[leish-l] Fwd: Articles found by RefScout for your requests

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    Date: Thu, 19 Aug 2004 01:08:06
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This is RefScout-Newsletter 34/2004






REQUEST: [ leishmaniasis ]

(38 articles match this request)



PMID: 15301974
 

TITLE: Canine visceral leishmaniasis: a histopathological study of lymph nodes.

AUTHORS: Wanderson Geraldo Lima, Marilene Suzan Marques Michalick, Maria Norma
de Melo, Washington Luiz Tafuri, Wagner Luiz Tafuri

AFFILIATION: Departamento de Patologia Geral, Instituto de Ciências Biológicas
(ICB), Universidade Federal de Minas Gerais (UFMG), Av. Antônio Carlos, 6627,
Campus Pampulha, Belo Horizonte MG31270-901, Brazil.

REFERENCE: Acta Trop 2004 Sep 92(1):43-53

Canine visceral leishmaniasis (CVL) is a zoonosis and a chronic systemic
 disease of the dog caused by a protozoan of the genus Leishmania. In 
the New World, the disease is caused by the species Leishmania (
Leishmania) chagasi. There are only a few studies on the histopathology 
of lymph nodes in canine leishmaniasis. In the present paper, we report 
a histopathological description of lymph nodes considering animals with 
a defined clinical status and the parasite burden of lymph node tissues
. Forty-eight mongrel dogs naturally infected with L. chagasi, were 
obtained from two endemic areas of Brazil. Cervical, axillary and 
popliteal lymph nodes were analyzed. The parasite burden, expressed as "
Leishman-Donovan units", was variable among the defined types of 
clinical condition. Asymptomatic dogs can show higher parasitism than 
oligosymptomatic or symptomatic animals. Grossly, a generalized 
lymphadenopathy was found, but it was mainly observed in cervical and 
popliteal nodes. Histologically, the increased number and size of 
lymphoid follicles, and the marked hypertrophy and hyperplasia of 
medullary macrophages (cords and sinus) explained the lymphadenopathy. 
In addition, the clinical status or the tissue parasitism load might not
 be directly related to the intensity of the lesions.








PMID: 15311290
 

TITLE: [Spacial modeling of cutaneous leishmaniasis risk zones]

AUTHORS: Cristina Aparicio, Marisa Dantas Bitencourt

AFFILIATION: Departamento de Ecologia, Instituto de Biociências, Universidade
de São Paulo, São Paulo, SP, Brasil.

REFERENCE: Rev Saude Publica 2004 Aug 38(4):511-6

OBJECTIVE: To spatially delimit cutaneous leishmaniasis (CL) human 
contact risk zones (CoRZ) using remote sensing and geoprocessing 
techniques. METHODS: A total of 27 CL cases reported from 1992 to 1997 
in the municipality Itapira, Brazil, were studied. The influence of some
 important environmental variables related to CL such as altitude and 
vegetation density measured by normalized difference vegetation index (
NDVI) images were analyzed for each CoRZ. RESULTS: The results showed 
that about 50% of the dwellings where CL cases were reported were 
located less than 200 meters from the limits of forest remnant area; 
more than 70% of the total area of the CoRZ, for each criterion, were in
 altitudes lower than 750 meters; and about 50% of the CoRZ, for each 
criterion, were located in a very dense green area (NDVI ranging from 0.
45 to 1.00). CONCLUSIONS: The study shows there may be three 
transmission modes in the study area: intra-forest, extra-forest (in 
this setting, transmission might be influenced by the vegetation density
 surrounding forest remnants); or domiciliary.




PMID: 15304181
 

TITLE: Spectrum of skin diseases in Yemen (Hajjah and adjacent region).

AUTHORS: Mishri Lal Khatri

AFFILIATION: Department of Dermatology, Saudi Hospital, Hajjah, Yemen.

