, Britt, W. J, Henderson, R. E, Walton, P. D, et al. Pri‐ mary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and

[3] Pass, R. F, Stagno, S, Myers, G. J, & Alford, C. A. Outcome of symptomatic congenital cytomegalovirus infection: results of long-term longitudinal follow-up. Pediatrics

[4] Kimberlin, D. W, Lin, C. Y, Sanchez, P. J, Demmler, G. J, Dankner, W, Shelton, M, et al. Effect of ganciclovir therapy on hearing in symptomatic congenital cytomegalovi‐ rus disease involving the central nervous system: a randomized, controlled trial. J Pe‐

[5] Yow, M. D, Williamson, D. W, Leeds, L. J, Thompson, P, Woodward, R. M, Walmus, B. F, et al. Epidemiologic characteristics of cytomegalovirus infection in mothers and

[6] Williamson, W. D, Demmler, G. J, Percy, A. K, & Catlin, F. I. Progressive hearing loss in infants with asymptomatic congenital cytomegalovirus infection. Pediatrics

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[8] Fowler, K. B, Mccollister, F. P, Dahle, A. J, Boppana, S, Britt, W. J, & Pass, R. F. Pro‐ gressive and fluctuating sensorineural hearing loss in children with asymptomatic

[9] Dahle, A. J, Fowler, K. B, Wright, J. D, Boppana, S. B, Britt, W. J, & Pass, R. F. Longi‐ tudinal investigation of hearing disorders in children with congenital cytomegalovi‐

[10] Fowler, K. B, Dahle, A. J, Boppana, S. B, & Pass, R. F. Newborn hearing screening: will children with hearing loss caused by congenital cytomegalovirus infection be

[11] Williamson, W. D, & Desmond, M. M. LaFevers N, Taber LH, Catlin FI, Weaver TG. Symptomatic congenital cytomegalovirus. Disorders of language, learning, and hear‐

[12] Rivera, L. B, Boppana, S. B, Fowler, K. B, Britt, W. J, Stagno, S, & Pass, R. F. Predictors of hearing loss in children with symptomatic congenital cytomegalovirus infection.

[13] Iwasaki, S, Yamashita, M, Maeda, M, Misawa, K, & Mineta, H. Audiological outcome of infants with congenital cytomegalovirus infection in a prospective study. Audiol

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congenital cytomegalovirus infection. J Pediatr (1997). , 130, 624-30.

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missed? J Pediatr (1999). , 135, 60-4.

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diatr (2003). , 143, 16-25.

(1992). , 90, 862-6.

[27] Usami, S, Wagatsuma, M, Fukuoka, H, Suzuki, H, Tsukada, K, Nishio, S, et al. The responsible genes in Japanese deafness patients and clinical application using Invad‐ er assay. Acta Otolaryingol (2008). , 128, 446-54.

[39] Kimberlin, D. W, Lin, C. Y, Sanchez, P. J, Demmler, G. J, Dankner, W, Shelton, M, et al. Effect of ganciclovir therapy on hearing in symptomatic congenital cytomegalovi‐ rus disease involving the cebtral nervous system: a randomized, controlled trial. J Pe‐

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[40] Kitajima, N, Sugaya, N, Futatani, T, Kanegane, H, Suzuki, C, Oshiro, M, et al. Ganci‐ clovir therapy for congenital cytomegalovirus infection in six infants. Pediatr Infect

[41] Meine Jansen CFToet MC, Rademaker CM, Ververs TH, Gerards LJ, van Loon AM. Treatment of symptomatic congenital cytomegalovirus infection with valganciclovir.

[42] Lackner, A, Acham, A, Alborno, T, Moser, M, Engele, H, Raggam, R. B, et al. Effect on hearing of ganciclovir therapy for asymptomatic congenital cytomegalovirus in‐

[43] Walter, S, Atkinson, C, Sharland, M, Rice, P, Raglan, E, Emery, V. C, et al. Congenital cytomegalovirus: association between dried blood spot viral load and hearing loss.

[44] Mizuno, T, Sugiura, S, Kimura, H, Ando, Y, Sone, M, Nishiyama, Y, et al. Detection of cytomegalovirus DNA in preserved umbilical cords from patients with sensori‐

[45] Boudewyns, A, Declau, F, Smets, K, Ursi, D, & Eyskens, F. Van den Ende J, et al. Cy‐ tomegalovirus DNA detection in Guthrie cards: role in the giagnostic work-up of

[46] Kimani, J. W, Buchman, C. A, Booker, J. K, Huang, B. Y, Castillo, M, Powell, C. M, et al. Seonsorineural hearing loss in a pediatric population: association of cobgenital cy‐ tomegalovirus infection with intracranial abnormalities. Arch Otolaryngol Head

[47] Adachi, N, Ito, K, Sakata, H, & Yamasoba, T. Etiology and one-year follow-up results of hearing loss identified by screening of newborn hearing in Japan. Otolaryngol

fection: four to 10 year follow up. J Laryngol Otol (2009). , 123, 392-6.

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[39] Kimberlin, D. W, Lin, C. Y, Sanchez, P. J, Demmler, G. J, Dankner, W, Shelton, M, et al. Effect of ganciclovir therapy on hearing in symptomatic congenital cytomegalovi‐ rus disease involving the cebtral nervous system: a randomized, controlled trial. J Pe‐ diatr (2003). , 143, 4-6.

