Journal of Preventive Medicine

Keywords

Dengue fever; Vitamin B₁₂; Thrombocytopenia

Introduction

Dengue is an endemic disease in Asia, America, Africa and the western pacific. Worldwide, dengue fever is an important arthropod-borne infection transmitted by Aedes female mosquitoes. Dengue fever cases around the globe have greatly increased over the past few decades. Dengue is RNA virus of flaviridae family, flavivirus genus. It has four serotypes: Dengue virus type 1 (DEN-1), DEN-2, DEN-3, and DEN-4. The most severe presentation of dengue virus is DHF (dengue hemorrhagic fever) characterized by high grade fever, leakage of plasma, bleeding manifestations and liver enlargement. Hypovolumic shock may occur in these patients because of plasma leakage. This lethal condition is known as Dengue Shock Syndrome (DSS). Dengue has been a problem in Pakistan for the past 20 years. In 1985, the first recorded case of dengue type 2 isolation occurred in a sero-epidemiological research for encephalitis. In 1994-1995, the first significant outbreak was documented in Karachi. In 2006, Karachi had another severe epidemic, after heavy rains and young adults were mainly affected [1].

Dengue causes thrombocytopenia that indicates a platelet count of less than 150 ×10³/L. For hemostasis to take place there must be an adequate number of healthy platelets in the blood; bleeding risks rise as platelet levels fall. With the aid of a comprehensive history, physical examination, and additional laboratory tests, the cause of thrombocytopenia is ascertained. Thrombocytopenia is a result of testing, not a medical condition.

Vitamin B₁₂ (also called cobalamin), plays a crucial part in cellular metabolism. B₁₂ deficiency is now recognized as a problem of global proportions that is frequently brought on by dietary inadequacy, especially in children and women of reproductive age. A healthy thrombopoiesis requires vitamin B₁₂. Patients having Vitamin B₁₂ deficiency have a 10% chance of developing thrombocytopenia. The purpose of this study was to examine the claim that some people's levels of vitamin B₁₂, could result in prolonged, serious thrombocytopenia [2].

Vitamin B₁₂ can effectively treat dengue-causing maturation arrest and thrombocytopenia, research has been made in India regarding this, so we decided to do an observational study in a tertiary care hospital of Karachi, Pakistan to find an association between vitamin B₁₂ and thrombocytopenia in dengue fever patient.

Aims and objectives: The study's objective was to determine an association between vitamin B₁₂ levels and the severity of thrombocytopenia in dengue patients [3].

Materials and Methods

Study design and period

An observational cross sectional study was done on both inpatients and outpatients of Abbasi Shaheed hospital Karachi, Pakistan during the period from October 2021 to March 2022 [4].

Study setting

Sample size of 81 patients with dengue fever positive for NS1 antigens with thrombocytopenia, including both inpatients and outpatients with informed consent.

Data collection and questionnaire

To obtain the required sample size, data were collected from voluntarily participating dengue fever patients using the simple, convenient sampling method. The data were gathered using a self-administered questionnaire under the supervision of a trained crew of data collectors. The participants received a brief introduction to the study's goals and directions for filling out the questionnaire before starting. Participants who required assistance were given it in order to finish the survey [5].

The questionnaire is composed of two parts, the first part consist of patients demographic data and the second part was patients clinical considerations i.e., presence of NS1 antigen, determination of acute or chronic dengue infection, thrombocytopenia either mild, moderate or severe and determination of vitamin B₁₂ level in blood [6].

Inclusion criteria

Patients with more than 13 years of age, positive NS1 antigen dengue fever cases and giving consent were included in the study.

Exclusion criteria

Patients with blood disorders, drugs induced thrombocytopenia, having cancer or under chemotherapy, sepsis and autoimmune disorders were excluded from the study [7].

Case definition

For the purpose of study, we defined case definitions. By dengue fever we mean NS1 antigen positive patients. Thrombocytopenia means platelets count less than 1.5 lac which is further classified into mild thrombocytopenia for platelets count in between 1 to 1.5 lac, moderate thrombocytopenia 50,000 to 1 lac platelets count and severe thrombocytopenia less than 50,000 platelets count. For Vitamin B₁₂ deficiency, less than 200 pg/ml is low and less than then 100 pg/ml is very low, borderline vitamin B₁₂ is 200 pg/ml to 300pg/ml and normal vitaminB₁₂ level is 300 to 950 pg/ml.

Statistical analysis

A pre-made study proforma contained all the data that was recorded. Statistical package for social sciences, version 25 (SPSS Inc.,) was used to analyze the data. Results for categorical variables are shown as a number; while those for continuous variables are presented as mean Standard Deviation (SD %). The paired t-test was used to compare quantitative data between the two groups. The level of significance was set at a p-value of 0.05 or lower. Where it was thought necessary, results were illustrated graphically [8].

