Suppressed transcript diversity and immune response in COVID-19 ICU patients: a longitudinal study.

Understanding the dynamic changes in gene expression during Acute Respiratory Distress Syndrome (ARDS) progression in post-acute infection patients is crucial for unraveling the underlying mechanisms. Study investigates the longitudinal changes in gene/transcript expression patterns in hospital-admitted severe COVID-19 patients with ARDS post-acute SARS-CoV-2 infection. Blood samples were collected at three time points and patients were stratified into severe and mild ARDS, based on their oxygenation saturation (SpO2/FiO2) kinetics over 7 d. Decline in transcript diversity was observed over time, particularly in patients with higher severity, indicating dysregulated transcriptional landscape. Comparing gene/transcript-level analyses highlighted a rather limited overlap. With disease progression, a transition towards an inflammatory state was evident. Strong association was found between antibody response and disease severity, characterized by decreased antibody response and activated B cell population in severe cases. Bayesian network analysis identified various factors associated with disease progression and severity, viz. humoral response, TLR signaling, inflammatory response, interferon response, and effector T cell abundance. The findings highlight dynamic gene/transcript expression changes during ARDS progression, impact on tissue oxygenation and elucidate disease pathogenesis.

Association of gut microbial dysbiosis with disease severity, response to therapy and disease outcomes in Indian patients with COVID-19.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is associated with systemic hyper-inflammation. An adaptive interaction between gut microbiota and host immune systems is important for intestinal homeostasis and systemic immune regulation. The association of gut microbial composition and functions with COVID-19 disease severity is sparse, especially in India. We analysed faecal microbial diversity and abundances in a cohort of Indian COVID-19 patients to identify key signatures in the gut microbial ecology in patients with severe COVID-19 disease as well as in response to different therapies. The composition of the gut microbiome was characterized using 16Sr RNA gene sequences of genomic DNA extracted from faecal samples of 52 COVID-19 patients. Metabolic pathways across the groups were predicted using PICRUSt2. All statistical analyses were done using Vegan in the R environment. Plasma cytokine abundance at recruitment was measured in a multiplex assay. RESULTS: The gut microbiome composition of mild and severe patients was found to be significantly different. Immunomodulatory commensals, viz. Lachnospiraceae family members and Bifidobacteria producing butyrate and short-chain fatty acids (SCFAs), were under represented in patients with severe COVID-19, with an increased abundance of opportunistic pathogens like Eggerthella. The higher abundance of Lachnoclostridium in severe disease was reduced in response to convalescent plasma therapy. Specific microbial genera showed distinctive trends in enriched metabolic pathways, strong correlations with blood plasma cytokine levels, and associative link to disease outcomes. CONCLUSION: Our study indicates that, along with SARS-CoV-2, a dysbiotic gut microbial community may also play an important role in COVID-19 severity through modulation of host immune responses.

Circulating Interleukin-8 Dynamics Parallels Disease Course and Is Linked to Clinical Outcomes in Severe COVID-19.

Severe COVID-19 frequently features a systemic deluge of cytokines. Circulating cytokines that can stratify risks are useful for more effective triage and management. Here, we ran a machine-learning algorithm on a dataset of 36 plasma cytokines in a cohort of severe COVID-19 to identify cytokine/s useful for describing the dynamic clinical state in multiple regression analysis. We performed RNA-sequencing of circulating blood cells collected at different time-points. From a Bayesian Information Criterion analysis, a combination of interleukin-8 (IL-8), Eotaxin, and Interferon-γ (IFNγ) was found to be significantly linked to blood oxygenation over seven days. Individually testing the cytokines in receiver operator characteristics analyses identified IL-8 as a strong stratifier for clinical outcomes. Circulating IL-8 dynamics paralleled disease course. We also revealed key transitions in immune transcriptome in patients stratified for circulating IL-8 at three time-points. The study identifies plasma IL-8 as a key pathogenic cytokine linking systemic hyper-inflammation to the clinical outcomes in COVID-19.

Clinical Trial Subgroup Analyses to Investigate Clinical and Immunological Outcomes of Convalescent Plasma Therapy in Severe COVID-19.