[27] Usami, S, Wagatsuma, M, Fukuoka, H, Suzuki, H, Tsukada, K, Nishio, S, et al. The responsible genes in Japanese deafness patients and clinical application using Invad‐

[28] Genser, B, Truschnig-wilders, M, Stunzner, D, Landini, M. D, & Halwachs-baumann, G. Evaluation of five commercial enzyme immunoassays for the detection of human cytomegalovirus-specific IgM antibodies in the absence of a commercially available

[29] De Vries, J. C, Claas, E. C, Kroes, A. C, & Vossen, A. C. Evaluation of DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infec‐

[30] Koyano, S, Inoue, N, Nagamori, T, Yan, H, Asanuma, H, Yagyu, K, et al. Dried um‐ bilical cords in the retrospective diagnosis of congenital cytomegalovirus infection as

[31] Lazzarotto, T, Gabrielli, L, Lanari, M, Guerra, B, Bellucci, T, Sassi, M, & Landini, M. P. Congenital cytomegalovirus infection: recent advances in the diagnosis of mater‐

[32] Abe, S, Yamaguchi, T, & Usami, S. Application of deafness diagnostic screening pan‐ el based on deafness mutation/gene database using invader assay. Genet Test

[33] Ramirez Inscoe JMNikolopoulos TP. Cochlear implantation in children deafened by cytomegalovirus: speech perception and speech intelligibility outcomes. Otol Neuro‐

[34] Lee, D. J, Lustig, L, Sampson, M, Chinnici, J, & Niparko, J. K. Effects of cytomegalovi‐ rus (CMV) related deafness on pediatric cochlear implant outcomes. Otolaryngol

[35] Pyman, B, & Blamey, P. Lacy P Clark G, Dowell R. The development of speech per‐ ception in children using cochlear implants: effects of etiologic factors and delayed

[36] Iwasaki, S, Nakanishi, H, Misawa, K, Tanigawa, T, & Mizuta, K. Cochlear implant in children with asymptomatic congenital cytomegalovirus infection. Audiol Neurotol

[37] Malik, V, Bruce, I. A, Broomfield, S. J, Henderson, L, Green, K, & Ramsden, R. T. Outcome of cochlear implantation in asymptomatic congenital cytomegalovirus

[38] Michaels, M. G, Greenberg, D. P, Sabo, D. L, & Wald, E. R. Treatment of children with congenital cytomegalovirus infection with gancicolovir. Pediatr Infect Dis J

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a cause of developmental delays. Clin Infect Dis (2009). e, 93-5.

**Chapter 2**

**Human Cytomegalovirus (HCMV) Infection in Sub-**

There have been over 25 published studies which present HCMV IgG seroprevalence data for sub-Saharan Africa patient groups and cohorts of healthy blood donors (Table 1). Up to eight different serology assays were used and older pre-ELISA methods might have slightly underestimated prevalence [1]. Antibodies to HCMV are generally present in high titres in seropositive individuals, so the use of different assays is unlikely to have had a major effect [2]. Hence, comparing these studies is primarily confounded by the diverse range of patient groups tested. Few studies stratify by age, or they do so using different groupings. Most of the studies use convenience samples, which do not provide accurate population-based estimates of

The most striking observation is that HCMV primary infection appears to be endemic in young infants. A population-based study in Zambia of 460 healthy infants showed 83% HCMV seroprevalence by 18 months of age [3](Table 1). This backs up much older studies from the Gambia [4] and Nigeria [5]. This differs from the results of larger studies in the USA (n = 30,000) where HCMV seroprevalence ranges from 36% in 6–11 year-olds to 91% in those over 80 years old. The cumulative incidence of HCMV primary infection was ~1% per year from adolescence [6]. In the USA, non-white ethnicity and lower socioeconomic status (SES) were linked with 10-30 percentage point increases in seroprevalence [7]. A study of over 20,000 women in the U.K attending antenatal clinics found similar results, with increasing parity also being linked

> © 2013 Bates et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

> © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Saharan Africa**

Alimuddin Zumla

**1. Introduction**

prevalence.

*1.1.1. HCMV seroprevalence*

http://dx.doi.org/10.5772/54907

Matthew Bates, Kunda Musonda and

Additional information is available at the end of the chapter

**1.1. HCMV epidemiology in Sub-Saharan Africa**

## **Human Cytomegalovirus (HCMV) Infection in Sub-Saharan Africa**

Matthew Bates, Kunda Musonda and Alimuddin Zumla

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54907

#### **1. Introduction**

#### **1.1. HCMV epidemiology in Sub-Saharan Africa**

#### *1.1.1. HCMV seroprevalence*

There have been over 25 published studies which present HCMV IgG seroprevalence data for sub-Saharan Africa patient groups and cohorts of healthy blood donors (Table 1). Up to eight different serology assays were used and older pre-ELISA methods might have slightly underestimated prevalence [1]. Antibodies to HCMV are generally present in high titres in seropositive individuals, so the use of different assays is unlikely to have had a major effect [2]. Hence, comparing these studies is primarily confounded by the diverse range of patient groups tested. Few studies stratify by age, or they do so using different groupings. Most of the studies use convenience samples, which do not provide accurate population-based estimates of prevalence.

The most striking observation is that HCMV primary infection appears to be endemic in young infants. A population-based study in Zambia of 460 healthy infants showed 83% HCMV seroprevalence by 18 months of age [3](Table 1). This backs up much older studies from the Gambia [4] and Nigeria [5]. This differs from the results of larger studies in the USA (n = 30,000) where HCMV seroprevalence ranges from 36% in 6–11 year-olds to 91% in those over 80 years old. The cumulative incidence of HCMV primary infection was ~1% per year from adolescence [6]. In the USA, non-white ethnicity and lower socioeconomic status (SES) were linked with 10-30 percentage point increases in seroprevalence [7]. A study of over 20,000 women in the U.K attending antenatal clinics found similar results, with increasing parity also being linked

© 2013 Bates et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

with increased HCMV seroprevalence. This supports the notion that seronegative adult women contract primary HCMV infection from children who are shedding virus [8]. Figure 1 presents a model for cumulative HCMV seroprevalence by age with respect to SES, showing more rapid uptake in low SES communities, and delayed uptake in high SES communities. Conversely an Israeli study found the effect of ethnicity persisted even when corrected for gender, education and SES [9], and high HCMV seroprevalence has been described in populations with high SES groups [10, 11].

