Diagnosis of visceral leishmaniasis by the polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan

We have evaluated the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool for Leishmania donovani using blood, bone marrow and lymph node samples from Sudanese patients with a confirmed infection. Forty patients were diagnosed by microscopic examination of bone marrow or lymph node samples. The PCR was able to detect parasite DNA in 37 out of 40 blood samples. In bone marrow and lymph node samples, the PCR was able to detect parasite DNA in all 7 and 6 samples, respectively. We suggest that the PCR should be considered as a valuable and sensitive tool for the diagnosis of L. donovani infection. However, if PCR diagnosis is to supplement or even replace microscopic diagnosis in developing countries, a large number of patients with no apparent signs of infection and patients with other diseases have to be tested in order to evaluate its true potential.     

Liver biopsy in the diagnosis of visceral leishmaniasis

Aim: To determine whether liver biopsy might be useful in the diagnosis of visceral leishmaniasis when bone marrow examination and serologic tests are inconclusive. Methods: Over a 10‐year period, liver biopsy was performed in five children with suspected visceral leishmaniasis when indirect hemagglutination tests and bone marrow aspirations were not diagnostic. Results: Leishmania amastigotes were seen in Kupffer cells in all patients. The accompanying liver histopathological findings were ischemic necrosis in two children, macrovesicular steatosis in two children, portal inflammatory inflammation in two children, and piecemeal necrosis in one child. During the study period, 32 additional pediatric visceral leishmaniasis cases were diagnosed by bone marrow examination. Conclusion: Liver biopsy can be recommended for diagnosing suspected visceral leishmaniasis in children when serology and bone marrow aspiration are inconclusive.     

Eosinophilic inflammation in lymph nodes of dogs with visceral leishmaniasis

Eosinophils are traditionally associated with the immune response against helminth parasites. However, several studies have demonstrated that these cells have a role regarding protective immunity in leishmaniasis. Here, we examined the relationship between the presence of eosinophils and parasite load in biopsy samples from dogs, obtained through fine needle puncture and aspiration of lymph nodes. Histological slides containing biopsy material from lymph nodes of dogs with canine visceral leishmaniasis and healthy dogs were used to obtain baseline eosinophil counts. Subsequently, scrapings were taken from slides for DNA extraction and determination of parasite load, using real‐time PCR (qRT‐PCR). Additionally, production of nitric oxide (NO) and reactive oxygen species (ROS) levels by eosinophils in the peripheral blood of dogs with canine visceral leishmaniasis and healthy dogs was measured. The eosinophil percentage were higher in lymph nodes of infected group, and the parasite load showed a significant negative correlation with the eosinophil count. The production of NO and ROS by eosinophils in the peripheral blood was higher in the dogs with canine visceral leishmaniasis. All the results together suggest that eosinophils may participate in antileishmanial immunity in canine visceral leishmaniasis.     

内脏利什曼病诊断技术研究进展

内脏利什曼病即黑热病 ,是由杜氏利什曼原虫感染所致的人兽共患寄生虫病。本病分布很广 ,亚、欧、非、拉丁美州均有本病流行。在我国 ,该病目前仍是严重危害人民健康的寄生虫病之一。尤其是在山丘疫区 ,且以儿童为主。因此 ,快速而准确的诊断成为防治该病的关键。诊断黑热病的传统方法 ,是以骨髓穿刺物涂片镜检或培养检查病原体 ,但骨髓穿刺不仅给患者造成痛苦 ,而且原虫密度低者易漏诊。因而发展高效、快速、准确的检测及鉴定技术仍是这一领域的重点研究课题。近年来关于黑热病的诊断在免疫学及分子生物学方面发展很快 ,主要进展如下 :1　…     

Sensitivity of bone marrow aspirates in the diagnosis of visceral leishmaniasis

Bone marrow aspirates are believed to provide a safer but less sensitive method in the diagnosis of visceral leishmaniasis (VL) compared with splenic aspirates. We examined the effect of the number of fields and the time of observation on bone marrow smear sensitivity and compared it to our experience with spleen aspiration. Bone marrow smears of 98 patients and splenic aspirates from 120 patients were examined. Among 87 patients with VL, the sensitivity of bone marrow aspirates was 40.2%, 65.5%, 89.7%, 92%, and 95.4% at 1, 5, 20, 30, and 60 minutes, respectively. The sensitivity of spleen aspirate examination was 93% for 114 patients. One patient died of shock after spleen aspiration. A bone marrow smear is very sensitive if examined thoroughly, reaching a sensitivity similar to that of spleen aspirate. We propose that a bone marrow smear be established as the technique of choice for the parasitologic diagnosis of VL.     

Utility of Wang needle aspiration in the diagnosis of actinomycosis

An 85-year-old man had a 4-year history of recurrent pneumonia with a persistent pleural effusion. He underwent repeated bronchoscopy that revealed a right bronchus intermedius mass, but bronchial washes and biopsies remained nondiagnostic. A repeat bronchoscopy was performed, and a Wang needle aspiration of the mass was obtained that showed sulfur granules, diagnosing actinomycosis. The patient was started on appropriate antibiotic therapy. Actinomycosis must be considered in a patient with recurrent pneumonia and an endobronchial mass. Wang needle aspiration via bronchoscopy may be an important diagnostic tool.     

Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan

We compared a strip test employing recombinant K39 (rK39) antigen and protein A/colloidal gold as read-out agents with the rK39 ELISA for IgM and IgG antibodies and the direct agglutination test (DAT) using 55 sera from patients with parasitologically confirmed visceral leishmaniasis (VL). The rK39 strip test was positive in 37/55 (67%), the DAT in 50/55 (91%) at > or = 1 : 1600 cut-off value and in 47/55 (85%) at > or = 1 : 6400 cut-off value. The rK39-ELISA gave positive IgG results for all sera; those who had a positive strip test had significantly higher IgG levels than those with a negative strip test (31.1 (SD=3.6) and 17.7 U/ml (SD=9.8), respectively, P < 0.0001). A total of 31/55 (56%) sera showed a positive IgM result; of these 27 (49%) had a positive strip test. We tested 115 apparently cured VL patients with the strip test during follow-up; 68 were also tested with DAT. In the strip test, 25-43% of patients had a positive result at time points 3, 6, 9 and 12 months after treatment; for DAT (cut-off > or = 1 : 1600) these results were 67-83%. In neither test did a significant decrease in positivity rates occur over time (P=0.37 for the strip test, P=0.17 for the DAT). No correlation (P=0.33) was found between a positive strip test and a positive DAT result (cut-off > or = 1: 1600), indicating that the strip test and DAT are complementary rather than interchangeable. Of 61 endemic controls two (3%) had a positive strip test result; both had a positive leishmanin skin test. The rK39 strip test has the ideal format for use in the field, but its sensitivity is limited; like DAT, but to a lesser extent, it remains positive after treatment.     

Accessibility of diagnostic and treatment centres for visceral leishmaniasis in Gedaref State, northern Sudan

OBJECTIVE: To evaluate the accessibility of visceral leishmaniasis (VL) treatment. METHOD: Community-based study using in-depth qualitative interviews and focus group discussions with key informants, as well as quantitative questionnaires with 448 randomly selected heads of households in nine representative villages in three geographical sub-regions. RESULTS: Despite the high incidence of the disease, most people in Gedaref State know little about VL, and help at a treatment centre is usually sought only after traditional remedies and basic allopathic drugs have failed. Factors barring access to treatment are: lack of money for treatment and transport, impassability of roads, work priorities, severe cultural restrictions of women's decision-making power and distance to the next health center. CONCLUSIONS: To provide more VL patients with access to treatment in this highly endemic area, diagnostic and treatment services should be decentralized. Health education would be a useful tool to rationalise people's health-seeking behaviour.     

Evaluation of complement fixation procedures for the diagnosis of visceral leishmaniasis

Three complement fixation (CF) procedures were evaluated for their ability to detect serum antibodies to visceral leishmaniasis. These tests differ in their use of buffers, volumes of complement and sensitized erythrocyte concentrations, incubation times and percentage haemolysis endpoints. Freeze-thawed sonicates of Leishmania donovani promastignotes were used as antigen. Test sensitivity was determined using sera from 46 Kenyans with parasitologically proven leishmaniasis. The frequencies of positive reactions in all three tests were 96-97% and positive antibody titres ranged from 1:16 to 1:4096. Specificity was determined with 20 sera from healthy individuals with no known exposure to leishmaniasis. The frequencies of false positive reactions were 0-10% in the control sera, with titres up to 1:16. No cross-reactions were observed with sera from patients with bacterial, fungal and other parasitic diseases. In replicate experiments, 99-100% of the sera tested were within one titre dilution of each other. All three CF procedures provide very good sensitivity, specificity and low cross-reactivity and are statistically similar in their capacity to diagnose visceral leishmaniasis.     

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.     

Evaluation of a mass distribution programme for fine-mesh impregnated bednets against visceral leishmaniasis in eastern Sudan

During an epidemic of visceral leishmaniasis (VL) in eastern Sudan, Médecins Sans Frontières distributed 357,000 insecticide-treated bednets (ITN) to 155 affected villages between May 1999 and March 2001. To estimate the protective effect of the ITN, we evaluated coverage and use of ITN, and analysed VL incidence by village from March 1996 to June 2002. We provided ITN to 94% of the individuals >5 years old. Two years later, 44% (95% CI 39-48%) of nets were reasonably intact. Because ITN were mainly used as protection against nuisance mosquitoes, bednet use during the VL transmission season ranged from <10% during the hot dry months to 55% during the beginning of the rainy season. ITN were put up from 9 to 11 p.m., leaving children unprotected during a significant period of sandfly-biting hours after sunset. Regression analysis of incidence data from 114 villages demonstrated a significant reduction of VL by village and month following ITN provision. The greatest effect was 17-20 months post-intervention, with VL cases reduced by 59% (95% CI: 25-78%). An estimated 1060 VL cases were prevented between June 1999 and January 2001, a mean protective effect of 27%. Although results need to be interpreted with caution, this analysis indicates a potentially strong reduction in VL incidence following a community distribution of ITN. The effectiveness of ITN depends on behavioural factors, which differ between communities.