Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

BACKGROUND: Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. METHODS: Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18 S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. RESULTS AND CONCLUSION: The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

Nyctanthes arbor-tristis alkaloids activates p53 independent cell death receptor and necroptosis pathways in HepG2 cells.

Nyctanthes arbor-tristis is a traditional medicinal plant with potential anti-cancer properties. In this study, crude and alkaloid extracts were prepared from different parts of the plant, and their cytotoxicity was evaluated on four different cancer cell lines. The alkaloid extracts from the leaf and fruit showed promising results, with the HepG2 cell line exhibiting significant cytotoxicity. The promising extracts were further studied for their apoptotic potential using various methods, including DNA fragmentation, TUNEL, Caspase-3 activity, Giemsa, and Hoechst staining. Our results indicated that the fruit extract had the highest apoptotic potential, with clear nuclear condensation, fragmentation, and apoptotic bodies observed. We also investigated the alteration of the Bax/Bcl-2 ratio both at the mRNA and protein levels. Our results showed a significant upregulation of the Bax gene and downregulation of the Bcl-2 gene for the fruit alkaloid extract. This indicates that the phenomenon of cell death expression might be following a p53-independent extrinsic pathway and Bax-activated caspase-independent AIF-mediated necroptosis in the HepG2 cancer cell line. Overall, our findings suggest that Nyctanthes arbor-tristis has potential as a therapeutic option for cancer treatment. The alkaloid extracts from the leaf and fruit may hold promise as a source of bioactive compounds for further development into anti-cancer agents. Further studies are needed to explore the underlying mechanisms of their cytotoxic and apoptotic effects and to evaluate their safety and efficacy in animal models and clinical trials.

Molecular genotyping of Indian blood group antigens amongst regular voluntary blood donors of Surat city, Gujarat, India.

BACKGROUND: There is paucity of data related to the prevalence of the rare blood group antigens amongst South Gujarat blood donor population due to unavailability and high cost of antisera. Therefore it is difficult to screen donors for such rare antigens by gold standard haemagglutination assay. The single nucleotide polymorphism (SNPs) of Ina and Inb antigens is the base of the PCR based detection methods that help to detect these alleles in regular voluntary blood donors. MATERIALS & METHODS: Blood samples of 200 unrelated regular voluntary blood donors wee collected. DNA was extracted using phenol-chloroform method and genotyped for Indian (Ina/IN*01, Inb/IN*02) blood group alleles by Sequence Specific PCR. Ina antigen positivity was confirmed by serology test. RESULTS: Four donors were found heterozygous for Ina antigen i.e. In (a + b+) by SS-PCR and their Ina positivity were confirmed by in-house polyclonal Anti-Ina reagent. SS-PCR was standardized using known heterozygous sample of a blood donor. The frequency of Ina antigen (2.0 %) was higher than Caucasians, lower than Iranians and Arabs while comparable to those reported among Indians of Mumbai city. CONCLUSION: In absence or unavailability of antisera particularly for low frequency alleles like Ina, such PCR based method would be extremely helpful to prepare rare donor registry by screening blood donors' at large scale. Red cells of Ina positive donors can be used as in-house reagent red cells for screening and identification of corresponding antibody.

Assessment of Long-Term in vitro Multiplied Human Wharton's Jelly-Derived Mesenchymal Stem Cells prior to Their Use in Clinical Administration.

