Chronic Chagas disease: Quantification of Trypanosoma cruzi in peripheral blood and dejections of Triatoma infestans fed by xenodiagnosis in patients with and without cardiopathy.

It is not currently known which individuals with chronic Chagas disease (ChD) will develop cardiopathy in a determined period and which will be maintained asymptomatic with normal routine laboratory tests all their lives. The parasite burden is a factor that could explain this different evolution. The objective of this study was to quantify Trypanosoma cruzi burden by real-time PCR in blood (qPCR-B) and dejections of triatomines fed by xenodiagnosis (qPCR-XD) in 90 individuals with chronic ChD untreated, classified according to XD results and the presence or absence of cardiopathy. All individuals came from hyperendemic areas of Chile and participated in the study under Informed Consent. The standard qPCR curves for qPCR-B and qPCR-XD were elaborated with a mixture of known concentrations of T. cruzi strains, performing DNA serial dilutions (1/10) with a dynamic range between 105 and 10-1 parasite equivalents/mL. The TaqManⓇ detection system was applied in a Stratagene Mx3000P thermocycler (Agilent Technologies, USA) with cruzi 1 and cruzi 2 satellite primers. 22.2% and 15.6% of cases with cardiopathy or without cardiopathy were XD positive. There was no significant difference between the groups. The positivity of qPCR-B and qPCR-XD in the positive XD group was 82.35% and 100%, respectively, while in the negative XD group was 55.26% and 42.10%, respectively. A superior qPCR value in chronic ChD patients with and without cardiopathy was determined for qPCR in cases with positive XD and positive qPCR-XD. The receiver operating characteristic (ROC) curve analyses show better accuracy for detecting parasite burden (area under the curve, AUC) for qPCR-XD in comparison to qPCR-B. That is to say, major performance in DNA samples obtained of positive XD (gold standard for viable T. cruzi) detected and quantified by qPCR-XD. A high percentage of cases with XD and qPCR-XD positive (80-100%) have result concordant with qPCR-B. In absence of XD, future challenges are especially related to the low parasitic load of chronic ChD patients treated with trypanocidal drugs and post-therapy parasitological evaluations by qPCR-B. Finally, no statistically significant differences were found between presence or absence of cardiopathy and XD, qPCR-B or qPCR-XD.

Xenodiagnosis Using Ixodes scapularis Larval Ticks in Humans.

Xenodiagnosis is the use of a natural vector to detect the presence of an organism, and xenodiagnosis using Ixodes ticks has long been used by entomologists in Lyme disease research to provide evidence of the host's infectious status with Borrelia burgdorferi. We developed the methodology and performed the first human research study using uninfected larval Ixodes scapularis ticks to assess evidence of B. burgdorferi infection. Here, we describe in detail the methodology used for the procedure. Xenodiagnosis using Ixodes ticks in humans remains an experimental method and must be performed under an approved clinical research protocol.

Sympatry influence in the interaction of Trypanosoma cruzi with triatomine.

INTRODUCTION: Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely distributed in nature, circulating between triatomine bugs and sylvatic mammals, and has large genetic diversity. Both the vector species and the genetic lineages of T. cruzi present a varied geographical distribution. This study aimed to verify the influence of sympatry in the interaction of T. cruzi with triatomines. Methods: The behavior of the strains PR2256 (T. cruzi II) and AM14 (T. cruzi IV) was studied in Triatoma sordida (TS) and Rhodnius robustus (RR). Eleven fifth-stage nymphs were fed by artificial xenodiagnosis with 5.6 × 103 blood trypomastigotes/0.1mL of each T. cruzi strain. Every 20 days, their excreta were examined for up to 100 days, and every 30 days, the intestinal content was examined for up to 120 days, by parasitological (fresh examination and differential count with Giemsa-stained smears) and molecular (PCR) methods. Rates of infectivity, metacyclogenesis and mortality, and mean number of parasites per insect and of excreted parasites were determined. RESULTS: Sympatric groups RR+AM14 and TS+PR2256 showed higher values of the four parameters, except for mortality rate, which was higher (27.3%) in the TS+AM14 group. General infectivity was 72.7%, which was mainly proven by PCR, showing the following decreasing order: RR+AM14 (100%), TS+PR2256 (81.8%), RR+PR2256 (72.7%) and TS+AM14 (36.4%). CONCLUSIONS: Our working hypothesis was confirmed once higher infectivity and vector capacity (flagellate production and elimination of infective metacyclic forms) were recorded in the groups that contained sympatric T. cruzi lineages and triatomine species.

