Dengue and malaria are some of the most common vector-borne diseases in the world and still remain a major problem in the public health especially in tropical and subtropical areas like Pakistan. Dengue is an infectious disease caused by the dengue virus and is passed on by Aedes mosquitoes, which have been on an increasing trend over the last ten years and has recurrent outbreaks in urban areas. Plasmodium species cause malaria and are transmitted by Anopheles mosquitoes, and continue to be endemic in most rural and peri-urban regions. These vectors coexist in the same geographical areas, hence providing a favorable environment to the simultaneous transmission of the two infections [1-3].

Dengue and malaria have a number of overlapping clinical manifestations, such as acute febrile disease, headache, myalgia, and gastrointestinal tract, which are hard to distinguish in the initial stages. Although some of the symptoms like retro-orbital pain and bleeding would be more indicative of dengue and chills with rigors are more indicative of malaria, these differences are not always conclusive. Consequently, there is a likelihood that the co-infection could be missed especially in low-resource based healthcare systems where the diagnostic centers are limited [4-6].

In recent years, the dengue-malaria co-infection has been in the spotlight because of its possible effect of aggravating the clinical outcome. It has been suggested that co-infected patients could have more severe disease, such as severe thrombocytopenia, dysfunction of multiple organs, an extended hospital stay, and increased mortality rates. The interaction between the two pathogens can be synergistic, which can increase immune responses and endothelial dysfunction, resulting in a more severe disease. Nevertheless, although it is clinically significant, the actual burden of co-infection is underreported in Pakistan, in part due to limited surveillance and absence of routine dual testing [7-9].

Understanding the prevalence and clinical implications of dengue and malaria co-infection is essential for improving patient management and reducing complications. Early identification of co-infection can guide appropriate therapeutic strategies and prevent delays in treatment. Therefore, this study was designed to investigate the prevalence and clinical outcomes of dengue and malaria co-infection in endemic regions of Pakistan, with the aim of providing evidence to support improved diagnostic and management approaches in clinical practice.

This multi-center cross-sectional study was conducted over a period of one year, from January 2025 to December 2025, to investigate the prevalence and clinical outcomes of dengue and malaria co-infection in endemic regions of Pakistan. Patients presenting with acute febrile illness and suspected of dengue, malaria, or both infections were enrolled from multiple tertiary care hospitals. The sample size was calculated using the WHO single population proportion formula:n = Z² × p (1 − p) / d², where Z = 1.96 at a 95% confidence level, p = 25% (anticipated prevalence of co-infection based on regional data), and d = 9% margin of error. The calculated sample size was approximately 80, which was rounded up to 82 participants. A consecutive non-probability sampling technique was employed to recruit eligible patients during the study period. The participants of the study were adult patients aged 1865 years of either gender who had a fever (>38 C) and laboratory-confirmed dengue infection (NS1 antigen and/or IgM serology) and/or malaria (peripheral blood smear and/or rapid diagnostic test). The patients were classified into three, namely dengue mono-infection, malaria mono-infection, and dengue-malaria co-infection. Other known infections (e.g., typhoid, COVID-19), chronic liver or renal disease, hematological diseases, or incomplete clinical data were not included in the study. The structured proforma was used to collect data and included demographic information, clinical presentation, and laboratory results. Symptoms like fever duration, headache, myalgia, rash, bleeding manifestations and hepatosplenomegaly were considered clinical variables. Laboratory parameters encompassed complete blood count (hemoglobin, platelet count, total leukocyte count), liver function tests (ALT, AST), and renal (serum creatinine) and disease-specific diagnostic tests. Indicators of disease severity, including shock, hemorrhage, organ dysfunction and ICU admission were also noted. Clinical outcomes such as length of stay, complications, recovery status and mortality were recorded. All data collected were entered and analyzed with IBM SPSS Statistics version 25. Continuous variables were reported in the form of mean standard deviation and categorical variables were reported in form of frequencies and percentages. Independent sample t-test or one-way ANOVA was used to compare mono-infection and co-infection groups on continuous variables and Chi-square was used to compare mono-infection and co-infection groups on categorical variables. A p-value of 0.05 or less was deemed to be statistically significant. In cases where necessary, 95 percent confidence intervals were determined to determine the accuracy of estimates

The number of patients was 82. The mean age was 34.7 ± 12.6 years, with the majority in the 21–40 years age group (45.1%). It was male dominated (65.9%). The majority of the respondents were urban (61.0%), with more tertiary care centers reporting. There was a relative equal recruitment of hospitals in multi-center distribution.

Table 1: Demographic Characteristics

Fever occurred in 100 percent of the patients. Myalgia (78.0%) and headache (70.7%) were common. More suggestive of malaria were chills and rigors (63.4%), whereas more suggestive of dengue were rash (29.3%), and bleeding (18.3%). There were overlapping symptoms in co-infected patients, which usually manifested more severely.

