Experimental infections of sand flies and geckos with Leishmania (Sauroleishmania) adleri and Leishmania (S.) hoogstraali.

BACKGROUND: Species belonging to the subgenus Sauroleishmania are parasites of reptiles, and traditionally considered to be non-pathogenic to mammals. Knowledge of the development of these parasites in sand flies and their mechanism of transmission is currently lacking. The main aim of this study was to test the susceptibility of various sand fly species to infection by two Sauroleishmania species, focusing on the localization of parasites in the sand fly intestinal tract. METHODS: The development of Leishmania (Sauroleishmania [S.]) adleri and Leishmania (S.) hoogstraali was studied in six sand fly species (Phlebotomus orientalis, P. argentipes, P. sergenti, P. papatasi, P. duboscqi, Sergentomyia schwetzi). Sand flies were fed through a chick-skin membrane on blood containing Sauroleishmania promastigotes, and they were dissected at various time intervals post blood meal (PBM). Guts were examined microscopically for the presence of parasites, and the intensity and localizations of infections were recorded. Morphological forms of both Sauroleishmania species developing in P. orientalis were analyzed. Experimental infections of geckos using sand fly-derived promastigotes were also performed, and the reptiles were repeatedly examined for Sauroleishmania infection by xenodiagnosis and PCR analysis. RESULTS: High infection rates for both Sauroleishmania species were observed in P. orientalis and P. argentipes, with the parasites migrating anteriorly and undergoing a peripylarian type of development, including colonization of the stomodeal valve. Conversely, the development of L. (S.) adleri in P. sergenti, P. papatasi and Se. schwetzi was restricted to the sand fly hindgut (hypopylarian type of development). Five morphological forms were distinguished for both Sauroleishmania species developing in P. orientalis. All experimentally infected geckos scored negative for Sauroleishmania based on xenodiagnosis and molecular analysis. CONCLUSIONS: The results showed that Sauroleishmania promastigotes can undergo either a peripylarian or hypopylarian type of development in the sand fly intestinal tract, depending on the sand fly species infected. We demonstrated that P. argentipes and P. orientalis, two sand fly species known as permissive vectors for mammalian parasites of subgenus Leishmania, are also highly susceptible to Sauroleishmania as the parasites developed mature late-stage infections, including colonization of the sand fly stomodeal valve. Thus, the role of Phlebotomus sand flies in transmission of Sauroleishmania should be reconsidered and further investigated.

Over-dispersed Trypanosoma cruzi parasite load in sylvatic and domestic mammals and humans from northeastern Argentina.

BACKGROUND: The distribution of parasite load across hosts may modify the transmission dynamics of infectious diseases. Chagas disease is caused by a multi-host protozoan, Trypanosoma cruzi, but the association between host parasitemia and infectiousness to the vector has not been studied in sylvatic mammalian hosts. We quantified T. cruzi parasite load in sylvatic mammals, modeled the association of the parasite load with infectiousness to the vector and compared these results with previous ones for local domestic hosts. METHODS: The bloodstream parasite load in each of 28 naturally infected sylvatic mammals from six species captured in northern Argentina was assessed by quantitative PCR, and its association with infectiousness to the triatomine Triatoma infestans was evaluated, as determined by natural or artificial xenodiagnosis. These results were compared with our previous results for 88 humans, 70 dogs and 13 cats, and the degree of parasite over-dispersion was quantified and non-linear models fitted to data on host infectiousness and bloodstream parasite load. RESULTS: The parasite loads of Didelphis albiventris (white-eared opossum) and Dasypus novemcinctus (nine-banded armadillo) were directly and significantly associated with infectiousness of the host and were up to 190-fold higher than those in domestic hosts. Parasite load was aggregated across host species, as measured by the negative binomial parameter, k, and found to be substantially higher in white-eared opossums, cats, dogs and nine-banded armadillos (range: k = 0.3-0.5) than in humans (k = 5.1). The distribution of bloodstream parasite load closely followed the "80-20 rule" in every host species examined. However, the 20% of human hosts, domestic mammals or sylvatic mammals exhibiting the highest parasite load accounted for 49, 25 and 33% of the infected triatomines, respectively. CONCLUSIONS: Our results support the use of bloodstream parasite load as a proxy of reservoir host competence and individual transmissibility. The over-dispersed distribution of T. cruzi bloodstream load implies the existence of a fraction of highly infectious hosts that could be targeted to improve vector-borne transmission control efforts toward interruption transmission. Combined strategies that decrease the parasitemia and/or host-vector contact with these hosts would disproportionally contribute to T. cruzi transmission control.

