Cuban parasitology in review: a revolutionary triumph

Review

Cuban parasitology in review: a revolutionary triumph La´zara Rojas Rivero1, Fidel Angel Nu´n˜ez Ferna´ndez1 and Lucy J. Robertson2 1

Subdirecccio´n de Parasitologı´a, Instituto de Medicina Tropical Pedro Kourı´, Autopista Novia del Mediodia km 61/2, Apartado Postal 601, Marianao 13, Ciudad de La Habana, Cuba 2 Parasitology Laboratory, Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, 0033 Oslo, Norway

Although the population of Hispaniola, Cuba’s most similar neighbour in the Caribbean, continues to be threatened by parasitic diseases (including malaria), many tropical parasitic infections in Cuba have been eliminated or controlled. However, some parasitic infections remain important in the Cuban population, and the occurrence of vectors and the high possibility of introduction of parasites mean that Cuban diagnosticians must remain alert. Some key aspects of human parasitology in Cuba are reviewed here, including historical information, comparative data from Hispaniola and Jamaica, and how Cuba strives to maintain and improve its control against parasitic infections. Data from recent key novel parasitology research conducted in Cuba are also described. Parasitological status in Cuba: history and Caribbean comparisons The Republic of Cuba is the largest and most populous island nation in the Caribbean and is located at the confluence of the Caribbean Sea, the Gulf of Mexico, and the Atlantic Ocean. Whereas in the most similar neighbouring island (Hispaniola, the second largest and second most populous island in the Caribbean), parasitic infections – including malaria – remain a threat to the inhabitants (see Table 1), the diagnosis, eradication and research of parasitological diseases are important facets of Cuban health services. Most important tropical parasitic diseases have been eliminated or prevented from establishing in Cuba, despite the presence of vectors and intermediate hosts [e.g. for malaria, schistosomiasis, leishmaniasis and Chagas disease (Table 1 and Box 1)] [1] and, also, despite the occurrence of these infections in neighbouring countries, from which many travellers arrive each year [2,3]. Malaria was eradicated in Cuba in 1967 after an extensive governmental campaign (National Service for the Eradication of Malaria), which started soon after the 1959 revolution; Cuba was officially certified as malaria free by the WHO in 1973, six years after the last case report [4] (Table 2). Autochthonous cases of other parasitic infections, such as lymphatic filariasis, have not been recorded for many years (Table 2). Nevertheless, despite enormous advances in Cuban parasitological status since the revolution (which are, Corresponding author: Robertson, L.J. ([email protected]).

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probably, largely due to the implementation of government initiatives to improve socioeconomic conditions, health, sanitation and water supplies [5]), some parasitic infections, particularly intestinal protozoa, are still important causes of morbidity among the Cuban population. This is particularly so in some high-risk groups, such as children attending day-care, and preschool children in rural mountainous areas [5–7]; these infections remain an important focus of parasitology research in Cuba today. During the ‘Special Period’ of the 1990s when economic hardships might have been expected to impact on Cuban parasitological status (Table 2), a survey of intestinal parasites on Isla de Juventud (the second island of the Cuban archipelago), indicated an overall improvement in parasitological status compared with the mid-1980s (Table 3). Another important focus for Cuban parasitology today is maintaining and improving diagnostic techniques and aptitudes in local health centres throughout the country, as various factors (Box 1) make it essential that Cuban diagnostic laboratories, including local and regional laboratories, remain fully alert and diagnostically capable. In addition, by means of the Programme for International Sanitary Control (which focuses particularly upon scholarship holders but, in addition, assesses other persons entering Cuba from endemic areas), the potential introduction of parasitic infections, or their vectors, is carefully monitored. In 1985, the first case of imported schistosomiasis was reported and, since then, sporadic imported cases have been reported annually. For example, between 1992 and 1997 in the Villa Clara province, 45 cases of Schistosoma haematobium (which were imported from five different African countries) and 42 cases of Schistosoma mansoni (which were imported from different African countries and from Yemen) were reported [8]. Additionally, during this period, three cases of Loa loa infection were imported from Africa [8]. To the outside world at least, Cuba currently seems to be at a time of transition, or poised on a cusp. Therefore, it seems timely to provide an overview of parasitology in Cuba today, including a look back at previous years (Table 2), and a comparison of the parasitological status of Cuba with that of other Caribbean islands (Table 1). As well as improving its own parasitological status, Cuban health authorities are determined to increase their own, and global, knowledge of parasitic infections, and recent data from research in Cuba are briefly described here. Table 4 provides an overview of the relevance of various