Sympatry influence in the interaction of Trypanosoma cruzi with triatomine

Abstract INTRODUCTION: Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely distributed in nature, circulating between triatomine bugs and sylvatic mammals, and has large genetic diversity. Both the vector species and the genetic lineages of T. cruzi present a varied geographical distribution. This study aimed to verify the influence of sympatry in the interaction of T. cruzi with triatomines. Methods: The behavior of the strains PR2256 (T. cruzi II) and AM14 (T. cruzi IV) was studied in Triatoma sordida (TS) and Rhodnius robustus (RR). Eleven fifth-stage nymphs were fed by artificial xenodiagnosis with 5.6 × 103 blood trypomastigotes/0.1mL of each T. cruzi strain. Every 20 days, their excreta were examined for up to 100 days, and every 30 days, the intestinal content was examined for up to 120 days, by parasitological (fresh examination and differential count with Giemsa-stained smears) and molecular (PCR) methods. Rates of infectivity, metacyclogenesis and mortality, and mean number of parasites per insect and of excreted parasites were determined. RESULTS: Sympatric groups RR+AM14 and TS+PR2256 showed higher values of the four parameters, except for mortality rate, which was higher (27.3%) in the TS+AM14 group. General infectivity was 72.7%, which was mainly proven by PCR, showing the following decreasing order: RR+AM14 (100%), TS+PR2256 (81.8%), RR+PR2256 (72.7%) and TS+AM14 (36.4%). CONCLUSIONS: Our working hypothesis was confirmed once higher infectivity and vector capacity (flagellate production and elimination of infective metacyclic forms) were recorded in the groups that contained sympatric T. cruzi lineages and triatomine species.

Chronic Chagas cardiopathy in Chile. Importance of Trypanosoma cruzi burden and clinical evaluation.

Currently there are no biological markers to indicate which individuals with chronic indeterminate period of Chagas disease develop heart disease and who will remain all his life in this phase. The aim of this survey was to determine if Trypanosoma cruzi burden is related to the presence of heart disease in patients with chronic Chagas disease. 200 patients who had not been treated, 100 with cardiopathy and 100 without, groups A and B respectively, were submitted to clinical study and electrocardiogram, Echo-Doppler was performed for group A in which all important known causes of cardiopathy were discarded. In both groups xenodiagnosis, conventional PCR and quantitative PCR were undertaken. The 100 cardiopaths had 133 electrocardiographic alterations most of them in grade II of the New York Heart Association classification. 98 cardiopaths were classified in grade I by Echo-Doppler and only 2 cases were in grade III due to low ejection fraction. The difference in average parasitemia in patients of group A and B was not significant and no statistically differences were observed between average parasitemia of cardiopaths grade II versus grade I of NYHA. This results allow to characterize same clinical, electrocardiographical and parasitological features in chagasic cardiopaths of Chile.

Xenodiagnosis of Leishmania donovani in BALB/c mice using Phlebotomus orientalis: a new laboratory model.

BACKGROUND: In areas endemic for visceral leishmaniasis (VL), the majority of infected hosts remain asymptomatic but potentially infectious to biting sand flies. Their infectiousness for sand fly vectors is crucial for the transmission of the disease and can be quantified only by xenodiagnosis. However, in the case of human hosts, xenodiagnosis can be problematic for ethical and logistic reasons. The BALB/c mouse model described in this paper was designed to enable xenodiagnostic studies on VL hosts circumventing the need for human volunteers, it permits xenodiagnosis using the same individual host repeatedly, over several months. METHODS: BALB/c mice were intradermally inoculated in the ear pinnae with Leishmania donovani, primarily metacyclic stages isolated from the thoracic midguts of experimentally-infected Phlebotomus orientalis females. Naïve sand flies were allowed to feed on anaesthetized mice in 1-3-weeks- interval, firstly on the site of inoculation of L. donovani (weeks 2-8 post infection, p.i.), later on the whole body of mice (weeks 9 - 15 p.i.). Infections of sand flies were evaluated microscopically or by PCR analysis. RESULTS: Although infected mice did not show any signs of disease, 19% (N = 876) of the P. orientalis females that fed at the site of inoculation, became infected. The majority of L. donovani-positive females (76%) had heavy infections with their stomodeal valves colonized by attached parasites. Inoculated mouse ears remained infective for sand flies until week 15 p.i. Females feeding on other parts of the body remained negative with exception of two groups feeding on contralateral ears by week 12 p.i. On week 15, however, these two mice returned negative at xenodiagnosis of the contralateral ears. In sacrificed mice, the highest parasite numbers were found in inoculated ears and their draining lymph nodes. Infections were detected also in the spleen, liver, blood and rarely in the contralateral ear. CONCLUSIONS: The study showed that BALB/c mice harbored parasites in sufficient numbers to promote heavy infections in P. orientalis and thus comprised a suitable laboratory model for xenodiagnoses of L. donovani. Parasites persisted in the inoculation site and were found transmissible for months to sand flies biting on the same site.

Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study.

BACKGROUND: Animal studies suggest that Borrelia burgdorferi, the agent of Lyme disease, may persist after antibiotic therapy and can be detected by various means including xenodiagnosis using the natural tick vector (Ixodes scapularis). No convincing evidence exists for the persistence of viable spirochetes after recommended courses of antibiotic therapy in humans. We determined the safety of using I. scapularis larvae for the xenodiagnosis of B. burgdorferi infection in humans. METHODS: Laboratory-reared larval I. scapularis ticks were placed on 36 subjects and allowed to feed to repletion. Ticks were tested for B. burgdorferi by polymerase chain reaction (PCR), culture, and/or isothermal amplification followed by PCR and electrospray ionization mass spectroscopy. In addition, attempts were made to infect immunodeficient mice by tick bite or inoculation of tick contents. Xenodiagnosis was repeated in 7 individuals. RESULTS: Xenodiagnosis was well tolerated with no severe adverse events. The most common adverse event was mild itching at the tick attachment site. Xenodiagnosis was negative in 16 patients with posttreatment Lyme disease syndrome (PTLDS) and/or high C6 antibody levels and in 5 patients after completing antibiotic therapy for erythema migrans. Xenodiagnosis was positive for B. burgdorferi DNA in a patient with erythema migrans early during therapy and in a patient with PTLDS. There is insufficient evidence, however, to conclude that viable spirochetes were present in either patient. CONCLUSIONS: Xenodiagnosis using Ixodes scapularis larvae was safe and well tolerated. Further studies are needed to determine the sensitivity of xenodiagnosis in patients with Lyme disease and the significance of a positive result. Clinical Trials Registration NCT01143558.

Comparison of field-based xenodiagnosis and direct membrane feeding assays for evaluating host infectiousness to malaria vector Anopheles gambiae.

Several techniques are currently being used to study host infectiousness to mosquitoes, including the experimental possibility of laboratory reared mosquitoes acquiring infections through membrane feeders or directly on host skin. Here, the relative performance of the laboratory-based membrane feeding method (DMFA) and the field-based xenodiagnosis (XD) of malaria infectious hosts using wild Anopheles mosquitoes were compared. A cross-sectional survey involving a sample of 70 children (aged 3-12 years) living in a malaria endemic area in Western Burkina Faso, was carried out to measure their infectiousness to Anopheles mosquitoes using two approaches. The first approach used the xenodiagnostic procedure in which children were exposed to mosquito bites overnight, being sleeping individually in different sentinel huts from 6 pm to 6 am (4 nights per child). Anopheles sp that had acquired blood-meal on each child were subsequently collected early in the morning, and examined for Plasmodium falciparum oocyst infection on day 7 post-feeding. In the second approach, the infectiousness of the same children was estimated by whole-blood membrane feeding procedure using F0 An. gambiae s.l. that emerged from field-collected larvae cohorts. In the DMFA, 41.4% of the children successfully infected at least one mosquito with the mean oocyst prevalence of only 4.6±1.1% in the 2171 mosquitoes that were examined (mean oocyst intensity: 2.0±(std error of mean) 0.3 oocysts per infected midgut). Comparatively 78.6% of children yielded oocysts infection in mosquitoes during the XD approach (Chi square=20.11, df=1; p<0.001), with a mean rate of 19.6±2.0 in the 3752 wild caught mosquitoes (mean intensity: 3.93±0.2 oocysts per infected mosquito). The DMFA failed to reveal a portion (n=26) of infectious individuals that were sharply evidenced by the XD, particularly at low gametocyte densities or at levels that could not be detected by the classical microscopic examination of blood smears. As opposed to the resource consuming DMFA, which is often mined by technical constraints, using the XD method could be an advantage in experimental investigations of host infectiousness in areas where anopheline species cannot be conveniently reared for the experimental studies. Ethical aspects of this approach, mainly related to exposure of the human subjects to potentially infectious mosquito bites are discussed.

