Genetic Markers of Adaptation of Plasmodium falciparum to Transmission by American Vectors Identified in the Genomes of Parasites from Haiti and South America.

The malaria parasite, Plasmodium falciparum, was introduced into Hispaniola and other regions of the Americas through the slave trade spanning the 16th through the 19th centuries. During this period, more than 12 million Africans were brought across the Atlantic to the Caribbean and other regions of the Americas. Since malaria is holoendemic in West Africa, a substantial percentage of these individuals carried the parasite. St. Domingue on Hispaniola, now modern-day Haiti, was a major port of disembarkation, and malaria is still actively transmitted there. We undertook a detailed study of the phylogenetics of the Haitian parasites and those from Colombia and Peru utilizing whole-genome sequencing. Principal-component and phylogenetic analyses, based upon single nucleotide polymorphisms (SNPs) in protein coding regions, indicate that, despite the potential for millions of introductions from Africa, the Haitian parasites share an ancestral relationship within a well-supported monophyletic clade with parasites from South America, while belonging to a distinct lineage. This result, in stark contrast to the historical record of parasite introductions, is best explained by a severe population bottleneck experienced by the parasites introduced into the Americas. Here, evidence is presented for targeted selection of rare African alleles in genes which are expressed in the mosquito stages of the parasite's life cycle. These genetic markers support the hypothesis that the severe population bottleneck was caused by the required adaptation of the parasite to transmission by new definitive hosts among the Anopheles (Nyssorhynchus) spp. found in the Caribbean and South America.IMPORTANCE Historical data suggest that millions of P. falciparum parasite lineages were introduced into the Americas during the trans-Atlantic slave trade, which would suggest a paraphyletic origin of the extant isolates in the Western Hemisphere. Our analyses of whole-genome variants show that the American parasites belong to a well-supported monophyletic clade. We hypothesize that the required adaptation to American vectors created a severe bottleneck, reducing the effective introduction to a few lineages. In support of this hypothesis, we discovered genes expressed in the mosquito stages of the life cycle that have alleles with multiple, high-frequency or fixed, nonsynonymous mutations in the American populations which are rarely found in African isolates. These alleles appear to be in gene products critical for transmission through the anopheline vector. Thus, these results may inform efforts to develop novel transmission-blocking vaccines by identifying parasite proteins functionally interacting with the vector that are important for successful transmission. Further, to the best of our knowledge, these are the first whole-genome data available from Haitian P. falciparum isolates. Defining the genome of these parasites provides genetic markers useful for mapping parasite populations and monitoring parasite movements/introductions.

In vivo study of Plasmodium falciparum chloroquine susceptibility in three departments of Haiti.

BACKGROUND: Malaria is considered a public health priority in Haiti, with a goal to eliminate by year 2020. Chloroquine is the first-line treatment recommended by the Ministry of Public Health and Population. In order to verify the suitability of chloroquine for uncomplicated malaria treatment, an in vivo study of susceptibility of Plasmodium falciparum to chloroquine was conducted from January 2013 to March 2015 in six localities in the south of Haiti. RESULTS: Sixty-one patients who presented with confirmed P. falciparum malaria were included in the study and followed until day 28 after having taken 25 mg/kg of chloroquine orally over 3 days. The sample included 28 children under the age of 10, 9 adolescents aged 10-19 years, and 24 adults aged 20 years and over. Among them, 30 were monitored on day 3 (49%) and 33 on day 28 (59%). Clinical and parasitological monitoring was carried out on day 7 on 28 subjects, on day 14 on 13 subjects and on day 21 on 18 subjects. Residual parasitaemia with presence of trophozoites was found in 7 of 30 subjects on day 3 (23%), and in 6 of 28 subjects on day 7 (21%) who had a temperature less than 37.5 °C. These patients can be considered as late parasitological failures. All monitoring performed on day 28 was negative. Gametocytes were found in 3 patients (9%) despite the use of primaquine. The continuing low parasitaemia on day 3 and 7 in more than one fifth of cases raises the question of the efficacy of chloroquine in southern Haiti. CONCLUSIONS: Results suggest a decrease of chloroquine susceptibility for treatment of P. falciparum malaria cases in southern Haiti. Consequently, there is a need to strengthen malaria treatment surveillance and to study the effectiveness of chloroquine in Haiti by monitoring patients after treatment.

Submicroscopic malaria infections in pregnant women from six departments in Haiti.