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Dominican Republic 9–10 million (2007 estimate)

Jamaica 2–3 million (2005 estimate)

Land area

109886 km 2

27750 km 2

48730 km 2

10991 km 2

Population density

102/km 2

335/km 2

201/km 2

252/km 2

Total GDP (US$ billion)

46.22 (2006 estimate)

16.51 (2007 estimate)

89.87 (2007 estimate)

11.69 (2005 estimate)

GDP per capita (US$)

4500 (2007 estimate)

1913 (2007 estimate)

9208 (2007 estimate)

4300 (2005 estimate)

Total health expenditure as % of GDP (source: WHO)

7.6%

6.2%

5.4%

4.7%

Population per physiciana (2004)

169 persons

4000 persons

532 persons

1176 persons

Prevalence of HIV per 100 000 population (in adults  15 years) (2005)b,c

52 b

3377

1036

1371

% deaths in children <5 years old because of diarrhoeal diseases (2000) c

1.3%

16.5%

11.7%

9.6%

% population with access to improved drinking water source (2006) c

91%

58%

95%

93%

Leishmaniasis

Autochthonous cases never recorded.

An autochthonous focus of cutaneous leishmaniasis recorded in Dominican Republic. Cases may also occur in Haiti, although none currently recorded.

No autochthonous cases recorded.

Lymphatic filariasis

No autochthonous cases reported for at least 50 years. Sporadic imported cases reported.

Not yet eliminated; in 2006, 560000 estimated infected in Haiti, and >50000 estimated infected in Dominican Republic. d

No autochthonous cases reported.

Malaria

Certified malaria free by WHO in 1973. Various imported infections reported annually from 1980 onwards (see Table 2) and some introduced infections (further spread of imported infections). Between 1980–2007, most (82%) cases of imported malaria are P.falciparum, but also P. vivax (14%), P.ovale (3%), and P. malariae (<1%), whereas most introduced cases are P. vivax (71%), although also P. falciparum (29%), and none of P. ovale and P. malariae. These data may indicate a greater suitability of the predominant Cuban anopheline species (A. albimanus) for P. vivax transmission. All infection foci have always been satisfactorily controlled, and there has been no suspension of malaria-free status.

Malaria remains endemic (P. falciparum and P. vivax). In 2003, 9837 cases and 16 deaths reported in Haiti, and 1296 cases and 16 deaths reported in the Dominican republice. In 2006, 32739 cases reported in Haiti, and 3525 cases reported in the Dominican republic. f

Certified malaria free in 1960s. In 2006– 2007, an outbreak of malaria occurred involving 280 confirmed cases and no deaths, and the temporary withdrawal of malaria-free status by WHO; malaria-free status was regained in September 2007 after a three-month period with no confirmed cases.

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Hispaniola Haiti 8–9 million (2007 estimate)

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Population

Cuba 11–12 million (2006 estimate)

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Table 1. Cuba, Hispaniola and Jamaica – a comparison of three Caribbean islands with respect to some aspects of human parasitology

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The 2004 value for Norway for this parameter is 281. The equivalent value for Norway for this parameter is 67 (see: http://www.who.int/whosis/whostat/EN_WHS08_Full.pdf). Data from WHO, available at: http://www.who.int/whosis/whostat/EN_WHS08_Full.pdf. d Data obtained from WHO, Global Programme to Eliminate Lymphatic Filiariasis, Weekly Epidemiological Record, 22, 221–232. http://www.filariasis.org/pdfs/WER_81_2006_221-232.pdf. e Data obtained from WHO global health atlas, available at: http://www.who.int/globalatlas. f Data obtained from Pan American Health Organization, Malaria Progress Report (May 2008), available at: http://www.paho.org/search/DbSReturn.asp. c

b

a

No autochthonous cases recorded. Transmission of S. mansoni still considered to occur in southeastern Dominican Republic. Data from Haiti unclear. Autochthonous cases never recorded. Imported cases reported annually since 1985. Schistosomiasis

No cases recorded. Might occur in Haiti; data scant and unreliable. No cases recorded. Onchocerciasis