REFERENCE: Int J Dermatol 2004 Aug 43(8):580-5

Abstract Background Epidemiological study of skin diseases from Yemen is
 not available, although some data has been published from nearby Arab 
countries. Objective To determine the pattern of skin diseases in Yemen 
and particularly in Hajjah and nearby regions. Methods This is a 
prospective study and analysis of the consecutive new patients attending
 the Dermatology Clinic of Saudi Hospital at Hajjah, Yemen, from January
 1997 to December 1999. The majority of the patients were from Hajjah 
and nearby regions but a substantial number of patients came from 
different distant regions of Yemen. The diagnosis was mainly based on 
clinical features but supported by laboratory investigations, diagnostic
 procedures and histopathology when needed. Results The total number of 
new patients was 13,840 with 14,259 diseases, with a male to female 
ratio of 1 : 0.81. Ninety-nine percent of the patients were Yemeni 
nationals. The leading group of diseases was dermatitis and eczematous 
disorders, followed by infections and infestations (including cutaneous 
leishmaniasis and mycetoma), followed by acne and acneiform disorders. 
Conclusion The spectrum of skin diseases in this analysis revealed that 
more than 50% of the diseases belonged to dermatitis and eczematous 
disorders and infectious diseases groups. Socioeconomic and 
environmental factors of the region are responsible for this. 
Implementation of public health programs, a proper health education, and
 inclusion of a suitable training program of dermatology in the 
undergraduate medical studies of this country may help to prevent and 
manage these problems rationally.




PMID: 15228491
 

TITLE: The recent emergence of Leishmania tropica in Jericho (A'riha) and its
environs, a classical focus of L. major.

AUTHORS: A Al-Jawabreh, L F Schnur, A Nasereddin, J M Schwenkenbecher, Z Abdeen,
F Barghuthy, H Khanfar, W Presber, G Schönian

AFFILIATION: Institute of Microbiology and Hygiene, University Medicine,
Charité, Berlin, Germany. islahjr at yahoo.com

REFERENCE: Trop Med Int Health 2004 Jul 9(7):812-6

Between 1997 and 2002, 49 strains of Leishmania were isolated from the 
cutaneous lesions of Palestinians living in and around Jericho. A 
polymerase chain reaction (PCR) amplifying the ribosomal internal 
transcribed spacer 1 (ITS1-PCR) was applied to their cultured 
promastigotes and to 207 individuals' skin scrapings spotted on filter-
papers, 107 of which proved positive for leishmanial DNA. Species 
identification was performed by restricting the ITS1-PCR amplification 
products from the cultured promastigotes and the amastigotes in the 
scrapings with the endonuclease HaeIII. Of the 49 cultures, 28 (57%) 
were L. major and 21 (43%) were L. tropica. Of the 107 dermal samples 
tested directly, 53 (49.5%) were infected with L. major, 52 (48.5%) with
 L. tropica and two remained unidentified. This is the first time L. 
tropica has been exposed in the population of the Jericho area and on 
such a large scale. The itinerant behaviour of some of this population 
precludes categorically declaring that L. tropica has recently become 
established in this classical focus of L. major. For this and although 
88.2% of the cases of L. tropica claimed not to have travelled out of 
the vicinity of Jericho, local infected sand fly vectors of L. tropica 
must be caught, identified and, if possible, shown to harbour infections
, and, if one exists, an animal reservoir host should also be exposed to
 endorse whether the cases caused by L. tropica were imported or 
autochthonous.




PMID: 15206465
 

TITLE: Cell differentiation and infectivity of Leishmania mexicana are inhibited
in a strain resistant to an ABC-transporter blocker.

AUTHORS: N Silva, N Camacho, K Figarella, A Ponte-Sucre

AFFILIATION: Laboratory of Molecular Physiology, Instituto de Medicina
Experimental, Facultad de Medicina, Universidad Central de Venezuela, Caracas,
Venezuela.

REFERENCE: Parasitology 2004 Jun 128(Pt 6):629-34

We analysed whether markers of cell differentiation and infectivity 
differed when compared to the parental sensitive strain [NR(Gs)] in an 
in vitro selected Leishmania strain [NR(Gr)] resistant to Glibenclamide
, an ATP-binding-cassette (ABC)-transporter blocker. The data show that 
the cell body area was larger in NR(Gr) compared to NR(Gs) and that 
functional characters associated with an infective metacyclic phenotype
, such as resistance to the lytic effect of the alternative complement 
pathway and expression of the Meta-1 protein, were reduced. The 
infectivity of NR(Gr) to J774.1 macrophages was also significantly 
reduced. These results suggest that resistance in Leishmania against 
Glibenclamide, a general blocker of P-glycoproteins, could produce 
functional modifications that may be relevant for Leishmania 
differentiation, infectivity and survival.




