Pneumocystis colonisation is common among hospitalised HIV infected patients with non-Pneumocystis pneumonia.

BACKGROUND: When Pneumocystis DNA is recovered from respiratory specimens of patients without Pneumocystis pneumonia (PCP), patients are said to be colonised with Pneumocystis, although the significance of this state is unknown. Understanding risk factors for and outcomes of colonisation may provide insights into the life cycle and transmission dynamics of Pneumocystis jirovecii. METHODS: We performed a cross sectional study of the prevalence and clinical predictors of Pneumocystis colonisation in 172 HIV infected, PCP negative inpatients undergoing diagnostic evaluation of 183 episodes of pneumonia at either the Medical Center of Louisiana at New Orleans between 2003 and 2005 or San Francisco General Hospital between 2000 and 2005. DNA was extracted from sputum and bronchoalveolar lavage specimens and amplified using a nested PCR assay at the mitochondrial large subunit (18S) ribosomal RNA locus. Colonisation was deemed present if Pneumocystis DNA was identified by both gel electrophoresis and direct DNA sequencing. RESULTS: 68% (117/172) of all patients were colonised with Pneumocystis. No strong associations with colonisation were identified for any demographic factors. Among clinical factors, having a CD4+ T cell count

Kinetics of Borrelia burgdorferi infection in larvae of refractory and competent tick vectors.

The acquisition of Borrelia burgdorferi by the larvae of competent and refractory ixodid ticks was assessed by quantitative polymerase chain reaction (PCR). Larvae were fed on infected mice, and the spirochete loads were determined during feeding and up to 93 d postfeeding. Amblyomma americanum (L.) was refractory to B. burgdorferi infection, with almost no detection of spirochete DNA during or postfeeding. In contrast, Ixodes scapularis Say supported high loads of spirochetes (10(3)-10(4) per larva). In Dermacentor variabilis (Say), B. burgdorferi uptake was reduced, with an average of 16 spirochetes per larvae acquired after 4 d of feeding, representing 1/195 of the counts in I. scapularis. However, during the first day postfeeding, the spirochete growth rate in D. variabilis reached 0.076 generations per hour, 7.7 times greater than the highest growth rate detected in I. scapularis. D. variabilis supported intense spirochete growth up to the fourth day postinfection, when the counts increased to an average of 282 spirochetes per larvae or 1/8.5 of the I. scapularis counts 4 d postfeeding. The kinetics of spirochete growth was unstable in D. variabilis compared with I. scapularis, and transmission of B. burgdorferi by D. variabilis could not be demonstrated. A cofeeding experiment indicated that I. scapularis feeding increased A. americanum spirochete uptake. These collective results indicate suboptimal conditions for B. burgdorferi uptake and colonization within A. americanum or the presence of anti-Borrelia factor(s) in this nonpermissive tick species.

Capillary feeding of specific dsRNA induces silencing of the isac gene in nymphal Ixodes scapularis ticks.

Ixodes scapularis transmits several pathogens including Borrelia burgdorferi. Bioactive compounds in tick saliva support tick feeding and influence pathogen transmission to the mammalian host. These studies utilized oral delivery of dsRNA to silence an anticomplement gene (isac) in I. scapularis nymphs. Silencing of isac significantly reduced fed-tick weight compared to delivery of control lacZ dsRNA, and immunoblots specific for FlaB protein indicated a reduction in spirochete load in isac-silenced infected nymphs. SDS-PAGE demonstrated that isac gene silencing affected expression of a number of salivary and non-salivary gland proteins in ticks. Finally, multiple isac cDNA homologues were cloned, and these may represent a new gene family coexpressed during tick feeding. This work presents a novel oral delivery approach for specific gene silencing in I. scapularis nymphs and characterizes the effect of isac on blood-feeding in an attempt to block transmission of B. burgdorferi.

Differential infectivity of the Lyme disease spirochete Borrelia burgdorferi derived from Ixodes scapularis salivary glands and midgut.

