Annals of Tropical Medicine and Public Health

: 2011  |  Volume : 4  |  Issue : 2  |  Page : 91--95

A fatal case of Trypanosoma lewesi in Maharashtra, India

PP Doke1, A Kar2,  
1 State Health System Resource Centre, University of Pune, Pune - 411 007, India
2 School of Health Sciences, University of Pune, Pune - 411 007, India

Correspondence Address:
P P Doke
State Health System Resource Centre, Parivartan Building, Arogya Bhavan, Alanid Road, Yerwada, Pune - 411 006


In the state of Maharashtra, India, in the three years 2004-05 to 2006-07, animal Trypanosoma parasites T. lewisi and T. evansi resulted in human illnesses in three different geographical regions. The first two cases have already been reported. The recent case was a man aged 57 years, residing in a small village in district Pune. Since September 2006 he had chronic intermittent fever, anemia, firm hepatosplenomegaly and edema on feet. The investigations in March 2007 revealed that it was a case of T. lewisi. The primary epidemiological investigations showed no other cases in the village. The investigations pointed to the probability of transmission through rat fleas. The case was brought under Suramin treatment in the B.J. Medical College and Sassoon General Hospital Pune. The patient expired after second dose in June 2007. His blood was sent for genetic study in the School of Health Sciences, University of Pune. The genetic study for mutation in the APOL 1 gene revealed that the four DNA fragments from the patient did not have any mutation in the amplified exonic fragments.

How to cite this article:
Doke P P, Kar A. A fatal case of Trypanosoma lewesi in Maharashtra, India.Ann Trop Med Public Health 2011;4:91-95

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Doke P P, Kar A. A fatal case of Trypanosoma lewesi in Maharashtra, India. Ann Trop Med Public Health [serial online] 2011 [cited 2020 Sep 20 ];4:91-95
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Human trypanosomiasis is a widely prevalent disease in all parts of Africa. There the causative parasite T. brucei and its antigenic variants cause sleeping sickness of varying chronicity and severity. In Central and South America a clinically different illness, called Chaga's disease is caused by T. cruzi. These are the only two strains of Trypanosome which are pathogenic to human beings. The existence of these two pathogenic strains is not known in other parts of the world. Besides man, all over the world the genus Trypanosoma is found in various animals causing different illnesses. In Asia the Trypanosoma are found in many animals including cattle (T. evansi) and rodents (T. lewisi). Animal parasites do not cause infection in human beings. There are only three reports of the accidental transmission of the Trypanosoma lewisi-like animal parasite to human beings in the South East Asia region excluding India. The first is a very old report way back in 1933 from Malaysia, [1] the second is from Thailand in 2005 [2] and the third from Gambia; [3] All these cases were infants suffering from febrile illness. The environmental conditions in India are conducive to the spread of the parasite from animals to human beings. A brief summary of reports of animal parasites causing illness in human beings from India and detailed investigations including genetic studies for this fatal case from the state of Maharashtra are presented here.

Human serum has an innate trypanolytic activity, first identified in 1900 by Laveran and Mesnil. [4] The protein responsible for this activity is a member of the serum apolipoprotein-L (apoL) family, termed apoL1. [5] This protein binds to a subset of high density lipoprotein (HDL) particles, which also contains another human protein, termed haptoglobin-related protein (Hpr). Studies with T. brucei have shown that the parasites possess a specific surface receptor for capturing hemoproteins that are essential for the survival of the host oxidative response. This receptor also binds Hpr-containing HDL particles; endocytosis of this receptor-ligand complex results in internalization of apoL1. [6] Within the lysosome, apoL1, a membrane pore-forming protein, is targeted to the lysosomal membrane, where, it induces an influx of chloride ions from the cytoplasm resulting in osmotic imbalance and death of the parasite. [7]

The parasites responsible for trypanosomiasis are resistant to lysis by normal human serum. Serum resistance is conferred by a variant surface glycoprotein termed serum resistance-associated protein (SRA). SRA is a lysosomal protein, and the N-terminal alpha-helix of SRA interacts strongly with a carboxy-terminal alpha-helix of apoL1. [8] Thus, apoL1 is the trypanosome lytic factor of normal human serum, and SRA confers resistance to lysis by interaction with apoL1 in the lysosome. Human susceptibility to trypanosomal infection is also known, since two genetic epidemiological studies conducted in Africa established that four single DNA polymorphisms located on genes coding for cytokines were correlated with a variable risk for development of the disease.

