S156
Indian Journal of Medical Microbiology
eighteenth day of illness when the IgM antibody has already ushered in the blood and CSF and neutralised the viral particles. So it could not be detected by RT-PCR method.
6.
The re-emerging CHIKV is of central-east African origin, replaced the previous Asian genotype from this country.[13] It is remain unclear whether the severity of the disease is due to the lack of immunity in the population or the changing of genotype. Detail study is required for this purpose.
7.
Ethical Approval The study was duly approved by the joint ethical committee of ICMR (Indian Council of Medical Research) virus unit and NICED (National Institute of Cholera and Enteric Diseases), Kolkata, India.
8.
9.
10.
Acknowledgement This work was supported by from the intramural funds of Indian Council of Medical Research, Government of India We express our sincere thanks to Dr. Sekhar Chakrabarti, Officer-In-Charge, ICMR Virus Unit, for allowing us to carry out the work. The enthusiastic help obtained from the physicians of District Hospitals in West Bengal, for providing us with the clinically suspected samples for this study, is gratefully acknowledged.
11. 12.
13.
References 1. 2.
3. 4. 5.
Powers AM, Logue CH. Changing patterns of chikungunya virus: Re-emergence of a zoonotic arbovirus. J Gen Virol 2007;88:2363-77. Shah KV, Gibbs CJ Jr, Banerjee G. Virological investigation of the epidemic of haemorrhagic fever in Calcutta: Isolation of three strains of Chikungunya virus. Indian J Med Res 1964;52:676-83. Padbidri VS, Gnaneswar TT. Epidemiological investigations of chikungunya epidemic at Barsi, Maharashtra state, India. J Hyg Epidemiol Microbiol Immunol 1979;23:445-51. Pavri K. Disappearance of Chikungunya virus from India and South East Asia. Trans R Soc Trop Med Hyg 1986;80:491. Ravi V. Re-emergence of chikungunya virus in India. Indian J Med Microbiol 2006;24:83-4.
vol. 33, Supplement 1
Taraphdar D, Sarkar A, Mukhopadhyay B, Chakrabarti S, Chatterjee S. Rapid spread of chikungunya virus following its resurgence during 2006 in West Bengal, India. Tran R Soc Trop Med Hyg 2012;106:160-6. Lewthwaite P, Vasanthapuram R, Osborne JC, Begum A, Plank JL, Shankar MV, et al. Chikungunya virus and central nervous system infections in children, India. Emerg Infect Dis 2009;15:329-31. Taraphdar D, Sarkar A, Chatterjee S. Mass scale screening of common arboviral infections by an affordable, cost effective RT-PCR method. Asian Pac J Trop Biomed 2012;2:97-101. Economopoulou A, Dominguez M, Helynck B, Sissoko D, Wichmann O, Quenel P, et al. Atypical chikungunya virus infections: Clinical manifestations, mortality and risk factors for severe disease during the 2005-2006 outbreak on Re' union. Epidemiol Infect 2009;137:534-41. Chandak NH, Kashyap RS, Kabra D, Karandikar P, Saha SM, Morey SH, et al. Neurological complications of chikungunya virus infection. Neurol India 2009;57:177-180. Rampal, Sharda M, Meena H. Neurological complications in chikungunya fever. J Assoc Physicians India 2007;55:765-9. Singh SS, Manimunda SP, Sugunan AP, Sahina, Vijayachari P. Four cases of acute flaccid paralysis associated with chikungunya virus infection. Epidemiol Infect 2008;136:1277-80. Pialoux G, Gaüzère BA, Jauréguiberry S, Strobel M. Chikungunya, an epidemic arbovirosis. Lancet Infect Dis 2007;7:319-27. Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.150946
How to cite this article: Taraphdar D, Roy BK, Chatterjee S. Chikungunya virus infection amongst the acute encephalitis syndrome cases in West Bengal, India. Indian J Med Microbiol 2015;33:153-6.
