From the Cover: A holistic picture of Austronesian migrations revealed by phylogeography of Pacific paper mulberry

The peopling of Remote Oceanic islands by Austronesian speakers is a fascinating and yet contentious part of human prehistory. Linguistic, archaeological, and genetic studies have shown the complex nature of the process in which different components that helped to shape Lapita culture in Near Oceania each have their own unique history. Important evidence points to Taiwan as an Austronesian ancestral homeland with a more distant origin in South China, whereas alternative models favor South China to North Vietnam or a Southeast Asian origin. We test these propositions by studying phylogeography of paper mulberry, a common East Asian tree species introduced and clonally propagated since prehistoric times across the Pacific for making barkcloth, a practical and symbolic component of Austronesian cultures. Using the hypervariable chloroplast ndhF-rpl32 sequences of 604 samples collected from East Asia, Southeast Asia, and Oceanic islands (including 19 historical herbarium specimens from Near and Remote Oceania), 48 haplotypes are detected and haplotype cp-17 is predominant in both Near and Remote Oceania. Because cp-17 has an unambiguous Taiwanese origin and cp-17–carrying Oceanic paper mulberries are clonally propagated, our data concur with expectations of Taiwan as the Austronesian homeland, providing circumstantial support for the “out of Taiwan” hypothesis. Our data also provide insights into the dispersal of paper mulberry from South China “into North Taiwan,” the “out of South China–Indochina” expansion to New Guinea, and the geographic origins of post-European introductions of paper mulberry into Oceania.The peopling of Remote Oceania by Austronesian speakers (hereafter Austronesians) concludes the last stage of Neolithic human expansion (1–3). Understanding from where, when, and how ancestral Austronesians bridged the unprecedentedly broad water gaps of the Pacific is a fascinating and yet contentious subject in anthropology (1–8). Linguistic, archaeological, and genetic studies have demonstrated the complex nature of the process, where different components that helped to shape Lapita culture in Near Oceania each have their own unique history (1–3). Important evidence points to Taiwan as an Austronesian ancestral homeland with a more distant origin in South China (S China) (3, 4, 9–12), whereas alternative models suggest S China to North Vietnam (N Vietnam) (7) or a Southeast Asian (SE Asian) origin based mainly on human genetic data (5). The complexity of the subject is further manifested by models theorizing how different spheres of interaction with Near Oceanic indigenous populations during Austronesian migrations have contributed to the origin of Lapita culture (1–3), ranging from the “Express Train” model, assuming fast migrations from S China/Taiwan to Polynesia with limited interaction (4), to the models of “Slow Boat” (5) or “Voyaging Corridor Triple I,” in which “Intrusion” of slower Austronesian migrations plus the “Integration” with indigenous Near Oceanic cultures had resulted in the “Innovation” of the Lapita cultural complex (2, 13).Human migration entails complex skills of organization and cultural adaptations of migrants or colonizing groups (1, 3). Successful colonization to resource-poor islands in Remote Oceania involved conscious transport of a number of plant and animal species critical for both the physical survival of the settlers and their cultural transmission (14). In the process of Austronesian expansion into Oceania, a number of animals (e.g., chicken, pigs, rats, and dogs) and plant species (e.g., bananas, breadfruit, taro, yam, paper mulberry, etc.), either domesticated or managed, were introduced over time from different source regions (3, 8, 15). Although each of these species has been shown to have a different history (8), all these “commensal” species were totally