ABSTRACT.
The global burden of dengue infections has increased dramatically. Early diagnosis of dengue infection is critical to proper medical management to avoid further complications in patients. This study was geared to assess the severity of dengue infections based on clinical and hematological examinations. A cross-sectional study was conducted among febrile patients with dengue infection in a teaching hospital in Pakistan. Blood samples were investigated for dengue-specific antibodies (IgM and IgG) and the nonstructural 1 antigen. The clinical findings of each subject were noted to assess the severity of the infection. Tests for hematological parameters were performed. Of 130 patients with confirmed dengue infection, 23 had severe and 107 had nonsevere dengue. Patients with severe dengue experienced mucosal bleeding (71.4%), fluid accumulation (57.1%), shock (35.7%), and gastrointestinal bleeding (28.6%). The most significant hematological findings among severe and nonsevere patients with dengue infection were thrombocytopenia, leukopenia, and a raised hematocrit level (P < 0.001). Patients with severe dengue infection showed marked thrombocytopenia, with a mean platelet count of 49.96 × 109 platelets/L. The clinical presentation of patients with dengue infection along with hematological markers are the most important clues for the diagnosis of, prognosis of, and therapy for dengue infection. Thrombocytopenia, leukopenia, and raised hematocrit levels were the most significant hematological parameters when assessing the severity of dengue infection.
INTRODUCTION
The virus that causes dengue infection is spread through the bite of female Aedes aegypti mosquitoes. In tropical and subtropical areas, this condition is prevalent. Four antigenically distinct dengue serotypes (dengue virus serotype 1 [DENV-1]–DENV-4) are reported; however, there was an addition of DENV-5 to the existing serotypes.1 Despite the ongoing COVID-19 pandemic, dengue infection has emerged in dengue-endemic countries, which creates an additional burden when coupled with COVID-19. Dengue infection has been reported in tropical and subtropical regions, with approximately 400 million cases and 22,000 deaths each year across the globe. Dengue virus epidemics are reported annually in Asia, Africa, America, and Australia.2 Since 1994, Pakistan has been included in dengue-endemic countries; it faced multiple outbreaks in 2005, 2010, 2011, and 2019.3 The last notable outbreak in Pakistan was reported in 2021, and included 48,906 cases and 183 deaths. The Punjab Province reported the greatest number of DENV cases, followed by Khyber Pakhtunkhwa and Sindh.4
Dengue infection is becoming a major concern, with increased mortality in tropical countries. Dengue infection leads to a varying spectrum of pathological conditions, which ranges from a mild febrile illness to a life-threatening situation. The most severe clinical picture associated with dengue shock syndrome includes plasma leakage, increased vascular fragility, and coagulation abnormalities, which may cause hypovolemic shock and multiorgan failure.5 In addition to severe and nonsevere dengue infection, there are three phases of dengue infection: the febrile, critical, and recovery phases. During the febrile phase, patients present with fever, dehydration, headache, febrile seizures, rash, anorexia, nausea, vomiting, myalgia, and arthralgia. Mucosal membrane and gastrointestinal bleeding may also be seen.6
A preexisting immunity to a particular DENV serotype protects individuals from infection with the same serotype.7 However, the risk of acquiring severe illness with a different serotype is increased substantially.8 Serotype-specific antibodies are the main factor in this protection failure, as antibodies cross-react and fail to neutralize other serotypes. Thus, reinfection with other DENV serotypes can predispose to more severe conditions and can enhance illness severity more than the primary infection.9 Antibody-dependent enhancement is the most important immunopathological phenomenon associated with DENV infection, coupled with vascular permeability.10 Different diagnostic techniques are used to diagnose dengue infection, including virus isolation and detection of viral nucleic acid, viral antigen, or host antibody. After the onset of illness, the nonstructural 1 (NS1) antigen is the most sensitive marker for detecting dengue infection.11
Several hematological and biochemical changes also occur during dengue infection. These changes are important to promoting early diagnosis and providing effective clinical management of dengue infection. Variations in hematological and biochemical parameters such as platelet count, albumin concentration, hematocrit level, aspartate aminotransferase ratio, leukocyte count, and lymphocyte count are associated with dengue infection.12,13 Hepatic involvement is also related to dengue infection characterized by increased aspartate transaminase and alanine transaminase, decreased serum albumin, and mildly raised serum total bilirubin. Similarly, cholesterol, calcium, and albumin levels are decreased during the plasma leakage phase of the illness. However, these changes may vary depending on disease severity.14 Even with improvements in diagnosis, managing patients with dengue infection still presents substantial hurdles clinically, particularly in the absence of trustworthy biomarkers that may predict the course of a severe illness accurately. The difficulty in making a clinical diagnosis of dengue might greatly rely on the patient’s presentation and the stage of the infection. There may be a variety of illness-causing organisms or disease states that can resemble the clinical spectrum resulting from dengue infection, depending on geographic location. Accurate clinical diagnosis of dengue is difficult because the clinical signs are so varied. As a result, it is crucial to combine clinical presentation evaluation with laboratory or point-of-care diagnostics.15 There is still disagreement regarding the clinical usefulness of hematological parameters. This investigation was undertaken to evaluate the severity of dengue infection using hematological and clinical parameters.
