Context : Human immunodeficiency virus (HIV) patients are at higher risk for Human cytomegalovirus (HCMV) infection. Aim: To identify HCMV and HIV co-infection among Nigerian patients for prompt therapeutic interventions. Materials and Methods: The study drew samples from the antiretroviral clinic of Nigerian Institute of Medical Research and patients’ informed consent was taken at enrollment. Variables collected included socio-demographic characteristics such as sex, occupation, marital status, educational status, income, and religion, while health-related variables were CD4 counts and HIV viral load. Genomic DNA from whole blood samples of 236 patients, 164 (69.5%) females and 72 (30.5%) males, was subjected to polymerase chain reaction (PCR) amplification of 2 genes within conserved immediate early (IE) and late (LA) transcribed regions of HCMV genome. Statistical Analysis: Statistical Package for Social Sciences was used to determine frequencies of HMCV infection, while Chi square was used to examine associations between patient’s characteristics and HMCV infection. Results: A total of 35 (14.8%) patients; 25 (10.6%) females and 10 (4.2%) males were positive for HCMV infection. Although there was variation in prevalence of HCMV in different marital status, it was statistically insignificant (P = 0.734; P > 0.05). Results also showed that 22 (62.9%) of HCMV positive patients had HIV viral load greater than 10,000, prevalence of HCMV decreased as CD4 counts increased, while 12 (34.3%) of HCMV positive patients had CD4 counts between 1 and 200. Conclusions: This study is the first molecular survey of HCMV/HIV co-infection in Nigeria and has provided valuable information for prompt therapeutic intervention to reduce morbidity among HIV patients.
Keywords: Co-infection, human immunodeficiency virus, human cytomegalovirus, Nigeria
According to the United States Agency for International Development (USAID), approximately 3.5 million Nigerian adults and children were living with human immunodeficiency virus/acquired immune deficiency virus (HIV/AIDS) by the end of 2001.  USAID also projected an increase in the number of new AIDS patients from 250,000 in 2000 to 360,000 by 2010. People living with HIV have compromised immune systems and are at a much greater risk for serious repercussions from common illnesses and opportunistic infections (OIs), which are caused by pathogens, bacterial, viral, fungal, or protozoan, in an immune compromised host and are the main culprits responsible for full blown AIDS and eventual death of HIV patients. Some of the common HIV-related OIs include (i) bacterial diseases such as tuberculosis, pneumonia, and septicemia, (ii) protozoan diseases such as microsporidiosis and leishmaniasis, (iii) fungal diseases such as candidiasis and cryptococosis, and (iv) viral diseases such as human cytomegalovirus (HCMV) and viral hepatitis.
HCMV is a species-specific deoxyribonucleic acid (DNA) herpes viral genus virus of the Herpesviridae family, in humans it is commonly known as human herpes virus 5 (HHV-5). HCMV belongs to the Betaherpesvirinae subfamily of Herpesviridae, which also includes Roseolovirus. Other herpesviruses fall into the subfamilies of Alphaherpesvirinae, including HSV 1 and 2 and varicella or Gammaherpesvirinae, including Epstein-Barr virus. Other HCMV viruses are found in several mammal species, but species isolated from animals differ from those from humans in terms of genomic structure. HCMV infection causes high morbidity and mortality, it is endemic worldwide and is more widespread in developing countries, particularly in areas of low socio-economic conditions.  HCMV infections are usually asymptomatic in healthy individuals. The virus initially enters the body through the epithelium of the upper alimentary, respiratory, or genitourinary tracts. After primary infection, it establishes a lifelong latency, called a nonproductive infectious state, in its host and can be reactivated by a number of different stimuli, including immune-suppression and inflammation. Latent HCMV can be found in mononuclear cells, lungs, spleen and cervix,  prostate, kidneys, and secretory glands.  Reactivation occurs mostly in immune-suppressed persons as well as in elderly because the immune system gets weaker with age  and can also occur in normal individuals without causing clinical disease. The infection often results in life threatening health conditions in individuals with impaired or underdeveloped immune systems including organ transplant recipients, HIV-infected individuals, and the fetus or newborn infants. In transplant recipients, HCMV is the single most important pathogen due to its frequent direct and indirect effects on both morbidity and mortality.  For HIV/AIDS patients, HCMV reactivations and re-infections are common problems, while it is a leading cause of congenital infection in newborns worldwide.  Immune-compromised individuals may develop pneumonitis, enterocolitis, encephalitis, polyradiculopathy, retinitis, hepatitis, myocarditis, pancreatitis, virus-induced host immune suppression, and allograft rejection.
