R. L. Ihalamulla

Sri Lankan cutaneous leishmaniasis is caused by Leishmania donovani zymodeme MON-37

Sri Lankan cutaneous leishmaniasis (CL), once considered sporadic, is fairly widespread in some parts of the country. Identification of 5 isolates from 4 CL patients by enzyme analysis during 2002 showed that they were all Leishmania donovani zymodeme MON-37, the parasite which also causes visceral leishmaniasis in India and East Africa.

Plasmodium vivax: isolation of mature asexual stages and gametocytes from infected human blood by colloidal silica (Percoll) gradient centrifugation

The densities of human erythrocytes infected with P. vivax obtained from infected patients were determined by isopycnic centrifugation in continuous gradients of Percoll. The approximate densities of erythrocytes infected with rings were 1.086 to 1.1, trophozoites (amoeboid stages) 1.053 to 1.086, schizonts and gametocytes 1.053 to 1.056 and of the other cellular elements of blood, uninfected erythrocytes, 1.086 to 1.1, polymorphonuclear leucocytes 1.073 to 1.086 and mononuclear cells 1.062 to 1.073 g/ml. Based on these values, a one-step gradient of 47% Percoll was devised to separate erythrocytes infected with the more mature stages (trophozoites, schizonts and gametocytes) from uninfected erythrocytes. By this method it is possible to obtain parasitaemias of 88% to 98% from blood with starting parasitaemias of less than 0.7%. This method is therefore being routinely used for immunological, biochemical and molecular biological studies on P. vivax.

Microculture for the isolation of Leishmania, modified to increase efficacy: a follow-up to a previous study

In studies on the leishmaniases, the identification of the parasites to species level, by iso-enzyme characterization or by most molecular biological techniques, requires the isolation of the parasites in culture. Novy, McNeal and Nicolle (NNN) and Evan’s modified Tobie’s (EMTb) are two conventional media commonly used for the isolation of Leishmania parasites from blood or tissue samples of patients. Both are biphasic, with a solid agar layer that has to be enriched with blood, and EMTb medium is based on relatively expensive reagents. The preparation of both of these media is laborious and time-consuming, and neither supports the multiplication of all Leishmania parasites from humans (Allahverdiyev et al., 2004; Ihalamulla et al., 2005). A monophasicmicroculture (MCC) method that only uses very small volumes of RPMI 1640 supplemented with 20% foetal calf serum (FCS) has recently been described by Ihalamulla et al. (2005). In the detection of human cutaneous leishmaniasis in Sri Lanka, this method, which involves the sealing with wax of a 1:1 mix of the medium and a sample of saline aspirate from a skin lesion in a microhaematocrit capillary tube, gives higher sensitivity than the culture of similar aspirates in NNN or EMTb medium (Ihalamulla et al., 2005). The possibility that the efficacy of the MCC method could be further improved, in effect by keeping all the parasites that are in the aspirate sample but replacing the saline with more medium, has now been explored.

Recovery of a species of Brugia, probably B. ceylonensis, from the conjunctiva of a patient in Sri Lanka.

A species of Brugia, probably B. ceylonensis, was recovered from the conjunctiva of a patient in Sri Lanka for the first time. This infection represents only the second record of Brugia in the human conjunctiva, and is clearly zoonotic, acquired from a dog. Brugia ceylonensis has a distinct head bulb like that of Wuchereria bancrofti and B. malayi. However, the parasite recovered was not W. bancrofti, as specific IFAT and DNA probes gave negative results, and B. malayi is believed to have been eradicated from Sri Lanka several years ago. The presence of a distinct head bulb excludes the possibility that the parasite was B. buckleyi.