MATERIAL AND METHODS
Study design.
A cross-sectional, hospital-based study was carried out in a hospital in Khyber-Pakhtunkhwa Dir Lower, Pakistan, from June 2021 to January 2022. The study population included febrile patients with dengue infection who were suspected of dengue viral infection according to the criteria of 2009 WHO classification.16 Cases such as dengue-like malaria, COVID-19, typhoid fever, kala-azar, and any other illnesses were excluded from the study.
Clinical data collection.
The clinical examination of each study participant was conducted by a physician. Demographic information and relevant medical profiles of the patients were noted using a data collection sheet. Patients with dengue infection were classified by clinicians as dengue with warning signs, dengue without warning signs, and severe dengue using clinical diagnosis based on the WHO 2009 dengue classification criteria.16
Laboratory procedures for hematological parameters.
After hospital admission, all study patients had 5 mL of blood drawn into EDTA tubes according to the CDC laboratory manual (NHANES 2007-2008). Hematological assays and other regular investigations were all carried out using an automated blood analyzer (CELL-DYN Ruby; Abbott Laboratories, Chicago, IL). The hematological parameters included segmented neutrophils, eosinophils, and basophils; white blood cell (WBC) count; mean platelet volume; number of monocytes, lymphocytes, and platelets; hemoglobin and hematocrit values, mean cell volume (MCV), mean cell hemoglobin (MCH), mean corpuscular hemoglobin concentration, and a red blood cell count.
Independent laboratory diagnosis of dengue infection.
Hospital staff performed a serological diagnosis to confirm dengue infection. The sensitivity of serological testing for DENV was 96.4% and the specificity was 98.9%. Serological diagnosis for dengue infection included IgM antibody testing against the dengue NS1 antigen. Using the previously published IgM capture enzyme-linked immunosorbent test, serum samples were used to identify acute dengue infection. The sample was also screened to detect the dengue NS1 antigen. Approximately 100 µL of the sample was used. After 20 minutes of incubation, the results were obtained for positive or negative samples, according to recommended procedures.
WHO 2009 classifications and hematological parameters.
The severity of dengue infection for all patients was evaluated based on hematological and clinical data. Severe and nonsevere dengue have been classified by the WHO.16 Plasma leakage, fluid buildup that causes shock or respiratory difficulty, severe bleeding, and/or serious organ damage are all signs of severe dengue. Patients with and without warning signals are separated into two groups in the nonsevere dengue group. For clinical data, the WHO 2009 dengue classification criteria16 were followed. As described earlier, all three groups were considered and all hematological parameters were assessed in all groups. We associated hematological findings with clinical data to determine the severity of DENV infection.
Statistical analysis.
We used SPSS (version 21; SPSS, Inc., Chicago, IL) and GraphPad’s Prism version 5 (San Diego, CA) for all statistical studies and graphics, respectively. Frequencies and percentages were calculated using descriptive statistics, and the mean of the hematological parameter was compared between patients with dengue infection using analysis of variance. Statistics were judged significant at P = 0.05.
