Schistosomiasis (Schistosoma spp.) - IgG antibodies; Molecular diagnosis (PCR); Molecular idetification of species (PCR and sequencing).

 

Information 13-10-2018.

Schistosomiasis is an acute and chronic parasitic disease caused by blood flukes (trematodes) of the genus Schistosoma, included within the Phylum Platyhelminthes, class Trematoda, subclass Digenea, family Schistosomatidae. There is evidence of the transmission of the disease in 78 countries and it is estimated that at least 206.5 million people needed preventive treatment in 2016, which should be repeated for a few years and that can reduce and prevent morbidity. However, preventive chemotherapy for schistosomiasis is only required in 52 endemic countries with moderate to high transmission.

The economic and health effects of schistosomiasis are considerable, since it causes disability. In children it can cause anemia, growth retardation and learning problems, although the effects are usually reversible with the treatment. Chronic schistosomiasis can affect the ability to work and in some cases can be fatal. The number of deaths attributable to schistosomiasis is difficult to calculate due to the existence of related hidden pathologies such as liver and kidney failure and bladder cancer. The WHO estimated that the figure was 200,000 deaths per year, but it may have declined significantly thanks to the expansion of large-scale prophylactic chemotherapy campaigns during the last decade.

Due to the globalized context in which we live, it is important to note the increase in cases of imported schistosomiasis as a consequence of international mobility. Whether by travelers, expatriates for work reasons or by South-North emigration, it is increasingly common to find cases of schistosomiasis in non-endemic countries.

So far, 20 Schistosoma species classified into 4 groups have been identified:

S. haematobium group: S. haematobium, S. intercalatum, S. bovis, S. matthei, S. curassoni, S. margrebowiei, S. leiperi.

S. mansoni group: S. mansoni, S. rodhaini, S. hippopotami, S. edwardiense.

 

S. japonicum group: S. japonicum, S. mekongi, S. malayensis, S. sinensium, S. ovuncatum.

S. indicum group: S. indicum, S. nasale, S. spindale, S. incognitum.

Among them are the five large species of blood staves that mainly affect people and that produce schistosomiasis in humans, each with its respective clinical manifestations:

Schistosoma mansoni and Schistosoma intercalatum - causative agents of intestinal schistosomiasis.

Schistosoma haematobium - cause of urinary schistosomiasis.

Schistosoma japonicum and Schistosoma mekongi - causing the Asian variety of schistosomiasis.

Epidemiology

Schistosomiasis is prevalent in tropical and subtropical regions, especially in poor communities without access to safe drinking water or adequate sanitation. It is estimated that at least 92% of people who need treatment for schistosomiasis live in Africa.

S. haematobium and S. mansoni are the most important species from a global point of view since between the two they represent more than 90% of the cases of schistosomiasis in the world. The first is the only causative agent of urogenital schistosomiasis and accounts for more than half of the cases of the disease. On the other hand, S. mansoni is the most important species in terms of morbidity and geographic distribution causing intestinal and hepatic schistosomiasis, being the only species present in the Americas.

For a correct transmission of schistosomiasis to occur, a series of conditions must occur. The first and most important is that there are populations of mollusks (snails) capable of maintaining the biological cycle and transmitting the disease. These hosts are resistant to drought, pollution, and climate changes. If a snail species that has natural resistance to infection is found in an area, the transmission rate will be lower. Normally, the populations of snails are stable and the disease has an endemic and local character. However, changes in hydrographic conditions such as floods or constructions of large dams (such as that of Aswan in Egypt or different dams in Nigeria) have allowed snails to proliferate and expand, bringing the disease to non-endemic areas of schistosomiasis. Another important characteristic for transmission is its association with underdevelopment. So that the life cycle of the parasite continues to be maintained, contamination of the water with feces and urine is required due to the lack of adequate sanitation.

Exposure to contaminated water is usually associated with daily and routine activities such as washing utensils and bathing, as well as activities related to agriculture, fishing or livestock. Contrary to what might be expected, the rural-urban demographic transition has not eliminated the disease, but the transmission of schistosomiasis is maintained in the peripheries of the large cities of South America and Africa.

Clinical characteristics of schistosomiasis

Schistosomiasis progresses in 3 distinct phases: phase of onset, acute phase and chronic phase.

Start phase

In the place of percutaneous penetration of the cercariae of the schistosome, a maculopapular, erythematous rash occurs, characterized by elevated lesions of 1 cm to 3 cm.

