INTRODUCTION
Herpesviridae, a family of viruses that infect humans, consists of eight separate species: herpes simplexvirus 1 (HSV-1), herpes simplex virus 2 (HSV2), varicella zoster virus (VZV), the Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8).1 Their genome contains double-stranded DNA. These viruses are recognized as causing a diversity of clinical syndromes ranging from minor cutaneous lesions to life-threatening illnesses, particularly in the immunocompromised host. Indeed, one of the most important characteristics of all herpes viruses is their ability to cause lifelong latent infection.2 Many clinical syndromes are associated with human herpes viruses (HHVs), including cutaneous lesions, mononucleosis, retinitis, pneumonitis, and hepatitis.3 However, primary herpesvirus infections in an immunocompetent patient, with the exception of VZV, are usually asymptomatic or with minor illness. Therefore, primary herpesvirus infections may remain clinically unrecognized. When symptoms do occur, primary infections in the immunocompetent patient are usually self-limiting and require only symptomatic treatment. Following the primary infections, herpes viruses establish latent infection in patient-specific cells, and then can reactivate, especially in immunocompromised individuals. Herpesvirus infections in the immunocompromised patients are frequently severe and sometimes life threatening. In patients who manifest symptoms with their herpesvirus infections, skin lesions (including the oral cavity) are very common.4
Measles is a highly infectious disease that is preventable with safe, effective, and inexpensive vaccine.5 The WHO African region aimed to eliminate measles by 2020.6,7 Many of these countries currently achieve high vaccination coverage rates7 alongside the disease surveillance programs. Measles is characterized by a generalized maculopapular rash, fever, cough, coryza (running nose), conjunctivitis, and photophobia.6 However, with the prospect of elimination, in many countries, the definition of the nature of the cutaneous rash is less acute, to avoid the missing of true measles cases. Hence, other etiologies are very probable. In this study, we assessed retrospectively, with molecular tools, the contribution of HHVs, including HSV, CMV, EBV, and VZV in measles-suspected cases’ sera collected between 2014 and 2017 in Senegal.
MATERIALS AND METHODS
Study population and patient data collection.
Case definition and clinical specimen.
In this retrospective study, sera were collected as part of the measles and rubella surveillance in Senegal. The WHO standard measles case definition (WHO, 2010) was used. Therefore, a suspected case of measles was defined as a person with fever, rash, and one of the following symptoms: cough, coryza, or conjunctivitis. Once a suspected measles case was identified, clinicians completed a case-based form in duplicate and collected a blood specimen for the measles reference laboratory testing. Blood samples were collected within 30 days of the rash onset and were shipped at a controlled temperature (4°C) and processed immediately on arrival at the laboratory for measles/rubella confirmation by the ELISA test. Aliquots of samples were also stored at −20°C for molecular investigations.
Serological studies for measles and rubella testing.
Serum specimens were tested in the laboratory for serological evidence of measles and rubella etiologies by detecting specific IgM antibodies by ELISA test with WHO-recommended kits (Enzygnost Anti-Measles-Virus/IgM from Dade Behring or Siemens) using manufacturers’ instructions. A measles or rubella laboratory-confirmed case was defined by an IgM absorbance value greater than 0.2 of the blank value.
DETECTION OF HERPES VIRUSES
DNA extraction.
DNA was extracted using QIAamp DNA Mini Kit from 140 µL of each clinical specimen following the manufacturer’s instructions. DNA was eluted with 60 μL nuclease-free water and stored at −80˚C until use.
Real-time PCR tests.
For molecular detection, each sample was tested for the following viruses: CMV, EBV, VZV, HSV, and HHV6. Tests were performed in a singleplex using specific primers and probe for each virus. The TaqMan Universal PCR Master Mix Kit (Applied Biosystems, Vilnius, Lithuania) was used for real-time amplifications. For each sample and virus, real-time PCR was carried out in a total reaction volume of 25 μL consisting of 6 μL nuclease-free water, 0.5 μL of each primer (diluted at 10 μM), 0.5 μL of probe (diluted at 10 μM), 12.5 μL of TaqMan 2X Universal PCR Master Mix, and 5 μL of DNA template under the following cycling conditions: initial denaturation step of 10 minutes at 95°C, followed by 40 PCR cycles of 15 seconds at 95°C and 1 minute at 55°C. For each test, controls (positive and negative) were included for validation.
LightCycler PCR.
