内脏利什曼病诊断方法研究进展

内脏利什曼病(黑热病)是在世界范围内流行的严重危害人类健康的重要寄生虫病.发展快速而又准确的诊断技术是治疗和控制该病的关键之一.随着免疫学技术、分子生物学技术的应用,黑热病的诊断技术不断取得进展,相应地我国黑热病诊断技术的研究和应用也在不断进步.该文就黑热病诊断技术研究取得的进展作一综述.     

Latent class analysis of diagnostic tests for visceral leishmaniasis in Brazil

Objective To estimate the sensitivities and specificities of different diagnostic tests for visceral leishmaniasis (VL) using latent class analysis (LCA). Methods This study was performed using data from a prospective study conducted in four Brazilian states from May 2004 to May 2007. Five diagnostic tests for VL were evaluated in 285 VL cases and 119 non‐cases: microscopy, indirect fluorescence antibody test (IFAT), enzyme‐linked immunosorbent assay using recombinant K39 antigen (rK39‐ELISA), direct agglutination test (DAT) and the rK39 rapid test. Results Microscopy showed sensitivity of 77.0% (CI: 71.5–81.5) and specificity of 99.0% (CI: 94.0–99.7). The IFAT and the DAT showed similar sensitivities, 88.3% (CI: 84.0–92.0) and 88.5% (CI: 84.1–92.0), respectively, but the DAT had a higher specificity (95.4%, CI: 89.2–98.1) than did the IFAT (83.0%, CI: 75.0–88.2). The rK39‐ELISA and the rK39 rapid test showed sensitivities of 99.0% (CI: 96.3–99.6) and 94.0% (CI: 90.1–96.3), and specificities of 82.5% (CI: 75.0–88.3) and 100% (CI: 97.0–100.0%), respectively. Conclusions Considering the lack of an adequate reference standard, LCA proved to be a useful tool in validating diagnostic methods for VL. The DAT and the rK39 rapid test showed better performance. Thus, clinically suspected cases of VL in a Brazilian endemic area could be treated based on the positivity of one of these tests.     

Oxidative damage of erythrocytes: a possible mechanism for premature hemolysis in experimental visceral leishmaniasis in hamsters

 Visceral leishmaniasis is accompanied by severe anemia and pancytopenia. Reactive oxygen species are known to contribute to the pathogenesis of several red blood cell (RBCs) disorders. The present study reveals the extent of oxidative stress and the efficacy of the primary antioxidant system in erythrocytes of hamsters in the progressive anemic response at different stages of leishmanial infection. Increased intracellular precipitation of Heinz bodies secondary to oxidative denaturation of hemoglobin and enhanced formation of malonyldialdehyde suggest oxidative damage of erythrocytes, both in the hemoglobin and cell membrane, respectively. Decreased activities of superoxide dismutase and catalase in the infected animals indicate the generation of O₂ ^* – and H₂O₂, which in turn may produce the highly reactive ^*OH species. Decreases in the reduced glutathione level along with the decreased activities of glutathione reductase and glutathione peroxidase point to a deficient antioxidant defense system during the post-infection period. Accentuated degradation of both cytoskeletal and integral membrane proteins after 3 months of infection may eventually lead to membrane destabilization and early lysis of erythrocytes in experimental visceral leishmaniasis. Received: 21 January 2000 / Accepted: 12 July 2000     

Epiglottis involvement in a visceral leishmaniasis

The visceral form of leishmaniasis caused by Leishmania infantum is frequently observed in Mediterranean countries, however otorhinolaryngeal involvement has only rarely been reported in the literature. We report here the case of a 23-year-old Cameroon man, resident in France since 1991, and presenting with recurrent epistaxis and an epiglottis tumoral lesion. The visceral form of leishmaniasis with hepatosplenomegaly and pancytopenia was diagnosed. Culture of the epiglottal lesion revealed L. infantum. Serological test for human immunodeficiency virus was negative and the patient was not on immunosuppressive drugs. Dramatic improvement was observed with antimony.     

A rapid and simple diagnostic test for active visceral leishmaniasis

We have developed an immunodot assay for the serodiagnosis of active visceral leishmaniasis (AVL) which utilizes protein A colloidal gold as the visualizing agent. The test is simple, requires few reagents, and can be completed in two hours. It is sensitive and specific for active visceral leishmaniasis, and generally correlates with the ELISA. Either whole blood or sera in minute quantities may be used as test antibody. In addition, the use of the protein A gold immunodot is shown to detect anti-leishmania antibodies in infected dogs.     

