Immunological stimulation for the treatment of leishmaniasis: A modality worthy of serious consideration

Instead of relying on drugs to reduce the parasite burden of leishmaniasis, and waiting for the effector immune response to develop in time to control the parasites, immunotherapy in conjunction with chemotherapy can rapidly induce the effector immune response. With a safe and potent drug plus an affordable therapeutic vaccine (immunostimulant), which remains to be developed, a single visit by patients with visceral or cutaneous leishmaniasis might be sufficient to induce a quick and lasting recovery. Drug toxicity and the emergence of resistance could also be dramatically reduced compared with present long-term monotherapy. Immunotherapy could be an effective addition to chemotherapy for leishmaniasi

HAEMATOGENOUS DISSEMINATION OF TUBERCULOUS LYMPHADENITIS

ABSTRACTObjective: To determine whether Mycobacterium tuberculosis infection spreads through the bloodto different lymph-node groups in patients with tuberculous lymphadenitis.Design: Prospective analytical study.Setting: The patients were recruited, managed and followed at the lymphodenopathy clinic, CentralPolice Hospital, Burr, Khartoum, Sudan.Subjects: Fifty two sequential patients were enrolled. Thirty patients with FNAC diagnosisof tuberculous lymphadenitis and positive PCR for M. tuberculosis complex had a mean ageof 26.9±11.2 years and similar male, female affection. Nine patients with FNAC tuberculouslymphadenitis, but negative PCR had a slightly higher mean age (32.6±18.2 years) with similarmale : female proportions. Patients with reactive lymphadenopathy (9/52) were older than patientswith tuberculous lymphadenitis with a mean age of 45±24.6 years.Results: None of the patients were positive for HIV or had clinical or radiological evidence ofpulmonary tuberculosis. M.tuberculosis DNA was detected in the blood samples of 30/39 (77%)patients with tuberculous lymphadenitis, but in none of the cases with reactive or malignantlymphadenopathy. The presence of M.tuberculosis DNA correlated strongly to multiple lymphnodeinvolvement [OR (odds ratio) = 96.7, 95% confidence interval (CI) 9.0 – 1,039] and to caseatinggranulomatousand predominantly necrotic cytomorphological categories [OR = 70, 95% confidenceinterval (CI) 7.0 – 703].Conclusion: M.tuberculosis most probably disseminates through the blood from one node groupto the other in patients with tuberculous lymphadenitis.East African Medical Journal Vol. 84 No. 1 January 2007HAEMATOGENOUS DISSEMINATION OF TUBERCULOUS LYMPHADENITISG.S. Sharafeldin, MSc, National Health Laboratory, Federal Ministry of Health, Khartoum, Sudan, E.A.G. Khalil, MBBS,FRCPath, Institute of Endemic Diseases, University of Khartoum, P.O. Box 45235, Khartoum, Sudan, I.A. El Hag,MBBS, PhD, PARAS Central Hospital, Sakaka Al-jouf, Saudi Arabia, K.E. Elsiddig, MBBS, MCS, FRCS, Departmentsof Surgery and Pathology, Faculty of Medicine, University of Khartoum, P.O. Box 45235, Khartoum, Sudan, M.E.M.O.Elsafi, MBBS, PhD, MD, Central Police Hospital, Khartoum, Sudan, A.S.A Aijafari MSc, A.A. Elnour MSc, Institute ofEndemic Diseases, University of Khartoum, P.O. Box 45235, Khartoum, Sudan, A.M. Hussein, MBBS, MD, Central PoliceHospital, Khartoum, Sudan, I.M. Elkhidir, MBBS, PhD, Department of Microbiology, Faculty of Medicine, University ofKhartoum, P.O. Box 45235, Khartoum, Sudan and A.M. El-Hassan, DKSM, PhD, FRCPath, Institute of Endemic Diseases,University of Khartoum, P.O. Box 45235, Khartoum, SudanRequest for reprints to: Prof. E.A.G. Khalil, Department of Clinical Pathology and Immunology, Institute of EndemicDiseases, University of Khartoum, P.O. Box 45235, Khartoum, SudanINTRODUCTIONTuberculosis remains one of the major healthproblems