Chronic Chagas disease: PCR-xenodiagnosis without previous microscopic observation is a useful tool to detect viable Trypanosoma cruzi.

UNLABELLED: We evaluate the elimination of the microscopic stage of conventional xenodiagnosis (XD) to optimize the parasitological diagnosis of Trypanosoma cruzi in chronic Chagas disease. To this purpose we applied under informed consent two XD cages to 150 Chilean chronic chagasic patients. The fecal samples (FS) of the triatomines at 30, 60 and 90 days post feeding were divided into two parts: in one a microscopic search for mobile trypomastigote and/or epimastigote forms was performed. In the other part, DNA extraction-purification for PCR directed to the conserved region of kDNA minicircles of trypanosomes (PCR-XD), without previous microscopic observation was done. An XD was considered positive when at least one mobile T. cruzi parasite in any one of three periods of incubation was observed, whereas PCR-XD was considered positive when the 330 bp band specific for T. cruzi was detected. 25 of 26 cases with positive conventional XD were PCR-XD positive (concordance 96.2%), whereas 85 of 124 cases with negative conventional XD were positive by PCR-XD (68.5%). Human chromosome 12 detected by Real-time PCR used as exogenous internal control of PCR-XD reaction allowed to discounting of PCR inhibition and false negative in 40 cases with negative PCR-XD. CONCLUSION: PCR-XD performed without previous microscopic observation is a useful tool for detection of viable parasites with higher efficiency then conventional XD.

Feeding of ticks on animals for transmission and xenodiagnosis in Lyme disease research.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Estudo da correlação entre a carga parasitária de cães com diferentes apresentações clínicas da leishmaniose visceral e a transmissão ao vetor da leishmania infantum / Study of correlation between parasite load of dogs with different clinical presentations of visceral leishmaniasis and transmission to the vector of Leishmania infantum

No Novo Mundo, a leishmaniose visceral (LV) é causada pela Leishmania infantum, que tem como vetor o inseto flebotomíneo Lutzomyia longipalpis. Os cães são considerados o principal reservatório urbano da infecção. Devido ao fato do controle da LV se basear, principalmente, no controle da leishmaniose visceral canina (LVC), é importante estudar o papel dos cães na transmissão da infecção. Foi demonstrado que cães apresentando diferentes apresentações clínicas da LV, inclusive os assintomáticos transmitem a infecção ao vetor flebotomíneo. Nenhum estudo sistemático avaliou a associação direta entre a carga parasitária em diferentes tecidos e a transmissão do parasito. A hipótese desse estudo é que cães com baixa carga parasitária na pele e no sangue não transmitem a infecção ao vetor flebotomíneo. O objetivo deste estudo foi analisar se há correlação entre a carga parasitária de cães com diferentes apresentações clínicas da LV e a transmissão ao vetor Lutzomyia longipalpis. Foram selecionados 35 cães de dois canis, locazidos em area endêmica (n=23) e não endêmica (n=12) para LV. Os animais foram classificados de acordo com o número de sinais clínicos em: assintomáticos (sem sinais; n=12), oligossintomáticos (1-3 sinais; n=15) e polissintomático (<3 sinais; n =8). Todos os 35 cães foram positivos em pelo menos um dos testes diagnósticos: ELISA (n=8), cultura de aspirado esplênico (n=9) e qPCR (n=35) dos tecidos avaliados. Diferentes tecidos (sangue periférico, aspirado esplênico e biópsia de pele) foram coletados para quantificação do DNA do parasito pela qPCR. Para avaliar a capacidade de transmissão dos cães, foi realizado xenodiagnóstico, seguido de determinação da carga parasitária em cada flebótomo utilizando qPCR. Finalmente, a capacidade de transmissão de Leishmania foi estimada pela determinação, após o xenodiagnóstico, da infectividade de cães ao flebótomo, da taxa de infecção de flebótomos, e da carga parasitária transmitida aos flebótomos. Baixa carga parasitária na pele e no sangue foi detectada em aproximadamente 85% dos cães assintomáticos. A infectividade de cães ao flebótomo variou de 60 a 90%, e foi similar entre animais apresentando diferentes números de sinais clínicos. Foi identificado que o maior percentual (51%) de cães transmite parasitos a um pequeno número de flebótomos (de 1 a 5 em 30 flebótomos utilizados no xenodiagnóstico). Entre os tecidos analisados, correlação positiva foi detectada entre a infectividade de cães ao vetor e a carga parasitária nas amostras de sangue (r = 0.50, p<0.01). Adicionalmente, foi observada, correlação positiva entre menor taxa de infecção dos flebótomos e baixa carga parasitária no sangue (r = 0.53, p<0.01). Em conjunto, estes dados mostram que cães com baixa carga parasitária são capazes de transmitir o parasito, porém a um pequeno número de flebótomos e com uma baixa carga parasitária.