Objective: To assess the clinical and immunological benefits of passive immunization using convalescent plasma therapy (CPT). Materials and Methods: A series of subclass analyses were performed on the previously published outcome data and accompanying clinical metadata from a completed randomized controlled trial (RCT) (Clinical Trial Registry of India, number CTRI/2020/05/025209). The subclass analyses were performed on the outcome data and accompanying clinical metadata from a completed RCT (patient recruitment between May 15, 2020 and October 31, 2020). Data on the plasma abundance of a large panel of cytokines from the same cohort of patients were also used to characterize the heterogeneity of the putative anti-inflammatory function of convalescent plasma (CP) in addition to passively providing neutralizing antibodies. Results: Although the primary clinical outcomes were not significantly different in the RCT across all age groups, significant immediate mitigation of hypoxia, reduction in hospital stay, and significant survival benefit were registered in younger (<67 years in our cohort) patients with severe coronavirus disease 2019 and acute respiratory distress syndrome on receiving CPT. In addition to neutralizing the antibody content of CP, its anti-inflammatory proteome, by attenuation of the systemic cytokine deluge, significantly contributed to the clinical benefits of CPT. Conclusion: Subgroup analyses revealed that clinical benefits of CPT in severe coronavirus disease 2019 are linked to the anti-inflammatory protein content of CP apart from the anti-severe acute respiratory syndrome coronavirus 2 neutralizing antibody content.

A phase 2 single center open label randomised control trial for convalescent plasma therapy in patients with severe COVID-19.

A single center open label phase 2 randomised control trial (Clinical Trial Registry of India No. CTRI/2020/05/025209) was done to assess clinical and immunological benefits of passive immunization using convalescent plasma therapy. At the Infectious Diseases and Beleghata General Hospital in Kolkata, India, 80 patients hospitalized with severe COVID-19 disease and fulfilling the inclusion criteria (aged more than 18 years, with either mild ARDS having PaO2/FiO2 200-300 or moderate ARDS having PaO2/FiO2 100-200, not on mechanical ventilation) were recruited and randomized into either standard of care (SOC) arm (N = 40) or the convalescent plasma therapy (CPT) arm (N = 40). Primary outcomes were all-cause mortality by day 30 of enrolment and immunological correlates of response to therapy if any, for which plasma abundance of a large panel of cytokines was quantitated before and after intervention to assess the effect of CPT on the systemic hyper-inflammation encountered in these patients. The secondary outcomes were recovery from ARDS and time taken to negative viral RNA PCR as well as to report any adverse reaction to plasma therapy. Transfused convalescent plasma was characterized in terms of its neutralizing antibody content as well as proteome. The trial was completed and it was found that primary outcome of all-cause mortality was not significantly different among severe COVID-19 patients with ARDS randomized to two treatment arms (Mantel-Haenszel Hazard Ratio 0.6731, 95% confidence interval 0.3010-1.505, with a P value of 0.3424 on Mantel-Cox Log-rank test). No adverse effect was reported with CPT. In severe COVID-19 patients with mild or moderate ARDS no significant clinical benefit was registered in this clinical trial with convalescent plasma therapy in terms of prespecified outcomes.

Development and Validation of a Treatment Benefit Index to Identify Hospitalized Patients With COVID-19 Who May Benefit From Convalescent Plasma.

