Rapid screening with a recombinant antigen (rK39) for diagnosis of visceral leishmaniasis using dipstick.

OBJECTIVE: To evaluate the diagnostic value of the recombinant antigen of 39 amino acid repeats encoded by a kinesin-like gene of Leishmania changasi (rK39) in serodiagnosis of visceral leishmaniasis (VL). METHODS: In Kashi, Xinjiang, 13 VL patients with splenomegaly and bone marrow aspirate culture positive were subjected to dipstick assay. A drop of whole blood or serum from patient was placed at the absorbing pad at the bottom of the dipstick. Flooding of the bottom protein with buffer allows serum proteins to migrate upwards, producing the positive band and Western blot analysis of rK39 subsequently performed with the sera collected. RESULTS: The end-point titers of anti-rK39 antibodies of these sera were determined by ELISA and found to fall within the range of 10(-2) to 10(-4), which were consistent with the intensity of their reaction with rK39 in dipstick assay. The positive sera could also recognize the specific rK39 band as analyzed by Western blot analysis. CONCLUSION: The rK39 dipstick assay is more rapid, specific, sensitive and less invasive than the conventional methods of diagnosis for VL in the areas of low endemicity.

Diagnosis of kala-azar by nested PCR based on amplification of the Leishmania mini-exon gene.

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.

Discovery and study of Leishmania turanica for the first time in China.

Reported are the results of multidisciplinary studies on Leishmania turanica, which was isolated from the auricular tissues of naturally infected great gerbils in Xinjiang, China. Discussed are the biology of the parasite, its molecular biology, its pathogenicity in rodents and humans and its vectors. This was the first time that L. turanica had been reported in China. L. turanica is highly pathogenic in BALB/c mice, with the resulting systemic infection being lethal, and it causes dermal lesions in Meriones unguiculatus. L. turanica parasitizes the macrophages in the interstitium of the testes of Cricetulus barabensis, and entirely destroys the Leydig's cells of severely infected animals. Inoculation of L. turanica can induce simian and human cutaneous leishmaniasis. The cell membrane and flagella of the promastigotes of L. turanica have rather active ACPase. The major vectors of L. turanica were Phlebotomus mongolensis and P. andrejevi.

Separation of Chinese Leishmania isolates into five genotypes by kinetoplast and chromosomal DNA heterogeneity.

Leishmaniasis remains endemic in China, especially in the west and northwest frontier regions in central Asia. Epidemic outbreaks of both visceral and cutaneous forms of the disease have become a serious concern in view of such events occurring in neighboring countries. In the present study, we have begun to characterize available parasites as an initial step in understanding the epidemiology of leishmaniasis in central Asia. Nineteen Leishmania isolates collected since the 1950s from epidemiologically different foci in China were separated into five genotypes (Groups I-V) based on their polymorphisms in both kinetoplast (kDNA) and nuclear (nDNA) DNAs. Both kDNA and nDNA are conserved in Group I, which consists of six isolates, i.e., five cases of human kala-azar and one case of canine leishmaniasis isolated from three distant foci more than 30 years apart. In contrast, both kDNA and nDNA are heterogeneous in Group II, consisting of 10 isolates scattered in the plain area from the eastern coast to the western desert. This group includes five kala-azar cases, one post-kala-azar dermal leishmaniasis case, two sand fly isolates, and two canine isolates. The remaining three groups (III-V), two from great gerbils (Rhombomys opimus) and one from a kala-azar case, differ among themselves and from the aforementioned groups. Groups I, II/III, IV, and V contain isolates that have been recognized epidemiologically or typed isoenzymatically as L. donovani s.l., L. infantum s.l., L. turinica, and L. gerbilli, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Leishmaniasis in Karamay. XI. The development of cutaneous leishmaniasis in monkey and man experimentally infected with Leishmania from Karamay big gerbil].