The first report of a suspected case of trypanosomiasis in India was from West Bengal in 1903. It describes many cases of irregular intermittent fever which were not malaria in an area called Dum Dum which is only seven miles away from Kolkata. The report cites a case of a patient who succumbed to the illness. [9]

The second report dates to 1974 and is from Raipur in Madhya Pradesh where Trypanosoma lewisi-like parasites in the peripheral blood of two cases, husband and wife, suffering from short febrile illness were detected. [10]

Almost after 30 years three cases in a short span of three years were reported in Maharashtra state which is home to about 10% of the population of the country. All these cases of human trypanosomiasis reported since 2004 come from three different geographical locations in the state. The first case infected by T. evansi reported in October 2004 was a male of 45 years, a cattle-breeder by profession. He was from village Shivni, block Sindewahi, District Chandrapur and responded to IV Suramin. [11] The village has a population of about 3000 and is very close to the Tadoba Reserve forest. The serum of this patient was markedly lacking in apoL1. Amplification of the complete APOL1 coding sequence revealed two different mutations, one allele, resulting in the absence of two bases, causing a frame shift mutation from residue 142 leading to a stop codon at position 149. For the other allele, the absence of a single base resulted in a frame shift mutation from residue 266 causing a premature stop codon at position 268. Both these mutations could be predicted to affect the pore-forming functions, explaining the lack of APOL1 activity in the serum. [12]

The investigations revealed that no other person from the village Shivni was suffering or was exposed to T. evansi. In the investigations it was found that a total of six bullocks had trypanosome parasite in their blood. The record of the animal husbandry department revealed that there was an outbreak of Sura (local name for the disease caused by T. evansi) in bovines in October 2004. There were 29 attacks and one death. The lack of trypanolytic factor, apoL-I might have caused the infection from the animal species of Trypanosoma. [11] On examination, a fresh scar on his right index finger was observed. The parasites may have entered through this wound, possibly while conducting cattle delivery. The other possibility may be mechanical transmission through Tabanid striatus flies like transmission of parasites in animals.

A serological survey was also carried out to assess the extent of the exposure of the community to T. evansi. A total of 410 (22.7%) people were positive by whole blood out of 1806 persons screened from the village. [13]

The second case was reported in September 2006 from Mumbai. The patient was a one month and twenty-two-days-old female infant infected by T. lewisi. She was given symptomatic treatment with antipyretics and is asymptomatic at the last follow-up on 22 December 2008. This patient might have got infected due to a bite by a flea carrying Trypanosoma on its feet. [14]

 Materials and Methods

The present case was first reported from a private hospital in Pune city in the month of March 2007. The patient was a 57-year-old man, a laborer from the water supply department at the Paud and resident of village Paud, Block Mulshi, District Pune. In his blood smear Trypanosoma parasites were found and hence the patient was referred to the B.J. Medical College and Sassoon General Hospital for further investigations and treatment. He was admitted on 16 March 2007 to the B.J. Medical College Pune. The parasite was suspected to be T. lewisi. The laboratory investigations and epidemiological investigations were carried out.

A 2-ml sample of blood from this patient was referred for genetic analysis of the APOL1 gene.

Sample and purification of DNA

DNA was isolated using Miller's method. Five ml of the patient's blood sample, collected in heparin-coated bottles was sent to the genetics laboratory from the hospital where the patient was admitted. Since heparin inhibits the polymerase chain reaction (PCR), the extracted DNA was purified by passing through a 10kDa Nanosep column to remove the heparin. Genomic DNA was also isolated from three normal individuals and the exonic sequences were amplified. The amplified fragments were sequenced and the sequences were aligned with the APOL1 gene sequences obtained from NCBI, National Centre for Biotechnology Information). [15] Amplification and sequencing was done at thrice for each fragment from the patients.

