Sequence Diversity in tRNA Gene Locus A-L among Iranian Isolates of Entamoeba dispar

Background

A number of methods for detecting diversity in Entamoeba have been described over the years. In the present study the genetic polymorphism of noncoding locus A-L was analyzed using PCR and sequencing in order to clarify the genotypic differences among E. dispar isolates.

Methods

A total of 28 E. dispar from patients with gastrointestinal symptoms were determined and the genomic DNA was extracted directly from stool. For genotype analysis; Locus A-L was amplified by PCR and PCR products were sequenced. The sequences obtained were edited manually and aligned using Gene Runner software.

Results

With sequencing of PCR products a reliable genetic diversity in size, number and position of the repeat units were observed among the Iranian E. dispar isolates in locus A-L gene. Sequences showed variation in length from 448bp to 507bp and seven distinct types were identified.

Conclusion

The genetic diversity of loci like A-L shows them to be suitable for epidemiological studies such as the characterization of the routes of transmission of these parasites in Iran.     

Molecular differentiation of Entamoeba spp. in a rural community of Loja province, South Ecuador

Although previous epidemiological surveys in Ecuador indicate the presence of Entamoeba histolytica, prevalence data of this parasite remain scarce. Most of the studies were based on microscopic examination, which does not allow a morphological differentiation from the non-pathogenic Ent. dispar and Ent. moshkovskii. In the present study, 674 stool samples from a South Ecuadorian rural community were screened for Entamoeba spp. Subsequently, molecular identification was performed on 101 samples containing Ent. histolytica/Ent. dispar/Ent. moshkovskii cysts. The study indicated the absence of Ent. histolytica in this South Ecuadorian community and confirmed the difficulty of differentiating Entamoeba spp. based on morphological features.     

An Annotated Checklist of the Human and Animal Entamoeba (Amoebida: Endamoebidae) Species- A Review Article

Background:

The number of valid of pathogen and non-pathogen species of Entamoeba has continuously increased in human and animals. This review is performed to provide an update list and some summarized information on Entamoeba species, which were identified up to the 2014.

Methods:

We evaluated the Entamoeba genus with a broad systematic review of the literature, books and electronic databases until February 2014. The synonyms, hosts, pathogenicity and geographical distribution of valid species were considered and recorded. Repeated and unrelated cases were excluded.

Results:

Totally 51 defined species of Entamoeba were found and arranged by the number of nuclei in mature cyst according to Levin’s grouping. Seven of these species within the 4 nucleate mature cysts group and 1 species with one nucleate mature cyst are pathogen. E. histolytica, E. invadence, E. rananrum and E. anatis causes lethal infection in human, reptiles, amphibians and brides respectively, four species causes non-lethal mild dysentery. The other species were non-pathogen and are important to differential diagnosis of amoebiasis.

Conclusion:

There are some unknown true species of Entamoeba that available information on the morphology, hosts, pathogenicity and distribution of them are still very limited and more considerable investigation will be needed in order to clarify the status of them.     

Emerging Intestinal Microsporidia Infection in HIV+/AIDS Patients in Iran: Microscopic and Molecular Detection

Background

Species of Microsporidia have been known as opportunistic obligate intracellular parasites particularly in immunocompromised patients. Enterocytozoon bieneusi is one of most prevalent intestinal microsporida parasites in HIV⁺/AIDS patients. In this study, intestinal microsporidia infection was determined in HIV⁺/AIDS patients using microscopic and molecular methods.

Methods

Stool samples were collected from HIV⁺/AIDS patients during 12 months. All of the stool specimens washed with PBS (pH: 7.5). Slim slides were prepared from each sample and were examined using light microscope with 1000X magnification. DNA extraction carried out in microscopic positive samples. DNA amplification and genus/species identification also performed by Nested-PCR and sequencing techniques.

Results

From 81 stool samples, 25 were infected with microsporidia species and E. bieneusi were identified in all of positive samples. No Encephalitozoon spp. was identified in 81 collected samples using specific primers.

Conclusion

E. bieneusi is the most prevalent intestinal microsporidia in immunocompromised patients of Iran. On the other hand, Nested-PCR using specific primers for ssu rRNA gene is an appropriate molecular method for identification of E. bieneusi.     

