A longitudinal study of antibody responses to selected host antigens in rheumatic fever and rheumatic heart disease.

Introduction. Group A streptococci can trigger autoimmune responses that lead to acute rheumatic fever (ARF) and rheumatic heart disease (RHD).Gap Statement. Some autoantibodies generated in ARF/RHD target antigens in the S2 subfragment region of cardiac myosin. However, little is known about the kinetics of these antibodies during the disease process.Aim. To determine the antibody responses over time in patients and healthy controls against host tissue proteins - cardiac myosin and peptides from its S2 subfragment, tropomyosin, laminin and keratin.Methodology. We used enzyme-linked immunosorbent assays (ELISA) to determine antibody responses in: (1) healthy controls; (2) patients with streptococcal pharyngitis; (3) patients with ARF with carditis and (4) patients with RHD on penicillin prophylaxis.Results. We observed significantly higher antibody responses against extracellular proteins - laminin and keratin in pharyngitis group, patients with ARF and patients with RHD when compared to healthy controls. The antibody responses against intracellular proteins - cardiac myosin and tropomyosin were elevated only in the group of patients with ARF with active carditis. While the reactivity to S2 peptides S2-1-3, 8-11, 14, 16-18, 21-22 and 32 was higher in patients with ARF, the reactivity in the RHD group was high only against S2-1, 9, 11, 12 when compared to healthy controls. The reactivity against S2 peptides reduced as the disease condition stabilized in the ARF group whereas the reactivity remained unaltered in the RHD group. By contrast antibodies against laminin and keratin persisted in patients with RHD.Conclusion. Our findings of antibody responses against host proteins support the multistep hypothesis in the development of rheumatic carditis. The differential kinetics of serum antibody responses against S2 peptides may have potential use as markers of ongoing cardiac damage that can be used to monitor patients with ARF/RHD.

Anti-retroviral therapy failure in HIV-1 infected pregnant women and its associated risk of HIV transmission.

BACKGROUND: The HIV perinatal transmission in India even after interventions is still high. The anti-retroviral therapy failure rate and the risk of HIV vertical transmission to infants from women with failed treatment during pregnancy also largely remains unevaluated. METHODS: This is a prospective, observational and follow-up study of 18 months to determine the association of ART failure in pregnant women and the subsequent risk of HIV transmission to their infants. A total of 81 mothers were evaluated for ART success/failure by analysing their viral loads. RESULTS: Analyses revealed that a high percentage (19.75%) of women on ART had high viral loads, while the overall HIV transmission rate to the infants was 8.64%. The rate of transmission from women with high viral load was significantly high compared to women with low viral load (37.5% vs. 1.54%; p = 0.0015). CD4 level was not associated with HIV transmission. However, CD4 levels in women, who had successful or failed ART, were significantly different (p = 0.0031). Factors such as mother's age, baby's sex and weight as well as delivery mode were not associated with HIV transmission, however, breastfeeding and viral loads were found to be independently associated with HIV transmission to the neonates. CONCLUSIONS: This study highlights that a significant proportion of women on ART had impaired viral load control. The rate of HIV transmission to infants was also significantly high among these women. This warrants viral load monitoring of HIV infected women to reduce the overall transmission to the infants.

Seroprevalence of Toxoplasma gondii in newly diagnosed HIV seropositive patients.

BACKGROUND & OBJECTIVES: Immunocompromised individuals mainly HIV infected patients are at a great risk for developing toxoplasmosis. The presence of toxoplasmosis among HIV-infected patients directly correlates with the prevalence of anti- Toxoplasma gondii antibodies and the degree of immunosuppression (measured by CD4 counts). The data regarding the seroprevalence of toxoplasmosis in HIV-infected patients are scarce in India. Therefore, this study was initiated to find out the seroprevalence of toxoplasmosis in treatment-naïve HIV seropositive patients and to determine its association with CD4 counts, if any. METHODS: Four hundred newly diagnosed antiretroviral therapy (ART) naïve adult HIV positive patients coming for CD4 count estimation were tested for the presence of anti- Toxoplasma IgG antibodies. Risk factors for acquisition of toxoplasmosis as well as the age, gender and CD4 counts of the patient were noted down. RESULTS: Toxoplasma IgG was positive in 292 (73%) patients, and the positivity was not related to their CD4 counts. The proportion of anti- Toxoplasma IgG positivity showed no significant association with age, gender and risk factors of the patients. INTERPRETATION & CONCLUSIONS: In the absence of any specific vaccine or prophylaxis for toxoplasmosis, it is pertinent to screen all HIV-positive patients for Toxoplasma IgG at diagnosis, irrespective of their CD4 counts, and sensitize them about the means to prevent either acquisition or activation of infection to avert the development of toxoplasmic encephalitis.