1

19

Whilst lower SES may be the main driver for endemic infant HCMV primary infection in sub-Saharan Africa, this is not the whole story. What factors, attributable to low SES, facilitate earlier HCMV transmission? HCMV is primarily transmitted through body fluids, being shed in urine, saliva [12, 13] and breast milk [14, 15]. In Nigeria, over-crowding was significantly associated with being HCMV seropositive, but source of drinking water, place of abode and type of toilet facility were not [16]. Some individuals remain seronegative into old age - even in sub-Saharan Africa where most people are infected in infancy. Human genetic variations may block or impair HCMV primary infection, as is seen with the CCR5 ∆32 mutation and HIV [17]. HCMV seronegative individuals have increased longevity, possibly linked with reduced clonal expansion of CD8 T cells and a larger reservoir of circulating naive T cells [18, 19] so early childhood primary infection with HCMV in sub-Saharan Africa may have profound effects. There is evidence linking early HCMV infections in sub-Saharan Africa with impaired physical and mental development [3], analogous to the known developmental CNS defects (hearing loss, mental retardation, cerebral palsy, seizures, chorioretinitis) caused by congenital HCMV infection [20, 21].

 Comparison of HCMV seroprevalence in different countries \* Percentages in bold are the averages within

**HIV+ Adults 80.0%**

**AIDS Patients**

**81.9%**

**HIV- Adults 79.3%**

each group, weighted by study size.

**Figure 1.** Model for the cumulative prevalence of primary HCMV seroconversion by age with respect to socioeconom‐ ic status (SES) groups.

with increased HCMV seroprevalence. This supports the notion that seronegative adult women contract primary HCMV infection from children who are shedding virus [8]. Figure 1 presents a model for cumulative HCMV seroprevalence by age with respect to SES, showing more rapid uptake in low SES communities, and delayed uptake in high SES communities. Conversely an Israeli study found the effect of ethnicity persisted even when corrected for gender, education and SES [9], and high HCMV seroprevalence has been described in

Whilst lower SES may be the main driver for endemic infant HCMV primary infection in sub-Saharan Africa, this is not the whole story. What factors, attributable to low SES, facilitate earlier HCMV transmission? HCMV is primarily transmitted through body fluids, being shed in urine, saliva [12, 13] and breast milk [14, 15]. In Nigeria, over-crowding was significantly associated with being HCMV seropositive, but source of drinking water, place of abode and type of toilet facility were not [16]. Some individuals remain seronegative into old age - even in sub-Saharan Africa where most people are infected in infancy. Human genetic variations may block or impair HCMV primary infection, as is seen with the CCR5 ∆32 mutation and HIV [17]. HCMV seronegative individuals have increased longevity, possibly linked with reduced clonal expansion of CD8 T cells and a larger reservoir of circulating naive T cells [18, 19] so early childhood primary infection with HCMV in sub-Saharan Africa may have profound effects. There is evidence linking early HCMV infections in sub-Saharan Africa with impaired physical and mental development [3], analogous to the known developmental CNS defects (hearing loss, mental retardation, cerebral palsy, seizures, chorioretinitis) caused by

**Figure 1.** Model for the cumulative prevalence of primary HCMV seroconversion by age with respect to socioeconom‐

populations with high SES groups [10, 11].

18 Manifestations of Cytomegalovirus Infection

congenital HCMV infection [20, 21].

ic status (SES) groups.

**Table 1.** Comparison of HCMV seroprevalence in different countries \* Percentages in bold are the averages within each group, weighted by study size.

1

#### *1.1.2. Molecular epidemiology*

HCMV has a large genome, predicted to encode at least 165 proteins. This includes hyper‐ variable segments [38-41] containing genes which encode membrane-bound glycoproteins. These are embedded in the virion envelope or presented on the surface of infected cells, making them candidate targets for the host immune response. Most published studies of polymor‐ phisms in these glycoproteins have concentrated on possible associations with and clinical disease or cellular tropism. No compelling connections have been reported in the literature to date, but much of the sequence data is from isolates from Europe, North America and Japan.

**2.2. Disease presentations and co-morbidity**

**2.3. HCMV as a cause of death**

0-8.5% of adult AIDS patients with ophthalmic conditions [77-83].

thological evidence of active HCMV infection post-mortem [92].

**3. HCMV pneumonia in HIV infected children**

infections and remain undiagnosed and untreated.

The most common presentation of HCMV infection in HIV-infected patients is HCMV pneumonia, where co-infection with other respiratory pathogens such a tuberculosis and *Pneumocystis jirovecci,* is almost ubiquitous [50, 60]. HCMV is an important HIV co-infection, also linked with a range diseases including meningitis [62], encephalitis [63], psychological disorders [64], malaria [65], various dermatological conditions [66, 67] and those affecting mucosal epithelia [68, 69], hypoadrenalism [70], adrenalitis [71], gastritis [72, 73] and other herpesvirus infections [74]. There has been a huge (possibly disproportionate) focus on HCMV as a cause of HIV-associated retinitis. Globally it has been estimated that 5-25% of AIDS patients will suffer from HCMV retinitis in their lifetime [75] leading to an 'epidemic of blindness' [76]. Whilst the cohorts and diagnostic methods vary in different studies, HIVassociated HCMV retinitis is less common in sub-Saharan Africa than elsewhere, seen in just