Results

During the course of the investigation, 81 patients with dengue fever were enrolled in the trial (Figure 1). Distribution of patient on the basis of age depicts the majority of the participants as being between the ages of 30 and 39; and Gender Distribution depicts 48 (59%) men and 33 (40.7%) women [9].

The experiment was not open to patients with chronic liver disease, hypersplenism, hematological diseases, autoimmune disorders, malignancies, sepsis, or who were taking drugs that produced thrombocytopenia [10].

Figure 1: Distribution of patients on the basis of age and gender distribution.

Thrombocytopenia is classified into three groups for the study: Mild, moderate, and severe. According to the results indicated in Figure 2 below, out of 81 patients, 28% had mild thrombocytopenia, 45.70% had moderate thrombocytopenia and 23% had severe thrombocytopenia [11].

Figure 2: No. of dengue patients having mild, moderate and severe thrombocytopenia.

Four categories of vitamin B₁₂ deficiency are recognized. Normal: 300 pg/ml; borderline: 200-300 Pg/ml; low: 200 Pg/ml; and extremely low: 100 pg/ml. Maximum 56 (69%) patients out of 81 were affected by low vitamin B₁₂ levels. Results of our analysis are displayed in Figure 3, which is provided below [12].

Figure 3: Vitamin B₁₂ level.

According to the Table 1 below, of the 81 dengue patients in our research, 24 had mild thrombocytopenia, 36 had moderate thrombocytopenia, and 21 had severe thrombocytopenia.

The association between vitamin B₁₂ levels with mild, moderate, and severe thrombocytopenia shows that out of 24 mild thrombocytopenic patients, 29% had very low vitamin B₁₂levels (less than 100 pg/ml), 62.5% had low vitamin B₁₂ levels (less than 200 pg/ml), and 7.4% had borderline vitamin B₁₂levels (between 200 and 300 pg/ml).

36 individuals with moderate thrombocytopenia were found to have low vitamin B₁₂ levels in 27.7% of cases (less than 100 pg/ml), low levels in 63.8% of cases (less than 200 pg/ml), and borderline levels in 8.3% of cases (between 200 and 300 pg/ml).

And among 21 patients with serious thrombocytopenia, 14% of patients had very low vitamin B₁₂ levels, 80.9% had low vitamin B₁₂evels, and 4.7% had borderline vitamin B₁₂ levels. Table 1 results are displayed [13].

Table 1: Association between vitamin B₁₂ and platelet count.

Standard deviation: When comparing platelet count with vitamin B₁₂ deficiency, we calculated the Standard Deviation (SD) in relation to its mean value to determine how dispersed the data is with respect to the mean. An SD of 42.29 means that 141.48 ± 42.29 indicates mild thrombocytopenia, an SD of 51.65 indicates moderate thrombocytopenia, and an SD of 40.25 shows 150.09 ± 40.25 severe thrombocytopenia deviations from the mean position. All of these findings are listed in Table 2 below [14].

Table 2: Comparison of platelet count with vitamin B₁₂ deficiency.

Correlation: A correlation coefficient, which ranges from -1 to 1, indicates the strength and direction of a connection between two variables. The correlation coefficient is typically represented by the symbol r.

In this study there are two main variables one is vitamin B₁₂ deficiency and other is platelet count (i.e., Thrombocytopenia). The Pearson correlation in SPSS analysis shows -0.162 which indicates negative correlation between these two variables. This result shows that decrease in Vitamin B₁₂ level increases the severity of thrombocytopenia in dengue fever patients.

And P value of 0.001 indicating significant results between these two variables (i.e., vitamin B₁₂evel and thrombocytopenia). All these results are shown in Table 3 given below [15].

Table 3: Correlations between vitamin B₁₂level and platelet count.

Regression: Regression analysis is a powerful statistical technique that enables to investigate the relationship between two or more relevant variables. Regression analysis is represented by b.

In this study Thrombocytopenia is dependent variable (constant) and vitamin B₁₂ level is independent variable. If any one of the regression value is negative then it indicates negative correlation between two variables as -0.230 indicates that indicates that vitamin B₁₂ level and thrombocytopenia are negatively correlated, decease in Vitamin B₁₂ level indicates higher cases of thrombocytopenia, results shown in Table 4 [16].

Table 4: Regression analysis (Coefficientsa).

Discussion

Since Pakistan's economy depends mostly on agriculture, people in Pakistan prefer plant-based meals to meat and dairy. The most common cause of B₁₂ insufficiency is a lack of availability to animal meat, fish, eggs and dairy products so, vegetarians and others at risk of deficiency may need to take supplements [17].

