Comparative analyses of whole genome sequences of Leishmania infantum isolates from humans and dogs in northeastern Brazil.

The genomic sequences of 20 Leishmania infantum isolates collected in northeastern Brazil were compared with each other and with the available genomic sequences of 29 L. infantum/donovani isolates from Nepal and Turkey. The Brazilian isolates were obtained in the early 1990s or since 2009 from patients with visceral or non-ulcerating cutaneous leishmaniasis, asymptomatic humans, or dogs with visceral leishmaniasis. Two isolates were from the blood and bone marrow of the same visceral leishmaniasis patient. All 20 genomic sequences display 99.95% identity with each other and slightly less identity with a reference L. infantum genome from a Spanish isolate. Despite the high identity, analysis of individual differences among the 32 million base pair genomes showed sufficient variation to allow the isolates to be clustered based on the primary sequence. A major source of variation detected was in chromosome somy, with only four of the 36 chromosomes being predominantly disomic in all 49 isolates examined. In contrast, chromosome 31 was predominantly tetrasomic/pentasomic, consistent with its regions of synteny on two different disomic chromosomes of Trypanosoma brucei. In the Brazilian isolates, evidence for recombination was detected in 27 of the 36 chromosomes. Clustering analyses suggested two populations, in which two of the five older isolates from the 1990s clustered with a majority of recent isolates. Overall the analyses do not suggest individual sequence variants account for differences in clinical outcome or adaptation to different hosts. For the first known time, DNA of isolates from asymptomatic subjects were sequenced. Of interest, these displayed lower diversity than isolates from symptomatic subjects, an observation that deserves further investigation with additional isolates from asymptomatic subjects.

The emergence of concurrent HIV-1/AIDS and visceral leishmaniasis in Northeast Brazil.

HIV has become increasingly prevalent in the Northeast region of Brazil where Leishmania infantum chagasi is endemic, and concurrent AIDS and visceral leishmaniasis (VL) has emerged. In this study, persons with HIV/AIDS and VL (n=17) had a mean age of 37.3 years (range 29-53 years) compared with 12.5 years (1-80 years) for persons with VL alone (n=2836). Males accounted for 88% of cases with concurrent VL and AIDS and 65% of those with VL alone. The mean CD4 count and antileishmanial antibody titre were lower and recurrence of VL and death were more likely with co-infection. Considering the prevalences of L.i. chagasi and HIV in the region, this may herald the emergence of an important public health problem.

Atypical American visceral leishmaniasis caused by disseminated Leishmania amazonensis infection presenting with hepatitis and adenopathy.

Leishmania amazonensis is widely recognised as a cause of cutaneous leishmaniasis in Latin America, but it can also disseminate to produce atypical visceral leishmaniasis with hepatitis and lymphadenopathy. The patient, an 8-year-old Brazilian boy, presented with a febrile illness and hepatosplenomegaly, elevated liver enzymes and generalised adenopathy. Serological tests using antigens of L. chagasi, the typical cause of visceral leishmaniasis in Latin America, were inconclusive. Leishmania amazonensis was eventually isolated in a culture of a lymph node. The patient recovered fully after treatment with meglumine antimoniate. As this case illustrates, L. amazonensis produces a spectrum of disease that includes atypical American visceral leishmaniasis with evidence of hepatocellular injury and generalised lymphadenopathy.

Identification and molecular characterisation of a peritrophin gene, peritrophin-48, from the myiasis fly Chrysomya bezziana.

