Letter to Editor

Filariasis is transmitted by the Culex mosquito and the adult worms live in the lymphatic vessels of the definitive host. The microfilariae are released into the peripheral circulation. Filariasis causes a spectrum of diseases including the following conditions: Asymptomatic microfilaria, acute lymphangitis and lymphadenitis, chronic lymphadenitis, edema of the limbs and genitalia, and tropical pulmonary eosinophilia.[4]

This will help in the institution of specific treatment, especially in unsuspected and asymptomatic cases in endemic areas. FNAC is extremely useful in identifying filarial infection at uncommon sites like the breast.

Srikanth Shastry Department of Pathology, Prathima Institute of Medical Sciences, Nagnur, Karimnagar, Andhra Pradesh, India. E‑mail: [email protected]

Despite a large number of people affected worldwide, it is quite unusual to find microfilaria in routine cytological smears and body fluids. There have been case reports of a single or a small number of cases of microfilaremia at the following sites: Bronchial aspirates, pericardial fluid, cervicovaginal smears, joint aspirates, and thyroid masses.

REFERENCES 1. Park K. Park’s textbook of preventive and social medicine, 18th ed. Jabalpur, India: Bhanot Publishers; 2005. p. 211‑6. 2. Sodhani P, Nayar M. Microfilariae in a thyroid aspirate smear: An incidental finding. Acta cytol 1989;33:942‑3. 3. Yenkeshwar PN, Kumbhalkar DT, Bobhate SK. Microfilariae in fine needle aspirate: A report of 22 cases. Indian J Pathol Microbiol 2006;49:365‑9. 4. Mcadam AJ, Sharpe HA. Infectious diseases. Robbins and Cotran pathologic basis of disease. In: Kumar V, Abbas AK, Fausto N, editors, 7 th ed. Philadelphia: Elsevier; 2007. 5. Rawat V, Rizvi G, Sharma N,  Pandey H. An unusual presentation of Wuchereria bancrofti infection. Indian J Med Microbiol 2009;27:382‑3.

Diagnosis of filarial infection is frequently made on clinical grounds in endemic areas, but demonstration of microfilariae in the circulating blood is the only means by which one can make a definitive diagnosis.[5] The main purpose of this case report is to raise the awareness that in tropical countries like India, where filariasis is endemic, it should always be considered as a differential diagnosis of swelling at any site.

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Our presentation revealed that microfilaria could even be present at rare sites like the breast. As the patient could not be followed up with a biopsy, the causal role of filariasis in the breast lump could not be established. However, considering such a kind of presentation, careful examination of cytological smears is very important for the prompt recognition of the disease.

www.tropicalparasitology.org DOI: 10.4103/2229-5070.129189 DOA: 23-09-2013 DOP: ***

cells is observed to be an important tool for the diagnosis of malaria and considered as one of the signs of severity.[3,4] These pigments can be detected by flow cytometry, spectrophotometer, polarizing microscope, etc., which found to be expensive and not very sensitive and specific.[5] Methylene blue dye remains a major component of Giemsa stain, but its use for the detection of hemozoin pigment and malaria parasite has not been studied much.

A simple and rapid staining method for detection of hemozoin pigment by methylene blue stain Sir,

A pilot study was carried out for detection of malaria parasite and hemozoin pigment using methylene blue stain and Giemsa stain. A  total of 30 blood samples positive for malaria by peripheral blood smear and rapid diagnostic tests were included in this study. The samples were divided into three groups based on parasitic count, i.e.,  >5000 parasites/µl (Group  A, n  =  8), ≥3000 to  ≤5000 parasites/µl (Group  B, n  =  8), 5000/µl (n=8) Giemsa stain Methylene blue stain Jan 2014 | Volume 4 | Issue 1 |