Some sera samples were tested with the LightCycler® Multiplex RNA Virus Master (Roche) using the LightCycler® 480 instrument II. For the test, a 5-μL aliquot of extracted nucleic acid was added to 15 μL of PCR mixture consisting of 10.5 μL of water PCR-grade, 0.5 μL of reagent mix (including primers and probes), and 4 μL of Roche Master for each sample. The multiplex PCR was carried out under the following cycling conditions: denaturation step of 5 minutes at 95°C followed by 45 PCR cycles of 05 seconds at 95°C, 15 seconds at 60°C, and 15 seconds at 72°C, and a cooling step of 30 seconds at 40°C.
Statistical analysis.
All data obtained from suspected cases and the laboratory results were compiled in an Excel spreadsheet. Subsequent analysis was performed using R statistical software version 3.0.1. Categorical variables and their association with herpes viruses were analyzed with Fisher’s exact or the χ2 test where appropriate. P-value < 0.05 was considered statistically significant.
Ethical considerations.
The surveillance protocol of measles/rubella was approved in its guidelines by the Senegalese National Ethical Committee of the Ministry of Health, and finalized with the support of Pasteur Institute in Dakar and the WHO in the perspective of the measles elimination policy. Anonymized data were collected as part of routine surveillance, and written consent is judged not necessary by the Senegalese National Ethics Committee. Therefore, the consent was verbal. The patients included in this study were of all ages (from one month to 60 years) and were consulted by local healthcare workers for a cutaneous rash.
RESULTS
Patient characteristics.
During the 4-year surveillance period (2014–2017), a total of 3,358 serum specimens from measles/rubella suspected cases have been tested for five herpes viruses (VZV, EBV, CMV, HSV, and HHV6). Of these 3,358 samples, 996 were collected in 2014 (29.7%), 806 in 2015 (24%), 1,015 in 2016 (30.2%), and 541 specimens in 2017 (16.1%). The patients’ ages varied from 1 month to 60 years, with a median age of 4 years 11 months and a mean age of 6 years 1 month. The male to female ratio was 1.23 (1829/1,483).
Regarding the age, nearly half of the overall suspected cases were children younger than 5 years (49.4%) followed by 5- to 10-year age-group with 33.05% and 10- to 20-year age-group with 12.4%. Patients older than 20 years represented 3.4% of suspected cases, and for 61 (1.8%) patients, the age was not documented.
The most common symptoms of the 3,358 enrolled patients were fever (96.6%) and cutaneous rash (95.6%), the primary inclusion criterions. Rhinitis and conjunctivitis were also recorded with, respectively, 40.9% and 26.3% of the overall suspected cases (Table 1).
Clinical and demographic characteristics of measles/rubella suspected cases in Senegal from 2014 to 2017
| Year | 2014 | 2015 | 2016 | 2017 | Total |
|---|---|---|---|---|---|
| Tested | N = 996 | N = 806 | N = 1,015 | N = 541 | N = 3,358 |
| Gender, n (%) | |||||
| Female | 457 (45.9) | 359 (44.54) | 467 (46.0) | 200 (40.0) | 1,483 (44.2) |
| Male | 525 (52.7) | 435 (54.0) | 529 (52.1) | 340 (62.8) | 1829 (54.5) |
| Missing data | 14 (1.4) | 12 (1.5) | 19 (1.9) | 1 (0.2) | 46 (1.4) |
| Age-group (years), n (%) | |||||
| 0–5 | 452 (45.4) | 405 (50.2) | 533 (52.5) | 268 (49.5) | 1,658 (49.4) |
| 5–10 | 367 (36.8) | 270 (33.5) | 307 (30.2) | 166 (30.7) | 1,110 (33.0) |
| 10–20 | 127 (12.7) | 103 (12. 8) | 109 (10.7) | 78 (14.4) | 417 (12.4) |
| > 20 | 35 (3.5) | 23 (2.8) | 32 (3.1) | 22(4.1) | 113 (3.4) |
| Missing | 15 (1.5) | 5 (0.6) | 34 (3.3) | 7 (1.3) | 61 (1.8) |
| Clinical signs, n (%) | |||||
| Fever | 971 (97.5) | 793 (98.4) | 951 (93.7) | 529 (97.8) | 3,244 (96.6) |
| Rhinitis | 335 (33.6) | 335 (41. 6) | 495 (48.8) | 209 (38.6) | 1,374 (40.9) |
| Skin rash | 981 (98.5) | 788 (97.8) | 930 (91.6) | 510 (94.3) | 3,209 (95.6) |
| Conjunctivitis | 209 (21.0) | 208 (25.8) | 326 (32.1) | 142 (26.2) | 885 (26.3) |
Measles and rubella serology confirmation results.