dependent upon humans for dispersal across major water gaps (6, 8, 16). The continued presence of these species as living populations far outside their native ranges represents legacies of the highly skilled seafaring and navigational abilities of the Austronesian voyagers.Given their close association to and dependence on humans for their dispersal, phylogeographic analyses of these commensal species provide unique insights into the complexities of Austronesian expansion and migrations (6, 8, 17). This “commensal approach,” first used to investigate the transport of the Pacific rat Rattus exulans (6), has also been applied to other intentionally transported animals such as pigs, chickens, and the tree snail Partula hyalina, as well as to organisms transported accidentally, such as the moth skink Lipinia noctua and the bacterial pathogen Helicobacter pylori (see refs. 2, 8 for recent reviews).Ancestors of Polynesian settlers transported and introduced a suite of ∼70 useful plant species into the Pacific, but not all of these reached the most isolated islands (15). Most of the commensal plants, however, appear to have geographic origins on the Sahul Plate rather than being introduced from the Sunda Plate or East Asia (16). For example, Polynesian breadfruit (Artocarpus altilis) appears to have arisen over generations of vegetative propagation and selection from Artocarpus camansi that is found wild in New Guinea (18). Kava (Piper methysticum), cultivated for its sedative and anesthetic properties, is distributed entirely to Oceania, from New Guinea to Hawaii (16). On the other hand, ti (Cordyline fruticosa), also a multifunctional plant in Oceania, has no apparent “native” distribution of its own, although its high morphological diversity in New Guinea suggests its origin there (19). Other plants have a different history, such as sweet potato, which is of South American origin and was first introduced into Oceania in pre-Columbian times and secondarily transported across the Pacific by Portuguese and Spanish voyagers via historically documented routes from the Caribbean and Mexico (17).Of all commensal species introduced to Remote Oceania as part of the “transported landscapes” (1), paper mulberry (Broussonetia papyrifera; also called Wauke in Hawaii) is the only species that has a temperate to subtropical East Asian origin (15, 20, 21). As a wind-pollinated, dioecious tree species with globose syncarps of orange–red juicy drupes dispersed by birds and small mammals, paper mulberry is common in China, Taiwan, and Indochina, growing and often thriving in disturbed habitats (15, 20, 21). Because of its long fiber and ease of preparation, paper mulberry contributed to the invention of papermaking in China in A.D. 105 and continues as a prime source for high-quality paper (20, 21). In A.D. 610, this hardy tree species was introduced to Japan for papermaking (21). Subsequently it was also introduced to Europe and the United States (21). Paper mulberry was introduced to the Philippines for reforestation and fiber production in A.D. 1935 (22). In these introduced ranges, paper mulberry often becomes naturalized and invasive (20–22). In Oceania, linguistic evidence suggests strongly an ancient introduction of paper mulberry (15, 20); its propagation and importance across Remote Oceanic islands were well documented in Captain James Cook’s first voyage as the main material for making barkcloth (15, 20).Barkcloth, generally known as tapa (or kapa in Hawaii), is a nonwoven fabric used by prehistoric Austronesians (15, 21). Since the 19th century, daily uses of barkcloth have declined and were replaced by introduced woven textiles; however, tapa remains culturally important for ritual and ceremony in several Pacific islands such as Tonga, Fiji, Samoa, and the SE Asian island of Sulawesi (23). The symbolic status of barkcloth is also seen in recent revivals of traditional tapa making in several Austronesian cultures such as Taiwan (24) and