A monkey (Macaca rhesus) was inoculated subcutaneously with 2 Leishmania isolates (MRHC/CN/88/KXG-2 and MRHO/CN/90/KXG-56) from Karamay big gerbils (Rhombomys opimus) into the forehead and cheek, 36 days later ulceration with a diameter of 0.2cm occurred at 2 sites. The lesions persisted for 60 and 95 days respectively and then healed spontaneously. However, Leishmania could be detected from the dermal tissue for more than one year. When MRHO/CN/88/KXG-2 isolate was inoculated into the forearm or ear of the monkey, no ulceration but swelling was revealed at the sites of inoculation. Leishmania was found to be latent in the subcutaneous tissue of the forearm for more than 103 days. The monkey was dissected one year after inoculation, no evidence of visceral involvement was noted. Another isolate (MRHO/CN/87/KXG-12) was inoculated subcutaneously into the forearm of 2 healthy volunteers. One of them developed cutaneous leishmaniasis. An ulcer with a diameter of 0.4cm and a nodule as large as 0.9 x 1.5cm around the ulcer occurred at the inoculation site 128 days later. Leishmania parasites were found harboring in macrophages of the dermis and in the epithelial cells of hair follicles. The results indicate that Leishmania parasite of the Karamay big gerbils is pathogenic to monkey and man, in addition to the fact that human cutaneous leishmaniasis and gerbil's Leishmania co-exist in Karamay, it seems that big gerbil is a potential reservoir of human cutaneous leishmaniasis in this area.

Recent advances in visceral leishmaniasis in China.

A review of major advances in investigations and laboratory work on visceral leishmaniasis in China is presented. In the eastern plain and northern mountainous regions, no new cases were detected, while sporadic appearance of the disease was noted in western mountain and desert regions. Considerable achievements were attained regarding epidemiology of visceral leishmaniasis, experimental research on disease control in mountainous regions, sandfly control and elimination as well as immuno-diagnosis of the disease. Noticeable consequences were also obtained from the exploration on the problem of geographical strains and biochemical study of the parasite. The intervention measures in sporadically occurring visceral leishmaniasis in the western mountain and desert regions are discussed.

Current status of kala-azar and vector control in China.

Kala-azar, which was prevalent in the vast area of China that lies to the north of the Yangtze River from the 1920s to the 1950s, is now effectively under control as a result of strenuous intervention since the founding of the People's Republic of China in 1949. Apart from 15-20 new cases that occur annually in the Keshi plain, Xinjiang Autonomous Region, the achievements of control practised in other former endemic areas in the plains have been significant and consolidated. In the mountainous areas in north-west China, where the vector, Phlebotomus chinensis, is abundant and canine visceral leishmaniasis is common, there are still sporadic cases of kala-azar. Also, in recent years, new infections have often occurred in the deserts of Xinjiang and western Inner Mongolia, although the reservoir of the infection has not been identified.

[Observation on the infectivity of Leishmania donovani from different areas to Phlebotomus alexandri from Xinjiang].

Hamsters or cotton rats were infected with 3 stocks of L. donovani, one of which had been isolated from an infected Ph. major wui in Xinjiang desert area and 2 from kala azar patients in a mountain area of Gansu and in a plain area of Henan respectively. The purpose of the study was to compare the infectivity of the parasites from different geographic areas through artificial infection of Ph. alexandri. Sandflies were dissected in 4, 6, 8 and 10 days after feeding on the infected animals to observe the growth and development of promastigotes of different isolates in their alimentary tracts. Evaluation was made on 3 aspects, i.e. infection rates, infectiosity and migration of promastigotes in sandflies. It was demonstrated that Xinjian isolate of L. donovani appeared to be particularly adapted to Ph. alexandri followed by Gansu isolate, the Henan isolate did not show good adaptation to this sandfly, because the number of promastigotes declined and the infection rate dropped after the digestion of the blood meal in the stomach of the sandflies. It seems that the phenomenon was related to the biological characteristics of L. donovani isolates. These results coincided with those of McAb dot ELISA (Qu,1987) and K-DNA dot hybridization (Lü & Hu 1988) for identification of L. donovani from different areas in China, and were also in parallel to the diverse epidemiological characteristics of different kala azar endemic areas (Guan, 1976). It is reasonable to infer that there may exist different geographical strains of L. donovani in China.