Polymerase chain reaction

The purified DNA was used as a template for amplification using the conditions reported by Vanhollebeke et al.[12] Each fragment was separately amplified three times. The amplified fragments were sequenced twice and the sequence obtained was aligned with the wild type gene sequence to detect mutations, if any.


A team visited the village and carried out epidemiological investigations in March 2007. The patient was not involved in grazing animals or washing clothes. The village population is 3196. Blood smears were collected from all 58 patients who had fever at the time of the survey. All were negative for malarial parasite and Trypanosomes. The animal count (buffalo, cattle and goat) was 176. The cattle sheds in 45 houses were adjacent to human dwellings. A total of 124 houses had rat infestation. The rat flea index was 0.8. Epidemiological investigations revealed that there were no other cases in that village. The blood and tissue smears from the rats, processed and screened at the National Institute of Communicable Diseases and Indian Veterinary Research Institute Izatnagar, have shown the presence of T. lewisi in 18% of the samples (six positive of the 36 examined).

From the month of June 2006 the patient had intermittent fever. In the month of January 2007 he was admitted to a private hospital. At the time of admission, he had history of fever with chills, dyspnoea on exertion. On examination he had anemia, firm splenomegaly and edema on feet. There he was diagnosed as a case of chronic malaria. He responded to antimalarial treatment. Again in the month of March he was admitted to another private hospital. In addition to the above clinical features, he has history of intermittent loose motions, loss of weight and appetite. On examination generalized edema was observed. In that hospital for the first time Trypanosome parasites were detected in the peripheral blood smear. His vital functions were stable when he was admitted to the B.J. Medical College. After consultation with experts from the World Health Organization (WHO) and the National Institute of Communicable Diseases, Delhi, it was decided to give intravenous Suramin. The results of his baseline investigations before the first dose of Suramin were; Hb-8.7 g., WBC-3,000, platelets- 135,000, Serum creatinine-0.7, urine albumin-traces, serum bilirubin 0.5, SGPT -11, alkaline phosphtase-273, apolipoprotein A1-54.06 mg/dl (n-60 to 230). Magnetic resonance imaging (MRI) revealed signs of cortical atrophy, ultrasonography showed splenomegaly. He was administered first dose of 1 g Suramin slowly as intravenous infusion. After six days of the first dose the results of his investigations were; Hemoglobin 7.7 g, white blood cell count-2800, platelets-47,000, serum creatinine-0.8, serum bilirubin 0.8, SGPT-96, alkaline phoshphatase-78, urine albumin positive. In view of bone marrow suppression in the form of moderate anemia and thrombocytopenia, second dose was postponed and supportive management was advised and patient was discharged. After 30 days second dose was planned giving adequate period for stabilization of the patient. The results of his investigations before the second dose were; Hb-9.3 g, WBC-4600, platelets-184,000, serum bilirubin- 0.6, SGPT- 32, alkaline phoshphatase 86, serum creratinine −0.9, serum protein-7.8, serum albumin- 3.1. He received the second dose of intravenous Suramin in the intensive care unit (ICU) at the B.J. Medical College, Pune under strict medical supervision. On 21 June 2007 he had massive hemetemesis during which he aspirated and there was flooding of respiratory passages. He did not respond to the treatment and expired on 21 June 2007. Postmortem was carried out. Trypanosome parasites were seen in the pericardial and ascetic fluid but the cerebrospinal fluid (CSF) did not show trypanosome. The postmortem impression was trypanosomiasis with benign thymoma, lobar pneumonia (left lung), testicular atrophy, esophageal and gastric ulceration and lymphocytic infiltrate in the thyroid, adrenals and intestine. The blood sample was sent to the Indian Veterinary Research Institute, Izatnagar. There, following morphometric identification of T. lewisi, the blood sample was also analyzed by PCR. The PCR assay result confirmed T. lewisi.