Human Cytomegalovirus (HCMV) Infection in Sub-Saharan Africa

http://dx.doi.org/10.5772/54907

21

It is unclear how much active HCMV infection is contributing to mortality in HIV infected people. One way to address this question is to measure mortality as a primary outcome. For example: a large longitudinal study of HIV infected miners in South Africa associated HCMV viraemia with a three-fold increase in mortality after just 11 months. The affect was weakened when controlling for CD4 T-cell count, WHO stage and HIV viral load– all conditions predic‐ tive of mortality [84]. A study of HIV-infected and -exposed Kenyan children found a strong correlation between HIV-1 and HCMV viral loads. Adjusting for maternal immunosuppres‐ sion and HIV-1 viral load, HCMV viraemia during pregnancy was linked with high risk of death for mothers and infants in the 2 years following delivery [85]. It is difficult to prove that HCMV viraemia is not simply a bystander and is actually involved in pathology. This requires post mortem studies, which are difficult due to cultural factors [86]. Paediatric post mortem studies from sub-Saharan Africa identify HCMV as a common cause of death [87, 88], espe‐ cially in HIV infected patients [89-91]. There is a need for new post-mortem data, from both prospective studies and routine cases, to better inform on the prevalence of active HCMV as a cause of death, and in particular, to calibrate HCMV viral loads pre-mortem with histopa‐

Pneumonia is the most common cause of death in children <5 yrs of age globally, accounting for 18% of all deaths [93]. In sub-Saharan Africa, pneumonia is the leading cause of death in HIV-infected and -exposed children [94-97]. Across the region antibiotics are cheap and widely available, yet pneumonia is still a major cause of paediatric mortality. This is likely in part due to antibiotic resistance [98], but also several viral pathogens cause lower respiratory tract

There is little information regarding HCMV genotypes in Africa. In an early study investigat‐ ing geographic differences in the frequency of certain HCMV genotypes from immunocom‐ promised patients, they found that the distribution differed between Zimbabwe, Italy and California [42]. This study was limited to UL55 (virion surface glycoprotein gB: involved in cell entry and signaling)[43] which is relatively conserved between strains [40] and not linked with more variable glycoproteins [44]. A study of HCMV strains from 19 Malawian Kaposi's sarcoma (KS) patients and 58 of their first-degree relatives detected HCMV readily in mouth rinse and urine specimens [45]. Two hypervariable glycoprotein genes were sequenced: gO (UL74) and gN (UL73) involved in promoting focal spread [46, 47] and virion morphogenesis and possible latency associated functions respectively [48, 49]. Studies from Zambia segregate variants of these two glycoproteins into eight linked groups [50, 51]. These studies and others from Africa have found evidence of co-infections with multiple HCMV strains [50, 52] and no evidence for geographic separation. This data contrasts with other herpesviruses such as HHV-6 [53] and KSHV [54]. The high prevalence of co-infections with multiple strains, in a broad range of patients, complicates genotyping studies and attempts to identify disease links with specific glycoprotein genotypes. New techniques combining PCR amplification with RFLP digestion could improve analysis of multiple strains and recombinants in pathological samples [55].

#### **2. HCMV infections and HIV in Sub-Saharan Africa**

#### **2.1. General considerations**

sub-Saharan Africa is at the epicentre of the HIV pandemic, with 1,900,000 new infections (18.9% children ≤ 14yrs of age) in 2010, and a total of 23.2 million people (13.4% children ≤ 14yrs of age) living with HIV. Progress is slow but new infections are down 16% on 2001, and HIV prevalence has declined in some sub-Saharan African countries. With the roll out of antiretroviral therapy (ART), the numbers of people dying from HIV is also down (30% decrease between 2004 and 2010) [56]. HCMV is an apex opportunistic pathogen, linked with HIV disease progression [57-59], so the HIV pandemic combined with over 80% of primary HCMV infections occurring during infancy, creates a unique environment. Active HCMV infections are common and present as complex co-infections with other viral, bacterial and fungal infections [60, 61]. A broader awareness of the frequency of co-infections and the complex interplay between different pathogens is needed.

#### **2.2. Disease presentations and co-morbidity**

*1.1.2. Molecular epidemiology*

20 Manifestations of Cytomegalovirus Infection

samples [55].

**2.1. General considerations**

**2. HCMV infections and HIV in Sub-Saharan Africa**

complex interplay between different pathogens is needed.

sub-Saharan Africa is at the epicentre of the HIV pandemic, with 1,900,000 new infections (18.9% children ≤ 14yrs of age) in 2010, and a total of 23.2 million people (13.4% children ≤ 14yrs of age) living with HIV. Progress is slow but new infections are down 16% on 2001, and HIV prevalence has declined in some sub-Saharan African countries. With the roll out of antiretroviral therapy (ART), the numbers of people dying from HIV is also down (30% decrease between 2004 and 2010) [56]. HCMV is an apex opportunistic pathogen, linked with HIV disease progression [57-59], so the HIV pandemic combined with over 80% of primary HCMV infections occurring during infancy, creates a unique environment. Active HCMV infections are common and present as complex co-infections with other viral, bacterial and fungal infections [60, 61]. A broader awareness of the frequency of co-infections and the

HCMV has a large genome, predicted to encode at least 165 proteins. This includes hyper‐ variable segments [38-41] containing genes which encode membrane-bound glycoproteins. These are embedded in the virion envelope or presented on the surface of infected cells, making them candidate targets for the host immune response. Most published studies of polymor‐ phisms in these glycoproteins have concentrated on possible associations with and clinical disease or cellular tropism. No compelling connections have been reported in the literature to date, but much of the sequence data is from isolates from Europe, North America and Japan. There is little information regarding HCMV genotypes in Africa. In an early study investigat‐ ing geographic differences in the frequency of certain HCMV genotypes from immunocom‐ promised patients, they found that the distribution differed between Zimbabwe, Italy and California [42]. This study was limited to UL55 (virion surface glycoprotein gB: involved in cell entry and signaling)[43] which is relatively conserved between strains [40] and not linked with more variable glycoproteins [44]. A study of HCMV strains from 19 Malawian Kaposi's sarcoma (KS) patients and 58 of their first-degree relatives detected HCMV readily in mouth rinse and urine specimens [45]. Two hypervariable glycoprotein genes were sequenced: gO (UL74) and gN (UL73) involved in promoting focal spread [46, 47] and virion morphogenesis and possible latency associated functions respectively [48, 49]. Studies from Zambia segregate variants of these two glycoproteins into eight linked groups [50, 51]. These studies and others from Africa have found evidence of co-infections with multiple HCMV strains [50, 52] and no evidence for geographic separation. This data contrasts with other herpesviruses such as HHV-6 [53] and KSHV [54]. The high prevalence of co-infections with multiple strains, in a broad range of patients, complicates genotyping studies and attempts to identify disease links with specific glycoprotein genotypes. New techniques combining PCR amplification with RFLP digestion could improve analysis of multiple strains and recombinants in pathological