Source of Support: Nil, Conflict of Interest: None declared
First case of Paragonimus westermani infection in a female patient in India *TS Singh, S Hiromu, KR Devi, WA Singh
Abstract Paragonimiasis is a foodborne parasitic zoonosis caused by lung fluke species of the genus Paragonimus. The Paragonimus westermani is the most common human pathogen in Asian countries. In northeast India, Paragonimus heterotremus has been documented as the only human pathogen in the earlier literature. In India, P. westermani infection in humans remained undetermined. Herein, we report a case of pulmonary paragonimiasis due to P. westermani in an adult female in Manipur. The diagnosis was made by morphological and molecular characterisation of the eggs in the sputum. This is the first confirmed case of paragonimiasis due to P. westermani in India. Key words: India, northeast India, Paragonimus westermani, pulmonary paragonimiasis www.ijmm.org
February
Case Reports
*Corresponding author (email: ) Department of Microbiology (TSS), Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim, India, Department of Parasitology (SH), National Institute of Infectious Diseases, Tokyo, Japan, Department of Microbiology (KRD), Department of Tuberculosis and Chest Medicine (WAS), Regional Institute of Medical Sciences, Imphal, Manipur, India Received: 23-05-2014 Accepted: 24-07-2014
Introduction Paragonimiasis or lung fluke infection is one of the neglected tropical foodborne parasitic zoonosis distributed worldwide. About 50 Paragonimus species including synonyms have been described from East and Southeast Asia, Africa and America.[1] Paragonimus westermani, the most common species in Asia, was first described by Kerbert from the lungs of a Bengal tiger, which was captured in India and died at a zoo in Amsterdam more than a century ago. However, very little attention has been paid to this parasite because paragonimiasis was never considered to be a public health problem in India until 39 cases were reported from Manipur, in 1986.[2] Thereafter, several endemic foci were discovered in Manipur,[3] Arunachal Pradesh[4] and Nagaland.[5] Most interestingly, P. heterotremus has been identified as the causative agent of human paragonimiasis in this part of India against the widely believed P. westermani, which was reported from many mammals in India and recently the metacercariae were isolated from the second intermediate crab hosts in northeast India.[6] Herein, we present a case of pulmonary paragonimiasis, which was initially diagnosed as smear negative pulmonary tuberculosis and was under anti-tubercular drugs. The diagnosis was based on the clinical features and laboratory findings of the patient, and molecular results demonstrated the causative species as P. westermani. This is the first case of P. westermani infection in humans in India confirmed by the morphological and molecular characterisation of Paragonimus eggs in the sputum specimen. Case Report A Manipuri tribal female, 50 years of age, was attended to the Regional Institute of Medical Sciences (RIMS), Imphal, with complaints of cough and recurrent hemoptysis for 2 years. She was initially diagnosed as smear negative pulmonary tuberculosis, at a primary health center and was under anti-tubercular therapy for a period of one and half years. As there was no clinical improvement, she was referred to RIMS. History of her dietary habits revealed frequent consumption of smoked and improperly cooked crabs collected from the mountain streams and bought from the market in Moreh, Manipur. She denied of associated fever, breathlessness, chest pain, and weight loss. Laboratory investigations of three consecutive sputum
S157
samples were negative for acid-fast bacilli, pyogenic bacteria, fungi, and malignant cells, but positive for Paragonimus eggs [Figure 1]. The chest roentgenogram showed multiple nodular opacities of varying sizes, some with breakdown (cavity) in the upper and middle zones of both lungs and in the right basal and para cardiac regions [Figure 2]. Both apices and costophrenic angles were clear. There was no evidence of calcification. Her complete blood count (CBC) revealed a total leucocyte count of 7200/l with 16% eosinophils and erythrocyte sedimentation rate (ESR) of 80 mm/1st hour (Westergren’s). The case was diagnosed as a chronic pulmonary paragonimiasis and was treated with praziquantel 25 mg/kg body wt., three times a day for 3 days. Sputum became free from blood and Paragonimus eggs on the second day following the chemotherapy. The clinical and laboratory data including radiological findings were critically reviewed, and identified P. westermani like eggs in the sputum smear, at the Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim. Molecular characterisation of a Paragonimus egg sample from the patient was performed by DNA isolation, amplification of the internal transcribed spacer 2 (ITS2) regions of the ribosomal DNA and sequencing as described by Sugiyama et al. (2002, 2013).