[Studies on the efficacy of five repellents against Phlebotomus alexandri].

The efficacy of five repellents were tested against Phlebotomus alexandri both in the laboratory and in the field in Meiyaogou, Turfan City, Xinjiang Uygur Autonomous Region during 1986-1987. A total of 3,301 female Ph. alexandri were used in the laboratory and 306 man-times of volunteers were tested in the field. At a dose of 0.25 microliters/cm2, the protective durations of mosquito repellent perfume (MRP), N, N-diethyl-mtaluamide (DETA), mosquito repellent liquid (MRL), dimethyl phthalate (DMP) and dibutyl phthalate (DBP) were 7.50 h, 5.00 h, 3.75 h, 1.25 h and 1.00 h, respectively. At a dose of 0.75 mg/cm2, the protective durations of the essential balms of DETA, DMP and DBP were 5.50 h, 3.50 h and 3.00 h, respectively. The repelling effect on volunteers in the field was the same as that tested in the laboratory. The relative efficacy of the five repellents was in the order of MRP greater than DETA greater than MRL greater than DMP greater than DBP. The above-mentioned repellents may be of practical use for individual protection against kala-azar transmission in the field.

[Identification of L. donovani promastigotes from Phlebotomus by monoclonal antibody].

It has been reported by the authors that monoclonal antibody L12G9 produced from target antigens of L. donovani promastigotes, was very useful for detecting promastigotes from artificially infected sandflies. In the present study, detection of promastigotes from artificially infected sandflies by McAb showed that the positive rate correlated with the infection duration of sandflies. 4 days after feeding on infected Chinese hamsters, the sandflies were lightly infected with L. donovani promastigotes with a positive rate of 15.9%, but 10 days later, the sandflies were heavily infected, the positive rate being 100%. Observation has been made on the relationship between the number of promastigotes and mouse blood dilution. The results showed that satisfactory results could be obtained by using monoclonal antibodies in the detection of L. donovani, the number of promastigotes should be over 1 x 10(7)/ml, and the blood meals in sandflies completely digested. If very few promastigotes were present in naturally infected sandflies before identification by monoclonal antibody, the parasites must be grown in NNN medium. Positive result could then be obtained.

The role of Phlebotomus alexandri Sinton, 1928 in the transmission of kala-azar.

Since 1968, kala-azar has been occurring sporadically in Meiyaogou, Turfan County, Xinjiang-Uygur Autonomous Region, where four species of sandflies are known to exist. The present study of sandflies collected in this area from May to August 1983 shows that Phlebotomus alexandri is the only anthropophilic and predominant species, accounting for 81.1% (7716/8843) of the sandfly population. After having been fed on cotton rats or hamsters infected with Leishmania donovani, 93.9% (230/245) of P. alexandri were infected, the promastigotes not only developing in the stomach but also invading the pharynx, buccal cavity and proboscis. Of the 643 female P. alexandri collected in the wild and in house, 13 (2.0%) were found to be naturally infected, with a distribution of promastigotes similar to that in the experimental infection. All the Chinese hamsters and golden hamsters that had been inoculated with 7 isolates of promastigotes from naturally infected sandflies developed visceral leishmaniasis. In addition, golden hamsters could be infected experimentally by the bite of P. alexandri which had an infection of promastigotes of L. donovani. This is the first demonstration of a species of Paraphlebotomus playing a role in the transmission of kala-azar in the Old World; studies for confirmation by biochemical identification of parasite isolates from naturally infected P. alexandri are now in progress.