PCR-RFLP Analysis of 28 SrDNA for Specification of Fasciola gigantica (Cobbold, 1855) in the Infected Lymnaea auricularia (Linnaeus, 1785) Snails from Northwestern Iran

Background

Fasciolosis in livestock is a crucial concern in the globe, mainly due to its impact on human health. The aim of this study was to determine the rate of infection with Fasciola gigantica (Cobbold, 1855) larvae in the field-collected snails of Lymnaea auricularia (Linnaeus, 1785) from northwestern Iran using a molecular approach.

Methods

A total of 6,759 pond snails were collected from 28 freshwater bodies in West Azarbaijan. PCR was performed to amplify a 618-bp fragment of the nuclear 28 SrRNA gene of Fasciola. The PCR products were digested by AvaII restriction enzyme to create restriction fragment length polymorphism (RFLP) patterns specific for the detection of F. gigantica.

Results

Of the total collected snails 496 (7.34 %) were L. auricularia, among which 4.64% (23 out of 496) were infected with a Fasciola species according to the PCR analysis. Only 2.22% (11 out of 496) of the infected snails were from the mountainous areas. The highest Fasciola infection rate recorded in the snails of a single site was 1.81% (9 out of 496 snails). Based on the RFLP pattern, F. gigantica accounted for 2.42% of the infection rates in the study sites.

Conclusion

Application of PCR-RFLP was proven to be a useful approach for valid and robust detection of the infection with F. gigantica in its intermediate host snails. These findings may therefore be applicable for establishment of the control programs against dissemination of the infection in different regions.     

Molecular and Parasitological Study of Cryptosporidium Isolates From Cattle in Ilam,West of Iran

Background

Cryptosporidiosis is one of the most important parasitic infections in human and animals. This study was designed for survey on the prevalence of Cryptosporidium infection in farms of Ilam, west of Iran, using parasitology method and genotyping by Nested PCR-RFLP.

Methods

Fecal samples of 217 cattle were collected fresh and directly from the rectum of cattle. All of the samples were examined by microscopic observation after staining with modified Ziehl-Neelsen (MZN). Genomic DNA extracted by using EURx DNA kit. A Nested PCR-RFLP protocol amplifying 825 bp fragment of 18s rRNA gene conducted to differentiate species and genotyping of the isolates using SspI and VspI as restriction enzymes.

Results

The prevalence of Cryptosporidium infection in cattle using both methods is 3.68%. Most of the positive cattle were calves under six months. Species diagnosis carried out by digesting the secondary PCR product with SspI that C. parvum generated 3 visible bands of 448, 247 and 106 bp and digested by VspI restriction enzyme generated 2 visible bands of 628 and 104bp. In this investigation all of the positive samples were Cryptosporidium parvum.

Conclusion

C. parvum (bovine genotype) detected in all positive cattle samples in Ilam, west of Iran. The results of the present study can help for public health care systems to prevention and management of cryptosporidiosis in cattle and the assessment of cattle cryptosporidiosis as a reservoir for the human infection.     

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA,ITS2)

Background

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.

Methods

The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species.

Results

The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.

Conclusion

The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.     

First Molecular Identification of Sarcocystis ovicanis (Protozoa,Apicomplexa) in the Brain of Sheep in Iran

Background

The objective of the present study was to survey the presence of Sarcocystis in sheep’s brain in North Khorasan Province.

Methods

In general, 80 samples of sheep’s brain were collected from slaughtered sheep in slaughterhouses of North Khorasan Province. Tissue digestion method was used for observing bradyzoites in tissues. Histopathological processing tracing Sarcocystis and ensuing structural change in the brain tissue were conducted. PCR analysis was conducted on all the brain samples. Sequencing was done for one PCR product. Genotype was identified by Blast search and homology analysis.

Result

Sarcocystis spp. was found in one of the brain samples (1.25%) using tissue digestion method. The presence of bradyzoite was also confirmed in the prepared histopathological sections. PCR analysis was positive in one of samples. Genotyping of one sample proved that Sarcocystis species was Sarcocystis ovicanis and the nucleotide sequence of this parasite was deposited in the GenBank database under accession number No.{"type":"entrez-nucleotide","attrs":{"text":"KF489431","term_id":"540198437","term_text":"KF489431"}}KF489431.

Conclusion

Sarcocystis ovicanis can involve brain tissue of sheep and consequently causes clinical symptoms.     

Isolation and Identification of Naegleria Species from Environmental Water in Changchun,Northeastern China

Background

Naegleria is a free-living amoeba, and pathogenic Naegleria may pose a health risk to people exposed to recreational water. Our objective in this study was to determine if there are pathogenic amoebae in environmental water samples from Changchun, Northeastern China.