Xenodiagnosis in four domestic cats naturally infected by Leishmania infantum.

Leishmaniasis is a neglected tropical disease that continues to pose a serious public health problem. Albeit dogs have long been held as the major reservoirs of Leishmania infantum, the involvement of domestic cats in the zoonotic cycle of visceral leishmaniasis has gained prominence. Here, 240 cats were evaluated by clinical signs and haematological/biochemical changes compatible with leishmaniasis and were diagnosed by serological, molecular, and parasitological techniques. Thus, four cats naturally infected by L. infantum were submitted to xenodiagnosis. A total of 203 females of Lutzomyia longipalpis were subjected to feeding on four cats, with all females completing the blood meal. Parasitological and molecular assays were carried out to evaluate the presence of L. infantum in the sand flies' midgut. Promastigotes were observed in 10 females (6.5%) that fed on one cat, and L. infantum DNA was detected in 17 (8.4%) females that fed on two cats. Our results strengthen the evidence that naturally infected cats are capable of transmitting L. infantum to sand flies.

Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism.

BACKGROUND: Dogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with mild to moderate disease showed significantly higher transmission to the vector than dogs with subclinical or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. CONCLUSIONS/SIGNIFICANCE: Together, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies.

Xenodiagnosis to evaluate the infectiousness of humans to sandflies in an area endemic for visceral leishmaniasis in Bihar, India: a transmission-dynamics study.

BACKGROUND: Visceral leishmaniasis, also known on the Indian subcontinent as kala-azar, is a fatal form of leishmaniasis caused by the protozoan parasite Leishmania donovani and transmitted by the bites of the vector sandfly Phlebotomus argentipes. To achieve and sustain elimination of visceral leishmaniasis, the transmission potential of individuals exposed to L donovani from across the infection spectrum needs to be elucidated. The aim of this study was to evaluate the relative infectiousness to the sandfly vector of patients with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, and individuals with asymptomatic infection. METHODS: In this prospective xenodiagnosis study done in Muzaffarpur district of Bihar, India, we included patients with clinically confirmed active visceral leishmaniasis or post-kala-azar dermal leishmaniasis who presented to the Kala-Azar Medical Research Center. These participants received treatment for L donovani infection. We also included asymptomatic individuals identified through a serosurvey of 17 254 people living in 26 high-transmission clusters. Eligible participants were aged 12-64 years, were HIV negative, and had clinically or serologically confirmed L donovani infection. During xenodiagnosis, the forearms or lower legs of participants were exposed to 30-35 female P argentipes sandflies for 30 min. Blood-engorged flies were held in an environmental cabinet at 28°C and 85% humidity for 60-72 h, after which flies were dissected and evaluated for L donovani infection by microscopy and quantitative PCR (qPCR). The primary endpoint was the proportion of participants with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, as well as asymptomatic individuals, who were infectious to sandflies, with a participant considered infectious if promastigotes were observed in one or more individual flies by microscopy, or if one or more of the pools of flies tested positive by qPCR. FINDINGS: Between July 12, 2016, and March 19, 2019, we recruited 287 individuals, including 77 with active visceral leishmaniasis, 26 with post-kala-azar dermal leishmaniasis, and 184 with asymptomatic infection. Of the patients with active visceral leishmaniasis, 42 (55%) were deemed infectious to sandflies by microscopy and 60 (78%) by qPCR before treatment. No patient with visceral leishmaniasis was found to be infectious by microscopy at 30 days after treatment, although six (8%) were still positive by qPCR. Before treatment, 11 (42%) of 26 patients with post-kala-azar dermal leishmaniasis were deemed infectious to sandflies by microscopy and 23 (88%) by qPCR. Of 23 patients who were available for xenodiagnosis after treatment, one remained infectious to flies by qPCR on the pooled flies, but none remained positive by microscopy. None of the 184 asymptomatic participants were infectious to sandflies. INTERPRETATION: These findings confirm that patients with active visceral leishmaniasis and patients with post-kala-azar dermal leishmaniasis can transmit L donovani to the sandfly vector and suggest that early diagnosis and treatment could effectively remove these individuals as infection reservoirs. An important role for asymptomatic individuals in the maintenance of the transmission cycle is not supported by these data. FUNDING: Bill & Melinda Gates Foundation.

