Tag Archive for genotype

Genotyping Hepatitis C Virus

The Hepatitis C virus (HCV) is a single-stranded RNA virus, with a genome of 9,500 nucleotides. The prevalence of HCV among the general population of Ethiopia is not known. Nonetheless, the impression at the lab is that prevalence is high. Egypt is know to have one of the highest HCV prevalence in the world. It is postulated that this is the result of mass treatment with praziquantel for schistosomiasis using contaminated syringes in the past. Mass treatment against schistosomiasis also occurred in Ethiopia, however, oral treatment were used. Intravenous drug users are also unknown of. Would it be facial tattoos, high number of cesarians, blood transfusions? The primarily mode of transmission is still a mystery.

About 75 – 85% of HCV-infected individuals develop chronic hepatitis, with up to 20% of chronically infected individuals developing cirrhosis. In patients with cirrhosis, the incidence of hepatocellular carcinoma is 1 – 4% per year. Genotype 1 and genotype 4 responds less well to interferon-based anti-HCV drug treatment than genotypes 2 and 3, especially in patients of African descent. Therefore, genotyping of HCV is recommended before the start of antiviral therapy.

Thanks to the lobbying of, among other PharmAccess, treatment for HCV is becoming more affordable in developing countries. Still, hepatitis C viral loads and genotyping is mandatory for managing the treatment.

The Abbott RealTime HCV genotype II assay is an RT-PCR assay for use with the Abbott mSample Preparation System reagents and with the Abbott m2000sp and m2000rt instruments for viral count and genotyping of the viral (HCV) RNA in human blood, serum plasma (EDTA) or tissue donors of HCV-infected individuals. The Abbott RealTime HCV assay is intended for use as an aid in the management of HCV-infected patients undergoing antiviral therapy. The assay measures HCV RNA levels at baseline and during treatment and can be utilized to predict sustained and non-sustained virological response to HCV therapy.

During the verification tests, some remarkable observations were made. There was failure of the internal controls and mismatch at low viral loads. But that’s not the interesting one.

The genotype 4e at the reference laboratory in Germany (most likely they use the Roche system) corresponds with a genotype 1 & 4 double reaction at our lab in Addis Ababa. Genotype 4e is a strain of which little is known. There are only few and partial RNA sequences available for this genotype. It is therefore hypothesized that the primers/probes for gt1 detection from the Abbott assay cross match with genotype 4e.

HCV genotyping

Conclusion

In case, with the Abbott system, a reaction occurs in which both genotype 1 & 4 are detected, the results should be interpreted as HCV genotype 4.

genosubtype-coverage

Cryptosporidium gp60 subtyping.

Cryptosporidium spp. are coccidian protozoan parasites that infect various vertebrate and invertebrate hosts. At least seven Cryptosporidium species have been associated with gastro-intestinal disease in humans: C. hominis, C. parvum, C. meleagridis, C felis, C. canis, C. suis and C. muris. Of these, C. hominis and C. parvum are the two species found most often in humans. Cryptosporidium-detection methods based on PCR has been an important instrument for studying the taxonomy and transmission of the parasite. For the differentiation of Cryptosporidium species/genotypes, different types of molecular tools have been used. In many studies the subtyping is performed using the Small SubUnit ribosomal RNA (SSU rRNA). Another genetic target is an oocyst wall protein (COWP) gene. However, the COWP gene is less useful because of its narrow specificity (Xiao, 2010). One gene that is becoming popular for subtyping Cryptosporidium is the 60 kDa glycoprotein (gp60). A Cryptosporidium subtype will end up with a name such as “IaA23R4” or “IIdA18G1”. In this blog I will explain the methodology behind this classification of Cryptosporidum subtypes based on gp60 analysis.

The gp60 gene has tandem repeats of the serine-coding trinucleotide: TCA, TCG or TCT. These tandem repeats have varying lengths. The DNA sequence outside these repeats also shows considerable differences; they are used to categorize C. parvum and C. hominis each into several subtype families. The name of gp60 subtypes start by designating “I” for C. hominis and “II” for C. parvum. Subsequently, each new subtype family is given a letter. Ia, Ib, Id, etc. for C. hominis and IIa, IIb, IIc, etc. for C. parvum. What follows then is the letter and number of the trinucleotide repeats. TCA is represented by the letter A, TCG is represented by the letter G and TCT by the letter T. The letter R is designated for any other repeat.

So lets look at some examples. In this text-file you may find some Cryptosporidium subtype DNA sequences that I used: three C. hominis subtypes and three C. parvum subtypes. 

cryp alignment Screen Shot 2013-08-13 at 22.27.46

By the way, here are two useful online tools for DNA sequence analyses, which I often use. For DNA aligment: Multalin …and to draw phylogenetic trees: ClustalW2

For the first example I took the DNA sequence with GenBank Accession number AF164502.