Blood fed nymphal Ixodes scapularis Say infected with Borrelia burgdorferi were dissected to obtain salivary gland and midgut extracts. Extracts were inoculated into C3H/HeJ mice, and ear, heart, and bladder were cultured to determine comparative infectivity. Aliquots of extracts were then analyzed by quantitative polymerase chain reaction to determine the number of spirochetes inoculated into mice. A comparative median infectious dose (ID50) was determined for both salivary gland and midgut extract inoculations. Our data demonstrated a statistically significant difference (P < 0.002) in the ID50 derived from salivary gland (average = 18) versus midgut (average = 251) extracts needed to infect susceptible mice. A rationale for the differential infectivity of salivary and midgut derived spirochetes is discussed.

Effect of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of P carinii pneumonia in patients with HIV-1: a prospective study.

BACKGROUND: Investigators have reported that patients infected with Pneumocystis carinii containing mutations in the DHPS (dihydropteroate synthase) gene have a worse outcome than those infected with P carinii containing wild-type DHPS. We investigated patients with HIV-1 infection and P carinii pneumonia to determine if DHPS mutations were associated with poor outcomes in these patients. METHODS: We compared presence of mutations at the DHPS locus with survival and response of patients to co-trimoxazole or other drugs. FINDINGS: For patients initially given co-trimoxazole, nine (14%) of 66 with DHPS mutant died, compared with nine (25%) of 36 with wild type (risk ratio50.55 [95% CI=0.24-1.25]; p=0.15). Ten (15%) of 66 patients with a DHPS mutant did not respond to treatment, compared with 13 (36%) of 36 patients with the wild type (0.42 [0.20-0.86]; p=0.02). For patients aged 40 years or older, four (14%) of 29 with the mutant and nine (56%) of 16 with the wild type died (0.25 [0.09-0.67]; p=0.005). INTERPRETATION: These results, by contrast with those of previous studies, suggest that patients with wild-type P carinii do not have a better outcome than patients with the mutant when given co-trimoxazole. Our results suggest that presence of a DHPS mutation should be only one of several criteria guiding the choice of initial drug treatment of P carinii pneumonia in patients with HIV-1 infection.

Genome size determination and coding capacity of Sodalis glossinidius, an enteric symbiont of tsetse flies, as revealed by hybridization to Escherichia coli gene arrays.

Recent molecular characterization of various microbial genomes has revealed differences in genome size and coding capacity between obligate symbionts and intracellular pathogens versus free-living organisms. Multiple symbiotic microorganisms have evolved with tsetse fly, the vector of African trypanosomes, over long evolutionary times. Although these symbionts are indispensable for tsetse fecundity, the biochemical and molecular basis of their functional significance is unknown. Here, we report on the genomic aspects of the secondary symbiont Sodalis glossinidius. The genome size of Sodalis is approximately 2 Mb. Its DNA is subject to extensive methylation and based on some of its conserved gene sequences has an A+T content of only 45%, compared to the typically AT-rich genomes of endosymbionts. Sodalis also harbors an extrachromosomal plasmid about 134 kb in size. We used a novel approach to gain insight into Sodalis genomic contents, i.e., hybridizing its DNA to macroarrays developed for Escherichia coli, a closely related enteric bacterium. In this analysis we detected 1,800 orthologous genes, corresponding to about 85% of the Sodalis genome. The Sodalis genome has apparently retained its genes for DNA replication, transcription, translation, transport, and the biosynthesis of amino acids, nucleic acids, vitamins, and cofactors. However, many genes involved in energy metabolism and carbon compound assimilation are apparently missing, which may indicate an adaptation to the energy sources available in the only nutrient of the tsetse host, blood. We present gene arrays as a rapid tool for comparative genomics in the absence of whole genome sequence to advance our understanding of closely related bacteria.

Sequence and organization of the mitochondrial genome of the Chagas disease vector, Triatoma dimidiata.

The 17 019 bp mitochondrial genome of Triatoma dimidiata is composed of thirteen protein coding sequences, twenty-two tRNAs, small and large ribosomal units, and a control region. The gene order and orientation are identical to that of Drosophila yakuba. The nucleotide composition is biased toward adenine and thymine (69.5% A + T). The 2.1 kb putative control region, known as the A + T rich region in most insects, has an A + T bias of 66%, but contains a 400 bp sequence that is 77.5% A + T and two other distinct regions: (1) one with a lower A + T bias (60.1%) and (2) a region of eight tandem repeat units. The identified 1.4 kb nuclear copy of mitochondrial sequences encompasses the string of Gs and the beginning of the cytochrome c oxidase 1 gene but lacks the 1.8 kb region spanning the eight tandem repeats and the 5' end of the NADH dehydrogenase subunit II gene.