[7,8] Sequence analysis of the polymerase chain reaction (PCR) products revealed that the alignments of the rDNA region spanning the ITS2 were 436 bp in length [Figure 3]. An asterisk indicates the presumed beginning of the actual ITS2. The 5’ end of the sequence is of 5.8S rDNA origin. The numbers refer to the alignment positions. A dot in the alignment of the P. westermani sequence with accession No. of JN656208 indicates a nucleotide identical to that of our sample: The lengths of 76 bp and 360 bp were estimated for the posterior portion of 5.8S rDNA and almost entire portion of the actual ITS2, respectively, in comparison with the sequence reported for the adult P. westermani. The nucleotide sequence of Paragonimus eggs from the patient determined in this study has been deposited in the DDBJ/EMBL/GenBank database under the accession number AB123456. Searches of the nucleotide databases revealed that the sequences were almost identical to that deposited in the GenBank/EMBL/DDBJ nucleotide database with the accession number of JN656208, which was for P. westermani isolated from Arunachal Pradesh as a metacercaria. Pairwise comparison of the sequences over an alignment length of 436 bp revealed just 1 (0.23%) base difference, which was probably due to the intraspecific sequence variation. We, therefore, identified the eggs discharged in a sputum sample of the patient as P. westermani. Discussion To date, five Paragonimus species viz. P. heterotremus, P. skrjabini, P. hueitu’ngensis, P. miyazaki manipurinus n. sub spp and P. westermani have been reported to infect
www.ijmm.org
S158
Indian Journal of Medical Microbiology
two fresh water crabs; Potamiscus manipurensis and Alcomon superciolosum, which serve as second intermediate
Figure 1: Saline wet mount preparation showing characteristic golden brown Paragonimus eggs in the sputum specimen of the patient. (40× magnification)
Figure 2: Chest roentgenogram posteroanterior (PA) view of the patient showing multiple nodular opacities, some with cavities in the upper and middle zones of both lungs, and in the right basal and para cardiac regions
Figure 3: An aligned nucleotide sequence of an ITS2 region obtained from P. westermani eggs from the patient by PCR amplification using the primer pair 3S (forward, 5æ-GGTACCGGTGGATCACTCGGCTCGTG-3æ) and BD2 (reverse, 5æ-GGGATCCTGGTTAGTTTCTTTTCCTCCGC-3æ). An asterisk indicates the presumed beginning of the actual ITS2. The 5æ end of the sequence is of 5.8S rDNA origin. The numbers refer to the alignment positions. A dot in the alignment of the P. westermani sequence with accession No. of JN656208 indicates a nucleotide identical to that of our sample
vol. 33, Supplement 1
hosts in Manipur.[6] The crabs, P. manipurensis are the most widely distributed as the host of P. heterotremus and other Paragonimus species occurring in Manipur and Nagaland. Recently, P. westermani metacercariae were isolated from the fresh water crabs, Alcomon collected at mountain streams in Moreh, Manipur. The P. westermani infection was also described in the two species of fresh water crab hosts, Maydelliathelphusa in Arunachal Pradesh and Meghalaya and Sartoriana spinigera in Assam.