Cryp subtype1.001

The dominant trinucleotide repeat is TCA (in red). The serine trinucleotide is repeated 23 times. Another repeat is with AAGACGGTGGTAAGG (in green). The sequence is repeated three times. However, the repeat is followed by a DNA sequence similar to the preceding one: AAACGGTGAAGG. In this case the researcher has decided that it should be part of the tandem repeat. So here we end up with a ‘Cryptosporidium hominis’ of the subtype family ‘Ia’ followed by ‘A23R4’.

Another one. Let’s take Accession number AY262034, C. parvum subtype family IIa.

Cryp subtype2.002

The dominant trinucleotide is TCA with 15 repeats (purple). In between are two TCG’s (green). Furthermore, this subtype also has one ‘R’ (other) repeat: ACATCA (orange). Huh? One repeat? Well, apparently there are similar subtypes with two copies of ACATCA. Hence, R=1 in this one, which makes IIaA15G2R1.

Ok, last one. Genbank number AF164491. A C. parvum beloning to subtype family IIc.

Cryp subtype3.003

Five times TCA (blue) and three times TCG (pink). In the IIc subtype family, all strains have A5G3 repeats. However, downstream in the 3’ region some strains are different. The diverged strains have subsequently an extension. In this case it’s IIcA5G3a.

Subtyping microbiological strains always had a slice of guesswork. How many nucleotide substitutions does it need to make the strain to be a new genotype? When would you denote a sequence to be a repeat? Molecular genotyping has provided many advantages over conventional typing methods. Sometimes it might seem that the resolution for differentiating strains with molecular methods are either to narrow or to broad. Still, as we see with the typing for Cryptosporidium strains, it takes a lot of effort, thought slowly a consensus is appearing.

Want to know more about sub typing Cryptosporidum strain? A good start is the article ‘Molecular epidemiology of cryptosporidiosis: An update‘ by Lihua Xiao in Experimental Parasitology (124 (2010) 80-90). Or if you have any specific questions, feel free to contact me.

Thesis: Molecular Detection of Intestinal Parasites for Clinical Diagnosis and Epidemiology

thesis

Summary

The detection of intestinal parasitic infections for routine diagnosis and for epidemiological research still depends mainly on microscopical examination of stool samples for the identification of helminth eggs and protozoan trophozoites and cysts. Because microscopy has several limitations, additional diagnostic methods (e.g. culture, antigen/antibody detection) have been accessed to surpass obstacles in detection and characterization of intestinal parasites. Although such additional methods increases sensitivity, the amount of hands-on time accumulates substantially.

During the last years remarkable progress has been made on another diagnostic methods that are based on Polymerase Chain Reaction (PCR) technique. DNA isolation from stool can be processed in a semi- or fully-automated system where after specific DNA of multiple targets can be simultaneously amplified, visualized and semi-quantified in a closed tube system with multiplex real-time PCR. The molecular diagnostic approach was merged with an alternative diagnostic strategy where clusters of patients with shared characteristics are routinely screened for a selected number of parasites species. This new diagnostic strategy was assessed for the routine diagnosis and epidemiology of intestinal parasites in patients consulting general practitioners.

Samples of a general practice patient group were processed on a real-time PCR panel for the detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium (HGC PCR). The retrospective analysis with HGC PCR was compared with the results obtained with those of routinely performed microscopy. The results revealed that significant numbers of G. lamblia and Cryptosporidium infections remained undetected with microscopy. On the other hand, the parasites that have been detected with microscopy but not with real-time PCR consisted mainly of non-pathogenic parasites and Dientamoeba fragilis, although the pathogenicity of the latter is disputed. The results showed that, compared to the molecular approach, microscopy provided limited additional value in routine diagnosis of general practice patients with gastro-intestinal complaints, even with the use of multiple sampling procedure of faecal specimens in combination with fixatives (also referred to as the triple faeces test (TFT) procedure). The introduction of the TFT method in Dutch laboratories resulted of D. fragilis being increasingly diagnosed. Although D. fragilis has been suggested to be a potential pathogen in children, more studies are needed to support this statement. The introduction of a D. fragilis real-time PCR in routine diagnostics can help to elucidate the pathogenicity of this parasite. Cases of clinical E. histolytica infections (amoebiasis) in The Netherlands are very rare and can be associated to travel in areas where the parasite is endemic. Still, in several cases the source of infection could not be explained. Unselected screening for this parasite can be appreciated because of its major clinical importance for the patient and its potential to spread among household members.

Many intestinal parasites species, such as hookworms or Cyclospora cayetanensis, have adapted their transmission pattern and life cycle to specific environmental factors. Being bound to these factors, these parasites appear only occasionally in The Netherlands and surrounding countries; usually they are taken along by travellers returning from countries where the parasites are endemic. Real time PCRs for more exotic parasites can be used as extensions of basic molecular assays in clinical diagnostic settings. The relative frequency of a wide variety of intestinal parasitic diseases has been assessed in a population of travellers (such as tourists, immigrants, expats, etc.) using two different diagnostic approaches. The “overall view” of the conventional approach (i.e. microscopy combined with antigen tests) was compared with the molecular diagnostic approach, where only those species that are targeted in the assay are detected. The molecular diagnostic method showed more effective in detecting the targeted parasites than the conventional diagnostic approach, in particular E. histolytica and Strongyloides stercoralis for which additional diagnostic techniques are needed op top of microscopy. Only few additional parasite species have been detected with microscopy, while many more unexplained causes of gastro-intestinal complaints might be resolved by the application of additional real-time PCR targets.

