Dengue Virus Serotypes 1 and 2 Responsible for Dengue Burden in Amazonas: Epidemiological and Clinical Features

ABSTRACT.

The increase in dengue cases in Amazonas poses a public health risk. This study aimed to examine the circulating serotypes in the region of Amazonas, Peru, from 2021 to 2023, along with the associated clinical and epidemiological characteristics. Serotype identification was determined for 420 serum samples using a multiplex real-time reverse transcription–polymerase chain reaction assay, revealing that 194 patients were infected by dengue virus (DENV)-1 and 226 by DENV-2. Both serotypes co-circulated in all affected provinces. Warning signs were more frequent in patients with DENV-2 (P = 0.004), and the manifestation of abdominal pain was correlated with this serotype (P = 0.004), highlighting its association with severe forms of the disease. In conclusion, this study emphasizes the widespread circulation of DENV-1 and DENV-2 in Amazonas. Furthermore, it underscores the importance of real-time molecular surveillance that could provide an early prognosis of disease severity.

Dengue, one of the most important arboviruses, causes around 390 million infections and 29,000 deaths annually.¹ There are four serotypes of the dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), and although initial exposure typically confers immunity to that specific serotype, subsequent heterologous infections result in a higher risk of developing a severe dengue disease that manifests as shock or respiratory distress, severe bleeding, and organ involvement.² The widespread distribution of the main vector, Aedes aegypti, and the concurrent circulation of various serotypes have contributed to an increased incidence of dengue across the Americas.³ In Peru, the reentry of DENV has been documented since 1990 with the presence of the DENV-1 serotype.⁴ The 1995–1996 epidemic revealed the emergence of the DENV-2 American genotype followed by the presence of the four serotypes by 2001, triggering outbreaks in different areas of the country.⁵

In 2018, Peru experienced 4,698 cases caused by DENV-1 genotypes III and V, as well as DENV-2 American and American/Asian genotypes. Since October 2019, a shift has been noted with the appearance of the DENV-2 Cosmopolitan genotype co-circulating with DENV-1 genotype V, resulting in 15,287 occurrences.^6,7 This trend continued, and in 2020 and 2021 Peru reported 47,932 and 44,971 cases, respectively, both representing about a 200% increase compared with 2019. In the following year, Amazonas experienced a peak with 3,571 cases, and by epidemiological week 36 of 2023, five out of seven provinces and 18 out of 84 districts were affected, reporting 3,062 cases with 361 presenting warning signs and eight classified as severe dengue cases. In addition, four deaths were recorded resulting in a fatality rate of 0.1%, and the cumulative incidence rate in the region was 700.73 cases per 100,000 inhabitants.^8,9 This study aimed to examine circulating serotypes in different provinces of Amazonas and the associated sociodemographic and clinical-epidemiological characteristics.

A total of 420 serum samples from dengue-confirmed patients by positive non-structual protein 1 tests, collected in five provinces of Amazonas between October 2021 and September 2023 as part of epidemiological surveillance by the Regional Health Directorate (DIRESA)-Amazonas, were included in the study (Figure 1). RNA was extracted using the Patho Gene-Spin DNA/RNA Extraction Kit (iNtRON, Kyungki-Do, South Korea). Dengue serotype detection was performed by a multiplex real-time reverse transcription–polymerase chain reaction (RT-PCR) assay using the TransScript II Multiplex Probe One-Step qRT-PCR SuperMix UDG kit (Transgen Biotech, Beijing, China) and serotype-specific primers and probes designed to amplify the envelope gene in a QuantStudio 5 thermocycler, based on a protocol established by the Peruvian National Institutes of Health.¹⁰ Demographic, clinical, and epidemiological data were obtained from DIRESA-Amazonas. Statistical analysis was performed using R software v. 4.2.2. This study was funded by the project with contract N° 050-2021-FONDECYT, which was approved by the Institutional Research Ethics Committee of the Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas.

Figure 1.

Geographical distribution of dengue serotypes of 420 dengue-confirmed patients collected between October 2021 and September 2023 in Amazonas. DENV = dengue virus.

Dengue samples were collected in Bagua (n = 59; 4,222.63 cases/100,000 inhabitants), Utcubamba (n = 188; 1,616.97 cases/100,000 inhabitants), Condorcanqui (n = 91; 2,023.64 cases/100,000 inhabitants), Chachapoyas (n = 43; 472.84 cases/10,000 inhabitants), and Bongará (n = 39; 14.29 cases/10,000 inhabitants) (Figure 1). Chachapoyas and Bongará experienced recent dengue outbreaks in 2021 and 2023, respectively.^9,10 The results of serotype-specific RT-PCR revealed that 194 of the patients were infected by DENV-1 and 226 by DENV-2. These serotypes have been reported to co-circulate in Amazonas since 2020.