The most common presentation of HCMV infection in HIV-infected patients is HCMV pneumonia, where co-infection with other respiratory pathogens such a tuberculosis and *Pneumocystis jirovecci,* is almost ubiquitous [50, 60]. HCMV is an important HIV co-infection, also linked with a range diseases including meningitis [62], encephalitis [63], psychological disorders [64], malaria [65], various dermatological conditions [66, 67] and those affecting mucosal epithelia [68, 69], hypoadrenalism [70], adrenalitis [71], gastritis [72, 73] and other herpesvirus infections [74]. There has been a huge (possibly disproportionate) focus on HCMV as a cause of HIV-associated retinitis. Globally it has been estimated that 5-25% of AIDS patients will suffer from HCMV retinitis in their lifetime [75] leading to an 'epidemic of blindness' [76]. Whilst the cohorts and diagnostic methods vary in different studies, HIVassociated HCMV retinitis is less common in sub-Saharan Africa than elsewhere, seen in just 0-8.5% of adult AIDS patients with ophthalmic conditions [77-83].

#### **2.3. HCMV as a cause of death**

It is unclear how much active HCMV infection is contributing to mortality in HIV infected people. One way to address this question is to measure mortality as a primary outcome. For example: a large longitudinal study of HIV infected miners in South Africa associated HCMV viraemia with a three-fold increase in mortality after just 11 months. The affect was weakened when controlling for CD4 T-cell count, WHO stage and HIV viral load– all conditions predic‐ tive of mortality [84]. A study of HIV-infected and -exposed Kenyan children found a strong correlation between HIV-1 and HCMV viral loads. Adjusting for maternal immunosuppres‐ sion and HIV-1 viral load, HCMV viraemia during pregnancy was linked with high risk of death for mothers and infants in the 2 years following delivery [85]. It is difficult to prove that HCMV viraemia is not simply a bystander and is actually involved in pathology. This requires post mortem studies, which are difficult due to cultural factors [86]. Paediatric post mortem studies from sub-Saharan Africa identify HCMV as a common cause of death [87, 88], espe‐ cially in HIV infected patients [89-91]. There is a need for new post-mortem data, from both prospective studies and routine cases, to better inform on the prevalence of active HCMV as a cause of death, and in particular, to calibrate HCMV viral loads pre-mortem with histopa‐ thological evidence of active HCMV infection post-mortem [92].

#### **3. HCMV pneumonia in HIV infected children**

Pneumonia is the most common cause of death in children <5 yrs of age globally, accounting for 18% of all deaths [93]. In sub-Saharan Africa, pneumonia is the leading cause of death in HIV-infected and -exposed children [94-97]. Across the region antibiotics are cheap and widely available, yet pneumonia is still a major cause of paediatric mortality. This is likely in part due to antibiotic resistance [98], but also several viral pathogens cause lower respiratory tract infections and remain undiagnosed and untreated.

HCMV pneumonia is very common in HIV-infected and –exposed in sub-Saharan Africa [99, 100] and is associated with rapid progression of HIV disease [101] and death [102-104]. A seminal post mortem study in 264 Zambian children who died of respiratory disease identified classical HCMV inclusions in the lung tissue of up to 22% of HIV-infected cases [60], and then follow-up molecular work found high loads of HCMV were virtually ubiquitous in the lung tissue of HIV-infected paediatric respiratory deaths [50]. HCMV pneumonia is virtually impossible to distinguish clinically from *Pneumocystis jirovecci* pneumonia and co-infections with both *Pneumocystis jirovecci* and tuberculosis are common in HIV-infected and -exposed infants [60, 61, 105]. In South Africa, HCMV pneumonia was more common than *Pneumocystis jirovecci* pneumonia and other viral pneumonias in HIV-infected children [106], and was histologically confirmed in 72% of HIV-infected and ventilated infant mortalities with severe pneumonia. The authors recommend empiric use of ganciclovir or other anti-HCMV drugs in HIV-infected children with severe pneumonia who are not responding to co-trimoxazole [107].

a study from Zambia has shown that HCMV seroprevalence in 18 month old infants is linked with impaired growth and mental development [3]. There is a need for more prospective studies to investigate the clinical significance of congenital HCMV infections in sub-Saharan

One of the greatest challenges for HCMV diagnosis in this region is to differentiate clinically active from sub-clinical infection. Serological tests for HCMV IgM are useful for diagnosing primary infections in infants, particularly congenital infections in neonates, but the majority of the disease burden is caused by re-activation or re-infections in immunocompromised patients. Detection of the virus itself was traditionally achieved using culture-based methods. These are time-consuming and require well-trained staff and a well-serviced diagnostic laboratory. Moreover, HCMV culture is not very sensitive. For these reasons, quantitative DNA-based molecular diagnostics are now commonly used to detect active HCMV infections. The required infrastructure is becoming commonly available at tertiary and secondary referral centres across sub-Saharan Africa, often donated by international research projects. However low level HCMV reactivations are common in a wide range of patients, linked with reduced