Hawaii (25). To make tapa, the inner bark is peeled off and the bark pieces are expanded by pounding (20, 21, 23). Many pieces of the bark are assembled and felted together via additional poundings to create large textiles (23). The earliest stone beaters, a distinctive tool used for pounding bark fiber, were excavated in S China from a Late Paleolithic site at Guangxi dating back to ∼8,000 y B.P. (26) and from coastal Neolithic sites in the Pearl River Delta dating back to 7,000 y B.P. (27), providing the earliest known archaeological evidence for barkcloth making. Stone beaters dated to slightly later periods have also been excavated in Taiwan (24), Indochina, and SE Asia, suggesting the diffusion of barkcloth culture to these regions (24, 27). These archaeological findings suggest that barkcloth making was invented by Neolithic Austric-speaking peoples in S China long before Han-Chinese influences, which eventually replaced proto-Austronesian language as well as culture (27).In some regions (e.g., Philippines and Solomon Islands), tapa is made of other species of the mulberry family (Moraceae) such as breadfruit and/or wild fig (Ficus spp.); however, paper mulberry remains the primary source of raw material to produce the softest and finest cloth (20, 23). Before its eradication and extinction from many Pacific islands due to the decline of tapa culture, paper mulberry was widely grown across Pacific islands inhabited by Austronesians (15, 20). Both the literature (15, 20) and our own observations (28–30) indicate that extant paper mulberry populations in Oceania are only found in cultivation or as feral populations in abandoned gardens as on Rapa Nui (Easter Island), with naturalization only known from the Solomon Islands (20). For tapa making, its stems are cut and harvested before flowering, and as a majority of Polynesian-introduced crops (16), paper mulberry is propagated clonally by cuttings or root shoots (15, 20), reducing the possibility of fruiting and dispersal via seeds. The clonal nature of the Oceanic paper mulberry has been shown by the lack of genetic variability in nuclear internal transcribed spacer (ITS) DNA sequences (31). With a few exceptions (30), some authors suggest that only male trees of paper mulberry were introduced to Remote Oceania in prehistoric time (15, 20). Furthermore, because paper mulberry has no close relative in Near and Remote Oceania (20), the absence of sexual reproduction precludes the possibility of introgression and warrants paper mulberry as an ideal commensal species to track Austronesian migrations (6, 30).To increase our understanding of the prehistoric Austronesian expansion and migrations, we tracked geographic origins of Oceanic paper mulberry, the only Polynesian commensal plant likely originating in East Asia, using DNA sequence variation of the maternally inherited (32) and hypervariable (SI Text) chloroplast ndhF-rpl32 intergenic spacer (33). We sampled broadly in East Asia (Taiwan, S China, and Japan) and SE Asia (Indochina, the Philippines, and Sulawesi) as well as Oceanic islands where traditional tapa making is still practiced. Historical herbarium collections (A.D. 1899–1964) of Oceania were also sampled to strengthen inferences regarding geographic origins of Oceanic paper mulberry. The employment of ndhF-rpl32 sequences and expanded sampling greatly increased phylogeographic resolution not attainable in a recent study (31) using nuclear ITS sequences (also see SI Text and Fig. S1) and intersimple sequence repeat (ISSR) markers with much smaller sampling.Open in a separate windowFig. S1.ITS haplotype network (n = 17, A–Q) and haplotype distribution and frequency. The haplotype network was reconstructed using TCS (34), with alignment gaps treated as missing data. The sizes of the circles and pie charts are proportional to the frequency of the haplotype (shown in parentheses). Squares denote unique haplotypes (haplotype found only in one individual).     