Acute schistosomiasis (Katayama syndrome)

Symptoms are mediated by the immune complex and usually begin with the deposition of schistosome eggs in host tissues. Symptoms may include fever, malaise, myalgias, fatigue, nonproductive cough, diarrhea (with or without blood), hematuria (S. haematobium), and right upper quadrant abdominal pain. Acute schistosomiasis is seen in people who are infected for the first time when they travel to endemic areas. In the case of S. japonicum, it is also associated with a superinfection or a hypersensitivity reaction in previously infected persons. The differential diagnosis includes gastroenteritis; Hepatitis A, B and C; infection with the human immunodeficiency virus (HIV); Salmonellosis and urinary tract infections.

Chronic and advanced disease

Mature schistosome infections are associated with a chronic local inflammatory response to schistosome eggs trapped in host tissues, which can lead to inflammation and obstruction of the urinary tract (S. haematobium) or to intestinal diseases, hepatosplenic inflammation, and fibrosis. hepatic (S mansoni, S intercalatum, S. japonicum, S. mekongi).

Main clinical forms of schistosomiasis:

Gastrointestinal and liver disease

The eggs retained in the wall of the intestine induce inflammation, hyperplasia, ulceration, the formation of microabscesses and polyposis. Symptoms of gastrointestinal disease include colic pain in the hypogastrium or pain in the left iliac fossa, diarrhea (especially in children), which may alternate with constipation, and hematochezia (blood in the stool). Severe chronic intestinal disease can lead to stenosis of the colon or rectum. Colonic polyposis can manifest as a protein losing enteropathy. An inflammatory mass in the colon can even mimic cancer.

The eggs of S. mansoni and S. japonicum embolize in the liver, where the granulomatous inflammatory response induces a presinusoidal inflammatory process and periportal fibrosis, associated with a persistent and severe infection, which may take many years to develop.

Genitourinary disease

Urinary tract disease develops after infection with S. haematobium and granulomatous inflammation is the response to the deposition of eggs in the tissues of the urinary tract (bladder and ureter). Hematuria appears 10 to 12 weeks after infection and is the first sign of established disease. Dysuria and hematuria occur both at the beginning and in the late stages of the disease. The final manifestations of the disease are proteinuria (often as nephrotic syndrome), calcification of the bladder, urethral obstruction, secondary bacterial infection of the urinary tract, renal colic, hydronephrosis and renal failure. In children there may be structural abnormalities of the urinary tract.

In Egypt and other parts of Africa, epidemiological studies have associated schistosomiasis with squamous cell carcinoma of the bladder. In recent decades, in Egypt, the incidence of bladder cancer has decreased in parallel with the decrease in the prevalence of schistosomiasis. Squamous cell carcinoma of the bladder associated with S. haematobium tends to be well differentiated and give local metastases. S. haematobium causes genital diseases in approximately one third of infected women.

Diagnosis

Currently, several diagnostic methods are available, including parasitological methods based on microscopy, immunology and molecular microbiology. They can be grouped into three main categories: (a) microscopic parasitological diagnosis, based on the observation of eggs of these helminths, (b) immunological diagnosis based on the detection of specific antibodies, and (c) molecular diagnosis based on nucleic acid detection.

a) Parasitological diagnosis

The parasitological methods were the first used for the diagnosis of schistosomiasis. They are based on the detection of eggs in feces (in the case of intestinal and hepatic schistosomiasis) or in urine (in the case of urogenital schistosomiasis). They are not appropriate for the early diagnosis of the disease, since egg laying begins at least at the fourth week post-infection.

For the microscopic detection of eggs in faeces, the Kato-Katz technique is used (currently recommended by the WHO as reference method -gold standard-). It is simple, low cost, very specific and allows to estimate the intensity of the infection. However, when the parasitic load is low the sensitivity of the test decreases markedly. However, the sensitivity can be improved by increasing the number of extensions examined in samples collected on consecutive days.

For the detection of S. haematobium eggs, filtration or sedimentation/centrifugation of the urine and subsequent microscopic observation are used. As in the detection of stool eggs, the main problem is low sensitivity, especially in mild infections. On the other hand, the presence of proteinuria and mainly hematuria are indicators that have been used in the diagnosis of urogenital schistosomiasis. However, hematuria is not a specific sign of schistosomiasis since it can occur in other pathologies.

b) Immunological diagnosis

The immunological diagnosis consists in the detection of anti-schistosome antibodies or the detection of schistosome antigens in different fluids (urine, serum, plasma or sputum). There is a wide variety of diagnostic tests with different levels of sophistication. These methods are particularly useful when parasitological tests are not effective, especially in situations where the parasitic load is very low.