Table 1 (Continued )

Cuba

Hispaniola Haiti

Dominican Republic

Jamaica

Box 1. Why it is essential that regional Cuban diagnostic laboratories remain fully alert and capable o Neighbouring countries, and other countries in the region, are endemic for various parasitic infections which are considered to have been eliminated in Cuba. o The high number of long-term visitors to Cuba from regions where such infections are endemic (especially students studying at Cuban institutions) means that the risk of introduction is considerable (there were >25000 such visitors in 2005). o Cuban professionals, such as medical personnel, often travel to endemic areas where they could acquire parasitic infections and return to Cuba with them, thereby increasing the risk of introduction. o Vectors for transmission of some parasitic infections occur, and might be widespread, in some regions of Cuba. Such vectors include anopheline mosquitoes (the main species recorded is Anopheles albimanus), triatomine bugs, sandflies of Lutzomyia genus and snails of Biomphalaria genus ( pallida, havanensis and helophila species). The onward transmission of imported malaria cases by Cuban vectors has been documented in over 70 cases. o Cuba is a key location for over 250 migratory bird species, and these might import infected vectors or intermediate host species such as Biomphalaria glabarata, which currently does not occur in Cuba. o Infrastructure difficulties may jeopardise the transport of samples and/or patients from outlying regions to a central ‘expert’ diagnostic laboratory.

parasitic diseases in Cuba as endemic or imported infections and/or as research subjects by Cuban parasitologists. The primary health care available in Cuba is one of the strengths of the free, publically funded Cuban health system. Not only is the ratio of persons per doctor much lower than that in neighbouring countries, being equivalent to (or lower than) some of the world’s wealthiest countries (Table 1), but also almost 50% of doctors are family practitioners who are based in small primary care centres [9]. Before the revolution, health care was inadequate, particularly in rural areas because it was based in the larger cities, and the situation regarding intestinal parasites in the Cuban population before 1959 has been described as ‘deplorable’ [5]. However, access to medical services has now equalised, and 99.7% of today’s population have access to a family doctor. Also, the relative lack of regional migration within Cuba enables doctors and nurses to have an in-depth knowledge of the families in their practices [9]. Undoubtedly, this is advantageous in ensuring that cases of parasitic infection are identified and endemic foci are recognized and eliminated. The low HIV prevalence in Cuba (Table 1) – which is the lowest in the Caribbean region by a significant margin (and is equivalent or lower to that of some of the world’s wealthiest countries) and is postulated to be due, in part, to an extensive testing policy [10] – presumably mitigates against the upsurge of various opportunistic parasitic infections that are associated with HIV/AIDS observed in other regions [11]. Intestinal protozoan infections: epidemiology, treatment and diagnosis Cryptosporidium, Cyclospora and Giardia are important aetiological agents of diarrhoea in Cuban children [5– 7,12,13] and AIDS patients [14,15], and have been the subjects of various investigative studies. Knowledge regarding these infections in Cuban children is significantly

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Table 2. Timeline – a pre´cis of Cuban history and malaria Date Up to 1492 1500–1865

1868–1878

Historical events Pre-colonial Cuba Spanish colonization of Cuba. Various skirmishes over Cuba between the Spanish and other colonial powers, including French, Dutch and English. First war of Cuban independence.

1895–1898 1898

Second war of Cuban independence Spanish–American war.

1899–1902

American occupation.

1902–1909

1903–1934 1935–1959 1959–1961

1962

1963

Cuban independence, although severely restricted by the Platt amendment. Fulgencio Batista effectively in power (although not always president). Much of Platt amendment withdrawn. Cuban revolution (1959). Fidel Castro elected as First Minister of the new government. Bay of Pigs invasion of Cuba organized by USA (1961). Cuban missile crisis.

Complete diplomatic and commercial embargo placed by USA on Cuba.

1964–1967

1968–1972 1973 1974–1979

Heavy reliance on Soviet economic aid.

US-Cuba relations remain low; bombing of civilian Cubana flight 455 in 1976.

1980–1990

Mariel boatlift in 1980; US invasion of Grenada in 1983, killing more than 25 Cubans, and expelling other Cubans present on the island.

1991–2000

Collapse of Soviet Union, upon which Cuba reliant for trade and goods (US total embargo still in place), brings period of economic hardship known as the ‘Special Period’ (80% of total exports, 80% of total imports and 90% of oil imports lost). Hardships compounded by Helms-Burton act (see below).

