INTRODUCTION
It has been estimated that 240 million people worldwide are infected with schistosomiasis, with an additional 700 million at risk of infection. Currently, 2 to 6 million individuals are considered to be infected in Brazil1.
Most of the infected individuals are asymptomatic, but in 5% to 10% of infected individuals, liver periportal fibrosis results in severe disease consisting of portal hypertension, splenomegaly, esophageal varices, and recurrent hematemesis2. Pathologically, dense bands of fibrosis around the portal tracts are typical of schistosomiasis3.
Schistosomal periportal fibrosis is usually assessed using imaging methods, and abdominal ultrasonography (US) has become the imaging technique of choice4–8. However, the identification of other non-invasive, inexpensive, and simple routine laboratory tests as surrogate markers is of interest. Such markers would facilitate the selection of patients with hepatosplenic schistosomiasis (HSS) in rural areas for further clinical and laboratory evaluations.
In the hepatosplenic form of schistosomiasis, portal hypertension is the most evident clinical complication, and splenomegaly with platelet sequestration in the spleen is the most accepted explanation for thrombocytopenia9,10.
However, thrombocytopenia has been shown to be a good marker of HSS in patients admitted to Brazilian hospitals. A group of Brazilian authors showed that low blood platelet counts were associated with portal hypertension, and others also found an association between low platelet counts and the intensity of liver fibrosis11,12.
This study aimed to determine whether low platelet counts in the blood can be used as a screening tool to identify patients with HSS in an area endemic for Schistosomiasis mansoni in Brazil.
METHODS
Patients
This was a cross-sectional, analytical study. Topázio, a small district in Teófilo Otoni, State of Minas Gerais, Brazil, with a population of 1,543 individuals and a 23% prevalence of schistosomiasis, was selected for this investigation. A total of 400 volunteers with ages ranging from 16 to 97 years (median=41) were enrolled in the study after signing informed consent forms. During one week in July 2008, a local health agent invited all volunteers to be examined in a local outpatient clinic; three individuals did not respond to our invitation, and 13 (3.3%) were excluded because they did not complete the examinations. Therefore, a total of 384 volunteers were ultimately included in the study.
Clinical examination
The 384 participants were subjected to anamnesis and physical examination by one of the authors (JRL). Particular attention was given to the abdominal examination; specifically, the right hepatic lobe was examined along the anterior axillary line, and the left hepatic lobe was examined along a line passing through the xiphoid process. The spleen was palpated and measured under the left costal margin with the patient in the dorsal decubitus position during deep inspiration13–19.
Ultrasound
All participants were submitted to abdominal US using a portable Medison Sonoace 1500 system with a 3.5-MHz probe (Samsung, Korea) and examined according to the proposed protocol of the World Health Organization (WHO) for US assessment of schistosomiasis-related morbidity20. In this study, we used a spleen size cut-off point of >13cm (longitudinal diameter) because spleens tend to be more enlarged in HSS than in other diseases that evolve with splenomegaly, such as cirrhosis and mononucleosis-like syndromes21–26.
Definition of hepatosplenic schistosomiasis
HSS was classified into four groups: Group 1 consisted of patients with a spleen >13cm and liver periportal fibrosis, as confirmed by US; Group 2 consisted of patients with a palpable spleen and spleen >13cm, as measured by US; Group 3 consisted of patients with a spleen >13cm using US; and Group 4 consisted of patients with a palpable spleen.
Laboratory studies
Stool samples from 1,543 individuals (2 slides each) were examined using the Kato-Katz technique. A 10-ml sample of blood was collected from a peripheral vein in a tube containing ethylenediamine tetraacetic acid (EDTA) as an anticoagulant, and a hemogram with platelet counts was performed (Sysmex, E 2100 D). Individuals positive for schistosomiasis were treated with a single oral dose of praziquantel (50mg/kg body weight)27.