Decision-Making at the Intersection of Risk and Pleasure: A Qualitative Inquiry with Trans Women Engaged in Sex Work in Lima,Peru

AIDS and Behavior - To inform culturally relevant HIV prevention interventions, we explore the complexity of sex work among Peruvian transgender women. In 2015, we conducted twenty in-depth...     

Case Report: Possible Vertical Transmission of Bartonella bacilliformis in Peru

A 22-day-old male was admitted with a 2-day history of irritability, dyspnea, jaundice, fever, and gastrointestinal bleeding. A thin blood smear was performed, which showed the presence of intraerythrocyte bacteria identified as Bartonella bacilliformis, and subsequently, the child was diagnosed with Carrion''s disease. The diagnosis was confirmed by specific polymerase chain reaction. The child was born in a non-endemic B. bacilliformis area and had not traveled to such an area before hospitalization. However, the mother was from an endemic B. bacilliformis area, and posterior physical examination showed the presence of a wart compatible with B. bacilliformis in semi-immune subjects. These data support vertical transmission of B. bacilliformis.Bartonella bacilliformis is an endemic pathogen from Andean areas at 600–3,000 m above sea level (masl), and its presentation has two phases. The first phase (acute phase) is the so-called Oroya Fever, in which the pathogen invades the red blood cells, causing severe anemia that may lead to death, especially in the absence of antibiotic treatment. Indeed, mortality rates had reportedly been as high as 90% in the pre-antibiotic era.^1,2 Oroya Fever mostly affects previously non-exposed people.³ Thus, young children are especially affected by this illness.^3,4 In the second phase of the illness, which may occur weeks to months after the acute phase (but may be present in the absence of previously described acute phase symptoms), the bacteria cause an abnormal proliferation of endothelial cells, producing the so-called Peruvian wart.^3,5 Additionally, the presence of healthy carriers, which may act as a natural bacterial reservoir, has also been described.⁶Natural transmission is mediated by a sandfly (different members of the Lutzomyia genera) bite, but vertical and post-transfusion transmissions have also been proposed as possible routes of infection.^7–10In this report, a neonatal case of Oroya Fever from a non-endemic coastal area of Peru is presented, and the possible routes of transmission are discussed.A 22-day-old male child was attended at the Hospital Regional Eleanor Guzman Barron (HREGB; Nuevo Chimbote, Peru) after 2 days of evolution of irritability and breathing difficulty, jaundice, fever, and gastrointestinal bleeding as reported by the mother. The child presented a fever of 38.4°C, and biochemical analysis showed hyperbilirubinemia (total bilirubin: 45.8 mg/dL; conjugate bilirubin: 13 mg/dL), creatinine levels of 1.7 mg/dL, and an erythrocyte count of 12% with hemoglobin levels of 4 g/dL. Despite no reports of B. bacilliformis in the area of the child''s origin, the thin blood smear showed the presence of coccoid (90%) and bacillar (10%) forms, leading to the diagnosis of Oroya Fever. During hospitalization, severe red blood cell hemolysis and digestive hemorrhage made a blood transfusion from the mother necessary because of the lack of a blood bank.Seven days after hospital admission, the child presented worsening clinical evolution, including hepatosplenomegaly and renal insufficiency, leading to his transfer to the Intensive Care Unit of the Instituto Nacional de Salud del Niño (INSN; Lima, Peru), in which an additional pericardic effusion was observed on cardiac echography. After admission to the INSN, the initial diagnosis of Oroya Fever was confirmed by direct blood polymerase chain reaction (PCR) (Figure 1) as previously described and posterior bacterial culture in 5% blood agar plates incubated at 28°C in 5% CO₂.^11,12 The microorganisms were further identified as B. bacilliformis by amplification and sequence of the 16s ribosomal RNA (rRNA) gene both directly from blood samples and from growing microorganisms.¹²Open in a separate windowFigure 1.Direct blood PCR detection of B. bacilliformis. CN = negative control; EC1 = positive control; EC125 = amplification of DNA extraction from neonatal blood; WM = molecular weight marker.After 10 days in the Intensive Care Unit, the child was transferred to the Infectious Diseases Department, in which concomitant pneumonia was also diagnosed.During the stay in the INSN, the child was treated with ciprofloxacin, ceftazidime, ampicillin, and vancomycin following the schedule and dosages presented in Open in a separate windowAntibiotic scheduleIn this case, several severe complications were observed, including pericarditis effusion and digestive hemorrhage as well as pneumonia. These complications have been previously described as relevant complications of severe Oroya Fever, especially in children.