Toxoplasmosis lymphadenopathy-A series of 8 cases diagnosed in a 2-year timeframe: Is it a needle in a haystack?

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite. Clinical presentation of infection depends on the age and immune status of the patient. In immunocompetent patient, it may present as a non-specific lymphadenopathy and self- limiting illness. In contrast, in immunocompromised patients it can be a life-threatening infection. We present a series of 8 cases of toxoplasma lymphadenitis diagnosed in our institute in last two years. Lymphadenopathy raises a suspicion of malignancy; however, diagnosis of reactive lymphadenitis often marks the end of diagnostic evaluation. Careful morphological evaluation, the classic triad, often can direct at a conclusive diagnosis. We are presenting a case series to draw attention to this entity as majority of the published literature is in the form of case report and serological surveys and very occasional study showing both the histopathology and serology in tandem.

Comparing two techniques of performing an epidural catheter-assisted epidural blood patch using a 20 ml syringe versus a 5 ml syringe and its effect on clotting time, the strength of clot retraction and haemolysis - A prospective in vitro study (EC-EBP study).

Background and Aims: Epidural blood patch (EBP) is performed by injecting autologous blood into the epidural space using a Tuohy needle. Certain clinical scenarios mandate an epidural catheter (EC)-assisted EBP. Collecting blood in a 20-ml versus 5-ml syringe appears to influence the quality of the clot. This in vitro study compared the techniques of performing the EC-assisted EBP using 20-ml versus 5-ml syringe on clotting time (CT), clot retraction (CR) and haemolysis. Methods: This in vitro study was performed in a haematology laboratory. Five consented adult healthy male volunteers donated blood. In the 5-ml syringe technique, blood was injected through an EC, and as it flowed out of the tip, it was collected at the beginning and the end of 1 min. With the 20-ml technique, blood was collected at the beginning and end of the first, second and third minute. The samples were tested for CT, CR and haemolysis by measuring the plasma-free haemoglobin (PFHb). Results: Five injections were made using a 5-ml syringe, and another five with a 20-ml syringe. Injection time was shorter in the 5-ml technique (80.80 ± 5.89 vs. 272 ± 28.4 s, P < 0.0001). With the 20-ml technique, CT progressively increased (>15 min), whereas, with the 5-ml syringe, the CT was normal. Both techniques caused mild, insignificant haemolysis (PFHb >0.005 g/dl), without affecting the quality of CR. Conclusion: EC-assisted EBP using a 5-ml syringe technique shortens the injection time and deposits fresh blood quickly without affecting CT and CR.

Laboratory characterization of obligate carriers of type 3 von Willebrand disease with a potential role for Platelet Function Analyzer (PFA-200).

INTRODUCTION: Type 3 von Willebrand disease (VWD) is a rare autosomal recessive disorder characterized by undetectable von Willebrand Antigen (VWF:Ag). Carriers of type 3 VWD carry one null allele and have von Willebrand factor (VWF) at about 50% of normal. The aim of this study was to characterize type 3 VWD carriers and to study the role of Platelet Function Analyzer (PFA-200) in this cohort. METHODS: This was a cross-sectional study where data were collected from carriers (parents/offspring) of type 3 VWD patients and evaluated with activated partial thromboplastin time, factor VIII, blood group, ristocetin cofactor assay (VWF:RCo), VWF:Ag, and closure time on PFA-200 with collagen/epinephrine (COL/EPI), and collagen/ADP (COL/ADP). RESULTS: One hundred carriers were included in the study of which 85 were included for PFA-200 analysis. The mean (SD) of VWF:Ag (IU/ml) and VWF:RCo (IU/ml) was 0.63 (0.24) and 0.61 (0.26), respectively. Among the 100 carriers, based on VWF levels (VWF:Ag and/or VWF:RCo) and bleeding history, there were 7 type 1 VWD, 10 type 2 VWD, 25 borderline VWF (0.30-0.50 IU/ml and no bleeding), and 58 normal VWF (>0.50 IU/ml). PFA-200 was prolonged in 71% of the carriers, all carriers with type 1 and type 2 VWD phenotype, 80% carriers with borderline VWF, and 59% with normal VWF. COL/EPI was more sensitive than COL/ADP and showed better correlation with VWF parameters than COL/ADP. CONCLUSION: Carriers of type 3 VWD can have a variable laboratory phenotype. PFA-200 showed good sensitivity among the carriers at VWF levels <0.50 IU/ml.

Breast milk contains red cell isohaemagglutinins: An observational study of 176 mothers.