Methods

During July to September 2012, a total of 70 water samples were collected from Changchun, Northeastern China, and Naegleria was enriched by in vitro culture and detected by PCR using Naegleria genus-specific primers. Resulting PCR products were sequenced and phylogenetically analyzed to identify Naegleria species.

Results

Naegleria was detected in 65 (92.9%) of 70 water samples. DNA sequence and phylogenetic analyses based on the internal transcribed spacer (ITS) rDNA sequences revealed four Naegleria species, including N. pagei (n = 24) and N. Australiensis (n = 18), N. clarki (n = 13) and N. gruberi (n = 10), in which N. australiensis is pathogenic to mice. But the pathogenic species N. fowleri was not detected.

Conclusion

This is the first report on Naegleria species in Northeastern China, showing that almost all environmental water samples were contaminated with Naegleria, including N. pagei, N. Australiensis, N. clarki and N. gruberi, which should be considered a potential public health threat.     

Potentially Pathogenic Free-Living Amoebae in Contact Lenses of the Asymptomatic Contact Lens Wearers

Background

Free-living amoebae (FLA) including Acanthamoeba spp. and Hartmannella spp. are the causative agents of serious corneal infection especially within contact lens wearers. Thus contact lenses and their storage case could be a suitable niche for potentially pathogenic amoebae. The main objective of the present study was to evaluate the contamination of contact lenses to free living amoebae using morphological and sequencing based methods.

Methods

Overall, 90 volunteers provided their contact lenses. All volunteers wore soft contact lenses. Both lenses were cultured in the same plate. Forty-eight of the volunteers were medical and dentistry student and 42 were ophthalmology attendees of hospitals in Tehran, Iran. All of the samples were inoculated to non-nutrient medium and monitored daily for the outgrowth of the amoebae. PCR and sequencing were performed using various primer pairs.

Results

Of the 90 volunteers, 9 (10%) were positive for free-living amoebae outgrowth. Morphological analysis revealed that 3 isolates were belonged to Hartmannella genus according to small round cysts and 6 isolates were belonged to Acanthamoeba genus based on the star shape of endocysts. Sequencing revealed that Acanthamoeba belonged to T4, T3 and T5 genotype. Hartmannella were also belonged to vermiformis species.

Discussion

The presence of potentially pathogenic free living amoebae including Acanthamoeba and Hartmannella could be a high risk for people using soft contact lenses. These results revealed that improved clarification and professional recommendations for contact lens wearers is of utmost importance.     

Acanthamoeba species in Swimming Pools of Cairo,Egypt

Background

The free-living amoebae Acanthamoeba spp. have been recognized as etiologic agents of amoebic encephalitis, keratitis, otitis, lung lesions and other skin infections mainly in immuno-compromised individuals. The purpose of this study is to detect the presence of Acanthamoeba in swimming pools in Egypt using a polymerase chain reaction (PCR) method.

Methods

Water samples were collected from 10 different swimming pools in Cairo, Egypt. Samples were cultured on non-nutrient agar for the detection of Acanthamoeba isolates that were confirmed by PCR amplification using genus specific primers. The molecularly confirmed Acanthamoeba isolates were morphologically identified to the species level.

Results

Members of genus Acanthamoeba were detected in 49.2% of the examined swimming-pool water samples. Morphologically, six Acanthamoeba species were isolated from the examined swimming pool water namely A. polyphaga, A.castellanii, A. rhysodes, A. mauritaniensis, A. royreba and A. triangularis. All the identified species of Acanthamoeba were molecularly confirmed to be related to the genus Acanthamoeba.

Conclusion

The isolated species of Acanthamoeba could provoke variable degrees of infections to the swimmers. The culture method is cheaper and easier than PCR techniques that are faster for the detection of free-living amoebae     

A Sensitive and Specific PCR Based Method for Identification of Cryptosporidium Sp. Using New Primers from 18S Ribosomal RNA

Background

The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidiosis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.

Methods

A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neelsen staining method. Total DNA was extracted by QIA amp DNA stool mini kit. PCR and nested-PCR was carried out by using designed primers.

Results

Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopically were positive. The described primary and nested PCR method could detect all Cryptosporidium positive samples from human and cattle. Regards to suspected negative samples in primary PCR examination, the Nested PCR could approve two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was negative in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100%.

Conclusion

Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.     