A PCR and RFLP-based molecular diagnostic algorithm for visceral leishmaniasis

Objective:To determine an algorithm for molecular diagnosis of visceral leishmaniasis(VL) by kinetoplast DNA(kDNA)(RV1/RV2) and internal transcriber spacer(ITS1)(LITSR/L5.8 S)polymerase chain reaction(PCR),complemented by ITS 1 PCR restriction fragment length polymorphism(RFLP),using peripheral blood or bone marrow aspirate from patients with suspected VL.Methods:Biological samples were submitted to the gold standard for the diagnosis of VL and molecular diagnosis represented by ITS 1 PCR,kDNA PCR,and ITS 1 PCR RFLP.The samples were obtained from seven groups:group Ⅰ,82 samples from patients with confirmed VL;group Ⅱ,16 samples from patients under treatment for VL;group Ⅲ,14 samples from dogs with canine visceral leishmaniasis(CVL);group Ⅳ,a pool of six experimentally infected sandflies(Lutzomya longipalpis);group V,18 samples from patients with confirmed tegumentary leishmaniasis(TL) and groupsⅥ and Ⅶ were from control groups without VL.Results:The following gold standard and molecular examination results were obtained for each of the seven groups:groupⅠ:parasitologic and immunochromatographic tests showed a sensitivity of 76.3%(61 of 80) and 68.8%(55 of 80),respectively,and a sensitivity of 97.6%(80 of 82) and 92.7%(76 of 82) by ITS1 and kDNA PCR,respectively.After ITS1 PCR RFLP(Hae Ⅲ)analysis of the 80 positive samples,52.5%(42 of 80) generated three fragments of 180,70,and 50 bp,corresponding to the pattern of Leishmania infantum infantum;group Ⅱ:negative for the parasitologic methods and positive for IrK39(100%,16 of 16),presented 12.5%(2 of 16) of positivity by ITS 1 PCR and 25.0%(4 of 16) by kDNA PCR;group Ⅲ:positive in the parasitologic and serologic tests(100%,14 of 14),presented 85.7%(12 of 14) of positivity by ITS1 PCR and kDNA PCR.ITS1 PCR RFLP showed that 83.3%(10 of 12) of the canine samples contained parasites with profiles similar to L.infantum;group Ⅳ presented amplifications by ITS1 PCR and kDNA PCR.ITS1 PCR products were analyzed by RFLP,generating a profile similar to that of L.infantum;group Ⅴ:positive in the parasitologic examination(100%,18 of 18),presented 72.2%(13 of 18) of the samples by ITS1 PCR positive.A total of 69.2%(9 of 13) showed profiles corresponding to a Viannia complex by ITS1 PCR RFLP;and group Ⅵ and groupⅥ were negative by ITS 1 and kDNA molecular tests.Comparing the molecular results with the parasitologic and serologic diagnosis from group Ⅰ,almost perfect agreement was found(κ both0.80,P0.001).ITS1 and RV1/RV2 PCR detected 90.2%(74 of 82) of the samples.Two samples positive by RV1/RV2 were negative by LITSR/L5.8 S,and six samples positive by LITSR/L5.8 S were negative by RV1/RV2.Therefore,these two systems complemented each other;they diagnosed 100% of the samples as belonging to the Leishmania genus.Conclusions:We suggest an algorithm for the molecular diagnosis of VL,which must consider previous parasitologic and serologic(immunochromatographic) diagnoses,and should combine kDNA and ITS1 to determine the Leishmania subgenus using RFLP as a complement method to define the L.infantum species.     

Wilson disease with visceral leishmaniasis: an extremely uncommon presentation

Visceral leishmaniasis (VL), which is caused by the protozoa Leishmania donovani and transmitted by the bite of the female sand fly Phlebotomus argentipes, is common in Bihar, India. Wilson disease is an autosomal recessive disorder of copper metabolism in which copper is deposited in the brain and liver. We report a case of an extremely uncommon combination of these diseases in a patient. Treatment options for such a combination of diseases are limited and difficult.     

Chemotherapy of leishmaniasis: recent advances in the treatment of visceral disease

New lipid formulations of amphotericin B--AmBisome, Amphotec, and Abelcet--have dramatically decreased the toxicity associated with amphotericin B and have made this group of agents the treatment of choice for visceral leishmaniasis. An agent of a completely different chemical class, the aminoglycoside aminosidine, was 97% curative in India. This agent too may be used for visceral leishmaniasis.     

