1. Introduction
Encephalitis is a severe neurological syndrome associated with high morbidity and mortality, long-term neurological sequelae, and a significant burden on the healthcare system [1]. It is a relatively common disease, with a global incidence of 1.5 - 14 cases per 100,000 people annually [2]. In Africa, the annual prevalence of encephalitis is between 10% and 20% [3]. Data on the causes of encephalitis in West Africa are sparse, with infectious pathogens of the central nervous system typically identified through isolated case reports [4] [5]. Information specific to Côte d’Ivoire is particularly lacking. Nonetheless, regional studies suggest a rising incidence of viral encephalitis, especially linked to herpesviruses, with cases documented in various hospitals. Recent studies in urban areas estimate a prevalence of 5 - 8 cases per 100,000 people annually, although this figure can fluctuate depending on the diagnostic capabilities of local laboratories [3].
Encephalitis can result from a wide range of infectious and non-infectious etiologies. Bacterial, fungal, and parasitic agents, such as Mycobacterium tuberculosis, Listeria monocytogenes, Cryptococcus neoformans, and Toxoplasma gondii, remain important, particularly in individuals with compromised immune systems. Autoimmune encephalitis and paraneoplastic syndromes have been increasingly recognized in clinical practice [6]. However, viruses remain the predominant cause worldwide, with herpesviruses responsible for most cases of encephalitis [7] [8].
Among herpesviruses, HSV-1, HSV-2, VZV, EBV, CMV, and HHV-6 are the most frequently implicated, particularly in high-resource settings where their diagnosis is well established [9]. In contrast, in low- and middle-income countries, including those in West Africa, the burden is unknown because of limited access to advanced diagnostic tools and treatments.
Real-time PCR has emerged as a powerful tool for the rapid, sensitive, and simultaneous detection of multiple pathogens directly from cerebrospinal fluid [8] [10] [11]. This technique significantly improves the diagnostic yield, especially in settings where empirical treatment is common and laboratory infrastructure is limited. The implementation of molecular methods in routine diagnostic processes could significantly improve the early detection and management of herpes virus-associated encephalitis in Côte d’Ivoire and other similar settings with limited resources.
This study aimed to determine the prevalence of Herpesviridae in patients with encephalitis who were hospitalized in tertiary care centers in Côte d’Ivoire. The findings of this study are expected to highlight the role of herpesviruses in encephalitis cases and support the implementation of molecular tests, such as real-time PCR, as routine diagnostic tools in the national surveillance and management of encephalitis.
2. Material and Methods
2.1. Study Design and Participants
It is a cross-sectional, multicenter study carried out between March 2024 and April 2025 in teaching hospital centers in two regions of Côte d’Ivoire (Bouaké and Abidjan), that receive the majority of patients from different areas in Côte d’Ivoire with signs of encephalitis (fever, convulsions, altered consciousness). Informed consent was obtained from all participants in this study, either directly from the patient when conscious or from a legal representative if the patient was unable to provide consent.
Socioepidemiological and clinical data were collected from the patients’ medical records using a data collection form. Participants’ sociodemographic data, such as age and sex, were collected using a survey form. The clinical data were extracted from the patient files. The questionnaire included exposure history, symptoms, and clinical signs of encephalitis.
2.2. Inclusion/Exclusion Criteria
This study focused on patients aged 15 years or older who were admitted to the hospital with clinical signs consistent with encephalitis, such as fever, confusion, convulsions, or neurological deficits, and who provided their informed consent directly or indirectly through a legal representative. Patients with a confirmed non-infectious diagnosis and those with contraindications for lumbar puncture were also excluded from the study.
2.3. Sample Collection
A volume of 500 µL of cerebrospinal fluid (CSF) was collected from all patients included in the study under strict sterile conditions by lumbar puncture performed by trained medical staff. Samples were collected in sterile tubes and immediately transported to the Molecular Biology Unit of the CHU Treichville Central Laboratory for further analyses.
In the laboratory, samples were stored at –20˚C until molecular analysis was performed.
2.4. Laboratory Analysis
Cerebrospinal fluid samples were thawed at room temperature and gently homogenized. Viral DNA was extracted from 200 µL of CSF using a commercial extraction kit (PureLinkTM Genomic DNA Mini Kit, Invitrogen) according to the manufacturer’s protocol. The extracted DNA was immediately used for amplification or stored at –20˚C until analysis.