BACKGROUND AND OBJECTIVES: Maternal antibodies are transferred to the child, predominantly IgG, via the transplacental route, and mostly IgA through breast milk. Cases reported by us and others have shown the transfer of red cell allo-antibodies through breast milk. This study was conducted to assess the presence of isohaemagglutinins in breast milk, the range of titres, and the correlation between breast milk and maternal plasma titres. MATERIALS AND METHODS: A total of 176 mothers were recruited in this study. Breast milk was collected after sufficient feeding was established and within 2-5 days of delivery in a sterile container without any anticoagulant. Antibody screen, identification and titres were performed on maternal plasma as well as breast milk. RESULTS: Anti-A and anti-B in breast milk corresponding to their respective maternal blood groups were found in all the samples. This study has shown titres in the breast milk of anti-A and anti-B ranging from 2 to 1024 in both saline and Coombs phases. There was no association between plasma and breast milk titres, thus making it impossible to predict which mother may potentially transfer a larger amount of these haemagglutinins. Isotypes of anti-A and anti-B were evaluated in both plasma and breast milk of 11 samples, which showed predominantly IgG in 7 (63.63%) and predominantly IgA in 4 (36.36%) samples. CONCLUSION: Our study demonstrates the presence of a wide range of titres for IgG antibodies of the ABO blood group system in breast milk. The clinical impact of this finding needs to be studied further, as it assumes great relevance in developing countries where anaemia continues to challenge young infants.

Diagnostic utility of flow cytometry based coated-platelets assay as a biomarker to predict thrombotic or hemorrhagic phenotype in acute stroke.

BACKGROUND: Coated-platelets are sub-population of platelets "coated" with highly procoagulant proteins and phosphatidylserine that sustains thrombin generation. They are produced upon dual agonist stimulation by collagen and thrombin. This study was conducted to assess if there was any difference in the levels of coated-platelets in patients with primary intracranial hemorrhage (PICH) and ischemic stroke due to large artery atherosclerosis (LAA) as compared to healthy controls, and to see if coated-platelet levels had any influence on the hemorrhagic transformation (HT) of ischemic stroke. METHODS: Coated-platelet levels were determined by flow cytometry using fluorescently tagged Annexin V antibody to identify phosphatidylserine exposed on the surface of platelets activated by dual agonists (convulxin and thrombin) in cross-sectional cohort of 75 patients with stroke and 34 controls. RESULTS: Patients with PICH (n = 35) had significantly lower coated-platelets than the controls (adjusted mean ± SE, 21.0 ± 1.9% vs. 36.1 ± 1.7%, p < 0.001), while patients with LAA (n = 30) had significantly higher coated-platelets than controls (adjusted mean ± SE, 51.9 ± 1.5% vs. 36.1 ± 1.7%, p < 0.001). Patients with subsequent HT of ischemic stroke (n = 10) had significantly lower coated-platelet levels at admission compared to those without HT (adjusted mean ± SE, 18.1 ± 2.6% vs. 51.9 ± 1.5%, p < 0.001). CONCLUSIONS: Coated-platelet levels are significantly different in patients with hemorrhagic and ischemic stroke as compared with controls. Lower levels of coated-platelets measured by flow cytometry may be earliest predictor of subsequent HT in patients with ischemic stroke even before the radiological changes suggestive of HT are visualized.

Plasma Von Willebrand Factor Levels Predict Survival in COVID-19 Patients Across the Entire Spectrum of Disease Severity.

BACKGROUND: Characterization of reticulo-endothelial activation in COVID-19 may guide treatment. OBJECTIVES: To assess reticulo-endothelial activation and its correlation with disease severity and death in patients across the entire spectrum of COVID-19 severity. METHODS: Consecutive hospitalized COVID-19 patients were studied, with similar number of patients in each disease severity category. Baseline serum ferritin, sCD163 (macrophage activation markers) and plasma von Willebrand factor (VWF) antigen (endothelial activation marker) levels were studied. Clinical parameters and plasma D-dimer levels were also studied. The study parameters were correlated with COVID-19 severity and survival. RESULTS: The 143 patients (104 males [80%], age 54 [42 - 65] years, median [inter-quartile range]) presented 4 (3-7) days after symptom onset. Thirty-four patients had mild disease, 36 had moderate disease, 36 had severe disease and 37 had critical disease at baseline. With increasing COVID-19 severity, ferritin, sCD163, VWF and D-dimer levels significantly increased at baseline, however, 139 patients had normal sCD163 levels. Of the reticulo-endothelial markers, VWF level independently correlated with COVID-19 severity and with survival. VWF level > 332.6 units/dl correlated with COVID-19 severity (odds ratio [OR]: 2.77 [95% confidence interval (C.I): 1.1 - 6.99], p value: 0.031) and in-hospital death (OR [95% CI]: 29.28 [5.2 - 165], p value < 0.001). CONCLUSIONS: Reticulo-endothelial activation markers increased incrementally with worsening COVID-19 severity. Baseline endothelial activation marker (VWF), and not macrophage activation markers, independently correlated with COVID-19 severity and death.

Sickle cells are not necessarily protective against falciparum- A case report.