The quantity of mesenchymal stem/stromal cells (MSCs) required for a particular therapy demands their subsequent expansion through ex vivo culture. During in vitro multiplication, they undergo replicative senescence which may alter their genetic stability. Therefore, this study was aimed to analyze cellular, molecular, and chromosomal alterations in Wharton's jelly-derived MSCs (WJ-MSCs) during their in vitro sequential passages, where WJ-MSCs were sequentially passaged up to P14 and cells were evaluated at an interval of P2, P6, P10, and P14. They were examined for their morphology, tumorigenicity, surface markers, stemness markers, DNA damage, chromosomal aberration, and telomere length. We have processed five full-term delivered human umbilical cord samples to obtain WJ-MSCs. Morphological appearance observed at initial stages was small fine spindle-shaped WJ-MSCs which were transformed to flat, long, and broader cells in later passages. The cell proliferation rate was gradually decreased after the 10th passage. WJ-MSCs have expressed stemness markers OCT-4 and NANOG, while they showed high expression of positive surface markers CD90 and CD105 and lower expression of CD34 and CD45. They were non-tumorigenic with slow cellular aging during subsequent passages. There was no chromosomal abnormality up to the 14th passage, while increase in comet score and decrease in telomere length were observed in later passages. Hence, our study suggests that early and middle passaged (less than P10) WJ-MSCs are good candidates for clinical administration for treatment.

Risk of hepatitis-E virus infections among blood donors in a regional blood transfusion centre in western India.

BACKGROUND: Hepatitis-E virus (HEV) is an emerging infectious threat to blood safety. The enormity of the transmission of HEV and its clinical consequence are issues currently under debate. This study aimed to evaluate the prevalence of HEV-RNA in blood donors in western India. MATERIALS AND METHODS: We screened 13 050 blood donors for HEV using HEV-RNA screening of 10 mini-pools using RealStar HEV RT-PCR Kit (95% limit of detection (LOD): 4.7 IU/ml). Furthermore, all HEV-RNA-positive donors were investigated for the presence of IgM/IgG antibody along with liver function tests. RESULTS: Of the 13 050 blood donations, 7 (0.53%) were found to be HEV-RNA positive, and the prevalence of HEV nucleic acid testing yield cases among blood donors was 1 in 1864. All seven HEV-RNA-positive samples were tested with anti-HEV IgM and anti-HEV IgG antibodies; this resulted in two (28.5%) positive anti-HEV IgM and two (28.5%) positive anti-HEV IgG antibodies. Hepatic activity was measured, with two of seven HEV-RNA-positive donors demonstrating abnormal serum glutamic oxaloacetic transaminase (SGOT) andserum glutamic pyruvic transaminase (SGPT). Two HEV-RNA-positive blood donors who had abnormal SGOT and SGPT were found to have a high HEV viral load. Furthermore, we were able to follow up two HEV-RNA donors, and both were HEV-RNA positive and had anti-HEV IgM and anti-HEV IgG antibodies; moreover, their liver function tests were also abnormal. One of the HEV-RNA donors with high viral load did show hepatitis-E-like virus on electron microscopy. CONCLUSION: Our studies indicate that there is a significant risk of blood-borne transmission of HEV. This finding may help to provide a direction towards the safety of blood transfusions in clinical settings in countries like India, which fall under the endemic category for HEV infection.

Comparison of serology and molecular detection of common red cell antigens in multitransfused thalassemia major and sickle cell disease patients.

BACKGROUND: Serological testing for extended RHCcEe, Kell, Kidd and Duffy blood grouping from multitransfused patients may not give correct blood grouping of the recipient. Hence molecular testing for these blood groups was compared with serological groups in a cohort of multitransfused thalassemia mjor and sickle cell anaemia patients. OBJECTIVE: Molecular genotyping of antigens of Rh (D, C, c, E, e), Kell (K, k), Duffy (Fya, Fyb) and Kidd (Jka, Jkb) blood group antigens by PCR and PCR-RFLP methods and comparison of predicted genotypes with their serological phenotypes. MATERIALS AND METHODS: A cohort of multitransfused thalassemia and sickle cell anemia patient were serologically and molecularly tested for RHCc, RHEe, K, k Fya, Fyb, Jka and Jkb antigens and compared. Serological testing was done by tube agglutination and molecular testing was done either by allele specific PCR or by RFLP technique just before next transfusion. RESULTS: In more than 80% of the cases recipient's molecular testing blood groups were at variance with serologically tested blood groups (p < 0.0001). Mixed field reactions in serological typing were common. In sickle cell anemia patients no discrepancy was found. Molecular technique results were checked by Sanger's sequencing. DISCUSSION: Extended phenotyping in multitransfused thalassemia patients by serological technique often donot detect the exact red cell phenotype of the recipient and molecular techniques for such grouping is preferable, especially in multitransfused thalassemia patients where red cells from previous transfusions continues to be present in significant numbers whenever the testing is done.