Statistical analysis
The questionnaire data were transferred to a databank using the EpiData 3.1 program (EpiData Association, Odense, Denmark) and analyzed using Statistical Package for Social Sciences (SPSS) 18.0 software (SPSS, IBM Company, Chicago, IL). Tables were constructed, and the percentages, median values, and mean values were calculated. Categorical variables were compared using the χ2 test; mean values were compared using Student’s t-test or analysis of variance (ANOVA); and median values were compared using the Kruskal-Wallis test. Additionally, Spearman correlation analysis was performed for continuous variables, and receiver operating characteristic (ROC) curves were used to define the cut-off value of platelet counts to aid in identifying the best combination of sensitivity and specificity. The areas under the ROC curves were calculated to define the accuracy of the platelet counts in the identification of patients with HSS. Differences between the groups were evaluated using the Mann-Whitney test. Statistical significance was accepted at the 5% level.
RESULTS
The comprehensive flow chart shown in Figure 1 summarizes the results of the present study. Demographic data and physical examination findings in the 384 individuals of this study are presented in Table 1. Eight patients in the group with HSS and 136 in the group without HSS (p=0.92) were male. Overall, the patients with HSS were older than the individuals without HSS (p<0.00).
FIGURE 1 – Flow chart of the study. Patients with spleens >13cm and liver periportal fibrosis were evaluated using ultrasound. HSS: hepatosplenic schistosomiasis.
TABLE 1 – Demographic and physical examination data of 384 individuals with and without hepatosplenic schistosomiasis in the district of Teófilo Otoni, State of Minas Gerais, Brazil, who were examined during a week in July 2012.
| Demographic and clinical data | Patients with hepatosplenic schistosomiasis (n=21) | Individuals without hepatosplenic schistosomiasis (n=363) | p value |
|---|---|---|---|
| Median (min-max) | Median (min-max) | ||
| Age | 51 (18-79) | 37 (18-97) | 0.00 |
| Weight | 59 (31-83) | 58 (29-103) | 0.99 |
| Height | 157 (134-169) | 158 (135-193) | 0.09 |
| Heart rate | 80 (52-124) | 80 (40-160) | 0.66 |
| Systemic systolic blood pressure | 140 (100-200) | 130 (85-250) | 0.27 |
| Systemic diastolic blood pressure | 88 (60-120) | 80 (50-140) | 0.49 |
The mean platelet counts in individuals with and without HSS were 152,952 ± 66,341/mm3 and 237,275 ± 84,345/mm3, respectively (p<0.01).
Using the ROC curve, we found that a platelet count <143,000/mm3 yielded the highest sensitivity and specificity for each HSS definition (Figure 1).
In Table 2, the area under the ROC curves (AUC), confidence intervals (CIs), and sensitivity and specificity of the ROC curves for a platelet cut-off point of <143,000/mm3 are presented for each definition of hepatosplenic schistosomiasis.
TABLE 2 – Areas under the ROC curves (AUCs), confidence intervals, and sensitivity and specificity of the ROC curves for a platelet cut-off point of <143,000/mm3 for each definition of hepatosplenic schistosomiasis.
| Definitions* | #AUC (%) | CI | Sensitivity (%) | Specificity (%) |
|---|---|---|---|---|
| Group 1 | 92.3 | 85.0 – 99.5 | 92.3 | 75.0 |
| Group 2 | 77.2 | 65.5 – 88.8 | 93.4 | 47.6 |
| Group 3 | 92.2 | 85.0 – 99.4 | 92.4 | 75.0 |
| Group 4 | 74.4 | 48.5 – 80.4 | 93.0 | 44.4 |
*Group 1: spleen >13cm + liver periportal fibrosis; Group 2: spleen >13cm + palpable spleen; Group 3: spleen >13cm; Group 4: palpable spleen.
#AUC: Area under the ROC curve. ROC: receiver operating characteristic; CI: confidence interval.
DISCUSSION
This report is the first fieldwork investigation demonstrating that thrombocytopenia can be used as a surrogate marker of HSS in areas highly endemic for schistosomiasis. Souza et al.11 showed that low blood platelet counts were related to portal hypertension in schistosomiasis. In addition, Lambertucci et al.12 also found that blood platelet counts less than 140,000/mm3 identified patients with HSS (a relationship was observed between the spleen size and liver periportal fibrosis grading). Both studies were performed in patients who were admitted to University Hospitals in large cities.
If the successful use of this simple and inexpensive marker for HSS in endemic areas is confirmed by others, then this marker would hasten the identification of morbidity in poor areas of the country. Patients selected based on a thrombocytopenia assessment would then have their diagnosis confirmed by other tests, such as US.