Chronic Chagas disease: PCR-xenodiagnosis without previous microscopic observation is a useful tool to detect viable Trypanosoma cruzi

We evaluate the elimination of the microscopic stage of conventional xenodiagnosis (XD) to optimize the parasitological diagnosis of Trypanosoma cruzi in chronic Chagas disease. To this purpose we applied under informed consent two XD cages to 150 Chilean chronic chagasic patients. The fecal samples (FS) of the triatomines at 30, 60 and 90 days post feeding were divided into two parts: in one a microscopic search for mobile trypomastigote and/or epimastigote forms was performed. In the other part, DNA extraction-purification for PCR directed to the conserved region of kDNA minicircles of trypanosomes (PCR-XD), without previous microscopic observation was done. An XD was considered positive when at least one mobile T. cruzi parasite in any one of three periods of incubation was observed, whereas PCR-XD was considered positive when the 330 bp band specific for T. cruzi was detected. 25 of 26 cases with positive conventional XD were PCR-XD positive (concordance 96.2%), whereas 85 of 124 cases with negative conventional XD were positive by PCR-XD (68.5%). Human chromosome 12 detected by Real-time PCR used as exogenous internal control of PCR-XD reaction allowed to discounting of PCR inhibition and false negative in 40 cases with negative PCR-XD. Conclusion: PCR-XD performed without previous microscopic observation is a useful tool for detection of viable parasites with higher efficiency then conventional XD.

Real-Time PCR in faecal samples of Triatoma infestans obtained by xenodiagnosis: proposal for an exogenous internal control.

BACKGROUND: The polymerase chain reaction (PCR) has proved to be a sensitive technique to detect Trypanosoma cruzi in the chronic phase of Chagas disease, which is characterized by low and fluctuating parasitemia. Another technique proposed for parasitological diagnosis in this phase of infection combines a microscopic search for motile trypomastigote forms in faecal samples (FS) obtained by xenodiagnosis (XD) with conventional PCR (XD-PCR). In this study we evaluate the use of human blood DNA as an exogenous internal control (EIC) for real time PCR (qPCR) combined with XD (XD-qPCR) using chromosome 12 (X12) detection. FINDINGS: None of the FS-XD evaluated by qPCR amplified for X12. Nevertheless, all the EIC-FS-XD mixtures amplified for X12. CONCLUSIONS: We determined that X12 is useful as an EIC for XD-qPCR because we showed that the FS-XD does not contain human DNA after 30 or more days of XD incubation. This information is relevant for research on T. cruzi by XD-qPCR since it allows ruling out inhibition and false negative results due to DNA loss during the process of extraction and purification.

Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis.

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺, led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.

Detection of Trypanosoma cruzi in untreated chronic chagasic patients is improved by using three parasitological methods simultaneously.