The relation between sickle cell disease (SCD) and malaria is captivating where sickling of the infected red blood cells (RBCs) causes premature hemolysis and parasite death. Although patients with sickle cell trait are relatively protected, malaria can often lead to marked anemia in them due to hemolysis. We report an unusual case of a child with homozygous SCD presenting with falciparum malaria and had hyper parasitemia and severe anemia which completely resolved following treatment. Clinical suspicion in our case arose considering the endemic nature of malaria in our country. The two overlapping injuries to spleen reduced the clearance of parasites by the spleen as evidenced by high parasite load. Our case report reinforces malaria as a cause of clinical worsening of SCD and highlights the importance of a multifactorial approach in the management of worsening anemia in SCD.

Factors associated with mortality among moderate and severe patients with COVID-19 in India: a secondary analysis of a randomised controlled trial.

OBJECTIVE: Large data on the clinical characteristics and outcome of COVID-19 in the Indian population are scarce. We analysed the factors associated with mortality in a cohort of moderately and severely ill patients with COVID-19 enrolled in a randomised trial on convalescent plasma. DESIGN: Secondary analysis of data from a Phase II, Open Label, Randomized Controlled Trial to Assess the Safety and Efficacy of Convalescent Plasma to Limit COVID-19 Associated Complications in Moderate Disease. SETTING: 39 public and private hospitals across India during the study period from 22 April to 14 July 2020. PARTICIPANTS: Of the 464 patients recruited, two were lost to follow-up, nine withdrew consent and two patients did not receive the intervention after randomisation. The cohort of 451 participants with known outcome at 28 days was analysed. PRIMARY OUTCOME MEASURE: Factors associated with all-cause mortality at 28 days after enrolment. RESULTS: The mean (SD) age was 51±12.4 years; 76.7% were males. Admission Sequential Organ Failure Assessment score was 2.4±1.1. Non-invasive ventilation, invasive ventilation and vasopressor therapy were required in 98.9%, 8.4% and 4.0%, respectively. The 28-day mortality was 14.4%. Median time from symptom onset to hospital admission was similar in survivors (4 days; IQR 3-7) and non-survivors (4 days; IQR 3-6). Patients with two or more comorbidities had 2.25 (95% CI 1.18 to 4.29, p=0.014) times risk of death. When compared with survivors, admission interleukin-6 levels were higher (p<0.001) in non-survivors and increased further on day 3. On multivariable Fine and Gray model, severity of illness (subdistribution HR 1.22, 95% CI 1.11 to 1.35, p<0.001), PaO2/FiO2 ratio <100 (3.47, 1.64-7.37, p=0.001), neutrophil lymphocyte ratio >10 (9.97, 3.65-27.13, p<0.001), D-dimer >1.0 mg/L (2.50, 1.14-5.48, p=0.022), ferritin ≥500 ng/mL (2.67, 1.44-4.96, p=0.002) and lactate dehydrogenase ≥450 IU/L (2.96, 1.60-5.45, p=0.001) were significantly associated with death. CONCLUSION: In this cohort of moderately and severely ill patients with COVID-19, severity of illness, underlying comorbidities and elevated levels of inflammatory markers were significantly associated with death. TRIAL REGISTRATION NUMBER: CTRI/2020/04/024775.

Prevalence of HLA-B*27 subtypes in the Tamil population of India with Ankylosing spondylitis and its correlation with clinical features.

INTRODUCTION: HLA-B*27 is strongly associated with Ankylosing spondylitis (AS). Its subtypes show considerable geographic and ethnic difference. The main aim of this study was to assess the frequency of subtypes of HLA-B*27 in the Indian Tamil AS patients. METHODS AND MATERIALS: Adult AS patients positive for HLA-B*27 were considered for the study. The high-resolution typing to define HLA-B*27 subtypes were done using Invitrogen B kits from One Lambda (SeCore® Sequencing Kits, Thermo Fisher, United States). RESULTS AND CONCLUSION: Prevalence of subtypes identified were HLA-B*27:04 (52.2%), HLA-B*27:05 (41.6%), HLA-B*27:07 (3.5%) and HLA-B*27:02 (2.7%). All subtypes showed disease predisposition for males. The most common extra articular manifestation seen was enthesitis in HLA-B*27:04 and HLA-B*27:05. Uveitis was mainly associated with HLA-B*27:05 and dactylitis with HLA-B*27:04. A significant peripheral joints involvement for female and axial joint involvement for males was seen in HLA-B*27:04. Our study establishes the prevalence of HLA-B*27 subtypes and the associated clinical phenotypes among the Indian Tamil population. Considering the variability of presentation, organ involvement, and disease course in different subtypes and across ethnicities it is critical to define these associations in the ethnic populations we treat for their appropriate care considering the significant negative health and socioeconomic effects of AS.