Detection of Trypanosoma cruzi DNA in false negative samples of collected triatomines, xenodiagnosis material, and biopsies of experimentally infected animals.

Direct test over the gut material from triatomine vectors and xenodiagnosis over mammalian hosts are classical techniques for Trypanosoma cruzi parasitological diagnosis. Nevertheless, negative results can be a source of uncertainty. Experimental models have allowed evaluating the tissue invasion of different strains of T. cruzi, but conventional techniques for tissue biopsies involve time-consuming and elaborated procedures and have low sensitivity. Gut material of collected triatomines (microscopically negative) (n = 114), material of mammal xenodiagnoses (microscopically negative) (n = 138), and biopsy material (microscopically negative) from experimentally infected animals (n = 34) with isolates from endemic areas of Chagas' disease from Venezuela were used for DNA extraction and PCR for the amplification of kinetoplast DNA (kDNA) and satellite DNA (sDNA) of T. cruzi. Positive PCR was observed in 53.6% of collected triatomine material, 15.8% of parasitological negative xenodiagnosis material, and 70.6% in biopsies, revealing underestimation by the parasitological tests and the valour of this analysis with preserved material. Anzoátegui was the state with the highest percentage of infection, and the triatomine species Rhodnius prolixus and Panstrongylus geniculatus had the highest percentages of infection. Didelphis marsupialis and Canis familiaris were the most infected by T. cruzi revealed by PCR of xenodiagnosis material. In addition, the PCR technique allowed demonstrating the invasion of T. cruzi in all tissues analyzed, constituting a molecular marker of tissue invasion.

Xenodiagnosis to address key questions in visceral leishmaniasis control and elimination.

Visceral leishmaniasis (VL) remains an important public health issue worldwide causing substantial morbidity and mortality. The Indian subcontinent accounted for up to 90% of the global VL burden in the past but made significant progress during recent years and is now moving towards elimination. However, to achieve and sustain elimination of VL, knowledge gaps on infection reservoirs and transmission need to be addressed urgently. Xenodiagnosis is the most direct way for testing the infectiousness of hosts to the vectors and can be used to investigate the dynamics and epidemiology of Leishmania donovani transmission. There are, however, several logistic and ethical issues with xenodiagnosis that need to be addressed before its application on human subjects. In the current Review, we discuss the critical knowledge gaps in VL transmission and the role of xenodiagnosis in disease transmission dynamics along with its technical challenges. Establishment of state of the art xenodiagnosis facilities is essential for the generation of much needed evidence in the VL elimination initiative.

A case report of Chagas disease in acute phase diagnosed by xenodiagnosis.

Chagas disease is a serious problem of parasitic disease in the American continent, this zoonosis is caused by the flagellated protozoan known as Trypanosoma cruzi and transmitted through trypomastigotes present in the blood of sick hosts or in the faeces of the triatomines. Metacyclic trypomastigotes were detected in the faeca material of Triatoma pallidepennis maintained in the laboratory after the xenodiagnosis study performed on a patient with suspected Chagas disease, which allowed to administer treatment in a timely manner in the acute phase of this disease.