HCMV infection is also strongly associated with an increased incidence of fungal infections  and bacterial infections particularly with Gram-negative organisms.  The infection in persons living with HIV can last for weeks or months and can be life threatening and may require many weeks of hospital treatment. HCMV retinitis is reported to have become one of the most common manifestations of the virus and has been shown to be the most common intraocular viral OI and a common cause of irreversible visual loss in patients with AIDS because the disease is usually progressive without treatment. ,, It has also been described as the neglected disease of the AIDS pandemic.  Autopsy studies  have also shown clinical/pathological evidence of HCMV in up to 81% of cases, while 32% had HCMV retinitis.
Laboratory methods for the diagnosis of HCMV infections are evolving and include serology, antigen detection, virus culture, tissue histopathology, and nucleic acid detection. However, its diagnosis remains difficult because of the issues of virus latency, virus infection versus clinical disease, and virus reactivation.  IgG avidity testing is a useful sero-diagnostic test to differentiate a new infection from a reactivation while culture assays have limited utility especially in immune compromised patients. Quantitative culture methods have been used to predict disease or follow a patient’s response to therapy. Recently, alternative approaches have emerged, of which the polymerase chain reaction (PCR) is especially promising for its high sensitivity, specificity, and relative simplicity.
Early diagnosis of HCMV co-infection in HIV/AIDS patients is therefore essential in improving the quality of life of people living with HIV/AIDS (PLWHA). There have been a few cases of sudden loss of vision in newly registering patients in the antiretroviral (ARV) clinic of Nigerian Institute of Medical Research (NIMR) Yaba, Lagos State, Nigeria. Some of these patients have been examined by ophthalmologists and subjected to laser surgery before being referred to the ARV clinic, however, with little or no benefit. This investigation therefore will be useful in identifying patients with a high risk of end organ damage from HCMV, for early commencement of highly active antiretroviral therapy (HAART), as a preemptive measure. In this study, we applied qualitative PCR to determine HCMV infection in HIV patients attending NIMR ARV clinic and applied statistical analysis to correlate HCMV infection with patients’ demographic data, CD4 counts, and HIV viral load. Positive cases were followed up for further ophthalmologic evaluation and initiation of preemptive therapy and early HAART.
Ethical/institutional approval and consent documentation
Ethical approval for the study was obtained from the Institutional Review Board (IRB) of the (NIMR-IRB). In addition, approval was obtained from the Harvard School of Public Health, USA prior to the study. Informed consent of patients attending the NIMR-ARV clinic was taken during enrollment at the clinic.
The study site was the ARV clinic of NIMR, Yaba, Lagos State, Nigeria. Laboratory analysis was performed at the Molecular Parasitology Research Laboratory, Public Health Division NIMR.
The study was cross sectional and involved 236 newly registered HIV infected patients who attended NIMR ARV clinic between June 2010 and January 2011.
Collection of socio-demographic, virological, hiv0 viral load and cd4 counts data
These data were extracted from the electronic database of patients attending ARV clinic in NIMR. To ensure confidentiality and privacy of the patients’ health records during data collection, data retrieved from the electronic database were de-linked from all identifiers, such as name, that could be used in tracing the data to the patients. Variables collected included socio-demographic characteristics such as sex, occupation, marital status, level of education, income, and religion. Health-related variables collected were CD4 counts and HIV-1 viral load. Every participant was allocated a unique study code for sample identification.
Genomic DNA extraction
Genomic DNA (gDNA) was extracted from whole blood samples collected from each of the 236 participants using GenSpin TM Genomic DNA Purification Kit (Whatman Incorporated, USA) according to the manufacturers’ instructions. The extracted gDNA was then stored at 4°C before PCR assay.
The extracted gDNA was subjected to PCR amplification using two pairs of primers – LA1 5′ CCG CAA CCT GGT GCC CAT GG 3′, LA2 5′ CGT TTG GGT TGC GCA GCG GG 3′; IE1 5′ GCT GCG GCA TAG AAT CAA GGA GCA C 3′ and IE2 5′ GGT TGG TGG TCT TAG GGA AGG CTG AG 3′, which are known to anneal to well conserved immediate early (IE) and late (LA) transcribed regions of HCMV genome.  PCR amplifications was performed using illustra™ pureTaq Ready-To-Go PCR Beads (Life Sciences, USA) in the Thermal Cycler (BioRad, USA). Amplified products were subjected to electrophoresis and analyzed in 2% agarose gels followed by photo documentation using Gel Documentation and analysis System (Clinx Science Instruments, USA).