[9,10] Although, P. westermani infection was found in many wild and domestic mammals, infection in humans remained undetermined in India. In the past P. westermani was presumed to be the causative agent of paragonimiasis in Manipur only on the observation of Paragonimus eggs in the clinical specimens even without describing the morphological characteristics of the eggs. Nevertheless, some morphological features of eggs such as shape, size and shell character can be used to discriminate Paragonimus species, for example the eggs of P. heterotremus have an almost uniform shell thickness and discernible at the operculated end in contrast to the P. westermani eggs that are asymmetrically ovoid, shell thickened mostly at the apopercular end and opercular end as prominent shoulders. However, these features are inconsistent as the eggs of many Paragonimus species have overlapping morphological features that make species identification impracticable. Therefore, the earlier reports that implicated P. westermani as the causative agent of human infection cannot be considered as valid. Recent advances in the molecular technology such as PCR and DNA sequencing and hybridisation have enabled to identify and discriminate Paragonimus species at various stages of development in the life cycle. Subsequently, morphological and molecular characterisations of eggs in the sputum specimens of several patients have revealed P. heterotremus as the common human pathogen in the northeast India.[10,11] In the present case, we confirmed P. westermani as the causative agent of the infection based on the morphological features and molecular characterisation of the eggs in the sputum specimen. This is the first record of P. westermani infection in human, in India. The low incidence of P. westermani infection in this region may be due to the restricted areas of distribution of the parasite, low host specificity, and paucity of suitable intermediate hosts. Further study will be needed to find some characteristic features of the eggs that can help identify and discriminate different Paragonimus species in the clinical specimens. Presently PCR and DNA sequencing of the eggs in the clinical specimens is the only option for species determination. Acknowledgements This study was supported in part by the Japan Society for the Promotion of Science, JSPS (KAKENHI: Grant No. 25305011) and by grants for Research on Emerging and Re-emerging Infectious
www.ijmm.org
February
Diseases (H23-Shinko-ippan-014 and H26-Shinko-ippan-009) from the Ministry of Health, Labor and Welfare of Japan.
References 1.
2. 3. 4. 5.
6. 7.
8.
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Case Reports
Miyazaki I. Lung flukes in the world: Morphology and life history. In: Sasa M, editor. A Symposium on Epidemiology of Parasitic Diseases. Tokyo: International Medical Foundation of Japan; 1974. p. 101-34. Singh TS, Mutum SS, Razaque MA. Pulmonary paragonimiasis: Clinical features, diagnosis and treatment of 39 cases in Manipur. Trans R Soc Trop Med Hyg 1986;80:967-71. Singh TS, Mutum S, Razaque MA, Singh YI, Singh EY. Paragonimiasis in Manipur. Indian J Med Res 1993;97:247-52. Narain K, Devi Rekha K, Mahanta J. Paragonimus and paragonimiasis-A new focus in Arunachal Pradesh, India. Curr Sci 2003;l84:985-7. Singh TS, Sugiyama H, Umehara A, Hiese S, Khalo K. Paragonimus heterotremus infection in Nagaland: A new focus of pragonimiasis in India. Indian J Med Microbiol 2009;27:123-7. Singh TS, Sugiyama H, Rangsiruji A. Paragonimus and paragonimiasis in India. Indian J Med Res 2012;136:192-204. Sugiyama H, Morishima Y, Kameoka Y, Kawanaka M. Polymerase chain reaction (PCR)-based molecular discrimination between Paragonimus westermani and P. miyazakii at the metacercarial stage. Mol Cell Probes 2002;16:231-6. Sugiyama H, Singh TS, Rangsiruji A. Paragonimus (ch 39). Molecular Detection of Human Parasitic Pathogens. 1st ed. Florida: CRC Press; 2013. p. 421-33.
9.
Devi KR, Narain K, Agatsuma T, Blair D, Nagataki M, Wickramasinghe S, et al. Morphological and molecular characterization of Paragonimus westermani in northeastern India. Acta Trop 2010;116:31-8. 10. Singh TS, Sugiyama H, Rangsiruji A, Devi KR. Morphological and molecular characterizations of Paragonimus heterotremus, the causative agent of human paragonmiasis in India. Southeast Asian J Trop Med Public Health 2007;38 suppl 1:82-6. 11. Devi KR, Narain K, Bhattacharya S, Negmu K, Agatsuma T, Blair D, et al. Pleuropulmonary paragonimiasis due to Paragonimus heterotremus: Molecular diagnosis, prevalence of infection and clinicoradiological features in an endemic area of northeastern India. Trans Roy Soc Trop Med Hyg 2007;101:786-92. Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.150950
How to cite this article: Singh TS, Hiromu S, Devi KR, Singh WA. First case of Paragonimus westermani infection in a female patient in India. Indian J Med Microbiol 2015;33:S156-9. Source of Support: Nil, Conflict of Interest: None declared.