PMID: 15214955
 

TITLE: Generalized Microsporum canis dermatophytosis in six Yorkshire terrier
dogs.

AUTHORS: Rosario Cerundolo

AFFILIATION: Department of Veterinary Clinical Sciences, Faculty of Veterinary
Medicine, Naples, Italy. cerundol at vet.upenn.edu

REFERENCE: Vet Dermatol 2004 Jun 15(3):181-7

Six Yorkshire terrier dogs with generalized, chronic dermatophytosis 
caused by Microsporum canis were seen over a 3-year period. Specific 
tests showed that they also had concurrent leishmaniosis (four cases), 
leishmaniosis and ehrlichiosis (one case) or diabetes mellitus (one case
). Although specific therapy for these infectious diseases was 
instituted and the dogs were treated systemically and topically with 
appropriate antifungal drugs, only partial clinical resolution of the 
dermatophytosis was achieved. M. canis infection resolved in the dog 
with diabetes mellitus after stabilizing the diabetes mellitus. Although
 immunological studies were not performed in these cases, it is 
theorized that the immune disregulation caused by leishmaniosis, 
ehrlichiosis or diabetes mellitus may have favoured generalization of 
the infection and prevented favourable responses to appropriate 
treatment of the M. canis infection.




PMID: 15305692
 

TITLE: [New drugs for the treatment of human parasitic protozoa]

AUTHORS: J Dupouy-Camet

AFFILIATION: Laboratoire de Parasitologie-Mycologie, Hôpital Cochin,
Université René Descartes, 27 Faubourg St Jacques, 75014, Paris, France.
jean.dupouy-camet at cch.ap-hop-paris.fr

REFERENCE: Parassitologia 2004 Jun 46(1-2):81-4

Whereas parasitic diseases are always a heavy burden for humanity, few 
are the new antiparasitic molecules marketed during the last 25 years. 
Thus on the 1393 new molecules marketed between 1975 and 1999, only 7 
have antiprotozoan properties. This talk will detail the progress made 
in the treatment of the intestinal protozoa, malaria, visceral 
leishmaniasis and toxoplasmosis, problems with which are especially 
confronted the European parasitologists. The treatment of Giardia and 
intestinal amoebas is based on 5-nitro-imidazoles derivatives. Single-
dose treatments can be used with tinidazole or secnidazole. Resistance 
to these compounds of Giardia were described and in these cases, 
treatment by quinacrine or nitazoxanide are possible alternatives. 
Nitazoxanide is marketed in the United States and in Australia. It seems
 to be a well tolerated antiparasitic agent with a broad spectrum 
because it is active on a lot of intestinal protozoa and helminths. It 
acts on the same metabolic way as the 5-nitro-imidazoles (inhibition of 
the ferredoxine reductase) but without synthesis of free radicals and 
DNA deterioration of the target cell. It is thus neither teratogenic nor
 mutagenic. Artemisinin derivatives allowed considerable progress in the
 treatment of malaria. They have short half-lifes, allowing a fast 
parasitic clearance and these derivatives do no provoke resistance. They
 are first line drugs for the treatment of malaria in areas of drug 
resistance. The arthemeter-lumefantrine association (Riamet, Coartem) 
ensures a rapid disappearance of the circulating parasites and is well 
tolerated. Atovaquone-proguanil (Malarone) is usable in the treatment of
 acute malaria but also in disease prevention with the advantage of 
continuing drug intake for only 7 days after having left the infected 
area. The treatment of leishmaniasis is always delicate and is 
characterized by the worrying development of antimony resistances, 
probably related in the European zones to the treatment of dogs. 
Liposomal amphotricin is an alternative of choice but remains very 
expensive. The heating of amphotericin to 70 degrees C during 20 minutes
 gives it experimental properties and efficacies comparable with that of
 liposomal amphotericin, but at a less cost. Miltefosine, an alkyl-
phospholipide antimetabolite, is very active on visceral leishmaniasis 
resistant to antimonial treatment. However, its long half-life could 
induce the emergence of resistances. Miltefosine induces much less side 
effects than conventional amphotericin B. The commonly used anti-
toxoplasmic drugs (sulphadiazine and pyrimethamine) were marketed some 
50 years ago and are only active on the rapid forms in multiplication. 
No drug is really efficient on the cysts although preliminary tests with
 atovaquone are encouraging to treat ophthalmologic forms in 
immunocompetent patients. To conclude, it is important to continue to 
search for new antiprotozoan molecules because, for some parasites, drug
 resistance is an important problem. Moreover, the treatment of the 
pregnant women, particularly during the first trimester, is often 
impossible and there is a lack of galenic forms easily usable in 
children. A better knowledge of the metabolic pathways of protozoa (
particularly the apicoplast of Apicomplexa parasites) would certainly 
open the posssibility to identify new drugs. To reduce and delay the 
appearance of resistances, mass-treatments of mass should be avoided and
 targeted treatments prefered as well as the use of associations of 
molecules having different modes of action.