The extracted DNA was used as template for PCR reactions targeting 6 viruses of the Herpesviridae family, namely, Cytomegalovirus, Varicella-zoster virus, Herpes simplex type 1 and 2, Epstein Barr virus and Human Herpesvirus type 6. Table 1 shows the nucleotide sequences of the primers and probes used for the target genes of each of the viruses.
Table 1. Probes, primers and target genes for the viruses tested.
Herpesvirus |
Target |
Primers and hydrolysis probe TaqMan (5’→3’) |
Reference |
HSV 1/2 |
gB |
F CCGTCAGCACCTTCATCGA |
[12] |
R CGCTGGACCTCCGTGTAGTC |
P (FAM) CCACGAGATCAAGGACAGCGGCC (TAMRA) |
CMV |
US8 |
F ACCAACATAAGGACTTTTCACACTTTT |
|
R GAATACAGACACTTAGAGCTCGGGGT |
P (FAM) CTGGCCAGCACGTATCCCAACAGCA (TAMRA) |
EBV |
BXLF1 |
F GGGGCAAAATACTGTGTTAG |
|
R CGGGGGACACCATAGT |
P (FAM) CGGCGCATGTTCTCCTCCAC (TAMRA) |
HHV6 |
U65_U66 |
F GACAATCACATGCCTGGATAATG |
|
R TGTAAGCGTGTGGTAATGGACTAA |
P (FAM) AGCAGCTGGCGAAAAGTGCTGTGC (TAMRA) |
VZV |
ORF63 |
F CGCGTTTTGTACTCCGGG |
|
R ACGGTTGATGTCCTCAACGAG |
P (FAM) TGGGAGATCCACCCGGCCAG (TAMRA) |
F = forward; R = reverse; P = probe.
PCR was performed in a total volume of 20 µL, consisting of 15 µL of reaction mixture (4 μL of HOT FIREPol ® Plus qPCR master mix (without ROX), 0.5 μl of primers and probe corresponding to each virus target gene, and 9.5 µL of sterile nuclease-free water) and 5 µL of extracted DNA.
The cycling conditions for the reaction were as follows: initial denaturation at 95˚C for 15 min, followed by a cyclic phase repeated 40 times, including a denaturation step at 95˚C for 15 s and a hybridization-elongation step at 60˚C for 1 min. The Bio-Rad CFX96 C1000 thermal cycler (Bio-Rad Laboratories Inc., USA) was used for cycling and analysis of PCR results. Negative and positive samples for the different targets of interest were included in the tests as negative and positive controls, respectively, to validate the results. Samples with cycle threshold (Ct) values lower than 38 were considered positive. Samples with a clear amplification curve but Ct values greater than 38 were considered negative.
2.5. Data Management and Statistical Analysis
Data collected from the form were recorded using Excel version 2019 (Microsoft Corporation, Washington, USA), and analysed with statistical software R version 4.2.1 (The R Foundation, Auckland, United States). Statistical analysis was used to assess the distribution of demographic and clinical characteristics and the associations between the various parameters.
A descriptive analysis was performed to summarize the data collected. Summary frequencies and proportions were used to describe all nominal characteristics, including sex and patient symptoms. The mean and standard deviation were used to describe quantitative variables. Comparisons between groups were made using the Chi2 test (or Fisher’s exact test when numbers were small) for categorical variables. For all analyses, a value (P < 0.05) was considered statistically significant.
2.6. Ethics
This study was conducted after administrative authorization was granted by the management of the various participating teaching hospitals, and approval was obtained from the Comité National d’Éthique des Sciences de la Vie et de la Santé (CNESVS), which examined and approved the study protocol with reference number 00211 24/MSHPCMU/CNESVS-km. Written informed consent was obtained from all the patients. For patients under the age of 18 years and those with altered consciousness, consent was obtained from their parents or legal representatives.
In addition, the study was conducted in accordance with the principles governing the conduct of research involving human subjects as set out in the Declaration of Helsinki [17].