1996

Helms-Burton act passed in USA, which limited trade with Cuba from non-US companies. Hugo Cha´vez becomes president in Venezuela and begins extensive cooperation with Cuban administration. Increased economic cooperation with other countries, particularly Venezuela, Bolivia, and China. Trade embargo from US and Helms-Burton act remain in place, although other countries, notably Mexico and Canada, have passed their own laws intended to counteract the effects of the Helms-Burton act. Rau´l Castro is elected president.

1999 2001–2007

2008

Malaria and other infectious diseases Yellow fever (especially) periodically decimates the population.

Santiago Ramo´n y Cajal, Spanish histologist, physician and Nobel laureate (1906), worked as an army doctor in Cuba (1874–1875) where he contracted malaria, which nearly killed him. Although yellow fever (and typhoid) had the most severe impact on troops, malaria and diarrhoeal diseases also had a devastating effect. First campaign against yellow fever and malaria began in Havana in 1901. According to recommendations of Cuban physician Carlos J. Finlay, Assistant Surgeon General William Gorgas of the US Army implemented anti-mosquito measures in Havana that involved burning pyrethrum, a natural insecticide derived from chrysanthemum flowers, inside sealed buildings. Mosquito populations were eliminated and decreased. Creation of the National Health Service, including various centralized bodies and institutions such as the National School of Hygienists. Carlos J. Finlay chief health officer of Cuba. The Ministry of Public Health was established, the first ever worldwide.

The Institute of Tropical Medicine founded in Havana by Dr Pedro Kourı´ (1937). Last reported case of lymphatic filariosis during 1940s. Creation of the Rural Social Medical Service (SMSR) (1960).

3519 cases of malaria reported. National Service for the Eradication of Malaria established based on vector control measures, epidemiological control of transmission foci and surveillance of febrile patients in the whole country including the rural areas. 833 cases of malaria reported. Following the national campaign for eradication of malaria, reported malaria cases decrease annually (1964, 624; 1965, 127; 1966, 36; and 1967, 7). No cases of malaria reported. DDT spraying banned in 1970 (two years before a similar ban in the USA). No cases of malaria reported. Certified as malaria free by the WHO. No cases of malaria reported. The ‘Pedro Kourı´’ Institute of Tropical Medicine supported by the Cuban government to work towards avoiding introduction of previously eradicated infectious diseases and control of those not yet eliminated. The Sanitary International Control Programme initiated by the Ministry of Public Health. 992 cases of malaria reported in the Cuban population, of which 960 (96.8%) were imported (Cubans travelling from endemic areas), but 32 cases were introduced. Despite shortage of various medical supplies, including pharmaceuticals and equipment consumables limiting medical interventions and laboratory testing, parasitological status apparently not greatly affected. Most significant health effects due to nutritional deficiencies (e.g. over 50 000 Cubans temporarily blinded by 1993 because of optical neuropathy from vitamin deficiencies), which indicates that pathology from parasite infections might have been compounded by nutritional deficiencies. 163 cases of malaria reported in the Cuban population, of which 145 (89%) were imported (Cubans travelling from endemic areas), but 18 cases were introduced.

170 cases of malaria reported in the Cuban population, of which 143 (84.1%) were imported (Cubans travelling from endemic areas), but 27 cases were introduced.

No autochthonous cases of malaria reported, although sporadic imported/introduced cases continue to be diagnosed periodically.

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Table 3. Intestinal parasites on Isla de la Juventud, Cuba; comparison of prevalence rates before and during the Special Perioda 1983 (n = 605) No.

%

1998 (n = 1200) No.

%

Helminths Ascaris lumbricoides Necator americanus b Trichuris trichiura b Taenia species

7 55 178 1

1.1% 9% 29.4% 0.1%

22 1 42 0

1.8% 0.1% 3.5% 0%

Protozoa Entamoeba histolytica/E. dispar Giardia intestinalis b

17 96

2.8% 15.8%

38 119

3.2% 9.9%

Parasite species

a

Data derived from a MSc study (Alvarez Almanza ED, Parasitismo Intestinal en la Isla de la Juventud, Ciudad de La Habana, Instituto de Medicina Tropical ‘Pedro Kourı´’ 1999). Statistically significant (P < 0.01) reduction in prevalence of parasitosis between 1983 and 1998.

b

Table 4. Status of some human parasitic infections in Cuba today; occurrence and research focus Parasitic disease Amoebiasis

Chagas disease

Cryptosporidiosis

Cyclosporiasis

Eosinophilic meningitis owing to Angiostrongylus cantonensis Fascioliasis

Giardiasis

Leishmaniasis

Occurrence in Cuba Relatively prevalent (8.3% prevalence, according to the most recent national survey), but might be overdiagnosed. Never recorded in Cuba.