PMID: 15305694
 

TITLE: [Highly Active AntiRetroviral Therapy and opportunistic protozoan
infections]

AUTHORS: E Pozio

AFFILIATION: Dipartimento di Malattie Infettive, Parassitarie e Immunomediate,
Istituto Superiore di Sanità , viale Regina Elena 299, 00161 Roma.

REFERENCE: Parassitologia 2004 Jun 46(1-2):89-93

Opportunistic parasite infections (OPIs) are an important cause of 
morbidity and mortality in persons infected with HIV. In industrialised 
countries, the use of Highly Active AntiRetroviral Therapy (HAART) 
results to be effective in suppressing the HIV viral load, with a 
quantitative and qualitative improvement in the CD4+ T-cell count 
followed by a strong reduction of opportunistic infections including 
those caused by parasites. These successes have been mainly attributed 
to the reconstitution of the cell immunity, which play the most 
important role in controlling OPIs. However, there are many clinical 
reports and several laboratory results, which suggest that the control 
of OPIs in HIV-positive persons under HAART is also induced by the anti-
HIV protease inhibitors (PIs), which inhibit the aspartyl proteases of 
the parasites. The non-conventional use of HIV-PIs seems to be an 
alternative way for the treatment of parasitic infections, which should 
be deeply investigated. Of five longitudinal studies carried out before 
and after the introduction of HAART, four studies showed a strong 
reduction of toxoplasmic encephalitis (TE) in HIV-positive persons under
 HAART, whereas in another study, no difference was observed in the 
incidence rate of TE before and after the introduction of HAART. The 
influence of HAART in reducing TE has been also confirmed in a 
randomised, controlled clinical trial, which showed that there is no 
increase in the risk of developing TE after beginning HAART, even though
 HIV-infected persons with TE had a discontinuing prophylaxis for 
Toxoplasma gondii. Four HIV protease inhibitors were tested against the 
T. gondii virulent RH strain in vitro, alone or in association with 
pyrimethamine or sulfadiazine. Ritonavir and nelfinavir were highly 
inhibitory for the parasite growth. Furthermore, none of the antiviral 
drugs negatively affected the anti-Toxoplasma activity of pyrimethamine 
or sulfadiazine. In HIV-Leishmania co-infections, a changing pattern has
 been observed in the HAART era, characterised by a high rate of 
relapses, which could be explained by the increased survival rate 
resulting from the effective antiretroviral therapy. A 64.8% decrease of
 the visceral leishmaniasis incidence was detected after HAART began to 
be used extensively in Spain. In a large cohort study carried out in ten
 European countries and in Australia, the relative risk to contract 
cryptosporidiosis as the first AIDS defining disease was reduced by 96% 
in the HAART era. In Italy, the relative risk of death for 
cryptosporidiosis reduced of 74% in the period 1997-98, when HIV-
positive persons received HAART. In a large study carried out in Italy, 
isosporiasis was included in the group of opportunistic infections, of 
which the relative hazards showed a reduction of 95% in the HAART era. 
Since 1997, there was the evidence that the use of HAART in persons with
 advanced HIV infection can improve chronic diarrhoea and lead to 
disappearance of Enterocytozoon bieneusi from the stools. Although the 
reconstitution of the cellular immunity seems to be the main factor 
influencing the reduction of OPIs in persons with AIDS who undergo HAART
, there are clinical and microbiological evidences, as well as in vitro 
and in vivo results, which indicate direct effects of HIV-PIs on the 
proteases of opportunistic parasites. These findings stress the 
existence of non-conventional unexpected benefits of PIs in HAART 
against protozoa. In addition, this benefit of PIs has been demonstrated
 also for Candida albicans secreted aspartyl proteases and for P. 
carinii acid proteases. In spite of these important results, HIV PIs are
 still very toxic for humans, specially in cases of very long treatment
, and no clinical trial has been carried out for persons at risk, such 
as children and pregnant women, because the priority was to reduce the 
severity of HIV and not the evaluation of possible side effects of the 
therapy. It follows that further researches are needed to establish the 
non-conventional use of HIV PIs. Furthermore, the study of PIs against 
specific aspartyl proteases of those opportunistic protozoa that cause 
severe and intractable diseases, could be considered an alternative way 
towards the development of new drugs that may prove effective against 
these infections.