¹³A questionnaire given to the mother revealed her origin as Huaraz, an endemic area of Carrion''s disease. Interestingly, during the third trimester of pregnancy, the mother presented a febrile syndrome, which was documented as a urinary tract infection and treated with an unspecified antibiotic plus paracetamol. In the questionnaire, the mother reported the presence of a wart, which was visually compatible with endothelium proliferation of B. bacilliformis, but no additional analyses or molecular determinations were made. These data strongly suggest that, in this case, vertical transmission of Carrion''s disease took place, especially considering the history of the mother, including a personal relationship with an endemic area, a gestational episode of fever, and the presence of a wart, in addition to the fact that the illness incubation time varies from 7 days to several months. Thus, possible external infection can probably be ruled out.¹³Three other methods of transmission should also be considered: natural (sandfly bite), transfusion, or breastfeeding. Nonetheless, although infection transmitted by transfusion could be ruled out in this case, because transfusion was done after the diagnosis of the disease in the neonate, a sandfly bite or breastfeeding transmission could not be ruled out. However, we need to note that this transfusion with maternal blood resulted in an increase in the neonate bacterial burden, playing a role in the aggravation of the clinical presentation, which led to the child''s transfer to the INSN. Regarding the transmission of Bartonella by breastfeeding, to the best of our knowledge, it has not been described to date, but in the absence of specific data, this possibility should be considered. With respect to a sandfly bite as the cause of the episode presented, this is unlikely, because Nuevo Chimbote is on the Peruvian coast, and the presence of these vectors has not been reported in this area (Lutzomyia vectors are located in areas higher than 600 masl). However, stable or unstable introduction of illness transmission vectors, which may result in the development of vector-borne diseases, has been largely described.¹⁴ Thus, possible sporadic introduction of Carrion''s disease vector in the area cannot be discarded. Furthermore, possible visits by relatives from Huaraz, who may act as involuntary vector carriers, should also be considered.Infection by B. bacilliformis during pregnancy has often been related to serious maternal or fetal complications, including miscarriage, fetal death, or pre-term birth among others.¹⁵ Mother-to-child transmission was first proposed by Tomas de Salazar in 1858,⁹ and it was also reported by both Malpartida⁷ and Colareta⁸ in the mid-1930s. Nonetheless, a bibliographic search showed very little data on the vertical transmission of B. bacilliformis, being mainly limited to sporadic cases showing or suggesting this route of transmission. Tuya and others¹⁶ reported the case of a 19-day-old child presenting Oroya Fever with 30% parasitemia, in which the mother also presented a positive thin blood smear. Tarazona and others¹³ reported a pre-term child with a mother presenting verrucous lesions, in which blood samples from the child collected at 90 minutes after birth resulted in a positive Bartonella culture.⁸ Regarding other Bartonella spp., to our knowledge, only perinatal transmission of B. vinsonii ssp. berkhoffii and B. henselae has been reported,¹⁷ which strongly suggests vertical transmission. Additionally, it is of note that vertical transmission of members of the Bartonella genus has been observed in naturally infected rodents.¹⁸This quasiabsence of data regarding the vertical transmission of B. bacilliformis might be because of the lack of reports in article format; also, it could be because the population at risk lives in remote rural areas, in which health facilities have many limitations, including the lack of diagnostic tools other than microscopy, which is strongly expertise-dependent. Social attitudes and practices may also result in delays or non-attendance to health centers during pregnancy or after childbirth. All of these factors may lead to an underestimation or misdiagnosis of mother-to-child B. bacilliformis transmissions.¹⁹ However, early adequate antibiotic treatment of pregnant women with Bartonella infection is effective to avoid or limit both maternal illness complications and fetal/newborn involvement,¹³ and it may, therefore, obviate or diminish vertical transmission.In summary, these data suggest the vertical transmission of B. bacilliformis, reinforcing the need for early detection and treatment of infected pregnant women.     