Cryptosporidium hominis common in diarrhoeal infants (0.1–11% prevalence), schoolchildren and AIDS patients (10% prevalence). Occurs in diarrhoeal paediatric patients (4% prevalence in one study), schoolchildren and AIDS patients (3% prevalence). Sporadic cases identified since 1981. Fasciola hepatica considered prevalent in cattle in Cuba, but only isolated human cases; some localized foodborne outbreaks. Over 50% prevalence in preschool children; less prevalent in diarrhoeal infants. Autochthonous cases never recorded in Cuba.

No autochthonous cases reported since 1940s. Malaria-free status awarded by WHO in Malaria 1973. Imported and introduced cases reported annually since 1980 (see Tables 1 and 2). Autochthonous cases never recorded in Schistosomiasis Cuba. Imported cases reported annually since 1985. Relatively prevalent (5–40% prevalence Soil-transmitted helminthiases (ascariasis, trichuriasis, strongyloidiasis depending on helminth and location) in school children, particularly those from and hookworm infections). (Also pinworm, although not soil-transmitted) rural, mountainous areas. Taenia saginata infection occasionally Taeniasis and cystiscercosis reported; occasional cases of autochthonous cysticercosis. Human infections (visceral larval migrans Toxocariasis and ocular larval migrans) occasionally reported. Considered prevalent, but mostly not Toxoplasmosis diagnosed as asymptomatic. Antibody testing of pregnant women has revealed over 70% prevalence. Occurs predominantly in high risk groups, Trichomoniasis such as HIV patients, and sexually active teenagers. Lymphatic filariasis

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Research focus Development of an alternative diagnostic test.

Refs [23,24,55]

[1,58–60] Exploring new immunogenic derivatives in a search for a vaccine candidate; morphometric and genetic studies of triatomine bugs to assess risk of transmission via indigenous vectors. Epidemiological studies, both traditional and [12–14,16] molecular.

Epidemiological studies.

[13–15]

Various immunological, malacological and clinical investigations. Diagnostics and immunology; characterisation of antibodies and immunisation. Malacological investigations.

[61]

Epidemiological studies, both traditional and molecular. Clinical trials.

[5–7,17–22]

[27–33]

[48–54,62] Exploring new immunogenic derivatives in a search for a vaccine candidate; candidate vector studies; treatment studies. Currently not a research focus. [1] Quality control for diagnosis; treatment studies in vitro and in animal models.

[4,45–47]

Malacological investigations (Biomphalaria species studies) and biocontrol.

[63,64]

Prevalence, epidemiological, and risk-factor studies. Investigation of associations between helminth infections and atopic diseases.

[5,6,25]

Currently not a research focus.

Survey work on soil and dogs.

[65]

Quality control issues. Epidemiological studies, both traditional and molecular, planned.

[66]

Prevalence studies; isolate characterisation studies; identification of genetic virulence markers; study of cytoadherence proteinase.

[34–43]