No. of slides positive for asexual forms

Mean asexual forms

No. of slides positive for gametocytes

Mean gametocytes

No. slides positive for pigmented leukocytes

14 13

1404 1480

9 11

1188 2444

1 1

8 8

3940 2828

8 8

2440 4968

0 2

8 8

10688 7868

8 8

4420 5544

2 3

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Tropical Parasitology

Letter to Editor

leucocytes. Therefore, the number of false positivity and false negativity was found lesser than Giemsa stain and screening rate of slides with methylene blue was found to be more rapid. During the evaluation period, we also observed that the gametocyte forms with methylene blue stain were easier to identify. However, the morphology of the asexual form was less appreciated in comparison to Giemsa. Hence, methylene blue can be added with Giemsa stain in the endemic areas as a screening test to screen the carrier stage. It can be useful to give accurate result in chronic conditions, relapsed cases and partially treated patients showing only leucocyte containing pigment. It is cost‑effective, easy to prepare and unlike Giemsa, do not need time for maturation. As per the result of our evaluation, gametocyte, pigment containing leukocytes and the hemozoin pigments were easily identified with this stain and need less expertise. However, the number of slides with pigment containing leucocytes was less, which is a limitation to the study. Further, study can be planned with more number of samples positive for pigment containing leucocytes to evaluate the efficacy of both stains for the detection of hemozoin pigments.

REFERENCES 1. Bejon P, Andrews L, Hunt‑Cooke A, Sanderson F, Gilbert SC, Hill AV. Thick blood film examination for Plasmodium falciparum malaria has reduced sensitivity and underestimates parasite density. Malar J 2006;5:104. 2. Murray CK, Gasser RA Jr, Magill AJ, Miller RS. Update on rapid diagnostic testing for malaria. Clin Microbiol Rev 2008;21:97‑110. 3. Grobusch MP, Hänscheid T, Krämer B, Neukammer J, May J, Seybold J, et al. Sensitivity of hemozoin detection by automated flow cytometry in non‑ and semi‑immune malaria patients. Cytometry B Clin Cytom 2003;55:46‑51. 4. Nguyen PH, Day N, Pram TD, Ferguson DJ, White NJ. Intraleucocytic malaria pigment and prognosis in severe malaria. Trans R Soc Trop Med Hyg 1995;89:200‑4. 5. Hänscheid T, Frita R, Längin M, Kremsner PG, Grobusch MP. Is flow cytometry better in counting malaria pigment‑containing leukocytes compared to microscopy? Malar J 2009;8:255. 6. Kiernan JA. On chemical reactions and staining mechanisms, subsection what is Giemsa stain and how does it color blood cells, bacteria and chromosomes? In: Kumar GL, Kiernan JA, editors. Education Guide‑Special Stains and H and E. 2 nd ed. Ch. 19. California: Dako North America, Carpinteria 2010; p. 167‑76. 7. Wilson TM. On the chemistry and staining properties of certain derivatives of the methylene blue group when combined with eosin. J Exp Med 1907;9:645‑70. 8. Bain BJ, Lewis SM. Preparation and staining methods for blood and bone marrow films. In: Lewis SM, Bain BJ, Bates I, editors. Dacie and Lewis Practical Haematology. 10th ed. London: Elseveir, Churchil Livingstone 2006; p. 59‑77.

In the present study, peripheral blood smear with methylene blue stain found to be an easier and better method for the identification of gametocyte of Plasmodum spp. and hemozoin pigments. It is accurate, rapid and cost‑effective in comparison to other methods available for pigment detection. Hence, it can be useful as an adjunct to Giemsa stain in mass survey for detection of malaria in low parasitemic conditions and identification of hemozoin pigments.

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Sarita Mohapatra, Daisy Sharma, Kavita Gupta, Manorama Deb, Rajni Gaind

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Department of Microbiology, Vardhaman Mahavir Medical College and Safdarjung Hospital, New Delhi, India. E‑mail: [email protected]

10.4103/2229-5070.129190 DOA: 21-09-2013, DOP: ***

APPENDIX 1

Excellent

Questionnaire for the evaluation of both the stains 1. How would you grade methylene blue stain in comparison to Giemsa for detection of gametocyte of malarial parasite? Excellent 0/11

Very good

Good

Fair

Poor

4/11

6/11

1/11

0/11

0/11

Very good

Good

Fair

Poor

0/11

4/11

1/11

6/11

3. How would you grade methylene blue stain in comparison to Giemsa for hemozoin pigment detection? Excellent 3/11

2. How would you grade methylene blue stain in comparison to Giemsa for detection of asexual forms of malarial parasite? Tropical Parasitology