Canine visceral leishmaniasis: performance of a rapid diagnostic test (Kalazar Detect) in dogs with and without signs of the disease

Current visceral leishmaniasis (VL) control programs in Brazil include the infected dog elimination but, despite this strategy, the incidence of human VL is still increasing. One of the reasons is the long delay between sample collection, analysis, control implementation and the low sensitivity of diagnostic tests. Due to the high prevalence of asymptomatic dogs, the diagnosis of these animals is important considering their vector infection capacity. Hence, a rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. In this study we evaluated the performance of rK39 antigen in an immunochromatographic format to detect symptomatic and asymptomatic Leishmania chagasi infection in dogs and compared the results with those using a crude antigen ELISA. The sensitivity of rK39 dipstick and ELISA were 83% vs. 95%, respectively, while the specificity was both 100%. Our results also demonstrated that the dipstick test was able to detect infected dogs presenting different clinical forms.     

Cystatin C as a marker of immune complex-associated renal impairment in a Sudanese population with visceral leishmaniasis

Renal function was studied in visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) by means of the specific marker cystatin C and related to circulating immune complexes and cytokine production. Forty patients with VL (23 with sub-acute disease and 17 with acute disease), 17 patients with PKDL, and 22 healthy controls were included. Cystatin C, but not creatinine, was significantly raised in VL (P = 0.004). The highest levels of cystatin C were found in those with acute disease (P < 0.0001). In VL, cystatin C levels were positively correlated to circulating immune complexes and production of granulocyte-macrophage colony stimulating factor (GM-CSF), but negatively correlated to aspartate aminotransferase and lactate dehydrogenase. We conclude that cystatin C is a superior marker of glomerular function in leishmaniasis and that immune complex deposition and GM-CSF are two functions that most likely are causally involved in the mechanisms leading to glomerular dysfunction in leishmaniasis.     

Evaluation of a new recombinant K39 rapid diagnostic test for Sudanese visceral leishmaniasis

A new rK39 rapid diagnostic dipstick test (DiaMed-IT-Leish) was compared with aspiration and a direct agglutination test (DAT) for diagnosis of visceral leishmaniasis (VL) in 201 parasitologically confirmed cases, 133 endemic controls, and in 356 clinical suspects in disease-endemic and -epidemic areas in Sudan. The sensitivity of the rK39 test in parasitologically confirmed VL cases was 90%, whereas the specificity in disease-endemic controls was 99%. The sensitivity of the DAT was 98%. In clinically suspected cases, the sensitivity of the rK39 test was 81% and the specificity was 97%. When compared with the diagnostic protocol based on the DAT and aspiration used by Médecins sans Frontières in epidemic situations, the positive predictive value was 98%, and the negative predictive value was 71%. This rK39 rapid diagnostic test is suitable for screening as well as diagnosis of VL. Further diagnostic work-up of dipstick-negative patients with clinically suspected VL is important. The ease and convenience of the dipstick test will allow decentralization and improved access to care in disease-endemic areas in Sudan.     

Hyponatremia in visceral leishmaniasis

There are few reports linking hyponatremia and visceral leishmaniasis (kala-azar). This is a study of 55 consecutive kala-azar patients and 20 normal individuals as a control group. Hyponatremia and serum hypo-osmolality were detected in 100% of kala-azar patients. High first morning urine osmolality (750.0 ± 52.0 vs. 894.5 ± 30.0mOsm/kg H?O, p < 0.05), and high 24-hour urine osmolality (426.0 ± 167.0 vs. 514.6 ± 132.0 mOsm/kg H?O, p < 0.05) demonstrated persistent antidiuretic hormone secretion. Urinary sodium was high (82.3 ± 44.2 vs.110.3 ± 34.7 mEq/L, p < 0.05). Low seric uric acid occurred in 61.8% of patients and increased fractional urinary uric acid excretion was detected in 74.5% of them. Increased glomerular filtration rate was present in 25.4% of patients. There was no evidence of extracellular volume depletion. Normal plasma ADH levels were observed in kala-azar patients. No endocrine or renal dysfunction was detected. It is possible that most hyponatremic kala-azar patients present the syndrome of inappropriate antidiuretic hormone secretion.