3. Results
3.1. Demographic and Clinical Characteristics of Included Patients
This study included 388 participants in total. The majority (76.03%, n = 295) of the participants were from Treichville Teaching Hospital, followed by 15.46% (n = 60) from Angré teaching hospital, 7.22% (n = 28) from Bouaké teaching hospital, and 1.29% (n = 5) from Cocody teaching hospital. The participants comprised 222 (57.22%) women and 166 men (42.78%), with a sex ratio (M/F) of 1.34. The age of the participants ranged from 15 to 79 years, with a mean age of 45.36 ± 13.56 years. With regard to symptoms, the principal clinical manifestations observed in patients suspected of suffering from herpesvirus encephalitis were altered consciousness (confusion), fever, and convulsions. Fever was the most common symptom, observed independently in 13 patients (3.35%), or in association with altered consciousness (AC) in 185 patients (47.68%), with convulsions in 23 patients (5.93%), or with both manifestations in 26 patients (6.70%). Other symptoms were observed in 141 (36.34 %) patients. In addition, 282 patients (72.68%) included in this study were HIV-infected. The demographic and clinical characteristics of the patients are shown in Table 2.
Table 2. Summary of demographic and clinical characteristics of the patients.
Variable |
Category |
N (%) |
Age |
[15 - 29[ |
53 (13.66%) |
[30 - 44[ |
150 (38.66%) |
[45 - 59[ |
111 (28.61%) |
≥60 |
74 (19.07%) |
Gender |
Men |
166 (42.78%) |
Women |
222 (57.22%) |
Provenance |
Treichville |
295 (76.03%) |
Angré |
60 (15.46%) |
Cocody |
5 (1.29%) |
Bouaké |
28 (7.22%) |
Symptoms |
Fever |
23 (5.93%) |
Fever + AC* |
185 (47.68%) |
Fever + AC* + Seizures |
13 (3.35%) |
Fever + Seizures |
26 (6.70%) |
Other symptoms |
141 (36.34%) |
HIV Status |
HIV+ |
282 (72.68%) |
HIV− |
11 (2.84%) |
Not available |
95 (24.48%) |
*AC = altered consciousness.
3.2. Viral Etiologies
PCR test results revealed the presence of viruses in sixty (60) patients, corresponding to a prevalence of 15.46%. The Epstein-Barr virus (EBV) was the most frequently detected virus. Among the patients with a positive PCR for Herpesviridae, 10 (16.67%) were co-infected with at least two Herpesviridae viruses. The co-infection associations were varied and mostly included CMV, EBV and VZV. In 70% of cases, the most frequent associations were CMV + EBV, EBV + HHV6, VZV + HHV6 and CMV + VZV. Only 30% of cases showed triple associations of CMV + HSV + VZV, CMV + EBV + HHV6 and CMV + VZV + HHV6. Table 3 shows the detection frequency of each targeted herpesvirus in the study population.
Table 3. Prevalence of viruses detected.
|
PCR+ |
Prevalence of positivity (n = 60) |
Overall prevalence (n = 388) |
Virus |
EBV |
33 |
55.00% |
8.50% |
CMV |
20 |
33.33% |
5.15% |
VZV |
12 |
20.00% |
3.09% |
HHV6 |
7 |
11.67% |
1.80% |
HSV1/2 |
1 |
1.67% |
0.26% |
3.3. Factors Associated with Viral Infection
Analysis of demographic variables revealed no statistically significant association between age (P = 0.09) or sex (P = 0.54) and PCR positivity for Herpesviridae. Comparison between hospital centers revealed variable detection rates: 25% at Bouaké teaching hospital (7/28), 16.61% at Treichville teaching hospital (49/295), 6.7% at Angré teaching hospital (4/60), and 0% at Cocody teaching hospital. However, these differences were not statistically significant (P = 0.08).
From a clinical aspect, a significant association was observed between the detection of Herpesviridae and the concomitant presentation of fever and disorders of consciousness (P = 0.005). In addition, HIV infection appeared to be a risk factor strongly associated with PCR positivity in this study. HIV-positive patients represented 91.67% (55/60) of herpesvirus PCR-positive patients, compared with 1.67% (1/60) of HIV-negative patients and 6.67% (4/60) of patients with unknown status. This correlation was statistically significant (OR = 3.9; 95% CI: [1.4 - 9.3]; P < 0.001). All associations between clinical and epidemiological characteristics and PCR results are presented in Table 4.
4. Discussion
The introduction of virological diagnostic techniques in the laboratory reduces the burden on both patients and health services. Indeed, some encephalitis etiologies have specific treatments, such as herpesviruses, for which there are well-established antiviral treatments [18]-[20]. Rapid identification of the etiology is essential, as these neurological infections are fatal and therefore require rapid and effective laboratory tests to diagnose the etiological agents. Once the etiology is established, therapeutic interventions are less difficult.
Table 4. Summary of factors associated with PCR status.