worldwide with 8.7 million new casesevery year and an estimated 1.7 million annualdeaths (1). Tuberculosis is a systemic diseaseaffecting almost all organs with two clinical forms;pulmonary and extra-pulmonary. The incidenceof extra-pulmonary tuberculosis is dramaticallyrising, largely because of HIV/AIDS pandemic (2).Tuberculous lymphadenitis is the most commontype of extra-pulmonary tuberculosis (3-6). Lymph4E A S T A F R I C A N M E D I C A L J O U R N A L January 2007nodes are usually involved as a component ofa primary complex. Less often lymphadenitis isseen in secondary tuberculosis but the nodes areusually smaller and firmer than in primary disease.Tuberculous lymphadenitis may involve a singlediscrete lymph-node, multiple lymph-nodes ormultiple sites of lymph-nodes and could even begeneralised. Nevertheless, the most commonlyaffected lymph-nodes are those of the cervicalregion (7,8). Since tuberculous lymphadenitis ispredominantly a primary disease, the question of howthe infection spreads from the primary lymph-nodeto other nodes arises. Using blood culture techniques,circulating M. tuberculosis has been found to be oneof the frequent causes of bloodstream infections(BSI) among febrile adults with advanced HIVinfection in sub-Saharan Africa. These patients haveradiological, microbiological or clinical evidence foractive pulmonary disease. Recently, non-tuberculousmycobacteria was detected by culture technique inimmunocompetent individuals (9-13).In this communication we provide evidenceto the probable route of M. tuberculosis spreadin peripheral tuberculous lymphadenitis bydemonstrating circulating M. tuberculosis DNA inimmune-competent Sudanese patients who had nosigns of pulmonary disease, using a highly sensitivemolecular technique (PCR).MATERIALS AND METHODSThe study proposal was scientifically and ethicallyreviewed by the Ethical Committee of the Instituteof Endemic Diseases, University of Khartoum. Fiftytwo sequential patients with lymphadenopathywere enrolled in this prospective study followinginformed consent. FNAC was performed onall patients as a routine diagnostic procedure.Following thorough clinical examination; bloodcell count, ESR, Mantoux test, HIV ELISA testand a chest X-ray were performed. Five millilitersof EDTA-blood were collected from all patientsand DNA was extracted from the mononuclearblood cells (PBMCs) using the phenol-chloroformiso-amyl alcohol (PCI) method. Peripheral bloodmononuclear cells (PMBCs) were collected usingdensity gradient centrifugation with FicolHypaque.The quality of the extracted DNA was checked by1.2% agarose gel electrophoress.Oligonucleotide primers: A single set of oligonucleotideprimers was used (MOL BIOL, Berlin, Germany®).The lyophilised primers were reconstituted asdescribed by the manufacturer. The target for PCRamplification was IS6110 (size 123bp), an insertionlikeelement found in Mycobacterium tuberculosiscomplex strains. The oligonucleotides primers usedwere:E1 (20 mer)(5’-CCTGCGAGCGTAGGCGTCGG-3’)E2 (5’ -CTCGTCCAGCGCCGCTTCGG-3’)DNA amplification: Following strict StandardOperating Procedures, DNA extraction andPCR were carried in separate rooms to reducecross contamination. PCR for the specimens wasperformed in a total volume of 50μl of the reactionmixture containing 10X PCR buffer, 2.25μM.Magnesium Chloride, dNTPs mixture (dATP, dGTP,dCTP & dTTP) 100 each (Boehringer Mannheim,Germany), E1 and E2 oligonucleotide primers 0.2μMeach and 2 U/μL of Taq polymerase (Finnzmes®,Vienna, Austria). The reaction mixture was overlaidwith mineral oil. The tubes were then subjected to40 thermal cycles in a programmable heat block(Biometra®, Göttingen, Germany). The cycle wasas follows: denaturation at 95ºC for five minutes,annealing at 65ºC for one