Of the 3,358 serum specimens tested by IgM ELIZA during the 4-year surveillance period, 227 (6.7%) were measles laboratory-confirmed cases and 39 (1.2%) measles equivocal cases, whereas 152 (4.5%) rubella laboratory-confirmed cases were observed and 101 (3%) equivocal cases (Table 2).
Measles and rubella serology confirmation results from suspected cases in Senegal during the study period (2014–2017)
| Year | 2014 | 2015 | 2016 | 2017 | Total |
|---|---|---|---|---|---|
| Total tested | N = 996 | N = 806 | N = 1,015 | N = 541 | N = 3,358 |
| IgM ELIZA | |||||
| Measles | 16 (1.6) | 51 (6.3) | 146 (14.4) | 14 (2.6) | 227 (6.7) |
| Equivocal | 15 (1.5) | 6 (0.7) | 16 (1.6) | 2 (0.4) | 39 (1.2) |
| Rubella | 108 (10.8) | 18 (2.2) | 14 (1.4) | 12 (2.2) | 152 (4.5) |
| Equivocal | 48 (4.8) | 23 (2.8) | 16 (1.6 | 14 (2.6) | 101 (3) |
Detection of herpes viruses.
During the study period, HHVs were detected in 1763 (52.5%) cases of the 3,358 sera analyzed for the five herpes viruses. The age of patients infected with HHVs in this study ranged from 1 month to 55 years, with mean and median ages of 6 years 1 month and 4 years 10 months, respectively, whereas the male to female ratio was 1.3 (993/749). Varicella zoster virus was the most frequently detected viral agent with 44.3% (1,487/3,358), followed by EBV with 10.7% (358/3,358), CMV 6.6% (221/3,358), HHV6 2.6% (87/3,358), and HSV 0.2% (8/3,358). Of the 1763 positive samples, single viral infection accounted for 80% (1,411/1763), whereas infections with multiple viruses were observed in 20% (352/1763), including 312 double infections, 34 triple infections, and six quadruple infections. The most common coinfections were VZV and EBV or VZV and CMV (169 and 81 cases, respectively). The highest detection rate was observed in 2015 (78.5%, 633/806) and the lowest in 2014 (29.4%, 293/996), whereas in 2016 and 2017, we noted herpes viruses in 48% (487/1,015) and 64.3% (348/541), respectively.
Varicella zoster virus, EBV, CMV, and HHV6 were mostly detected in male patients, with more than half of the overall positive cases (55.2%, 59.8%, 66.8%, and 56.3%, respectively), whereas HSV was more frequently observed in female patients, with 62.5% (5/8).
With regard to the viral detection per age-group, VZV, EBV, CMV, and HHV6 were more frequently observed in children younger than 5 years (41%, 58.4%, 73.6%, and 70.1%, respectively), whereas HSV was only detected in young children (0–5 and 5–10 years with, respectively, 37.5% and 50%). In adult patients (≥ 20 years), VZV and EBV were detected at similar rates with, respectively, 3.2% and 3.3%, whereas CMV and HHV6 were encountered only in three (1.3%) and one (1.1%) cases, respectively.
Taking into account the clinical symptoms and viral detection, the majority of the 1763 positive patients experienced fever and skin rash (97% and 95.6%, respectively). Rhinitis (36.7%) and conjunctivitis (23.4%) were also observed in relatively high proportions among the positive cases (Table 3).