Distribution of human leucocyte antigen-C in HIV-1-infected patients in Mumbai, India.

HIV pathogenesis is known to be highly influenced by host genetic factors, such as human leucocyte antigens (HLAs) HLA-A and HLA-B. However, the role of HLA-C remains largely unexplored. We evaluated HLA-C distribution in 186 HIV-1-infected individuals and compared them to ethnically matched data derived from the Allele Frequency Net Database using Chi-square test with Fisher's exact two-tailed test. The frequency of HLA-C*05 and HLA-C*15 was higher in infected group, whereas the frequency of HLA-C*04 and HLA-C*14 was higher in control group. HLA-C*17, a rare allele, was significantly higher in infected group. These data could be useful in designing and testing vaccines in Indian population.

Single-dose and Steady-state Pharmacokinetics of Vancomycin in Critically Ill Patients Admitted to Medical Intensive Care Unit of India.

RATIONALE: Vancomycin remains the standard of care for gram-positive bacterial infections, though there are significant developments in newer antibacterial agents. Efficacy can be improved by linking pharmacokinetic with pharmacodynamic principles, thus leading to optimum antibiotic exposure. There is scarcity of pharmacokinetic data in Indian intensive care unit (ICU) population. MATERIALS AND METHODS: Fifteen subjects with suspected or proven gram-positive bacterial infection of either gender between 18 years and 65 years of age were enrolled. Vancomycin at the dose of 1 g every 12 hours was administered over 1-hour period and pharmacokinetic assessments performed on blood samples collected on days 1 and 3. Vancomycin concentrations were measured on validated liquid chromatography mass spectrometry method. Pharmacokinetic parameters were calculated using Winnonlin (Version 6.3; Pharsight, St. Louis, MO). RESULTS: The mean C max, elimination half-life, AUC0-12hours, volume of distribution, and clearance of single dose were 36.46 µg/mL (±14.87), 3.98 hours (±1.31), 113.51 µg/mL (±49.51), 52.01 L (±31.31), and 8.90 mL/minute (±3.29), respectively, and at steady state were 40.87 µg/mL (±19.29), 6.27 hours (±3.39), 147.94 µg/mL (±72.89), 56.39 L (±42.13), and 6.98 mL/minute (±4.48), respectively. The elimination half-life increased almost two-fold at steady state. The steady state mean AUC0-24 was 295.89 µg/mL (±153.82). Out of 45 trough levels, 32 (71.11%) concentrations were below recommended range. CONCLUSION: Recommended AUC0-24hours and trough concentrations were not achieved in majority of patients with current dosing, suggesting reevaluation of current vancomycin dosing. Individualized treatment based on close monitoring of vancomycin serum concentrations in critically ill patients is imperative. HOW TO CITE THIS ARTICLE: Mali NB, Deshpande SP, Wandalkar PP, Gupta VA, Karnik ND, Gogtay NJ, et al. Single-dose and Steady-state Pharmacokinetics of Vancomycin in Critically Ill Patients Admitted to Medical Intensive Care Unit of India. IJCCM 2019;23(11):513-517.

Carba NP as a simpler, rapid, cost-effective, and a more sensitive alternative to other phenotypic tests for detection of carbapenem resistance in routine diagnostic laboratories.