Variable |
Category |
PCR+ (n = 60) |
PCR− (n = 328) |
P-value |
Age |
[15 - 29[ |
8 (15.09%) |
45 (84.91%) |
0.09 |
[30 - 44[ |
17 (11.33%) |
133 (88.67%) |
[45 - 59[ |
25 (22.52%) |
86 (77.48%) |
≥60 |
10 (13.51%) |
64 (86.51%) |
Gender |
Men |
23 (13.86%) |
143 (86.14%) |
0.54 |
Women |
37 (16.67) |
185 (83.33%) |
Provenance |
Treichville |
49 (16.61%) |
246 (83.39%) |
0.08 |
Angré |
4 (6.67%) |
56 (93.33%) |
Cocody |
0 (0.0%) |
5 (100.0%) |
Bouaké |
7 (25.0%) |
21 (75.0%) |
Symptoms |
Fever |
2 (8.70%) |
21 (91.30%) |
0.005 |
Fever + AC |
42 (22.70%) |
143 (77.30%) |
Fever + AC + Seizures |
0 (0.00%) |
13 (100.00%) |
Fever + Seizures |
7 (26.92%) |
19 (73.08%) |
Other symptoms |
9 (6.38%) |
132 (93.62%) |
HIV Status |
HIV+ |
55 (19.50%) |
227 (80.50%) |
< 0.0015 |
HIV− |
1 (9.1%) |
10 (90.90%) |
Not available |
4 (4.2%) |
91 (95.79%) |
In our study, encephalitis predominantly occurred in adult patients. This observation differs from those of studies conducted in other countries, such as Brazil [21], Australia [22] or Sweden [23]. In these studies, encephalitis was more frequent in the juvenile population. The mean age of the patients in the present study was 45.36 ± 13.57 years, and the predominance of females (57.22%) suggests a trend that diverges slightly from the global data. Indeed, viral encephalitis most often affects children and the elderly, in contrast to the results of the present study, which predominantly involved young adults. This difference could be explained by the medical history of the patients included in this study. Indeed, 91.67% of those infected with Herpesvirus were also HIV-positive, highlighting an interaction between these pathogens. By weakening the immune system, HIV promotes the reactivation of latent herpesviruses, which can lead to serious complications, such as encephalitis [24]. This observation is consistent with the literature, where it is well documented that HIV patients have an increased risk of herpesvirus encephalitis, particularly EBV, CMV, and HHV6 [25]. Young adults are more affected by HIV and are more vulnerable to opportunistic infections, including viral encephalitis. The hypothesis of co-infection favoring viral reactivation could explain this difference [26].
In terms of clinical signs, fever, disturbances of consciousness, and convulsions are characteristic of herpesvirus encephalitis, as described in other studies [27]. The frequencies observed in the present study were consistent with the results of other studies on viral encephalitis [28] [29]. PCR revealed that 15.46% of patients were positive for at least one herpesvirus. This rate is consistent with the expected figures for opportunistic infections in immunocompromised patients [30] [31]. However, the viral etiologies observed differed from those in other parts of the world. In this study, Epstein-Barr virus (EBV) was the most frequent pathogen, accounting for 55.00% of positive cases, followed by Cytomegalovirus (CMV), Varicella zoster virus and Human Herpesvirus type 6 (HHV6), and Herpes simplex virus 1 and 2, with 20.00%, 11.67%, and 1.67%, respectively.
This distribution is unusual in the literature. Indeed, Herpes Simplex virus (HSV) has been described in numerous studies as the most frequent cause of herpetic encephalitis, particularly in high-income countries [32]. However, this virus had the lowest rate in the present study. This low rate of HSV could be explained by local factors (vaccination, local viral prevalence, and influence of co-infections) or epidemiological factors specific to Côte d’Ivoire. Indeed, the high frequency of EBV in the present study could also be linked to the prevalence of HIV in the patients tested, since this virus is strongly associated with complications such as lymphoma in immunocompromised individuals [33].
The results of this study also highlight several significant associations between clinical and demographic factors, and the detection of Herpesviridae by PCR, with important clinical and epidemiological implications. The significant association between HIV infection and Herpesviridae detection (OR = 3.9; IC 95%: [1.4 - 9.3]; P < 0.0015) confirms the key role of immune status in Herpesviridae infections. This result corroborates global data highlighting the role of immunosuppression in the reactivation of latent herpesviruses, notably CMV and EBV [34] [35]. Previous studies have reported a higher prevalence of Herpesviridae infections in immunocompromised patients, sometimes reaching up to 20% - 30% in patients hospitalized for encephalitis [36]. The prevalence observed in this 55/282 study (19.50%) is therefore consistent with these data, underlining the importance of systematic screening for Herpesviridae in HIV + patients with neurological signs. In the sub-Saharan context, where HIV remains endemic, this association reinforces the need for systematic screening for Herpesviridae in HIV-positive patients, in line with WHO recommendations [3].