Human herpes virus detection rates, age, and clinical distribution of measles/rubella suspected cases in Senegal, 2014–2017
| Pathogens | Varicella zoster virus | Epstein-–Barr virus | Cytomegalovirus | Herpes simplex virus | Human herpesvirus 6 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Positive | P value | Positive | P value | Positive | P value | Positive | P value | Positive | P value | |
| Gender, n (%) | ||||||||||
| Male | 821 (55.2) | 0.506* | 213 (59.8) | 1* | 147 (66.8) | 1* | 3 (37.5) | 1* | 49 (56.3) | 1* |
| Female | 646 (43.5) | 0.488† | 143 (40.2) | 0.1† | 74 (33.6) | 0.001† | 5 (62.5) | 0.313* | 37 (42.5) | 0.827* |
| Missing data | 19 (1.3) | – | 0 (0.0) | – | 0 (0.0) | – | 0 (0.0) | – | 1 (1.1) | – |
| Age-group (years), n (%) | ||||||||||
| 0–5 | 610 (41.0) | 0.000* | 208 (58.4) | 0.0005* | 162 (73.6) | 0.000* | 3 (37.5) | 0.726* | 61 (70.1) | 0.0001† |
| 5–10 | 577 (38.8) | 0.000* | 105 (29.5) | 0.12* | 45 (20.5) | 0.000* | 4 (50) | 0.452* | 16 (18.4) | 0.002* |
| 10–20 | 223 (15) | 0.000* | 31 (8.6) | 0.02* | 6 (2.7) | 0.000* | 0 (0.0) | 0.606* | 7 (8) | 0.279† |
| ≥ 20 | 48 (3.2) | 0.846* | 12 (3.3) | 1* | 3 (1.3) | 0.134† | 0 (0.0) | 1* | 1 (1.1) | 0.368* |
| Missing data | 29 (2.0) | – | 2 (0.6) | – | 5 (2.3) | – | 1 (12.5) | – | 2 (2.3) | – |
| Clinical signs, n (%) | ||||||||||
| Fever | 1,445 (97.2) | 0.048* | 343 (96.3) | 0.57† | 215 (97.7) | 0.70* | 8 (100) | 1* | 85 (97.7) | 0.769* |
| Rhinitis | 501 (33.7) | 0.000* | 159 (44.7) | 0.16† | 105 (47.7) | 0.04* | 3 (37.5) | 1* | 40 (46.0) | 0.322* |
| Skin rash | 1,433 (96.4) | 0.02* | 334 (93.8) | 0.05* | 210 (95.5) | 0.738* | 7 (87.5) | 0.312* | 79 (90.8) | 0.0598* |
| Conjunctivitis | 314 (21.1) | 0.000* | 94 (26.4) | 1* | 71 (32.3) | 0.048* | 2 (25) | 1* | 32 (36.8) | 0.035* |
| Year, n (%) | ||||||||||
| 2014 | 264(17.7) | – | 36(10) | – | 16 (7.3) | – | 2 (25) | – | 2 (2.3) | – |
| 2015 | 537(36.1) | – | 175(49) | – | 119 (54) | – | 3 (37.5) | – | 45 (51.7) | – |
| 2016 | 421(28.3) | – | 78(21.8) | – | 40 (18.2) | – | 2 (25) | – | 15 (17.2) | – |
| 2017 | 265(17.8) | – | 68 (19) | – | 45 (20.4) | – | 1 (12.5) | – | 25 (28.7) | – |
| Total, n (%) | 1,487 (44.3) | – | 357 (10.6) | – | 220 (6.5) | – | 8 (0.2) | – | 87 (2.6) | – |
P-values in bold are considered statistically significant.
* Fisher’s exact test.
† Chi-square test.
Seasonal distribution.
The temporal distribution pattern on a monthly basis of the detected viruses was also investigated and is shown in the figure. Varicella zoster virus, CMV, and EBV were detected throughout the study period, with a clear seasonal pattern. These herpes viruses were mostly detected from February through April, with a detection peak in March each year. However, with regard to HHV6, the infection occurred intermittently with no clear temporal pattern. The number of HSV-positive cases was very low, ranging from zero to one case per month, which appeared insufficient to allow for the description of seasonal pattern.