IL‐27 stimulates human NK‐cell effector functions and primes NK cells for IL‐18 responsiveness

IL‐27, a member of the IL‐12 family of cytokines, is produced by APCs, and displays pro‐ and anti‐inflammatory effects. How IL‐27 affects human NK cells still remains unknown. In this study, we observed that mature DCs secreted IL‐27 and that blockade of IL‐27R (CD130) reduced the amount of IFN‐γ produced by NK cells during their coculture, showing the importance of IL‐27 during DC–NK‐cell crosstalk. Accordingly, human rIL‐27 stimulated IFN‐γ secretion by NK cells in a STAT1‐dependent manner, induced upregulation of CD25 and CD69 on NK cells, and displayed a synergistic effect with IL‐18. Preincubation experiments demonstrated that IL‐27 primed NK cells for IL‐18‐induced IFN‐γ secretion, which was associated with an IL‐27‐driven upregulation of T‐bet expression. Also, IL‐27 triggered NKp46‐dependent NK‐cell‐mediated cytotoxicity against Raji, T‐47D, and HCT116 cells, and IL‐18 enhanced this cytotoxic response. Such NK‐cell‐mediated cytotoxicity involved upregulation of perforin, granule exocytosis, and TRAIL‐mediated cytotoxicity but not Fas‐FasL interaction. Moreover, IL‐27 also potentiated Ab‐dependent cell‐mediated cytotoxicity against mAb‐coated target cells. Taken together, IL‐27 stimulates NK‐cell effector functions, which might be relevant in different physiological and pathological situations.     

Soluble tumor necrosis factor (TNF) receptors (sTNF-R1 and -R2) in pregnant women chronically infected with Trypanosoma cruzi and their children

Most congenital transmissions of Trypanosoma cruzi are not detected. As the levels of mediators regulating the immune response might be different in the absence or in the presence of transmission, we explored the levels of tumor necrosis factor (TNF) and soluble TNF receptors TNF-R1 and -R2 in T. cruzi-infected pregnant women and the neonates. We previously found that the circulating levels of TNF were higher in non-transmitting than in transmitting pregnant women. This observation has now been extended to the spontaneous release of TNF by peripheral blood leukocytes (PBLs) that was also higher in non-transmitting than in transmitting pregnant women. As their mothers, non-infected neonates had higher circulating levels of TNF than congenitally infected children. The circulating levels of sTNF-R1 increased in non-transmitting and transmitting mothers and in infected and non-infected neonates. The circulating levels of sTNF-R2 were approximately 60% higher in infected than in non-infected neonates (1,635 +/- 101 and 1,027 +/- 100 pg/mL, respectively) and remained higher at 1 year of age. This important increase, only observed in infected neonates, could be useful to orientate to the presence of vertical transmission of T. cruzi infection.     

Withholding or withdrawing of life-sustaining therapy in older adults (≥ 80 years) admitted to the intensive care unit

Purpose

To document and analyse the decision to withhold or withdraw life-sustaining treatment (LST) in a population of very old patients admitted to the ICU.

Methods

This prospective study included intensive care patients aged?≥?80 years in 309 ICUs from 21 European countries with 30-day mortality follow-up.

Results

LST limitation was identified in 1356/5021 (27.2%) of patients: 15% had a withholding decision and 12.2% a withdrawal decision (including those with a previous withholding decision). Patients with LST limitation were older, more frail, more severely ill and less frequently electively admitted. Patients with withdrawal of LST were more frequently male and had a longer ICU length of stay. The ICU and 30-day mortality were, respectively, 29.1 and 53.1% in the withholding group and 82.2% and 93.1% in the withdrawal group. LST was less frequently limited in eastern and southern European countries than in northern Europe. The patient-independent factors associated with LST limitation were: acute ICU admission (OR 5.77, 95% CI 4.32–7.7), Clinical Frailty Scale (CFS) score (OR 2.08, 95% CI 1.78–2.42), increased age (each 5 years of increase in age had a OR of 1.22 (95% CI 1.12–1.34) and SOFA score [OR of 1.07 (95% CI 1.05–1.09 per point)]. The frequency of LST limitation was higher in countries with high GDP and was lower in religious countries.

Conclusions

The most important patient variables associated with the instigation of LST limitation were acute admission, frailty, age, admission SOFA score and country.

Trial registration

ClinicalTrials.gov (ID: NTC03134807).