Review assisted by regular biannual (i.e. occurring in May and October) surveillance, in which a stool sample is analysed from all asymptomatic children attending selected day care centres (which are attended by 15% of children aged 1–4 years) [9]. Clinical and epidemiological aspects of cryptosporidiosis and cyclosporiasis in symptomatic children have been compared and their key clinical differences have been described [13]. In collaboration with Norwegian and Dutch colleagues (from the Norwegian School of Veterinary Science, Oslo and the Medical Microbiology Twente Achterhoek, Enschede, respectively), studies are in progress investigating Cryptosporidium infections (including species, sub-types and clinical and epidemiological aspects) in children [16]. To date, most Cuban studies on intestinal protozoan infections have focused upon Giardia. A longitudinal study in Havana identified a group of children with an apparently increased likelihood of being re-infected with Giardia, who were also more prone to developing clinical manifestations [7]. A case-control study demonstrated that this group was also more likely to drink water that has not been boiled and have poor hygiene habits regarding the preparation of vegetables [17]. These results indicate that, despite the high proportion of the population with improved water supplies (Table 1), water probably has a role in giardiasis transmission in Cuba, and this deserves investigation. In addition, gerbils inoculated with Giardia isolates from the children who have an apparent predisposition to symptomatic giardiasis had more prolonged infections and elevated cyst excretion than gerbils inoculated with isolates obtained from less frequently infected, asymptomatic children, which confirms that the spectrum of symptoms associated with Giardia infection is affected by both host and parasite [18]. Further study, using random amplified polymorphic DNA (RAPD) technique, of 18 Giardia isolates from these children indicated a role for parasite genetic background in clinical variability and the genesis of susceptibility to infection [19]. Further studies, in collaboration with colleagues in Norway and the Netherlands, describe associations between sequencespecific Giardia isolate descriptions, and epidemiological and clinical characteristics [20]. Treatment trials for giardiasis have also been conducted in Cuba because some infections do not respond to treatment with 5-nitroimidazolic drugs (e.g. metronidazole). Clinical trials in children demonstrated a high cure rate (91.5%) with aminosidine sulphate, and an ethanolic extract of propolis was also considered to be an alternative therapeutic option [21]. Further trials compared treatment efficacies of albendazole, chloroquine and tinidazole. In this randomized treatment trial, which involved 165 Cuban children with confirmed giardiasis, both tinidazole (91% cure) and chloroquine (86% cure) were significantly (P < 0.01) more effective at curing giardiasis than albendazole (62% cure) [22]. To date, research concerned with amoebiasis in Cuba has focused upon the development of an effective diagnostic technique as an alternative to microscopy. Although stool microscopy remains a mainstay of the parasite diagnostician because of its low cost and capacity for detecting

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different microorganisms in the same assay [23], a survey in the Cienfuegos region of Cuba [24] demonstrated that amoebiasis might be overdiagnosed, possibly owing to the misidentification of leukocytes in stool samples [23]. An enzyme-immunoassay, ENZYMEBA, based on immunoenzymatic capture of histolysaine, a cysteine proteinase of Entamoeba, has been developed [23]; this enzyme-immunoassay has high sensitivity and specificity, has no crossreactivity with other parasite genuses, requires only one stool sample, and is also effective on frozen samples. However, an important limitation is that it does not distinguish between Entamoeba histolytica and Entamoeba dispar. Intestinal helminthiases: infection and atopic diseases As well as intestinal protozoa being frequently identified in Cuban children (20–50% prevalences) [5,6], intestinal helminthiases are also common (15–40% prevalences), especially in children from rural areas [5,6]. Because intestinal helminthiases are often widespread in developing countries, but atopic diseases are not, and the converse applies in developed countries (i.e. atopic diseases are relatively common, but helminth infections are rare), an inverse relationship between these two phenomena has been postulated [25]. A study in school children from two Cuban municipalities [26] with 20% helminth infection prevalence [5] investigated this theory and examined not only asthma and atopy but also atopic dermatitis and allergic rhinoconjunctivitis, in relation to past and current intestinal helminth infections. Interactions between intestinal helminth infection and atopic disease were shown to depend upon type of atopic disease, the helminth species, and whether the helminth infestation was current or had occurred previously [26]. Particular findings were that whereas current infection with Ascaris lumbricoides apparently protects against atopic dermatitis, infection with Enterobius vermicularis and hookworm are risk factors for developing allergic rhinoconjunctivitis and/or atopic dermatitis later in childhood [26]. Fasciola hepatica: improving diagnosis Although fascioliasis is infrequently diagnosed in the human population of Cuba, it is of considerable veterinary importance and, therefore, the potential exists for further transmission to human hosts; as well as isolated human cases, food-borne outbreaks (which have been attributed to contaminated watercress and lettuce) have occurred. Diagnosis of fascioliasis remains a debated subject, largely because of the nonspecific, elusive nature of the clinical picture, the prolonged prepatent period, irregular egg excretion by adult flukes and the lack of a standardized diagnostic protocol including patient-history details (www.who.int/neglected_diseases/preventive_chemotherapy/WHO_CDS_NTD_PCT_2007.1.pdf); previous and ongoing parasitological research in Cuba has contributed to improving this situation. For example, immunological methods for Fasciola hepatica diagnosis, using ES antigens and the monoclonal antibody (mAb) ES-78 have been developed to detect antibodies in sera [27], and antigens in stools and sera [28]. Both procedures have been used in animals [29] and humans [30]. The mechanism of action of 445