PMID: 15305696
 

TITLE: [Nitric oxide and anti-protozoan chemotherapy]

AUTHORS: L Gradoni, P Ascenzi

AFFILIATION: Reparto di Malattie Trasmesse da Vettori e Sanità Internazionale,
Dipartimento MIPI, Istituto Superiore di Sanità , Roma, Italy.

REFERENCE: Parassitologia 2004 Jun 46(1-2):101-3

Constitutive nitric oxide (NO) is generated by constitutively expressed 
types of NO-synthase enzymes (NOS-I and -III), being involved in 
physiological processes such as nervous transmission and vasodilatation
. Inducible NO, synthesized by the NO-synthase isoform NOS-II, is an 
anti-pathogen and tumoricidal agent. However, inducible NO production 
requires a tight control because of cytotoxic and immune-modulation 
activity. NO produced by human and canine macrophages has long been 
demonstrated to be involved in the intracellular killing of Leishmania. 
Mechanisms of parasite survival and persistence in the host have been 
throughly investigated, and include suppression of NOS-II and the 
parasite entry into NOS-II negative cells. Both intracellular and 
extracellular morphotypes of Trypanosoma cruzi are killed by NO in vitro
 and in vivo, although a role of NO in the pathogenesis of heart disease
 has been reported. Killing of extracellular protozoa such as 
Trichomonas vaginalis and Naegleria fowleri by activated macrophages is 
also mediated by NO. The main control of Plasmodium spp infection in 
human and murine hepatocytes, and in human monocytes is achieved by NO-
mediated mechanisms. Protection from severe malaria in African children 
has been found associated with polymorphisms of the NOS-II promoter; 
however, a pathogenic role of endogenous NO has been documented in 
cerebral malaria. Although several macromolecules are putative NO 
targets, recent experimental work has shown that NO-releasing compounds 
inhibit cysteine proteases (CP) of P. falciparum, T. cruzi and L. 
infantum in a dose-dependent manner. CPs are present in a wide range of 
parasitic protozoa and appear to be relevant in several aspects of the 
life cycle and of the parasite-host relationships. Comparative analysis 
of 3-D amino acid sequence models of CPs from a broad range of living 
organisms, from viruses to mammals, suggests that the Sy atom of the Cys
 catalytic residue undergoes NO-dependent chemical modification (S-
nytrosilation and disulfide bridge formation), with the concomitant loss
 of enzyme activity. The NO-donor S-nitroso-N-acetilpenicillamine (SNAP
) was shown to kill T. cruzi epimastigotes and L. infantum promastigotes
 in culture, while a combination of nitrite plus acid organic salts was 
highly effective against L. major amastigotes in mouse macrophages. A 
parasitostatic effect--with both encystation and excystation inhibition
--of S-nitrosoglutathione and spermine-NONOate was documented in 
trophozoite cultures of Giardia duodenalis. Recently, a novel 
formulation of metronidazole bearing a NO-releasing group was found to 
enhance significantly the in vitro killing of Entamoeba histolytica 
trophozoites, compared to metronidazole. So far, only two clinical 
studies were performed on human patients, suffering from cutaneous 
leishmaniasis. In one study, 16 Ecuadorean patients were treated with a 
SNAP cream administered on lesions for 10 days. All lesions were 
parasitologically cured and clinically healed by day 30. In the second 
study, a different NO-producing cream (basically nitrite in acidic 
environment) was employed to treat 40 Syrian patients. Only 28% of them 
showed improvement and 12% were cured by day 60. In conclusion, despite 
the wide evidence that NO can be regarded as a natural anti-protozoal 
weapon, little efforts have been made to develop and test NO-based drugs
 in human medicine. This is mainly due to the difficulty in designing 
suitable chemical carriers able to release the right amount of NO, in 
the right place and in the right time, to avoid toxic effects against 
non-target host cells.