Phylogenetic Location of the Spirometra Sparganum Isolates from China,Based on Sequences of 28S rDNA D1

Background

The phylogenetic location of Chinese Spirometra sparganum isolates remains unclear. The aim of this study was to explore the phylogenetic location of the Spirometra sparganum isolates from China.

Methods

The 28S ribosomal DNA (rDNA) D1 sequences of 14 Spirometra sparganum isolates collected from thirteen locations in China were analyzed by using Neighbor-Joining (NJ), maximum parsimony (MP) and Bayesian inference (BI), respectively. To investigate the deep variance of 28S rDNA D1 region among included species, the secondary structure of 28S rDNA D1 region was also calculated using the program RNA structure.

Results

The genus Spirometra as a monophyletic group was evidenced by two inference methods (MP and BI). All sequences within the genus Spirometra had a bulge of a cytosine residue (Bulge C) in the stem 13 of the secondary structure model of 28S rRNA D1 region. Varietal sites in sequences from all thirteen Chinese isolates were appeared in loops. In loops, adenine was the most abundant base (averagely 41.9%) followed by guanine (averagely 30.0%), and cytosine (averagely 15.1%). In stems, the average percentage of G + C (58.3%) was higher than the percentage of A + T (41.7%).

Conclusion

The ‘Bulge C’ in the stem 13 of the 28S rDNA D1 secondary structure could be as a suitable mark to identify the Spirometra species.     

Molecular Detection of Malaria in South Punjab with Higher Proportion of Mixed Infections

Background

Malaria is well known for its fatalities worldwide, Plasmodium vivax and the Plasmodium falciparum are the two important species of malaria reported from Pakistan and creating lots of morbidities across the country.

Method

Study was conducted to determine the Surveillance of malaria in South Punjab by microscopy and Polymerase chain reaction (PCR).

Result

samples out of 100 patients were found positive for malarial parasites. One patient was found with mixed infection, whereas P. falciparum and P. vivax infections were detected in 17 and 22 patients, respectively. In nested PCR, genus-specific primers for Plasmodium species. in round 1 and species-specific primers for P. falciparum and P. vivax in round 2 were used. By the application of PCR 41% were found to be infected by Plasmodium spp. Among Plasmodium positive patients: mixed, P. falciparum and P. vivax infection were detected in 10, 15 and 16 patients, respectively. Thirty nine microscopically positive patients confirmed to have Plasmodium spp. One negative by PCR, 2 microscopically negative patients had shown Plasmodium spp. infection (P. falciparum and P. vivax) by PCR. In total samples, P. falciparum, P. vivax and mixed infection accounted for 36.6%, 39.0% and 24.3%, respectively.

Conclusion

Microscopy was found deficient for interpretation of mixed infections, low parasitaemia, and species specific diagnosis. The sensitivity, specificity and efficacy of nested PCR was calculated 95%, 98% and 97%, respectively, showing PCR as a more effective and efficient diagnostic tool for malaria.     

Seroprevalence and Associated Risk Factors of Toxoplasma gondii in Pregnant Women Attending in Northwest Ethiopia

Background

Toxoplasmosis is a major public health problem among immuno-compromised individuals. This study aimed to determine the seroprevalence and associated risk factors of Toxoplasma gondii infection among pregnant women with and out HIV infections.

Methods

This cross sectional study was conducted among consecutive 385 pregnant women attended Antenatal Clinic from May 2010 to October 2011 at the Gondar University Teaching Hospital, Northwest Ethiopia. Venous blood was collected from each pregnant woman for testing HIV-1/2 and anti- Toxoplasma antibodies using rapid test kits. Data were entered and analyzed using SPSS version 20 statistical package.

Results

The overall magnitude of T. gondii and HIV was 88.6% (341/385) and 11.2% (43/385), respectively. The seroprevalence of T. gondii was not different among HIV infected and non-infected pregnant women (88.4%, 38/ 43 vs 88.6%, 303/342). Keeping cats in house showed statistically significant association with seropositivity of toxoplasmosis (P<0.05).

Conclusion

Irrespective of HIV infection, high rate of T. gondii was detected among pregnant women. These high prevalences indicate the need for an intensified public health awareness to reduce both infections.     

Cryptosporidium Infection in Patients with Gastroenteritis in Sari,Iran

Background

Cryptosporidiosis is a common coccidian parasite infection in patients with diarrhea that has worldwide distribution especially in developed countries. Therefore, the aim of this study was to determine the occurrence of Cryptosporidium infection in patients with gastroenteritis admitted to hospitals of Mazandaran University of Medical Sciences by parasitological and molecular methods in Sari, Iran.