Review ES-78 has been studied [31], its epitopes have been partially characterized [32] and immunization has conferred passive protection against fascioliasis in mice [33]. Further research is under way, with Norwegian collaboration, with the intention of developing a simple and user-friendly dipstick test for use in remote, rural areas where this infection has greatest impact. Trichomonas vaginalis: occurrence and key research Trichomoniasis is an important sexually transmitted disease, which might increase HIV transmission risk. Prevalence studies among different risk groups have been conducted in Cuba and have provided data on where education and prevention controls should be focused. In HIV patients, a trichomoniasis prevalence of 16% was found [34], in couples with fertility problems the prevalence found was 10.5% [35], in apparently healthy women it was 8% [36], and in teenagers it was 13.7% [37]. An in vitro T. vaginalis cytoadherence assay – which requires neither radioisotope use nor the necessary associated specialized, expensive equipment – has been developed in Cuba [38]. This assay has demonstrated a strong correlation between adhesion to HeLa cells and severity of clinical manifestations [38]. Biological characterization studies in an experimental model have indicated correlation between clinical severity of vaginal trichomoniasis in adolescents and isolate virulence in NMRI mice [39]. Further investigations of parasite and clinical presentation associations, which were conducted using RAPD technique on 40 different isolates [40], showed that isolates grouped genetically according to clinical manifestations and revealed a possible marker for virulence [40,41]. Other research on Trichomonas in Cuba has established the role of a 62-kDa excretion–secretion (ES) proteinase in parasite cytoadherence [42] and that this molecule is a possible vaccine candidate because intranasal immunisation of BALB/c mice, in conjunction with adjuvants, enhanced parasite elimination after intravaginal challenge [43]. Treatment for malaria and leishmaniasis Although neither malaria nor leishmaniasis are endemic in Cuba, there is a very real possibility of these infections being imported and, perhaps, establishing (Tables 1 and 2); the search for new treatments for these parasitoses, which has been partly driven by the spread of treatment-resistant Plasmodium falciparum strains, has been another important line of recent research in Cuba. In the search for new drugs against Plasmodium, haemoglobin (Hb)-hydrolyzing enzymes have been identified as promising targets. Most of these enzymes are aspartyl proteases, called plasmepsins (Plm), of which PlmI and PlmII are perhaps the best characterised [44]. Occurring in the parasite digestive vacuole, Plm are essential in the initial stages of Hb degradation, which is necessary for the parasites’ growth and maturation. The search for new inhibitors depends on high-throughput screening of the most attractive candidates. Most assays rely on peptidic substrates whose sequences reproduce the Hb a-chain F33–L34 cleavage site that is recognized by Plm I and Plm II. Continuous spectrophotometric and fluorometric assays have been used effec446