OBJECTIVES: This study compared three parasitological methods applied simultaneously in individuals with untreated chronic Chagas' disease in order to determine their individual and combined performances. METHODS: From a total of 100 chronic chagasic patients from endemic areas of Chile, with informed consent, we extracted 2 mL of peripheral venous blood for PCR (PCR-B) and applied two xenodiagnosis (XD) boxes with seven uninfected Triatoma infestans nymphs each for microscopic examination and PCR of faecal samples of the triatomines fed on each patient (PCR-XD). The PCR-B and PCR-XD reactions were performed with oligonucleotides 121 and 122, which anneal to the four constant regions of the minicircles of Trypanosoma cruzi kinetoplasts. The 330 bp PCR product was analysed by electrophoresis in a 2% agarose gel and visualized by staining with ethidium bromide. RESULTS: PCR-B detected T. cruzi in 58% of the cases, while PCR-XD proved to be more sensitive than XD (67% versus 14%, respectively) (P = 0.0001). There was no difference between the detection power of PCR-B and PCR-XD (P = 0.222). The percentage detected as positive was much greater when the three tests were considered (84%) (P = 0.00001). CONCLUSIONS: The simultaneous application of more than one technique for the parasitological diagnosis of Chagas' disease in untreated individuals increases the possibility of detection of T. cruzi.

A qPCR-based multiplex assay for the detection of Wuchereria bancrofti, Plasmodium falciparum and Plasmodium vivax DNA.

The purpose of this study was to develop real-time multiplex quantitative PCR (qPCR) assays for the simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes, estimated by PoolScreen 2 software, for Wb, Pf and Pv were 4.9%, 2.7% and 2.1%, respectively. Parasite DNA rates were consistently higher in blood-fed mosquitoes than in host-seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases.

Persistence of Borrelia burgdorferi following antibiotic treatment in mice.

The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis. Mice were treated with ceftriaxone or saline solution for 1 month, commencing during the early (3 weeks) or chronic (4 months) stages of infection with Borrelia burgdorferi. Tissues from mice were tested for infection by culture, PCR, xenodiagnosis, and transplantation of allografts at 1 and 3 months after completion of treatment. In addition, tissues were examined for the presence of spirochetes by immunohistochemistry. In contrast to saline solution-treated mice, mice treated with antibiotic were consistently culture negative, but tissues from some of the mice remained PCR positive, and spirochetes could be visualized in collagen-rich tissues. Furthermore, when some of the antibiotic-treated mice were fed on by Ixodes scapularis ticks (xenodiagnosis), spirochetes were acquired by the ticks, as determined based upon PCR results, and ticks from those cohorts transmitted spirochetes to naïve SCID mice, which became PCR positive but culture negative. Results indicated that following antibiotic treatment, mice remained infected with nondividing but infectious spirochetes, particularly when antibiotic treatment was commenced during the chronic stage of infection.

Xenodiagnosis: use of mosquitoes for the diagnosis of arboviral infections.

The arboviruses have a worldwide distribution and, mosquitoes and ticks contribute principally in their transmission. In the last two decades, arboviral diseases have been recognised due to their resurgence and spread in newer geographic areas. Surveys to determine the prevalence of arboviruses in any region largely depend on the isolation attempts from the arthropods along with the serosurveys. Xenodiagnosis means use of insects for the diagnosis of infectious diseases affecting human being. The present communication discusses the application of mosquitoes for propagation and assays of arboviruses, the technique of mosquito inoculation and importance of xenodiagnosis.

The PCR-based detection of Trypanosoma cruzi in the faeces of Triatoma infestans fed on patients with chronic American trypanosomiasis gives higher sensitivity and a quicker result than routine xenodiagnosis.

In the xenodiagnosis (XD) of American trypanosomiasis (Chagas disease), Trypanosoma cruzi in the triatomine bugs fed on the patient can now be detected using PCR (XD-PCR) as well as by microscopy (XD-M). In a study to compare XD-PCR with XD-M, triatomine bugs were fed on 50 cases of chronic American trypanosomiasis, of whom only 25 were ever found positive by XD-M. Overall, the bugs fed on 34 of the patients (all 25 cases found positive by XD-M and nine of the other patients) were found PCR-positive, giving a 330-bp fragment corresponding to part of the hyper variable region of the kinetoplast DNA of T. cruzi. Of the 25 patients who were ever found positive by XD-M, 20 gave bugs that were smear-positive on day 90 and a similar number (24; P=0.125) gave bugs that were PCR-positive at this time. On day 30, however, the bugs fed on only 11 of these 25 patients were found positive by microscopy, whereas 23 of these patients were found positive by XD-PCR (P=0.0016). Thus, not only was XD-PCR more sensitive than XD-M but it was also quicker, revealing more cases within 30 days than detected using XD-M over a period of 90 days.