minute, extension at 72ºCfor one minute and final extension at 72ºC for tenminutes.Detection of amplified DNA: The PCR products wereanalysed by ethidium bromide-stained agarosegel electrophoresis on 1.5% agarose gel. Positive,negative controls and a 100 bp DNA marker wereincluded with every electrophoresis run.Statistical analysis: Odds ratio were computed onAcaStat statistical software. If any of the four valuesin the contingency table were zero, one is added toall values before calculating the odds ratio. RESULTSBaseline characteristics, laboratory findings, FNACand PCR results were summarised in Table l.Cervical lymph nodes were the most commonlyaffected group and were seen in 91% of patientswith lymphadenopathy. The cytological diagnosiswas; tuberculous lymphadenitis in 39 cases (75%),reactive in nine (17.3%) and malignant in four casesJanuary 2007 E A S T A F R I C A N M E D I C A L J O U R N A L 5(7.7%). Based on the cytological findings, cases oftuberculous lymphadenitis were categorised intothree groups; necrotising-granulomatous (21/39;54%), predominantly necrotic (6/39; 15.3%) andgranulomatous (12/39; 30.7%). Multiple lymphnodeswere seen in 28 cases, but a single discrete nodewas seen in 11 cases of tuberculous lymphadenitis. Allcases in the caseating-granulomatous (21/21) and thepredominantly necrotic (6/6) categories had multiplelymphnodes. Single lymph-node presentation wasmainly associated with granulomatous lesion(11/12). An ear, nose, throat examination revealedno abnormalities.None of the patients were HIV positive or hadradiological or clinical evidence of pulmonary ormiliary disease.The ESR was high in the tuberculous groupespecially the granulomatous type, while it wasvariable in patients with reactive and malignantgroups. A significant Mantoux reactivity (> l 5mm) was seen in all patients with tuberculouslymphadenitis, while it was < 10 mm in the reactiveand the malignant groups.Circulating M. tuberculosis DNA was detectedin 30 out of 39 cases (77%) with tuberculous lymphnodes, but in none of the cases with reactive ormalignant nodes. The PCR positive rates variedamong tuberculous patients with different smearfinding, while it was 100% (27/27 cases) in thecaseating-granulomatous and the predominantlynecrotic categories; the positivity rate was 25%(3/12 cases) among patients with granulomatouslesions. The computed odds ratio was 70 with a95% confidence interval CI between 7.0 and 703.Circulating M. tuberculosis DNA was detected inall of the 28 patients (100%) with multiple lymphnodes, but in 2 out of 11 cases (18%) with single nodeinvolvement. The odds ratio was 96.7 with a 95%confidence interval CI between 9.0 and l,039.DISCUSSIONTuberculous lymphadenitis is characterisedby painless enlargement of lymph nodes andoccasional constitution symptoms like nocturnalfever and sweating. The cervical group is the mostcommonly affected group. Peripheral tuberculouslymphadenitis usually develops as a part of a primarycomplex i.e. a primary focus and regional glands.Involvement of lymph-nodes as a manifestation ofa generalised tuberculous infection is rather rare(14). For tuberculous lymphadenitis of the neck,the primary focus would be mainly in the tonsilsand mouth, however recent or previous seedingof the lymph nodes from an occult site cannot beruled out. None of our cases had shown clinical orradiological evidence of another primary complexor military disease and the lymph-nodes werelarge and soft. These findings probably support theprimary nature of the disease in our cases. Therefore,the demonstration of circulating mycobacteria insuch cases would suggest spread by blood stream.Patients with multiple nodes were considered ashaving bulky disease.Table 1Baseline characterisitics, cytomorphological patterns