PURPOSE: Resistance to carbapenems due to carbapenemases has been increasingly noticed in Enterobacteriaceae. Clinical and Laboratory Standards Institute (CLSI) has recommended the latest Carba NP (CNP) test as a confirmatory test for carbapenemase production in Enterobacteriaceae. Low sensitivity of disk diffusion (DD) and modified Hodge test (MHT) may result in missing out of resistant strains which can adversely affect clinical management. The present study compares three phenotypic tests - CNP test, DD, and MHT for detection of carbapenemase production. MATERIALS AND METHODS: Four hundred consecutive, nonduplicate Enterobacteriaceae isolates were tested for carbapenem resistance using ertapenem disc (10 µg) by Kirby-Bauer DD method, MHT, and CNP. These tests were performed and interpreted as per the CLSI standards. CNP was considered to be the reference test for comparison. Sensitivity, specificity, and accuracy rates for ertapenem DD and MHT were calculated. RESULTS: One hundred and six out of 400 strains were positive by CNP test. Of the 294 CNP-negative strains, 28 were resistant by DD and 18 were resistant by MHT. Of the 106 CNP-positive strains, 82 were resistant and 16 were intermediate by DD while 76 were positive by MHT ertapenem DD had a sensitivity and specificity of 66.04% and 90.48%, respectively. Sensitivity and specificity of MHT were 54.72% and 93.88%, respectively. There was considerable discordance between all the three tests. CONCLUSION: As a rapid, simple, and cost-effective test with a greater capability greater to detect carbapenemase producers, CNP can be implemented in routine diagnostic laboratories, thereby benefiting patient care and antimicrobial stewardship.

LED fluorescence microscopy: Novel method for malaria diagnosis compared with routine methods.

Rapid and accurate diagnosis of malaria is the need of hour for effective management and controlling drug resistance. The conventional and gold-standard method, Light microscopy (LM), is time-consuming, requires trained staff and well-maintained equipments. The newly developed, rapid diagnostic tests (RDT) are fast and reliable, but give only qualitative results, are expensive and have short shelf life. Light Emission Diode fluorescence microscopy (LED FM) may provide a reliable alternative which can be used for routine diagnosis. In order to assess the effectiveness of LED fluorescence microscopy in malaria diagnosis, a cross-sectional study was conducted at a tertiary care teaching hospital in Mumbai. 2-3ml of blood of 300 patients, who were clinically suspected of having malaria but were not on anti-malarial treatment, was collected in EDTA vials. These specimens were processed to diagnose malaria by three methods, namely-Peripheral smear examination with LM, Peripheral smear examination with LED FM and RDT. The results of all the 3 tests were compared, taking Light Microscopy as the gold standard method. Of the 300 specimens, LM, LED FM and RDT reported 111 (37%), 86 (28.67%) and 107 (35.67%), respectively, as positive. The sensitivity and specificity were respectively 71.2% and 96.3% for LED FM and 91% and 96.8% for RDT. Of the LM positive cases, 53 (47.75%) had parasitic index (PI) <1% and 58 (52.25%) had PI ≥1%. LED FM was found to be only moderately sensitive but highly specific in comparison to Light microscopy. In order to improve the performance of this technique, more precise training in fluorescence staining and reading of the slides, will be required.

Chronic Giardiasis in a Case of Common Variable Immunodeficiency (CVID): A Case Report.

Common Variable Immunodeficiency (CVID) is a primary immunodeficiency characterized by low antibody levels and recurrent infections. This makes an individual more prone to recurrent respiratory and gastrointestinal tract infections. In cases where there is persistent positive finding of intestinal parasites in stool, a high index of suspicion should be raised to rule out immunodeficiency state. Early diagnosis of such cases will help in reducing the morbidity and better management of the patient. A case of CVID in 18-year-old male with recurrent lower respiratory tract infection and chronic diarrhoea due to Giardia lamblia is reported herewith.

Seropositivity for Antibodies to DRS-G, a Virulence Factor from Streptococcus dysgalactiae subsp. equisimilis, Is an Independent Risk Factor for Poststreptococcus Glomerulonephritis and Chronic Kidney Disease in Mumbai, India.