     

Temporal Fluctuation of Infection with Different Trypanosoma cruzi Genotypes in the Wild Rodent Octodon degus

We identified and followed-up for two years Octodon degus rodents infected with Trypanosoma cruzi genotypes by using xenodiagnosis with two vector species (Mepraia spinolai and Triatoma infestans), polymerase chain reaction DNA-based detection of insect dejections, Southern blot analysis, and minicircle hybridization with genotype-specific probes. Results show temporal fluctuations of infection with four parasite lineages (TCI, TCII, TCV, and TCVI) in one co-infected O. degus. Results are discussed in the context of parasitemia level and infection control in mammal hosts.Chagas disease is a vector-borne zoonosis caused by the protozoa Trypanosoma cruzi. This taxon had been described as composed of two lineages (TCI and TCII) and five subgroups (IIa–IIe), but a recent study reported six lineages or discrete typing units (DTUs) (T. cruzi I–VI).¹ These lineages are defined as sets of stocks that are genetically more related to each other than to any other stock and are identifiable by common genetic molecular and immunologic markers.²Trypanosoma cruzi populations circulate in nature in multiple T. cruzi genotypes that coexist in different hosts, including Octodon degus rodents.^3–5 After a short acute or primary infection, the mammal host sustains subclinical infections, which are microscopically undetectable in peripheral blood during the undetermined and chronic phases. Conversely, parasitemia in those phases is detected only by polymerase chain reaction (PCR). The classic parasitologic diagnostic method for Chagas disease xenodiagnosis, which can amplify T. cruzi after feeding on infected hosts.⁶ Although xenodiagnosis is specific, it lacks sensitivity and is limited to high levels of parasitemia.⁷ The epidemiology of Chagas disease and clinical symptoms are associated with the infective T. cruzi genotypes.⁸ Therefore, would be useful to know the dynamics of these genotypes.In the present study, we assess the occurrence of temporal fluctuations of T. cruzi DTUs in peripheral blood of two naturally infected wild reservoir specimens of O. degus by using a combination of two diagnosis methods: 1) xenodiagnosis with domestic and sylvatic vectors (Triatoma infectans and Mepraia spinolai), respectively, and 2) PCR DNA-based detection specific for minicircles and hybridization analyses with T. cruzi genotype-specific probes.Ten nymphs (stages II and III) of each vector species were allowed to feed simultaneously on anesthetized O. degus rodents for 30 minutes or until engorgement on the rodent (mean ± SD weight of ingested blood = 0.2 ± 0.05 mg). After 30 days, feces and intestinal contents of the triatomines were observed under a light microscope. The minimal theoretical parasitemia detected under these conditions is approximately 5 parasites/mL (1 parasite/0.2 mL). However, because several but not all insects (2–5) were parasite positive by visual examination, the estimated parasitemia would be > 10–25 parasites/mL.After microscopic inspection, the intestinal contents of each vector species pool was collected and PCR was performed as reported.⁹ Amplicons were subjected to electrophoresis on an agarose gel and transferred to nylon membranes. Copies of these membranes were hybridized separately with each probe under high stringency conditions.³ Construction of genotype-specific probes was performed as described.¹⁰ Different T. cruzi clones were used as templates to generate DNA probes to determine parasite genotypes. The probes were P³²-labeled.⁴A total of 35 O. degus were captured at the field and analyzed. Overall, only two O. degus showed infection with both vector species and six were positive only for M. spinolai.⁹ The two O. degus samples positive for both vector species were subjected to serial xenodiagnosis to determine the genotype of the T. cruzi population circulating at different times: time 0, one year, two years, and two and a half years. Results for O. degus sample 5, which was infected with a one genotype (TCI), are shown in Figure 1. This result was confirmed with both vector species at different times. Results obtained with O. degus sample 8 showed mixed infection with DTUs TCI, TCII, and TCVI at time zero for M. spinolai, but only TCII for T. infestans. However, one year later, both vectors showed mixed infections with lineages TCI and TCV. After two years, both