Molecular tools and triatomine systematics: a public health perspective.

Triatomines, or kissing bugs, are vectors of Chagas disease to humans. This disease is a substantial public health problem affecting up to 12 million people throughout the Americas, and its control relies mainly on the insecticide treatment of triatomine-infested houses within villages. In this article, Fernando Monteiro, Ananias Escalante and Ben Beard review how molecular markers have been used to clarify triatomine systematics, and give examples of how our understanding of triatomine population structure and accurate vector identification can be used to optimize vector control.

Bacterial symbiosis and paratransgenic control of vector-borne Chagas disease.

The triatomine vectors of Chagas disease are obligate haematophagous insects, feeding on vertebrate blood throughout their entire developmental cycle. As a result of obtaining their nutrition from a single food source, their diet is devoid of certain vitamins and nutrients. Consequently, these insects harbour populations of bacterial symbionts within their intestinal tract, which provide the required nutrients that are lacking from their diet. We have isolated and characterised symbiont cultures from various triatomine species and developed a method for genetically transforming them. We can then reintroduce them into their original host species, thereby producing stable paratransgenic insects in which we are able to express heterologous gene products. Using this methodology, we have generated paratransgenic Rhodnius prolixus that are refractory for infection with Trypanosoma cruzi. Two examples of potentially refractory genes are currently being expressed in paratransgenic insects. These include the insect immune peptide cecropin A and active single chain antibody fragments. We have also developed an approach that would allow introduction of genetically modified bacterial symbionts into natural populations of Chagas disease vectors. This approach utilises the coprophagic behaviour of these insects, which is the way in which the symbionts are transmitted among bug populations in nature. The production and ultimate release of transgenic or paratransgenic insects for public health applications is potentially very promising but also worthy of much careful consideration with respect to environmental, political, and human safety concerns.

Pneumocystis carinii cytochrome b mutations are associated with atovaquone exposure in patients with AIDS.

This retrospective cohort study was conducted to determine whether Pneumocystis carinii cytochrome b gene mutations in patients with AIDS and P. carinii pneumonia (PCP) are associated with atovaquone exposure. Portions of the P. carinii cytochrome b genes that were obtained from 60 patients with AIDS and PCP from 6 medical centers between 1995 and 1999 were amplified and sequenced by using polymerase chain reaction. Fifteen patients with previous atovaquone prophylaxis or treatment exposure were matched with 45 patients with no atovaquone exposure. Cytochrome b coenzyme Q binding site mutations were observed in 33% of isolates from patients exposed to atovaquone, compared with 6% from those who were not (P=.018). There was no difference in survival 1 month after treatment between patients with or without cytochrome b mutations (P=.14). Thus, cytochrome b mutations are significantly more common in patients with AIDS and PCP with atovaquone exposure, but the clinical significance of these mutations remains unknown.

Sulfa or sulfone prophylaxis and geographic region predict mutations in the Pneumocystis carinii dihydropteroate synthase gene.

To determine factors associated with mutations in the Pneumocystis carinii dihydropteroate synthase (DHPS) gene, a prospective study of human immunodeficiency virus (HIV)-infected patients with confirmed P. carinii pneumonia was conducted in Atlanta, Seattle, and San Francisco. Clinical information was obtained from patient interview and chart abstraction. DHPS genotype was determined from DNA sequencing. Overall, 76 (68.5%) of 111 patients had a mutant DHPS genotype, including 22 (81.5%) of 27 patients from San Francisco. In multivariate analysis, sulfa or sulfone prophylaxis and study site were independent predictors of a mutant genotype. Fourteen (53.8%) of 26 patients who were newly diagnosed with HIV infection and had never taken prophylaxis had a mutant genotype. The significance of geographic location as a risk factor for mutant genotype and the high proportion of mutant genotypes among persons never prescribed prophylaxis, including those newly diagnosed with HIV infection, provide indirect evidence that these mutations are transmitted from person to person either directly or through a common environmental source.