The disease spectrum caused by Streptococcus dysgalactiae subsp. equisimilis resembles that of S. pyogenes (group A streptococcus [GAS]). These two bacterial species are closely related and possess many common virulence characteristics. While some GAS strains express virulence factors called streptococcal inhibitor of complement (SIC) and distantly related to SIC (DRS), some S. dysgalactiae subsp. equisimilis isolates express an orthologue of DRS, which is referred to as DRS-G. We reported previously that seropositivity for either anti-SIC or anti-DRS antibodies (Abs) is associated with poststreptococcal glomerulonephritis (PSGN). However, only seropositivity for anti-SIC Abs is associated with chronic kidney disease (CKD). We now extend the study to test whether seropositivity for anti-DRS-G Abs is also associated with these renal diseases. Stored serum samples collected for our previous study were tested by an enzyme-linked immunosorbent assay (ELISA) for Abs to DRS-G. The samples represented sera from 100 CKD adult patients, 70 adult end-stage renal disease (ESRD) patients, 25 PSGN pediatric patients, and corresponding age-matched control subjects. The proportion of PSGN, CKD, and ESRD patients who showed seroreaction to anti-DRS-G Abs was significantly higher than that of the corresponding age-matched controls, who in general exhibited seropositivity rates commensurate with the isolation rate of drsG-positive S. dysgalactiae subsp. equisimilis in the community during this study period. Since higher rates of seropositivity for anti-DRS-G Abs in the renal disease categories are resultant of previous infections with DRS-G-positive S. dysgalactiae subsp. equisimilis strains, we conclude the seropositivity is an additional risk factor for these renal diseases. In this regard, anti-DRS-G Abs have attributes similar to those of the anti-SIC Abs.

Antibodies to group A streptococcal virulence factors, SIC and DRS, increase predilection to GAS pyoderma.

BACKGROUND: Streptococcus pyogenes (group A streptococcus; GAS) is an etiological agent for pharyngitis, pyoderma, and invasive infections in humans. Pharyngitis and pyoderma may lead to serious immune sequelae such as rheumatic heart disease and post-streptococcal glomerulonephritis (PSGN). Streptococcal Inhibitor of Complement (SIC) and its orthologue, distantly related to SIC (DRS), are virulence factors expressed by only four of more than 100 M types of GAS. These four types (M1, M57, M12 and M55) are among the M types, which are associated with PSGN. In several populations PSGN has been shown to be a risk factor for chronic kidney disease (CKD) and end-stage renal disease (ESRD). Previous studies showed SIC or DRS antibody-prevalence was associated with PSGN, and seroprevalence of SIC antibodies is significantly high among CKD and ESRD patients in Mumbai. METHODS: Streptococcal isolates recovered from GAS pyoderma cases were typed. Seropositivity for SIC and DRS antibodies in subjects with pyoderma, PSGN pediatric cases, age matched healthy controls and non-GAS pyoderma cases were determined. RESULTS: We confirm in this study an association between seroprevalence to SIC and DRS antibodies, and PSGN in Mumbai population despite low point prevalence of M1, M12, M55 and M57. In addition we extended the study to GAS-pyoderma and non-GAS pyoderma cases. To our surprise, we found a positive association between the seroprevalence to SIC and DRS antibodies, and GAS-pyoderma owing to infection with diverse M types. The mechanism of increased predisposition to pyoderma owing to infection by diverse GAS among SIC or DRS antibody-positive population is not clear. Nonetheless, our findings could be explained by a phenomenon akin to antibody-dependent enhancement (ADE). CONCLUSIONS: This is the first report showing a small number of GAS M types conferring predisposition to pyoderma by diverse types. Implications of this ADE-like phenomenon are discussed in the light of evolutionary advantage to GAS, vaccine design and control of renal diseases.

Hepatitis B Virus (HBV) Infection in Liver Disease Patients in Mumbai, India with Special Reference to Hepatitis B Surface Antigen (HBsAg) Mutant Detection.