Report of the Fifth Post-Kala-Azar Dermal Leishmaniasis Consortium Meeting, Colombo, Sri Lanka, 14-16 May 2018.

The 5th Post-Kala-Azar Dermal Leishmaniasis (PKDL) Consortium meeting brought together PKDL experts from all endemic areas to review and discuss existing and new data on PKDL. This report summarizes the presentations and discussions and provides the overall conclusions and recommendations.

Human infectiousness and parasite load in chronic patients seropositive for Trypanosoma cruzi in a rural area of the Argentine Chaco.

A key parameter in the transmission of vector-borne infections, including Chagas disease, is the ability of the different host species to transmit the parasite to the vector (infectiousness). Here, we determined infectiousness to the vector of Trypanosoma cruzi-seropositive humans examined by artificial xenodiagnosis (XD), established its relationship with T. cruzi DNA levels (a surrogate of intensity of parasitemia) quantified by real-time PCR (qPCR), and assessed whether infectiousness was associated with the body mass index (BMI), age, ethnic background and parasite genotype. XD was performed to 117 T. cruzi-seropositive residents from Pampa del Indio and parasite load was quantified in 81 of them. Using optical microscopy (OM) 33.6% of the seropositive people tested were infectious and this fraction nearly doubled (66.0%) when XD triatomines were examined by kDNA-PCR. The mean infectiousness (defined as the percentage of all infected triatomines detected by OM at any time point among the total number of insects examined by OM 30 days post-feeding) was 5.2%, and the mean parasite load was 0.51 parasite equivalents per ml. Infectiousness to the vector was associated negatively with age and BMI, and positively with the detection of parasitemia by kDNA-PCR, and parasite load by qPCR in bivariate analysis. Patients with a positive XD by OM exhibited a significantly higher mean parasite load. Using multiple regression, infectiousness was associated with parasite load (positively) and with the household presence of T. infestans and Qom ethnic group (negatively); no significant association was observed with age or its interaction with ethnicity. We did not find significant associations between identified DTUs and infectiousness or parasite load. Infectiousness was aggregated: 18% of the people examined by XD generated 80% of the infected triatomines. Detecting and treating the super-infectious fraction of the infected human would disproportionally impact on domestic transmission risks. Nonetheless, treatment of all eligible infected people who meet the inclusion criteria regardless of their parasitemia should be ensured to improve their prognosis.

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.

Use of miltefosine to treat canine visceral leishmaniasis caused by Leishmania infantum in Brazil.

BACKGROUND: Visceral leishmaniasis (VL) is an infectious disease with a variety of clinical signs. The main form of parasite transmission to humans and other mammalian hosts is through the bite of infected arthropod females with Lutzomyia longipalpis serving as the main vector in the Americas. Dogs are the main urban domestic reservoirs of the parasite and the main source of vector infection due to their high prevalence in endemic areas and the large number of parasites in the skin of infected animals. Although miltefosine has been used in Europe since 2002 for treatment of VL infected dogs, in the Americas the treatment of dogs has not been recommended. Therefore, this study aimed to evaluate efficacy of miltefosine observing a reduction of clinical signs in infected dogs and the infectiveness to the vector by Leishmania (L.) infantum. METHODS: To our knowledge, this is the first controlled study using qPCR and xenodiagnosis to evaluate the efficacy of miltefosine (Milteforan®, Virbac) as a single treatment in Brazil. Thirty-five adult dogs with canine visceral leishmaniasis (CVL), confirmed by clinical and laboratory tests, were included in this study. They received miltefosine at a dose of 2 mg/kg every 24 h for 28 days. The dogs were observed over a three-month period, during which clinical evaluations based on a scoring system were conducted at pre-established times. Parasite load was assessed by cytology and real-time polymerase chain reaction (qPCR). Transmissibility to the vector was evaluated by xenodiagnosis. RESULTS: At the end of the period, the following were observed: (i) the remission of clinical signs with a reduction in clinical scores for 94.2% of the animals; (ii) a statistically significant reduction (98.7%) in parasitic load by qPCR; and (iii) a reduction in infectivity to sand flies. After treatment, 74.2% of the animals remained or had become non-infectious. CONCLUSIONS: Our study indicates that the use of miltefosine administered orally for 4 weeks contributes to a clinical improvement and reduction in infectivity of dogs to L. infantum.