Four of the five exonic sequences of the APOL1 gene were examined for presence of variations. The amplified fragments [Figure 1] were sequenced at least twice and the sequence obtained was aligned with the wild type gene sequence to detect mutations, if any. There were no mutations detected in the amplified fragments.{Figure 1}


The trypanosome parasite was first discovered by David Bruce in 1895 and he suggested it to be the cause of Nagana disease in horses and cattle. Forde, in 1902, demonstrated motile Trypanosomes in the blood of a man suffering from fever. The parasite was subsequently named T. gambiense in the same year by J. E. Dutton as the patient was from Gambia. In the next year itself there was a report of suspected Trypanosome infection in India. In Dum Dum near Kolkata there were many cases of chronic intermittent fever which was not malaria. A fatal case was described with autopsy findings of microscopic examination of tissues. In the report briefly it is also described why these cases miss diagnosis. Again after more than hundred years a serious concern is being expressed about the threat to human health in South East Asia. [16] Till 2005 there was no death and all the patients described in the studies referred above resolved or responded to the standard drug Suramin. Trypanosoma parasites were recently found in the blood of a patient in the Canning subdivision of West Bengal in the district of South 24 Paraganas near Kolkata. The presence of the parasite was confirmed by the protozoology experts of the School of Tropical Medicine Kolkata. The patient expired in a private nursing home, within few days of admission. [17] Similarly a sero survey from W. Bengal revealed that, in different geographical areas sero-positive results for T. evansi varied from 0-70%. There is no significant sex difference regarding positive results, however, there is a distinct seasonal variation with increased positive results between July to September. [18] These findings certainly indicate that the problem of animal parasite causing human illness is not very infrequent. Whilst genetic susceptibility (such as APOL1 polymorphisms) could account for one reason, changes in the parasite could also be responsible for increased invasiveness of the parasite.

In the present report, the clinical manifestations of the patient were similar to the first diagnosed patient from Maharashtra. Both had history of intermittent fever for some months. Although negative, they were treated with antimalarial and responded to it for a short period, only to relapse again. The first patient was cured completely after Suramin treatment, however, the present patient expired after a second dose of Suramin. Even after two doses of the medicines the Trypanosomes were present in the pericardial and ascetic fluid. It is difficult to comment on whether this is treatment failure or not. It is also difficult to quantify the drug toxicity in this patient.

Particularly in the state of Maharashtra the concern is more serious because one case of T. evansi and two cases of T. lewisi have been reported in a span of three years. Moreover, the last case expired. The investigations indicate that the present third case also might have got infected due to a possible bite by a flea carrying Trypanosomes on its feet.

As expected from the earlier case the serum was deficient in Apolipoprotein A1. Further genetic investigations were carried out to determine whether the infection in the patient was due to mutations in APOL1 gene. This gene was selected because earlier report of first diagnosed patient with trypanosomiasis had demonstrated no serum APOL1 activity. That patient showed two different frame shift mutations, predicted to disrupt APOL1 protein function. [12] Therefore, in this study we investigated whether a mutation in the APOL 1 gene could be responsible for trypanosomiasis in the patient. Polymerase chain reaction technique was used for amplification of the fragments followed by sequencing. The PCR amplification conditions were standardized using three normal volunteers. Sequences of all three individuals showed identical match to the wild type gene sequence. Analysis of the sequences of the four DNA fragments from the patient did not reveal any mutations in the amplified exonic fragments.

More than 10 million blood smears are examined for the presence of malarial parasites from febrile patients or those having history of fever in Maharashtra state. The majority are acute viral respiratory infections. About 50,000 are diagnosed as malaria cases. About 5000 are dengue cases. In many patients conclusive diagnosis is not reached. Among these, quality staining for Trypanosomes and examination by a fully trained microbiologist may yield a few more cases at least in areas where T. evansi and T. lewisi are frequently found. This strategy will still give better results if supported earlier by sero surveillance. Studies have already shown high sero-positivity in areas reported human trypanosomiasis.


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