RESULTS
A total of 450 patients with febrile illnesses were admitted to the medical ward during the study period, and their dengue IgM antibody and NS1 antigen levels were assessed. Of these patients, 152 tested positive for dengue. The study included 130 DENV-positive individuals; the other 22 were not included because they had concomitant conditions (Figure 1).
Figure 1.
Flow chart of the study.
Ninety patients were male (69.2%) and 40 (30.8%) were female. The age distribution varies, with 7 subjects younger than 18 years old and the remaining enrolled participants were older than 18 (Table 1). Of the 130 patients, 117 (90%) had IgM antibody positivity, 13 (10%) had IgG antibody positivity, and 95 (73.1%) had NS1 antigen positivity (Figure 2). The WHO’s 2009 classification criteria16 determined that 107 patients (82.30%) had dengue infection with warning signs, 9 (6.92%) did not have any warning signals, and 14 (10.76%) had symptoms of severe dengue infection.
Table 1.
Baseline characteristics of patients with dengue infection (N = 130)
| Variable | n | % |
|---|---|---|
| Gender | ||
| Male | 90 | 69.2 |
| Female | 40 | 30.8 |
| Age, years | ||
| < 18 | 7 | 05.38 |
| ≥ 18 | 123 | 94.62 |
| Previous dengue infection | 9 | 6.9 |
Figure 2.
Results for IgM and IgG antibodies, and the nonstructural 1 (NS1) antigen.
Clinical characteristics of patients.
The clinical characteristics of patients with confirmed DENV infection are represented in Table 2. Fever was a commonly noted symptom in severe and nonsevere dengue infections. The most common clinical features noted in patients with dengue infection who presented with warning signs were headache (n = 56, 52.3%), nausea (n = 59, 55.1%), vomiting (n = 51, 47.7%), tenderness (n = 28, 26.1%), and abdominal pain (n = 21, 19.6%), followed by arthralgia (n = 30, 28%) and myalgia (n = 18, 16.8%). Dengue patients without warning signs presented with skin rash (n = 8, 88.9%), headache (n = 4, 44.4%), nausea (n = 2, 22.2%), and vomiting (n = 2, 22.2%). Patients with severe DENV infection experience mucosal bleeding (n = 10, 71.4%), fluid accumulation (n = 8, 57.1%), shock (n = 5, 35.7%), gastrointestinal bleeding (n = 4, 28.6%) coupled with other common symptoms (Table 2).
Table 2.
Clinical presentations of the patients with dengue infection (N = 130)
| Signs and symptoms | Dengue with a warning sign (n = 107), n (%) | Dengue without a warning sign (n = 9), n (%) | Severe dengue (n = 14), n (%) | Total (N = 130), n (%) | P value* |
|---|---|---|---|---|---|
| Fever | 107 (100) | 09 (100) | 14 (100) | 130 (100) | – |
| Headache | 56 (52.3) | 4 (44.4) | 9 (64.3) | 69 (63.1) | 0.60 |
| Abdominal pain | 21 (19.6) | 0 (0) | 5 (35.7) | 26 (20) | 0.11 |
| Nausea | 59 (55.1) | 2 (22.2) | 6 (42.9) | 67 (51.5) | 0.13 |
| Vomiting | 51 (47.7) | 2 (22.2) | 9 (64.3) | 62 (47.7) | 0.14 |
| Rash | 6 (5.6) | 8 (88.9) | 2 (14.3) | 16 (12.3) | 0.01 |
| Tenderness | 28 (26.1) | 0 (0) | 5 (35.7) | 33 (25.4) | 0.01 |
| Mucosal bleeding | 13 (12.1) | 0 (0) | 10 (71.4) | 23 (17.7) | 0.01 |
| Myalgia | 18 (16.8) | 0 (0) | 8 (57.1) | 26 (20%) | 0.01 |
| Arthralgia | 30 (28) | 0 (0) | 4 (28.6) | 34 (26.2) | 0.18 |
| Shock | 0 (0) | 0 (0) | 5 (35.7) | 5 (3.8) | 0.01 |
| Gastrointestinal bleeding | 0 (0) | 0 (0) | 4 (28.6) | 4 (3.1) | 0.01 |
| Fluid accumulation | 0 (0) | 0 (0) | 8 (57.1) | 8 (6.2) | 0.01 |
The χ2 test was used for the comparison of clinical characteristics across three groups.