PMID: 15305704
 

TITLE: [The serodiagnosis of parasitic infections]

AUTHORS: F Bruschi, B Castagna

AFFILIATION: Dipartimento di Patologia Sperimentale, Biotecnologie Mediche,
Infettivologia ed Epidemiologia, Università degli Studi di Pisa e U.O. di
Microbiologia Universitaria, Azienda Ospedaliera Universitaria Pisana, Pisa.

REFERENCE: Parassitologia 2004 Jun 46(1-2):141-4

Recently, the term of clinical immunoparasitology has been coined to 
indicate the application of immunological methods to the laboratory 
diagnosis of parasitic infections. In particular, serological diagnosis
 (indirect diagnosis) is useful especially in the cases of toxocarosis, 
trichinellosis, echinococcosis, cysticercosis, toxoplasmosis, amoebic 
abscess, some filariasis, visceral leishmaniasis, schistosomiasis. When 
possible, for infections caused by protozoa or helminths, the "gold 
standard" is represented by direct diagnosis performed by microscopic 
and/or macroscopic observation of the parasite. In any case, 
immunological results must be interpreted in consideration of the 
clinical picture of the patient and confirmed possibly by finding the 
parasite or its genome, even using molecular methods. Furthermore, since
 the presence of specific antibodies can reveal an acquired infection, 
but not necessarily a disease, it is particularly helpful, in addition 
to a qualitative evaluation, a quantitative one, by determining the 
serum antibody titre. After recovery, the antibody levels decrease, 
however, they may persist for long periods, for this reason they do not 
help in evaluating the treatment outcome. Interpretation of serological 
results may be difficult when the patients originate from areas where 
the suspected infection is endemic, in that case, a serum positivity 
could reflect an old exposition to the parasite, therefore it is not 
related to the present clinical status. Furthermore, serology may 
frequently result falsely negative in not immunocompetent subjects (
organ transplanted, HIV positive individuals, premature babies, 
diabetics). Clinicians can interpret correctly the serological results 
only if the Parasitology laboratory inform them about the significant 
diagnostic values, the sensitivity and the specificity of the test in 
use. At present time, many diagnostic kits for immunoparasitology are 
commercially available, and industries are developing newer and newer 
ones (which are not always validated). In relation to this aspect, it 
should be helpful, for each of parasitic infection, to establish 
reference centers, not only to control the quality of commercial kits, 
but also as a reference point to those laboratories which use "in house
" kits. To this regard, the recent establishment of a European Centre 
for Control of Infectious Diseases will help. The antigen 
characteristics (crude, E/S, recombinant, synthetic) for assays 
searching for antibodies (IHA, IFA, EIA, WB) of different classes, the 
controls to choose for these assays, the specimen requirements will be 
discussed. The recent findings on the serological diagnosis of 
intestinal protozoa infections, malaria, leishmaniasis, echinococcosis, 
cysticercosis, trichinellosis, toxocariasis, schistosomiasis, 
strongyloidiasis will be presented.




PMID: 15305709
 

TITLE: [Quantitative PCR in the diagnosis of Leishmania]

AUTHORS: M Mortarino, A Franceschi, F Mancianti, C Bazzocchi, C Genchi, C Bandi

AFFILIATION: Dipartimento di Patologia Animale, Igiene e Sanità Pubblica
Veterinaria, Università di Milano.