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Indian Journal of Medical Microbiology
eighteenth day of illness when the IgM antibody has already ushered in the blood and CSF and neutralised the viral particles. So it could not be detected by RT-PCR method.
6.
The re-emerging CHIKV is of central-east African origin, replaced the previous Asian genotype from this country.[13] It is remain unclear whether the severity of the disease is due to the lack of immunity in the population or the changing of genotype. Detail study is required for this purpose.
7.
Ethical Approval The study was duly approved by the joint ethical committee of ICMR (Indian Council of Medical Research) virus unit and NICED (National Institute of Cholera and Enteric Diseases), Kolkata, India.
8.
9.
10.
Acknowledgement This work was supported by from the intramural funds of Indian Council of Medical Research, Government of India We express our sincere thanks to Dr. Sekhar Chakrabarti, Officer-In-Charge, ICMR Virus Unit, for allowing us to carry out the work. The enthusiastic help obtained from the physicians of District Hospitals in West Bengal, for providing us with the clinically suspected samples for this study, is gratefully acknowledged.
11. 12.
13.
References 1. 2.
3. 4. 5.
Powers AM, Logue CH. Changing patterns of chikungunya virus: Re-emergence of a zoonotic arbovirus. J Gen Virol 2007;88:2363-77. Shah KV, Gibbs CJ Jr, Banerjee G. Virological investigation of the epidemic of haemorrhagic fever in Calcutta: Isolation of three strains of Chikungunya virus. Indian J Med Res 1964;52:676-83. Padbidri VS, Gnaneswar TT. Epidemiological investigations of chikungunya epidemic at Barsi, Maharashtra state, India. J Hyg Epidemiol Microbiol Immunol 1979;23:445-51. Pavri K. Disappearance of Chikungunya virus from India and South East Asia. Trans R Soc Trop Med Hyg 1986;80:491. Ravi V. Re-emergence of chikungunya virus in India. Indian J Med Microbiol 2006;24:83-4.
vol. 33, Supplement 1
Taraphdar D, Sarkar A, Mukhopadhyay B, Chakrabarti S, Chatterjee S. Rapid spread of chikungunya virus following its resurgence during 2006 in West Bengal, India. Tran R Soc Trop Med Hyg 2012;106:160-6. Lewthwaite P, Vasanthapuram R, Osborne JC, Begum A, Plank JL, Shankar MV, et al. Chikungunya virus and central nervous system infections in children, India. Emerg Infect Dis 2009;15:329-31. Taraphdar D, Sarkar A, Chatterjee S. Mass scale screening of common arboviral infections by an affordable, cost effective RT-PCR method. Asian Pac J Trop Biomed 2012;2:97-101. Economopoulou A, Dominguez M, Helynck B, Sissoko D, Wichmann O, Quenel P, et al. Atypical chikungunya virus infections: Clinical manifestations, mortality and risk factors for severe disease during the 2005-2006 outbreak on Re' union. Epidemiol Infect 2009;137:534-41. Chandak NH, Kashyap RS, Kabra D, Karandikar P, Saha SM, Morey SH, et al. Neurological complications of chikungunya virus infection. Neurol India 2009;57:177-180. Rampal, Sharda M, Meena H. Neurological complications in chikungunya fever. J Assoc Physicians India 2007;55:765-9. Singh SS, Manimunda SP, Sugunan AP, Sahina, Vijayachari P. Four cases of acute flaccid paralysis associated with chikungunya virus infection. Epidemiol Infect 2008;136:1277-80. Pialoux G, Gaüzère BA, Jauréguiberry S, Strobel M. Chikungunya, an epidemic arbovirosis. Lancet Infect Dis 2007;7:319-27. Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.150946
How to cite this article: Taraphdar D, Roy BK, Chatterjee S. Chikungunya virus infection amongst the acute encephalitis syndrome cases in West Bengal, India. Indian J Med Microbiol 2015;33:153-6.