Novel Deleterious Sequence Change in the NLRP12 Gene in a Child with the Autoinflammatory Syndrome, Joint Hypermobility and Cutis Laxa from India.

An otherwise healthy male child of 9 years presented with paroxysmal fever and diffuse abdominal pain along with the loss of appetite and nausea lasting for 3-4 days every 4-6 weeks in the last two years. He also has stretchable skin and hypermobile joints, inherited from his mother who never suffered any paroxysmal attack of the kind. Work up for acute intermittent porphyria, lead poisoning, and familial Mediterranean fever was negative. A novel harmful sequence change in the NLRP12 gene was detected, and a diagnosis of NLRP12 associated autoinflammatory syndrome was made. This sequence change within the NLRP12 gene causing disease has not yet been reported in the literature and is the first such a case reported from India.

Prevalence of Macrothrombocytopenia in Healthy College Students in Western India.

Macrothrombocytopenia is being increasingly described across the globe. There is paucity of data on the prevalence of this condition from different parts of India. 10,047 healthy college students from the city of Surat in western India were investigated for macrothrombocytopenia i.e. those with Mean platelet Volume of > 11 fL and platelet count of less than 150 × 109/L. ABO blood groups, complete blood counts, peripheral smear examination and haemoglobinopathy work up was also done. Siblings and parents of the macrothrombocytopenic individuals were also studied when available. Bleeding assessment tool of International society of thrombosis and haemostasis were applied to see if there were excessive bleeding in macrothrombocytropenia patients. One hundred and ninety-six students (1.95%) had asymptomatic macrothrombocytopenia. More female students (P < 0.0001) had this condition and blood group A was under represented (P = 0.019) with this condition. Prevalence of macrothrombocytopenia was not related to ethnic subgroups to which the students belonged to, nor was it linked to presence of any haemoglobinopathy gene. In 38 of the 52, 1st degree relatives studied macrothrombocytopenia was confirmed at least in one of them. Excessive bleeding in none of the individuals with macrothrombocytopenia was noted. Asymptomatic macrothrombocytopenia is rare in western parts of India and affects 1.95% of the healthy population. Females were over represented with this condition raising a suspicion of X linked dominant inheritance. Underrepresentation of blood group A in this condition requires further study.

A multiplex ARMS PCR approach to detection of common ß-globin gene mutations.

BACKGROUND: ß-thalassaemia is a group of inherited single-gene disorders worldwide. Each ethnic population has its own common mutations. Heterogeneity of ß-thalassaemia mutations in multi-ethnic population of Surat, makes molecular diagnosis expensive and time consuming. METHODS: Specific primers were used to differentiate four common mutations, IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G), by a simple PCR involving a multiplex amplification refractory mutation system. RESULTS: Several high prevalence ß-Thalassemia trait groups constituted by Muslims, Patels, Sindhis, ModhBanias, and Mahayavanshi. Four most common mutations detected in them are IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G). We identified each of these ß-thalassemia mutations in multiplexed ARMS from positive control samples. Our multiplex-ARMS-PCR system was first standardized on positive DNA samples with above known four most common ß-thalassemia mutations, and these positive samples had been diagnosed with ß-thalassemia and also all these samples belonged to Surat ethnic groups. The system was subsequently tested on 110 blood samples from different ethnic backgrounds with unknown ß-thalassemia mutations which were in all specimens. CONCLUSION: The ARMS multiplex system was found reliable, cost effective, fast and most applicable for mutation screening of Thalassemia in Surat populations.