Many of the immunological techniques have been developed for the detection of antibodies against the different phases of the parasite and are capable of diagnosing S. mansoni and S. haematobium infections, being useful in areas of low endemicity.

Circumoval precipitation test (COPT), detects serum antibodies from infected patients using lyophilized parasite eggs. It has turned out to be sensitive and specific and using it together with microscopy has greatly improved the effectiveness of the diagnosis. The main disadvantages of the COPT are the complexity of the technique and that the result continues to be positive once the parasite has been eliminated from the organism.

Indirect immunofluorescence test (IIF), detects antibodies in serum using sections of adult helminths, eggs or cercariae as an antigen and observing the results by fluorescence microscopy. It has been used alone or in combination with other diagnostic methods in areas of low prevalence. This test is difficult to apply since this type of microscopy and the reagents represent a barrier for its use in endemic areas.

Indirect hemagglutination test (IH), is able to detect antibodies from infected patients using erythrocytes coated with schistosome antigens. Due to its simplicity, this test has been used to study prevalence and surveillance in endemic areas. However, in addition to the elevated antibody titer after treatment, it has high cross reactivity with other helminth species, which implies low specificity.

ELISA tests, which used at first raw extracts of adult helminths or eggs as antigens, giving cross-reactivity with other helminths. Subsequently, ELISA tests were developed with proteins or fractions of purified proteins to improve their specificity. This test shows the same sensitivity as the Kato-Katz test (K-K) for the diagnosis of S. mansoni and the urine leakage test for S. haematobium. However, it is necessary to evaluate this test in other types of situations, such as in areas of low endemicity or after a post-treatment campaign.

 Currently there are different commercial kits based on the detection of antibodies (ELISA and IHA), used in clinical practice particularly in non-endemic countries, to diagnose cases of imported schistosomiasis. However, the specificity of these kits remains low.

c) Diagnosis based on the molecular detection of nucleic acids

The molecular diagnosis consists in the detection of nucleic acids (DNA) by means of amplification techniques, mainly the polymerase chain reaction (PCR) with all its variants. The detection of schistosome DNA in stool samples, urine or tissue biopsies has been used as highly sensitive and specific diagnosis.

Conventional PCR is more sensitive than microscopy, both in feces for the diagnosis of S. mansoni, and in urine for the diagnosis of S. haematobium. Common sequences can be amplified from specific primers for the genes coding for the 28S nucleic acid of the ribosomal RNA of S. haematobium, S. mansoni and S. intercalatum, in urine samples, in order to make the diagnosis of the 3 imported species of schistosomiasis. Recent studies show a high diagnostic efficiency to detect DNA of all schistosome species in urine and feces samples but there seems to be less efficacy in serum samples.

In turn, the sensitivity of the test has been increased by studying mitochondrial genes, due to the high number of copies that exist in each cell. Being unique sequences of each species, they present a high degree of specificity. The mitochondrial gene of cytochrome oxidase 1 (Cox1) is used as a molecular marker of the eggs of S. haematobium and S. mansoni in urine and feces samples.

Tests carried out in IVAMI:

  • Microscopic examination.
  • Molecular detection of the Schistosoma by amplification of the mitochondrial gene of cytochrome oxidase 1 (Cox1).
  • Identification of the species of Schistosoma spp. by sequencing the coding region of the mitochondrial gene of cytochrome oxidase 1 (Cox1).

Recommended sample:

  • Microscopic exam: stool or urine.
  • Molecular diagnosis: biopsies of tissues, urine, feces.

Conservation and shipment of the sample:

  • Samples must be received refrigerated or frozen and their transport time must be less than 48 hours.

Delivery of results:

  • Microscopic examination: 24 hours.
  • Molecular detection of Schistosoma spp. DNA: 24 to 48 hours.
  • Identification of the species of Schistosoma spp. by sequencing the coding region of the mitochondrial gene of cytochrome oxidase 1 (Cox1): 48 to 72 hours.

Cost of the test:

  • Sequencing of the coding region of the mitochondrial gene of cytochrome oxidase 1 (Cox1): Consult to ivami@ivami.com.