Univariate analysis revealed significant associations between dengue serotypes and the following variables: sex, year of diagnosis, location, and clinical classification (Table 1). A higher proportion of men were diagnosed with DENV-1 (58.3%, P = 0.006) compared with women. Notably, the distribution of DENV-1 and DENV-2 changed over time (P = 0.005). Both serotypes displayed significantly different distributions across the locations, as depicted in Figure 1 (P <0.001); DENV-1 predominated in Bagua (56%) and Bongará (90%), whereas DENV-2 prevailed in Chachapoyas (74%), Condorcanqui (59%), and Utcubamba (59%) (Figure 1).

Table 1.

Sociodemographic characteristics of patients stratified by dengue serotype

Variables	Total (N = 420)	DENV-1 (N = 194)	DENV-2 (N = 226)	P-Value*
	n (%)	n (%)	n (%)
Sex
Male	213 (50.71)	113 (58.25)	100 (44.25)	0.006
Female	207 (49.29)	81 (41.75)	126 (55.75)
Age Groups (years)
≤10	53 (12.62)	29 (14.95)	24 (10.62)	0.102
11–20	117 (27.86)	57 (29.38)	60 (26.55)
21–30	76 (18.10)	27 (13.92)	49 (21.68)
31–40	72 (17.14)	37 (19.07)	35 (15.49)
41–50	45 (10.71)	23 (11.86)	22 (9.73)
51–60	29 (6.90)	8 (4.12)	21 (9.29)
>60	28 (6.67)	13 (6.70)	15 (6.64)
Mean (±SD)	29.18 (±17.78)	28.33 (±18.01)	29.92 (±17.57)	0.363
Year of Diagnosis
2021	17 (4.04)	3 (1.54)	14 (6.19)	0.005
2022	26 (6.19)	7 (3.61)	19 (8.40)
2023	377 (89.77)	184 (94.85)	193 (85.41)
Location
Bagua	59 (14.05)	33 (17.01)	26 (11.50)	<0.001
Bongará	39 (9.29)	35 (18.04)	4 (1.77)
Chachapoyas	43 (10.24)	11 (5.67)	32 (14.16)
Condorcanqui	91 (21.67)	37 (19.07)	54 (23.89)
Utcubamba	188 (44.76)	78 (40.21)	110 (48.67)
Fever Day at Presentation, Mean (SD)	2.63 (±1.73)	2.59 (±1.79)	2.67 (±1.69)	0.622
Dengue Classification†
Dengue without Warning Signs	378 (90.00)	184 (94.85)	194 (85.84)	0.004
Dengue with Warning Signs	40 (9.52)	10 (5.15)	30 (13.27)
Severe Dengue	2 (0.48)	0 (0)	2 (0.89)

DENV = dengue virus. Values in bold are statistically significant (P-value <0.05).

^*Across DENV serotypes (Pearson’s χ² test, Fisher’s exact test, Welch two-sample t-test).

^† According to clinical manifestation: dengue without warning signs presents with nausea, rash, headache, muscle or joint pain, and/or leukopenia; dengue with warning signs presents with intense abdominal pain, persistent vomiting, fluid accumulation, mucosal bleed, lethargy, and/or increase in hematocrit; severe dengue presents with shock or respiratory distress, severe bleeding, and/or severe organ involvement.

Dengue infections with warning signs were more frequently observed in patients with DENV-2 compared with DENV-1 (P = 0.004). Additionally, severe dengue cases were exclusively reported in DENV-2 infections, highlighting its association with severe forms of the disease. Conversely, age and days of fever on admission did not exhibit significant differences by serotype. The mean age of patients was 29 years, and the average duration of fever on admission was three days.