and circulating mycobacterial DNA of the study patientsCytomorphological typeof lymphadenopathy M: F Mean age Mean ESR Mean TBCs Mean mantouxmm mmPCR positive (n = 30) 1:1.5 26.9 ± 11.2 87 ± 31 5.3 ± 1.6 22 ± 6Necrotising Tb (n = 6) 1:5 23 ± 8.6 69 ± 24.9 6.8 ± 2.3 19.2 ± 5.5Granulomatous Tb (n = 3) 1:2 43.7 ± 15.1 113 ± 25.1 4.6 ± 0.7 36.3 ± 7.6Necro/Granul.Tb (n = 21) 3:4 25.1 ± 9.2 69.3 ± 32.4 4.6 ± 0.6 21.4 ± 6PCR negative (n = 22) 2:1 34.6 ± 21.6 77 ± 30 5.9 ± 3.2 13 ± 10.8TB lymphadenitis (n=9) 2:1 32 ± 18.2 90.4 ± 27.8 7.3 ± 4.5 19.3 ± 8.2Reactive (n = 9) 2:1 45 ± 24.6 76 ± 35.1 5.3 ± 1.7 5.3 ± 9.2Malignancy (n = 4) 1:0 26.8 ± 25.5 56.7 ± 23.1 4.3 ± 0.6 8.5 ± 6.6Continuous variables are expressed as means ±SD6 E A S T A F R I C A N M E D I C A L J O U R N A L January 2007For the detection of circulating M. tuberculosis,a highly sensitive molecular technique (PCR)was used in this study. PCR has proven to bemore sensitive compared to the conventionalmicrobiological methods (LJ culture and ZNstaining for acid fast bacilli in smears) in theidentification of mycobacterium (15-19). However,PCR is known to be associated with high falsepositive results, with rates ranging from 3 – 20%,mainly due to cross-contamination (20, 21). Toprevent cross-contamination we followed strictlythe manufacturer instructions, DNA extraction andamplification were carried out in different roomsand the sequences of the process were adequatelymonitored. Positive and negative controls wereincluded as extra quality assurance measure withevery run. We have recently shown that, over 96%of cases of tuberculous lymphadenitis in Sudanare caused by M. tuberculosis (18). This made theuse of a single primers set that amplifies a 123bp sequence common to all M. tuberculosis moreappropriate for this study. Circulating M. tuberculosisDNA could be demonstrated in most patientswith tuberculous lymphadenitis in this study. Thehighest positive rate was reported among cases withcaseating-granulomatous or predominantly necroticchanges. The positive rate among patients withgranulomatous lesion was low. This agrees well withprevious studies demonstrating lower mycobacterialload in granulomatous lesions which was reflectedin scantier mycobacteria in ZN smears and lowerpositive rates in LJ culture (22,23). Althoughcirculating M. tuberculosis DNA was seen in patientswith multiple lymph-node involvement or showingcaseating-granuloma tous or predominantly necroticcytological pictures, the confidence intervals werewide probably indicating weak association. Thegranulomatous pattern is associated with singlenode involvement (91.6%) and a low positive rate(25%) for circulating M. tuberculosis DNA. Manystudies showed that bloodstream infections (BSI) byM. tuberculosis do occur. However, it affects mainlyimmuno-compromised adults with advanced HIVinfection with radiological, microbiological and/or clinical evidence of pulmonary tuberculosis.The demonstration of circulating M. tuberculosisDNA in immuno-competent patients, mainly inassociation with multiple lymph node involvementprobably suggests haematogenous spread. Lack ofmultiple organ involvement by the disease in-spiteof haematogenous spread can be explained bycompartmentalisation of mycobacterial infection.Garcia de Viedma et al (24), demonstrated thatinfection by more than one mycobacterial strains isvery rare and when occurs, the co-infecting strainsare not equally distributed at pulmonary andextra-pulmonary sites. Circulating M. tuberculosisDNA in our patients could explain the nocturnalfever that was reported by more than 80% of ourpatients (unpublished data). The pattern of thefever could be explained by episodic release of themycobacteria and its antigens into the blood stream.Demonstration of circulating