OBJECTIVES: To describe the epidemiology of malaria in pregnancy in Haiti. METHODS: Cross-sectional study among pregnant women in six departments of Haiti. After obtaining informed consent, whole blood samples and demographic surveys were collected to investigate malaria prevalence, anaemia and socio-behavioural risk factors for infection, respectively. A total of 311 pregnant women were screened for Plasmodium falciparum infection using a rapid diagnostic test (RDT), microscopy and a novel, quantitative reverse transcriptase polymerase chain reaction method (qRT-PCR). RESULTS: Overall, 1.2% (4/311) of pregnant women were tested positive for malaria infection by both microscopy and RDT. However, using the qRT-PCR, 16.4% (51/311) of pregnant women were positive. The prevalence of malaria infection varied with geographical locations ranging between 0% and 46.4%. Additionally, 53% of pregnant women had some form of anaemia; however, no significant association was found between anaemia and submicroscopic malaria infection. The socio-behavioural risk factors identified to be protective of malaria infection were marital status (P < 0.05) and travel within one month prior to screening (P < 0.05). CONCLUSION: This study is the first to document the high prevalence of submicroscopic malaria infections among pregnant women in Haiti and identify social and behavioural risk factors for disease transmission.

T cell subtypes and reciprocal inflammatory mediator expression differentiate P. falciparum memory recall responses in asymptomatic and symptomatic malaria patients in southeastern Haiti.

Asymptomatic Plasmodium falciparum infection is responsible for maintaining malarial disease within human populations in low transmission countries such as Haiti. Investigating differential host immune responses to the parasite as a potential underlying mechanism could help provide insight into this highly complex phenomenon and possibly identify asymptomatic individuals. We performed a cross-sectional analysis of individuals who were diagnosed with malaria in Sud-Est, Haiti by comparing the cellular and humoral responses of both symptomatic and asymptomatic subjects. Plasma samples were analyzed with a P. falciparum protein microarray, which demonstrated serologic reactivity to 3,877 P. falciparum proteins of known serologic reactivity; however, no antigen-antibody reactions delineating asymptomatics from symptomatics were identified. In contrast, differences in cellular responses were observed. Flow cytometric analysis of patient peripheral blood mononuclear cells co-cultured with P. falciparum infected erythrocytes demonstrated a statistically significant increase in the proportion of T regulatory cells (CD4+ CD25+ CD127-), and increases in unique populations of both NKT-like cells (CD3+ CD8+ CD56+) and CD8mid T cells in asymptomatics compared to symptomatics. Also, CD38+/HLA-DR+ expression on γδ T cells, CD8mid (CD56-) T cells, and CD8mid CD56+ NKT-like cells decreased upon exposure to infected erythrocytes in both groups. Cytometric bead analysis of the co-culture supernatants demonstrated an upregulation of monocyte-activating chemokines/cytokines in asymptomatics, while immunomodulatory soluble factors were elevated in symptomatics. Principal component analysis of these expression values revealed a distinct clustering of individual responses within their respective phenotypic groups. This is the first comprehensive investigation of immune responses to P. falciparum in Haiti, and describes unique cell-mediated immune repertoires that delineate individuals into asymptomatic and symptomatic phenotypes. Future investigations using large scale biological data sets analyzing multiple components of adaptive immunity, could collectively define which cellular responses and molecular correlates of disease outcome are malaria region specific, and which are truly generalizable features of asymptomatic Plasmodium immunity, a research goal of critical priority.

High prevalence of asymptomatic malaria infections: a cross-sectional study in rural areas in six departments in Haiti.

BACKGROUND: Public health measures are poised for transition from malaria control to malaria elimination on the island of Hispaniola. Assessment of the reservoir of asymptomatic infections from which acute malaria cases may derive is critical to plan and evaluate elimination efforts. Current field technology is ill suited for detecting sub-microscopic infections, thus highly sensitive survey methods capable of detecting virtually all infections are needed. In this study the prevalence of infection with Plasmodium falciparum was determined in patients seeking medical care primarily for non-febrile conditions in six departments in Haiti using a newly designed qRT-PCR-based assay. METHODS: Three different methods of parasite detection were compared to assess their utility in approximating the prevalence of P. falciparum infections in the population: malaria rapid diagnostic test (RDT) designed to detect histidine-rich protein 2 (HRP2), thick smear microscopy, and a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based upon the small sub-unit ribosomal RNA. The limit of detection of the qRT-PCR assay utilized was 0.0003 parasite/µL of blood. Venous blood was obtained from a total of 563 subjects from six departments in Haiti, all of whom were seeking medical attention without complaints consistent with malaria. Each subject was questioned for knowledge and behaviour using demographic and epidemiological survey to identify risk factors for disease transmission. RESULTS: Among the 563 samples tested, ten and 16 were found positive for malaria by RDT and microscopy, respectively. Using the qRT-PCR test to assess the infection status of these subjects, an additional 92 were identified for a total of 108. Based upon the qRT-PCR assay results, a wide variation in prevalence of infection in asymptomatic subjects was seen between geographic locations ranging from 4-41%. The prevalence of infection was highest in the Grand Anse, Nord and Sud-Est Departments, and demographic data from questionnaires provide evidence for focal disease transmission. CONCLUSIONS: The qRT-PCR assay is sufficiently sensitive to identify an unexpectedly large number of asymptomatic, submicroscopic infections. Identifying and clearing these infections presents a significant challenge to both control and elimination efforts, but the qRT-PCR assay offers a reliable method to identify them.