Importance: Identifying which patients with COVID-19 are likely to benefit from COVID-19 convalescent plasma (CCP) treatment may have a large public health impact. Objective: To develop an index for predicting the expected relative treatment benefit from CCP compared with treatment without CCP for patients hospitalized for COVID-19 using patients' baseline characteristics. Design, Setting, and Participants: This prognostic study used data from the COMPILE study, ie, a meta-analysis of pooled individual patient data from 8 randomized clinical trials (RCTs) evaluating CCP vs control in adults hospitalized for COVID-19 who were not receiving mechanical ventilation at randomization. A combination of baseline characteristics, termed the treatment benefit index (TBI), was developed based on 2287 patients in COMPILE using a proportional odds model, with baseline characteristics selected via cross-validation. The TBI was externally validated on 4 external data sets: the Expanded Access Program (1896 participants), a study conducted under Emergency Use Authorization (210 participants), and 2 RCTs (with 80 and 309 participants). Exposure: Receipt of CCP. Main Outcomes and Measures: World Health Organization (WHO) 11-point ordinal COVID-19 clinical status scale and 2 derivatives of it (ie, WHO score of 7-10, indicating mechanical ventilation to death, and WHO score of 10, indicating death) at day 14 and day 28 after randomization. Day 14 WHO 11-point ordinal scale was used as the primary outcome to develop the TBI. Results: A total of 2287 patients were included in the derivation cohort, with a mean (SD) age of 60.3 (15.2) years and 815 (35.6%) women. The TBI provided a continuous gradation of benefit, and, for clinical utility, it was operationalized into groups of expected large clinical benefit (B1; 629 participants in the derivation cohort [27.5%]), moderate benefit (B2; 953 [41.7%]), and potential harm or no benefit (B3; 705 [30.8%]). Patients with preexisting conditions (diabetes, cardiovascular and pulmonary diseases), with blood type A or AB, and at an early COVID-19 stage (low baseline WHO scores) were expected to benefit most, while those without preexisting conditions and at more advanced stages of COVID-19 could potentially be harmed. In the derivation cohort, odds ratios for worse outcome, where smaller odds ratios indicate larger benefit from CCP, were 0.69 (95% credible interval [CrI], 0.48-1.06) for B1, 0.82 (95% CrI, 0.61-1.11) for B2, and 1.58 (95% CrI, 1.14-2.17) for B3. Testing on 4 external datasets supported the validation of the derived TBIs. Conclusions and Relevance: The findings of this study suggest that the CCP TBI is a simple tool that can quantify the relative benefit from CCP treatment for an individual patient hospitalized with COVID-19 that can be used to guide treatment recommendations. The TBI precision medicine approach could be especially helpful in a pandemic.

Plasma Gradient of Soluble Urokinase-Type Plasminogen Activator Receptor Is Linked to Pathogenic Plasma Proteome and Immune Transcriptome and Stratifies Outcomes in Severe COVID-19.

Disease caused by SARS-CoV-2 coronavirus (COVID-19) led to significant morbidity and mortality worldwide. A systemic hyper-inflammation characterizes severe COVID-19 disease, often associated with acute respiratory distress syndrome (ARDS). Blood biomarkers capable of risk stratification are of great importance in effective triage and critical care of severe COVID-19 patients. Flow cytometry and next-generation sequencing were done on peripheral blood cells and urokinase-type plasminogen activator receptor (suPAR), and cytokines were measured from and mass spectrometry-based proteomics was done on plasma samples from an Indian cohort of COVID-19 patients. Publicly available single-cell RNA sequencing data were analyzed for validation of primary data. Statistical analyses were performed to validate risk stratification. We report here higher plasma abundance of suPAR, expressed by an abnormally expanded myeloid cell population, in severe COVID-19 patients with ARDS. The plasma suPAR level was found to be linked to a characteristic plasma proteome, associated with coagulation disorders and complement activation. Receiver operator characteristic curve analysis to predict mortality identified a cutoff value of suPAR at 1,996.809 pg/ml (odds ratio: 2.9286, 95% confidence interval 1.0427-8.2257). Lower-than-cutoff suPAR levels were associated with a differential expression of the immune transcriptome as well as favorable clinical outcomes, in terms of both survival benefit (hazard ratio: 0.3615, 95% confidence interval 0.1433-0.912) and faster disease remission in our patient cohort. Thus, we identified suPAR as a key pathogenic circulating molecule linking systemic hyperinflammation to the hypercoagulable state and stratifying clinical outcomes in severe COVID-19 patients with ARDS.

Isolation, characterization and acyclovir susceptibility of herpes simplex virus isolates among immunocompromised patients.

INTRODUCTION: Herpes simplex virus (HSV) Type 2 primarily causes genital herpes, while HSV Type 1 is responsible for oral and facial lesions. The objective of this study was to isolate and characterize HSV from herpetic lesions among human immunodeficiency virus (HIV) infected patients and to evaluate their acyclovir susceptibility pattern. MATERIALS AND METHODS: Blister fluid and swabs from ulcers were collected from patients with clinical diagnosis of HSV infection among patients attending the HIV clinic of two tertiary care centers - Medical College, Kolkata, and School of Tropical Medicine, Kolkata. These samples were cultured in the Vero cell line. Growth of virus was noted by observing the characteristic cytopathic effect of HSV, which was further confirmed by immunofluorescence and polymerase chain reaction (PCR). These isolates were then subjected to the Vero cells with serial dilutions of acyclovir for determining the susceptibility pattern. RESULTS: Among the 52 samples received, 8 (15.38%) showed growth of HSV. After confirmation by immunofluorescence and PCR, all seven isolates from genital samples were identified as HSV-2 and the lone isolate from oral lesion was confirmed as HSV 1. Out of the eight isolates, 25% showed resistance to acyclovir. The overall isolation rate was more from genital blister than genital ulcer which was 46.15% and 2.86%, respectively. CONCLUSION: HSV was isolated in 15.38% of cases of clinical herpes. There was a higher isolation rate of virus from blister fluid as compared to ulcer scrapings. Acyclovir resistance in 25% of cases is alarmingly high.