Most studies of HCMV viral loads with respect to disease outcomes are in the transplant field,

be indicative of active disease, depending on the specific patient group [125]. An autopsy study

value of 100% for HCMV disease, making the commercial assay used (COBAS AMPLICOR CMV Monitor test - Roche) better for 'ruling in', than 'ruling out' [126]. There is a need for prospective studies in sub-Saharan Africa to monitor HCMV viral loads in patients with HIVassociated pneumonia, and infants with congenital HCMV infection, the two major HCMV disease groups in the region – although there are also transplant recipients in sub-Saharan Africa [127]. HCMV is shed in high loads in both urine and saliva (non-invasive specimens ideal for low income settings) and detection of virus DNA in these specimens should be

Several drugs are licensed for the treatment of HCMV infections, although they are expensive and broadly unavailable in sub-Saharan Africa. At some tertiary referral centres in South Africa, intravenous ganciclovir is used to treat HCMV pneumonia in HIV-infected and exposed children failing antibiotic or anti-mycobacterial therapy. Decisions are largely consultant led but two descriptive studies have reported dramatic reductions in mortality due to ganciclovir [106, 107]. Readers are advised to look up the latest guidelines on treatment of HCMV and to check the correct doses, side effects and dosing schedules. In South African centres, PCR or culture-proven HCMV disease is typically treated with 5mg/kg intravenously every 12hrs for 14-21 days, and then daily maintenance therapy at 5mg/kg [94]. But there is an

to 106 copies/ml whole blood have been suggested to

Human Cytomegalovirus (HCMV) Infection in Sub-Saharan Africa

http://dx.doi.org/10.5772/54907

23

copies/ml whole blood, gave a specificity and positive predictive

immune surveillance due to other infections, illness or malnutrition.

evaluated versus viraemia, as potentially useful markers of active disease.

Africa.

**5. HCMV diagnosis and treatment**

where viral loads within the range of 104

found that a cut off of 104

**in Sub-Saharan Africa**

#### **4. HCMV Congenital Infection in sub-Saharan Africa**

Congenital HCMV is generally defined by the detection of viral DNA and/or IgM antibody in infant sera within the first 3 weeks post-partum [108]. It is a damaging infection initiated by either primary or reactivated infection in the mother during pregnancy, although congenital HCMV infections transmitted from mothers with pre-existing immunity can be less severe [109]. Congenital HCMV is the major viral cause of mental and physical disability in children, infecting 0.2-2.2% of newborns [110, 111]. Around 7-11% of infected foetuses are then born with symptoms [112, 113], with a neonatal mortality rate of 20-30% [114, 115]. Of those congenitally infected (both symptomatic and asymptomatic), up to 28% will develop late sequelae [116]. Symptoms include growth retardation, hepatosplenomegaly, jaundice, pneumonia, gastrointestinal, and neurological disease such as sensorineural hearing loss, mental retardation, chorioretinitis, seizures [117] and cerebral palsy [118].

Congenital HCMV infection was considered rare in populations with high adult seropreva‐ lence [33]. A study of 2032 newborn infants in the Ivory Coast cultured HCMV from urine and showed congenital HCMV infection in 1.4% of all births [119]. In sub-Saharan Africa, congen‐ ital HCMV largely reflects maternal reactivations or re-infections, which may not result in severe disease in the child [109]. However, a few studies from the region suggest congenital HCMV maybe a significant cause of morbidity and mortality. A study from Zambia associated HCMV antibody titres above 1:1024 with still births [120]. HCMV IgM antibodies were detected in 24% of 99 newborn babies who were jaundiced, died within a few days of birth or showed gross congenital abnormalities [121]. Cervical shedding of HCMV is very common in HIV-infected women, and is readily detected in amniotic fluid collected at C-section [122, 123]. A Gambian study found the prevalence of congenital CMV among healthy neonates was 5.4%, at least 2-fold higher than reported in industrialized countries. Congenitally infected children were more often first born babies, more frequently born in crowded compounds and active placental malaria was more prevalent. During the first year of follow up, mothers of congenitally infected children reported more health complaints for their child [124]. Recently a study from Zambia has shown that HCMV seroprevalence in 18 month old infants is linked with impaired growth and mental development [3]. There is a need for more prospective studies to investigate the clinical significance of congenital HCMV infections in sub-Saharan Africa.

### **5. HCMV diagnosis and treatment in Sub-Saharan Africa**

HCMV pneumonia is very common in HIV-infected and –exposed in sub-Saharan Africa [99, 100] and is associated with rapid progression of HIV disease [101] and death [102-104]. A seminal post mortem study in 264 Zambian children who died of respiratory disease identified classical HCMV inclusions in the lung tissue of up to 22% of HIV-infected cases [60], and then follow-up molecular work found high loads of HCMV were virtually ubiquitous in the lung tissue of HIV-infected paediatric respiratory deaths [50]. HCMV pneumonia is virtually impossible to distinguish clinically from *Pneumocystis jirovecci* pneumonia and co-infections with both *Pneumocystis jirovecci* and tuberculosis are common in HIV-infected and -exposed infants [60, 61, 105]. In South Africa, HCMV pneumonia was more common than *Pneumocystis jirovecci* pneumonia and other viral pneumonias in HIV-infected children [106], and was histologically confirmed in 72% of HIV-infected and ventilated infant mortalities with severe pneumonia. The authors recommend empiric use of ganciclovir or other anti-HCMV drugs in HIV-infected children with severe pneumonia who are not responding to co-trimoxazole [107].