Hematological characteristics of patients.
The most common hematological finding was thrombocytopenia, which was noted in most of the patients with platelet counts of < 150 × 109/L. Severe thrombocytopenia (< 100 × 109/L) was noted in 92% of patients with severe dengue infection, whereas one third of patients with nonsevere dengue infection also presented severe thrombocytopenia. Collectively, 84 patients had low WBC counts (< 5 × 109/L), along with 65 patients (60.74%) with dengue infection with warning signs, 6 patients (66.66%) with dengue without warning signs, and 13 patients (92.85%) with severe dengue infection. Most of the patients with severe dengue infection had elevated monocyte levels (64%). Increased hematocrit level was another predominant feature of patients with dengue infection (n = 108, 83%). All patients with severe dengue had increased hematocrit levels, with half of them demonstrating a ≤ 10% increase and the other half showing a ≤ 20% increase. In patients with nonsevere dengue, most had ≤ 10% elevated levels of hematocrit (Table 3).
Table 3.
Distribution of hematological parameters among patients with dengue infection
| Parameter | Dengue with warning sign (n = 107), n (%) | Dengue without warning sign (n = 9), n (%) | Severe dengue (n = 14), n (%) | Total (N = 130), n (%) |
|---|---|---|---|---|
| Platelets | ||||
| < 100 × 109/L | 38 (35.51) | 3 (33.33) | 13 (92.85) | 57 (43.84) |
| < 150 × 109/L | 46 (42.99) | 3 (33.33) | 1 (07.14) | 50 (38.46) |
| Leukocytes, < 5 × 109/L | 65 (60.74) | 6 (66.66) | 13 (92.85) | 84 (64.61) |
| Lymphocytes, < 1.5 × 109/L | 79 (73.83) | 7 (77.77) | 14 (100) | 100 (76.92) |
| Monocytes, > 0.8 × 109/L | 34 (31.77) | 2 (22.22) | 9 (64.28) | 4 (03.07) |
| Eosinophils, > 0.44 × 109/L | 48 (44.85) | 1 (11.11) | 5 (35.71) | 54 (41.53) |
| Neutrophils, > 1.5 × 109/L | 22 (20.56) | 3 (33.33) | 6 (42.85) | 31 (23.84) |
| Hematocrit level | ||||
| ≤ 10% increase | 72 (67.28) | 4 (44.44) | 7 (50) | 83 (63.84) |
| ≤ 20% increase | 16 (14.95) | 2 (22.22) | 7 (50) | 25 (19.23) |
The results of the hematological investigation revealed that patients with severe dengue infection had a very low mean platelet count (49.96 × 109/L), whereas the mean platelet counts of patients with dengue infection with and without warning signs were 115.7 × 109/L and 90.0 × 109/L, respectively, which was statistically significant (P < 0.001) (Table 4). Leukopenia was another statistically significant (P < 0.001) result of the study. The mean value of leukocytes in patients with severe dengue infection was 2.63 × 109/L. Of note, the patients with dengue infection without warning signs had a mean leukocyte count of 3.48 × 109/L, and patients with warning signs had a 3.81 × 109/L WBC count. The mean hematocrit level showed a marked increase from the reference range (male, > 52%; female, > 48%) in patients with severe and nonsevere dengue (P = 0.001). In contrast to nonsevere dengue infection, severe dengue infection exhibited a more pronounced hematocrit level that was considerably higher (P = 0.001). According to Table 4, there were no discernible variations between the patterns of monocyte, eosinophil, neutrophil, MCH, MCV, and hemoglobin in patients with severe and nonsevere dengue.