M. tuberculosis DNAwas previously shown by Mirza et al (25).It has long being claimed that the mantouxtest can help to differentiate between tuberculousand non-tuberculous lymphadenitis (26,27). Allour patients with tuberculous lymphadenitishad strongly positive mantoux test (induration>15mm).We conclude that the presence of circulatingM. tuberculosis DNA in the blood of most patientswith tuberculous lymphadenitis, especially thosewith multiple lymph node involvement indicatesthat heamatogenous spread is the probable routeof mycobacterial dissemination in tuberculouslymphadenitis.REFERENCES1. World Health Organisation. (2002). Global TuberculosisControl: Surveillance, Planning, Financing. WHO report,Geneva, Switzerland, 2000 WHO/CDC/TB/; 295.2. Fanning A. Tuberculosis: Extrapulmonary disease. CMAJ.1999; 160: 1597-1603.3. Jha B.C., Dass A., Nagarkar N.M., Gupta R. and Singhal S.Cervical tuberculous lymphadenopathy: changing clinicalpattern and concepts in management. Postgrad. Med. J. 2001;77: 185-187.4. Chao S.S., Loh K.S., Tan K.K. and Chong S.M. Tuberculousand nontuberculous cervical lymphadenitis: a clinical review.Otolaryngol. Head Neck Surg. 2002; 126: 176-179.5. Shenoy R., Kapadi S.N., Pai K.P., et al. Fine needle aspirationdiagnosis in HIV-related lymphadenopathy in Mangalore,India. Acta. Cytol. 2002; 46: 35-39.6. El_Hassan A.M. and Tag El Deen M. Primary intestinaltuberculosis in the Sudan. Ann. Trop. Med. Parasitol. 1982; 76:317-322.7. Bandapat M.C., Mishra B.M., Dash S.P. and Kar P.K.Peripheral lymph-node tuberculosis: A review of 80 cases.Brit. J. Surg. 1990; 77: 911-912.January 2007 E A S T A F R I C A N M E D I C A L J O U R N A L 78. Yassin M.A., Olobo J.O., Kidane D., et al. Diagnosis oftuberculous lymphadenitis in Butjira, Rural Ethiopia. Scand.J. Infect. Dis. 2003; 35: 240-243.9. Archibald L.K., den Dulk M.O., Pallangayo K.J. and PellerL.B. Fatal Mycobacterium tuberculosis bloodstream infectionsin febrile hospitalised adults in Dar es Salaam, Tanzania. Clin.Infect. Dis. 1998; 26: 290-296.10. Archibald L.K., McDonald L.C., Nwanyanwu O., et al. Ahospital-based prevalence survey of bloodstream infectionsin febrile patients in Malawi: Implications of diagnosis andtherapy. J. Infect. Dis. 2000; 181: 1414-1420.11. Waddell R.D., Lishimpi K., von Reyn C.F., et al. Bacteraemiadue to M. tuberculosis or Mbovis, Bacille Calmette Guerin(BCG) among HIV-positive children and adults in Zambia.AIDS. 2001; 15: 55-60.12. Lai C.C., Lee L.N., Ding L.W., Yu C.J., Hsueh P.R. andYang P.C. Emergence of disseminated infections due tonontuberculous mycobacteria in non-HIV-infected patients,including immunocompetent and immunocompromisedpatients in a university hospital in Taiwan. J. Infect. 2005;[Epub ahead of print].13. MacGregor R.R., Hafner R., Wu J.W., et al. ACTG Protocol341 Team. Clinical, microbiological, and immunologicalcharacteristics in HIV-infected subjects at risk for disseminatedMycobacterium avium complex disease: an AACTG study.AIDS Res. Hum. Retroviruses. 2005; 21: 689-695.14. Miller F.J.W. and Cashman J.M. The natural history ofperipheral tuberculous lymphadenitis associated with avariable primary focus. Lancet. 1955; 268: 1286-1289.15. Singh K.K., Muralidhar M., Kumar A., et al. Comparisonof in house polymerase chain reaction with conventionaltechniques for the detection of Mycobacterium tuberculosisDNA in granulomatous lymphadenopathy. J. Clin. Pathol.2000; 53: 355-361.16. Goel M.M., Ranjan V., Dhole T.N., et al. Polymerase chainreaction vs. conventional diagnosis in fine needle aspirationof tuberculous lymph nodes. Acta. Cytol. 