Website:

Very good

Good

Fair

Poor

5/11

3/11

0/11

0/11

4. How would you grade methylene blue stain in comparison to Giemsa for the detection of pigment‑containing leucocytes? 61

Jan 2014 | Volume 4 | Issue 1 |

Letter to Editor

Excellent 0/11

Very good

Good

Fair

Poor

Faster

2/11

8/11

1/11

0/11

9/11

5. How often would you report false negative report in methylene blue stain in comparison to Giemsa? More frequent

Less frequent

Infrequent

Equal

9/11

1/11

0/11

1/11

Less frequent

Infrequent

Equal

7/11

2/11

1/11

1/11

1/11

1/11

Easy

Difficult

Equal

7/11

2/11

2/11

9. How would you grade the utility of methylene blue stain for detection of malarial pigment in peripheral smear in comparison to Giemsa? Less useful 3/11

7. How would you grade methylene blue stain for the rate of screening of peripheral smears in comparison to Giemsa?

More useful 8/11

asthenic. The patient’s wife had lymphatic filariasis 2 years back and was treated with diethylecarbamazine. At the time of presentation the patient was febrile and there was severe pallor and angular stomatitis. On the examination of the abdomen, a massive hepato‑splenomegaly was found. Rest of the clinical examination was within normal limits. The patient’s preliminary blood investigations revealed pancytopenia with relative lymphocytosis. Ultrasonogram of the abdomen showed massive hepato‑splenomegaly with minimal free fluid [Table 1].

A case of mixed infection with filariasis and visceral leishmaniasis Sir, Leishmaniasis and lymphatic filariasis are important differential diagnoses in patients presenting with prolonged fever in Indian sub‑continent. [1] Both the parasitic infections are endemic in many parts of India. Leishmania donovani, the parasite causing visceral leishmaniasis (Kala Azar) is a hemo‑flagellate, transmitted by the bite of infected sand flies. [2] Wuchereria bancrofti, the most common agent causing lymphatic filariasis in India, is a nematode, transmitted by female Culex, Anopheles and Aedes mosquitoes.[3] Although both the parasites singly are commonly encountered in cases of pyrexia of unknown origin  (PUO), especially in patients from endemic areas; mixed infection by both of these parasites is seldom reported. We are reporting a rare and interesting case of mixed infection with L. donovani and W. bancrofti. To the best of our knowledge, this is the second confirmed case of mixed infection by these two parasites.[4]

Bone marrow aspiration was done on the 3rd  day of hospitalization and was sent to the microbiology laboratory, All India Institute of Medical Sciences, New  Delhi, India. Examination of Acridine‑orange and Giemsa stained smears of bone marrow revealed the presence of amastigote forms of L.  donovani ‑ Leishman‑Donovan bodies, grade  3+ [Figure  1]. Serum sample was tested for specific recombinant‑Kinesin (rK)‑39 anti‑leishmania antibodies and was found to be positive (grade 4+). The quantitative buffy coat  (QBC) examination was done to exclude malaria; which revealed the presence of motile microfilariae [Figure  2a]. A  microfilarial count was done from the QBC, which came out to be 123/ml. Speciation of microfilaria, as W. bancrofti was done in QBC on the basis of distinct nuclei, cephalic space being as long as broad, tail end being pointed and free of nuclei, smooth body curves and absence of secondary kinks. In a Giemsa stained smear of concentrated blood specimen, speciation was confirmed [Figure 2b‑d].

A 30‑year‑old male patient, resident of Bihar, India, presented with fever and loose motions for 2 months; and lump in the left side of the abdomen for one and ½ month duration. During the course of illness, he lost around 7 kg of body weight, became anorexic and Jan 2014 | Volume 4 | Issue 1 |

Equal

8. How would you grade methylene blue stain for the rate of screening of peripheral smears in comparison to Giemsa?

6. How often would you report an artefact to be falsely positive in methylene blue stain in comparison to Giemsa? More frequent

Slower

62

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A simple and rapid staining method for detection of hemozoin pigment by methylene blue stain.

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