INTRODUCTION
Visceral leishmaniasis (VL) (also called kala-azar) is a notable neglected tropical disease. More than 90% of the cases occur in India, Bangladesh, and Nepal. In Bihar, India, 33 of 38 districts are VL endemic, particularly their low socioeconomic communities. Humans are the only known reservoir of Leishmania donovani parasites in India.1 The signs and symptoms of VL are similar to other commonly occurring infectious diseases in the endemic region such as malaria, typhoid fever, and tuberculosis. Visceral leishmaniasis most commonly presents with fever, weight loss, hepatosplenomegaly, and lymphadenopathy (particularly in Sudan).2
The rK39 rapid diagnostic test (RDT) is considered ideal for the screening of VL, but it gives false-positive results with other diseases, namely, malaria, tuberculosis, and liver cirrhosis.3 The present case series describes a rare occurrence of false-positive rK39 test results in five confirmed chronic myeloid leukemia (CML) cases in India.
MATERIALS AND METHODS
rK39 RDT.
Qualitative detection of IgG antibodies to Leishmania donovani was made in the patients’ blood samples using the rK39 RDT. One or two drops of blood were placed on the absorbent pad on the stripped bottom. After that, two or three drops of the buffer solution were added to the absorbent pad area of the test kit. After 5–10 minutes, if a red band appeared in the test region (T) and in the control region (CR), the test result was considered positive. If only one band appeared in the CR, then the test result was considered negative. If control line did not appear, then the test was considered invalid and it was repeated using a fresh rK39 RDT.
The Institutional Ethics Committee of the Indian Council of Medical Research (ICMR)-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna, Bihar, India, approved this study, and a written informed consent was obtained from all participants. All participants were admitted to the ICMR-RMRIMS in the year 2018, with signs and symptoms of VL (i.e., all patients had a fever of > 15 days duration, loss of appetite, weight loss, hepatomegaly, and massive splenomegaly). None of the patients had a history of VL. At admission, all of them tested positive for the rK39 RDT. Therefore, they were admitted to confirm the diagnosis of VL.
Before performing the gold standard procedure for the diagnosis of VL (i.e., splenic aspiration/bone marrow aspiration for LD bodies), pertinent pathological and biochemical tests were performed in the Central Diagnostic Laboratory, ICMR-RMRIMS, Patna (National Accreditation Board for Testing and Calibration Laboratories accredited). Thereafter, splenic aspiration was performed in all the patients but found to be negative for LD bodies. The blood picture including the peripheral smear was suggestive of CML. Thus, the patients were subjected to bone marrow aspiration, which was also negative for LD bodies. However, bone marrow examination and peripheral blood film slides were suggestive of CML in all cases. Therefore, they were referred to a cancer detection and treatment center where BCR-ABL gene was found to be positive in all the cases. The patients were treated with imatinib mesylate.
RESULT AND DISCUSSION
Clinical and laboratory investigation details of the patients are summarized in Table1. The common presentations of CML, namely, fever, anemia, decreased appetite, weight loss, abdominal fullness, and massive splenomegaly are similar to VL. The rK39 RDT has been recommended as the main diagnostic tool at the field level in India by the National Vector Borne Disease Control Programme in the VL elimination program, which aims to reduce the number of VL cases to < 1/10,000 population in the endemic area at the block level.3 This RDT is considered as efficient and reliable for VL and post–kala-azar dermal leishmaniasis diagnosis at the field level. False positivity of rK39 has been reported with malaria, tuberculosis, and liver cirrhosis.3 However, this is the first case series of five confirmed cases of CML from the VL-endemic area of Bihar where rK39 was found to be false positive. Our data are comparable with the previous findings of Mandal et al.,4 where false-positive rK39 test results were reported in four CML and two myelofibrosis cases.
Clinical and laboratory profiles of study patients
| Parameter | Case 1 | Case 2 | Case 3 | Case 4 | Case 5 | |
|---|---|---|---|---|---|---|
| Age (years) | 35 | 40 | 40 | 60 | 55 | |
| History of fever (months) | 3 | 3 | 6 | 2 | 7 | |
| rK39 strip test | Positive | Positive | Positive | Positive | Positive | |
| Splenic aspiration | LD bodies not found | LD bodies not found | LD bodies not found | LD bodies not found | LD bodies not found | |
| Bone marrow aspiration | LD bodies not found | LD bodies not found | LD bodies not found | LD bodies not found | LD bodies not found | |
| Hypercellular, mostly myeloid cells, that is, myelocytes, metamyelocytes, stab and segmented cells, and blast cells. The M/E ratio was markedly increased | Hypercellular, mostly myeloid cells, that is, myelocytes, metamyelocytes, stab and segmented cells, and blast cells. The M/E ratio was markedly increased | Hypercellular, mostly myeloid cells, that is, myelocytes, metamyelocytes, stab and segmented cells, and blast cells. The M/E ratio was markedly increased | Hypercellular, mostly myeloid cells, that is, myelocytes, metamyelocytes, stab and segmented cells, and blast cell. The M/E ratio was markedly increased | Hypercellular, mostly myeloid cells, that is, myelocytes, metamyelocytes, stab and segmented cells, and blast cells. The M/E ratio was markedly increased | ||