2001; 45: 333-340.17. Hirunwiwatkul P., Tumwasorn S., Chantranuwat C. andSirichai U. A comparative study of diagnostic tests fortuberculous lymphadenitis: polymerase chain reaction vshistopathology and clinical diagnosis. J. Med. Assoc. Thai.2002; 85: 320-326.18. Aljafari A.S., Khalil E.A., Elsiddig K.E.G., et al. Diagnosisof tuberculous lymphadenitis by FNAC, microbiologialmethods and PCR: A comparative study. Cytopathol. 2004;15: 44-48.19. Jain A., Verma R.K., Tiwari V. and Goel M.M. Dot-ELISA vs.PCR of fine needle aspirates of tuberculous lymphadenitis: aprospective study in India. Acta. Cytol. 2005; 49: 17-21.20. Noordhoek G.T., Kolk A.H., Bjune G., et al. Sensitivityand specificity of PCR for detection of mycobacteriumtuberculosis: A blind comparison study among sevenlaboratories. J. Clin. Microbiol. 1994; 32: 277-284.21. Noordhoek G.T., Mulder S., Wallace P. and Van LoonA.M. Multicentre quality control study for the detection ofmycobacterium tuberculosis in clinical samples by nucleicamplification methods. Clin. Microbiol. Infect. 2004; 10: 295-301.22. Gupta S.K., Chagh T.D., S.heikh Z.A. andal-Rubah N.A. 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Pattern of haemopoietic progenitor cells differentiation and differentiation enhancing effect of Telfairia occidentalis extract in irradiated guinea-pig

Ionizing radiation is essentially considered a viable treatment modality for human malignancies. Acute and chronic toxicities with severe morbidity and mortality are well documented. Experimental and clinical approaches to correcting radiation haemopoietic syndrome deficiencies have focused on cytokine activity with same documented toxicities. Limited data is available on differentiation enhancing effects of plant extracts on haemopoietic progenitor cells following radiation injury. This study aimed to determine the potential haemopoietic progenitor cells differentiation effects of Telfairia occidentalis in irradiated guinea pigs. Bone marrow cells from irradiated guinea pigs were harvested and treated with varying concentrations of 0.313-100% of the extracts of the Telfairia occidentalis and the degree of differentiation of the cultured guinea-pig bone marrow haemopoietic stem cells determined. The concentration of the extract ranges from 0.313-100%. This showed Mean ± SD of 3.54±0.30, 0.18±0.01 and 0.88±0.05 for the extract treated, non-extract treated (-ve control) and GM-CSF (+ve control) treated culture plates respectively [p=0.000]. Extract concentrations corrected positively with differentiation of the cultured guinea-pig bone marrow haemopoietic stem cells at 'r' 0.835 [r= 0.812; p=0.001]. Conclusively, Telfairia occidentalis extract has positive differentiation enhancing effects on haemopoietic progenitor cells with deflection in favour of erythroid lineage

Uncommon clinical presentations of cutaneous leishmaniasis in Sudan

International audienceCutaneous leishmaniasis in Sudan is caused by Leishmania major zymodeme LON1. Self-healing usually occurs within 1 year but occasionally its duration is prolonged and treatment is required. The clinical forms are ulcers, nodules and noduloulcerative lesions. Here we describe seven patients with uncommon lesions that were difficult to recognize as Leishmania infections. These included mycetoma-like lesions, lesions that resembled L. tropica infection and others. One HIV/AIDS patient had Kaposi's sarcoma with Leishmania parasites in the Kaposi lesions. Most of these uncommon clinical forms were difficult to treat. The diagnosis depended on a high degree of suspicion and the demonstration of parasites in smears or culture. PCR was used to characterize parasites from the patients described here. Leishmania major was found by kDNA PCR in all patients, except one, who had a leishmanioma due to L. donovani. In three patients, including one with a L. tropica like-lesion, the parasites were confirmed as L. major by gp63 PCR-RFLP