OBJECTIVES: To determine the prevalence of Hepatitis B Surface antigen (HBsAg) in patients attending the Hepatology Out Patient Department (OPD) of a tertiary care hospital and to compare the routinely used HBsAg detection kit with the mutant detection kit to find out the presence of mutants in a given setting. MATERIALS AND METHODS: A cross-sectional study was carried out in adult patients with liver disease attending the Hepatology OPD, of a tertiary care hospital in Mumbai, India. Age, gender and clinical history of the patient were recorded. Blood specimen was tested for HBsAg (Microscreen(TM) ELISA, Span diagnostics, India) and HBsAg mutants (Hepanostika HBsAg Ultra(TM) ELISA, Biomerieux, France). The samples with discordant results between these two ELISAs were confirmed by Hepatitis B Virus (HBV) Deoxyribonucleic Acid (DNA) Polymerase Chain Reaction (PCR) (Cobas Taqman(TM), Roche Molecular Systems, USA). RESULTS: Seven hundred and eighteen patients were enrolled in the study. The mean age of patients in the study group was 41 years (range 17 to 69 years). Four hundred and ninety seven (69.22%) were males and remaining were females. The prevalence of HBsAg was found to be 17.4%. The positivity amongst the male population was 18.1% which was higher than the female population (15.8%). Of the 718 samples tested, 120 were positive for HBsAg by Microscreen(TM) ELISA and 132 were positive by Hepanostika HBsAg ultra(TM). Of the 12 discordant samples, HBV DNA was detected in five samples indicating 0.7% prevalence of mutants. CONCLUSION: Hepatitis B is prevalent in liver disease patients. The mutant detecting assay is recommended in set-ups where missing HBsAg in patients would have tremendous impact on the outcome such as in blood donors, organ or tissue donors and antenatal screening of mothers. It is also helpful in chronic liver disease patients where the routine HBsAg detection test is negative and the other causes of chronic liver disease have been ruled out. However, it is not recommended for use in routine diagnostic set-ups where high false positivity would lead to over-diagnosis of the condition.

HIV-2 Infection: Where Are We Today?

CONTEXT: The choice of antiretroviral therapy for HIV-2 differs from that for HIV-1, underscoring the importance of differentiating between the two. AIMS: The current study was planned to find out the prevalence of HIV-2 infection at our center and to find out the utility of the current diagnostic algorithm in identifying the type of HIV infection. SETTING AND DESIGN: Retrospective analysis in a tertiary care teaching institute over a period of three years. MATERIALS AND METHODS: All patients diagnosed as HIV infected using NACO/WHO HIV testing strategy III were included in the study. They were classified as HIV-1 infected, HIV-2 infected and HIV-1 and HIV-2 co-infected based on their test results. For discordant samples, immunoblotting result from National Reference Laboratory was considered as final. STATISTICAL ANALYSIS USED: Comparison between HIV-1, HIV-2 and HIV-1+2 positive groups for age, gender, route of transmission was made using chi squared test. P value < 0.05 was considered as significant. RESULTS: Of the total of 66,708 patients tested, 5,238 (7.9%) were positive for HIV antibodies. 7.62%, 0.14%, 0.08% and 0.004% were HIV-1, HIV-2, HIV-1 and HIV-2 co-infected and HIV type indeterminate (HIV-1 Indeterminate, 2+) respectively. The current algorithm could not differentiate between the types of HIV infection (as HIV-1 or HIV-2) in 63 (1.2%) cases. CONCLUSION: In areas like the Indian subcontinent, where epidemic of both HIV-1 and HIV-2 infections are ongoing, it is important to modify the current diagnostic algorithms to diagnose and confirm HIV-2 infections.

Human leukocyte antigen B distribution in HIV discordant cohort from India.