Congenital HCMV is generally defined by the detection of viral DNA and/or IgM antibody in infant sera within the first 3 weeks post-partum [108]. It is a damaging infection initiated by either primary or reactivated infection in the mother during pregnancy, although congenital HCMV infections transmitted from mothers with pre-existing immunity can be less severe [109]. Congenital HCMV is the major viral cause of mental and physical disability in children, infecting 0.2-2.2% of newborns [110, 111]. Around 7-11% of infected foetuses are then born with symptoms [112, 113], with a neonatal mortality rate of 20-30% [114, 115]. Of those congenitally infected (both symptomatic and asymptomatic), up to 28% will develop late sequelae [116]. Symptoms include growth retardation, hepatosplenomegaly, jaundice, pneumonia, gastrointestinal, and neurological disease such as sensorineural hearing loss,

Congenital HCMV infection was considered rare in populations with high adult seropreva‐ lence [33]. A study of 2032 newborn infants in the Ivory Coast cultured HCMV from urine and showed congenital HCMV infection in 1.4% of all births [119]. In sub-Saharan Africa, congen‐ ital HCMV largely reflects maternal reactivations or re-infections, which may not result in severe disease in the child [109]. However, a few studies from the region suggest congenital HCMV maybe a significant cause of morbidity and mortality. A study from Zambia associated HCMV antibody titres above 1:1024 with still births [120]. HCMV IgM antibodies were detected in 24% of 99 newborn babies who were jaundiced, died within a few days of birth or showed gross congenital abnormalities [121]. Cervical shedding of HCMV is very common in HIV-infected women, and is readily detected in amniotic fluid collected at C-section [122, 123]. A Gambian study found the prevalence of congenital CMV among healthy neonates was 5.4%, at least 2-fold higher than reported in industrialized countries. Congenitally infected children were more often first born babies, more frequently born in crowded compounds and active placental malaria was more prevalent. During the first year of follow up, mothers of congenitally infected children reported more health complaints for their child [124]. Recently

**4. HCMV Congenital Infection in sub-Saharan Africa**

22 Manifestations of Cytomegalovirus Infection

mental retardation, chorioretinitis, seizures [117] and cerebral palsy [118].

One of the greatest challenges for HCMV diagnosis in this region is to differentiate clinically active from sub-clinical infection. Serological tests for HCMV IgM are useful for diagnosing primary infections in infants, particularly congenital infections in neonates, but the majority of the disease burden is caused by re-activation or re-infections in immunocompromised patients. Detection of the virus itself was traditionally achieved using culture-based methods. These are time-consuming and require well-trained staff and a well-serviced diagnostic laboratory. Moreover, HCMV culture is not very sensitive. For these reasons, quantitative DNA-based molecular diagnostics are now commonly used to detect active HCMV infections. The required infrastructure is becoming commonly available at tertiary and secondary referral centres across sub-Saharan Africa, often donated by international research projects. However low level HCMV reactivations are common in a wide range of patients, linked with reduced immune surveillance due to other infections, illness or malnutrition.

Most studies of HCMV viral loads with respect to disease outcomes are in the transplant field, where viral loads within the range of 104 to 106 copies/ml whole blood have been suggested to be indicative of active disease, depending on the specific patient group [125]. An autopsy study found that a cut off of 104 copies/ml whole blood, gave a specificity and positive predictive value of 100% for HCMV disease, making the commercial assay used (COBAS AMPLICOR CMV Monitor test - Roche) better for 'ruling in', than 'ruling out' [126]. There is a need for prospective studies in sub-Saharan Africa to monitor HCMV viral loads in patients with HIVassociated pneumonia, and infants with congenital HCMV infection, the two major HCMV disease groups in the region – although there are also transplant recipients in sub-Saharan Africa [127]. HCMV is shed in high loads in both urine and saliva (non-invasive specimens ideal for low income settings) and detection of virus DNA in these specimens should be evaluated versus viraemia, as potentially useful markers of active disease.

Several drugs are licensed for the treatment of HCMV infections, although they are expensive and broadly unavailable in sub-Saharan Africa. At some tertiary referral centres in South Africa, intravenous ganciclovir is used to treat HCMV pneumonia in HIV-infected and exposed children failing antibiotic or anti-mycobacterial therapy. Decisions are largely consultant led but two descriptive studies have reported dramatic reductions in mortality due to ganciclovir [106, 107]. Readers are advised to look up the latest guidelines on treatment of HCMV and to check the correct doses, side effects and dosing schedules. In South African centres, PCR or culture-proven HCMV disease is typically treated with 5mg/kg intravenously every 12hrs for 14-21 days, and then daily maintenance therapy at 5mg/kg [94]. But there is an urgent need for further descriptive studies to identify patients for treatment. Randomized controlled clinical trials are needed to evaluate safety and efficacy.

types, although IE gene transcripts have not been detected in T- or B-lymphocytes [145, 146]. Despite this, HCMV influences cell-mediated immunity. T-cell populations in HCMV-infected infants in the Gambia showed higher levels of differentiation [147, 148] and similar HCMVinduced differentiation in elderly non-African populations is associated with depleted naive T-cell populations and impaired vaccine responses [149]. HCMV infection is also associated with a decline in naive T cells and impaired T-cell reconstitution in HIV infected adults initiating HAART [150]. But naive T-cell populations appear unaffected by HCMV infection in African children, and infection was not linked with impaired T-cell responses to measles virus vaccination [151], with HCMV activated T-helpers possibly improving measles antibody response [152]. A study in older African children, found that HIV-negative Malawian teen‐ agers had a lower percentage of naïve T cells, higher memory T and higher CD28- memory Tcells, compared to age-matched UK teenagers. Whilst all of the adolescents tested in Malawi were seropositive for HCMV, seroprevalence was just 36% in the UK group, and was associated with a reduced percentage of naïve T cells and an increased percentage of CD28- memory T

Human Cytomegalovirus (HCMV) Infection in Sub-Saharan Africa

http://dx.doi.org/10.5772/54907

25

Whilst more evidence is required, these studies suggest early infant infection with HCMV, and maybe a general higher burden of infectious disease, contribute to a more rapid ageing of the immune system in sub-Saharan Africa. Whilst access to anti-HCMV drugs would likely significantly reduce morbidity and mortality in acute HCMV infections, such as congenitally infected infants or HIV/AIDS patients with pneumonia or disseminated HCMV, the implica‐ tions of a successful HCMV vaccine have potentially far-reaching benefits across the region. Future studies evaluating vaccine efficacy in sub-Saharan Africa should stratify by HCMV serostatus, and where facilities permit, include work on HCMV genotypes and flow cytometric