REFERENCE: Parassitologia 2004 Jun 46(1-2):163-7

Polymerase chain reaction (PCR) is a sensitive and rapid method for the 
diagnosis of canine Leishmania infection and can be performed on a 
variety of biological samples, including peripheral blood, lymph node, 
bone marrow and skin. Standard PCR requires electrophoretic analysis of 
the amplification products and is usually not suitable for 
quantification of the template DNA (unless competitor-based or other 
methods are developed), being of reduced usefulness when accurate 
monitoring of target DNA is required. Quantitative real-time PCR allows 
the continuous monitoring of the accumulation of PCR products during the
 amplification reaction. This allows the identification of the cycle of 
near-logarithmic PCR product generation (threshold cycle) and, by 
inference, the relative quantification of the template DNA present at 
the start of the reaction. Since the amplification product are monitored
 in "real-time" as they form cycle-by-cycle, no post-amplification 
handling is required. The absolute quantification is performed according
 either to an internal standard co-amplified with the sample DNA, or to 
an external standard curve obtained by parallel amplification of serial 
known concentrations of a reference DNA sequence. From the 
quantification of the template DNA, an estimation of the relative load 
of parasites in the different samples can be obtained. The advantages 
compared to standard and semi-quantitative PCR techniques are reduction 
of the assay's time and contamination risks, and improved sensitivity. 
As for standard PCR, the minimal components of the quantitative PCR 
reaction mixture are the DNA target of the amplification, an 
oligonucleotide primer pair flanking the target sequence, a suitable DNA
 polymerase, deoxynucleotides, buffer and salts. Different technologies 
have been set up for the monitoring of amplification products, generally
 based on the use of fluorescent probes. For instance, SYBR Green 
technology is a non-specific detection system based on a fluorescent 
dsDNA intercalator and it is applicable to all potential targets. TaqMan
 technology is more specific since performs the direct assessment of the
 amount of amplified DNA using a fluorescent probe specific for the 
target sequence flanked by the primer pair. This probe is an 
oligonucleotide labelled with a reporter dye (fluorescent) and a 
quencher (which absorbs the fluorescent signal generated by the reporter
). The thermic protocol of amplification allows the binding of the 
fluorescent probe to the target sequence before the binding of the 
primers and the starting of the polymerization by Taq polymerase. During
 polymerization, 5'-3' exonuclease activity of Taq polymerase digests 
the probe and in this way the reporter dye is released from the probe 
and a fluorescent signal is detected. The intensity of the signal 
accumulates at the end of each cycle and is related to the amount of the
 amplification product. In recent years, quantitative PCR methods based 
either on SYBR Green or TaqMan technology have been set up for the 
quantification of Leishmania in mouse liver, mouse skin and human 
peripheral blood, targeting either single-copy chromosomal or multi-copy
 minicircle sequences with high sensitivity and reproducibility. In 
particular, real-time PCR seems to be a reliable, rapid and noninvasive 
method for the diagnosis and follow up of visceral leishmaniasis in 
humans. At present, the application of real-time PCR for research and 
clinical diagnosis of Leishmania infection in dogs is still foreseable. 
As for standard PCR, the high sensitivity of real-time PCR could allow 
the use of blood sampling that is less invasive and easily performed for
 monitoring the status of the dogs. The development of a real-time PCR 
assay for Leishmania infantum infection in dogs could support the 
standard and optimized serological and PCR methods currenly in use for 
the diagnosis and follow-up of canine leishmaniasis, and perhaps 
prediction of recurrences associated with tissue loads of residual 
pathogens after treatment. At this regard, a TaqMan Real Time PCR method
 developed for the quantification of Leishmania infantum minicircle DNA 
in peripheral blood of naturally infected dogs sampled before and at 
different time points after the beginning of a standard antileishmanial 
therapy will be illustrated.








PMID: 15305715
 

TITLE: [Monitoring of canine leishmaniasis in northern Italy: an update from a
scientific network]

AUTHORS: G Capelli, R Baldelli, E Ferroglio, C Genchi, L Gradoni, M Gramiccia, M
Maroli, M Mortarino, M Pietrobelli, L Rossi, M Ruggiero

AFFILIATION: Università di Padova.

REFERENCE: Parassitologia 2004 Jun 46(1-2):193-7

Canine leishmaniasis (CanL) due to Leishmania infantum is a disease of 
great veterinary importance and a serious public health problem. In 
humans, L. infantum causes visceral (VL) and cutaneous leishmaniasis (CL
) and the distribution of VL overlaps that of CanL. Currently, VL is 
considered by WHO as an emerging zoonosis in southern Europe. The dog is
 the only domestic reservoir of the infection and phlebotomine sandflies
 are the only proven vectors of leishmaniasis for dogs and humans. CanL 
is endemic in Italy, particularly in central and southern regions, 
including islands. Until 1983, all regions of northern Italy but Liguria
 and some territories of Emilia Romagna were considered free from CanL.