Using the molecular hight throughput screening system in combination with a simple collection procedure for stool specimens, valuable data has been produced that has yielded new insights in the epidemiology of several parasitic diseases.

The prevalence of Cryptosporidium in Dutch patients with gastro-intestinal complaints attending their general practitioner has so far exceeded the figures in previous studies. The highest infection rate has been detected among children aged under five years with a peak in the month of September; almost one-third of them had been infected with Cryptosporidium. Microscopic examination for Cryptosporidium, which requires an additional staining procedure of the faecal smear, was specifically requested by the general practitioner twenty-one times and was found positive in 13 cases once, whereas with HGC PCR 80 cases were detected. The lack of request for additional diagnostic procedures is not the only reason for missing infections. Basic microscopic stool examination has often not been requested, leaving a substantial number of gastro-intestinal parasitic infections undiagnosed.

Symptoms of G. lamblia infections are highly variable, and can range from asymptomatic to the presence of severe gastro-intestinal complaints. Data in this thesis showed that, although the G. lamblia Cycle-threshold (Ct)-values correlated with the number of specified gastro-intestinal complaints, the parasite was still detected in 4.7% of persons who did not have complaints. The reasons for the clinical heterogeneity of G. lamblia infections are not fully understood. Although the epidemiological role of G. lamblia assemblages (i.e. group of genotypes) had been suggested as an important factor associated with gastro-intestinal complaints, this could not be proven for adults. Nevertheless, in children the assemblages might still be relevant for the clinical presentation of the patient.

In this thesis, additional real-time PCRs have been evaluated to cover the most important intestinal parasite species for epidemiological studies and to be used as extensions of basic molecular diagnostic assays in clinical settings. Isospora belli (recently renamed as Cystoisospora belli), Encephalitozoon intestinalis and Enterocytozoon bieneusi are opportunistic pathogens that can cause life-threatening diarrhoea and malabsorption, in immuno-compromised patients. With increased awareness, infections with E. bieneusi are diagnosed not only in immune-compromised patients, but also showed presence in asymptomatic persons. Furthermore, the high prevalence of weak infections or low shedding of spores (high Ct-values) have been observed among populations in sub-Saharan- and East-African countries. The phylogenetic study on microsporidia infections in persons with different clinical backgrounds indicated a dynamic evolutionary process between genotypes of E. bieneusi. One specific genotype was restricted to transplantation patients receiving immuno-supressives and another genotype showed its preferential habitat in patients living with HIV/AIDS, which further emphasizes the predisposition for specific hosts by different E. bieneusi isolates.

Last but not least, real-time PCRs were developed for the detection of Schistosoma mansoni and Schistosoma haematobium. Both Schistosoma species could successfully be detected in stool samples with Ct-values correlating with the results of quantitative microscopy. S. haematobium could also be detected in DNA isolated both from urine and from cervico-vaginal lavages. Results indicated that real-time PCR may potentially serve as a gold standard to determine the prevalence and intensity of Schistosoma infections in surveys. The real-time PCR may even be provided as a diagnostic tool for urine, stool and other clinical samples. This might prove helpful in particular for the difficult to diagnose female genital schistosomiasis. The semi-quantitative outcome of the PCR might be used as a predictor in the disease pathology and help to determine if observed lesions are caused by the parasite eggs.

Full-text PDF

Chapter 8 – Characterization of Genotypes of Enterocytozoon bieneusi in Immunosuppressed and Immunocompetent Patient Groups.

Abstract

A retrospective phylogenetic analysis was performed on isolates of Enterocytozoon bieneusi to characterize the genotypes in different patient cohorts. Fifty-seven isolates, collected from patients living in Malawi and The Netherlands, were classified by age and immune status of the hosts. Sequence analysis of the internal transcribed spacer (ITS) region identified 16 genotypes; nine have not previously been described. Genotypes K and D were most prevalent among patient groups, whereas genotype C was restricted to transplantation patients receiving immunosupressives and genotype B showed a predisposition towards patients living with HIV/AIDS. Different genotypes showed more dispersion among isolates from Malawi compared with those from The Netherlands. A constructed map estimating the genealogy of the ITS region reveals a dynamic evolutionary process between the genotypes.

Full-text PDF

Robert J. ten Hove, Lisette van Lieshout, Mike B.J. Beadsworth, M. Arantza Perez, Klaartje Spee, Eric C.J. Claas, Jaco J. Verweij. The Journal of Eukaryotic Microbiology (2009)