The peritrophic matrix lines the midgut of most insects and has important roles in digestion, protection of the midgut from mechanical damage and invasion by micro-organisms. Although a few intrinsic peritrophic matrix proteins have been characterised, no direct homologues of any of these proteins have been found in other insect species, even closely related species, suggesting that the peritrophic matrix proteins show considerable sequence divergence. We now report the identification of the cDNA and genomic DNA sequences of a Chrysomya bezziana homologue of the Lucilia cuprina intrinsic peritrophic matrix protein, peritrophin-48. The gene for C. bezziana peritrophin-48 spans 1315 bp and consists of three exons (65, 560 and 690 bp, respectively) separated by introns of 566 and 72 bp. The transcriptional start site, identified by a consensus of cDNA clones and primer extension analysis, is probably located 58 bp upstream from the start codon. However, there may be multiple start sites for transcription. Two potential TATA boxes and a consensus arthropod transcription initiator are located within 134 bp of sequence upstream of the putative transcriptional start site suggesting that this region contains the gene promoter. Immuno-fluorescence localization demonstrated that C. bezziana peritrophin-48 was localised to the larval peritrophic matrix. Protein fold recognition analysis indicated structural similarities between peritrophin-48 and wheatgerm lectin. As wheatgerm lectin binds chitin, this result suggested that C. bezziana peritrophin-48 may also bind chitin, a constituent of the peritrophic matrix. Chitin binding studies with a recombinant peritrophin-48 protein confirmed that it binds chitin. A Drosophila melanogaster homologue of peritrophin-48 encoded in an EST and a genomic sequence was also identified. The pairwise percentage identities of the deduced amino acid sequences for the peritrophin-48 homologues from the three higher Dipteran species were relatively low, ranging between 32 and 42%. Despite this sequence variability, the predicted structure of these proteins, dictated by five domains, each containing a characteristic distribution of six cysteines, was strictly conserved. It is concluded that considerable sequence variation can be tolerated in this protein because of the constraints imposed on the structure of the protein by an extensive disulphide bonded framework.

Cerebral malaria and developmental biology: making parasites grow-up.

Erythrocytes infected with the protozoan parasite Plasmodium falciparum, in the final stages of asexual development, sequester and adhere to the lining of capillaries in the brain; killing 1-3 million (mostly children under the age of 5) each year. I will argue here that the rapidly evolving tools of developmental biology be employed to find a way of inducing gametocytogenesis, thereby making the parasite 'grow-up' prematurely, and in large numbers alleviating the severe symptoms in the brain.

Delusions of intestinal parasitosis.

Delusions of parasitosis, though uncommon, are an important cause of distress for affected patients and frequently of frustration for their physicians. They occur primarily in middle-aged or older women, who have the delusional belief that they are infested with parasites. Although the vast majority of cases involve dermatologic manifestations, some patients may have delusions of intestinal infection, as illustrated by this case.

Insect chitin synthase cDNA sequence, gene organization and expression.

Chitin is a major component of the cuticle of arthropods. However, the synthesis of chitin is poorly understood. Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration-dependent mortality of the larvae (LD50 = 280 nM). This result demonstrates that chitin is an essential component of this insect. The complete cDNA and deduced amino-acid sequences of the first arthropod chitin synthase-like protein, LcCS-1, from the larvae of the insect L. cuprina have been determined. The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717). Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes. Strikingly, LcCS-1 contains 15-18 potential transmembrane segments, indicating that the protein is an integral membrane protein. Two alternative topographical models of LcCS-1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles. LcCS-1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea. In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument. cDNA or genomic sequences that are highly related to fragments of LcCS-1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin-producing organisms. Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS-1, DmCS-1.

Distribution of phlebotomine sand flies (Diptera: Psychodidae) in the state of Rio Grande do Norte, Brazil.

Visceral and cutaneous leishmaniasis are major endemic diseases in northeast Brazil. The objective of the current study was to determine the species and geographic distribution of potential sand fly vectors of Leishmania in the state of Rio Grande do Norte. Sand flies were captured using CDC light traps in 30 municipalities distributed throughout the 8 geographic zones of the state. Twelve Lutzomyia species were identified. Lutzomyia longipalpis Lutz & Neiva was the most prevalent and accounted for 85.59% of the sand fly captured. The remaining species were distributed as follows: L. evandroi Costa Lima & Antunes (10.83%), L. oswaldoi Mangabeira (0.99%), L. sallesi Galvão & Coutinho (0.58%), L. intermedia Lutz & Neiva (0.53%), L. lenti Mangabeira (0.53%), L. migonei França (0.49%), L. walkeri Newstead (0.24%), L. goiana Martins, Falcão & Silva (0.15%), L. samueli Deane (0.04%), and L. capixaba Dias, Falcão, Silva & Martins (0.03%), and L. peresi Mangabeira (0.01%). L. longipalpis, which is known to be a vector of Leishmania chagasi Cunha & Chagas (L. donovani chagasi), was captured in 93% of municipalities distributed across all geographical areas of the state and its distribution was independent of obvious climatic and topographic parameters. It was identified in all municipalities where human visceral leishmaniasis had been reported. In contrast, climate and topography appeared to be important for other Lutzomyia species. For example, L. intermedia and L. migonei, which are known to transmit Leishmania braziliensis Viana, were geographically restricted. They were captured in municipalities where cases of cutaneous and mucosal leishmaniasis had been reported. The widespread distribution of L. longipalpis, its adaptation to peridomicillary settings, and its ability to transmit L. (d.) chagasi suggest that a large number of persons may be at risk of acquiring visceral leishmaniasis in the state of Rio Grande do Norte, Brazil.