In the present study, the patients with HSS were found to be older than the individuals without HSS (p<0.00). A possible explanation for this observation is the initiation of a mass chemotherapy program for schistosomiasis in the late 1970s in Brazil. As the program was directed especially toward school children, it can be seen that the mean age of patients with HSS has shifted to an older population (age-cohort effect).
The median platelet counts were significantly lower in individuals with HSS (p<0.01), and platelet counts <143,000/mm3 identified patients with HSS with a sensitivity >92%. The prevalence of the disease was 23% in the study area. As expected, with a platelet count <143,000/mm3, sensitivities and specificities for the diagnosis of HSS varied depending on the definition of HSS. Based on the HSS definition of a palpable spleen and a spleen >13cm measured by US, 21 individuals (5.5% of the people enrolled in the study) had HSS, with a sensitivity of 93.4% and specificity of 47.6%. Some individuals with enlarged spleens most likely had other diseases, which would explain the low specificity. In addition, errors that occur during abdominal palpation or US examination cannot be excluded. When the HSS definition consisted of a spleen >13cm that was associated with periportal fibrosis as confirmed by US, 8 (2.1%) patients had HSS, with a sensitivity of 92.3% and specificity of 75%. This definition appears to be more accurate because it includes the characteristic schistosomal liver periportal fibrosis.
A group of 20 individuals with periportal fibrosis confirmed by US but with normal spleens was identified in our study, and the platelet counts were normal in this group. Symmer’s periportal fibrosis without splenomegaly has been reported previously2,13,28. This finding demonstrates the importance of enlarged spleens in schistosomal thrombocytopenia, and this condition has been referred to as the hepatic form of the disease. Knowing whether such patients have esophageal varices would be interesting. It is of interest to know whether these individuals bleed similarly to patients with HSS. Severe upper digestive bleeding in patients without esophageal varices but with duodenal varices has been reported29,30. An upper digestive endoscopy may partially address this issue in such patients. If this hypothesis is confirmed, this clinical form of schistosomiasis may be classified as hepatic with or without esophageal varices.
To confirm the field diagnosis, we brought 22 individuals with thrombocytopenia to our University Hospital in Belo Horizonte to undergo examination. Every week, two patients were admitted to the hospital for five days. During the first two weeks, we found one patient who presented a variable number of platelet counts: 35,000, 27,000, 93,000, 37,000, and zero. In addition, platelet clumping was observed in a peripheral blood smear. After changing the anticoagulant (EDTA to heparin), the platelet count increased to 160,000/mm3. The patient was diagnosed with pseudothrombocytopenia (an in vitro error) and was reallocated to the group with normal platelet counts. Pseudothrombocytopenia has been reported to occur in as many as 17% of patients who visit hematology units to seek a diagnosis for their thrombocytopenia31–36. We are planning a new study in rural areas of Brazil that are endemic for schistosomiasis to assess the frequency of pseudothrombocytopenia. Thrombocytopenia is a nuisance in the life of patients with HSS. Minor surgical, dental, and diagnostic procedures have been refused to these patients by misinformed healthcare professionals due to the possibility of bleeding because of low platelet counts. A percentage of such patients may have pseudothrombocytopenia, an artifact that can be easily avoided.
The major mechanisms associated with reduced platelet counts are the following: 1) decreased production (e.g., due to diseases or toxic drugs that affect the bone marrow); 2) increased destruction (caused by cytotoxic agents and other drugs, viral infections, and vitamin B12 or folic acid deficiency); 3) dilutional thrombocytopenia (caused by blood transfusions with packed red blood cells); 4) distributional thrombocytopenia (caused by splenomegaly); 5) increased platelet destruction [caused by autoimmune diseases, use of heparin, and diseases such as infectious mononucleosis, cytomegalovirus, and acquired immune deficiency syndrome (AIDS)]. Therefore, all these situations should be kept in mind and considered when analyzing thrombocytopenia in areas endemic for schistosomiasis.
We concluded that in areas endemic for schistosomiasis with a prevalence of 23%, platelet counts <143,000/mm3 identified HSS patients with good sensitivity and moderate specificity and can be used as a screening tool to identify severe disease in endemic areas. However, one question that remains to be answered is whether similar results will occur in areas with different schistosomiasis prevalences (5%, 10%, or 40%).