New human isolates of Trypanosoma cruzi confirm the predominance of hybrid lineages in domestic transmission cycle of the Argentinean Chaco.

Trypanosoma cruzi, the etiological agent of Chagas disease, was initially classified into 6 Discrete Typing Units (DTUs). The hybrid DTUs TcV and TcVI are the most frequent in domestic transmission cycles throughout the Southern Cone countries of South America. Here, we genotyped parasite isolates from human residents in Pampa del Indio municipality, Chaco, to further characterize the structure of T. cruzi populations, and to assess the degree of overlapping between the domestic and sylvatic transmission cycles. Artificial xenodiagnostic tests were performed to blood samples from 125 T. cruzi-seropositive people (age range, 3-70 years) who represented 14.3% of all seropositive residents identified. Parasites were obtained from feces of T. cruzi-infected Triatoma infestans examined 30 or 60 days after blood-feeding, and grown in vitro. The cultured parasites were genotyped by means of two PCR-based protocols. DTUs were determined from 39 (31%) patients residing in 28 dwellings. The only DTUs identified were TcV (92%) and TcVI (8-36%). Households with more than one parasite isolate consistently displayed the same DTU. Further sequencing of a fragment of the TcMK gene from selected samples argue against the occurrence of mixed TcV-TcVI infections in the study population. Sequencing data revealed an unexpected degree of genetic variability within TcV including two apparently robust subgroups of isolates. Our results for human residents confirm the predominance of hybrid lineages (TcV and to a much lesser extent TcVI) and the absence of sylvatic genotypes (TcI and TcIII) in (peri)domestic transmission cycles in the Argentinean Chaco area. 245 words.

The Potential Role of Humans in the Transmission Cycle of Leishmania major (Kinetoplastida: Trypanosomatidae), the Causative Agent of the Old World Zoonotic Cutaneous Leishmaniasis.

Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major (Yakimoff and Schokhor, 1914) is known as a neglected tropical disease, transmitted by Phlebotomus papatasi (Scopoli, 1786) in North Africa and the Middle East. The main reservoirs of ZCL are desert gerbils and the role of humans as a reservoir host of the disease is not clearly defined and is therefore investigated in this study. In order to achieve this objective, Ph. papatasi sand flies were allowed to take blood through indirect blood sources (human, Rhombomys opimus (Lichtenstein, 1823) (great gerbil), and BALB/c mice) using artificial feeding or direct xenodiagnoses from ZCL patients. To detect Leishmania promastigotes inside digestive canal, blood-fed sand flies were dissected and examined under light microscope and then confirmed by nested-PCR. In indirect xenodiagnoses, promastigote forms of Leishmania parasite were observed in 7.1%, 12%, and 13.6% of sand flies which fed on Rh. opimus, BALB/c mice, or human blood sources, respectively. In direct xenodiagnoses, among 93 female lab-reared sand flies which fed on active L. major lesion(s), 2.1% Leishmania infection was subsequently observed in the sand flies. No infection was detected in those sand flies that fed on nonlesioned skin of CL patients. Humans can serve as a reservoir of ZCL since the data indicate that Ph. papatasi is able to acquire L. major parasites from an active lesion of ZCL patients and the parasites can complete metacyclogenesis in the sand fly.

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.