Table 4.
Distribution of hematological parameters in severe and nonsevere groups of patients with dengue infection
| Parameter | Dengue with warning sign (n = 107), n ± SD | Dengue without warning sign (n = 9), n ± SD | Severe dengue (n = 14), n ± SD | P value* |
|---|---|---|---|---|
| Mean platelets, ×109/L | 115.7 ± 3.84 | 90.0 ± 7.83 | 49.96 ± 7.41 | < 0.001 |
| Mean leukocytes, ×109/L | 3.81 ± 0.88 | 3.48 ± 0.28 | 2.63 ± 0.11 | < 0.001 |
| Mean lymphocytes, ×109/L | 1.26 ± 0.46 | 1.16 ± 0.21 | 1.03 ± 0.86 | 0.226 |
| Mean monocytes, ×109/L | 0.76 ± 0.02 | 0.68 ± 0.06 | 0.71 ± 0.068 | 0.610 |
| Mean eosinophils, ×109/L | 0.39 ± 0.01 | 0.31 ± 0.05 | 0.39 ± 0.03 | 0.386 |
| Mean neutrophils, ×109/L | 2.56 ± 0.08 | 2.43 ± 0.33 | 2.24 ± 0.30 | 0.467 |
| Mean cell volume, fL | 86.78 ± 0.39 | 88.32 ± 1.34 | 87.95 ± 1.16 | 0.380 |
| Mean cell hemoglobin, pg | 30.29 ± 0.29 | 30.47 ± 1.02 | 29.57 ± 0.65 | 0.685 |
| Mean hemoglobin, g/L | ||||
| Male | 13.85 ± 0.20 | 14.00 ± 0.60 | 14.30 ± 0.40 | 0.277 |
| Female | 12.55 ± 0.24 | 11.25 ± 0.75 | 13.15 ± 0.05 | |
| Mean hematocrit, % | ||||
| Male | 55.66 ± 0.59 | 55.31 ± 3.40 | 61.80 ± 0.84 | < 0.001 |
| Female | 54.82 ± 0.75 | 52.05 ± 8.55 | 62.50 ± 2.70 | |
One-way analysis of variance was used for the comparison of the means of the hematological parameters among patients with dengue infection.
DISCUSSION
Dengue patients present with distinct clinical features and unpredictable clinical outcomes, as some patients may develop severe dengue and others may have neither severe infection nor may require hospitalization. The WHO issues diagnostic criteria for dengue infection to classify patients into severe and nonsevere cases, which may provide proper patient management and may reduce hospital workloads.17,18 Previous studies have shown comparisons of WHO 1997 and WHO 2009 classification schemes and found pros and cons of both classification systems, and suggested the involvement of some new biochemical factors for the classification of severity of dengue infection. The WHO 2009 classification scheme has the advantage for clinicians to identify patients with dengue hemorrhagic fever and severe dengue infection.16 Nonstructural antigen 1 and polymerase chain reaction are sensitive diagnostic methods, but only show positive within the first 48 hours. Moreover, these methods can only diagnose the infection and may not distinguish between dengue fever and dengue hemorrhagic fever, which may lead to substantial plasma leakage in severe illness. Hence, we focused on hematological changes associated with the clinical course of DENV infection.
The most common symptom in all our patients (with dengue infection with warning signals, without warning signs, and severe dengue illness) was fever. Additional symptoms in patients with dengue infection who had warning indications included headache, nausea, vomiting, soreness, abdominal pain, arthralgia, and myalgia. A small number of study participants in our study had dengue infection without warning signs and experienced fever, headache, nausea, and skin rash, as reported in a previous study.18 Mucosal bleeding, fluid accumulation, shock, and gastrointestinal bleeding were the hallmarks of severe dengue infection in our current study, as documented in the WHO 2009 classification.16 This difference in the clinical spectrum of dengue infection might be helpful in the diagnosis of dengue infection and assessment of disease severity.