The challenge of using the virtual crossmatch as a singular tool for the detection of Anti-HLA antibodies- A study from a tertiary care institute from South India.

INTRODUCTION: Detection of donor specific antibodies (DSA) is critical in both solid organ and mismatched haematopoietic stem cell transplants. The single antigen bead assay (SAB) is widely used as a virtual crossmatch in these settings. However, HLA allele variation across ethnicities and differing genetic backgrounds is a well-known and acknowledged fact and representation of alleles prevalent in a population is key while using a virtual crossmatch as a sole decision making tool. Against this background, this study was performed to assess the feasibility of using the SAB as a single tool to identify DSA in our population. MATERIALS AND METHODS: The HLA alleles identified in the study population were analysed to assess their representation on SAB panels from two different vendors. RESULTS: The study population comprised of a total of 966 subjects for whom 6 loci high resolution HLA typing was done. A total of 241 different alleles were assigned in the population. Among the 241 alleles identified in our study population, 48.55% (n = 117) alleles were represented in the SAB A panel and 48.13% (n = 116) represented in the SAB B panel. Unrepresented alleles were 51.45% (n = 124) in panel A and 51.87% (n = 125) in panel B. All the twelve alleles were represented for 16.05% (n = 155) and 16.25% (n = 157) of study population in panel A and in panel B respectively. The remaining individuals (83.95%, (n = 811) in panel A and 83.75%, (n = 809) in panel B) had at least one allele unrepresented. CONCLUSION: Our study addresses an important limitation in utilizing the SAB as a single tool to identify DSA, owing to non-representation of locally prevalent / unique alleles in our population. More than 50% of alleles were unrepresented in both the SAB assays we studied, which included alleles from both Class I and Class II. We recommend therefore that, until a comprehensive coverage of alleles is provided, or epitope matching becomes robust, that the SAB be combined with a physical crossmatch when mismatched alleles are not represented.

Association of prior sensitizing events with anti-human leukocyte antigen antibodies: An analysis of renal transplant recipients in a tertiary care centre in South India.

Traditionally, sensitizing events such as previous pregnancies, previous transfusions and prior transplants result in the production of anti-Human Leukocyte Antigen (HLA) antibodies. However, it has been observed that, anti-HLA antibodies have been detected in many patients with no prior history of sensitizing events. This retrospective study analysed the most recent 100 consecutive Single Antigen Bead (SAB) assay results performed on 100 patients. The SAB assay is used routinely to detect anti-HLA antibodies in transplant recipients. Results of the SAB assay were analyzed and subsequently studied to see if a correlation existed between sensitizing events, the type of events and presence of antibody. Analysis showed that 77% (77/100) had anti-HLA antibodies. 61 out of 100 patients had prior sensitizing events while the remaining 39 had none. Both these groups showed an almost equal percent of patients with anti-HLA antibodies 77% (47/61) and 76.9% (30/39) respectively. A single sensitizing event was seen in 54.1% (33/61) patients including previous transfusions in 29.5% (18/61), pregnancies in 11.4% (7/61) and prior transplant in 13.1% (8/61). Our study suggests that irrespective of whether patients have prior sensitizing events or not, patients run the risks of alloimmunization, and therefore appropriate screening tests should be included in the pre-transplant compatibility algorithm.

Do red cell alloantibodies continue to challenge breast fed babies?

BACKGROUND: Newborns have limited specific immune capability at birth, owing to delayed and constrained development of adaptive immunity. To supplement this period the mother passively transfers antibodies to the child either transplacentally or through breast milk. When maternal alloimmunisation occurs through foreign or fetal red cell surface antigens, stimulating the production of immunoglobulin G (IgG) antibodies, these IgG antibodies can cross the placenta and cause haemolytic disease of the fetus and the newborn. OBJECTIVE: We present two case reports of a neonate and an infant in whom IgG red cell alloantibodies were transferred through maternal breast milk. METHODS: Maternal serum, baby's serum and expressed breast milk samples were tested for the presence of red cell alloantibodies using gel card. Antibody screening, antibody identifications and titres alongside monospecific direct antiglobulin test, IgG subtypes were performed using the standard methods. RESULTS: In the first case, a 6-month-old child was incidentally found to have positive antibody screen. Anti-KELL1 was identified, which was also present in maternal serum and breast milk. The second neonate was evaluated for haemolysis and was found to have anti-D. Anti-D was also detected in the maternal serum and breast milk. Both babies did not have any sensitising events. The first baby was asymptomatic, but the second baby had ongoing haemolysis until 1 month. CONCLUSION: We report that maternal anti-KELL1 and anti-D antibodies were present in breast milk and were capable of being transferred to a feeding child. Our case report also raises interesting and unanswered immunologic fundamentals that should be considered in neonates with unexplained anaemia or delayed and persistent haemolysis.