An important number of cases of MAYV were detected in patients with AFI that attended outpatient health-centers during this study. Approximately, a fifth of the total cases were diagnosed with MAYV, demonstrating that this pathogen may be circulating during this region and that peri-urban transmission may be ongoing. Moreover, approximately 6.4% of the total cases were co-infection between MAYV and DENV.
The association between MAYV and DENV is a topic of great interest for the scientific community, as both pathogens share common characteristics and can co-exist in a specific region. However, only two studies have previously reported co-infections of DENV and MAYV in the scientific literature, a child with an unspecified febrile illness, serologically diagnosed with MAYV, , and during an outbreak in Brazil . Our study reports the first clinical characterization of patients co-infected with MAYV and DENV during surveillance of AFI near the Peruvian Amazon basin for eight months. The frequency of unspecific symptoms among patients with mono-infection and co-infeccion were similar, including fever, myalgia, arthralgia, retroorbital pain, and headache. This finding further confirms the majority of symptoms amongst arboviral infections are non-specific, even when presenting as co-infections. [9, 24, 36, 37].
Pinheiro et al.  previously reported that the rash associated with MAYV infection appears on the fifth day of illness, suggesting its association with of humoral antibody appearance. In contrast, we found patients with a rash on the 3rd day of illness for MAYV, second day for co-infected patients, and the first day for DENV mono-infected patients.
Serologic evaluations for the detection of alphaviruses have shown cross-reactivity  as they belong to the Semliki complex serologic group [6, 7], denoting the importance of sensitive and specific molecular diagnostic methods such as RT-PCR. Previous studies report that viremia is detectable for up to 5 days post-infection . However, we evidenced RT-PCR detection of MAYV up to 9 days in mono-infected patients and up to 7 days in co-infected patients. Our findings evidence that the window of detection for MAYV by RT-PCR could be longer than reported previously. Altogether, these characteristics make the RT-PCR an excellent diagnostic tool for the detection of MAYV during outbreaks.
Furthermore, our studied population showed no significant difference of positive cases between genders. A previous study determined that being male poses a risk for arboviral infections, given the higher occupational exposure . Given that spillover zoonosis is considered the main source of recent arboviral outbreaks, this could have led to the peri-urban transmission of the disease . Similarly, to our study, another outbreak in Brazil caused by peri-urban transmission of MAYV, showed that both genders were affected equally .
Additionally, we found a greater number of cases of both DENV and MAYV in May and July. This could be explained by some meteorological factors that could influence the vector expansion, behavior and biology. According to the meteorological national service (SENAMHI) the temperature and rainfall peak during the months of March and April; however, are still high during May and June, with a further decline in the later months. These factors altogether could enable a more easily transmission of the disease. Considering previous studies on the adaptability of alphaviruses to novel vectors , evidence of effective MAYV transmission by more urban anthropophilic mosquitoes in laboratory studies [30, 31, 35], and high aedic index reported in the studied region (aedic index 1–4%) , these findings may suggest that a common vector could be responsible for the transmission of both viruses during this outbreak. Further demographic and on-site vector studies are necessary to determine if the urbanization of MAYV is ongoing.
In conclusion, this study provides the first clinical characterization of patients co-infected with MAYV and DENV and also reports the first outbreak of MAYV-DENV co-infections, confirmed by molecular diagnostic methods. Our findings also provide further evidence that symptoms in co-infected patients are non-specific and that disease severity may not be associated with co-infections.