Nature and Dimensions of Systemic Hyperinflammation and its Attenuation by Convalescent Plasma in Severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), has led to significant morbidity and mortality. While most suffer from mild symptoms, some patients progress to severe disease with acute respiratory distress syndrome (ARDS) and associated systemic hyperinflammation. METHODS: First, to characterize key cytokines and their dynamics in this hyperinflammatory condition, we assessed abundance and correlative expression of a panel of 48 cytokines in patients progressing to ARDS as compared to patients with mild disease. Then, in an ongoing randomized controlled trial of convalescent plasma therapy (CPT), we analyzed rapid effects of CPT on the systemic cytokine dynamics as a correlate for the level of hypoxia experienced by the patients. RESULTS: We identified an anti-inflammatory role of CPT independent of its neutralizing antibody content. CONCLUSIONS: Neutralizing antibodies, as well as reductions in circulating interleukin-6 and interferon-γ-inducible protein 10, contributed to marked rapid reductions in hypoxia in response to CPT. CLINICAL TRIAL REGISTRY OF INDIA: CTRI/2020/05/025209. http://www.ctri.nic.in/.

A prospective study on COVID-19 convalescent plasma donor (CCP) recruitment strategies in a resource constrained blood centre.

Background and Objectives: The COVID-19 pandemic has spread across 87 million people with more than 1·8 million deaths in the world. As there is no definite treatment modality, the use of convalescent plasma has become increasingly popular worldwide. This study aimed to identify an appropriate strategy of donor recruitment and to evaluate the appropriateness of pre-set plasma donation guidelines. Material and Methods: In this prospective study conducted from May to September 2020, the donors were recruited under the following two circumstances: Group I, patients in the post-COVID-19 follow-up in the clinic, and Group II, patients recovered from COVID-19 recruited through mass and electronic media. A pre-set donor selection criteria and laboratory investigation was designed according to national and international guidelines. Approximately 500 ml of COVID-19 convalescent plasma (CCP) was collected from recovered individuals in each group by two different cell separators. The overall donor's attendance rate, deferral rate, adverse events and donor compliance was analysed and compared between the two groups. Results: There was a significant difference in attendance in relation to registration between the groups (P < 0·0001). Donor deferral was significantly higher in group II compared with group I. The single most frequent cause of donor deferral was low antibody index (P = 0·0001). The total donor adverse event rate in CCP donation was significantly lower compared with routine plateletpheresis procedures. The donor's compliance to blood centre's protocol was satisfactory in both the groups. Conclusion: Recruitment of patients in the post-COVID-19 follow-up in the clinic was more effective than the general recruitment through mass and electronic media for convalescence plasma donation in a resource-constrained blood centre.

Nontuberculous mycobacteria: A report of eighteen cases from a tertiary care center in India.

CONTEXT: Nontuberculous mycobacteria (NTM) are ubiquitous mycobacteria present in environment and generally affect patients with either structural lung disease or immunosuppression and commonly involve lungs, lymph node, or skin. MATERIALS AND METHODS: Between July 2016 and February 2019, 18 cases of NTM were diagnosed and their relevant clinical, diagnostic, and treatment details were recorded after taking informed consent. RESULTS: We report 18 cases of NTM involving lungs (n = 11), skin and soft tissue (n = 3), joint (n = 2), genitourinary (n = 1), and central nervous system (n = 1). History of immunosuppression was present in two patients, whereas history of some form of intervention was seen in six patients. Mycobacterium fortuitum group (n = 5) was the most commonly isolated organism, followed by Mycobacterium avium complex (n = 4), Mycobacterium abscessus (n = 3), Mycobacterium kansasii (n = 2), and Mycobacterium chelonae (n = 1). In two patients, M. chelonae and M. abscessus were isolated in succession. Of these 18 patients, clinical response was present in 15 of the patients. Diagnosis and treatment of NTM in resource limited settings is extremely challenging. CONCLUSION: Most of the patients with NTM are misdiagnosed and are treated as tuberculosis in India, sometimes with a multidrug resistance regimen, which results in significant morbidity and mortality. We present these cases to shed some light on the epidemiology of NTM in this part of India.