Molecular epidemiology of metronidazole resistance in a population of Trichomonas vaginalis clinical isolates.

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women, where it may be asymptomatic or cause severe vaginitis and cervicitis. Despite its high prevalence, the genetic variability and drug resistance characteristics of this organism are poorly understood. To address these issues, genetic analyses were performed on 109 clinical isolates using three approaches. First, two internal transcribed spacer (ITS) regions flanking the 5.8S subunit of the ribosomal DNA gene were sequenced. The only variation was a point mutation at nucleotide position 66 of the ITS1 region found in 16 isolates (14.7%). Second, the presence of a 5.5-kb double-stranded RNA T. vaginalis virus (TVV) was assessed. TVV was detected in 55 isolates (50%). Finally, a phylogenetic analysis was performed based on random amplified polymorphic DNA data. The resulting phylogeny indicated at least two distinct lineages that correlate with the presence of TVV. A band-sharing index indicating relatedness was created for different groups of isolates. It demonstrated that isolates harboring the virus are significantly more closely related to each other than to the rest of the population, and it indicated a high level of relatedness among isolates with in vitro metronidazole resistance. This finding is consistent with the hypothesis that drug resistance to T. vaginalis resulted from a single or very few mutational events. Permutation tests and nonparametric analyses showed associations between metronidazole resistance and phylogeny, the ITS mutation, and TVV presence. These results suggest the existence of genetic markers with clinical implications for T. vaginalis infections.

Research ethics. Managing risks of arthropod vector research.

Field research with vectors is an essential aspect of vector biology research and vector-borne disease prevention and control. This type of research, which brings experimental vector manipulations into endemic areas, can present risks to human populations. This paper seeks to stimulate a full discussion within the medical entomology community of the risks associated with vector field research. Such discussions will promote development of a consensus, among investigators, sponsoring agencies and the communities within which the work is done, so that appropriate steps can be taken to minimize and manage the risks, and adequate oversight can be maintained.

Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission.

To study transmission patterns of Pneumocystis carinii pneumonia (PCP) in persons with AIDS, we evaluated P. carinii isolates from patients in five U.S. cities for variation at two independent genetic loci, the mitochondrial large subunit rRNA and dihydropteroate synthase. Fourteen unique multilocus genotypes were observed in 191 isolates that were examined at both loci. Mixed infections, accounting for 17.8% of cases, were associated with primary PCP. Genotype frequency distribution patterns varied by patients' place of diagnosis but not by place of birth. Genetic variation at the two loci suggests three probable characteristics of transmission: that most cases of PCP do not result from infections acquired early in life, that infections are actively acquired from a relatively common source (humans or the environment), and that humans, while not necessarily involved in direct infection of other humans, are nevertheless important in the transmission cycle of P. carinii f. sp. hominis.

Phylogeny and molecular taxonomy of the Rhodniini derived from mitochondrial and nuclear DNA sequences.

Eleven species of Rhodnius and one of Psammolestes were compared by DNA sequence analysis of fragments of the mitochondrial large subunit ribosomal RNA (mtlsurRNA), the mitochondrial cytochrome b (mtCytb), and the D2 variable region of the 28S nuclear RNA (D2), totaling 1,429 base pairs. The inferred phylogeny, using Triatoma infestans as an outgroup, revealed two main clades within the Rhodniini--one, including the prolixus group of species (Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, and Rhodnius nasutus) together with Rhodnius domesticus and Rhodnius neivai, and the other comprising two groups formed by Rhodnius pictipes plus Rhodnius brethesi, and Rhodnius ecuadoriensis plus Rhodnius pallescens. Psammolestes tertius appeared most closely related to the prolixus group. The analysis strongly supports the validity of R. robustus as a species distinct from others of the prolixus group, but suggests higher genetic structuring of R. robustus populations compared to the other species. Although R. robustus has been found naturally infected by Trypanosoma cruzi, the fact that it is apparently entirely sylvatic and unable to establish in homes suggests that it is of no great importance as a Chagas disease vector in humans.