In our study, patients with dengue infection without warning signs had a greater proportion of rashes than those with severe dengue. The finding that patients with dengue infection without warning signs had a greater proportion of rashes than those with warning signs and severe dengue could be attributed to several factors, including immune response. In mild cases of dengue, the immune system may react more strongly, leading to a greater incidence of rashes compared with severe cases, in which the immune system may be overwhelmed or suppressed.19,20 Patients with severe dengue may have a greater viral load, leading to more severe symptoms and consequences. Patients with mild dengue and no warning signals, on the other hand, may have a reduced viral load, resulting in milder symptoms such as rashes. Patients with severe dengue infection frequently complain of headache, stomach pain, soreness, and myalgia (muscle pain). The reason these symptoms are more common in severe dengue cases is unknown, but various variables may contribute to their occurrence, such as the capacity of the virus to damage blood vessels, resulting in greater vascular permeability. This causes blood vessels to leak, allowing fluids to enter bodily tissues, particularly the abdomen, producing pain and tenderness.21,22 Severe dengue can also affect organs, including the liver, producing stomach pain and tenderness. The virus can also cause muscle inflammation, which can contribute to myalgia. There is an increased risk of bleeding and shock with severe dengue infection, which can result in additional symptoms such as abdominal pain and tenderness.23,24
The main hematological abnormalities in patients with severe dengue infection were thrombocytopenia resulting from increased destruction of platelets (mean platelet count, 49.96 × 109/L). Several factors might be involved in severe thrombocytopenia, such as peripheral destruction of platelets, infected megakaryocytes, and cross-reaction of antibodies.25 Antibody-dependent enhancement is likely to be associated with cross-reactive antibodies, which are produced by the first dengue infection, followed by a secondary infection from another dengue serotype. The increased level of antibodies fails to neutralize the virus, but reacts with platelets, blood clotting factors, and vascular endothelium, which form immune complexes.26 Importantly, most of the patients with severe dengue infection showed a history of previous infection, which might be associated with severe thrombocytopenia. There was also thrombocytopenia in patients with nonsevere dengue infection, but their platelet count was high, unlike in severe dengue infection; a similar pattern was also observed by other studies.14,27 The marked difference in platelet count is helpful to distinguish nonsevere dengue infection from severe dengue infection.
Leukopenia was another notable result of our study, which distinguished severe dengue from nonsevere dengue. More than 90% of patients with severe dengue infection experienced severe leukopenia compared with the number of patients with nonsevere dengue infection. A decrease in granulocytes such as neutrophils and infected leukocytes might have contributed to leukopenia in patients with dengue infection.28 Leukopenia may also be the result of DENV-mediated suppression of WBC production in the bone marrow.29 Leukopenia is common in dengue infection and could be a useful indicator for the prediction of disease severity.
With regard to increased hematocrit levels, prominent statistical significance was found among all three classified dengue cases. All patients with severe dengue infection had elevated hematocrit levels by as much as 10% to 20%, which is consistent with another study.30 Increased hematocrit levels were common in patients with severe dengue resulting from plasma leakage.31 These parameters can be taken into account when distinguishing between severe and nonsevere dengue infection.
The hematological and clinical profiles of patients with dengue infection with warning signals, without warning signs, and with severe infection were studied for the first time. Our study has certain drawbacks. Dengue cases with severe and nonsevere illness did not undergo serotype analysis. A small sample size is another limitation of our study. Regardless of the depicted limitations, the hematological changes that occurred during nonsevere DENV infection and severe DENV infections could be used to predict the severity and course of illness. This will aid clinicians in establishing an accurate diagnosis based on hematological parameters coupled with clinical presentation.
CONCLUSION
In dengue infection, clinical presentation along with hematological markers are the most important guide for diagnosis of, prognosis of, and therapy for dengue infection. Thrombocytopenia, leukopenia, and increased hematocrit levels were found to be significant hematological parameters in our study. Fluid accumulation, shock, bleeding, severe thrombocytopenia, a low WBC count, and increased hematocrit levels indicated a severe form of dengue infection.
ACKNOWLEDGMENTS
We thank all the colleagues and health-care workers who helped conduct the study. The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.
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