Source of Support: Nil, Conflict of Interest: None declared
First case of Paragonimus westermani infection in a female patient in India *TS Singh, S Hiromu, KR Devi, WA Singh
Abstract Paragonimiasis is a foodborne parasitic zoonosis caused by lung fluke species of the genus Paragonimus. The Paragonimus westermani is the most common human pathogen in Asian countries. In northeast India, Paragonimus heterotremus has been documented as the only human pathogen in the earlier literature. In India, P. westermani infection in humans remained undetermined. Herein, we report a case of pulmonary paragonimiasis due to P. westermani in an adult female in Manipur. The diagnosis was made by morphological and molecular characterisation of the eggs in the sputum. This is the first confirmed case of paragonimiasis due to P. westermani in India. Key words: India, northeast India, Paragonimus westermani, pulmonary paragonimiasis www.ijmm.org
February
Case Reports
*Corresponding author (email: ) Department of Microbiology (TSS), Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim, India, Department of Parasitology (SH), National Institute of Infectious Diseases, Tokyo, Japan, Department of Microbiology (KRD), Department of Tuberculosis and Chest Medicine (WAS), Regional Institute of Medical Sciences, Imphal, Manipur, India Received: 23-05-2014 Accepted: 24-07-2014
Introduction Paragonimiasis or lung fluke infection is one of the neglected tropical foodborne parasitic zoonosis distributed worldwide. About 50 Paragonimus species including synonyms have been described from East and Southeast Asia, Africa and America.[1] Paragonimus westermani, the most common species in Asia, was first described by Kerbert from the lungs of a Bengal tiger, which was captured in India and died at a zoo in Amsterdam more than a century ago. However, very little attention has been paid to this parasite because paragonimiasis was never considered to be a public health problem in India until 39 cases were reported from Manipur, in 1986.[2] Thereafter, several endemic foci were discovered in Manipur,[3] Arunachal Pradesh[4] and Nagaland.[5] Most interestingly, P. heterotremus has been identified as the causative agent of human paragonimiasis in this part of India against the widely believed P. westermani, which was reported from many mammals in India and recently the metacercariae were isolated from the second intermediate crab hosts in northeast India.[6] Herein, we present a case of pulmonary paragonimiasis, which was initially diagnosed as smear negative pulmonary tuberculosis and was under anti-tubercular drugs. The diagnosis was based on the clinical features and laboratory findings of the patient, and molecular results demonstrated the causative species as P. westermani. This is the first case of P. westermani infection in humans in India confirmed by the morphological and molecular characterisation of Paragonimus eggs in the sputum specimen. Case Report A Manipuri tribal female, 50 years of age, was attended to the Regional Institute of Medical Sciences (RIMS), Imphal, with complaints of cough and recurrent hemoptysis for 2 years. She was initially diagnosed as smear negative pulmonary tuberculosis, at a primary health center and was under anti-tubercular therapy for a period of one and half years. As there was no clinical improvement, she was referred to RIMS. History of her dietary habits revealed frequent consumption of smoked and improperly cooked crabs collected from the mountain streams and bought from the market in Moreh, Manipur. She denied of associated fever, breathlessness, chest pain, and weight loss. Laboratory investigations of three consecutive sputum
S157
samples were negative for acid-fast bacilli, pyogenic bacteria, fungi, and malignant cells, but positive for Paragonimus eggs [Figure 1]. The chest roentgenogram showed multiple nodular opacities of varying sizes, some with breakdown (cavity) in the upper and middle zones of both lungs and in the right basal and para cardiac regions [Figure 2]. Both apices and costophrenic angles were clear. There was no evidence of calcification. Her complete blood count (CBC) revealed a total leucocyte count of 7200/l with 16% eosinophils and erythrocyte sedimentation rate (ESR) of 80 mm/1st hour (Westergren’s). The case was diagnosed as a chronic pulmonary paragonimiasis and was treated with praziquantel 25 mg/kg body wt., three times a day for 3 days. Sputum became free from blood and Paragonimus eggs on the second day following the chemotherapy. The clinical and laboratory data including radiological findings were critically reviewed, and identified P. westermani like eggs in the sputum smear, at the Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim. Molecular characterisation of a Paragonimus egg sample from the patient was performed by DNA isolation, amplification of the internal transcribed spacer 2 (ITS2) regions of the ribosomal DNA and sequencing as described by Sugiyama et al. (2002, 2013).