| RBC (3.9–5.7 million/mm3) | 3.63 | 2.91 | 2.61 | 3.33 | 3.00 | |
| Hemoglobin (11.0–16.2 gm/dL) | 10.5 | 7.8 | 8.37 | 8.9 | 9 | |
| Total WBC count (4,000–10,500/mm3) | 125,000 | 160,000 | 492,000 | 127,000 | 190,000 | |
| Differential WBC count (%) | N | Stab cell: 12 | Stab cell: 06 | Stab cell: 08 | Stab cell: 21 | Stab cell: 18 |
| N: 40–70 | Segmented: 38 | Segmented: 37 | Segmented: 20 | Segmented: 36 | Segmented: 31 | |
| Promyelocyte: 01 | Promyelocyte: 03 | Promyelocyte: 11 | Promyelocyte: 03 | Promyelocyte: 05 | ||
| Myelocyte: 28 | Myelocyte: 33 | Myelocyte: 40 | Myelocyte: 20 | Myelocyte: 19 | ||
| Metamyelocyte: 8 | Metamyelocyte: 05 | Metamyelocyte: 06 | Metamyelocyte: 10 | Metamyelocyte: 06 | ||
| Blast: 01 | Blast: 03 | Blast: 03 | Blast: 01 | Blast: 04 | ||
| L: 15–40 | L | 06 | 05 | 05 | 04 | 07 |
| M: 01–06 | M | 03 | 02 | 01 | 01 | 02 |
| E: 01–05 | E | 02 | 05 | 03 | 02 | 05 |
| B: 00–01 | B | 01 | 01 | 03 | 02 | 03 |
| Platelet count (140,000–450,000/mm3) | 155,000 | 233,000 | 320,000 | 655,000 | 180,000 | |
| Peripheral blood smear comments | RBCs showed mild to moderate anisopoikilocytosis, immature cells, polychromasia, and nRBCs. WBC count markedly increased suggestive of CML | RBCs showed mild to moderate hypochromasia and mild anisocytosis. Immature myeloid cell count increased, suggestive of CML | RBCs showed mild hypochromasia and moderate anisocytosis. WBC count markedly increased, as well the immature cells. Basophils also increased, suggestive of CML | RBCs showed mild to moderate hypochromasia and mild anisocytosis. WBC count increased, as well as immature myeloid cells, suggestive of CML | Few microcytes, mild anisocytosis and hypochromasia, few polychromatic cells, few Barr cells. WBC count increased, as well as immature myeloid cells, suggestive of CML | |
| Hepatitis B test | Nonreactive | Nonreactive | Nonreactive | Nonreactive | Nonreactive | |
| Hepatitis C test | Nonreactive | Nonreactive | Nonreactive | Nonreactive | Nonreactive | |
| HIV I and II test | Nonreactive | Nonreactive | Nonreactive | Nonreactive | Nonreactive | |
B = basophils; CML = chronic myeloid leukemia; E = eosinophils; L = lymphocytes; LD = Leishmania donovani; M = monocytes; M/E = myeloid:erythroid; N = neutrophils; RBC = red blood cell; WBC = white blood cell.
All cases in the present study had a history of fever for more than 2 months, and rK39 positivity indicated them as possible VL subjects. However, both splenic and bone marrow aspirate examinations ruled out the possibility of VL. The total white blood cell (WBC) counts in all the screened patients were in between 125,000 and 492,000/mm3, which was significantly higher than the normal range. Fascinatingly, differential WBC counts showed abnormal cells, namely, stab cells, segmented cells, promyelocytes, myelocytes, metamyelocytes, and blast cells. Furthermore, anisopoikilocytosis in red blood cells was observed among all cases. Interestingly, all cases showed a markedly increased myeloid-to-erythroid precursor ratio in the bone marrow. It is quite evident that escalated myeloid:erythroid ratio is found in infections, chronic myelogenous leukemia, or erythroid hypoplasia; a decreased ratio may mean a depression of leukopoiesis or normoblastic hyperplasia.5,6 Notably, all five patients had signs and symptoms of VL; however, their high blood WBC counts were not suggestive of VL. This together with other symptoms, particularly severe splenomegaly, will usually guide physicians to think about CML or other similar disorders. Therefore, one should go first for bone marrow examination and not for spleen aspiration, which is a very risky procedure.
Visceral leishmaniasis has to be differentiated from malaria, typhoid fever, CML, and myelofibrosis, as the clinical features are almost similar (fever, hepatomegaly, and splenomegaly). However, as shown, the rK39 positivity in all these cases can lead to a fallacious diagnosis of VL. Hence, CML may also be taken into consideration once a case of fever with hepatomegaly and massive splenomegaly turns up at a medical clinic in VL-endemic regions. Notably, this study did not include those CML cases who were rK39 negative; this is one of the limitations of this study. Hence, the findings of this study should be read cautiously, as rK39-positive result in CML cases was a serendipitous occurrence; this should be validated further to determine the utility of the rK39 test in the differential diagnosis of VL with CML. The rK39 strip test alone is not reliable for determining the diagnosis/prognosis of VL. Therefore, the use of quantitative ELISA or other diagnostic approaches is recommended to minimize the occurrence of false diagnosis of VL.
ACKNOWLEDGMENTS
We are highly thankful to the study participants who provided their consent and enthusiastically participated in the study. We are also thankful to Mary Shanti Grace Thithio, Surya Suman, Sushil Kumar, Rajendra Kumar, Tathagat Gupta, and Rakhi Kumari for their valuable support during nursing care, field visit, and laboratory work.
REFERENCES
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