Risk of needle stick and sharp object injuries among medical students.

BACKGROUND: Much is known about sharp object and needle stick injuries among employee health care workers, but relatively little attention has been directed to exposures among medical students. METHOD: The frequency and mechanisms of needle stick and sharp object injuries were determined retrospectively by surveying students in their fourth year of medical school. Students were questioned about the number of percutaneous injuries that they had sustained during their clinical years. Descriptive information was collected on their most recent injury. RESULTS: Of 137 students in the class, 106 (77%) responded. Thirty-five (33%) of the students who responded sustained one or more injuries; 24 (69%) were injured while on a surgical service, and 60% of the injuries occurred in an operating room. Suturing was the procedure most frequently associated with injury. In 34% of cases, the injury was caused by a needle or device being used by another person. The most frequent site of injury was the hand (97%). Ninety-four percent of students were wearing gloves at the time of the injury. None of the injuries was associated with recapping needles. Only 43% of students reported their injuries to proper authorities. CONCLUSION: Medical students frequently sustain needle stick and sharp object injuries during their clinical training. Concerted efforts are needed to protect them.

Molecular cloning and expression in Escherichia coli of an aquaporin-like gene from adult buffalo fly (Haematobia irritans exigua).

A gene fragment encoding a putative member of the aquaporin gene family was amplified using cDNA prepared from unfed adult buffalo fly poly(A)+ RNA and degenerate PCR primers designed from highly conserved regions of amino acids found in all members of the aquaporin gene family. This PCR product was labelled with digoxigenin-dUTP and used as a probe to screen a lambdagt-11 cDNA library constructed from unfed adult buffalo fly. One positively hybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide of predicted Mr = 26 163 Da. Comparison of the AqpBF1 deduced protein sequence with the GenBank database revealed significant homology to many aquaporin genes, including 72% identity with a partial DNA sequence encoding a member (DRIP) of the MIP protein family isolated from Drosophila melanogaster. The most closely related, full-length, GenBank sequence was an aquaporin gene isolated from the digestive tract of the sap-sucking insect Cicadella viridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence. The full-length coding sequence of AqpBF1 was cloned into the (His)6-fusion vector, pQE10, and the recombinant protein was expressed in Escherichia coli following induction by IPTG. The recombinant (His)6-fusion protein was localized predominantly in the membrane fraction of E. coli. The protein was solubilized from E. coli membranes with n-octyl beta-D-glucopyranoside and purified by affinity chromatography on a Ni++-sepharose column in the presence of detergent.

Infected physicians and invasive procedures: national policy and legal reality.

Recent reports of the transmission of hepatitis B, hepatitis C, and HIV from physicians to patients during invasive procedures have again raised the question of whether physicians infected with bloodborne pathogens should perform invasive procedures that place patients at risk, and if so, under what conditions. Attempts to formulate a national policy on this subject must consider the competing interests of the patient's welfare versus the physician's livelihood. A review of the legal aspects of this topic is provided to assist policy makers and to serve as a foundation for the recommended establishment of a multidisciplinary committee to develop a uniform national policy. Both legal and medical realities call for the formulation of a clear policy to guide those who must make the decisions on this issue.