A Poisson regression with robust variance was used to explore the relationship between clinical manifestations and DENV serotypes. Fever (DENV-1: 93.8% and DENV-2: 88.9%) and headache (DENV-1: 88.1% and DENV-2: 87.2%) were the most prevalent symptoms (Figure 2). Severe abdominal pain or tenderness exhibited a significant correlation with DENV-2; individuals infected with this serotype were more likely to exhibit this warning sign than were those with DENV-1 (prevalence ratio: 3.11; 95% CI: 1.42–6.81; P = 0.004). Other symptoms such as fever, headache, myalgia, arthralgia, retroocular pain, lumbar pain, nausea, rash, conjunctivitis, chest pain, and persistent vomiting were not significantly associated with a specific serotype. Moreover, six patients with DENV-1 infection and five with DENV-2 were hospitalized. It is noteworthy that the two patients with severe dengue were infected with DENV-2 and manifested warning signs such as serous effusion, hypothermia, diuresis decrease, hepatomegaly, jaundice, altered mental status, and hematocrit increase, as well as severe signs such as hypovolemic shock and severe bleeding, and one of them showed a Glasgow coma scale score of 3.

Figure 2.

Relationship between clinical manifestations and DENV serotypes using a Poisson regression with robust variance. Values in bold are statistically significant (P <0.05). *Reference. CI = confidence interval; DENV = dengue virus; PR = prevalence ratio.

This study confirmed the concurrent circulation of DENV-1 and DENV-2 in Amazonas, as the Peruvian National Institutes of Health reported, with the genotypes V and Cosmopolitan, respectively; however, we have a difference in the serotype distribution between some provinces that could be attributed to the sample size analyzed.^8,9 These two serotypes are widespread across all affected provinces, including the recently affected areas of Chachapoyas (DENV-1: 26%, DENV-2: 74%) and Bongará (DENV-1: 90%, DENV-2: 10%). The emergence of the virus in these provinces may have occurred through various means, including infected human migration and transportation of mosquitoes or desiccated eggs in vehicles.¹¹ Given that these provinces are part of the main terrestrial corridor of Amazonas, with connecting routes for trade and tourism, they are linked to dengue-endemic regions in both northwestern Peru and Loreto, facilitating the spread of DENV.¹⁰

Despite the relatively limited sample size from 2021 and 2022, the higher proportion of DENV-2 cases aligns with reports suggesting that the new genotype of DENV-2 may have replaced the previously circulating genotype and expanded into new areas of Amazonas. This phenomenon could be attributed to its heightened adaptability to both humans and vectors across a broader geographical range, leading to outbreaks characterized by a rapid and notable increase in DENV-2 cases.^11,12 Such outbreaks may be associated with increased clinical severity in Brazil, Colombia, and Venezuela^13–15 and an elevated dengue-related mortality rate, especially in the Amazon region of Peru, reminiscent of the crisis in health service demand experienced in 2010 with the introduction of the American/Asian genotype DENV-2.¹⁶

The enhanced pathogenicity of the DENV-2 serotype could be due to its efficient and fast replication, leading to a higher viral load. Furthermore, its ability to stimulate higher levels of nitric oxide production compared with other serotypes may contribute to toxic and inflammatory effects, resulting in complex symptoms.¹⁷ Clinical observations have revealed that DENV-2 infections often exhibit warning signs and severe manifestations, such as persistent vomiting, abdominal pain, severe bleeding, and dengue shock. Notably, the DENV-2 Cosmopolitan genotype has been highly prevalent among patients experiencing abdominal pain and has also been associated with dengue shock,¹⁸ which is related to the results shown in Figure 2 and is evidenced by the two severe cases.

Although information regarding past dengue infections was not provided, the concurrent presence of both dengue virus serotypes, even in nonendemic areas, raises significant concern about secondary infections with DENV-2 after previous infection with DENV-1, which may result in greater severity contributed by the waning cross-protection.^19,20 In addition, it is important to mention that Ae. aegypti has been documented at elevations reaching up to 1,980 meters above sea level, as observed in control initiatives conducted in localities of Amazonas. Despite the current absence of reported dengue cases in these areas, their climatic conditions remain optimal for the potential transmission of dengue.

Nevertheless, this study has limitations. Uneven sample size distribution across provinces may have influenced serotype prevalence findings. Insufficient data on prior dengue infections hindered understanding of secondary infection dynamics and their association with severe cases. Variability in serum sample quality could also have affected result accuracy. These limitations highlight the importance of broader studies to expand upon our findings.

In conclusion, this study emphasizes the widespread circulation of DENV-1 and DENV-2 in Amazonas, highlighting the necessity to understand its transmission dynamics. Furthermore, it underscores the importance of real-time molecular surveillance that could provide an early prognosis of disease severity. The risk of primary and particularly secondary infections with DENV-2, often associated with warning signs or severe manifestations, poses significant public health concerns. Future research should prioritize investigating genotype distribution, transmission routes, and the impact of co-circulating serotypes to develop more effective control measures.