The efficacy of conjunctival swab (CS) as a sampling method for visceral leishmaniasis (VL) diagnosis by PCR of asymptomatic dogs was evaluated. The CS was compared to blood samples (B) and skin biopsies (SB), two less invasive samples potentially useful for massive screening of dogs. Thirty asymptomatic dogs, with serological and parasitological positive tests, were used. The samples were analyzed by two PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The DNA sample volume used was of 1.0 microL and 10.0 microL respectively. Using CS samples the kDNA PCR-hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR-hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. The kDNA PCR-hybridization and ITS-1 nPCR methods showed similar sensitivities for CS and SB samples. On the other hand, for blood samples, the positivity of ITS-1 nPCR was significantly higher than the one obtained by the kDNA PCR-hybridization, indicating that sensitivity of PCR methods can vary according to the biological sample examined. Our results showed that CS was suitable to detect Leishmania DNA in asymptomatic animals when comparing to other low-invasive samples. The CS sensitivities obtained in this study were similar to the ones observed in other studies for VL diagnosis in symptomatic dogs. We concluded that the use of CS for regular screenings of dogs by PCR should be considered.

Background We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively. Methodology/Principal Findings Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti- Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups. Conclusions The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.

Recebido em 22/1/04; aceito em 13/8/04; publicado na web em 2/2/05 BISPHOSPHONATES: SYNTHESIS, CHEMICAL ANALYSIS AND PHARMACOLOGICAL APPLICATIONS. Bisphosphonates are drugs that have been widely used in different bone diseases, and have recently been used successfully against many parasites. Various synthetic routes to prepare different types of bisphosphonates have been described, with distinct potency and pharmacological activity. A number of analytical techniques are currently being used to analyze these drugs; among these, the high performance liquid chromatography (HPLC), with different systems of detection, is worth highlighting. However, the development of more sensitive methods is still necessary, once they are essential for bioavailability and bioequivalence studies. This paper reports the major synthesis routes, chemical analysis methodologies and pharmacological applications of bisphosphonates.Keywords: bisphosphonates; bone diseases; high performance liquid chromatography. INTRODUÇÃOOs bisfosfonatos (BP's) formam uma classe de substâncias quí-micas que apresenta uma ligação P-C-P em sua estrutura, e agem como inibidores da reabsorção óssea, mediada pelos osteoclastos. Estes compostos são extensivamente utilizados no tratamento de várias doenças ósseas, destacando-se a doença de Paget, a hipercalcemia maligna, a osteoporose e a doença metastática e osteolítica.São análogos químicos da substância endógena denominada ácido pirofosfórico (Figura 1), que no organismo se encontra como pirofosfato, um inibidor natural da reabsorção óssea. No entanto, essa substância não pode ser utilizada como agente terapêutico no tratamento de doenças ósseas, pois sofre uma rápida hidrólise enzimática. Os bisfosfonatos são seus análogos sintéticos, onde o átomo central de oxigênio é substituído por um de carbono. Essa modificação faz com que os BP's sejam mais resistentes à degradação enzimática, e possuam uma meia-vida biológica maior, suficiente para influenciar o metabolismo ósseo 1 .Diferentes substituintes ligados ao carbono central dão características únicas para cada fármaco (Figura 2). O grupo R1 fornece a afinidade dos BP's pelos cristais ósseos, enquanto o grupo R2 é responsável pela potência e atividade farmacológicas 2 . Segundo Shinoda 3 , a adição de um grupo hidroxila ao átomo de carbono na posição 1 aumenta a potência anti-reabsortiva. Derivados com um grupo amino no final da cadeia lateral são extremamente ativos. Além disso, o comprimento da cadeia lateral é também muito importante, sendo a maior atividade encontrada em compostos com quatro carbonos, como o alendronato. Mais recentemente tem sido demonstrado que o grupo amino não tem que estar localizado necessariamente no fim da cadeia. No entanto, ainda não está bem clara a relação existente entre estrutura e atividade, podendo-se dizer apenas que a estrutura P-C-P é fundamental para a atividade e que a intensidade do efeito é intensamente dependente da cadeia lateral. Os BP's são sintetizados por meio de uma grande variedade de rotas sintéticas. Estas...

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The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil once the dog is the main reservoir host of the disease. The aim of this study was to evaluate the conjunctival swab (CS) as a mass-screening tool for CVL molecular diagnosis in an endemic area classified as priority for the Brazilian Ministry of Healthy for surveillance action. A total of 1350 domiciled dogs were screened. The animals were evaluated by serological tests (enzyme-linked immunosorbent assay (ELISA) as screening and immunofluorescence antibody test (IFAT) for confirmation) and by CS associated to real-time PCR, using primers addressed to kinetoplast DNA (kDNA) minicircles and SYBR Green. Canine β-globin gene amplification was used to evaluate the sample DNA integrity. A subgroup of 484 animals was also submitted to clinical evaluation. Among the 1350 dogs screened, 369 (27.3%) were positive by CS real-time PCR and 126 (9.3%) tested positive by ELISA. Thirty-one percent (39/126) of the ELISA-positive dogs were confirmed by IFAT. CS real-time PCR was able to detect infection in dogs independently of the symptomatology degree (p > 0.05), while ELISA was more sensitive in the group of dogs that present three or more clinical signs related to CVL. The results demonstrated that CS real-time PCR was able to detect a higher number of infected dogs than ELISA and that the prevalence of canine infections has been underestimated by the serological assays. The use of sensitive molecular diagnostic methods like CS real-time PCR, mainly in endemic areas, could greatly contribute to disease control.

Rapid methods for etidronate, clodronate, pamidronate, and alendronate assays are presented. The methods are based on ion chromatography with indirect UV detection, which avoids the need for chemical derivatization procedures. Each compound is analyzed on an individual basis. There is no need for having separation among these analytes because the aim of the proposed methods is to analyze each compound separately either in bulk material or pharmaceuticals. Phenosphere (150 x 2.0-mm, 5 microm) and Sphereclone (250 x 2.0-mm, 5 microm) anion exchange columns were employed with sodium citrate (20mM) as the mobile phase. The methods are simple, rapid (analysis time of 5 min for etidronate and clodronate and 7 min for alendronate and pamidronate), and demonstrate precision (relative standard deviation was lower than 2.0% in all concentrations), accuracy, and specificity. Calibration curves are linear with r(2) > 0.99 over the concentration range of 50 to 400 microg/mL for etidronate and clodronate, and of 100 to 500 microg/mL for pamidronate and alendronate. Furthermore, they employed silica-based columns, which are cheaper than polymeric columns frequently used in previous reported methods.