Data was analyzed using Statistical Package for Social Sciences (SPSS) (Windows version 16.0; SPSS Inc, Chicago, IL, US), to determine the frequencies of HMCV infections. Chi Square Test of Association was also used to examine associations between patient’s characteristics and the infection.
Background characteristics of patients
[Table 1] shows that 72 (30.5%) of the patients were males, 164 (69.5%) were females, majority were Christians (89.4%), 6.4% and 0.4% were Muslims and traditionalists, respectively, while 9 (3.8%) of them did not have any record concerning their religion. The distribution of patients by marital status shows that more of married people 125 (53.0%) were examined, 64 (27.1%) were single, 7 (3.0%) were separated, 6 (2.5%) were divorced, 22 (9.3%) were widowed, while 12 (5.1%) of the patients had no record of their marital status. Four (1.7%) of the patients had no form of education, 43 (18.2%) had primary education, 99 (41.9%) had secondary education, while 64 (27.1%) had tertiary education and 26 (11.0%) had no record about their level of education. The participants were of diverse occupational status 0.28.0% were into business or trading, 4.7% were artisan, 2.5% of the participant were house wives, 5.1% were civil servant, 0.8% were private sector employee, 5.9% of the respondents were students, 9.3% were unemployed, and 0.4% were clergies. [Table 2] also shows that 219 (92.8%) of the participants fall within ages 21-50 years.
Prevalence of HCMV infection
[Table 3] shows that there was a positive correlation between HCMV prevalence and sex (r = -0.001) as more females than males were positive for HCMV co-infection, while the difference was not statistically significant (X 2 = 0.989; P > 0.05). [Table 3] shows that, though there was variation in prevalence of HCMV in different marital status, but the variation was not statistically significant (X 2 = 0.208; P > 0.05). [Table 4] shows that most of the participants that were positive for HCMV were also within the age bracket of 21-50 years and the difference in the prevalence of the infection among the age groups was statistically significant (t = 2.36; P = 0.05). There was significant positive correlation between the age groups and the prevalence of HCMV (r = 0.988; P = 0.0001; P < 0.005).
HCMV infection versus patients’ health related characteristics
[Figure 1] shows that most of the patients who were positive for HCMV had HIV viral load greater than 10,000. Although, there was a positive correlation between HCMV and HIV viral load (r = 0.025), the relationship was not statistically significant (P = 0.734; P > 0.05) in this study. However, the HCMV prevalence dereased as the CD4 count increased [Figure 2] and 10 (32.3%) of HCMV positive patients had CD4 counts between 1 and 200 showing a positive correlation between HCMV prevalence and CD4 counts (r = 0.613).
HCMV is a severe pathogen especially for immuno-compromised individuals and can be found globally, while in developing countries, the study of HCMV co-infection in HIV infected patients remains neglected. Whereas in laboratories equipped with PCR facilities, detection of HCMV DNA may be of value to detect patients at high risk of the disease. Although qualitative PCR is a sensitive technique and is the gold standard diagnostic tool for predicting HCMV disease, it does not necessarily correlate with active HCMV infection, since latently present viral DNA or incomplete viral genomes may be amplified. , However, the detection of HCMV late mRNA may improve the ability to identify patients at risk for the development of HCMV disease, as it directly reflects viral transcriptional activity. 
Following reports that a positive HCMV PCR result is a strong predictor of the disease by different studies conducted before and after HAART in other countries, ,,, we applied a qualitative PCR technique to amplify both the immediate early (IE) and late (LA) transcribed genes located within conserved regions of HCMV genome so as to estimate the prevalence of HCMV in whole blood samples of newly diagnosed HIV infected Nigerian patients. The results of our study were generally consistent with those of other reports from developing countries where HCMV infection occurs particularly in immune-compromised individuals. ,,,, We therefore report for the first time in Nigeria, a study evaluating the prevalence of HCMV infection by qualitative PCR in HIV infected patients.
In this study, the prevalence of HCMV infection, which was 14.8% in newly diagnosed HIV patients, indicated the endemicity of HCMV infection among the patients and may be possibly due to the fact that their immune status was already compromised by the HIV infection. Also the PCR method used in this study seemed to be sensitive because as all positive PCR samples were repeatedly positive, which makes the assay ideal for clinical diagnostic use and corroborated previous observations by other researchers that PCR is a rapid, sensitive, and specific test for detecting HCMV DNA in blood samples of patients. Thirty five (14.8%) HIV infected patients who were found to be co-infected with HCMV were referred to the clinician and ophthalmologist for further investigations and treatment.
Source of Support: Nigerian Institute of Medical Research, Yaba, Lagos State, Nigeria, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]