Table 2: Clinical Presentation

There was considerable thrombocytopenia (mean platelets 92,000 ± 38,500/µL). Liver enzymes were also high, particularly in cases of co-infection. In dengue, leukopenia was more prevalent whereas in malaria, anemia was more eminent.

Table 3: Laboratory Findings

Dengue mono-infection was predominant (41.5) among 82 patients followed by malaria (32.9) with co-infection being found in 25.6, which is a significant burden.

Table 4: Prevalence of Infection Types

Co-infected patients had a high prevalence of severe disease. This group had higher rates of ICU admission (19.5%), and complications.

Table 5: Disease Severity

The average length of stay in the hospital was 5.8 days with a standard deviation of 2.3. Co-infected patients were admitted longer and had more complications. The total death rate was 6.1, predominately in co-infections.

Table 6: Clinical Outcomes

Co-infected patients were significantly less in the number of platelets, length of stay and complication rates. These were statistically significant differences.

Table 7: Comparative Analysis (Co-infection vs Mono-infection)

This multi-center study evaluated the prevalence and clinical outcomes of dengue and malaria co-infection in endemic regions of Pakistan. The findings demonstrated that co-infection was present in 25.6% of cases, highlighting a considerable overlap between these two vector-borne diseases in endemic settings. This prevalence is consistent with recent regional evidence reporting co-infection rates ranging between 15–30%, particularly during peak transmission seasons where Aedes and Anopheles vectors coexist. The relatively high burden observed in our study may be attributed to climatic conditions, poor vector control strategies, and increased urbanization, which facilitate simultaneous transmission of both pathogens [10, 11].

Regarding clinical presentation, our study revealed that co-infected patients had overlapping clinical presentation, such as fever, myalgia, headache, and gastrointestinal symptoms, which were difficult to differentiate early. Nevertheless, some aspects like chills and rigors were more indicative of malaria and rash and bleeding manifestations were more indicative of dengue, which is consistent with the published clinical profiles. Notably, co-infected individuals were more likely to have more severe and unusual manifestations, which confirmed the hypothesis that dual infection can enhance the severity of the disease by acting synergistically through pathophysiological mechanisms [12].

The severity of co-infection was also supported by laboratory findings in this study. Our study found a large reduction in platelet counts in co-infected patients (p < 0.001) than in mono-infection groups. Thrombocytopenia is a characteristic of dengue infection, but its increased manifestation in co-infection could be indicative of synergistic effects of bone marrow suppression, immune-mediated destruction, and endothelial dysfunction. Moreover, high liver enzymes and mild renal dysfunction were more evident in co-infected cases, which means that it is a multi-organ involvement, which has been also reported in the recent literature. These results highlight the significance of extensive lab testing of febrile patients in endemic areas [13-15].

One of the main results of this research was the much poorer clinical outcome related to co-infection. The dengue-malaria co-infection patients experienced longer hospitalization, more complications (42.9%), and more deaths (14.3%), which were statistically significant compared to the mono-infection groups. These findings are consistent with recent research that has shown that co-infected individuals are at risk of severe disease, such as shock, hemorrhage, and organ dysfunction. The increased ICU admission rate in our study also highlights the clinical burden and implications of co-infection on healthcare resources [16-18].

The reason behind the observed higher mortality rate among co-infected patients could be due to late diagnosis, similar clinical presentation, and under-identification of dual infections in standard clinical practice. Clinicians in resource-limited environments, where diagnostic resources might be limited, tend to concentrate on one etiology, which may overlook co-occurring infections. Thus, our results indicate that regular dual screening of dengue and malaria in the endemic areas is necessary, especially in patients with severe or unusual manifestations [19, 20].

Although it has its strengths, such as a multi-center design and a thorough clinical and laboratory assessment, this study has some limitations. The sample size (n=82) is relatively small, which can restrict the extrapolation of the results. Also, non-probability sampling can be subjected to selection bias. No molecular diagnostic techniques were used and this would have enhanced diagnostic accuracy. Moreover, no long-term outcomes were evaluated, which restricted the knowledge of the long-term disease effects. It is suggested that future large-scale prospective studies including modern diagnostics and follow-up data should be implemented,

Dengue and malaria co-infection represents a significant and clinically important entity in endemic regions of Pakistan, with a prevalence of 25.6% in this study. Co-infected patients exhibit more severe clinical manifestations, greater laboratory derangements, prolonged hospitalization, and higher rates of complications and mortality compared to mono-infected individuals. These findings emphasize the need for early recognition, routine dual diagnostic screening, and aggressive clinical management to reduce morbidity and mortality. Strengthening surveillance systems and improving vector control strategies are essential to mitigate the growing burden of co-infection in endemic settings

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