Dengue itself causes thrombocytopenia either due to bone marrow suppression or because of direct damage to the platelets, pathophysiology is still controversial. Also vitamin B₁₂ deficiency is common in Pakistan so, we started this study to test the hypothesis that vitamin B₁₂ level may have correlation with severity of thrombocytopenia in dengue fever patients, as thrombocytopenia is often seen in dengue patients [18].

In the bone marrow, vitamin B₁₂ functions as a co-factor when cells are being synthesized. Therefore, thrombocytopenia is brought on by vitamin B12 insufficiency. However, host nutritional status (vitamin B12) as adjuvant therapy could lower the likelihood of progression from DENV infection to reduce disease severity in patients. Modulation of vitamin B12 can act as supportive therapy to assist patients suffering from dengue infection, and this is the main focus of the literature reviewed here in this manuscript, even though there is no substitute for an effective dengue vaccine.

In our study, we reached out to 81 dengue fever patients who had been tested positively for NS1 Ag and we compared their platelet counts to blood vitamin B₁₂levels. The majority of patients (39%) were between the ages of 30 -39, with 59% of patients being men and 41% being women, as shown in Figure 1.

Paliwal et al., from India found that 67.33% of patients had vitamin B12 levels greater than 200 pg/ml had mean platelet count of 40,564.35 ± 6104.78 per ml, whereas 32.67% had levels less than 200 pg/ml had mean platelet count of 38,653.06 ± 6362.49 per ml. Their P-value obtained was 0.07 which determined that their study showing link between vitamin B₁₂ deficiency and platelet count was insignificant.

Comparing their results to our study the majority 45.70% of individuals were having moderate thrombocytopenia, 28%suffered from mild and 23% were having severe thrombocytopenia. Table 1 shows the association of serum vitamin B₁₂ level with mild, moderate, and severe thrombocytopenia. These results indicates the significant relationship between serum vitamin B₁₂ level and severity of thrombocytopenia because individuals who lacked vitamin B₁₂ experienced considerably moderate to severe thrombocytopenia (p<0.05). Mean vitamin B₁₂ level was lower in dengue patients with moderate thrombocytopenia as represented in Table 2.

While there are numerous publications published in India that indicate significant results between vitamin B₁₂ deficiency and the severity of thrombocytopenia, we evaluate some of those articles with high citations and compare all of their results.

Tak et al., from India found that 40% of subjects were having B₁₂ level less than 200 pg/ml, and 72% of patients had B₁₂ level less than 300pg/ml, their correlation with platelet count had p value of <0.05 showing significant correlation between these 2 parameters.

Kansara et al., from India worked on 75 individuals with dengue fever, 61 (81%) have thrombocytopenia with vitamin B₁₂ deficiency (B₁₂ level 300 pg/l), while 22 (44%) have thrombocytopenia with vitamin B₁₂ level normal (B₁₂ level 201-300). Vitamin B12 levels of 190 pg/ml were reported in 47 (94%) of the patients with severe thrombocytopenia and 32 (74%) of the patients with mild thrombocytopenia, showing positive correlation between severity of thrombocytopenia and vitamin B₁₂ deficiency (negative correlation between vitamin B₁₂ deficiency and platelet count). Comparing with our correlation results between vitamin B₁₂ deficiency and severity of thrombocytopenia shown in Table 3 indicates negative correlation (r=-0.162) which means that decreasing level of vitamin B₁₂ also decreases platelet count (positive correlation) which worsens thrombocytopenia in dengue fever patients comparing these results to other previous related articles also shows the same results Their results shows correlation between vitamin B₁₂ level and platelet count is positive (r=0.5748).

According to the regression analysis in Table 4, also indicates that the severity of thrombocytopenia (extremely low platelet count) correlates negatively with vitamin B₁₂ level, meaning that a decline in serum vitamin B₁₂ level worsens the condition.

Thus, a significance ratio of 0.05 and a negative correlation between the blood vitamin B₁₂ level and thrombocytopenia indicated a relationship between the two variables; a low platelet count brought on by a serum vitamin B₁₂ deficiency in dengue patients results in serious thrombocytopenia [20].

Conclusion

Patients with dengue fever who were vitamin B₁₂ deficient developed moderate to severe thrombocytopenia. Vitamin B₁₂ levels in dengue fever patients should be checked, and the adequate dose and dosage of vitamin B₁₂ supplements should be administered depending on how mild or serious their deficiencies are.

Limitations

This research had a small sample size and was conducted in a single tertiary care Hospital; additional multicenter studies with a larger population are needed to confirm the results.

And in addition to the vitamin B₁₂ discussed here, future study should look into possible associations between DENV infection and other vitamins and micronutrients.