[7,8] Sequence analysis of the polymerase chain reaction (PCR) products revealed that the alignments of the rDNA region spanning the ITS2 were 436 bp in length [Figure 3]. An asterisk indicates the presumed beginning of the actual ITS2. The 5’ end of the sequence is of 5.8S rDNA origin. The numbers refer to the alignment positions. A dot in the alignment of the P. westermani sequence with accession No. of JN656208 indicates a nucleotide identical to that of our sample: The lengths of 76 bp and 360 bp were estimated for the posterior portion of 5.8S rDNA and almost entire portion of the actual ITS2, respectively, in comparison with the sequence reported for the adult P. westermani. The nucleotide sequence of Paragonimus eggs from the patient determined in this study has been deposited in the DDBJ/EMBL/GenBank database under the accession number AB123456. Searches of the nucleotide databases revealed that the sequences were almost identical to that deposited in the GenBank/EMBL/DDBJ nucleotide database with the accession number of JN656208, which was for P. westermani isolated from Arunachal Pradesh as a metacercaria. Pairwise comparison of the sequences over an alignment length of 436 bp revealed just 1 (0.23%) base difference, which was probably due to the intraspecific sequence variation. We, therefore, identified the eggs discharged in a sputum sample of the patient as P. westermani. Discussion To date, five Paragonimus species viz. P. heterotremus, P. skrjabini, P. hueitu’ngensis, P. miyazaki manipurinus n. sub spp and P. westermani have been reported to infect
www.ijmm.org
S158
Indian Journal of Medical Microbiology
two fresh water crabs; Potamiscus manipurensis and Alcomon superciolosum, which serve as second intermediate
Figure 1: Saline wet mount preparation showing characteristic golden brown Paragonimus eggs in the sputum specimen of the patient. (40× magnification)
Figure 2: Chest roentgenogram posteroanterior (PA) view of the patient showing multiple nodular opacities, some with cavities in the upper and middle zones of both lungs, and in the right basal and para cardiac regions
Figure 3: An aligned nucleotide sequence of an ITS2 region obtained from P. westermani eggs from the patient by PCR amplification using the primer pair 3S (forward, 5æ-GGTACCGGTGGATCACTCGGCTCGTG-3æ) and BD2 (reverse, 5æ-GGGATCCTGGTTAGTTTCTTTTCCTCCGC-3æ). An asterisk indicates the presumed beginning of the actual ITS2. The 5æ end of the sequence is of 5.8S rDNA origin. The numbers refer to the alignment positions. A dot in the alignment of the P. westermani sequence with accession No. of JN656208 indicates a nucleotide identical to that of our sample
vol. 33, Supplement 1
hosts in Manipur.[6] The crabs, P. manipurensis are the most widely distributed as the host of P. heterotremus and other Paragonimus species occurring in Manipur and Nagaland. Recently, P. westermani metacercariae were isolated from the fresh water crabs, Alcomon collected at mountain streams in Moreh, Manipur. The P. westermani infection was also described in the two species of fresh water crab hosts, Maydelliathelphusa in Arunachal Pradesh and Meghalaya and Sartoriana spinigera in Assam.