In sub-Saharan Africa, HCMV infection is endemic in young infants where it is linked with impaired physical and mental development [3], giving the infection a unique epidemiology across the region, with a potentially broad-reaching impact on the health of southern African populations. Studies conducted in sub-Saharan Africa and elsewhere, have shown that HCMV is a serious cause or morbidity and mortality, in both immunocompromised groups and congenitally infected children. In a region where 23.2 million people are living with HIV and most of the population are infected with HCMV in infancy [124], more prospective studies are required to better characterise the impact of HCMV in sub-Saharan Africa. This will lay the foundations for future clinical trials of anti-HCMV drugs in patient sub-sets in whom there is strong evidence that they might be effective. Drugs such as ganciclovir are already used in South Africa as life-saving treatment for HIV-infected children with severe pneumonia that is not responsive to antibiotic or anti-mycobacterial therapy. Furthermore, the clinical impact and importance of HCMV infections in sub-Saharan Africa may increase over the next decade for several reasons: Wider access to ART is resulting in increasing numbers of older HIV infected patients; Cancer incidence is forecast to increase by 32% across sub-Saharan Africa

analysis to further characterise the effect of infant HCMV infection on immunity.

cells in the periphery [153].

**7. Summary**

The introduction of expensive antiviral treatments in low income settings is always problem‐ atic; Ethics review boards may state that it is unethical to trial antiviral drugs which are unaffordable and inaccessible to the majority of the affected patients. The path to new treatments has to start somewhere, and as scientists we favour evidence as the basis for action. The case of CD4 testing and antiretroviral therapy has proven that resources can be mobilized from a range of stake holders, including governments, NGOs and private enterprise [128-130]. A second ethical dilemma is that if the drug is being used successfully in South Africa to treat HIV-associated HCMV pneumonia, is it ethical for Ganciclovir trials to administer placebos? When answering such ethical questions we should note that HCMV affects a broad range of patient groups across sub-Saharan Africa, including congenitally infected neonates, HIV infected infants, children and adults causing pneumonia, specific organ disease (eg. retinitis, encephalitis, gastritis) and disseminated infection. Furthermore, malnutrition and co-infection with other common pathogens (Malaria, Tuberculosis, *Pneumocystis Jirovecci* etc..) are preva‐ lent. For this diverse patient group, the evidence base for the optimal dose, duration and route of administration is poor [131].

#### **6. Effect of HCMV on vaccine efficacy and immune senescence in Sub-Saharan Africa**

Infant vaccination programmes are a central component of national paediatric disease prevention strategies in sub-Saharan Africa [132], but they are less effective than equivalent programmes in high income populations. For example: The efficacy of live attenuated measles virus vaccine in Europe and North America is over 90% [133-135] whereas in West Africa it is below 70% [136-138]. This could be partly due to the higher infectious disease burden in sub-Saharan Africa, which may affect antibody [139, 140] and cytokine [141] responses to vacci‐ nation, and also reduced vaccine performance in HIV-infected children [132]. With 3.1 million children living with HIV/AIDS across the region [56], vaccine safety and efficacy must be independently assessed in this significant and vulnerable patient group. HIV-infected children can generally seroconvert in response to both live-attenuated and inactivated/subunit vac‐ cines, but the immune response is generally weaker with lower antibody levels and seropro‐ tection rates in HIV-infected children [142-144]. The weaker immune response in HIV-infected children could be due to defective antigen presentation, defective B-cell priming or impaired differentiation into memory cells, impaired primary response due to low CD4, loss of protec‐ tive antibodies or loss of immunological memory of T and B cells after priming [143].

Most HIV-infected children in sub-Saharan Africa will also be infected with HCMV, which encodes over thirty genes with potential immunomodulatory functions. These genes may affect classical and non-classical major histocompatibility complex (MHC) protein function, leukocyte migration and activation, cytokine responses and host cell susceptibility to apoptosis [43]. HCMV can infect and initiate gene expression in an extraordinarily broad range of cell types, although IE gene transcripts have not been detected in T- or B-lymphocytes [145, 146]. Despite this, HCMV influences cell-mediated immunity. T-cell populations in HCMV-infected infants in the Gambia showed higher levels of differentiation [147, 148] and similar HCMVinduced differentiation in elderly non-African populations is associated with depleted naive T-cell populations and impaired vaccine responses [149]. HCMV infection is also associated with a decline in naive T cells and impaired T-cell reconstitution in HIV infected adults initiating HAART [150]. But naive T-cell populations appear unaffected by HCMV infection in African children, and infection was not linked with impaired T-cell responses to measles virus vaccination [151], with HCMV activated T-helpers possibly improving measles antibody response [152]. A study in older African children, found that HIV-negative Malawian teen‐ agers had a lower percentage of naïve T cells, higher memory T and higher CD28- memory Tcells, compared to age-matched UK teenagers. Whilst all of the adolescents tested in Malawi were seropositive for HCMV, seroprevalence was just 36% in the UK group, and was associated with a reduced percentage of naïve T cells and an increased percentage of CD28- memory T cells in the periphery [153].

Whilst more evidence is required, these studies suggest early infant infection with HCMV, and maybe a general higher burden of infectious disease, contribute to a more rapid ageing of the immune system in sub-Saharan Africa. Whilst access to anti-HCMV drugs would likely significantly reduce morbidity and mortality in acute HCMV infections, such as congenitally infected infants or HIV/AIDS patients with pneumonia or disseminated HCMV, the implica‐ tions of a successful HCMV vaccine have potentially far-reaching benefits across the region. Future studies evaluating vaccine efficacy in sub-Saharan Africa should stratify by HCMV serostatus, and where facilities permit, include work on HCMV genotypes and flow cytometric analysis to further characterise the effect of infant HCMV infection on immunity.

#### **7. Summary**

urgent need for further descriptive studies to identify patients for treatment. Randomized