Phlebotomine sand fly survey in the focus of leishmaniasis in Madrid, Spain (2012-2014): seasonal dynamics, Leishmania infantum infection rates and blood meal preferences.

BACKGROUND: An unusual increase of human leishmaniasis cases due to Leishmania infantum is occurring in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys have shown that Phlebotomus perniciosus is the only potential vector. Direct xenodiagnosis in hares (Lepus granatensis) and rabbits (Oryctolagus cuniculus) collected in the focus area proved that they can transmit parasites to colonized P. perniciosus. Isolates were characterized as L. infantum. The aim of the present work was to conduct a comprehensive study of sand flies in the outbreak area, with special emphasis on P. perniciosus. METHODS: Entomological surveys were done from June to October 2012-2014 in 4 stations located close to the affected area. Twenty sticky traps (ST) and two CDC light traps (LT) were monthly placed during two consecutive days in every station. LT were replaced every morning. Sand fly infection rates were determined by dissecting females collected with LT. Molecular procedures applied to study blood meal preferences and to detect L. infantum were performed for a better understanding of the epidemiology of the outbreak. RESULTS: A total of 45,127 specimens belonging to 4 sand fly species were collected: P. perniciosus (75.34%), Sergentomyia minuta (24.65%), Phlebotomus sergenti (0.005%) and Phlebotomus papatasi (0.005%). No Phlebotomus ariasi were captured. From 3203 P. perniciosus female dissected, 117 were infected with flagellates (3.7%). Furthermore, 13.31% and 7.78% of blood-fed and unfed female sand flies, respectively, were found infected with L. infantum by PCR. The highest rates of infected P. perniciosus were detected at the end of the transmission periods. Regarding to blood meal preferences, hares and rabbits were preferred, although human, cat and dog blood were also found. CONCLUSIONS: This entomological study highlights the exceptional nature of the Leishmania outbreak occurring in southwestern Madrid, Spain. It is confirmed that P. perniciosus is the only vector in the affected area, with high densities and infection rates. Rabbits and hares were the main blood meal sources of this species. These results reinforce the need for an extensive and permanent surveillance in this region, and others of similar characteristics, in order to control the vector and regulate the populations of wild reservoirs.

Within-host temporal fluctuations of Trypanosoma cruzi discrete typing units: the case of the wild reservoir rodent Octodon degus.

BACKGROUND: Chagas disease caused by Trypanosoma cruzi is considered a major public health problem in America. After an acute phase the disease changes to a chronic phase with very low parasitemia. The parasite presents high genetic variability with seven discrete typing units (DTUs): TcI-TcVI and Tc bat. The aim of this work is to evaluate fluctuation of parasitemia and T. cruzi DTUs in naturally infected Octodon degus. METHODS: After animal capture parasitemia was obtained by qPCR and later the animals were evaluated by three serial xenodiagnoses using two insect vector species, Mepraia spinolai and Triatoma infestans. The parasites amplified over time by insect xenodiagnosis were analyzed by conventional PCR and after that the infective T. cruzi were characterized by means of hybridization tests. RESULTS: The determination of O. degus parasitemia before serial xenodiagnosis by qPCR reveals a great heterogeneity from 1 to 812 parasite equivalents/ml in the blood stream. The T. cruzi DTU composition in 23 analyzed animals by xenodiagnosis oscillated from mixed infections with different DTUs to infections without DTU identification or vice versa, this is equivalent to 50% of the studied animals. Detection of triatomine infection and composition of T. cruzi DTUs was achieved more efficiently 40 days post-infection rather than after 80 or 120 days. CONCLUSION: Trypanosoma cruzi DTUs composition fluctuates over time in naturally infected O. degus. Three replicates of serial xenodiagnosis confirmed that living parasites have been studied. Our results allow us to confirm that M. spinolai and T. infestans are equally competent to maintain T. cruzi DTUs since similar results of infection were obtained after xenodiagnosis procedure.