Limited reports are available on association of HLA-B with HIV infection from India, a home to the third largest population of HIV infected people in the world. This emphasizes the need to have more information specifically the genetic constitution of HIV serodiscordant couples (DCs), where one spouse is seropositive (HSP) while the other remains seronegative (HSN) even after repeated exposure. Hence, aim of this study was to document association of HLA-B with HIV infection in DCs living in Mumbai, India. A cohort was designed to enroll DCs attending the ICTC/Shakti Clinic of KEM Hospital, Mumbai. A group of unexposed volunteers were also enrolled as healthy controls (HC). HLA-B alleles were typed using sequence-specific oligonucleotide probes. Allele frequency comparison was done using 2×2 contingency tables. Results were considered significant, when p<0.05 with two-tailed Fisher's exact test. At HLA-B locus, the frequencies of HLA-B*40;-B*35;-B*07;-B*15;-B*51;-B*44;-B*52;-B*37 and -B*57 were found in decreasing order in the population. Frequency of HLA-B*35 allele was significantly higher (HSP vs HSN; p<0.02 and HSP vs HC; p<0.04) in HSP. HLA-B*40 (HSN vs HSP; p<0.01 and HC vs HSP; p<0.01) and HLA-B*18 (HSN vs HSP; p<0.02) were significantly associated with HSN. Both HSN and HC had similar HLA-B*35 and -B*40 allele frequency. HLA-B*57 allele was observed in 15 individuals (3.69%). However, HLA-B*57:01 which is known to be associated with adverse reactions against Abacavir was observed in 7 of them. HLA-B*39 was observed exclusively in HSP. Our observation in DCs confirmed the association of HLA-B*35 with susceptibility while HLA-B*40 (specifically *B40:06), -B*18 with protection. These identified alleles can be used as possible marker associated with HIV transmission. In India, HLA screening is not carried out before initiation of HIV treatment. However, the presence of HLA-B*57:01 in the population emphasizes the importance of such screening to predict/avoid Abacavir hypersensitivity.

Pharmacokinetics of colistin in critically ill patients with multidrug-resistant Gram-negative bacilli infection.

PURPOSE: Colistin, which had not been used widely because of nephrotoxicity and neurotoxicity, has gained clinical importance in recent times due to the resurgence of multidrug-resistant Gram-negative bacilli. Very few studies, especially pharmacokinetic studies, have been performed with intravenous colistimethate sodium, and none in India. The aim of our study was to study the single-dose and steady-state pharmacokinetics of colistin in patients with multidrug-resistant Gram-negative bacilli infections. METHOD: This was a prospective open-label pharmacokinetic study done in an intensive care unit in a tertiary care hospital on 15 critically ill patients with proven multidrug-resistant Gram-negative bacilli infection. Colistimethate sodium was injected as intermittent intravenous infusions in accordance with the recommendations on the package insert. For patients weighing ≥ 60 kg with a normal renal function or with a creatinine clearance (CL(CR)) of between 20 and 50 ml/min, the drug was administered at 2 million international units (MIU) every 8 h; for those with a CL(CR) of 10-20 ml/min, the dose was 2 MIU every 12 h. Those patients who weighed <60 kg were administered 50,000 IU/kg/day in three divided doses at 8-h intervals. Both single-dose and steady-state pharmacokinetics of colistin were determined and correlated with clinical outcomes. RESULTS: A wide inter-individual variation was observed in pharmacokinetic parameters. The median (range) of the maximum plasma drug concentration/minimum inhibitory concentration (C(max)/MIC) ratio for Acinetobacter spp. was 13.4 (1.3-40.3) following the administration of a single dose of colistimethate sodium and 26.3 (0.9-64.9) at steady-state. For Pseudomonas spp., these values were 3.18 (1.6-23.1) following the single dose and 3.82 (2.3-10.9) at steady-state. For those patients whose cultures grew Acinetobacter spp., an optimum value of the C(max)/MIC ratio of >8 was achieved in seven of nine patients after the single dose and in seven of eight patients at steady-state. For those patients whose cultures grew Pseudomonas spp, only one patient after the single dose and one patient at steady-state achieved a C(max)/MIC ratio of >8. A significant association was noted between dose and survival, and a trend was observed with patients weighing ≤ 60 kg (who received 50,000 IU/kg/day instead of 6 MIU/day for those >60 kg) having an increased mortality. CONCLUSION: The pharmacokinetic parameters of colistin were comparable to those reported in previous studies in critically ill patients. However, the recommended dose may be inadequate to maintain the C(max)/MIC ratio to an optimal level-at least in patients infected with Pseudomonas spp. The dose recommendation should be based only on creatinine clearance and not body weight.