In this study, the rate of PCR positivity varied significantly between university hospitals. In Abidjan, the highest rate was observed at Treichville University Hospital (16.61%), compared to Angré (6.67%) and Cocody (0%). These disparities could reflect differences in the influx of patients or the severity of cases admitted. Treichville University Hospital, being a national referral center, probably receives more severe patients or cases referred from other establishments, which would explain the high rate of positive PCRs results [37]. However, the absence of PCR-positive cases at the CHU de Cocody could be due to low recruitment rates. Bouaké University Hospital (7 PCR + /28 patients) had a small number of patients (n = 28), which could limit statistical analysis, and is a frequent challenge in multicenter African studies [38]. The high rate of positive cases observed at Bouaké University Hospital (25%, 7/28) calls for particular attention. Bouaké, being located in a central area of Côte d’Ivoire, could reflect a higher prevalence in rural populations where access to early care is limited, or a higher concentration of untreated HIV + patients.
No statistically significant association was found between patient age (P = 0.09), sex (P = 0.54), and Herpesviridae detection. These results are consistent with previous studies, which indicate that Herpesviridae infections are not strictly linked to age groups or sex differences, although some studies report a slight male predominance for certain viral encephalitides, probably due to behavioral or exposure factors [39]. Clinically, the complete absence of PCR + in patients presenting with fever, altered consciousness, and convulsions is an intriguing finding. This result contrasts with Asian studies, where HSV-1 is often associated with convulsions [40]. This triad could be linked to bacterial, parasitic, or autoimmune causes, which are dominant in this subgroup [41]. The classic triad usually associated with herpetic encephalitis was absent in all PCR-positive patients. This suggests that Herpesviridae infections may present with varied or atypical clinical manifestations, particularly in immunocompromised patients. Studies have shown that fever alone, or associated with disturbances of consciousness without convulsions, can represent a significant proportion of clinical presentations [6] [42].
Bivariate analysis confirmed that HIV infection was an independent risk factor for PCR positivity (OR 3.9; IC 95%: [1.4 - 9.3]; P = 0.0015). This highlights the importance of integrating Herpesviridae screening into the management of HIV + patients, particularly those with neurological symptoms.
These results highlight the importance of tailored care for patients with neurological disorders, depending on their HIV status and their place of origin. Disparities between university hospital centers highlight the need to strengthen diagnostic capabilities and access to care in the country’s underserved areas. A longitudinal evaluation of Herpesviridae encephalitis cases and their associated factors is essential for improving prevention and management strategies.
5. Conclusion
This study highlights the high burden of herpesvirus encephalitis in immunocompromised patients in Ivorian hospitals. The high proportion of EBV and CMV viruses and the low rate of HSV suggest local epidemiological specificities or are probably linked to HIV infection. These results underline the importance of rapid detection, notably by PCR, for the appropriate management of herpesvirus encephalitis to reduce the morbidity and mortality associated with these infections.
Acknowledgements
We would like to express our deep gratitude to all the structures and institutions that contributed to this study. In particular, we would like to thank the following:
To Institut Pasteur de Côte d’Ivoire for expertise and scientific collaboration.
To Hospitals and Universities for help in collecting samples and participating in the study.
Medical teams from the Tropical Infectious Diseases Department provided logistical support and assistance in patient care.
Authors’ Contributions
Aboubacar Bamba, designed the study, drafted the protocol and the manuscript.
Aboubacar Bamba and Kobina Amandze Adams Kofi carried out the laboratory analyses.
Flora Ahonzo, Arouna Coulibaly, Sodji Emilie K N’Goran, Pacôme Monemo, Mbodje Ophélia Gnamon for collecting the samples.
Kalpy Julien Coulibaly, Eric Essoh Akpa, for reading the document.
Edgard Valery Adjogoua, Zélica Diallo, Pacôme Monemo, for coordinating activities on their behalf.
Kouadio Stephane Koffi validated the protocol and revised the document.
All authors have read and approved the manuscript.