[9,10] Although, P. westermani infection was found in many wild and domestic mammals, infection in humans remained undetermined in India. In the past P. westermani was presumed to be the causative agent of paragonimiasis in Manipur only on the observation of Paragonimus eggs in the clinical specimens even without describing the morphological characteristics of the eggs. Nevertheless, some morphological features of eggs such as shape, size and shell character can be used to discriminate Paragonimus species, for example the eggs of P. heterotremus have an almost uniform shell thickness and discernible at the operculated end in contrast to the P. westermani eggs that are asymmetrically ovoid, shell thickened mostly at the apopercular end and opercular end as prominent shoulders. However, these features are inconsistent as the eggs of many Paragonimus species have overlapping morphological features that make species identification impracticable. Therefore, the earlier reports that implicated P. westermani as the causative agent of human infection cannot be considered as valid. Recent advances in the molecular technology such as PCR and DNA sequencing and hybridisation have enabled to identify and discriminate Paragonimus species at various stages of development in the life cycle. Subsequently, morphological and molecular characterisations of eggs in the sputum specimens of several patients have revealed P. heterotremus as the common human pathogen in the northeast India.[10,11] In the present case, we confirmed P. westermani as the causative agent of the infection based on the morphological features and molecular characterisation of the eggs in the sputum specimen. This is the first record of P. westermani infection in human, in India. The low incidence of P. westermani infection in this region may be due to the restricted areas of distribution of the parasite, low host specificity, and paucity of suitable intermediate hosts. Further study will be needed to find some characteristic features of the eggs that can help identify and discriminate different Paragonimus species in the clinical specimens. Presently PCR and DNA sequencing of the eggs in the clinical specimens is the only option for species determination. Acknowledgements This study was supported in part by the Japan Society for the Promotion of Science, JSPS (KAKENHI: Grant No. 25305011) and by grants for Research on Emerging and Re-emerging Infectious
www.ijmm.org
February
Diseases (H23-Shinko-ippan-014 and H26-Shinko-ippan-009) from the Ministry of Health, Labor and Welfare of Japan.
References 1.
2. 3. 4. 5.
6. 7.
8.
S159
Case Reports
Miyazaki I. Lung flukes in the world: Morphology and life history. In: Sasa M, editor. A Symposium on Epidemiology of Parasitic Diseases. Tokyo: International Medical Foundation of Japan; 1974. p. 101-34. Singh TS, Mutum SS, Razaque MA. Pulmonary paragonimiasis: Clinical features, diagnosis and treatment of 39 cases in Manipur. Trans R Soc Trop Med Hyg 1986;80:967-71. Singh TS, Mutum S, Razaque MA, Singh YI, Singh EY. Paragonimiasis in Manipur. Indian J Med Res 1993;97:247-52. Narain K, Devi Rekha K, Mahanta J. Paragonimus and paragonimiasis-A new focus in Arunachal Pradesh, India. Curr Sci 2003;l84:985-7. Singh TS, Sugiyama H, Umehara A, Hiese S, Khalo K. Paragonimus heterotremus infection in Nagaland: A new focus of pragonimiasis in India. Indian J Med Microbiol 2009;27:123-7. Singh TS, Sugiyama H, Rangsiruji A. Paragonimus and paragonimiasis in India. Indian J Med Res 2012;136:192-204. Sugiyama H, Morishima Y, Kameoka Y, Kawanaka M. Polymerase chain reaction (PCR)-based molecular discrimination between Paragonimus westermani and P. miyazakii at the metacercarial stage. Mol Cell Probes 2002;16:231-6. Sugiyama H, Singh TS, Rangsiruji A. Paragonimus (ch 39). Molecular Detection of Human Parasitic Pathogens. 1st ed. Florida: CRC Press; 2013. p. 421-33.
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Devi KR, Narain K, Agatsuma T, Blair D, Nagataki M, Wickramasinghe S, et al. Morphological and molecular characterization of Paragonimus westermani in northeastern India. Acta Trop 2010;116:31-8. 10. Singh TS, Sugiyama H, Rangsiruji A, Devi KR. Morphological and molecular characterizations of Paragonimus heterotremus, the causative agent of human paragonmiasis in India. Southeast Asian J Trop Med Public Health 2007;38 suppl 1:82-6. 11. Devi KR, Narain K, Bhattacharya S, Negmu K, Agatsuma T, Blair D, et al. Pleuropulmonary paragonimiasis due to Paragonimus heterotremus: Molecular diagnosis, prevalence of infection and clinicoradiological features in an endemic area of northeastern India. Trans Roy Soc Trop Med Hyg 2007;101:786-92. Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.150950
How to cite this article: Singh TS, Hiromu S, Devi KR, Singh WA. First case of Paragonimus westermani infection in a female patient in India. Indian J Med Microbiol 2015;33:S156-9. Source of Support: Nil, Conflict of Interest: None declared.
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