DISCUSSION
In the last two decades, PCR-based diagnostics have been used as a valuable tool to investigate viral diseases.8 The present study was conducted to evaluate retrospectively by RT-qPCR the contribution of five HHVs, including VZV, EBV, CMV, HHV6, and HSV from suspected cases in Senegal after four consecutive years of measles and rubella surveillance period (2014–2017). Like in many other studies,9,10 60.1% (2019/3,358) of the suspected cases had been experimentally confirmed. However, the distribution of detected viral pathogens differed. Our study showed that only 6.7% and 4.5% of the 3,358 suspected cases during the study period were laboratory confirmed by IgM ELIZA for measles and rubella, respectively. However, 52.5% of the overall positives cases were attributable to HHVs, suggesting that cutaneous rash illness in Senegal is often caused by viruses other than measles and rubella. Our data show that the number of measles and rubella cases observed during the study period was relatively low when compared with the findings of Dia et al.11 and Dromigny et al.12 Indeed, Dia et al.11 demonstrated that 21.4% measles laboratory cases were confirmed between 2004 and 2013, and the study of Dromigny et al.12 confirmed that 90.1% rubella cases were found in a seroepidemiological survey carried out from January 1996 to December 2001. Our low number in comparison to the two previous studies is not particularly surprising because the combined measles and rubella vaccine has been integrated into the Expanded Program on Vaccination (EPV) in Senegal since 2013. Varicella zoster virus was the most common viral agent encountered, with 44.3% of the overall positive patients. This high prevalence of VZV is in agreement with that of Schmutzhard et al.13 in Sweden, with a detection rate of 46%. However, the VZV overall positive in our study is high compared with the 17.4% reported by Espy et al.14 or the 2.7% noted by Zhou et al.15 Unlike our results, most similar studies found that EBV was the most prevalent viral pathogen.16,17 In this report, EBV was the second most common herpes virus among the suspected cases after VZV, with 10.7% of the total number of detected viruses. This overall prevalence is consistent with reports from other studies15,18 but lower than rates reported in previous works conducted in other geographical areas, including Mexico with 20%19 and Germany with 16.4%.20 Consistent with many other results,10,16,21 the detection rates of CMV (6.6%), HHV6 (2.6%), and HSV (0.2%) were relatively low. Coinfections were relatively common in this study (20%), and the most frequently co-detected viruses were VZV/EBV or VZV/CMV. However, it should be pointed out that the use of different diagnostic methods explains some discrepancies in rates of detection in different areas: primarily in their sensitivity and second in the number of targeted viruses. Moreover, differences in detection rates can also be attributed to geographical differences in overall burden, differences in study populations (outpatients or hospitalized patients), the number of patients tested, the sampling period, and even the duration of the study.
Our study confirmed the clear influence of the patients’ gender on HHV infection. Varicella zoster virus, EBV, CMV, and HHV6 were mostly detected in male patients, whereas HSV was more frequently observed in female patients. This finding is in line with reports from other studies.16,22
Regarding the distribution of the targeted HHV per age-group, VZV, EBV, CMV, and HHV6 positive cases were found in all age-groups, whereas HSV was only encountered in patients aged between 0 and 10 years. As expected, children younger than 5 years were the most infected group with HHVs. Consistent with our findings, several studies have reported this sensitivity of children younger than 5 years to human herpes virus infection.23,24 Nonetheless, it is important to note that this highest detection rate of HHVs in children younger 5 years can be explained by the bias in the composition of our study population (nearly 50% of children younger than 5 years).
The study also investigated the circulation pattern of HHVs during the 4-year study period. Varicella zoster virus, CMV, and EBV showed a similar circulation profile with a clear seasonality, whereas HHV6 and HSV showed no seasonality. Indeed, VZV, CMV, and EBV were detected among suspected cases throughout the study period, with the highest incidence from February through April each year.
CONCLUSION
In summary, this is the first study in Senegal to characterize five common HHVs in suspected cases during four consecutive years of measles and rubella surveillance period. Using RT-qPCR, herpes viruses were detected in 52.5%, with VZV (44.3%) as the leading viral agent. Our study confirmed children younger than 5 years as being more susceptible to HHV infection. Globally, our findings also identified a clear seasonal pattern of VZV, EBV, and CMV, with the highest incidence between February and April each year.
Results of this investigation provide more information on cutaneous rash syndrome etiologies in patients sampled in the framework of measles/rubella surveillance in Senegal, which is useful for the guidance of both case definition revision and clinical practice, as well as for public health policy.
Monthly distribution of detected herpes viruses during the study period (2014–2017). Bars represent the number of positive cases for each month.
Citation: The American Journal of Tropical Medicine and Hygiene 104, 6; 10.4269/ajtmh.20-1444
Monthly distribution of detected herpes viruses during the study period (2014–2017). Bars represent the number of positive cases for each month.
Citation: The American Journal of Tropical Medicine and Hygiene 104, 6; 10.4269/ajtmh.20-1444
Monthly distribution of detected herpes viruses during the study period (2014–2017). Bars represent the number of positive cases for each month.
Citation: The American Journal of Tropical Medicine and Hygiene 104, 6; 10.4269/ajtmh.20-1444
Acknowledgments:
We acknowledge the Senegalese ministry of health for their help in implementing Measles surveillance.
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