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tively, but they are expensive, and spectrophotometric assays are rather insensitive. An Anson assay has been adapted for measuring PlmII activity using C14-labelled Hb, but its intrinsic low throughput and the requirement for radioactive materials are problematic. Thus, a primary objective of Cuban researchers was to develop a cheap, high-throughput, heterogeneous enzymatic assay for measuring PlmII activity to use as a tool in investigating new inhibitors of this enzyme [45]. In the assay developed, PlmII acts upon a solid-phase bound synthetic peptide (DU2), whose sequence comprises the cleavage site F33– L34 present in the Hb a-chain, and a classical ELISA-based procedure is used to quantify peptide surface density [45]. Because of the intrinsic advantages of the ELISA format, this assay is applicable for high-throughput screening of potential PlmII inhibitors. Growth of Plasmodium in vitro and in animal models has also been used to investigate the potential of derivates from marine invertebrates [46] and algae [47] as antimalarials. Of 27 aqueous extracts from different marine invertebrates from the Cuban coast, the three tunicate species Microcosmus goanus, Ascidia sydneiensis, and Phallusia nigra showed notable antimalarial properties, both in vitro and in animal models, and should be investigated further. Similarly, ethanolic extracts of the algal species Laurencia obtusa and L. corallopsis indictated antiplasmodial activity; several molecules (terpenoids, lactonic/coumarine compounds, alkaloids, free-reducing sugars, saponins and flavonoids) were detected, and research is in progress to identify and isolate the active components. Although pentavalent antimonials remain the mainstay for leishmaniasis therapy in most countries (with amphotericin B, aminosidine and pentamidine recommended as secondary drugs), Cuban researchers believe that the disadvantages associated with these treatments – including parenteral administration mode, prolonged therapy duration, toxic effects, cost and parasite resistance – mean that new drugs are essential. Research on thiadiazine derivatives [48,49] has demonstrated that they have antiproliferative action on all developmental stages of Leishmania amazonensis in vitro, causing irreversible inhibition of promastigote growth and reducing infection with amastigotes by 12–89%. However, these compounds exhibited high toxicity and inhibited phagocytosis in the murine host cell [49]. Essential oils derived from the herb Epazote (Chenopodium ambrosioides), a plant native to Central and South America, have also been evaluated for their efficacy against L. amazonensis and Leishmania donovani, in vitro [50–53] and in animal models [54], and yielded promising results, as well as an apparent lack of toxicity in mice. Clearly, further research is warranted here. Quality control of diagnosis in Cuba The significant potential for parasitic infections to occur in the Cuban population and, additionally, infrastructure difficulties, which mean the transport of patients or samples to a central parasitology laboratory is problematic, have resulted in considerable effort being directed towards ensuring that local laboratories across Cuba are able to make accurate diagnoses (Box 1). In a study con-

Review ducted in Cienfuegos province, overdiagnosis of intestinal amoebiasis was observed. After a series of corrective actions, a repeat survey was conducted one year later and demonstrated considerable improvements in cognitive aspects, although some technical and organizational deficiencies required further correction [55]. Although a regular national programme for quality control (QC) in Cuban parasitology has yet to be established and approved by the Cuban Ministry of Public Health, various individual initiatives have been implemented. To improve diagnostic capabilities nationwide, a national training project on the diagnosis of intestinal parasites began in 1997. Four courses were developed, with educational materials and practical evaluations, involving all provincial laboratories and training 62 individuals. This intervention was rapidly demonstrated to have been successful, but it was agreed that the only way to sustain these results is by periodic mandatory training and the establishment of national programmes for external quality assessment in parasitology [56]. In this respect, Cuba’s central parasitology laboratory, at Instituto de Medicina Tropical Pedro Kourı´, Havana, has participated in the regional CAREC (Caribbean Epidemiological Centre) programme and the WHO programme from the Centre for External Quality Assessment in Parasitology (Paris, France). Cuba: a collaborative partner and global actor Much of the research outlined above has been conducted solely by Cuban researchers, with collaboration between Instituto de Medicina Tropical Pedro Kourı´ and Cuban university departments, hospitals and other Cuban institutes. However, Cuban parasitologists are also global players. They often have been requested to assume, or have offered, advisory roles on specific topics, particularly in Venezuela, Guatemala, the Dominican Republic and the Republic of The Gambia. For example, a four-person multidisciplinary group from Cuba provided substantial input into the National Malaria Control Programme in Gambia from 2000 onwards, successfully contributing to clinical case management, QC for malaria diagnosis, vector control and epidemiological surveillance [57]. The overall goal was to reduce morbidity, mortality and socioeconomic impacts of malaria, and ensure early diagnosis and prompt treatment in specific target groups [57]. With respect to research, Cuban parasitologists have collaborative links with, among others, institutes in Belgium, Brazil, Canada, France, Libya, Mexico, Netherlands and Norway, and the 8th Central American and Caribbean Congress on Parasitology and Tropical Medicine, which was held in Cuba (in December 2007), provided an indication of the potential for interaction, with 490 delegates attending from around the world (see ‘Cryptosporidium and Giardia in drinking water: not just a problem for industrialised countries’ at http://cabiblog.typepad.com/hand_picked/2008/01/cryptosporidi-2.html). Health and social services, upon which a country’s parasitological status is based, are invariably tied to politics and governmental policies and, at present, Cuba might well be on a cusp. In the field of parasitology, however, if the research ideals and the commitment to the exchange of ideas, information and knowledge continue as

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