Epidemiologic survey of Mansonella ozzardi in Corail, Haiti.

An epidemiologic (cross-sectional study) survey on 462 inhabitants in Corail, Haiti showed that 16.5% were infected with Mansonella ozzardi. This finding was determined from a single 20-µL sample of finger prick blood from each person. Among children, < 2% had a detectable microfilaremia. In persons > 15 years of age, the prevalence of infection for males and females was 23% and 21%, respectively. In general, the microfilaremias were low and 70% of positive persons had < 10 microfilariae per 20 µL of blood; only 5% had > 50 microfilariae. This study shows that persons living near mangrove marshes that are breeding sites for Culicoides furens and C. barbosai biting midges, which are recognized vectors of M. ozzardi in Haiti, are consequently more frequently infected than those living in downtown area of Corail or inland.

Cryptosporidium contamination of surface and water supplies in Haiti.

Cryptosporidiosis is one of the most frequent causes of diarrhea in Haiti. Transmission in children less than five years-old, HIV-infected individuals, and people living in low socio-economic conditions is frequently due to consumption of water or food contaminated by Cryptosporidium oocysts. This study examined the circulation of Cryptosporidium oocysts in surface waters and in public water supplies in the district of Port-au-Prince. Data were gathered from December 2000 to June 2002 in 37 sites. In the district of Port-au-Prince, 24/37 samples of water collected (65%) were contaminated by Cryptosporidium oocysts and 10/11 (91%) of those collected in reservoirs used by people living in peripheral areas. The rate of contamination was 7/13 (54%) in water from public standpipes provided by the public company of water distribution. All surface water (4/4) collected was highly contaminated.

Cryptosporidium hominis and Isospora belli diarrhea in travelers returning from West Africa.

Travel-related diarrhea is common among tourists to developing countries. We report two cases of diarrhea due to Cryptosporidium hominis and Isospora belli, respectively, in a child and an adult returning from Africa, without other associated microorganisms. We emphasize the need to detect underdiagnosed coccidiosis in diarrheic travelers with specific methods.

The emergence of Trichophyton tonsurans in Port-au-Prince, Haiti.

The occurrence of the anthropophilic dermatophyte Trichophyton tonsurans as a frequent causative agent of tinea capitis in several developed countries has been associated with a global rise in its isolation during recent years. While T. tonsurans was never found in Haiti before 1988, a sharp increase in the number of isolates of this species from scalp lesions began to be observed in 2005 in Port-au-Prince, Haiti. A prospective study was conducted in Port-au-Prince from May to November 2006 of 64 children presenting with tinea capitis at the dermatological outpatient clinic of the university hospital. Forty-five (70%) were male and 19 female (30%), with an average age at presentation of 6.1 years (age range 1-16 years). Direct microscopic examination of scalp hair using 10% potassium hydroxide was positive in 93.8% and culture confirmation was established in 55 cases (85.9%). Five species of dermatophytes were identified, with the anthropophilic dermatophyte T. tonsurans, accounting for the majority or 35 (63.6%) of all cases of tinea capitis. Other dermatophyte species identified included T. mentagrophytes (14.5%), Microsporum audouinii (12.7%), T. rubrum (7.3%) and in one case, the geophilic M. gypseum (1.8%). In two cases caused by T. tonsurans skin involvement on other areas of the body was recorded. The most frequent pathogen in tinea capitis is now T. tonsurans in Port-au-Prince. We speculate that the recent emergence of T. tonsurans in Haiti is linked to the dramatically increasing mobility of Haitian Diaspora.

Presence of Enterocytozoon bieneusi associated with intestinal coccidia in patients with chronic diarrhea visiting an HIV center in Haiti.