vectors contained only genotype TCII. After two and a half years, vectors were still infected with TCII.Open in a separate windowFigure 1.Hybridization patterns of xenodiagnosis samples from Mepraia spinolai (sp) and Triatoma infestans (i) and staining with ethidium bromide (EB) and Southern blot analyses with specific probes (TCI, TCII, TCV, and TCVI) for xenodiagnosis samples at A, time 0 (i.e., immediately after capture); B, one year later, C, two years later, and D, two and a half years later. Numbers 5 and 18 correspond to identification numbers for Octodon degus rodents.Trypanosoma cruzi colonizes several tissues and evades the immune response by a concomitant low parasitemia level not detectable by several diagnosis methods.¹¹ Parasites circulate as mixed infections. This finding is common for T. cruzi because several mammals and vectors are infected with more than one T. cruzi genotype,^4,5 which results in recombination and hybrid genotypes.⁸We report that infection of rodents can show temporal fluctuations with different T. cruzi genotypes, which is probably the result of fluctuation of relative proportions of parasite loads of different genotypes in peripheral blood. We detected infections in this O. degus with at least three of the four T. cruzi genotypes during the complete follow-up (xenodiagnosis at time 0). Two genotypes (TCII and TCVI) disappeared, and another one (TCV) appeared one year later. During the second year, only one genotype (TCII) was detected and maintained. A different scenario was detected for O. degus sample 5, which showed infection with only TCI during the entire sampling period.In this study, we preferentially detected genotype TCII in both vector species. This genotype was likely circulating at high parasitemia levels in O. degus sample 18 because experimental infections in T. infestans with different T. cruzi DTUs indicated that genotype TCII is transmitted at a low rate; genotype TCI is transmitted at a high rate.¹² Our results for T. cruzi genotypes in these two animals are consistent with local prevalence in the study area.⁴ Recent studies of T. cruzi genotypes circulating in the wild vector in this disease-endemic area showed that TCI and TCII are the most prevalent genotypes.⁵We suggest that both rodent species showed moderate or high levels of parasitemia. We used xenodiagnosis with two triatomine species because insect vectors amplify T. cruzi in the midgut, which enables easy detection. Our results indicate fluctuation in specific genotype infections in a T. cruzi-infected sylvatic rodent.The temporal fluctuation of the four T. cruzi genotypes could be explained by at least two hyptheses that are not mutually exclusive. First, colonization of different tissues with T. cruzi described in patients and experimentally infected animals with organ damage^11,13 releases T. cruzi into the vascular system; these parasites then colonize other tissues. Second, infection is controlled by the immune system. Both processes might reach an equilibrium and explain the low parasitemia levels observed in immunocompetent patients in the chronic phase of Chagas disease. Future parasitologic studies of molecular pathogenesis may be necessary to understand the mechanisms underlying infection control in naturally infected hosts.     

Coexistence of Trypanosoma cruzi genotypes in wild and periodomestic mammals in Chile

Epidemiologic evidence suggests a preferential association of Trypanosoma cruzi genotypes TCI and TCII with marsupials and placental mammals, respectively. We identify T. cruzi genotypes from 117 infected mammals. Minicircle DNA amplified by polymerase chain reaction and hybridization with a panel of four specific probes showed frequencies for the T. cruzi genotypes TCI, TCIIb, TCIId, and TCIIe of 38%, 41%, 26%, and 9%, respectively, in wild mammals. In peridomestic mammals, frequencies for the same clones were 29%, 33%, 43%, and 14%, respectively. As a whole, mixed infections are found in more than 31% of the cases, which indicates the coexistence of multiclonal strains circulating in nature, and the absence of specific associations between T. cruzi genotypes and reservoir hosts, including marsupials. The direct characterization of parasite genotypes emphasizes the importance of obtaining unbiased epidemiologic information from parasite-endemic areas. Results are discussed in the context of competition or facilitation of T. cruzi genotypes within hosts.