Impact of trainee-driven Antimicrobial Stewardship Program in a high burden resource-limited setting.

Antimicrobial Stewardship Program (ASP) is one of the most critical interventions required to halt the growing global antimicrobial resistance. The study aimed to evaluate the effect of trainee driven ASP implementation with limited available resources on outcome variables. An ASP team comprising of infectious diseases trainees and consultants was constituted to conduct stewardship activities in the Department of Medicine, All India Institute of Medical Sciences, a tertiary care apex institute in north India. Prospective audit and feedback were conducted by the team, and the following outcome variables were recorded and analyzed: the practice of sending cultures, appropriateness of prescribed empiric antibiotics, gross antimicrobial consumption and mortality. ASP intervention led to an increase in blood culture positivity rates by two folds (p<0.001). The trend of empiric prescription choices gradually shifted over time towards the use of more effective antibiotics according to the local antibiogram. Redundant usage of antibiotics substantially reduced over time. There was no impact of the antimicrobial stewardship program on the all-cause mortality rate. ASP had a significant effect on the practice of sending cultures and appropriateness of antibiotic usage. In resource-limited settings, trainee-driven antimicrobial stewardship program can succeed in inculcating rational practices among fellow residents and practicing physicians.

Choosing the therapy for neurological infection with rapidly growing mycobacteria.

The management of neurological infections due to non-tubercular mycobacteria is extremely challenging because of scarce literature, issues with penetration, lack of easily available susceptibility platforms and adverse effects associated with long term therapy. We report a case of a young girl with neurological infection due to rapidly growing mycobacteria to discuss the factors that should be considered while choosing the therapy for such rare and persistent infections.

Risk Benefit Analysis in Reference to use of LMWH in COVID-19.

Venous thromboembolism (VTE) and disseminated intravascular coagulation (DIC) are frequent cardiovascular and/or respiratory complications among hospitalized patients of COVID-19 infection. A relatively high mortality of severe coronavirus disease 2019 (COVID-19) is worrying, and the application of heparin in COVID-19 has been assessed and recommended with some expert consensus because of the risk of DIC and venous thromboembolism. However, "Risk Benefit Analysis" on the aspect of safety in using low molecular weight heparin (LMWH) in COVID-19 patients for thrombosis prophylaxis has been explained below with a few case studies and detailed information from various clinical evidence. COVID-19 infection has been associated with inflammation and a prothrombotic state, with increase in fibrin, fibrin degradation products, fibrinogen, and D-dimers. Heparin treatment including unfractionated and low molecular weight heparin appears to be associated with better prognosis in severe COVID-19 patients with coagulopathy. Major studies since the onset of this pandemic, found better prognosis in severe COVID-19 patients meeting SIC criteria or with markedly elevated D-dimer, by approaching thrombosis prophylaxis with LMWH.

Brain abscess in patients with chronic kidney disease: A case-based approach to management in resource-limited settings.

The management of patients with brain abscess poses a significant challenge to clinicians in patients with chronic kidney disease. Obtaining a biopsy sample from the affected area is the mainstay in the diagnosis, but it is often unavailable. In most cases, therapy is guided by clinical findings and imaging alone. We discuss three cases of brain abscess- each with a different scenario and discuss the issues faced in management. The first case was a 32-year-old post-renal transplant male patient with a brain abscess due to dematiaceous fungi and was treated with amphotericin. The second case was a 42-year-old female patient with stage 5 chronic kidney disease on maintenance hemodialysis who presented with a brain abscess due to suspected fungal infection based on imaging findings and was managed with antibiotics and voriconazole. The third case was a 42-year-old post-renal transplant male patient who presented with a brain abscess due to nocardiosis and was managed with cotrimoxazole, meropenem and linezolid. We also summarize the approach to the management of brain abscess in resource-limited settings.