Methods

Stool samples were collected from 348 patients with gastroenteritis admitted to the hospitals of Medical University in the Sari and Ghaemshahr cities in Mazandaran Province, Northern Iran in 2010-2011. Oocysts of Cryptosporidium identified using Formalin-Ether concentration method and stained by Aacid-fast staining (AFS) and Auramine phenol fluorescence (APF). Genomic DAN extracted from microscopically positive samples and nested PCR -RFLP by using SSU rRNA that identifies of the species of cryptosporidium.

Results

In 348 patients with gastroenteritis, the most clinical symptoms were diarrhea, nausea, vomiting, dehydration, fever and weight loss. 2.3% (8 cases) of diarrheal samples tested by both microscopy and molecular methods were positive for the presence of cryptosporidium. Nested PCR products yielded unique bands of 846 bp, correspond to cryptosporidium. Species diagnosis carried out by digesting the secondary PCR product with SspI restriction enzyme, which noted 3 clearly bands of 449, 254, and 108 bp correspond to Cryptosporidium spp.

Conclusion

The results of present study on Cryptosporidium spp. in this area can make a background data for control programs and further molecular analyses. Thus, further work needs to determine the origin of Cryptosporidium species in this area.     

Genotyping of Echinococcus granulosus Isolates from Human Clinical Samples Based on Sequencing of Mitochondrial Genes in Iran,Tehran

Background

The present study was aimed to investigate molecular diversity of Echinococcus granulosus isolates collected from human clinical samples using two mitochondrial genes cox1 and nad1 in Iran.

Methods

Forty seven human hydatid cysts were collected through surgery from two hospitals in Tehran during 2010-2012. To determine the fertility of protoscoleces, the cyst fluids were subjected to morphological microscopic examinations. Protoscoleces were removed from each cyst and their total genomic DNAs were extracted. PCR was performed to amplify fragments of 450 and 400 base pair (bp) for cox1 and nad1 genes, respectively. Genotype diversity and sequence variation of the strains were studied by bioinformatics software and in comparison with those mtDNA sequences already deposited in GenBank.

Results

Sixteen, (53.3%), 13 (43.3%), and 1 (3.3%) samples were related to lung, liver, and spleen, respectively. The remained 17 unfertile samples were excluded from the study. From the 29 isolates, 86.7% (n=26) and 10% (n=3) were related to G1, and G3 genotypes, respectively. The sole isolate with G6 genotype was obtained from lung sample. Analysis of concatenated sequences of cox1+nad1 indicated the presence of 11 haplotypes among our strains that were related to genotypes G1 (n=9), G3 (n=1) and G6 (n=1).

Conclusion

In consistent to other reports from Iran, genotypes G1, G3, and G6 were observed in our human isolates. The rate of G3 genotype was however higher than other studies implying that human can be considered as a new appropriate host for G3 genotype. Further studies with more sample size from different geographic areas of Iran are needed for E. granulosus mapping.     

Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples

Background

Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples.

Methods

A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar''s χ² test, with consideration of a P-value of <0.05 as indication of significant difference.

Results

In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture.

Conclusion

Single PCR method amplifying a short (100bp) target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.     

Genetic Diversity of Human Blastocystis Isolates in Khorramabad,Central Iran

Background

There are some genetic differences in Blastocystis that show the existence of species or genotypes. One of these genes that help in identifying Blastocystis is SSUrRNA. The aim of this study was assessment of genetic diversity of Blastocystis by PCR with seven pairs of STS primers.

Methods

This study was done on 511 stool samples collected from patients referred to the health care centers of Khorramabad, Central Iran, in 2012. Genomic DNA was extracted and in order to determine the Blastocystis subtype in contaminated samples, seven pairs of primers STS (subtype specific sequence-tagged site) were used.

Results

Out of 511 samples, 33 (6.5%) samples were infected with Blastocystis. Subtype (ST) of 30 samples was identified and three subtypes 2, 3 and 4 were determined. Mix infection was reported 10% which 3.33% of the infection was for the mixture of ST 3 and ST5 and 6.67% was for the mixture of ST 2 and ST 3.

Conclusion

The predominant subtype was ST3 that is the main human subtype. The dominance of ST2 and 5 are important in this study. This superiority has been reported in some of the studies in ST 2 which is different from the studies in other countries, because they have announced priorities of the ST1 and ST6 after ST3.