Molecular Diagnosis of Congenital Cytomegalovirus Infection Among Neonates in Tertiary Health Institutions in Jos: A Cross-Sectional Study

2. Materials and Methods

2.1. Study Design and Setting

This cross-sectional molecular study was conducted between January 2021 and December 2022 among neonates attending the Special Care Baby Unit and immunization units of three tertiary health facilities in Jos metropolis, Plateau State, Nigeria: Jos University Teaching Hospital (JUTH), Plateau State Specialist Hospital (PSSH), and Bingham University Teaching Hospital (BhUTH).

2.2. Study Population and Sample Size

Neonates aged ≤21 days presenting to the selected health facilities were eligible for inclusion. Written informed consent was obtained from parents or guardians prior to enrolment. Neonates were consecutively recruited, and no samples were excluded from analysis.

The minimum sample size was calculated using the formula

N = (Z² × p × q × D) / d²,

where Z = 1.96 (95% confidence level), p = 0.038 (prevalence of congenital CMV infection reported by Olusanya et al., 2015), q = 1 − p, d = 0.05 (margin of error), and D = 2 (design effect). This yielded a minimum sample size of 112 neonates. To improve statistical power, the final sample size was increased to 180 neonates.

2.3. Data Collection

Sociodemographic, clinical, and maternal data were collected using a structured data collection form. Information obtained included neonatal sex, age, anthropometric measurements, clinical features, maternal demographic characteristics, and ward of recruitment.

2.4. Sample Collection and DNA Extraction

Buccal swab samples were collected under aseptic conditions by brushing the inner cheek with a sterile flocked swab. Samples were transported under cold-chain conditions to the molecular laboratory and stored at −80 °C until DNA extraction.

Genomic DNA was extracted using the Zymo-Spin™ IIC column–based protocol (Zymo Research, USA) in accordance with the manufacturer’s instructions. DNA was eluted in ≥50 µL of elution buffer and stored at −20 °C until polymerase chain reaction (PCR) analysis.

2.5. PCR Detection of Human Cytomegalovirus

Human cytomegalovirus (HCMV) DNA was detected using an in-house nested PCR assay targeting the glycoprotein B (gB) gene, employing primers adopted from previously published studies (Choudhary et al., 2015). The outer primer pair (gB1138/gB1638) amplified a 661-bp fragment, while the inner primer pair (gB1276/gB1524) produced a 249-bp fragment (Table 1).

PCR amplification was performed for 35 cycles under standard conditions. HCMV strain AD169 was included as a positive control, and nuclease-free water served as a negative control. PCR products were resolved on 1.5% agarose gel and visualized under ultraviolet illumination using a molecular size ladder.

2.6. Sequencing and Phylogenetic Analysis

Purified PCR products were subjected to Sanger sequencing using the ABI Prism BigDye Terminator v3.1 Cycle Sequencing Kit and analyzed on an ABI 3730/3730xl DNA Analyzer, following the manufacturer’s instructions. Sequence similarity analysis was performed using the Basic Local Alignment Search Tool (BLAST). Phylogenetic analysis was conducted using MEGA version 11. The generated nucleotide sequence was deposited in the GenBank database.

2.7. Statistical Analysis

Data were analyzed using standard statistical software. Continuous variables were summarized as means and standard deviations, while categorical variables were presented as frequencies and percentages. The molecular prevalence of congenital CMV infection was calculated with exact 95% confidence intervals using the Clopper–Pearson method. Due to the low number of PCR-positive cases, inferential statistical analyses were not performed, and results were reported descriptively.

2.8. Ethical Approval

Ethical approval for the study was obtained from the institutional ethics committees of the participating hospitals. Written informed consent was obtained from parents or guardians prior to enrolment. All data were anonymized to ensure participant confidentiality.

3. Results

3.1. Study Population Characteristics

Between January 2021 and August 2022, a total of 180 neonates aged ≤21 days were enrolled from three healthcare facilities in Jos, Nigeria: Jos University Teaching Hospital, Plateau State Specialist Hospital, and Bingham University Teaching Hospital. Of the enrolled neonates, 102 (56.7%) were male and 78 (43.3%) were female.

The majority of neonates (145/180; 80.6%) were aged 1–5 days, while 21 (11.7%) were aged 6–10 days, and 14 (7.8%) were older than 10 days. The age and sex distribution of the study population is summarized in Table 2.

3.2. Detection of Congenital CMV Infection and Maternal Characteristics

Out of the 180 neonatal samples analyzed, one neonate (0.6%) tested positive for CMV DNA by PCR. Owing to the presence of only a single PCR-positive case, statistical comparisons between CMV-positive and CMV-negative groups were not performed.

Maternal demographic characteristics, including age group, marital status, parity, occupation, educational level, and ward of recruitment, are therefore presented descriptively according to neonatal CMV PCR status in Table 3. The CMV-positive neonate was born to a mother aged ≤25 years, in a monogamous marriage, with parity of 1–2, secondary education, and recruited from the Special Care Baby Unit (SCBU).

3.3. Molecular Detection of CMV

PCR amplification targeting the cytomegalovirus glycoprotein B (gB) gene yielded a positive result in one sample (Sample 144). Agarose gel electrophoresis demonstrated a distinct band at approximately 661 bp, corresponding to the expected size of the outer PCR amplicon. No amplification was observed in the remaining samples or in the negative control (Plate 1).

3.4. Sequence Analysis and Phylogenetic Characterization

The CMV-positive PCR product was successfully sequenced using the Sanger method. BLAST analysis of the nucleotide sequence showed 98.9% similarity to Human herpesvirus 5 strain HAN22. The sequence was deposited in GenBank under the accession number PV668598.

Phylogenetic analysis performed using MEGA version 11 revealed that the study isolate clustered within the HAN22 lineage, showing close genetic relatedness to CMV strains previously reported from southern Africa, including Zambia. In the phylogenetic tree, the study sequence is labeled “ISOLATE BELLO” to distinguish it from reference strains (Figure 1).