Nipah virus disease: A rare and intractable disease.

Nipah virus, an enveloped ribonucleic acid virus, has been a major cause of encephalitis out-breaks with high mortality, primarily in the Indo-Bangladesh regions. Except for the first outbreak in Malaysia-Singapore, which was related to contact with pigs and the outbreak in Philippines associated with horse slaughter, most other outbreaks have affected the Indo- Bangladesh regions. The Indo-Bangladesh outbreaks were associated with consumption of raw date palm sap contaminated by fruit bats and had a very high secondary attack rate. The patient usually presents with fever, encephalitis and/or respiratory involvement with or without thrombocytopenia, leukopenia and transaminitis. Diagnosis can be confirmed by isolation and nucleic acid amplification in the acute phase or antibody detection during the convalescent phase. Treatment is mostly limited to supportive care and syndromic management of acute encephalitis syndrome. Ribavirin, m102.4 monoclonal antibody and favipiravir are the only anti-virals with some activity against Nipah virus. Standard precautions, hand hygiene and personal protective equipments are the cornerstone of comprehensive infection prevention and control strategy. With the recent outbreaks affecting newer geographical areas, there is a need for physicians to be aware of this disease and keep abreast of its current detection and management strategies.

SLE in a Male Patient Presented Initially as Rowell's Syndrome.

A 22 year old male Indian patient presented with high grade fever, multiple joint pain, low back pain, generalized body ache since 6 months and erythematous pruritic rashes and atypical annular target like lesions over face, arm, leg and back and ulcers on hard palate and buccal mucosa for 2 months. Laboratory investigations showed a speckled pattern anti-nuclear antibody with a titer >1:160 and positive SS-A, dsDNA auto-antibodies and Rheumatoid factor. Diagnosis of Rowell's syndrome was made based on clinical and laboratory finding and the patient was treated with oral prednisolone (50 mg/day), hydroxychloroquine (200 mg q12h) and pulse cyclophosphamide (700 mg) chemotherapy. Majority of skin lesions and oral ulcerations subsided after 4 weeks of therapy. Till date only 11 male patients out of the total 71 cases of Rowell's syndrome were reported in the world's literature.

Nosocomial pneumonia: Search for an empiric and effective antibiotic regimen in high burden tertiary care centre.

The clinical practice guidelines on nosocomial pneumonia recommends an empirical regimen that would work in 95% of the patients based on the local antibiogram. The aim of the study was development of an antibiogram for guiding empiric therapy in settings with high prevalence of multi-drug resistant organisms. A retrospective review of electronic health records (e-hospital portal) was done to analyze all respiratory isolates from patients admitted in medical wards and intensive care unit between May 2016 and May 2017. The samples included brocho-alveolar lavage (BAL), mini broncho-alveolar lavage (mini-BAL) and endotracheal aspirate. The sensitivity pattern (combined and individual) of all bacterial isolates were analysed for commonly used antibiotics and their combinations. Out of the 269 isolates, the most common organisms were Pseudomonas aeruginosa (125, 46%), Acinetobacter baumanni (74, 27%) and Klebsiella pneumoniae (50, 19%). Cefoperazone-sulbactam (43%) had the best sensitivity pattern overall. Cefoperazone-sulbactam plus amikacin (56%) was the combination with the best combined sensitivity overall. There is a high prevalence of resistance in the commonly implicated organisms to the available antibiotics. There is an urgent need for implementation of effective anti-microbial stewardship programmes and development of newer antimicrobials.

Scleroderma-like Initial Presentation of Multiple Myeloma.

Systemic sclerosis (SSc) is a multisystem connective tissue disease affecting skin and internal organs. Certain drugs, environmental toxins and some viruses have been implicated in SSc-like illnesses. Scleroderma may be associated with some connective tissue disorders or autoimmune diseases but coexistence of scleroderma with multiple myeloma (MM) is an unusual finding. We here report a case of a 59 years old female patient with 5 months history of progressive thickening of skin all over the body. Multiple myeloma was diagnosed by osteolytic lesion in skull X-ray, increase in clonal plasma cells by bone marrow biopsy, very high Kappa light chain in serum light chain assay and detection of M band by serum protein electrophoresis.