This study investigated the presence of Enterocytozoon bieneusi as a possible cause of chronic diarrhea in Haitian patients attending the GHESKIO AIDS clinic in Port-au-Prince, Haiti. Coccidian oocysts were found by polymerase chain reaction (PCR) in the stools of 58/74 patients with chronic diarrhea and included the following agents: 45 (60%) Cryptosporidium spp., 27 (34%) Cyclospora cayetanensis, and 11 (15%) Isospora belli. Four patients (5.5%) were co-infected with E. bieneusi and one (1.4%) had E. bieneusi alone. The PCR-restriction fragment length polymorphism (RFLP) method made it possible to document the presence in human feces of E. bieneusi in Haiti. As in sub-Saharan Africa, the association of E. bieneusi with coccidian parasites found in Haitian patients with diarrhea is probably caused by the high level of fecal contamination of soils and surface waters usually associated with countries with low hygienic standards.

[Human cryptosporidiosis and Cryptosporidium spp. in Haiti]. / Cryptosporidiose humaine et espèces en cause en Haïti.

Contamination by water-born infectious diseases is closely linked to urban slums conditions such as overcrowding and high level of faecal pollution by animal and human excreta. In this environment, cryptosporidiosis is a major cause of acute diarrhoea in children and chronic persistent diarrhoea in AIDS patients, resulting in increased morbidity and mortality in both populations. The aims of this study conducted in Port-au-Prince, Haiti were to: (i) determine the frequency of Cryptosporidium infection in two populations of patients with diarrhoea, children and AIDS patients, and the existence of Cryptosporidium carriage in healthy adults living in close contact with them; (ii) identify by molecular genotyping the Cryptosporidium species involved; and (iii) evaluate the viability of Cryptosporidium oocysts isolated from human stools. From January 2000 to January 2001, 158 of 1529 diarrhoea stool samples collected from 93 patients with diarrhoea, 57 adults followed at Centres GHESKIO and 36 children admitted at the University Hospital in Port-au-Prince contained Cryptosporidium oocysts (10.3%). The majority of adult patients (98%) were HIV-infected whereas the majority of children (81%) tested negative for HIV. Cryptosporidium was documented in only 1/102 healthy persons living in contact with Cryptosporidium infected patients and infection was with the same genotype as that of the contact patient. Among the 69 Cryptosporidium isolates studied for genotyping, three species were identified: C. hominis (59%), C. parvum (38%) and C. felis (3%). The two C. felis cases are the first reported from AIDS patients in the Caribbean. Most of the children regardless of their HIV status were infected with C. hominis (72%), whereas AIDS patients were more likely to be infected by either human or animal genotypes. These data confirm that immunocompromised individuals are susceptible to a wide range of Cryptosporidium spp. Viability of Cryptosporidium oocysts were determined in an experimental mouse model for 17/18 specimen studied including in 12/13 C. hominis, 4/4 C. parvum and 1/1 C. felis. Infectivity in newborn mice was found to be dose-dependent and more effective with C. parvum than the other two genotypes. Cryptosporidiosis remains a frequent hazard for both AIDS patients and young children in Haiti because of poor hygiene, particularly contaminated water and overcrowded conditions associated with urban slums.

Hematologic and serum biochemistry values in two species of free-ranging porcupines (Coendou prehensilis, Coendou melanurus) in French Guiana.

Individuals of two porcupine species (Coendou prehensilis and Coendou melanurus) were translocated during the flooding of a forest at a hydroelectric dam site in French Guiana. Blood samples were collected for 11 mo to determine hematologic and serum chemistry reference mean values and ranges and to look for blood parasites. Male C. prehensilis had significantly higher hemoglobin levels, packed cell volumes, and creatinine and potassium values than did females of the same species. Coendou prehensilis had significantly lower amylase levels but higher creatinine, calcium, lactate dehydrogenase, and gamma glutamyl transferase levels than C. melanurus.

[Presence of Angiostrongylus cantonensis in Haiti]. / Presence d'Angiostrongylus cantonensis en Haïti.

In the West Indies Angiostrongylus cantonensis was found first in Cuba in 1973, then in Puerto Rico (1986), in the Dominican Republic (1992) and recently in Jamaica (2002). Concurrently, the presence of the parasite was detected in the Bahamas and in New Orleans (LA, USA) in 1990. To assess the occurrence of A. cantonensis in Haiti, we investigated a number of rats in Port-au-Prince in 2002. Preliminary results among 23 captured and examined rats show that 75% (three of four) Rattus norvegicus and 21% (four of 19) R. rattus harboured A. cantonensis in their cardiopulmonary systems. Haiti is, for the first time, recognized as a new enzootic area for A. cantonensis in the Caribbean. This emerging zoonosis should be considered as a new public health hazard in Haiti.